异丙酚前药HX0891和HX0892在大鼠体内的初步药效学评价

收稿日期:20052032301基金项目:甘肃省自然科学基金项目(ZR 2972068)1硒酸酯多糖的免疫调节与抗肿瘤作用张哲文1,魏虎来2,苏海翔2(11兰州大学基础医学院;21兰州大学医学实验中心,甘肃兰州　730000) 硒(Se )是人类必需的微量元素之一,多年来国内外对硒的生物学功能进行了大量研究,尤其是硒与肿瘤的关系,已成为硒生物学功能研究中最为令人关注的领域之一,并取得了重大进展[1]。

流行病学调查、动物实验和临床应用研究证实硒是许多肿瘤强有力的预防剂、抑制剂和治疗辅助剂,能够减轻化疗药物的毒副作用,增加机体对抗肿瘤药物的耐受性而不影响其抗癌活性。

近年的研究表明,与亚硒酸钠等无机硒相比,硒酸酯多糖、硒酵母、富硒麦芽等有机硒化合物具有生物活性高,硒的生物利用度高,不易蓄积,毒性极低等特点,更适合于人体生理和临床应用。

随着生命科学的发展,多糖和硒元素在生命过程中的重要作用日渐引人注目,本文重点综述一种全新的有机硒化合物硒酸酯多糖的免疫调节作用和抗肿瘤效应。

1　硒酸酯多糖的生物特性卡拉胶是从红藻中提取的天然植物胶。

分为Kappa 型(由鹿角藻中提取)和Iota 型(由麒麟菜中提取),其化学本质为硫酸酯多糖。

硒酸酯多糖又称硒化卡拉胶(Kappa 2Selenocarrageenan ,KSC ),是人工制备的卡拉胶的硒化产物。

卡拉胶是β2D 2吡喃半乳糖与3,62内醚222D 2吡喃半乳糖以C1,3键和C1,4键交替连接构成的无分支链型硫酸酯多糖。

其结构中的硫部分地被硒取代,即形成硒酸酯多糖。

此有机结构能够有效地提高硒的生物利用度和生理功能,且毒副作用比无机硒大大降低,是一种安全、有效、健康的富硒化合物[2]。

KSC 具有硒和多糖的双重功效,具有优势互补,彼此协同增效的特点,具有广泛的生理活性,兼具有机硒和多糖的诸多生理功能,已证明KSC 在体内外具有显著的抗肿瘤免疫调节作用,可抑制肿瘤细胞增殖、增强免疫功能和降低化疗药物的毒副作用等。

精氨酸右布洛芬在大鼠体内的药物动力学研究徐远成;许军【期刊名称】《现代中西医结合杂志》【年(卷),期】2011(020)025【摘要】目的应用高效液相色谱法(HPLC)测定大鼠血浆中精氨酸右布洛芬浓度,研究单次口服给药后在大鼠体内的药代动力学.方法 SD大鼠分别按照体质量单次口服精氨酸右布洛芬50mg/kg后,采用HPLC测定血浆中精氨酸右布洛芬药物的浓度,绘制血药浓度-时间曲线,计算其药代动力学参数.结果单次口服精氨酸右布洛芬药-时曲线均符合口服吸收的一级动力学二室模型,主要药代动力学参数:t1/2α为(1.956±1.136)h,t1/2β为(5.551±3.981)h,Cmax为(7.365±2.627)μg/L,AUC(0-12)为(18.807±4.007)μg/(L·h),AUC(0-∞)为(23.852±7.767)μg/(L·h).结论 HPLC 法能够准确地测定精氨酸右布洛芬血药浓度,能满足药代动力学的研究需求.【总页数】2页(P3139-3140)【作者】徐远成;许军【作者单位】江西中医学院,江西,南昌,330004;江西中医学院,江西,南昌,330004【正文语种】中文【中图分类】R-332【相关文献】1.布洛芬异构体在大鼠体内的药代动力学研究 [J], 曾文学;周春阳;何林2.布洛芬颗粒剂在大鼠体内的时辰药物动力学 [J], 哈娜;杜智敏;郭美华3.丹参提取物及益心舒片中丹酚酸B在大鼠体内的药物代谢动力学研究 [J], 刘云峰;李海岛;李正;蔡芳;徐飞鹏4.酸模素大鼠体内药物代谢动力学研究 [J], 李文;罗莉丹;陈达林;廖禹东5.催产素在妊娠晚期及剖宫产术后大鼠体内药物代谢动力学研究 [J], 曲星铭;张纪;徐辉;张雷因版权原因，仅展示原文概要，查看原文内容请购买。

