迟缓爱德华氏菌 铁蛋白 Dps(DNA-binding proteins from starved cells)论文

ReviewThe tumor suppressor kinase LKB 1:lessons from mouse modelsSaara Ollila and Tomi P.Ma¨kela ¨*Institute of Biotechnology,University of Helsinki,Viikki Biocenter,Viikinkaari 9B,FIN-00014,Helsinki,Finland*Correspondence to:Tomi P.Ma¨kela ¨,E-mail:tomi.makela@helsinki.fiMutations in the tumor suppressor gene LKB 1are important in hereditary Peutz–Jeghers syndrome,as well as in sporadic cancersincluding lung and cervical cancer.LKB 1is a kinase-activating kinase,and a number of LKB 1-dependent phosphorylation cascades regulate fundamental cellular and organismal processes in at least metabolism,polarity,cytoskeleton organization,and prolifer-ation.Conditional targeting approaches are beginning to demonstrate the relevance and specificity of these signaling pathways in development and homeostasis of multiple organs.More than one of the pathways also appear to contribute to tumor growth fol-lowing Lkb 1deficiencies based on a number of mouse tumor models.Lkb 1-dependent activation of AMPK and subsequent inacti-vation of mammalian target of rapamycin signaling are implicated in several of the models,and other less well characterized pathways are also involved.Conditional targeting studies of Lkb 1also point an important role of LKB 1in epithelial–mesenchymal interactions,significantly expanding knowledge on the relevance of LKB 1in human disease.Keywords:LKB 1,tumor suppressor,mouse model,AMPKIntroductionCancer arises as a result of accumulating genetic and epige-netic changes,which compromise the cell’s ability to control its identity and proliferation.Many identified tumor suppressors play a well-established role in regulation of cell growth and div-ision (e.g.Rb,APC,p 21,PTEN)and genome maintenance (e.g.p 53,BRCA 1-2,ATM,ATR,MLH 1,MSH 2),providing a logical link between the loss of gene product and promotion of carcinogen-esis.An interesting exception is the serine /threonine kinase gene LKB 1(also known as STK 11),which has in recent years taken a prominent position among tumor suppressors.Heterozygous germline mutations in LKB 1predispose to Peutz–Jeghers syndrome (PJS)where patients develop benign polyps in the gastrointestinal (GI)tract and are in high risk of developing malignant tumors in GI tract,breast,and gyneco-logical organs (Giardiello et al .,2000).Importantly,somatic LKB 1mutations are found at least in lung (Ji et al.,2007)and cervical cancer (Wingo et al .,2009).Through phosphoryl-ation of several cellular kinases LKB 1has been implicated in control of cellular and organismal metabolism,cell polarity,and a variety of other functions ranging from proliferation and migration to senescence,apoptosis,DNA damage responseand differentiation (Vaahtomeri and Ma¨kela ¨,2011).Despite these many functions attributed to LKB 1,their specific contri-butions to the maintenance of tissue homeostasis in vivo and tumor growth are only sketchily appearing with thedevelopment of LKB 1mouse models.This work is important to enable rational treatment strategies to LKB 1-deficient tumors.The LKB 1kinase acts in a trimer with a pseudokinase STRAD and the scaffold protein MO 25to phosphorylate at least 14kinases with conserved activation sites (Katajisto et al.,2007).A well-known substrate of LKB 1is AMPK,which is the master reg-ulator of cellular and organismal metabolism,providing a putative downstream pathway to LKB 1-mediated tumor suppression (Shackelford and Shaw,2009).In mouse studies,AMPK requires LKB 1for activation in vivo in most tissues (Sakamoto et al .,2005;Shaw et al .,2005;Contreras et al .,2008;Hezel et al .,2008).AMPK senses the energy state of cells through monitoring AMP levels as a sensitive readout for ATP.AMPK is activated following exercise,hypoxia,or glucose deprivation,after which it phosphor-ylates multiple targets to increase energy uptake and catabolic processes such as glucose uptake and fatty acid oxidation,and suppress anabolic processes such as lipogenesis and cholesterol synthesis (Hardie et al.,2003).AMPK is the potential candidate to mediate LKB 1’s effects in cell growth via the mammalian target of rapamycin (mTOR)signal-ing (Corradetti et al .,2004;Shaw et al .,2004),which is the pathway monitoring the availability of nutrients in regulation of cell size and protein synthesis as well as proliferation (Zoncu et al.,2011).Increased mTOR signaling is common in cancer (Guertin and Sabatini,2007)and also present in at least some Lkb 1-deficient tumors (Shaw et al.,2004;Ji et al.,2007;Contreras et al.,2008;Hezel et al.,2008;Shackelford et al.,2009).An additional link between LKB 1and mTOR pathway#The Author (2011).Published by Oxford University Press on behalf of Journal ofMolecular Cell Biology ,IBCB,SIBS,CAS.All rights reserved.doi:10.1093/jmcb /mjr 016Journal of Molecular Cell Biology (2011),Vol no.0,1–11|1Journal of Molecular Cell Biology Advance Access published September 15, 2011 at Shihezi University on September 27, 2011 Downloaded frommay lie in regulation of PI 3K-Akt pathway inhibitor PTEN by LKB 1(Mehenni et al .,2005).Loss of cell polarity is commonly noted in cancer,and LKB 1is an important factor for cell polarity in different organisms.In C.elegans ,the orthologs for LKB 1(par-4)and MARK s (par-1)were identified in a panel of six partitioning (par )mutants which disrupted the polarity of the early embryos (Kemphues et al.,1988).In Drosophila ,Lkb 1is required for proper oocyte polarity (Martin and St Johnston,2003).In mammalian cells,in both 2D and 3D cell culture models and in vivo ,LKB 1is known to regulate polarity (Baas et al .,2004;Partanen et al .,2007;Hezel et al .,2008).Polarity defects are,however,not seen in all Lkb 1-deficient tumors (Contreras et al.,2008,2010).Several of the LKB 1substrates have been reported to mediate the regulation of cell polarity through regulating the cytoskeleton and formation of cell–cell junctions.MARK kinases are implicated in the stability of microtubules by phosphorylating and thereby dissociation microtubule-associated proteins (MAPs),for example the tau protein,from microtubules (Drewes et al .,1997;Stoothoff and Johnson,2005).Neuronal polarity and axon formation are regu-lated by LKB 1at least partially via BRSK kinases (Kishi et al.,2005;Barnes et al.,2007).To what extent LKB 1acts as a polarity protein in mammalian non-neuronal cells still remains to be deter-mined,although at least in both exo-and endocrine pancreas Lkb 1loss leads to polarity defects in vivo (Hezel et al .,2008;Granot et al .,2009).As formation of stress fibers is essential incell contractility,recent studies associate LKB 1with cell motility via NUAK 1and NUAK 2,which have been implicated in regulation of myosin light chain phosphorylation (Vallenius et al.,2010;Zagorska et al.,2010).For detailed information of the molecular signaling pathways of Lkb 1,the reader is recommended recent reviews more focused on that topic (Katajisto et al.,2007;Hezel and Bardeesy,2008;Vaahtomeri and Ma¨kela ¨,2011).Role of Lkb 1in development and tissue homeostasis in miceAlthough LKB 1is a tumor suppressor,inactivation of Lkb 1through homologous recombination or ‘knock-out’(KO)does not always lead to tumors.This is due partly to essential functions of Lkb 1in development and partly demonstrates the tissue-specificity of Lkb 1functions,where in some cell types biallelic deletion is detrimental to cells or affects specific functions in metabolism as summarized in Figure 1and discussed below.Role of Lkb 1in embryogenesisGeneration of full KO revealed that Lkb 1is essential for embry-ogenesis;no viable Lkb 12/2embryos were seen after E 11.Analysis of the E 8.5–E 9.5embryos revealed severe developmen-tal defects including impaired neural tube closure and somitogen-esis,mesenchymal tissue cell death,and defective vasculature.The extra-embryonic tissues (yolk sac and placenta)were also deformed.VEGF signaling was highly upregulated in theKOFigure 1Non-tumorigenic phenotypes following Lkb 1targeting in mice.Phenotypes (green)are grouped according to tissue type,cell typeaffected /analyzed (blue),and alleles used for targeting.When appropriate,activator of deletor is indicated in purple.Noted signaling change(s)indicated in red.Alleles as displayed in original publications except for Lkb 1flox 2h /flox 2h hypomorphic Lkb 1(Sakamoto et al,2005).(1)Londesborough et al.,2008;(2)Ohashi et al.,2010;(3)Cao et al.,2010;Tamas et al.,2010;(4)Shorning et al.,2009;(5)Woods et al.,2011;(6)Shaw et al.,2005;(7)Sun et al.,2010a ;(8)Sun et al.,2011;(9)Granot et al.,2009;Fu et al.,2009;(10)Koh et al.,2006;(11)Sakamoto et al.,2005;(12)Sakamoto et al.,2006;Jessen,et al.,2010;(13)Ikeda et al.2009;(14)Gurumurthy et al.,2010;Nakada et al.,2010;(15)Gan et al.,2010;(16)Barnes et al.,2007;(17)Ylikorkala et al.,2001.tam,tamoxifen;b -NF,b -naphtoflavone;pIpC,polyinosinic–polycytidylic acid;iv,intravenous.2|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromembryos,possibly relating to the vascular phenotype (Ylikorkala et al .,2001).Embryonic lethality,no embryonic turning,and small somites were also shown in another report of Lkb 1full KO (Jishage et al .,2002).The severe developmental defect was not a result of the abnormal extra-embryonic tissues,since epiblast-specific conditional inactivation of Lkb 1using Mox 1-Cre resulted in very similar embryonic lethal phenotype to full KO (Londesborough et al .,2008).The important role of Lkb 1in development and maintenance of neurons,mesenchymal cells,and vascularization has been recapitulated in tissue-specific Lkb 1KOs.Role of Lkb 1in angiogenesisLondesborough et al .(2008)further dissected the role of Lkb 1in endothelia by deleting Lkb 1in vascular endothelial cells using Tie 1-Cre (Figure 1).The mice died at E 12.5and displayed dilated embryonic vessels and pericardial swelling.The vessels were irre-gular and distorted and suffered from inadequate supportive vas-cular smooth muscle cell layer.Since Tgf b signaling was reduced both in Lkb 1-deficient mouse yolk sacs and human umbilical vein endothelial cells (HUVECs)where LKB 1expression was silenced by siRNA,the vascular phenotype was suggested to result from a loss of supporting vascular smooth muscle cells as a conse-quence of attenuated Tgf b signaling from endothelial cells (Londesborough et al .,2008).Another report also described mice lacking Lkb 1in endothelial cells,deleted using Tie 2-Cre driver (Ohashi et al.,2010)(Figure 1).This study repeated the finding that endothelial Lkb 1is essential for proper embryonic development and no homozygous mutants were born.Analysis of heterozygous Tie 2-Cre;Lkb 1flox /+mice revealed that the mice,including vasculature,seemed phenotypically normal,but displayed reduced revascularization after hind-limb ischemia.Studies in mouse tissues,primary mouse endothelial cells,and HUVECs implemented that the phenotype was mediated via AMPK (Ohashi et al.,2010).In this study,the authors did not address the contribution of Tgf b signaling to the observed phenotype.In the Tie 2-Cre model,Lkb 1–AMPK axis seemed to mediate proangiogenetic signaling as Lkb 1heterozygosity resulted in reduction of revascularization in adult mice (Ohashi et al.,2010).In developing embryo,increased VEGF signaling upon Lkb 1loss would suggest the opposite,antiangiogenic role for Lkb 1(Ylikorkala et al .,2001).Also in the context of PJS polyps where a loss of Lkb 1leads to increased HIF 1a and vasculature,Lkb 1seems to be rather antiangiogenic (Shackelford et al .,2009).However,reduced capillary density was reported in mice where Lkb 1was conditionally deleted from the heart (Ikeda et al .,2009).In 3D culture system where endothelial cells are embedded in Matrigel,both over-expression (Xie et al .,2009)and inhibition (Ohashi et al.,2010)of Lkb 1have been reported to inhibit network formation,suggesting proper expression of LKB 1is essential for angiogenesis.Thus,the precise role of Lkb 1in angiogenesis seems to be dependent on the tissue type and /or the developmental phase,varying from inhibition to promotion.Role of Lkb 1in liverThe finding that Lkb 1functions upstream of AMPK (Shaw et al .,2004)led to interest to study its effects in liver,where many path-ways of carbohydrate and lipid metabolism,including glycogen-esis,glycogenolysis,gluconeogenesis,lipogenesis,and cholesterol synthesis take place.Tail-vain injection of Adeno-Cre to mice carrying conditional Lkb 1allele led to hepatocyte-specific Lkb 1deletion since Adeno-Cre has high tropism for hepatocytes (Shaw et al .,2005)(Figure 1).Lkb 1loss resulted in nearly complete abolishment of AMPK activation in liver,and the glucose metabolism of the mice was impaired demonstrated by elevated blood glucose.CRTC 2phosphorylation was reduced in the livers of the mice,leading to elevated CREB-mediated transcription,including expression of PGC 1a and other gluconeogenetic genes.Also lipogenetic genes were over-expressed.Metformin,the diabetes drug which reduces blood glucose levels via AMPK pathway (Zhou et al .,2001),did not lower blood glucose in the liver-specific Lkb 1KO mice,demonstrating that AMPK activity induced by Lkb 1in liver is required for the effects of metformin in vivo .In another report of liver-specific Lkb 1knockout using Alb-Cre driver,Woods et al.(2011)reported defective bile ducts in liver,leading to accumulation of bile in liver and serum (Figure 1).Bile salt export pump was not located in canalicular membrane of the bile canaliculi,indicating possible defects in cell polarity.The mice also suffered from cholestasis (Woods et al.,2011).These reports of liver-targeted deletions of Lkb 1demonstrate the critical requirement of Lkb 1in glucose,lipid,bile,and cholesterol metab-olism.Furthermore,they show that in liver,Lkb 1is the main acti-vator of AMPK,and its activity is required for the AMPK-mediated suppression of lipogenesis and gluconeogenesis to take place.Role of Lkb 1in muscleMuscles are highly energy-consuming tissues whose glucose homeostasis needs to be regulated both in response to insulin after blood sugar increase,and to exercise-mediated deficiency of glucose storage.Sakamoto et al.(2005)provided the first genetic evidence that Lkb 1is required for AMPK activation in vivo in skeletal muscle.They generated conditional Lkb 1mice in which cDNA of Lkb 1exons 5–7fused with neomycin resistance cassette,surrounded by loxP sites,was inserted between exons 4and 8in the genomic Lkb 1locus.The resulting mice were hypomorphic and expressed only 10%–20%of normal levels of Lkb 1in the absence of Cre -mediated ing MCK-Cre driver to create muscle-specific Lkb 1KO,they found that AMPK a 2(one of the two alternative catalytic subunits of AMPK)activation either by the AMP analog AICAR,muscle con-traction or phenformin,a similar blood glucose lowering drug to metformin,was lost and AMPK a 1activation greatly reduced.Upon contraction,glucose transport to muscle cells was abol-ished (Sakamoto et al.,2005).In another study using the same muscle-specific MCK-Cre with another (non-hypomorphic)con-ditional Lkb 1line,effects of Lkb 1loss in muscle to levels of blood glucose were investigated (Koh et al.,2006)(Figure 1).Interestingly,glucose metabolism seemed to be enhanced in these mice,demonstrated by reduced fasting blood glucose and blood insulin concentrations,improved glucose tolerance,and increased muscle glucose uptake.This phenotype,indicating that Lkb 1in muscle functions as a negative regulator of glucose metabolism,was suggested to be resulting from improved muscle glucose uptake,mediated by increased phosphorylation of Akt and reduced the gene expression of the Akt inhibitor TRB 3.Lkb 1loss abolished the activity of AMPK a 2,but notLessons from LKB 1mouse modelsJournal of Molecular Cell Biology |3at Shihezi University on September 27, 2011 Downloaded fromAMPK a 1in muscle cells.Also MARK 4,but not MARK 2/3activitywas significantly reduced.Based on this study,the metabolic effects mediated by Lkb 1in muscle seem to oppose those of the liver,at least in terms of blood glucose levels (Koh et al.,2006).Recently,the Lkb 1substrate NUAK 2was proposed to be a mediator of contraction-stimulated glucose transport by skel-etal muscle (Koh et al.,2010).Also cardiac muscle lacking Lkb 1has been investigated.Sakamoto et al.(2006)studied the effect of Lkb 1deficiency in heart using the MCK-Cre driver,which deletes Lkb 1in both skel-etal and cardiac myocytes and found that Lkb 1inactivation did not lead to overt cardiac dysfunction,although the weight of the heart was reduced and the atria enlarged;however,the study revealed that cardiac Lkb 1is required for activation of AMPK a 2both in basal conditions and in response to ischemia (Figure 1).Also Jessen et al.(2010)used the MCK-Cre driver but the Lkb 1allele was not hypomorphic as in the Sakamoto et al.(2006)study.They showed that ablation of Lkb 1in heart leads to impaired cardiac function both in basic conditions and post-ischemia and suggested that failure to downregulate mTOR sig-naling by AMPK a 2activation underlined the phenotypes.Ikeda et al.(2009)used a -MHC-Cre to delete Lkb 1specifically from the heart,and a more severe phenotype was observed:the mice displayed hypertrophy and impaired function of the heart,reduction of cardiac capillary density,and increased fibrosis and collagen content and died by 6months of age.The differ-ences between these phenotypes may reflect differences in the timing of Cre activity,specificity of the Cre recombination,and /or the conditional Lkb 1allele used.However,it seems clear that Lkb 1is needed for the normal function of heart both in basal and ischemic conditions.Role of Lkb 1in pancreasPancreatic b -cells secrete insulin and are thus important mediators of whole-body glucose metabolism.As Lkb 1–AMPK axis is important in regulation of liver metabolism and muscle glucose homeostasis,it is of interest to study whether Lkb 1has an effect on the insulin release.Granot et al.(2009)used the Pdx 1-CreER driver to delete pancreatic Lkb 1in 6-week-old mice by tamoxifen injection (Figure 1).In response to glucose injection,the mutant mice secreted more insulin than control mice,which carried the conditional Lkb 1allele but were not subjected to tamoxifen injection.Deletion of Lkb 1led to increased size of b -cells together with disrupted polarity.Increased mTOR signal-ing seemed to mediate the cell size increase,while the polarity defect took place at least partially through MARK 2.Increased insulin secretion was partially dependent on AMPK (Granot et al .,2009).Fu et al .(2009)used the same Pdx 1-CreER system to delete Lkb 1in adult b -cells and also found that the mice showed improved glucose tolerance,b -cells mass had increased,and mTOR pathway was activated (Figure 1).These results place Lkb 1as an important regulator of pancreatic b -cell size,polarity,and function,further highlighting its essence in regulation of organismal metabolism.Sun et al.(2010a)investigated pancreatic b -cells with the Rip 2-Cre driver,which activates Cre -mediated recombination in pancreatic b -cells and some hypothalamic neurons,and found that the mice displayed diminished food intake and weight gain,enhanced insulin secretion,and improved glucose tolerance (Figure 1).Also here,mTOR pathway was activated.However,the study by the same group where both AMPK a subunits were deleted in b -cells using the same Rip 2-Cre showed decreased insulin secretion (Sun et al.,2010b ).This suggests that Lkb 1loss regulates mTOR signaling in b -cells partially independent of AMPK,or that the hypothalamic Lkb 1and AMPK have different functions,impacting the feeding behavior and hormonal balance.Role of Lkb 1in immune systemThree recent studies elegantly demonstrated that Lkb 1regu-lates the quiescence and maintenance of hematopoietic stem cells (HSCs)using conditional Lkb 1alleles with Mx 1-Cre followed by injections of polyinosinic–polycytidylic acid (pIpC),or Rosa 26-CreERt 2followed by tamoxifen injections (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010)(Figure 1).Both approaches resulted in a similar phenotype:increased pro-liferation followed soon by decline in HSC number,resulting in loss of all immune cell types (pancytopenia)and death.Transplantation experiments demonstrated that Lkb 1-deficient HSCs were not able to reconstitute the bone marrow of irradiated wild-type (wt)mice,nor were they able to compete with wt donor cells,demonstrating that the effect was cell-autonomous;mito-chondrial defects and decreased ATP levels,as well as altered long-chain fatty acid and nucleotide metabolite levels suggested metabolic defects to underlie the phenotypes noted (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).Interestingly,only minor similarities in mitochondrial phenotypes were found when mice defective for both AMPK a subunits were compared with Lkb 1KO mice (Nakada et al.,2010),implicating other Lkb 1substrates in these phenotypes.Consistent with this,rapamycin or AMPK activators AICAR and A 769662did not rescue the phenotype in any of the studies.Immune cell apopto-sis was increased,and Lkb 1-deficient HSCs also demonstrated increased autophagy in bone marrow,and inhibiting this further decreased immune cell survival (Gan et al.,2010;Gurumurthy et al.,2010;Nakada et al.,2010).This would suggest that Lkb 1in this context is suppressing autophagy,whereas previously it has been reported to activate it following elevation of reactive oxygen species (Alexander et al.,2010).Yet another phenotype potentially decreasing HSC viability was the noted increase in supernumerary centrosomes,aberrant mitotic spindles,and aneuploidy (Nakada et al.,2010),which could be due to compro-mised BRSK 2activity (Alvarado-Kristensson et al.,2009).Recently,two groups generated mice where Lkb 1expression is specifically abolished in the T cell progenitors using the proximal p 56lck-Cre promoter.The studies demonstrate severe deficiency in survival and proliferation of T cell progenitors and mature T cells in the absence of Lkb 1(Cao et al.,2010;Tamas et al.,2010)(Figure 1).Also the survival of isolated peripheral T cells in vitro was dependent on Lkb 1(Tamas et al.,2010).Transfection of thymo-cytes with constitutively active AMPK a 2partially rescued the thy-mocytes from cell death,indicating that thymocyte survival is mediated at least via AMPK pathway (Cao et al.,2010).Thus,the common hematopoietic cell precursors and T cell precursors seem to have different requirement for AMPK signaling,although cell sur-vival is defective in both cell types in the absence of Lkb 1.The studies in hematopoietic cells have revealed an interesting aspect4|Journal of Molecular Cell Biology Ollila and Ma¨kela ¨ at Shihezi University on September 27, 2011 Downloaded fromof Lkb 1biology:although being a tumor suppressor in some tissues,in others Lkb 1is required for survival.Role of Lkb 1in nervous systemLkb 1KO embryos exhibit severe deficiencies in development of neuronal tissues (Ylikorkala et al .,2001).Since LKB 1orthologs in nematodes and fruit flies have been identified through their indis-pensable role in establishing polarity (Kemphues et al .,1988;Martin and St Johnston,2003)and LKB 1regulates polarity also in some mammalian cells (Baas et al .,2004;Partanen et al .,2007),it was of interest to generate models which would reveal the in vivo relevance of Lkb 1in establishing the axon-dendrite polarity in neuronal cells.Barnes et al.(2007)deleted Lkb 1in cer-ebral cortex of developing mice using Emx-Cre driver and showed that Lkb 1and its substrates BRSK 1and BRSK 2are required for axon specification in the studied neurons.This finding confirmed the previously described role of BRSK kinases in neuronal polar-ization (Kishi et al .,2005),and placed Lkb 1as the upstream kinase required for the polarization to take place.Lkb 1-activated BRSKs were shown to modify the cytoskeleton by phosphorylating MAPs (Barnes et al.,2007).Studies in rat hip-pocampal neurons in vitro and developing rat cortical neurons in vivo agreed with the finding that Lkb 1is essential in establishing neuronal polarity;there,lack of either Lkb 1or STRAD prevented axon differentiation (Shelly et al.,2007).Interestingly,over-expression of Lkb 1and STRAD resulted in formation of multiple axons.PKA-mediated phosphorylation of Lkb 1Ser 431was shown to be required for the axon specification (Barnes et al.,2007;Shelly et al.,2007).Thus,Lkb 1activity is modulated by upstream factors in a tissue-and context-specific manner.Not only axon specification but also maintenance seems to be regulated via Lkb 1in some systems.Sun et al.(2011)reported,using the pancreatic and hypothalamic Rip 2-Cre ,that the mice developed hind-limb paralysis due to axon degeneration in thor-acic spinal cord neurons at about 7–8weeks of age (Figure 1).The Rip 2-Cre was found to be active also in spinal cord,especially in the thoracic segments.Deleting both AMPK a subunits did not result in axon degeneration or paralysis,and the authors specu-lated that in the absence of Lkb 1,the neuronal polarization and axon degeneration defects might be mediated by BRSK kinase pathways (Sun et al.,2011).PJS and its mouse modelsLKB 1was linked to human disease when its mutations were found to be causative for PJS (Hemminki et al .,1998;Jenne et al .,1998).A major manifestation in PJS is the appearance of large occluding hamartomatous polyps in the GI tract (Giardiello and Trimbath,2006).Mice carrying one inactivated allele of Lkb 1(Lkb 1+/2)recapitulate PJS by developing hamartomatous GI polyps which are indistinguishable from PJS patient polyps (Bardeesy et al .,2002;Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)(Figure 2),although in mice polyps appear more in the stomach and less in the small intestine.Polyps appear at 4–6months (Udd et al.,2010),and lead to lethality at an average age of 11months due primarily to obstructions.Biallelic loss of wt Lkb 1is not a prerequisite for polyp formation,indicating that Lkb 1is a haploinsufficient tumor suppressor at least in the context of PJS polyps (Jishage et al .,2002;Miyoshiet al .,2002;Rossi et al .,2002).Strong up-regulation of COX 2has been identified in the mouse and also PJS patient polyps (Rossi et al .,2002),and COX 2inhibitors have been shown to be efficient suppressors of PJS polyps (Udd et al .,2004).PJS is associated with elevated risk of cancer,especially in the GI tract,and also in breast,pancreas and gynecological cancers (Giardiello and Trimbath,2006;Hearle et al .,2006;Mehenni et al .,2006).Lkb 1+/2mice in turn have been reported to have increased frequency of cancer in liver (Nakau et al .,2002),bones (Robinson et al .,2008),and endometrium (Contreras et al .,2008)(Figure 2).Polyposis in Lkb 1+/2mice is accelerated in a p 53-deficient background (our unpublished data)(Wei et al .,2005;Takeda et al .,2006)(Figure 2),and p 53mutations are detected in the GI cancers of PJS patients (Miyaki et al .,2000).Despite these observations,progression of the benign hamarto-matous polyps to dysplasia or carcinoma is not clearly estab-lished possibly due to the rapid growth of the hamartomatous polyps leading to GI occlusions.As haploinsufficiency of Lkb 1is sufficient for polyp initiation (Jishage et al .,2002;Miyoshi et al .,2002;Rossi et al .,2002)though biallelic loss has been noted (Bardeesy et al .,2002),loss of the remaining allele of Lkb 1may represent a progression step,although it has also been suggested that the loss of Lkb 1is associated with the resist-ance to progression in this context (Bardeesy et al.,2002).Mesenchymal Lkb 1loss leads to PJS-type polyposis in mice PJS polyps are classified as hamartomatous polyps thought to contain all the cell types of the surrounding tissue.However,it was recently noted that epithelial differentiation is disrupted in gastric and small intestinal polyps in Lkb 1+/2mice (Udd et al.,2010),but the model did not enable distinguishing whether this was a cell autonomous function of Lkb 1in epithelial cells.Biallelic disruption of Lkb 1in GI epithelia lead to imbalanced differentiation and positioning of epithelial cells (Shorning et al .,2009)(Figure 1),but was not reported to be associated with tumorigenesis.Polyps in both PJS patients and Lkb 1+/2mice harbor a large component of smooth muscle tissue.Remarkably,in a mouse model,where Lkb 1deficiency was restricted to the smooth muscle lineage by using a tamoxifen-inducible SM 22-CreERt 2line,PJS type polyps appeared in stomachs of the mice with the hetero-and homozygous Lkb 1mutants (Katajisto et al .,2008)(Figure 2).The polyps appeared later than those in the Lkb 1+/2mice,suggesting either that tamoxifen-induced Lkb 1loss at 6weeks of age delayed the poly-posis,or that mesenchymal loss of Lkb 1signaling is sufficient to drive hyperproliferation of epithelial tissue,but that coexisting epithelial mutations accelerate the process.This interesting aspect of Lkb 1signaling in tissue interactions is discussed later.Other Lkb 1tumor mouse modelsInactivating LKB 1mutations are associated with the develop-ment of cancer in several tissues.Various strategies of targeted inactivation of Lkb 1in mice,sometimes in combination of other tumorigenic mutations,have led to the development of various types and grades of tumors in multiple tissues,sometimes mod-eling human cancers in very useful ways as discussed below and summarized in Figure 2.Lessons from LKB 1mouse modelsJournal of Molecular Cell Biology |5at Shihezi University on September 27, 2011 Downloaded from。