Toxicology237(2007)126–133Toxic effects of di(2-ethylhexyl)phthalate on mortality,growth, reproduction and stress-related gene expression inthe soil nematode Caenorhabditis elegansJi-Yeon Roh,In-Ho Jung,Jai-Young Lee,Jinhee Choi∗Faculty of Environmental Engineering,College of Urban Science,University of Seoul,90Jeonnong-dong,Dongdaemun-gu,Seoul130-743,Republic of KoreaReceived16March2007;received in revised form19April2007;accepted2May2007Available online18May2007AbstractIn this study,di(2-ethylhexyl)phthalate(DEHP)toxicities to Caenorhabditis elegans were investigated using multiple toxic endpoints,such as mortality,growth,reproduction and stress-related gene expression,focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP monitoring.The possible use of C.elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conducting the experiment on the exposure of nematode tofield soil. Twenty-four-hour median lethal concentration(LC50)data suggest that DEHP has a relatively high potential of acute toxicity to C. elegans.Decreases in body length and egg number per worm observed after24h of DEHP exposure may induce long-term alteration in the growth and reproduction of the nematode population.Based on the result from the C.elegans genome array and indicated in the literatures,stress proteins,metallothionein,vitellogenin,xenobiotic metabolism enzymes,apoptosis-related proteins,and antioxidant enzyme genes were selected as stress-related genes and their expression in C.elegans by DEHP exposure was analyzed semi-quantitatively.Expression of heat shock protein(hsp)-16.1and hsp-16.2genes was decreased by DEHP exposure.Expression of cytochrome P450(cyp)35a2and glutathione-S-transferease(gst)-4,phase I and phase II of xenobiotic metabolism enzymes, was increased by DEHP exposure in a concentration-dependent manner.An increase in stress-related gene expressions occurred concomitantly with the deterioration on the physiological level,which suggests an increase in expression of those genes may not be considered as a homeostatic response but as a toxicity that might have physiological consequences.The experiment with the soil from the landfill site suggests that the potential of the C.elegans biomarker identified in laboratory conditions should be calibrated and validated for its use in situ.©2007Elsevier Ireland Ltd.All rights reserved.Keywords:Caenorhabditis elegans;Di(2-ethylhexyl)phthalate;Stress-related gene expression;Ecotoxicity monitoring1.IntroductionDi(2-ethylhexyl)phthalate(DEHP)is widely used in flexible polyvinyl chloride(PVC)as a plasticizer to∗Corresponding author.Tel.:+82222105622;fax:+82222442245.E-mail address:jinhchoi@uos.ac.kr(J.Choi).soften plastic materials.Owing to its utility and cost effectiveness,DEHP has been used in a broad range of applications,such as wires and cables,floor tiles,garden hoses,containers,footwear and clothing(De Jonge et al., 2002;Chao and Cheng,2007).Due to the widespread use of DEHP,it is ubiquitous in the environment and many environmental species are thus exposed to vari-ous levels of DEHP in their natural habitat.At present,0300-483X/$–see front matter©2007Elsevier Ireland Ltd.All rights reserved. doi:10.1016/j.tox.2007.05.008J.-Y.Roh et al./Toxicology237(2007)126–133127hazard or risk assessments of DEHP conducted by inter-national authorities are available(WHO,1992;EPA, 1999;EU,2001;ATDSR,2002),some of which include an assessment of the ecological risk of DEHP to aquatic life.However,few studies have been performed on the ecotoxicity of soil organism.Caenorhabditis elegans,a free-living nematode that lives mainly in the liquid phase of soils,is thefirst multicellular organism to have its genome completely sequenced.The genome showed an unexpectedly high level of conservation with the vertebrate genome,which makes C.elegans an ideal system for biological studies, such as those in genetics,molecular biology and devel-opment biology(Brenner,1974;Bettinger et al.,2004; Leacock and Reinke,2006;Schafer,2006;Schroeder, 2006).C.elegans is also a good animal model for the study of ecotoxicology.Due to its abundance in soil ecosystems,its convenient handling in the labora-tory,and its sensitivity to different kinds of stresses, C.elegans is frequently used in ecotoxicological stud-ies utilizing various exposure media,including soil and water(Peredney and Williams,2000;Willams et al., 2000;Boyd and Williams,2003a).The range of C.ele-gans studies in ecotoxicology focuses on organism-level endpoints,such as mortality,behavior,growth or repro-duction.However,using these classical test endpoints, it is difficult tofind significant effects.Therefore,more specific and sensitive systems than classical ecotoxico-logical tests are needed.In this study,to identify a suitable tool to develop a screening system for ecotoxicity monitoring,DEHP toxicities to C.elegans were investigated using multiple toxic endpoints,such as mortality,growth,reproduc-tion and stress-related gene expression,focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP toxicity.C.elegans whole genome microarray was conducted for screen-ing the differentially expressed gene list by DEHP exposure.Tested stress-related genes were selected according to the results of microarray data and liter-ature.Alteration on stress-related gene expression by DEHP exposure was examined in a semi-quantitative manner.The response of greenfluorescent protein (gfp)transgenic nematode,incorporated full-length heat shock protein(hsp)-16.2and hsp-16.48genes to DEHP exposure was also examined to test a possibility of transgenic worm as a biosensor of environmen-tal monitoring.Additionally,in situ application of C. elegans toxicity indicator was investigated on the nema-todes exposed to soil from landfill sites,using the same endpoints applied for the exposure of laboratory condition.2.Materials and methodsanismsThe wild-type C.elegans Bristol strain N2was used in this study.C.elegans were maintained on nematode growth medium(NGM)plates seeded with Escherichia coli strain OP50,at20◦C,using the standard method previously described by Brenner(1974).Young adults(3days old)from an age-synchronized culture were used in all the experiments.Worms were incubated at20◦C for24h without a food source,and were then subjected to the analysis.2.2.Sample preparationFour types of endpoints(mortality,growth,reproduction, and stress-related gene expression)were assessed for exposure to DEHP.Pure analytical-grade DEHP(Sigma–Aldrich Chem-ical,St.Louis,MO,USA)was used in the experiment and it was dissolved in dimethyl sulfoxide(DMSO,Sigma–Aldrich Chemical).Nematodes were exposed to DEHP prepared in a K-medium(0.032M KCl,0.051M NaCl)(Williams and Dusenbery,1990).Three replicates for each concentration and a control were conducted for all the test types.The DEHP concentrations in a K-medium were nominal values.Soil toxicity testing with C.elegans was performed as described in the American Society for Testing and Materials (ASTM,2001).Briefly,for each sample,2.33g of soil as loaded in to a35mm×10mm petridish.The moisture was adjusted to35%(dry weight)using K-medium,worms were added and were incubated at20◦C for24h without a food source.The worms were then recovered with centrifugation/flotation using Ludox(Sigma–Aldrich).Three replicates were conducted for all the test types.2.3.Lethal toxicity testsEach test consisted of four concentrations and a control,in which10±1of young C.elegans adults were transferred onto 24-well tissue culture plates containing1ml of the test solution for each of thefive wells.The worms were exposed for24h at20◦C.After the24h,the numbers of live and dead worms were determined through visual inspection and by probing the worms with a platinum wire under a dissecting microscope.2.4.Measurement of growth and reproductionFollowing the24h incubation with exposure to sub-lethal concentrations of DEHP,growth and reproduction were assessed.Growth was assessed by measuring the length of the worms that had been killed by the heat through microscopy, with a scaled lens in each sample.The average length of the unexposed control worm was in the range of1.0–1.2mm. Reproduction was assessed by counting the eggs of each worm through the microscopic inspection of the transparent C.ele-gans body in each sample.Although this procedure differs from128J.-Y.Roh et al./Toxicology237(2007)126–133more commonly used reproduction tests of offspring counting from an age-synchronized single worm,this simple detection method seems appropriate for the rapid screening of the repro-duction effect(Roh et al.,2006).The average number of eggs per worm in the unexposed controls was in the range of10–25. One hundred worms were examined per treatment for growth and reproduction experiments.2.5.RNA extractionFollowing the24h incubation with exposure to sub-lethal concentrations of DEHP,nematodes were harvested for the preparation of RNA.Total RNA was prepared by phenol–chloroform extraction,according to the manufacturer’s standard protocol.RNA concentrations were determined by the absorbance at260nm.The quality of total RNA was estimated based on the ratio of the optical densities from RNA samples measured at260and280nm.2.6.Microarray analysisFive micrograms of the total RNA extracted from nema-todes exposed