亚铁螯合酶铁死亡引言亚铁螯合酶（Subtilisin-like proprotein convertase）是一类重要的酶，参与多种细胞生理过程，其中之一是铁离子的调节。

铁离子在细胞内扮演着关键的角色，参与氧气运输、DNA合成和能量代谢等生物过程。

铁死亡是指细胞内铁离子水平异常降低，导致细胞功能受损甚至死亡的现象。

本文将详细探讨亚铁螯合酶在铁离子调节中的作用以及铁死亡的机制。

亚铁螯合酶的功能亚铁螯合酶是一类蛋白酶，主要参与蛋白质的剪切和修饰。

在铁离子调节中，亚铁螯合酶起到了重要的作用。

它能够识别和结合细胞内的亚铁离子，并将其释放到细胞质中。

亚铁离子在细胞内参与多个铁依赖性酶的活性中心，从而调节细胞的生理功能。

亚铁螯合酶的调节亚铁螯合酶的活性受到多种因素的调节，包括基因表达、翻译后修饰和亚铁离子的浓度。

在细胞内，亚铁螯合酶的基因表达受到转录因子的调节，其中最为重要的是铁调控蛋白（iron regulatory proteins，IRPs）的作用。

IRPs能够结合亚铁螯合酶基因的调控区域，促进其转录和翻译。

同时，IRPs还能够调节亚铁螯合酶的翻译后修饰，进一步调控其活性。

亚铁螯合酶与铁死亡的关系铁死亡是指细胞内铁离子水平异常降低，导致细胞功能受损甚至死亡的现象。

亚铁螯合酶在铁死亡中起到了重要的作用。

当细胞内的亚铁离子浓度显著下降时，亚铁螯合酶的活性会受到抑制，导致铁离子无法被有效地释放到细胞质中。

这会导致细胞内铁离子水平的进一步下降，最终导致细胞功能的受损和死亡。

铁死亡的机制铁死亡的机制涉及多个细胞生理过程，包括细胞膜的通透性改变、线粒体功能的受损和细胞凋亡的激活等。

以下是铁死亡的具体机制：1. 细胞膜通透性改变当细胞内的亚铁离子水平显著下降时，细胞膜的通透性会发生改变。

亚铁离子的减少会导致细胞膜上的离子通道关闭，从而影响细胞内外的离子平衡。

这会导致细胞内外浓度差的增大，进一步加剧细胞功能的受损。

2. 线粒体功能的受损亚铁离子在线粒体中参与细胞呼吸链的电子传递过程，维持线粒体的正常功能。

微生物群体中耐药滞留菌的研究进展郭静;张晟;亓庆国【摘要】Treated with a fatal dose of antibiotic,most microbial population will soon die,while some sub-population cannot be elimi-nated and thus survive.Different from the traditional resistant mutants,these bacterial strains do not change as to their gene sequence. After the collection and re-incubation of the bacterial strains,it's been found that their minimal inhibitory concentration ( MIC) remains the same.The sub-population that cannot be eliminated thoroughly is known as persisters.More and more studies suggest that persisters seem to play the main role in the recalcitrance of chronic infections.This paper reviewed the biological characteristics and the generation mechanism of persisters and their tolerance mechanism to antibiotic,as well as the strategy to eliminate them.%微生物群体在受到抗微生物药物冲击后，绝大多数很快死亡，但是有小部分的微生物亚群（ sub-population）并不能被彻底清除，与传统意义上的耐药菌（ resistant mutants）不同，这部分菌株并没有发生基因序列上的变化，因此收集这部分菌株再培养后，发现其最低抑菌浓度（MIC）并没有变化，这部分不能被彻底清除的微生物亚群被称为是滞留菌（persisters）。

Erchen Decoction improves iron homeostasis in mice with non-alcoholic fatty liver disease by regulating iron transport capacity in the spleenDENG Guanghui 1,2,JIA Hui 1,LI Yunjia 1,LI Junjie 1,WU Chaofeng 1,SHI Hao 1,QIN Mengchen 1,ZHAO Jiamin 1,LIU Chang 1,LIAO Yuxin 1,GAO Lei 1,21School of Traditional Chinese Medicine,Southern Medical University,Guangzhou 510515,China;2Department of Gastroenterology,Integrated Hospital of Traditional Chinese Medicine,Southern Medical University,Guangzhou 510315,China摘要：目的探讨二陈汤对非酒精性脂肪性肝病铁稳态的影响以及调控脾脏细胞铁离子转运能力的机制。

方法将36只雄性C57BL/6J 小鼠，随机分为对照组、模型组、二陈汤低剂量组（7.5g/kg ）、二陈汤中剂量组（15g/kg ）、二陈汤高剂量组（30g/kg ）、多烯磷脂酰胆碱组（9.12mg/kg ），6只/组。

对照组给予低脂低糖饮食，其余组给予高脂饲料饲养12周。

药物组于第7周开始灌胃给药，其余组灌胃等体积饮用水，3次/周。

采用HPLC-MS 检测二陈汤的活性成份；HE 染色和尼罗红染色评估肝脏脂质累积情况；普鲁士蓝染色观察脾脏铁含量；蛋白印迹法、免疫组织化学法以及免疫荧光染色法检测铁转运蛋白（Fpn1）、转铁蛋白受体（TfR ）、前列腺六跨膜上皮抗原3（Steap3）、血红素加氧酶1（HO-1）、Ter-119、CD163和CD68的表达。

ＤＯＩ:１０.３９６９/ｊ.ｉｓｓｎ.１６７１－４６９５.２０２０.０１０.００９㊀㊀文章编号:１６７１－４６９５(２０２０)０１０－１０３７－０４铁调素在肝纤维化中的表达特点及对肝星状细胞的作用杨爱婷㊀李为雨㊀严旭禛㊀陈巍㊀尤红∗㊀(首都医科大学附属北京友谊医院科研实验中心北京市临床医学研究所㊀国家消化系统疾病临床医学研究中心㊀北京㊀１０００５０)㊀㊀ʌ摘要ɔ㊀目的㊀观察铁调素在肝纤维化过程中的表达特点ꎬ评估铁调素对肝星状细胞(ＨＳＣ)的作用及其机制ꎮ方法㊀腹腔注射四氯化碳与橄榄油混合物诱发肝纤维化ꎬ于第０㊁４㊁８㊁１２周后处死大鼠ꎬ动态观察铁调素在血清和肝组织中的表达特点ꎮ用不同浓度的铁调素－２５多肽刺激大鼠星状细胞系(ＨＳＣ－Ｔ６)４８ｈꎬ检测纤维化相关指标如α－平滑肌肌动蛋白(α－ＳＭＡ)㊁金属蛋白酶组织抑制因子－１(ＴＩＭＰ－１)和Ｉ型胶原在基因和蛋白质水平的表达ꎮ数据比较采用单因素方差分析与Ｔｕｋｅｙ检验ꎮ结果㊀成功建立四氯化碳诱导的大鼠肝纤维化模型ꎮ对铁调素的检测发现ꎬ随着纤维化进展ꎬ肝组织中铁调素的基因水平在四氯化碳注射后逐渐降低(Ｐ<０.０５)ꎻ四氯化碳混合物注射后第４㊁８㊁１２周铁调素的相对表达分别为０.７５ʃ０.１２㊁０.５２ʃ０.２０㊁０.２０ʃ０.１０ꎮ血清中铁调素在四氯化碳混合物注射０㊁４㊁８㊁１２周分别为(３５１.４５ʃ５１.１２)ｐｇ/ｍｌ㊁(２５４.２４ʃ４９.４１)ｐｇ/ｍｌ㊁(２１１.４５ʃ４２.２８)ｐｇ/ｍｌ㊁(１８９.２３ʃ３０.２４)ｐｇ/ｍｌꎮ体外实验表明ꎬ外源性补充铁调素(１０ｎｇ/ｍｌ㊁１００ｎｇ/ｍｌ)可以直接抑制ＨＳＣ中纤维化相关基因(α－ＳＭＡꎬＴＩＭＰ－１和Ｉ型胶原)的表达ꎬ且具有剂量依赖性ꎮ进一步我们发现铁调素可以通过抑制ＴＧＦβ１信号中ＳＭＡＤ４的表达阻断ＴＧＦβ１诱导的星状细胞活化和Ｉ型胶原的分泌ꎮ结论㊀铁调素水平与纤维化进展负相关ꎬ体外补充铁调素能够抑制ＨＳＣ的活化并减少Ｉ型胶原的分泌ꎮʌ关键词ɔ㊀铁调素㊀肝星状细胞㊀肝纤维化Ｈｅｐｃｉｄｉｎｄｅｃｒｅａｓｅｄｈｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌｓａｃｔｉｖａｔｉｏｎ.ＹＡＮＧＡｉ－ｔｉｎｇꎬＬＩＷｅｉ－ｙｕꎬＹＡＮＸｕ－ｚｈｅｎꎬｅｔａｌ.ＥｘｐｅｒｉｍｅｎｔａｌａｎｄＴｒａｎｓｌａｔｉｏｎａｌＲｅ￣ｓｅａｒｃｈＣｅｎｔｅｒꎬＢｅｉｊｉｎｇＦｒｉｅｎｄｓｈｉｐＨｏｓｐｉｔａｌꎬＣａｐｉｔａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙꎬＢｅｉｊｉｎｇＣｌｉｎｉｃａｌＩｎｓｔｉｔｕｔｅꎬＮａｔｉｏｎａｌＣｌｉｎｉｃａｌＲｅｓｅａｒｃｈＣｅｎｔｅｒｏｆＤｉｇｅｓｔｉｖｅＤｉｓ￣ｅａｓｅｓꎬＢｅｉｊｉｎｇ１０００５０ꎬＣｈｉｎａ.ʌＡｂｓｔｒａｃｔɔ㊀Ｏｂｊｅｃｔｉｖｅ㊀Ｔｏｅｖａｌｕａｔｅｔｈｅｔｈｅｒａｐｅｕｔｉｃｐｏｔｅｎｔｉａｌｏｆｈｅｐｃｉｄｉｎｉｎｈｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌ(ＨＳＣ)ꎬｗｈｉｃｈｈａｖｅａｐｉｖｏｔａｌｒｏｌｅｉｎｌｉｖｅｒｆｉｂｒｏｓｉｓ.Ｍｅｔｈｏｄｓ㊀Ｔｏｅｓｔａｂｌｉｓｈａｌｉｖｅｒｆｉｂｒｏｓｉｓｍｏｄｅｌꎬｗｅｉｎｔｒａｐｅｒｉｔｏｎｅａｌｌｙｉｎｊｅｃｔｅｄａｍｉｘｔｕｒｅｏｆｃａｒｂｏｎｔｅｔｒａｃｈｌｏｒｉｄｅ(ＣＣｌ４)ａｎｄｏｌｉｖｅｏｉｌｔｏｉｎ￣ｄｕｃｅｌｉｖｅｒｆｉｂｒｏｓｉｓ.Ｒａｔｓｗｅｒｅｓａｃｒｉｆｉｃｅｄａｆｔｅｒ０ꎬ４ꎬ８ａｎｄ１２ｗｅｅｋｓꎬａｎｄｈｅｐｃｉｄｉｎｗａｓｏｂｓｅｒｖｅｄｄｕｒｉｎｇｔｈｅｐｒｏｇｒｅｓｓｉｏｎｏｆｆｉｂｒｏｓｉｓ.Ｍｅａｎｔｉｍｅꎬｗｅｕｓｅｄｄｉｆｆｅｒｅｎｔｃｏｎｃｅｎｔｒａｔｉｏｎｓｏｆｈｅｐｃｉｄｉｎ－２５ｐｅｐｔｉｄｅｔｏｓｔｉｍｕｌａｔｅＨＳＣｄｉｒｅｃｔｌｙｉｎｓｅｒｕｍ－ｆｒｅｅｍｅｄｉｕｍｆｏｒ４８ｈｏｕｒｓｉｎｖｉｔｒｏꎬａｎｄｔｈｅｎｄｅｔｅｃｔｆｉｂｒｏ￣ｓｉｓ－ｒｅｌａｔｅｄｉｎｄｉｃａｔｏｒｓα－ｓｍｏｏｔｈｍｕｓｃｌｅａｃｔｉｎ(α－ＳＭＡ)ꎬｔｉｓｓｕｅｉｎｈｉｂｉｔｏｒｏｆｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ－１(ＴＩＭＰ－１)ａｎｄｔｙｐｅＩｃｏｌｌａｇｅｎｅｘｐｒｅｓｓｉｏｎａｔｔｈｅｇｅｎｅａｎｄｐｒｏｔｅｉｎｌｅｖｅｌｓｔｏｏｂｓｅｒｖｅｄｔｈｅｉｎｆｌｕｅｎｃｅｏｆｈｅｐｃｉｄｉｎｏｎｆｉｂｒｏｇｅｎｅｓｉｓ.Ｄａｔａｗｅｒｅａｎａｌｙｚｅｄｂｙｏｎｅ－ｗａｙＡＮＯＶＡａｎｄＴｕｋｅｙｔｅｓｔ.Ｒｅｓｕｌｔｓ㊀ＴｈｅＣＣｌ４－ｉｎｄｕｃｅｄｌｉｖｅｒｆｉｂｅｒｍｏｄｅｌｗａｓｓｕｃｃｅｓｓｆｕｌｌｙｅｓｔａｂｌｉｓｈｅｄ.ＴｈｅｄｅｔｅｃｔｉｏｎｏｆｈｅｐｃｉｄｉｎｆｏｕｎｄｔｈａｔｗｉｔｈｔｈｅｐｒｏｇｒｅｓｓｏｆｆｉｂｒｏｓｉｓꎬｔｈｅｇｅｎｅｌｅｖｅｌｏｆｈｅｐｃｉｄｉｎｉｎｌｉｖｅｒｔｉｓｓｕｅｄｅｃｒｅａｓｅｄａｆｔｅｒＣＣｌ４ｉｎｊｅｃｔｉｏｎ(Ｐ<０.０５).Ｔｈｅｒｅｌａｔｉｖｅｅｘｐｒｅｓｓｉｏｎｏｆｈｅｐｃｉｄｉｎａｔ４ꎬ８ａｎｄ１２ｗｅｅｋｓａｆｔｅｒＣＣｌ４ｍｉｘｔｕｒｅｉｎｊｅｃｔｉｏｎｗａｓ０.７５ʃ０.１２ꎬ０.５２ʃ０２０ꎬａｎｄ０.２０ʃ０.１０.Ｔｈｅｓｅｒｕｍｈｅｐｃｉｄｉｎｗａｓ(３５１.４５ʃ５１.１２)ｐｇ/ｍｌꎬ(２５４.２４ʃ４９.４１)ｐｇ/ｍｌꎬ(２１１.４５ʃ４２.２８)ｐｇ/ｍｌꎬ(１８９.２３ʃ３０.２４)ｐｇ/ｍｌ.Ｉｎｖｉｔｒｏｅｘｐｅｒｉｍｅｎｔｓｓｈｏｗｔｈａｔｅｘｏｇｅｎｏｕｓｈｅｐｃｉｄｉｎｓｕｐｐｌｅｍｅｎｔａｔｉｏｎ(１０ｎｇ/ｍｌꎬ１００ｎｇ/ｍｌ)ｃａｎｄｉｒｅｃｔｌｙｉｎｈｉｂｉｔｔｈｅｅｘｐｒｅｓｓｉｏｎｏｆｆｉｂｒｏｓｉｓ－ｒｅｌａｔｅｄｇｅｎｅｓ(α－ＳＭＡꎬＴＩＭＰ－１ａｎｄｔｙｐｅＩｃｏｌｌａｇｅｎ)ｉｎＨＳＣꎬａｎｄｉｔｉｓｄｏｓｅｄｅｐｅｎｄｅｎｔ.ＦｕｒｔｈｅｒｗｅｆｏｕｎｄｔｈａｔｈｅｐｃｉｄｉｎｃａｎｂｌｏｃｋＴＧＦβ１－ｉｎｄｕｃｅｄｓｔｅｌｌａｔｅｃｅｌｌａｃｔｉｖａｔｉｏｎｂｙｉｎｈｉｂｉｔｉｎｇｔｈｅｅｘｐｒｅｓｓｉｏｎｏｆＳｍａｄ４ｉｎＴＧＦ－β１ｓｉｇｎａｌｉｎｇ.ＦｕｒｔｈｅｒｗｅｆｏｕｎｄｔｈａｔｈｅｐｃｉｄｉｎｃａｎｉｎｈｉｂｉｔＴＧＦ－β１－ｉｎｄｕｃｅｄＨＳＣｆｉｂｒｏｇｅｎｅｓｉｓｂｙｓｕｐｐｒｅｓｓｉｎｇｔｈｅｅｘｐｒｅｓｓｉｏｎｏｆＳＭＡＤ－４.Ｃｏｎｃｌｕｓｉｏｎ㊀Ｈｅｐ￣ｃｉｄｉｎｉｎｈｉｂｉｔｓａｃｔｉｖａｔｉｏｎｏｆＨＳＣａｎｄｉｎｃｒｅａｓｅｓａｎｔｉｏｘｉｄａｔｉｖｅｓｔｒｅｓｓ.Ｔｈｅｓｅｄａｔａｓｕｇｇｅｓｔａｐｏｔｅｎｔｉａｌｒｏｌｅｆｏｒｈｅｐｃｉｄｉｎｉｎｔｈｅｔｒｅａｔｍｅｎｔｏｆｌｉｖｅｒｆｉｂｒｏｓｉｓ.ʌＫｅｙｗｏｒｄｓɔ㊀ＨｅｐｃｉｄｉｎｓꎻＨｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌꎻＦｉｂｒｏｓｉｓ基金项目:国家自然科学基金资助项目(编号:８１５００４５６)∗通讯作者:尤红ꎬＥ－ｍａｉｌ:ｙｏｕｈｏｎｇ３０＠ｓｉｎａ.ｃｏｍ㊀㊀在慢性肝脏疾病中ꎬ继发性铁超载非常常见ꎬ表现为血清铁指数(转铁蛋白饱和度㊁铁蛋白)和肝铁浓度的升高[１]ꎮＬｕｄｗｉｇ等[１]研究表明ꎬ１４５/４４９(３２.４％)的肝硬化患者肝组织中铁染色呈阳性ꎻ此外ꎬ９１/４４９(２０.３％)的肝硬化患者肝脏铁浓度升高ꎮ肝组织铁超载是活性氧的来源之一ꎬ可以引起肝星状细胞(ＨｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌꎬＨＳＣ)的激活并且促进胶原的表达增高ꎬ从而导致甚至加重了肝纤维化[３ꎬ４]ꎮ铁调素是主要由肝细胞在铁含量失衡㊁炎症或缺氧状态下ꎬ分泌的一种铁调节肽[７ꎬ８]ꎮ研究表明ꎬ铁调素敲除的小鼠的肝脏㊁胰腺和心脏会发生铁超载ꎻ而过表达铁调素的小鼠则表现出严重的铁缺乏和贫血[９ꎬ１０]ꎮ铁调素在维持体内铁平衡中起着重要的作用ꎬ但铁调素在肝纤维化中的动态变化及对ＨＳＣ的作用尚不清楚ꎮ１㊀材料与方法１.１㊀动物与分组㊀雄性清洁级Ｓｐｒａｇｕｅ–Ｄａｗｌｅｙ大鼠２０只ꎬ体重为１６０~１８０ｇꎬ购自斯贝福(北京)生物技术有限公司ꎬ生产许可证编号:ＳＣＸＫ(京)２０１４－０００６ꎮ放在常规笼中ꎬ处于２０~２２ħ的房间中ꎬ进行１２ｈ的明暗７３０１ 临床和实验医学杂志㊀２０２０年５月㊀第１９卷㊀第１０期循环ꎬ可自由饮水进食ꎮ采用随机数值表法分成四氯化碳混合物注射０㊁４㊁８㊁１２周ꎬ共４组(每个时间点ｎ＝５)ꎮ１.２㊀试剂㊀ＱｉａｇｅｎＲＮｅａｓｙＭｉｎｉ(７４１０４ꎬＱｉａｇｅｎ公司ꎬ美国)试剂盒ꎬＩｍＰｒｏｍ－ＩＩＴＭ反转录系统试剂盒(Ａ３５００ꎬＰｒｏｍｅｇａ公司ꎬ美国)ꎬＡＢＩＰｏｗｅｒＳＹＢＲＧｒｅｅｎＰＣＲＭａｓ￣ｔｅｒＭｉｘ试剂盒(Ａ２５７４２ꎬＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ公司ꎬ美国)ꎬＢＣＡ蛋白质测定试剂盒(ＰｉｅｒｃｅꎬＲｏｃｋｆｏｒｄＩＬ公司ꎬ美国)ꎬ铁调素(ａｂ３１８７６ꎬＡｂｃａｍ公司ꎬ英国)ꎬ转化生长因子－β１(Ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒ－１ꎬＴＧＦ－β１)(ＡＦ－１００－２１ＣꎬＰｅｐｒｏＴｅｃｈ公司ꎬ美国)ꎬ抗β－肌动蛋白抗体(β－ａｃｔｉｎ)(Ａ５４４１ꎬＳｉｇｍａ–Ａｌｄｒｉｃｈ公司ꎬ美国)ꎬ抗α－ＳＭＡ抗体(ＭＡＢ１４２０－ＳＰꎬＲ＆ＤＳｙｓｔｅｍｓ公司ꎬ美国)ꎬ抗金属蛋白酶组织抑制剂－１(Ｔｉｓｓｕｅｉｎｈｉｂｉｔｏｒｏｆｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ－１ꎬＴＩＭＰ－１)抗体(ＡＦ９８０ꎬＲ＆ＤＳｙｓ￣ｔｅｍｓ公司ꎬ美国)ꎬ抗ＳＭＡＤ－４抗体(４６５３５ＴꎬＣｅｌｌＳｉｇｎａ￣ｌｉｎｇＴｅｃｈｎｏｌｏｇｙ公司ꎬ美国)ꎬ１０％胎牛血清(１０２７０ꎬＧｉｂ￣ｃｏ公司ꎬ美国)ꎬＥａｇｌｅ完全培养基(１１９６００４４ꎬＧｉｂｃｏ公司ꎬ美国)ꎬ四氯化碳(国药集团化学试剂有限公司ꎬ中国)ꎬ橄榄油(中国上海麦克林化工厂)ꎬ通用型组织固定液(武汉谷歌生物科技有限公司ꎬ中国)ꎮ１.３㊀仪器㊀冷冻离心机(５４２４ＲꎬＥｐｐｅｎｄｏｒｆ公司ꎬ德国)ꎬ分光光度计(ＮａｎｏｄｒｏｐＮＤ－１００ꎬＴｈｅｒｍｏＳｃｉｅｎｔｉｆｉｃ公司ꎬ美国)ꎬ实时荧光定量ＰＣＲ(７５００ꎬＡＢＩ公司ꎬ美国)ꎬ倒置显微镜(Ｍ－ＰＢ２０ꎬＯＬＹＭＰＵＳ公司ꎬ日本)ꎮ１.４㊀方法１.４.１㊀四氯化碳诱导肝纤维化模型㊀将雄性Ｓｐｒａｇｕｅ–Ｄａｗｌｅｙ大鼠每周２次(３~４ｄ/次)腹腔注射１０μｌ/ｇ的四氯化碳和橄榄油(２︰３)混合物ꎬ诱发肝纤维化ꎬ持续１２周ꎮ分别在开始注射后第０㊁４㊁８㊁１２周后处死大鼠ꎬ处死后心脏取血约１.０ｍｌꎬ放于１.５ｍＬ离心管中ꎬ静置１ｈ后ꎬ３０００ｒｐｍ/ｍｉｎ离心１５ｍｉｎꎬ离心半径８.５ｃｍꎬ将血清吸入新的１.５ｍｌ离心管中ꎬ冻存于－８０ħ冰箱中备用ꎮ同时留取各组肝脏组织标本ꎬ亦冻存于﹣８０ħ冰箱中备用ꎮ所有涉及实验动物的程序均经首都医科大学附属友谊医院动物保健和使用委员会审批(批号:１５－２００４)ꎮ１.４.２㊀细胞培养㊀ＨＳＣ－Ｔ６细胞系是来自美国纽约州西奈山医学中心的馈赠ꎮ将细胞接种在含１０％胎牛血清的Ｄｕｌｂｅｃｃｏ改良的Ｅａｇｌｅ完全培养基中ꎮ细胞贴壁后ꎬ换成无血清培养基饥饿１６ｈ后ꎬ根据实验目的分别用含铁调素(１０或１００ｎｇ/ｍｌ)㊁ＴＧＦ－β１(１０ｎｇ/ｍｌ)㊁铁调素(１０ｎｇ/ｍｌ)＋ＴＧＦ－β１培养基继续培养４８ｈ后ꎬ提取总ｍＲＮＡ和蛋白质ꎮ１.５㊀实时荧光定量ＰＣＲ(Ｑｕａｎｔｉｔａｔｉｖｅｒｅａｌ－ｔｉｍｅＰＣＲꎬＲＴ－ｑＰＣＲ)㊀利用ＱｉａｇｅｎＲＮｅａｓｙＭｉｎｉ试剂盒提取细胞总ｍＲＮＡꎮ用ＮａｎｏｄｒｏｐＮＤ－１００分光光度计测量ｍＲ￣ＮＡ浓度ꎮ使用ＩｍＰｒｏｍ－ＩＩＴＭ反转录系统试剂盒生成互补的ＤＮＡꎮ通过ＡＢＩＰｒｉｓｍ７５００快速序列检测器和ＡＢＩＰｏｗｅｒＳＹＢＲＧｒｅｅｎＰＣＲＭａｓｔｅｒＭｉｘ试剂盒进行ＲＴ－ｑＰＣＲ反应ꎮ由ＡＢＩＰｒｉｓｍ７５００－ｆａｓｔ的内置算法确定的公式２－ΔΔＣｔ给出了相对于内源参比(甘油醛３－磷酸脱氢酶)和校准品(未经处理的培养ＨＳＣ－Ｔ６细胞)标准化的靶标量ꎮ系统软件使用自适应基线确定Ｃｔꎮ１.６㊀细胞蛋白质表达检测㊀采用Ｗｅｓｔｅｒｎｂｌｏｔ法ꎬ检测蛋白表达ꎮ提取细胞总蛋白ꎬ使用ＢＣＡ蛋白质测定试剂盒测量蛋白质浓度ꎮ蛋白质煮沸１０ｍｉｎ后ꎬ将裂解物在１２％聚丙烯酰胺凝胶电泳分离ꎮ将蛋白质转移到硝酸纤维素膜上ꎬ后在含有５％脱脂奶粉的ＴＴＢＳ缓冲液中在室温下封闭２ｈꎮ然后将膜与抗α－ＳＭＡ的抗体ꎬＴＩＭＰ－１和ＳＭＡＤ－４在４ħ过夜孵育ꎬ洗涤３次后ꎬ将膜与辣根过氧化物酶耦联的兔抗小鼠或山羊抗兔二抗(分别以１︰８０００和１︰９０００稀释)孵育１ｈꎮ然后使用Ｘ射线胶片对膜进行化学发光检测和荧光照相ꎮ而后ꎬ将膜与抗β－肌动蛋白抗体再次孵育ꎬ作为上样对照ꎮ实验进行了３次ꎮ１.７㊀统计学处理㊀采用ＳＰＳＳ１７.０统计软件对数据进行处理ꎮ计量资料以均数ʃ标准差( ｘʃｓ)ꎬ多组间比较采用单因素方差分析ꎬ组内两组间的比较使用Ｔｕｋｅｙ检验ꎮＰ<０.０５为差异有统计学意义ꎮ２㊀结果２.１㊀铁调素在大鼠肝纤维化肝脏中表达下调㊀为了研究铁调素在肝纤维化中的作用ꎬ首先观察了铁调素在正常和纤维化肝脏中的表达ꎮ大鼠注射四氯化碳混合物后ꎬ肝组织铁调素的基因水平(图１Ａ)在注射后４周逐渐下降ꎬ差异有统计学意义(Ｆ＝５６.０７㊁Ｐ<０.０５)ꎻ与未注射四氯化碳混合物组(０周)大鼠相比ꎬ铁调素的基因水平在四氯化碳混合物注射后第４㊁８㊁１２周后分别下降了约２５％㊁５０％㊁８０％ꎬ差异均有统计学意义(ｑ＝５.４９㊁１０.５５㊁１７.５９ꎬＰ<０.０５)ꎮ与基因表达类似ꎬ血清中铁调素的水平ꎬ同样随纤维化进展逐渐下降(Ｐ<０.０５)ꎬ见图１Ｂꎮ铁调素在四氯化碳混合物注射后４㊁８㊁１２周分别为(２５４.２４ʃ４９.４１)㊁(２１１.４５ʃ４２.２８)㊁(１８９.２３ʃ３０.２４)ｐｇ/ｍｌꎻ四氯化碳混合物注射后４周铁调素与未注射四氯化碳混合物组(０周)大鼠[(３５１.４５ʃ５１.１２)ｐｇ/ｍｌ]相比ꎬ差异无统计学意义(ｑ＝４.