to DEHP and the control was used for reverse and in vitro transcription followed by application to a GeneChip®C.elegans Genome Array(Affymetrix,Santa Clara,CA, USA),which contains22,500probe sets against22,150unique C.elegans transcripts.The arrays were scanned with the GeneChip scanner3000(Affymetrix),controlled by GeneChip Operating Software(GCOS,Affymetrix).2.7.Semi-quantitative reverse transcription-polymerase chain reactionThe two-step reverse transcription-polymerase chain reac-tion(RT-PCR)method was used with RT Premix(Bioneer Co., Seoul,Korea)and PCR Premix kits(Bioneer Co.),using a PTC-100thermal cycler(MJ Research,Lincoln,MA,USA). The primers were designed on the basis of the sequences retrieved from the C.elegans database(). Actin mRNA was used for expression-level normalization of the studied genes.The PCR products were separated through electrophoresis on1.5%agarose gel(Promega,Madison,WI, USA)and were visualized with ethidium bromide(Bioneer Co.).All the tests were replicated at least three times,and the relative densities of each band were determined with the use of a Kodak EDAS290image analyzer(Kodak,Rochester,NY, USA),with a TFX-20.M UV transilluminator(Vilber Lourmat, Marne la Vallee,France).2.8.Detection of greenfluorescence protein transgenic C. elegansThe transgenic strains(hsp-16.2::gfp and hsp-16.48::gfp)of C.elegans were developed as previously described by Hong et al.(2004).Transgenic C.elegans were incubated for24h with 2mg/l of DEHP,as well as,with soil from landfill sites,and the fluorescence signal was examined from20independent trans-genic worms per treatment.Fluorescence was observed using a Leica DM IRB microscope(Leica,Wetzlar,Germany),and the image was taken using a Leica DC300FX camera(Leica). Levamisole(Sigma–Aldrich Chemical)treatment(2mM)was used to take pictures of the live worms.2.9.Analysis of DEHP from soil of Korean landfill sitesSoil was collected from Korean landfill sites,namely Jeonju (J),Gwangju(G)and Mokpo(M).Reference soil(R)was sampled from the clean area.Soil samples were dried in the air at room temperature.DEHP was extracted using the Pres-surized Solvent Extraction(SPE)method and was analyzed using6890Gas Chromatography–5973N Mass Spectroscopy systems(Agilent,Santa Clara,CA,USA).2.10.Data analysisMedian lethal concentration(LC50)were derived through Probits analysis.The statistical differences between the con-trol and treated worms were determined with the aid of the parametric t-test.3.ResultsAcute toxicity of DEHP on C.elegans was investigated using mortality as endpoint(Table1). Twenty-four-hour LC50of DEHP in C.elegans was estimated as22.55mg/l.Based on the results of the acute toxicity test,three concentrations corresponding to 1/1000,1/100,and1/10of the24h LC50were selected for laboratory sublethal exposure conditions,that is, 0.02,0.2and2mg/l.The changes in the worms’body lengths and in the number of eggs per worm were investigated as a growth and a reproduction indicator,respectively(Fig.1).The worms,which had been exposed to0.02and0.2mg/l of DEHP,showed a decrease in their body length,as well as,in the number of eggs per worm.Suitability of the DNA microarray technique for the ecotoxicological approach was investigated in C. elegans exposed to0.2mg/l of DEHP.DEHP-induced genes expression was screened using C.elegans Genome Arrays.Major up-and down-regulated known genes by Table1Estimation of24h LC10,LC50and LC90of DEHP in C.elegans24-h LC(mg/l)Interval of confidence(95%) LC10 3.6500.016<LC10<11.33LC5022.55 4.200<LC50<56.63LC90139.455.75<LC90<5611LC means lethal concentration.J.-Y.Roh et al./Toxicology237(2007)126–133129Fig.1.Growth and reproduction indicators examined in the young adult of Caenorhabditis elegans exposed to DEHP for24h.Growth was assessed by measuring the length of the worms that had been killed by the heat through microscopy,with a scaled lens in each sample.Reproduction was assessed by counting the eggs of each worm through the microscopic inspection of the transparent C.elegans body in each sample.DEHP exposure in C.elegans were shown in Table2.For stress-related gene expression profiling analysis,based on the result from the microarray and what is found in the literature,we investigated alteration on the gene expression of heat shock proteins(hsp-16.1,hsp-16.2, hsp-16.48,hsp-70),metallothioneins(mt-1,mt-2),vitel-logenins(vit-2,vit-6),xenobiotic metabolism enzymes (cyp35a2,gst-4),tumor suppressor and apoptosis pro-teins(cep-1,ape-1),and antioxidant enzymes(sod-1, ctl-2)in C.elegans by DEHP exposure.The stress-related gene expression profile was inves-tigated in the young C.elegans adults exposed to DEHP for24h(Fig.2).Expression of hsp-16.1and hsp-16.2decreased at0.02and0.2mg/l of DEHP expo-sure.Expression of mt-2increased at DEHP-treated C.elegans.However,due to high data variation,sta-tistical significance was not observed.Expression of cyp35a2and gst-4,phase I and phase II of xenobiotic metabolism enzymes,was increased by DEHP expo-sure in a concentration-dependent manner.Expression of genes related to vitellogenes,apoptosis,or antioxidant enzymes was not changed by DEHP exposure.As shown in Fig.3,the hsp-16.2and hsp-16.48 gene expression levels were assayed using gfp-basedTable2Screening of DEHP induced gene expression using C.elegans whole genome microarrayUp-regulated genes Down-regulated genesWormbase no.Sequence description Flod change Wormbase no.Sequence description Flod change K02D7.3Cuticular collagen17Y71D11A.5Ion channel protein0.03C39E9.3Collagen 6.4W03G1.7Sphingomyelin phosphodiesterase0.03R08F11.7Peroxidase 6.2F36D3.9Cysteine protease0.07B0365.6C-type lectin domain 5.8C06A8.9Glutamate receptor0.07CEC2033Major sperm protein 5.0F21F8.2Protease0.08K08E7.9Multidrug resistance protein 4.2C58111GTP-binding protein0.08C33A12.6UDP-glucuronysltransferase 3.6C52B9.9AMP-binding protein0.1K09F5.2vit-1 3.0C37H5.1Annexin0.13F09G2.3Permease 2.9C08H9.1Serine carboxypeptidase0.15T11F9.3Zinc metalloprotease 2.9T07G12.1Calmodulin0.16F34H10.1Ubiquitin/ribosomal protein 2.7K08C7.5Flavin-containing monoxygenase0.16C03G6.15Cytochrome P450 2.7F53B7.2G-protein coupled receptor0.18F28A12.4Peptidase 2.6F48E3.7Acetylcholine receptor0.18F22D6.10Collagen 2.6Y46H3A.3Heat shock protein0.19ZK1248.17Major sperm protein 2.6C42C1.2Protein phosphatase0.19F08A8.3Acyl-coenzyme A oxidase 2.5E02H9.5Glycosyl hydrolase0.23R07B1.3Membrane glycoprotein 2.5T27E4.8Heat shock protein HSP16-10.28W06D12.3Fatty acid desaturase 2.4T27E4.9Heat shock protein0.3K08B4.3Glucuronosyltransferase 2.4K03A1.4Calmodulin calcium-binding sites0.31F44G3.2Arginine kinase 2.4AU113943Carbonic anhydrase0.31130J.-Y.Roh et al./Toxicology 237(2007)126–133Fig.2.Stress-related gene expression profiling in the young adult of C.elegans exposed to DEHP for 24h.Stress related gene mRNA was amplified by RT-PCR method using the primers designed on the basis of the sequences retrieved from the C.elegans database ( ).Actin mRNA was used for expression-level normalization of the studied genes.The PCR products were separated through electrophoresis on 1.5%agarose gel and were visualized with ethidium bromide.All the tests were replicated at least threetimes.Fig.3.Response of hsp-16.2::gfp and hsp-16.48::gfp transgenic C.elegans exposed to DEHP for 24h.Transgenic C.elegans were incubated for 24h with 2mg/l of DEHP and the fluorescence signal was examined from 20independent transgenic worms per treatment.Fluorescence was observed using a Leica DM IRB microscope,and the image was taken using a Leica DC 300FX camera.reporter transgenic nematodes.Transgenic nematodes were exposed to 2mg/l of DEHP and the fluorescence signals from both gfp transgenic lines increased after DEHP exposure.The response of hsp-16.48::gfp,how-ever,was greater than that of hsp-16.2::gfp.Concomitantly with bioassay using C.elegans ,the analysis of DEHP was conducted on three Korean landfill sites (Table 3).The contamination levels of DEHP in soil from landfill sites were between 6and 20mg/kg soil.DEHP was not detected in the soil from the referencesite.Fig.4.Mortality,growth and reproduction indicators (A),stress-related gene expression profiling measured in the young adult of C.elegans exposed for 24h to DEHP contaminated soil from Korean landfill sites (B).Response of hsp-16.2::gfp and hsp-16.48::gfp transgenic C.elegans exposed for 24h to DEHP contaminated soil from Korean landfill sites (C).The soils were named as R,G,M,J (R:reference site soil,G:Gwangju landfill soil,M:Mokpo landfill soil,J:Jeonju landfill soil).J.-Y.Roh et al./Toxicology237(2007)126–133131Table3Analysis of DEHP on soil from Korean landfill sites(number=3; mean±standard error of the mean)R a G b M c J dDEHP(mg/kg)ND e 6.27±0.6520.05±1.3410.73±1.37a R:Reference soil site.b G:Gwangju landfill site.c M:Mokpo landfill site.d J:Jeonju landfill sites.e ND:non detected.Fig.4shows the response of C.elegans on mortal-ity,growth,reproduction,stress-related gene expression and the response of transgenic C.elegans exposed to soil from Korean landfill sites for24h.Increase in mortality occurred in the most severe extent(about30%)in soil from the M landfill site,which showed the highest DEHP contaminated level among the three sites(20mg/kg). Growth parameters did not change in the worm exposed