１３８ꎬＰ>０.０５)ꎻ铁调素在四氯化碳混合物注射后８㊁１２周分别与未注射四氯化碳混合物组(０周)比较ꎬ差异均有统计学意义(ｑ＝６.４６３㊁７.３９６ꎬＰ<０.０５)ꎮ这些结果表明ꎬ铁调素可能在肝纤维化中起重要作用ꎮ２.２㊀铁调素对ＨＳＣ具有抗纤维化作用㊀ＨＳＣ活化是纤维化形成过程中的核心环节ꎬ在纤维发生中发挥重要作用ꎬ进一步研究了体外补充铁调素是否可以直接抑制大鼠星状细胞系－Ｔ６(ＨＳＣ－Ｔ６)的活化ꎮ结果表明ꎬ不同剂量的铁调素(０㊁１０㊁１００ｎｇ/ｍｌ)刺激ＨＳＣ－Ｔ６４８ｈ８３０１ ＪｏｕｒｎａｌｏｆＣｌｉｎｉｃａｌａｎｄＥｘｐｅｒｉｍｅｎｔａｌＭｅｄｉｃｉｎｅＶｏｌ.１９ꎬＮｏ.１０㊀Ｍａｙ.２０２０后ꎬ铁调素不仅可以在基因水平上抑制ＨＳＣ－Ｔ６活化(α－ＳＭＡ)ꎬ铁调素０㊁１０㊁１００ｎｇ/ｍｌ组分别为１.００ʃ０.７５㊁０.７１ʃ０.２０㊁０.４０ʃ０.０５ꎻ铁调素０组与铁调素１０ｎｇ/ｍｌ组比较ꎬ差异无统计学意义(ｑ＝３.９２ꎬＰ>０.０５)ꎻ与铁调素１００ｎｇ/ｍｌ组比较ꎬ差异有统计学意义(ｑ＝８.１９ꎬＰ<０.０５)ꎮ其他纤维化相关指标也被抑制ꎬ基因水平ＴＩＭＰ－１的相对表达在铁调素０㊁１０㊁１００ｎｇ/ｍｌ组分别为１.１０ʃ０.０６㊁０.９３ʃ０.１１㊁０.６５ʃ.０３８ꎻ铁调素０组与铁调素１０ｎｇ/ｍｌ组比较ꎬ差异无统计学意义(ｑ＝３.８２ꎬＰ>０.０５)ꎻ与铁调素１００ｎｇ/ｍｌ组比较ꎬ差异有统计学意义(ｑ＝１０.１２ꎬＰ<０.０５)ꎮⅠ型胶原在铁调素０㊁１０㊁１００ｎｇ/ｍｌ组分别为１.１０ʃ０.１５㊁０.５２ʃ０.２９㊁０.３３ʃ０.３４ꎬ铁调素０组与铁调素１０ｎｇ/ｍｌ组比较ꎬ差异无统计学意义(ｑ＝３.６９ꎬＰ>０.０５)ꎻ与铁调素１００ｎｇ/ｍｌ组比较ꎬ差异有统计学意义(ｑ＝４.９０ꎬＰ<０.０５)ꎮＴＧＦ－β１在铁调素０㊁１０㊁１００ｎｇ/ｍｌ组分别１.００ʃ０.０２㊁０.５２ʃ０.０５㊁０.２４ʃ０.１１ꎬ铁调素０组与铁调素１０㊁１００ｎｇ/ｍｌ组比较ꎬ差异均有统计学意义(ｑ＝１１.７８㊁１８.６５ꎬＰ<０.０５)ꎻ并且具有剂量依赖性(图２Ａ~２Ｅ)ꎮ重要的是ꎬ铁调素可以抑制ＨＳＣⅠ型胶原的分泌ꎬ在铁调素０㊁１０㊁１００ｎｇ/ｍｌ组分别为(２９.８９ʃ１.００)㊁(２６.１５ʃ２.６１)㊁(２２.２５ʃ１.８４)ｐｇ/ｍｌꎬ铁调素０组与铁调素１０ｎｇ/ｍｌ组比较ꎬ差异无统计学意义(ｑ＝３.３５ꎬＰ>０.０５)ꎻ与铁调素１００ｎｇ/ｍｌ组比较ꎬ差异有统计学意义(ｑ＝６.８５ꎬＰ<０.０５)(图２Ｆ)ꎮ图１㊀在四氯化碳诱导的大鼠肝纤维化中ꎬ铁调素水平显着降低注:纤维化形成过程中铁调素的表达ꎮ铁调素在肝组织中的基因表达(Ａ)ꎻ通过ＥＬＩＳＡ检测血清中铁调素(Ｂ)ꎻ与四氯化碳混合物注射后０周相比ꎬａＰ<０.０５图２㊀铁调素对ＨＳＣ活化的影响注:铁调素１０㊁１００ｎｇ/ｍＬ刺激ＨＳＣ４８ｈ后ꎬ通过ＲＴ－ｑＰＣＲ检测ＨＳＣ中α－ＳＭＡ(Ａ)ꎬＴＩＭＰ－１(Ｂ)ꎬⅠ型胶原(Ｃ)和ＴＧＦβ１(Ｄ)的基因表达ꎮ通过蛋白质印迹ꎬ检测星状细胞α－ＳＭＡ和ＴＩＭＰ－１(Ｅ)的表达ꎮ通过ＥＬＩＳＡ检测ＨＳＣ分泌Ⅰ型胶原(Ｆ)的水平ꎮａ与对照组相比ꎬＰ<０.０５图３㊀铁调素阻断ＴＧＦ－β１对ＨＳＣ的活化作用相差显微镜观察ＨＳＣ的形态学变化ꎬ发现各组间细胞成典型的梭形(Ａ㊁Ｂ㊁Ｃ㊁Ｄ)ꎮ蛋白质印迹检测星状细胞ａ－ＳＭＡꎬＴＩＭＰ－１和ＳＭＡＤ４的表达(Ｅ)ꎬ以β－ａｃｔｉｎ作为内对照２.３㊀铁调素抑制ＴＧＦ－β１诱导的ＨＳＣ中ＳＭＡＤ４的表达㊀由于铁调素会降低ＨＳＣ中ＴＧＦ－β１的表达ꎮ因此ꎬ我们推测铁调素对纤维的作用是通过阻断ＴＧＦ－β１刺激的ＨＳＣ激活介导的ꎮ分析ＨＳＣ空白对照组㊁铁调素处理组㊁ＴＧＦ－β１刺激组和铁调素＋ＴＧＦ－β１刺激组ꎬ发现四组间ＨＳＣ均成典型的梭形ꎬ形态上没有明显变化(图３Ａ~Ｄ)ꎮ通过检测４组间纤维化相关指标我们发现ꎬＴＧＦ－β１可以促进ＨＳＣ中α－ＳＭＡ和ＴＩＭＰ－１的表达增加ꎬ这一作用可以被铁调素阻断(图３Ｅ)ꎮ为了明确铁调素是否影响ＨＳＣ中ＴＧＦ－β１信号通路ꎬ进一步我们检测了ＳＭＡＤ－４的表达ꎮ结果发现铁调素(１００ｎｇｍＬ)可以阻断ＴＧＦ－β１诱导的ＳＭＡＤ－４在蛋白水平的表达(图３Ｅ)ꎮ以上结果表明ꎬ铁调素对星状细胞的抑制作用可能通过阻断ＴＧＦ－β１/ＳＭＡＤ－４信号发挥作用的ꎮ３㊀讨论ＨＳＣ活化是肝纤维化的中心环节ꎬ肝组织内铁超载引起的活性氧增加可以促进ＨＳＣ活化ꎬＨＳＣ的活化使肠道内铁的吸收和巨噬细胞对于铁的释放相对不平衡[４ꎬ１１ꎬ１２]ꎬ进一步加重肝纤维化ꎮ铁调素是由肝脏产生的一种肽类激素ꎬ是维持铁稳态的关键物质[７ꎬ１３ꎬ１４]ꎬ亦能在一定程度上在急性肝损伤中起到保护作用[１５ꎬ１６]ꎮ然而ꎬ尚无研究报道铁调素与ＨＳＣ之间有直接相互作用ꎮ本研究在四氯化碳诱导的大鼠纤维化模型中观察到铁调素表达的显著下降ꎬ同时Ⅰ型胶原的表达也显著增加ꎬ提示低浓度的铁调素水平可能加重纤维化的进展ꎮ当不同浓度的铁调素刺激ＨＳＣ４８ｈ后ꎬ发现铁调素可以明显抑制ＨＳＣ活化(α－ＳＭＡ表达降低)和细胞外基质相关指标ꎬ如Ⅰ型胶原㊁ＴＩＭＰ－１和ＴＧＦ－β１的表达ꎮ铁调素除了能降低ＨＳＣ中ＴＧＦ－β的表达外ꎬ还可以抑制ＴＧＦ－β１诱导的ＨＳＣ的活化和细胞外基质的产生ꎮＴＧＦ－β１是致肝纤维化的经典细胞因子ꎬ大量研究表明ＴＧＦ－β１与纤维化显著相关ꎮ在ＨＳＣ中ꎬＴＧＦ－９３０１ 临床和实验医学杂志㊀２０２０年５月㊀第１９卷㊀第１０期β１促进活化的ＨＳＣ向成肌纤维细胞样细胞转化ꎬ刺激ＥＣＭ蛋白的合成并抑制其降解[１７ꎬ１８]ꎮ在实验模型中ꎬ阻断ＴＧＦ－β１合成和/或信号传导途径的实验已证明可明显减少纤维化ꎮ综上所述ꎬ我们的研究显示ꎬ铁调素可阻断ＴＧＦ－β１诱导星状细胞活化和细胞外基质的产生ꎮ这些研究有助于我们进一步了解慢性性肝病中铁代谢调控的机制ꎮ随着相关研究的进一步深入进行ꎬ铁调素或许可以成为肝纤维化和其他慢性肝脏疾病的潜在治疗方法ꎮ参考文献[１]㊀ＬｕｄｗｉｇＪꎬＨａｓｈｉｍｏｔｏＥꎬＰｏｒａｙｋｏＭＫꎬｅｔａｌ.Ｈｅｍｏｓｉｄｅｒｏｓｉｓｉｎｃｉｒｒｈｏ￣ｓｉｓ:ａｓｔｕｄｙｏｆ４４７ｎａｔｉｖｅｌｉｖｅｒｓ[Ｊ].Ｇａｓｔｒｏｅｎｔｅｒｏｌｏｇｙꎬ１９９７ꎬ１１２(３):８８２－８８８.[２]㊀ＤｅｕｇｎｉｅｒＹＭꎬＬｏｒéａｌＯꎬＴｕｒｌｉｎＢꎬｅｔａｌ.Ｌｉｖｅｒｐａｔｈｏｌｏｇｙｉｎｇｅｎｅｔｉｃｈｅｍｏｃｈｒｏｍａｔｏｓｉｓ:ａｒｅｖｉｅｗｏｆ１３５ｈｏｍｏｚｙｇｏｕｓｃａｓｅｓａｎｄｔｈｅｉｒｂｉｏｃｌｉｎｉｃａｌｃｏｒｒｅｌａｔｉｏｎｓ[Ｊ].Ｇａｓｔｒｏｅｎｔｅｒｏｌｏｇｙꎬ１９９２ꎬ１０２(６):２０５０－２０５９.[３]㊀ＷａｎｇＭꎬＬｉｕＲꎬＬｉａｎｇＹꎬｅｔａｌ.Ｉｒｏｎｏｖｅｒｌｏａｄｃｏｒｒｅｌａｔｅｓｗｉｔｈｓｅｒｕｍｌｉｖｅｒｆｉｂｒｏｔｉｃｍａｒｋｅｒｓａｎｄｌｉｖｅｒｄｙｓｆｕｎｃｔｉｏｎ:Ｐｏｔｅｎｔｉａｌｎｅｗｍｅｔｈｏｄｓｔｏｐｒｅｄｉｃｔｉｒｏｎｏｖｅｒｌｏａｄ－ｒｅｌａｔｅｄｌｉｖｅｒｆｉｂｒｏｓｉｓｉｎｔｈａｌａｓｓｅｍｉａｐａｔｉｅｎｔｓ[Ｊ].ＵｎｉｔｅｄＥｕｒｏｐｅａｎＧａｓｔｒｏｅｎｔｅｒｏｌＪꎬ２０１７ꎬ５(１):９４－１０３.[４]㊀ＮｅｌｓｏｎＪＥꎬＷｉｌｓｏｎＬꎬＢｒｕｎｔＥＭꎬｅｔａｌ.Ｒｅｌａｔｉｏｎｓｈｉｐｂｅｔｗｅｅｎｔｈｅｐａｔ￣ｔｅｒｎｏｆｈｅｐａｔｉｃｉｒｏｎｄｅｐｏｓｉｔｉｏｎａｎｄｈｉｓｔｏｌｏｇｉｃａｌｓｅｖｅｒｉｔｙｉｎｎｏｎａｌｃｏｈｏｌｉｃｆａｔｔｙｌｉｖｅｒｄｉｓｅａｓｅ[Ｊ].Ｈｅｐａｔｏｌｏｇｙꎬ２０１１ꎬ５３(２):４４８－４５７.[５]㊀ＤｅｕｇｎｉｅｒＹꎬＴｕｒｌｉｎＢꎬＲｏｐｅｒｔＭꎬｅｔａｌ.Ｉｍｐｒｏｖｅｍｅｎｔｉｎｌｉｖｅｒｐａｔｈｏｌｏｇｙｏｆｐａｔｉｅｎｔｓｗｉｔｈβ－ｔｈａｌａｓｓｅｍｉａｔｒｅａｔｅｄｗｉｔｈｄｅｆｅｒａｓｉｒｏｘｆｏｒａｔｌｅａｓｔ３ｙｅａｒｓ[Ｊ].Ｇａｓｔｒｏｅｎｔｅｒｏｌｏｇｙꎬ２０１１ꎬ１４１(４):１２０２－１２１１.ｅ３.[６]㊀ＪｉｎＨꎬＴｅｒａｉＳꎬＳａｋａｉｄａＩ.Ｔｈｅｉｒｏｎｃｈｅｌａｔｏｒｄｅｆｅｒｏｘａｍｉｎｅｃａｕｓｅｓａｃｔｉ￣ｖａｔｅｄｈｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌｓｔｏｂｅｃｏｍｅｑｕｉｅｓｃｅｎｔａｎｄｔｏｕｎｄｅｒｇｏａｐｏｐｔｏｓｉｓ[Ｊ].ＪＧａｓｔｒｏｅｎｔｅｒｏｌꎬ２００７ꎬ４２(６):４７５－４８４.[７]㊀ＤｒａｋｅｓｍｉｔｈＨꎬＰｒｅｎｔｉｃｅＡＭ.Ｈｅｐｃｉｄｉｎａｎｄｔｈｅｉｒｏｎ－ｉｎｆｅｃｔｉｏｎａｘｉｓ[Ｊ].Ｓｃｉｅｎｃｅꎬ２０１２ꎬ３３８(６１０８):７６８－７７２.[８]㊀ＳｐｉｖａｋＩꎬＡｒｏｒａＪꎬＭｅｉｎｚｅｒＣꎬｅｔａｌ.ＬｏｗＳｅｒｕｍＨｅｐｃｉｄｉｎＩｓＡｓｓｏｃｉａｔｅｄＷｉｔｈＲｅｄｕｃｅｄＳｈｏｒｔ‐ＴｅｒｍＳｕｒｖｉｖａｌｉｎＡｄｕｌｔｓＷｉｔｈＡｃｕｔｅＬｉｖｅｒＦａｉｌ￣ｕｒｅ[Ｊ].Ｈｅｐａｔｏｌｏｇｙꎬ２０１９ꎬ６９(５):２１３６－２１４９.[９]㊀ＬｕｎｏｖａＭꎬＳｃｈｗａｒｚＰꎬＮｕｒａｌｄｅｅｎＲꎬｅｔａｌ.Ｈｅｐｃｉｄｉｎｋｎｏｃｋｏｕｔｍｉｃｅｓｐｏｎｔａｎｅｏｕｓｌｙｄｅｖｅｌｏｐｃｈｒｏｎｉｃｐａｎｃｒｅａｔｉｔｉｓｏｗｉｎｇｔｏｃｙｔｏｐｌａｓｍｉｃｉｒｏｎｏ￣ｖｅｒｌｏａｄｉｎａｃｉｎａｒｃｅｌｌｓ[Ｊ].ＪＰａｔｈｏｌꎬ２０１７ꎬ２４１(１):１０４－１１４.[１０]ＬｕｎｏｖａＭꎬＧｏｅｈｒｉｎｇＣꎬＫｕｓｃｕｏｇｌｕＤꎬｅｔａｌ.Ｈｅｐｃｉｄｉｎｋｎｏｃｋｏｕｔｍｉｃｅｆｅｄｗｉｔｈｉｒｏｎ－ｒｉｃｈｄｉｅｔｄｅｖｅｌｏｐｃｈｒｏｎｉｃｌｉｖｅｒｉｎｊｕｒｙａｎｄｌｉｖｅｒｆｉｂｒｏｓｉｓｄｕｅｔｏｌｙｓｏｓｏｍａｌｉｒｏｎｏｖｅｒｌｏａｄ[Ｊ].ＪＨｅｐａｔｏｌｏｇｙꎬ２０１４ꎬ６１(３):６３３－６４１.[１１]ＤｅｕｇｎｉｅｒＹꎬＢｒｉｓｓｏｔＰꎬＬｏｒéａｌＯ.Ｉｒｏｎａｎｄｔｈｅｌｉｖｅｒ:ｕｐｄａｔｅ２００８[Ｊ].ＪＨｅｐａｔｏｌꎬ２００８ꎬ４８:Ｓ１１３－Ｓ１２３.[１２]ＧｕｙａｄｅｒＤꎬＴｈｉｒｏｕａｒｄＡＳꎬＥｒｄｔｍａｎｎＬꎬｅｔａｌ.ＬｉｖｅｒｉｒｏｎｉｓａｓｕｒｒｏｇａｔｅｍａｒｋｅｒｏｆｓｅｖｅｒｅｆｉｂｒｏｓｉｓｉｎｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓＣ[Ｊ].Ｊｈｅｐａｔｏｌꎬ２００７ꎬ４６(４):５８７－５９５.[１３]ＨａｎＣＹꎬＫｏｏＪＨꎬＫｉｍＳＨꎬｅｔａｌ.ＨｅｐｃｉｄｉｎｉｎｈｉｂｉｔｓＳｍａｄ３ｐｈｏｓｐｈｏｒｙｌ￣ａｔｉｏｎｉｎｈｅｐａｔｉｃｓｔｅｌｌａｔｅｃｅｌｌｓｂｙｉｍｐｅｄｉｎｇｆｅｒｒｏｐｏｒｔｉｎ－ｍｅｄｉａｔｅｄｒｅｇｕｌａ￣ｔｉｏｎｏｆＡｋｔ[Ｊ].Ｎａｔｕｒｅｃｏｍｍｕｎｉｃａｔｉｏｎｓꎬ２０１６ꎬ７(１):１－１４.[１４]ＤíａｚＶꎬＧａｍｍｅｌｌａＥꎬＲｅｃａｌｃａｔｉＳꎬｅｔａｌ.Ｌｉｖｅｒｉｒｏｎｍｏｄｕｌａｔｅｓｈｅｐｃｉｄｉｎｅｘｐｒｅｓｓｉｏｎｄｕｒｉｎｇｃｈｒｏｎｉｃａｌｌｙｅｌｅｖａｔｅｄｅｒｙｔｈｒｏｐｏｉｅｓｉｓｉｎｍｉｃｅ[Ｊ].Ｈｅｐａ￣ｔｏｌｏｇｙꎬ２０１３ꎬ５８(６):２１２２－２１３２.[１５]赵卫华ꎬ王燕红ꎬ丛敏ꎬ等.铁和铁调素在四氯化碳肝纤维化小鼠模型中的表达[Ｊ].临床和实验医学杂志ꎬ２０１７ꎬ１６(４):３１３－３１６.[１６]庞国进ꎬ谌双君ꎬ赵卫华ꎬ等.铁调素在ＣＣｌ４诱导的小鼠肝纤维化模型中的动态变化[Ｊ].临床肝胆病杂志ꎬ２０１５ꎬ３１(３):４１８－４２３.[１７]ＳｃｈｕｐｐａｎＤꎬＡｓｈｆａｑ－ＫｈａｎＭꎬＹａｎｇＡＴꎬｅｔａｌ.Ｌｉｖｅｒｆｉｂｒｏｓｉｓ:Ｄｉｒｅｃｔａｎｔｉｆｉｂｒｏｔｉｃａｇｅｎｔｓａｎｄｔａｒｇｅｔｅｄｔｈｅｒａｐｉｅｓ[Ｊ].ＭａｔｒｉｘＢｉｏｌｏｇｙꎬ２０１８ꎬ６８－６９:４３５－４５１.[１８]ＬｉｕＸꎬＨｕＨꎬＹｉｎＪＱ.ＴｈｅｒａｐｅｕｔｉｃｓｔｒａｔｅｇｉｅｓａｇａｉｎｓｔＴＧＦ－βｓｉｇｎａｌｉｎｇｐａｔｈｗａｙｉｎｈｅｐａｔｉｃｆｉｂｒｏｓｉｓ[Ｊ].ＬｉｖｅｒＩｎｔｅｒｎａｔｉｏｎａｌꎬ２００６ꎬ２６(１):８－２２.(收稿日期:２０２０－０２－２０)㊀㊀ＤＯＩ:１０.３９６９/ｊ.ｉｓｓｎ.１６７１－４６９５.２０２０.０１０.０１０㊀㊀文章编号:１６７１－４６９５(２０２０)０１０－１０４０－０５黄芪多糖抑制氧化应激致大鼠软骨细胞损伤及凋亡的作用机制分析肖剑伟１㊀周唯践２㊀蔡旭１㊀郭粉莲１㊀陈新鹏１㊀洪易炜１㊀曾苗雨１㊀叶志中１∗㊀(１深圳市福田区风湿病专科医院风湿科㊀广东㊀深圳㊀５１８０００ꎻ２云南省中医医院风湿科㊀云南㊀昆明㊀６５００００)基金项目:国家自然科学基金(编号:８１１０２２６６)ꎻ广东省医学科学技术研究基金(编号:Ａ２０１８０８９)ꎻ深圳市医疗卫生三名工程(编号:ＳＺＳＭ２０１６０２０８７)ꎻ２０１８福田区卫生公益性科研项目(编号:ＦＴＷＳ２０１８０６６)∗通讯作者:叶志中ꎬＥ－ｍａｉｌ:ｙｅｚｈｉｚｈｏｎｇ０８２３＠１６３.ｃｏｍ㊀㊀ʌ摘要ɔ㊀目的㊀探究黄芪多糖(ａｌｐ)抑制氧化应激致大鼠软骨细胞损伤及凋亡的作用机制ꎮ方法㊀选取ＳＰＦ级雌性３周龄大鼠ꎬ分为活性氧自由基培养组(ＲＯＳ组)㊁黄芪多糖组(ＲＯＳ＋ａｌｐ组)ꎬ另设空白对照组ꎮＲＯＳ组取其软骨细胞ꎬ加入活性氧自由基培养ꎻＲＯＳ＋ａｌｐ组在ＲＯＳ组的基础上加入浓度为３０ｍｇ/Ｌ的黄芪多糖处理ꎻ空白对照组不作处理ꎮ使用ＣＣＫ－８法测定不同浓度(０ｍｇ/Ｌ㊁２０ｍｇ/Ｌ㊁５０ｍｇ/Ｌ㊁１００ｍｇ/Ｌ㊁２００ｍｇ/Ｌ)黄芪多糖对大鼠细胞生存率的影响ꎻ采用蛋白质印记法(ＷｅｓｔｅｒｎＢｌｏｔ)检测大鼠软骨细胞中Ｂｃｌ－２相关Ｘ蛋白(Ｂａｘ)㊁Ｂ淋巴细胞瘤－２基因(Ｂｃｌ－２)㊁基质金属酶蛋白３(ＭＭＰ３)㊁ＭＭＰ１３㊁ＣＬＯ２蛋白表达情况ꎻ采用Ｈｏｅｃｈｓｔ３３３４２染色法测定软骨细胞凋亡率ꎮ结果㊀ａｌｐ０ｍｇ/Ｌ时ꎬ细胞大量凋亡ꎬ生存率不到４０％ꎬ随着ａｌｐ的浓度的增加ꎬ大鼠软骨细胞存活率显著升高ꎻ与空白对照组相比ꎬＲＯＳ组大鼠细胞Ｂｃｌ－２㊁ＣＬＯ２蛋白表达显著下降(Ｐ<０.０１)ꎬＭＭＰ３㊁ＭＭＰ１３㊁Ｂａｘ蛋白表达显著上升(Ｐ<０.０１)ꎻ与０４０１ ＪｏｕｒｎａｌｏｆＣｌｉｎｉｃａｌａｎｄＥｘｐｅｒｉｍｅｎｔａｌＭｅｄｉｃｉｎｅＶｏｌ.１９ꎬＮｏ.１０㊀Ｍａｙ.２０２０。