to soil from the landfill site.The number of eggs per worm slightly increased in the nematode that had been exposed to soil from the J landfill site.Differently from that in Fig.2,worms that had been exposed to soil from landfill sites did not show any statistically significant change in studied stress-related gene expression.Theflu-orescence signals from both gfp transgenic lines slightly increased in transgenic nematode exposed to soil from the landfill site.4.DiscussionsC.elegans is an attractive animal model for the study of the ecotoxicological relevance of chemical-induced molecular-level responses(Menzel et al.,2005;Reichert and Menzel,2005).In this study,the utility of molecu-lar parameters,such as stress-related gene expression, as biomarkers in C.elegans were investigated to iden-tify specific and sensitive tools to develop a screening system for ecotoxicity monitoring.And the possible use of C.elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conduct-ing the experiment on the exposure of nematode tofield soil.Although DEHP is a widely used environmental compound,little study has been performed on its eco-toxicological properties.Twenty-four-hour LC50data (Table1)suggest that DEHP has a relatively higher potential of acute toxicity to C.elegans than previ-ously studied metals have(Roh et al.,2006).Mortality is a reliable ecotoxicological endpoint.However,such a high level of exposure hardly occurred in the real environment.More sensitive indicators,physiological-level alterations,such as growth,reproduction,feeding, movement,or behavior,have been used as endpoints for chemical-induced toxicity testing in C.elegans(Dhawan et al.,2000;Anderson et al.,2001,2004;Kohra et al., 2002;Tominaga et al.,2003).The effects of xenobiotics on the growth and reproduction of the test organisms are broadly accepted test parameters,and were found to be more sensitive indicators of toxicity than lethality,as also shown in this study(Fig.1).The decreases in body length and egg number per worm observed after24h of DEHP exposure may induce alteration in the growth and reproduction of the nematode population in the long term.However,due to the low concentration of xenobi-otics in the environment,it is hard tofind a correlation between the occurrence of contaminants and a physio-logical effect of a test organism in the environment,even when using reliable ecotoxicological endpoints,such as,growth or reproduction,which emphasizes the need for understanding the sublethal effects at the biochem-ical and molecular levels where the toxicant-induced responses are initiated.The effect of DEHP to the C.elegans whole genome gene expression was ing concentra-tion corresponding to the1/100of LC50value,which was0.2mg/l of DEHP,it was possible to show that a strong and differential gene induction or repression was detectable in response to DEHP exposure;they were cyp superfamily,hsp,vit,multidrug-resistance protein, etc.(Table2).In case of some genes,such as C-type lectin,collagene,and major sperm protein genes,it is unclear what physiological meaning the over-expression has.Stress-related genes were selected from the array results and previously reported literature.Expression of selected stress-related genes to DEHP exposure was investigated at three different sublethal concentrations, using the semi-quantitative RT-PCR method.It is obvi-ous that members of the cyp family,gst,hsp,and vit genes should be included in a selection of stress-related genes.Among the group of genes,which encode proteins belonging to different metabolic pathways,the met-allothionein,apoptosis,and antioxidant enzyme genes were also included in the stress-related gene selection. As screened in the microarray result,the expression of hsp-16.1and hsp-16.2genes was decreased by DEHP exposure.DEHP exposure led to increases in the expres-sion of some stress-related genes tested,including mt-2, cyp35a2and gst-4.In particular,it has been reported that almost all cyp35forms in C.elegans are moder-ately or strongly inducible by different xenobiotics in a cyp450gene-expression screening experiment(Menzel et al.,2001,2005).Increase in stress-related gene expres-sions occurred concomitantly with this deterioration on132J.-Y.Roh et al./Toxicology237(2007)126–133the physiological level,which suggests that the increase in the expression of those genes may not be considered as a homeostatic response but as toxicity that might lead to physiological consequences(Figs.1and2).C.elegans also offers the advantage of transgenetic approaches,which may allow the development of a sensi-tive biosensor for environmental monitoring(Stringham and Candido,1994;Jones et al.,1996;Chu et al.,2005). In our previous study(Roh et al.,2006),gfp trans-genic nematode,incorporated full-length hsp-16.2and hsp-16.48genes were developed,since hsps are thought to play roles in various stress conditions.As shown in their response to the exposure to four metals(Roh et al.,2006),semi-quantitatively assayed using gfp-based reporter hsp-16.2and hsp-16.48transgenic nematodes were not very sensitive towards DEHP exposure.Even though,transgenic nematode seems to have a consider-able potential as a biosensor for toxicity monitoring,to develop a sensitive biosensor using transgenic C.ele-gans,the responses of a broad range of stress-related gene promoters to various classes of chemicals should be screened and validated with environmentally relevant low-concentration samples.Bioassay methods for soil toxicity monitoring have also been developed and frequently used in C.ele-gans(Peredney and Williams,2000;Boyd and Williams, 2003b).As a soil-dwelling organism,C.elegans has a potential as a bioindicator for the detection of soil con-tamination because of its abundance in soil ecosystems and its sensitivity to different kinds of stresses,which was tested in this study using the same endpoints used for laboratory K-media exposure condition(Fig.4).Increase in egg number per worm in the nematode exposed to soil from landfill sites suggests a mixture of contaminants, including DEHP,might have a stimulatory effect on the reproduction of the nematode.Expression of stress-related genes did not affect C.elegans exposed to soil from the landfill site,which were different from the response of nematodes exposed to DEHP prepared in K-media in laboratory conditions.The common feature of responses to DEHP of C.elegans between labora-tory andfield conditions was the increase in gst-4gene expression.But,as gst is known to use a broad range of substrates,it is difficult to consider it as to DEHP-specific biomarker.Increase influorescence signal of hsp-16.2::gfp and hsp-16.48::gfp transgenic nematodes might be interpreted as a response of heat-shock protein upon exposure to mixture contaminants.The experiment with the soil from the landfill site suggests that the poten-tial of the C.elegans biomarker identified in laboratory conditions should be calibrated and validated for their use in situ.The data obtained from this study can comprise an important contribution to the knowledge of the toxi-cology of DEHP in C.elegans,about which little data are available.A link or correlation between a vali-dated toxicity endpoint(e.g.,growth and reproduction) and upstream-induced gene expression is interest-ing,particularly for ecotoxicological purposes.Direct experimental demonstrations of the wider relationships between molecular-/biochemical level effects and their subsequent consequences at higher levels of biologi-cal organization are needed in order to establish causal relationships.The characterization of the causal rela-tionships between the molecular level responses and the effects at higher biological levels will help to define the sublethal hazards of chemicals in C.elegans. AcknowledgementThis work was accomplished through the generous support of the Korea Research Foundation Grant funded by the Korean Government(MOEHRD)(Grant No. KRF-2005-204-D00009).Appendix A.Supplementary dataSupplementary data associated with this arti-cle can be found,in the online version,at doi:10.1016/j.tox.2007.05.008.ReferencesAnderson,G.L.,Boyd,W.A.,Williams,P.L.,2001.Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans.Environ.Toxicol.Chem.20,833–838. Anderson,G.L.,Cole,R.D.,Williams,P.L.,2004.Assessing behav-ioral toxicity with Caenorhabditis elegnas.Environ.Toxicol.Chem.23,1235–1240.ASTM,2001.Standard Guide for Conducting Laboratory Soil Toxi-city Test with the Nematocde Caenorhabditis elegans.E2172-01.American Society for Testing and Materials,West Conshohocken, PA.ATDSR,2002.Toxicological Profile for Di-(2-ethylhexyl)phthalate (DEHP).Department of Health and Human Services,Public Health Service,Agency for Toxic Substances and Disease Registry, Atlanta.Bettinger,J.C.,Carnell,L.,Davies,A.G.,McIntire,S.L.,2004.The use of Caenorhabditis elegans in molecular neuropharmacology.Int.Rev.Neurobiol.62,195–212.Boyd,W.A.,Williams,P.L.,2003a.Availability of metals to the nematode Caenorhabditis elegans:toxicity based on total concen-trations in soil and extracted fractions.Environ.Toxicol.Chem.22,1100–1106.Boyd,W.A.,Williams,P.L.,parison of the sensitivity of three nematode species to cooper and their utility in aquatic and soil toxicity tests.Environ.Toxicol.Chem.22,2768–2774.。