铁死亡相关蛋白在AD小鼠海马中的表达李燕新;吕占云;李维;郝延磊【摘要】目的探讨铁死亡相关蛋白在阿尔茨海默病(Alzheimer disease,AD)小鼠海马组织中的表达情况.方法构建PSEN1 p.G378E敲入的AD小鼠,比较铁死亡相关蛋白谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)、溶质载体家族7成员11(solute carrier family 7,member 11,SLC7A11)、长链酯酰辅酶A合成酶4(long-chain fattyacyl-CoA synthetase 4,ACSL4)和磷脂酰乙醇胺结合蛋白1(phosphatidylethanolamine-binding protein1,PEBP1)在纯合(PSEN1p.G378E-/-)、杂合(PSEN1 p.G378E-/+)和野生(wild type,WT)小鼠海马组织的表达情况.结果同PSEN1 p.G378E-/+和WT小鼠相比,PSEN1 p.G378E-/-组小鼠海马组织中GPX4、PSEN1、SLC7A11和ACSL4蛋白的表达明显升高(P<0.05);PSEN1 p.G378E-/+组海马组织GPX4和PEBP1的蛋白表达也明显高于WT小鼠(P<0.05),但SLC7A11和ACSL4的蛋白表达在两组之间无统计学差异(P>0.05).结论 PSEN1 p.G378E敲入的AD小鼠海马组织中铁死亡相关蛋白表达升高.【期刊名称】《中国神经精神疾病杂志》【年(卷),期】2018(044)012【总页数】5页(P727-731)【关键词】阿尔茨海默病;PSEN1 p.G378E基因;铁死亡相关蛋白【作者】李燕新;吕占云;李维;郝延磊【作者单位】山东大学齐鲁医学院济南250012;浙江大学医学院;济宁医学院附属医院神经内科;济宁医学院附属医院神经内科【正文语种】中文【中图分类】R749.1+6铁死亡(ferroptosis)是一种铁离子依赖的、以脂质过氧化产物和致死活性氧的积累为特点的非典型的细胞死亡方式[1]。

畜牧兽医学报 2023,54(6):2280-2287A c t a V e t e r i n a r i a e t Z o o t e c h n i c a S i n i c ad o i :10.11843/j.i s s n .0366-6964.2023.06.008开放科学(资源服务)标识码(O S I D ):铁死亡在细菌性感染中的研究进展毛 鹏1,2,3,王志浩1,2,3,李建基1,2,3,崔璐莹1,2,3,朱国强1,2,3,孟 霞1,2,3,董俊升1,2,3,王 亨1,2,3*(1.扬州大学兽医学院江苏省动物重要疫病与人兽共患病防控协同创新中心,扬州225009;2.江苏高校动物重要疫病和重要人兽共患病防控技术国际合作联合实验室,扬州225009;3.农业与农产品安全教育部国际联合研究实验室,扬州225009)摘 要:铁死亡是一种新型程序性细胞死亡模式,其主要特点为铁依赖的脂质过氧化物蓄积㊂在细菌感染宿主细胞的过程中,铁代谢紊乱和氧化应激起到了重要作用,并且已经证明某些细菌可通过铁死亡的方式诱导宿主细胞死亡㊂本文就细菌感染对宿主铁代谢的影响以及细菌诱导铁死亡的研究现状进行阐述,以期为防控动物细菌感染提供新的思路㊂关键词:铁死亡;细菌感染;铁代谢;氧化应激中图分类号:S 855.1 文献标志码:A 文章编号:0366-6964(2023)06-2280-08收稿日期:2022-07-21基金项目:国家自然科学基金(32273070);江苏省自然科学基金面上项目(B K 20211324);江苏现代农业产业技术体系建设专项资金(J A T S[2022]499);江苏省333高层次人才培养工程资助项目;高等学校学科创新引智计划资助(D 18007);江苏高校优势学科建设工程资助项目;江苏高校动物重要疫病和重要人兽共患病防控技术国际合作联合实验室资助;江苏高校品牌专业建设工程资助项目作者简介:毛 鹏(1996-),男,甘肃酒泉人,博士生,主要从事动物临床疾病研究,E -m a i l :m a o p e n g 96@163.c o m *通信作者:王 亨,主要从事动物临床疾病诊疗和发病机制研究,E -m a i l :s d a u l e l l o w@163.c o mR e s e a r c h P r o g r e s s o f F e r r o pt o s i s i n B a c t e r i a l I n f e c t i o n MA O P e n g1,2,3,WA N G Z h i h a o 1,2,3,L I J i a n j i 1,2,3,C U I L u y i n g 1,2,3,Z HU G u o q i a n g 1,2,3,M E N G X i a 1,2,3,D O N G J u n s h e n g 1,2,3,WA N G H e n g1,2,3*(1.J i a n g s u C o -i n n o v a t i o n C e n t e r f o r P r e v e n t i o n a n d C o n t r o l o f I m p o r t a n t A n i m a l I n f e c t i o u s D i s e a s e s a n d Z o o n o s e s ,C o l l e g e o f V e t e r i n a r y M e d i c i n e ,Y a n g z h o u U n i v e r s i t y ,Y a n gz h o u 225009,C h i n a ;2.I n t e r n a t i o n a l R e s e a r c h L a b o r a t o r y o f P r e v e n t i o n a n d C o n t r o l o f I m p o r t a n t A n i m a l I n fe c t i o u s D i s e a s e s a n d Z o o n o t i c D i s e a s e s of J i a ng s u H i gh e r E d u c a ti o n I n s t i t u t i o n s ,Y a n gz h o u 225009,C h i n a ;3.J o i n t I n t e r n a t i o n a l R e s e a r c h L a b o r a t o r y o f A g r i c u l t u r e a n d A g r i -P r o d u c t S a f e t y ,t h e M i n i s t r y o f E d u c a t i o n ,Y a n gz h o u 225009,C h i n a )A b s t r a c t :F e r r o p t o s i s i s a n e w l y d i s c o v e r e d p r o g r a mm e d c e l l d e a t h p a t t e r n c h a r a c t e r i z e d b yi r o n -d e p e n d e n t a c c u m u l a t i o n o f l i p i d p e r o x i d a t i o n .I r o n m e t a b o l i s m d i s o r d e r a n d o x i d a t i v e s t r e s s p l a ya n i m po r t a n t r o l e i n b a c t e r i a l i n f e c t i o n ,a n d i t h a s b e e n p r o v e d t h a t s o m e b a c t e r i a c o u l d i n d u c e h o s t c e l l d e a t h b y f e r r o p t o s i s .I n t h i s p a pe r ,t h e ef f e c t s o f b a c t e r i a l i n f e c t i o n o n h o s t i r o n m e t a b o -l i s m a n d t h e m e c h a n i s m o f b a c t e r i a -i n d u c e d f e r r o pt o s i s w e r e r e v i e w e d ,i n o r d e r t o p r o v i d e n e w r e s e a r c h i d e a s f o r t h e p r e v e n t i o n o f b a c t e r i a l i n f e c t i o n i n a n i m a l s .K e y wo r d s :f e r r o p t o s i s ;b a c t e r i a l i n f e c t i o n ;i r o n m e t a b o l i s m ;o x i d a t i v e s t r e s s *C o r r e s p o n d i n g au t h o r :WA N G H e n g ,E -m a i l :s d a u l e l l o w@163.c o m6期毛鹏等:铁死亡在细菌性感染中的研究进展铁死亡是由各种因素导致细胞内铁离子代谢障碍,活性氧(r e a c t i v e o x y g e n s p e c i e s,R O S)和脂质过氧化物蓄积所引起的细胞程序性死亡,其主要特征为谷胱甘肽过氧化物酶4(g l u t a t h i o n e p e r o x i d a s e 4,G P X4)活性下降,细胞内游离铁水平增高,脂质过氧化物蓄积[1]㊂研究证实,铁死亡参与癌症[2-3]㊁退行性脑病[4]㊁缺血再灌注损伤[5]和镉致鸡的肝损伤[6]等疾病的发生和发展㊂由于机体在免疫防御过程中,以及细菌自身的代谢均可生成R O S[7],且细菌还可干扰宿主细胞对铁的代谢,因此,铁死亡在细菌感染诱导宿主细胞损伤的过程中发挥作用㊂1铁死亡概述2003年,铁死亡作为一种新型的细胞程序性死亡方式被首次发现和报道[8],于2012年最终命名[1]㊂目前研究发现3种途径可引发铁死亡:第一,用E r a s t i n[9]㊁柳氮磺胺吡啶[10]㊁丁硫氨酸亚砜亚胺[11]等物质抑制胱氨酸/谷氨酸反向转运体(c y s-t i n e/g l u t a m a t e a n t i p o r t e r,s y s t e m X C-),通过减少细胞内谷胱甘肽(g l u t a t h i o n e,G S H)的含量,进而引发细胞氧化还原失衡导致铁死亡;第二,通过R a s选择性致死化合物(R a s-s e l e c t i v e l e t h a l c o m-p o u n d s,R S L s)[12]直接抑制G P X4活性,最终导致细胞脂质过氧化物堆积,诱发铁死亡;第三,非依赖于G P X4的铁死亡通路,细胞膜上铁死亡抑制蛋白1(f e r r o p t o s i s s u p p r e s s o r p r o t e i n1,F S P1)通过利用烟酰胺腺嘌呤二核苷酸磷酸(n i c o t i n a m i d e a d e-n i n e d i n u c l e o t i d e p h o s p h a t e,N A D P H),形成还原型辅酶Q10(r e d u c e d c o e n z y m e Q10),以此降解细胞膜上的脂质过氧化物,从而抑制铁死亡[13]㊂其中第一和第二种途径,均基于G S H的抗氧化防御系统失衡,导致脂质过氧化物大量蓄积,引发铁死亡㊂在铁死亡通路中,关键调控因子s y s t e m X C-由溶质载体家族7成员11(s o l u t e c a r r i e r f a m i l y7 m e m b e r11,S L C7A11)和溶质载体家族3成员2 (s o l u t e c a r r i e r f a m i l y3m e m b e r2,S L C3A2)组成[14]㊂在正常生理条件下,s y s t e m X C-将胞外胱氨酸转运至胞内,同时将胞内的谷氨酸排出㊂细胞内的胱氨酸转变为半胱氨酸后,在谷氨酸半胱氨酸连接酶和谷胱甘肽合成酶的作用下生成G S H[15]㊂G P X4作为一种硒蛋白[16],以G S H作为底物来实现过氧化酶的作用,将细胞膜中的有毒脂质过氧化物转化为无毒脂质醇[17],以此抑制铁死亡㊂作为一种细胞程序性死亡方式,早期认为铁死亡在形态学上与凋亡㊁坏死和自噬不同,具有自己独特的形态特征,如细胞膜破裂,细胞体积减小,线粒体膜固缩,但细胞核膜完整[1]㊂但随着研究的深入,发现细胞出现铁死亡后,表现出类似坏死的形态学变化[18],如细胞膜断裂或出泡㊁细胞质与细胞器肿胀㊁染色质凝集等,但线粒体形态学变化与坏死不同,可见线粒体萎缩㊁膜密度增加㊁嵴减少或缺失,以及线粒体外膜破裂等[19]㊂在某些情况下,铁死亡还伴随着细胞的分离与聚集,以及自噬体的增加[20]㊂此外,发生铁死亡的细胞可向周围未暴露于铁死亡诱导剂的细胞转移脂质过氧化物,从而导致周围细胞铁死亡[21]㊂2细菌性感染中发现的铁死亡现象在细菌感染过程中,铁代谢紊乱与脂质过氧化发挥了重要的作用㊂已有研究证实,在脂多糖(l i-p o p o l y s a c c h a r i d e,L P S)刺激或病原菌侵袭致病的过程中,铁死亡参与其中㊂2.1L P S及大肠杆菌与铁死亡L P S是革兰阴性菌细胞壁的主要成分之一,可在一定程度上模拟革兰阴性菌的感染㊂研究证实, L P S通过T o l l样受体4(t o l l-l i k e r e c e p t o r4, T L R4),激活核因子κB(n u c l e a r f a c t o r k a p p a-B, N F-κB)信号通路,并促进下游包括白细胞介素1β(i n t e r l e u k i n-1β,I L-1β)㊁白细胞介素6(i n t e r l e u k i n-6,I L-6)㊁肿瘤坏死因子α(t u m o r n e c r o s i s f a c t o rα, T N F-α)等炎性细胞因子的产生[22]㊂目前,L P S已被证明可在多种组织中诱导铁死亡㊂在肺损伤模型中,L P S与人支气管上皮细胞共孵育后,S L C7A11和G P X4蛋白水平下降,丙二醛(m a l o n d i a l d e h y d e,M D A)㊁4-羟基壬烯醛(4-h y d r o x y n o n e n a l,4-H N E)和铁离子含量升高,使用铁死亡抑制剂 铁抑素-1(f e r r o s t a t i n-1,F e r-1)后,可改善L P S造成的细胞损伤,并且在L P S诱导的小鼠肺损伤模型中,F e r-1治疗显著改善了肺部损伤[23],该研究说明,L P S可导致肺部组织发生铁死亡㊂在另一项小鼠肺损伤模型中,也得出了相似的结果,并且该研究还证明L P S可导致肺部前列腺素内过氧化物合酶(p r o s t a g l a n d i n-e n d o p e r o x i d e s y n t h a s e2,P T G S2)表达量升高,核因子E2相关因子(n u c l e a r f a c t o r E2r e l a t e d f a c t o r2,N r f2)表达量降低[24],这些结果说明,铁死亡发生时的脂质过氧1822畜牧兽医学报54卷化是通过上调P T G S2和抑制N r f2通路造成的㊂此外,铁死亡与肺部炎症也存在密切关系,抑制L P S诱导的铁死亡后,可降低支气管上皮细胞炎性因子I L-1β㊁I L-6以及T N F-α的表达[25],还可减少小鼠肺部中性粒细胞浸润[26],以上研究说明,抑制铁死亡可作为治疗肺部炎症的潜在手段㊂在L P S诱导脓毒症引起小鼠心组织损伤的研究中,小鼠心组织收缩和射血功能减弱,外周血中肌酸激酶同工酶M B(c r e a t i n e k i n a s e-M B,C K-M B)㊁乳酸脱氢酶和谷草转氨酶活性显著升高,病理组织学发现心肌中铁及脂质过氧化物水平升高,透射电镜观察到细胞内线粒体固缩,使用F e r-1治疗小鼠后,可显著减少心肌损伤,增加小鼠的存活率㊂此外,该研究还证明核受体共激活因子4(n u c l e a r r e-c e p t o r c o a c t i v a t o r4,N C O A4)不仅可介导铁蛋白自噬,导致细胞内游离铁水平升高,还可通过上调s i d e r o f e x i n(S F X N1)线粒体锚定蛋白[27]表达,将细胞内的铁离子转运进入线粒体内,加重L P S诱导的心肌细胞损伤[28]㊂同时,研究也证实,L P S可导致小鼠心肌中铁转运蛋白1(f e r r o p o r t i n1,F P N1)表达量降低,敲除F P N1后,可促进L P S诱导的心肌细胞内铁离子和脂质过氧水平升高,加重心肌损伤[29]㊂有文献表明,F P N1的表达下调,会导致细胞内过量的不稳定铁池(l a b i l e i r o n p o o l,L I P)无法从细胞内排出,形成恶性循环,进一步增加细胞内铁的含量㊂而过量的L I P可通过芬顿反应,产生大量的R O S,最终导致细胞脂质过氧化[30],进而导致铁死亡㊂以上结果证明L P S可通过铁死亡的方式造成心肌组织损伤,并且在这一过程中铁外排受阻与N C O A4介导的铁蛋白自噬发挥了重要作用㊂L P S除了在以上两种组织中可导致铁死亡,在其他组织中也同样可导致铁死亡:在山羊乳腺炎模型中,L P S处理山羊乳腺上皮细胞后,细胞内F e2+㊁R O S与M D A水平升高,G S H与G P X4降低,透射电镜观察发现细胞内线粒体嵴减少,膜密度升高,炎性因子I L-6与T N F-α表达升高,而使用F e r-1处理,可降低F e2+水平与炎性因子表达,上调G P X4表达,增加细胞存活率,以上结果说明L P S可导致山羊乳腺上皮细胞铁死亡[31]㊂经L P S处理后,人滑膜细胞内M D A和铁含量升高,转铁蛋白受体(t r a n s f e r r i n r e c e p t o r,T F R)与N C O A4蛋白水平升高,S L C7A11㊁G P X4与N r f2蛋白水平降低,这些结果说明L P S可诱导滑膜细胞发生铁死亡[32]㊂大肠杆菌(E s c h e r i c h i a c o l i)为革兰阴性菌,是常见的致病菌,其在胞外感染的方式下,与草鱼红细胞共孵育,可导致血红素加氧酶1(h e m e o x y g e n a s e 1,H O-1)㊁自噬相关基因5(a u t o p h a g y-r e l a t e d g e n e5,A T G5)和铁蛋白的基因表达上调,F P N1基因表达被抑制[33]㊂这些结果表明,大肠杆菌可通过铁死亡的方式诱导草鱼红细胞死亡㊂2.2铜绿假单胞菌与铁死亡铜绿假单胞菌(P s e u d o m o n a s a e r u g i n o s a)为革兰阴性菌,可胞内感染细胞[34]㊂文献报道,L O X可选择性氧化细胞膜上的花生四烯酸磷脂酰乙醇胺(a r a c h i d o n i c a c i d p h o s p h a t i d y l e t h a n o l a m i n e s,A A-P E)来导致铁死亡[35]㊂铜绿假单胞菌感染人支气管上皮细胞时,可以合成脂氧合酶(l i p o x y g e n a s e, L O X)将支气管上皮细胞膜中的A A-P E氧化为15-羟基二十碳四烯酸(15-h y d r o p e r o x y e i c o s a t e t r a e n o i c a c i d,15-H E T E),导致15-H E T E这种脂质过氧化物蓄积[36],使人支气管上皮细胞铁死亡㊂在小鼠肺部感染铜绿假单胞菌模型中也得到了相同的结论,并且,在使用铁死亡抑制剂后,可减轻细胞损伤与肺部炎症[37],以上结果说明铜绿假单胞菌感染可通过合成脂氧合酶,氧化宿主细胞多不饱和脂肪酸来引发铁死亡㊂2.3结核分枝杆菌与铁死亡结核分枝杆菌(m y c o b a c t e r i u m t u b e r c u l o s i s)为胞内感染菌,在其感染人巨噬细胞时,G S H和G P X4活性降低,亚铁离子㊁线粒体超氧化物和脂质过氧化物水平增加,且使用F e r-1和铁螯合剂后,死亡细胞比例明显下降㊂在动物试验中,结核分枝杆菌感染小鼠肺部导致肺组织中G P X4活性降低,脂质过氧化物蓄积;而使用F e r-1治疗后,小鼠各器官的载菌量都明显降低,肺部损伤也减轻[38],故铁死亡在结核分枝杆菌致病的过程中起到了关键作用㊂2.4金黄色葡萄球菌与铁死亡金黄色葡萄球菌(S t a p h y l o c o c c u s a u r e u s)为革兰阳性胞内感染菌㊂目前,虽无文献表明其可通过铁死亡的方式诱导宿主细胞死亡,但其感染过程中造成的铁水平及脂质过氧化的变化可能与铁死亡密切相关[39]:金黄色葡萄球菌在感染细胞时能够进入宿主细胞中[40],并且其铁摄取调节子(f e r r i c u p t a k e r e g u l a t o r,F u r)可通过感知菌体外铁水平来调节自身毒素的释放,当铁缺乏时,F u r可通过增加溶血素的分泌,裂解红细胞,导致血红蛋白释放,从而使组28226期毛 鹏等:铁死亡在细菌性感染中的研究进展织中铁水平升高[41];其次,金黄色葡萄球菌感染小鼠肺部可导致严重的氧化应激,使肺部发生脂质过氧化,降低G S H 水平[42],由此可见,金黄色葡萄球菌感染可能会促进铁死亡发生㊂另一方面,有文献报道宿主细胞中的花生四烯酸和脂质过氧化产物可杀死金黄色葡萄球菌[43],金黄色葡萄球菌产生的S t a p h yl o f e r r i n A 和B 可螯合宿主细胞内的F e2+[39],会减少芬顿反应的发生,从而抑制铁死亡的发生㊂综合以上结果表明,铁死亡可能参与了金黄色葡萄球菌致病过程,但具体机制仍需进一步研究㊂2.5 细菌感染诱导铁死亡的机制基于以上报道,L P S 及其他细菌诱导铁死亡的过程中,其机制如图1所示㊂其中,铁的过载主要依靠以下途径:①N C O A 4通过选择性自噬铁蛋白,释放其中的亚铁离子;②上调T F R 的表达,增强细胞对铁的摄取;③抑制F P N 1的表达,阻碍细胞铁的外排㊂脂质过氧化主要通过:①依靠抑制N r f 2/G P X 4/F P N 1轴,细胞内依赖G S H 的抗氧化系统受损,导致细胞清除脂质过氧化物的能力下降;②通过损伤线粒体,产生过量的R O S ,最终导致脂质过氧化物蓄积㊂图1 细菌感染诱导铁死亡机制示意图F i g .1 S c h e m a t i c d i a g r a m o f t h e m e c h a n i s m o f f e r r o p t o s i s i n d u c e d b y ba c t e r i a 3 铁代谢与细菌感染的关系由于在细菌感染中铁代谢紊乱促进了铁死亡的发生,故铁代谢可能参与了细菌感染的进程㊂正常情况下,机体循环系统中铁以转铁蛋白(t r a n s f e r -r i n ,T F )和非转铁蛋白结合铁(n o n -t r a n s f e r r i nb o u n d i r o n ,N T B I )两种形式存在㊂当T F 与T F R结合后,T F 在胞内囊泡酸化使F e 3+与T F 解离,然后通过前列腺六段跨膜上皮抗原3(s i x -t r a n s m e m -b r a n e e p i t h e l i a l a n t i ge n of p r o s t a t e 3,S T E A P 3)还原为F e 2+;而N T B I 通过锌转运蛋白(z i n c t r a n s -po r t ,Z I P )中的Z I P 8或Z I P 14进入细胞[44]㊂铁进入细胞后以两种形式储存:一种是铁与蛋白质结合形成铁蛋白;另一种是铁离子弱结合,形成L I P [45]㊂当细胞内的铁超过正常水平时,多余的铁通过F P N 1排出细胞[46]㊂3.1 细菌感染对铁吸收的影响细菌感染可导致细胞因子的过度释放,从而上调T F R 的表达,促进铁进入细胞[47]㊂在L P S 诱导小鼠炎症模型的研究中发现,小鼠的血清铁和T F浓度显著降低,与T F 结合的铁通过T F R 转运进细胞内[48],肝细胞和神经细胞内铁调素表达增加,促进T F R 表达[49]㊂神经系统的炎症还可上调二价金属转运蛋白1(d i v a l e n t m e t a l t r a n s po r t e r 1,D MT 1)的表达,使细胞外的铁通过D MT 1转移到细胞内[50]㊂在小鼠盲肠结肠结扎穿刺导致的败血症模型[51],以及L P S 刺激小鼠模型研究中发现,Z I P 14表达上调[52],表明细胞内N T B I 转运增强㊂以上结果说明细菌感染会增强细胞对铁的吸收,导致细胞内铁水平的升高,为铁死亡的发生提供基础㊂3.2 细菌感染对铁储存的影响细胞内铁水平受到铁反应元件-铁调节蛋白(i -r o n -r e s p o n s i v e e l e m e n t s -i r o n r e g u l a t o r y pr o t e i n s ,3822畜牧兽医学报54卷I R E s-I R P s)系统的翻译后调控[53]㊂细胞内铁浓度升高会加速铁蛋白m R N A的翻译,使游离铁通过铁蛋白的形式储存,从而降低细胞内的游离铁水平㊂当铁蛋白被降解或细胞应激而激活非选择性自噬时,细胞内游离铁水平升高[54]㊂研究证明,使用牙龈卟啉单胞菌产生的L P S刺激人牙周膜细胞可促进铁蛋白的表达[55],牙龈卟啉单胞菌感染牙周膜成纤维细胞12h后,铁蛋白水平升高,但在感染24h 候后铁蛋白水平下降,且随着感染时间延长, N C O A4介导的铁蛋白自噬被激活,增加细胞内的游离铁水平,敲除N C O A4后可减少炎性因子的生成[56],该研究说明,细菌在长时间感染细胞后,铁蛋白自噬是细胞内游离铁的重要来源㊂铁是L O X和P T G S的辅助因子,随着铁水平的上升可增强L O X 和P T G S的活性,导致脂质R O S和炎性因子释放,进而加剧脂质过氧化和铁水平的进一步升高[57],从而促进铁死亡的发生㊂线粒体是铁代谢的主要场所,虽然线粒体可以合成线粒体铁蛋白(m i t o c h o n-d r i a l f e r r i t i n,F T MT),但F T MT易被N C O A4介导的自噬降解[58]㊂并且,细菌感染导致的R O S会显著降低线粒体的铁代谢能力,使铁潴留在线粒体内,从而产生过量的R O S[44],最终导致细胞脂质过氧化,发生铁死亡㊂3.3细菌感染对铁外排的影响F P N1是唯一可将细胞内的铁运输到细胞外的蛋白[59]㊂当L P S与T L R结合,F P N1的转录会被下调[60],故L P S可通过抑制F P N1导致铁潴留在细胞内,加重氧化损伤,促进铁死亡的发生㊂研究发现,当豚鼠感染结核分枝杆菌后,N r f2蛋白表达量升高,但并没有从细胞质进入细胞核发挥作用,且其下游抗氧化蛋白表达量也呈下降趋势,说明结核分枝杆菌可抑制N r f2通路的激活[61],该研究虽没有检测F P N1的表达,但由于F P N1的表达受N r f2通路的调控[62],故在结核分枝杆菌感染后,F P N1表达也可能被抑制,导致细胞内的铁在外排时受阻,使细胞内铁水平升高,促进了铁死亡的发生㊂4存在的问题与展望近些年,随着铁死亡的研究不断深入,其作用机制不断被揭露,但仍有许多问题亟待解决㊂脂质过氧化被认为是铁死亡发生的标志,但并非所有脂质过氧化造成的损伤都能引起铁死亡[63],故需要更深入的研究探讨脂质过氧化物在铁死亡中的作用㊂有文献指出,可以将多不饱和脂肪酰磷脂(p o l y u n s a t-u r a t e d f a t t y a c y l p h o s p h o l i p i d s,P U F A-P L s)和氧化还原活性铁(r e d o x a c t i v e i r o n)作为铁死亡的生物学标志[64],但氧化还原活性铁与P U F A-P L s具体通过怎样的机制导致细胞死亡还需更进一步阐明㊂在兽医领域中,已对细菌感染疾病中的脂质过氧化进行了较多的研究,如在奶牛生产繁殖过程中,细菌感染是奶牛乳腺炎[65]和子宫内膜炎[66]的主要病因之一,患有大肠杆菌性乳腺炎的荷斯坦奶牛血液㊁乳汁及尿液中的脂质过氧化物呈显著上升趋势[67],而在乳房灌注铁螯合剂后可减轻乳腺损伤[68],由此可见,大肠杆菌感染奶牛乳腺过程中,铁与脂质过氧化物的蓄积发挥了重要作用㊂有文献表明,巴黎链球菌(S t r e p t o c o c c u s l u t e t i e n s i s)感染奶牛乳腺上皮后,可导致R O S大量生成,并能抑制N r f2通路蛋白表达[69],使奶牛乳腺上皮细胞脂质过氧化物蓄积㊂L P S还能导致奶牛子宫内膜细胞发生严重的氧化应激[70],造成细胞脂质过氧化㊂以上结果表明,细菌感染可导致奶牛乳腺与子宫发生脂质过氧化,但铁死亡是否参与其中尚未见报道㊂综上所述,靶向抑制铁死亡可能成为动物细菌性疾病的潜在治疗手段,通过更深入研究细菌与铁死亡的关系,能为动物生产养殖中预防和治疗细菌性疾病提供更多的理论支持,并最终为提高动物饲养管理水平做出科学指导㊂参考文献(R e f e r e n c e s):[1] D I X O N S J,L E M B E R G K M,L AM P R E C H T M R,e t a l.F e r r o p t o s i s:A n i r o n-d e p e n d e n tf o r m o fn o n a p o p t o t i c c e l l d e a t h[J].C e l l,2012,149(5):1060-1072.[2] L I N R Y,Z HA N G Z H,C H E N L F,e t a l.D i h y d r o a r t e m i s i n i n(D HA)i n d u c e s f e r r o p t o s i s a n dc a u s e s c e l l c y c l e a r r e s t i n h e ad a n d ne c k c a r c i n o m ac e l l s[J].C a n c e r L e t 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·综述·鲍曼不动杆菌和铁离子及铁离子转运系统徐 晓， 许 巍 关键词： 铁载体； 亚铁离子转运系统； 鲍曼不动杆菌中图分类号：R378.99 文献标识码：A 文章编号：1009-7708 ( 2021 ) 01-0101-04DOI: 10.16718/j.1009-7708.2021.01.020Iron and Feo system in Acinetobacter baumanniiXU Xiao, XU Wei （Pediatric ICU, Shengjing Hospital of Chinese Medical University, Shenyang 110000, China ）基金项目： 国家自然科学基金（81771621，81270726）；辽宁省重点研发指导计划（2019JH8/10300023）；辽宁省自然科学基金（20170541023）。

作者单位： 中国医科大学附属盛京医院儿童重症监护室，沈阳110000。

第一作者简介： 徐晓（1996—），女，硕士研究生，主要从事耐碳青霉烯类鲍曼不动杆菌临床特征和相关基因的研究。

通信作者：许巍，E-mail ：*****************。

鲍曼不动杆菌是一种机会致病菌，主要感染重症患者及免疫力低下人群。

鲍曼不动杆菌与其他革兰阴性病原体相比感染率较低，但在全球范围内，约有45%该菌分离株具有多重耐药性，拉丁美洲和中东地区的多重耐药率高达70%。

并且鲍曼不动杆菌的多重耐药率比其他革兰阴性病原体（如铜绿假单胞菌和肺炎克雷伯菌）高出近4倍 [1]。

在重症监护室（ICU ）鲍曼不动杆菌感染相关病死率高达45%～60% [2]。

目前所用的抗菌药物，如抗假单胞菌头孢菌素、氟喹诺酮类、氨基糖苷类、碳青霉烯类和替加环素对该菌的作用有限。

因此需要深入了解细菌的毒力因子和耐药机制，找寻新的可替代的治疗方法。

铁离子是鲍曼不动杆菌生长所必须的金属离子，铁的吸收能增强菌株的致病性和毒力，细胞内铁离子浓度低于10-6 mol/L 时称为铁饥饿现象 [3]，不利于细菌生存，人体内游离铁离子浓度通常不能满足细菌生长需求，为了抵抗这种恶劣坏境，细菌衍生出了许多直接或者间接的机制来获取铁离子。

铁死亡调节剂与顺铂毒性研究的最新进展摘要：顺铂（CDDP）是第一种用于抗肿瘤药的重金属化合物，但它所导致的顺铂毒性（cisplatin-induced toxicity）使患者的生活质量大打折扣，这一点严重局限了CDDP的临床应用。

故预防和治疗肿瘤化疗后顺铂毒性成为了一个重要课题。

铁死亡(ferroptosis)是科学家们近几年发现的一种新型的非凋亡调控的细胞死亡方式，其特点是铁依赖的脂质过氧化物的积累。

越来越多的文献报道铁死亡参与了多种恶性肿瘤顺铂化疗过程，应用铁死亡调节剂（ferroptosis regulator）可以在一定程度上影响顺铂毒性。

因此，本文将对铁死亡调节剂的相关分子机制及其在顺铂诱导的耳毒性、肾毒性和细胞毒性研究中的应用和影响进行综述，旨在为临床实践中预防和治疗顺铂毒性寻找理论依据和靶标。

关键词：顺铂毒性；铁死亡调节剂；分子机制众所周知，顺铂(CDDP)是一种广泛应用于临床的抗肿瘤药物，在卵巢癌，乳腺癌，骨肉瘤，肺癌，头颈癌等恶性肿瘤都表现出显著疗效[1]。

伴随着化疗剂量的升高，顺铂的治疗效果越来越好，但其毒副作用也越来越强，这严重影响癌症患者的愈后，大大限制了它的临床应用。

顺铂毒性（cisplatin-induced toxicity）主要表现为肾毒性（nephrotoxicity）、耳毒性（ototoxicity）和细胞毒性（cytotoxicity）[2,3,4]。

导致顺铂毒性很难治愈或预防的主要原因就是它的具体分子机制尚不清楚。

因此，进一步探究顺铂毒性发生机制，发现新的治疗靶点，寻找有效的防治方法和药物已经迫在眉睫。

2012年Dixon等人提出了一种在形态学，生物化学和遗传学上与细胞凋亡和坏死不同的新型细胞死亡方式—铁死亡(ferroptosis)，它是铁依赖性的，由活性氧（ROS）和脂质过氧化物（LPO）的积累介导[5]。

铁死亡可以由许多小分子化合物诱导和抑制，涉及参与许多疾病，在恶性肿瘤顺铂化疗过程中也发挥重大作用。

ＴＢＰ相关凶ｒ３（ＴＲＦ３）在小鼠早期胚胎和细胞系中的表达及功能分析图１ＴＢＰ家族成员之间的进化关系㈣１．Ｚ．１ＴＲＦｌ第‘个ＴＢＰ相关因子ＴＲＦｌ首先在果蝇中发现，并且迄今为止也仅发现在果蝇中存在““。

ＴＲＦｌ的Ｃ末端与ＴＢＰＣ末端核心区域具有很高的同源性，可以与ＴＡＴＡ元件结合。

ＴＲＦｌ主要在胚胎、成体神经系统和雄性生殖细胞中表达。

ＴＲＦｌ也可以和ＴＦＩＩＡ和ＴＦＩＩＢ结合。

Ｈａｎｓｅｎ等（１９９７）“２１最初发现ＴＲＦｉ可能参与ＰｏｌＩＩ对部分组织特异性基因的转录，但同时他们也发现，多线染色体染色时，ＴＲＦＩ弓ＴＢＰ在染色体上具有不同的定位，ＴＦＲｌ集中在ＰｏｌＩＩＩ转录位点。

随后的生化分析表明，果蝇胚胎核抽提物中去除ＴＲＦｌ（而非ＴＢＰ）会导致ＰｏｌＩＩＩ的转录抑制”３，说明ＴＲＦｌ在ＰｏｌＩＩＩ的转录过程中起重要作用。

令人疑惑的是６图２不同物种ＴＲＦ３序列比较及与人ＴＢＰ序列比较…３黑色表示两个物种以上的ＴＲＦ３序列与ＴＢＰ保守，有变化的地方为灰色；蓝色表示３个物种以上的ＴＲＦ３序列保守。

ＴＢｐ序列中与ＴＡＴＡＴ元件（Ｉ）、ＴＦＩＩＡ（Ｖ）和ＴＦＩＩＢ（●）结合的位置分别在图中表示出。

２．早期胚胎的基因激活２．１卵母细胞和精子的成熟生长中的卵母细胞有一个很大的生发泡（有时也被称为生殖泡）（ｇｅｒｍｉｎａｌｙｅｓｉｅｌｅ，ＧＶ），相当于细胞的核，因此该阶段的卵母细胞也被称为ＧＶ期卵母细胞。

ＧＶ期卵母细胞存在活跃的转录，但随着卵母细胞的成熟，转录水平逐渐降低””。

充分生长的卵母细胞在激素的刺激下发生生发泡破裂（ＧＶｂｒｅａｋｄｏｗｎ），染色体浓缩，进行减数分裂，并最终停在第一次减数分裂的中期即ＭＩＩ期。

此时的染色体高度浓缩，转录沉默。

精子发生的最后一个阶段即拉长阶段，通过多步骤的蛋白交换过程，精子细胞核内的组蛋白最终被精子所特有的精蛋白代替。

成熟精子中ＤＮＡ与精蛋白并不形成核小体结构，而是形成晶体样结构，该结构比中期染色体的紧密程度还有高出６倍。

ＡｄｖａｎｃｅｓｉｎｔｈｅｓｔｕｄｙｏｆｎａｔｕｒａｌｓｍａｌｌｍｏｌｅｃｕｌｅｓｔｏｐｒｏｔｅｃｔｔｈｅｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｔｉｇｈｔｊｕｎｃｔｉｏｎｂａｒｒｉｅｒｉｎｉｎｆｌａｍｍａｔｏｒｙｂｏｗｅｌｄｉｓｅａｓｅＺＨＯＮＧＹｕ ｔｉｎｇ１，ＷＡＮＧＺｈｉ ｂｉｎ２，ＺＨＡＮＧＬｉ ｃｈａｏ１（１．ＤｅｐｔｏｆＰａｒｍａｃｙＳｈａｎｇｈａｉＭｕｎｉｃｉｐａｌＨｏｓｐｉｔａｌｏｆＴｒａｄｉｔｉｏｎａｌＣｈｉｎｅｓｅＭｅｄｉｃｉｎｅＡｆｆｉｌｉａｔｅｄｔｏＳｈａｎｇｈａｉＵｎｉｖｅｒｓｉｔｙｏｆＴｒａｄｉｔｉｏｎａｌＣｈｉｎｅｓｅＭｅｄｉｃｉｎｅ，Ｓｈａｎｇｈａｉ　２０００７１，Ｃｈｉｎａ；２．ＤｅｐｔｏｆＣｒｉｔｉｃａｌＣａｒｅＭｅｄｉｃｉｎｅ，ＳｃｈｏｏｌｏｆＡｎｅｓｔｈｅｓｉｏｌｏｇｙ，ＮａｖａｌＭｅｄｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｓｈａｎｇｈａｉ　２００４３３，Ｃｈｉｎａ）Ａｂｓｔｒａｃｔ：Ｉｎｆｌａｍｍａｔｏｒｙｂｏｗｅｌｄｉｓｅａｓｅ（ＩＢＤ），ａｓａｎｉｄｉｏｐａｔｈｉｃｉｎｆｌａｍｍａｔｏｒｙｄｉｓｅａｓｅｏｆｔｈｅｉｎｔｅｓｔｉｎａｌｔｒａｃｔ，ｃｏｎｓｉｓｔｉｎｇｍａｉｎｌｙｏｆＣｒｏｈｎ＇ｓｄｉｓｅａｓｅａｎｄｕｌｃｅｒａｔｉｖｅｃｏｌｉｔｉｓ，ｗｈｉｃｈｃａｎｉｎｖｏｌｖｅｔｈｅｒｅｃ ｔｕｍ，ｃｏｌｏｎａｎｄｉｌｅｕｍ，ａｎｄｗｈｏｓｅｐａｔｈｏｇｅｎｅｓｉｓｉｓｓｔｉｌｌｎｏｔｆｕｌｌｙｕｎｄｅｒｓｔｏｏｄ．ＴｈｅｉｎｉｔｉａｔｉｏｎｏｆｉｎｔｅｓｔｉｎａｌｉｎｆｌａｍｍａｔｉｏｎａｓｓｏｃｉａｔｅｄｗｉｔｈＩＢＤａｎｄｉｔｓｃｈｒｏｎｉｃｉｔｙｂｅｇｉｎｓｗｉｔｈｉｎｃｒｅａｓｅｄｉｎｔｅｓｔｉｎａｌｐｅｒ ｍｅａｂｉｌｉｔｙｃａｕｓｅｄｂｙｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｂａｒｒｉｅｒｄｉｓｒｕｐｔｉｏｎ．Ｔｈｅａｎｔｉ ｐｅｒｍｅａｂｉｌｉｔｙｏｆｔｈｅｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｂａｒｒｉｅｒｉｓｍａｉｎｔａｉｎｅｄｂｙｔｉｇｈｔｊｕｎｃｔｉｏｎｉｎｔｈｅａｐｉｃａｌｒｅｇｉｏｎｏｆｔｈｅｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ，ａｎｄｄｉｓｒｕｐｔｉｏｎｏｆｔｈｅｔｉｇｈｔｊｕｎｃｔｉｏｎｓｔｒｕｃｔｕｒｅｉｓｃｌｏｓｅｌｙａｓ ｓｏｃｉａｔｅｄｗｉｔｈｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｂａｒｒｉｅｒｄａｍａｇｅａｎｄｔｈｅｄｅｖｅｌ ｏｐｍｅｎｔｏｆＩＢＤ．Ｔｈｅｒｅｆｏｒｅ，ｉｔｉｓｓｉｇｎｉｆｉｃａｎｔｔｏｆｉｎｄｄｒｕｇｓｆｏｒｔｈｅｐｒｅｖｅｎｔｉｏｎａｎｄｔｒｅａｔｍｅｎｔｏｆＩＢＤｕｓｉｎｇｔｉｇｈｔｊｕｎｃｔｉｏｎｓａｓｒｅｇｕｌａｔｏ ｒｙｔａｒｇｅｔｓ．Ｉｎｒｅｃｅｎｔｙｅａｒｓ，ｍａｎｙｓｍａｌｌｍｏｌｅｃｕｌｅｓｏｆｎａｔｕｒａｌｐｒｏｄｕｃｔｏｒｉｇｉｎｈａｖｅｂｅｅｎｒｅｐｏｒｔｅｄｔｏｉｍｐｒｏｖｅｔｈｅｅｆｆｅｃｔｓｏｆＩＢＤ．Ｉｎｐａｒｔｉｃｕｌａｒ，ｗｅｒｅｖｉｅｗｔｈｅｃｏｍｐｏｕｎｄｓｔｈａｔｈａｖｅｔｈｅｆｕｎｃｔｉｏｎｏｆｒｅｐａｉｒｉｎｇｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｂａｒｒｉｅｒａｎｄｐｒｏｔｅｃｔｉｎｇｔｉｇｈｔｊｕｎｃｔｉｏｎｓｔｒｕｃｔｕｒｅ，ｉｎｏｒｄｅｒｔｏｐｒｏｖｉｄｅｒｅｓｅａｒｃｈｉｄｅａｓｆｏｒｔｈｅｄｅｓｉｇｎａｎｄｄｅｖｅｌｏｐｍｅｎｔｏｆｎｅｗｄｒｕｇｓｆｏｒｔｈｅｐｒｅｖｅｎｔｉｏｎａｎｄｔｒｅａｔｍｅｎｔｏｆＩＢＤ．Ｋｅｙｗｏｒｄｓ：ｉｎｆｌａｍｍａｔｏｒｙｂｏｗｅｌｄｉｓｅａｓｅ；ｉｎｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｂａｒｒｉｅｒ；ｔｉｇｈｔｊｕｎｃｔｉｏｎｓ；ｎａｔｕｒａｌｓｍａｌｌｍｏｌｅｃｕｌｅｃｏｍｐｏｕｎｄｓ；ｉｎ ｔｅｓｔｉｎａｌｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ；ｉｎｔｅｓｔｉｎａｌｐｅｒｍｅａｂｉｌｉｔｙ网络出版时间：２０２３－１２－０１１０：４８：４０　网络出版地址：ｈｔｔｐｓ：／／ｌｉｎｋ．ｃｎｋｉ．ｎｅｔ／ｕｒｌｉｄ／３４．１０８６．Ｒ．２０２３１１３０．１３１８．００８铁死亡及其在炎症性肠病中对肠上皮细胞作用机制的研究进展陈双兰１，刘青松１，胡双元１，张　怡１，刘　蓉２（成都中医药大学１．附属医院消化内科、２．药学院，四川成都　６１００７２）ｄｏｉ：１０．１２３６０／ＣＰＢ２０２２０５１０７文献标志码：Ａ文章编号：１００１－１９７８（２０２３）１２－２２１０－０６中国图书分类号：Ｒ ０５；Ｒ３２２ ４５；Ｒ３４９ １；Ｒ５７４；Ｒ５９１ １摘要：炎症性肠病（ｉｎｆｌａｍｍａｔｏｒｙｂｏｗｅｌｄｉｓｅａｓｅ，ＩＢＤ）主要表现为慢性进行性胃肠道炎症，损害胃肠道黏膜。

铁死亡的相关机制及其在临床疾病中的研究进展苏　展,王蕴凤,单奕迪,葛敬岩　(吉林大学基础医学院生理学系,吉林　长春　130021)[关键词]　铁死亡;ROS ;谷胱甘肽;GPX4;神经退行性疾病;肿瘤通讯作者:葛敬岩 调节性细胞死亡(regulated cell death,RCD)对于生物体的正常发育和体内稳态的维持至关重要㊂RCD 过多或不足都会导致疾病,包括神经退行性疾病,自身免疫性疾病和癌症等㊂最近,越来越多的RCD 被证明涉及非凋亡途径,包括坏死性凋亡㊁细胞焦亡㊁PARP-1依赖性细胞死亡㊁自死亡和铁死亡等[1]㊂这些非凋亡细胞死亡途径的性质㊁调控和功能仍然是人们非常感兴趣的话题㊂ 铁死亡(ferroptosis)是一种非凋亡性的新的细胞死亡形式,其特征是细胞内铁依赖的脂质过氧化物蓄积引起的细胞死亡,在形态学㊁生物化学和遗传学上与典型的坏死㊁凋亡和自噬不同,具有独特性㊂铁螯合剂㊁亲脂性抗氧化剂㊁脂质过氧化抑制剂和多聚不饱和脂肪酸的消耗可以抑制铁死亡㊂铁死亡与多种生物过程密切相关,包括氨基酸㊁铁和多聚不饱和脂肪酸的代谢,以及谷胱甘肽㊁磷脂㊁NADPH 和辅酶Q10的生物合成等㊂铁死亡与神经系统退行性疾病㊁肿瘤等多种疾病的发生发展相关,为临床疾病的治疗提供了新的思路和方法㊂ 2003年在大规模筛选各种化合物对肿瘤细胞杀伤作用的研究实验中,发现了一种新的化合物,即erastin,可以导致RAS 突变的肿瘤细胞以不同于传统细胞凋亡的方式死亡[2];2008年Stockwell 等人发现了两种新的化合物RSL3和RSL5,与erastin 具有相同的作用,他们还发现铁螯合剂去铁胺B-甲磺酸盐(DFOM)和抗氧化剂维生素E 可以抑制细胞死亡[3];2012年Stockwell 等人用 ferroptosis”描述由铁依赖性脂质过氧化物蓄积引起的这种类型细胞死亡,即 铁死亡”[4]㊂目前认为铁死亡与阿尔茨海默症㊁帕金森病等退行性疾病以及肿瘤㊁中风㊁缺血再灌注损伤和肾细胞变性等病理性细胞死亡相关,铁死亡亦具有抑制肿瘤的功能,因而有望成为肿瘤新的治疗方法㊂1　铁死亡过程及其特征 铁死亡是近年来发现的一种氧化性㊁非凋亡的细胞死亡途径㊂在小分子物质诱导下通过不同的信号通路直接或间接地影响谷胱甘肽过氧化物酶(glutathione peroxidase,GPXs),从而导致细胞抗氧化能力下降㊁氧化还原水平失衡㊁活性氧(re⁃active oxygen species,ROS)累积过多而发生膜脂质过氧化反应,从而损伤细胞膜的完整性,导致细胞死亡㊂铁死亡在形态学上的特征主要表现为线粒体体积变小,线粒体膜密度增加,线粒体嵴减少或消失,线粒体外膜破裂,而细胞核大小正常,无核浓缩或染色质边缘化现象[4-5],这是铁死亡区别于凋亡㊁坏死和自噬的主要形态学特征㊂铁死亡的生化特点主要表现为细胞内铁和ROS 的积累,MAPKs 信号传导系统的激活,胱氨酸/谷氨酸转运蛋白系统的抑制以及NADPH 氧化增加等[6]㊂2　铁死亡的相关机制及其调节 越来越多的研究表明,铁代谢和脂质过氧化是介导铁死亡的关键因素,本文主要从铁代谢和脂质过氧化及其调节对铁死亡的机制进行概述㊂2.1　铁代谢:铁和铁的衍生物(如血红素等)对线粒体电子传递链产生活性氧的酶和亚单位的功能至关重要,如NADPH 氧化酶㊁黄嘌呤氧化酶㊁脂氧合酶㊁细胞色素P450等㊂过多的铁能够通过Fenton 反应生成ROS 导致铁死亡的发生㊂循环中的铁主要是三价铁离子(Fe 3+),以与转铁蛋白(transferrin)结合的形式存在,通过细胞膜转铁蛋白受体1(transferrin receptor 1,TFR1)结合内吞进入细胞内,形成核内体㊂在核内体中,Fe 3+通过铁还原酶STEAP3被还原为二价铁离子(Fe 2+)㊂二价金属转运体1(divalent metal transporter 1,DMT1,也称为SLC11A2)最后将Fe 2+从核内体释放到细胞质不稳定的铁池中,过量的铁储存在铁蛋白(ferritin)中㊂细胞膜铁转运蛋白(ferroportin,也称为SLC11A3)能够将Fe 2+氧化成Fe 3+,并将细胞内的铁转运至细胞外[7]㊂在Ras 基因突变的铁死亡敏感细胞中,TFR1表达增加,而储存铁的铁蛋白表达降低[3]㊂铁代谢的主要转录因子铁反应元件结合蛋白2(iron response ele⁃ment binding protein 2,IREB2)基因沉默后能够使铁蛋白重链和轻链表达增加,降低细胞内铁含量,抑制erastin 诱导的铁死亡[4],表明铁的摄取增加和储存减少导致的铁超载有助于铁死亡的发生㊂因此,调控细胞铁的摄取㊁贮存和利用是调控铁死亡的重要节点㊂2.2　脂质代谢:ROS 是部分还原的含氧分子,包括超氧化物(O -2)㊁过氧化物(H 2O 2和ROOH )和自由基(HO㊃和RO ㊃)[5]㊂虽然低水平和可控的ROS 对正常的细胞和机体功能起重要作用,但ROS 的异常积累与许多急性器官损伤和慢性退行性疾病有关㊂线粒体是最重要的细胞器之一,通过电子传递链从正常的代谢产能中产生大量ROS㊂铁死亡可以被各种抗氧化剂和ROS 清除剂抑制,因此ROS 在铁死亡中发挥重要作用㊂目前普遍认为,铁死亡的最终执行者是脂质过氧化物,ROS 与脂膜上的多聚不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)发生反应,生成过量的脂质过氧化物导致细胞膜损伤,最终导致铁死亡发生㊂细胞膜胱氨酸/谷氨酸反向转运体(System Xc-)㊁GPX4以及NADPH氧化酶等也可通过影响脂质ROS生成进而调控铁死亡的发生[8]㊂ 细胞膜System Xc-能够将细胞外的胱氨酸摄入细胞内,同时将细胞内谷氨酸转运至细胞外㊂胱氨酸是细胞合成谷胱甘肽(glutathione,GSH)的原料,GSH是细胞内重要的抗氧化剂,对清除自由基维持细胞内外氧化还原平衡起到重要的作用㊂铁死亡促进剂erastin和柳氮磺胺吡啶主要通过抑制细胞膜system Xc-对胱氨酸的摄取,使内源性抗氧化剂谷胱甘肽耗竭,导致铁依赖的脂质ROS蓄积[4]㊂GPX4是GSH依赖性酶,能将还原型谷胱甘肽转化为氧化型谷胱甘肽(GSSG),同时将脂质氢过氧化物(L-OOH)还原为脂质醇(L-OH),或将游离过氧化氢转化为水,对抗依赖铁和O2的脂质过氧化㊂Erastin 通过消耗谷胱甘肽来抑制GPX4的活性,而RSL3可以直接抑制GPX4的活性[9]㊂GPX4目前被认为是多种铁死亡诱导剂如erastin㊁RSL3等引发铁死亡的关键靶点㊂NADPH是生物体中主要的还原剂,参与多种代谢反应㊂GSH脱氢形成GSSG,在NADPH存在下,谷胱甘肽还原酶将GSSH还原为GSH[10]㊂3　铁死亡与相关临床疾病进展 明确铁死亡发生的机制及其调节能够为涉及铁代谢紊乱㊁氧化损伤等相关疾病提供新的研究思路和治疗手段,具有重要的临床意义㊂3.1　铁死亡与神经系统疾病:铁死亡已被证明与多神经系统疾病相关㊂研究发现铁死亡与帕金森病(Parkinson's disease, PD)有关㊂铁死亡是多巴胺能神经元的一个重要的细胞死亡途径,铁死亡抑制剂ferrostatin-1可以抑制体外和体内的神经元细胞死亡[11-12]㊂阿尔茨海默病(Alzheimer disease,AD)是由学习和记忆所需的神经元退化引起的,在患有AD的大脑中发现脂质过氧化和铁的失调㊂水迷宫实验表明,敲除特定大脑皮层和海马神经元GPX4的小鼠表现出明显的认知障碍,以及海马神经元的退化,给予维生素E或铁死亡抑制剂liproxstatin-1后,神经退行性病变的程度降低[13-14]㊂这些研究表明,铁死亡在学习和记忆相关的神经元中发挥重要作用㊂缺血性中风是由于颈内动脉㊁大脑中动脉或椎体/基底动脉阻塞而引起的局部供血受限,氧气和营养物质的消耗,从而导致氧化应激㊁线粒体损伤,最终导致细胞死亡㊂研究表明,在大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型中,铁死亡抑制剂可以保护小鼠免于缺血-再灌注的损伤,表明铁死亡可以导致缺血性脑卒中后神经元的死亡[15]㊂3.2　铁死亡与肿瘤:铁死亡与多种肿瘤的发生发展有关,激活铁死亡途径能够抑制癌细胞增殖,因此,铁死亡为肿瘤的治疗和新药研发提供了新途径㊂许多研究已证实铁死亡在杀死癌细胞和抑制癌细胞生长中的重要作用㊂阿糖胞苷㊁顺铂㊁阿霉素㊁替莫唑胺等化疗药物联合应用铁死亡诱导剂对其抗肿瘤活性有显著的协同作用㊂与正常细胞相比,癌细胞对铁的依赖性更强,对铁的敏感性也更强㊂Erastin作为铁死亡的诱导剂,能进一步促进顺铂对非小细胞肺癌细胞的治疗作用[16]㊂肝细胞癌是最常见的肝癌类型,索拉非尼是治疗肝癌的有效药物,铁螯合剂去铁胺能显著抑制肝癌细胞抵抗索拉非尼引起的铁死亡,因此认为铁死亡是索拉非尼治疗肝癌的潜在机制之一[17]㊂胰腺癌恶性度高,存活率较低㊂青蒿素作为一种抗疟化合物,可诱导胰腺导管腺癌细胞发生铁死亡[18]㊂此外,铁死亡抑制剂ferrostatin‐1能显著抑制青蒿素诱导的卵巢癌细胞ROS产生和铁死亡㊂弥漫性大B细胞淋巴瘤对erastin诱导的铁死亡最敏感,而对其他致死性化合物不敏感,这种敏感性的增加可能是由于某些类型的白血病和淋巴瘤缺乏硫传递途径[9],GPX4过表达也能够抑制ROS诱导的弥漫性大B细胞淋巴瘤中细胞死亡[19]㊂GPX4抑制剂RSL3可以诱导乳腺癌细胞发生铁死亡,此外,溶酶体破坏剂(西拉美新)和酪氨酸激酶抑制剂(拉帕替尼)通过增加转铁蛋白表达,抑制铁转运蛋白1的表达对诱导乳腺癌细胞铁死亡具有协同作用,铁死亡抑制剂ferrastatin-1能抑制该效应[20]㊂这些研究表明铁死亡在抑制肿瘤中发挥重要的作用㊂因此深入研究铁死亡通路的分子机制,对肿瘤的治疗和抗癌药物的研发均具有重要意义㊂4　小结 铁死亡的发生涉及多种基因的表达㊁调控以及不同的信号通路,产生一系列复杂的生化反应,主要表现为代谢的失衡和氧化还原稳态的破坏㊂大量的半胱氨酸和NADPH的存在对GPX4的抗氧化功能至关重要,GPX4的失活有助于脂质过氧化,进一步导致铁死亡㊂因此,铁死亡中的代谢过程不是独立的,而是复杂代谢网络的一部分㊂虽然铁死亡的生理功能尚未明确,但已确定其在人类疾病中的作用㊂因此,更好地了解相关临床疾病的发生以及与铁死亡的关系无疑将促进对各种疾病的潜在治疗㊂5　参考文献[1]　Galluzzi L,Vitale I,Aaronson SA,et al.Molecular mecha⁃nisms of cell death:recommendations of the Nomenclature Commit⁃tee on Cell Death2018[J].Cell Death Differ,2018,25(3):486-541.[2]　Dolma S,Lessnick SL,Hahn WC,et al.Identification of genotype-selective antitumor agents using synthetic lethal chemi⁃cal screening in engineered human tumor cells[J].Cancer Cell, 2003,3(3):285-296.[3]　Yang WS,Stockwell BR.Synthetic lethal screening identi⁃fies compounds activating iron-dependent,nonapoptotic cell death in oncogenic-RAS-harboring cancer cells[J].Chem Biol,2008, 15(3):234-245.[4]　Dixon S,Lemberg K,Lamprecht M,et al.Ferroptosis:an i⁃ron-dependent form of nonapoptotic cell death[J].Cell,2012,149 (5):1060-1072.[5]　Stockwell BR,Friedmann Angeli JP,Bayir H,et 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Cancer,2013,133(7):1732-1742.[18]　Eling N,Reuter L,Hazin J,et al.Identification of artesu⁃nate as a specific activator of ferroptosis in pancreatic cancer cells.Oncoscience,2015,2(5):517-532.[19]　Kinowaki Y,Kurata M,Ishibashi S,et al.Glutathione per⁃oxidase 4overexpression inhibits ROS-induced cell death in dif⁃fuse large B-cell lymphoma[J].Lab Invest,2018,98(5):609-619.[20]　Ma S,Henson ES,Chen Y,et al.Ferroptosis is induced following siramesine and lapatinib treatment of breast cancer cells [J].Cell Death Dis,2016,7:e2307.[收稿日期:2018-12-23　编校:王丽娜]Caprini 风险评估的临床应用对ICU 预防静脉血栓栓塞(VTE )的影响研究进展李缘婷,米雪纯,田　莹　(昆明医科大学第一附属医院,云南　昆明　650032)[关键词]　ICU ;VTE ;Caprini ;风险评估工具基金项目:云南省应用基础研究(昆医联合专项)[项目编号:2015FB036]通讯作者:田　莹 静脉血栓栓塞(venous thromboembolism,VTE),包括深静脉血栓形成(deep vein thrombosis,DVT)和/或肺血栓栓塞(plumonary embolism,PE)两种主要形式[1],在住院患者中有很高的发病率,是导致院内非预期死亡和围手术期死亡的主要因素之一㊂2008年,美国外科医生发布了防止DVT 和PE 的行动呼吁,该呼吁报告揭示了美国每年因DVT(60,000-350,000)和PE(>10万)死亡的大量患者[2]㊂VTE 也是重症监护室(ICU)患者比较严重的并发症之一[3],重症患者发生VTE 的风险高于其他住院患者,很多因素是发病率升高的主要来源,包括患者因素㊁创伤㊁败血症㊁固定㊁中心静脉导管等㊂VTE 的致死率极高,因此ICU 医护人员必须做好防控工作[4]㊂Caprini 风险评估量表被广泛应用于呼吸科(肺栓塞)㊁妇科恶性肿瘤㊁颅脑手术患者㊁国内综合医院住院患者㊁骨科及外科大手术等患者的VTE 预防治疗过程,而ICU 患者因其特殊性,更需要高效且适宜的早期风险评估工具㊂本文旨在综述Caprini 风险评估工具在临床各科室的使用情况,结合目前其在ICU 的应用,以期为ICU 患者的VTE 防范工作提供参考㊂1　Caprini 风险评估工具1.1　概述:Caprini 风险评估量表由美国外科博士Joseph A.Caprini 设计,Caprini 博士基于临床经验和研究结果设计了该量表[5]㊂自20世纪80年代后期以来,Caprini 博士及其小。

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufficiently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitrification,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the first,rate-limiting step of nitrification;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clarification of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantification of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingflocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theflocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min influent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min effluent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the influent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andfiltered on quantitative filter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer specific16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples werefixed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andfinally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in paraffin(m.p.56e58 C)by serial immer-sion in1:1xylene-paraffin for30min at60 C,followed by 100%paraffin.After solidification in paraffin,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Paraffin was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andfluorescently labeled with fluorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to afinal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed briefly with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andfinal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efficiently.98%of NH 4þ-N and 100%of TOC were removed from the influent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reflects the slow andgradual process of sludge granulation,i.e.,from flocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small fluctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE profile of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reamplified,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic affiliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantification of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules fluctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge flocs contribute to lower denitrification rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efficiency raises fluctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efficiency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large fluctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in biofilter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE profile of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green fluorescence (B,E,H),and NOB appear as red fluorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an influence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitrification processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen affinity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also confirms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitrificans(such as Tb.deni-trificans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efficiency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneficial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination 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