• 140 •毒理学杂志2021 年4 月第35 卷第2 期J Toxicol April 2021 Vol. 35 No. 2中图分类号：R965. 1 ;R99;R114文献标识码：A文章编号：1002-3127( 2021 )02-0丨40-06•实验研究•姜黄素联合黄芩苷对乙醇诱导的大鼠胆汁淤积性肝损伤的保护作用研究王晓霞\占海兵'，高青\张琼\杨蒙蒙\李盛2,常旭红\孙应彪1(1.兰州大学公共卫生学院，甘肃兰州730030; 2.兰州市第一人民医院，甘肃兰州730050)【摘要】目的探讨姜黄素和黄芩苷对乙醇诱导的大鼠胆汁淤积性肝损伤的保护作用及其潜在机制。

方法将50只Wistai■雄性大鼠随机分为正常对照组、乙醇模型组（50%v/v)、姜黄素干预组（50m g/k g)、黄芩苷干预组（50m g/k g)及姜黄素（50 m g/k g)和黄芩苷（50 m g/k g)联合干预组。

酶比色法检测大鼠血清和肝总胆汁酸（T B A)水平，蛋白免疫印迹法检测大鼠肝C Y P7A1、C Y P8B1、B S E P、M R P2、M R P3、M R P4、C Y P3A4和S U L T2A1的蛋白表达水平。

结果与对照组比较，大鼠血清和肝组织中T B A水平均增加（P<0. 05)，肝组织中胆汁酸合成酶C Y P7A1和C Y P8B1及胆汁酸转运蛋白M R P4的蛋白表达水平均上调，而胆汁酸转运蛋白B S E P和M R P2及胆汁酸解毒酶C Y P3A4和S U L T2A1的蛋白表达水平均下调（尸<0.05)。

与乙醇模型组比较，联合干预组大鼠血清和肝T B A水平均降低（P<0.05);肝组织中C Y P7A1、C Y P8B1和M R P4的蛋f t表达水平均下调（P<0.05)，而B S E P、M R P2、C Y P3A4和S U L T2A1蛋白表达水平均上调（P<0.05)。

麝香通心滴丸预处理对大鼠心肌缺血再灌注损伤的保护作用机制李蒙1,刘用2,张健真2,郝学增1,张立晶1摘要目的:观察麝香通心滴丸预处理对大鼠心肌缺血再灌注(I/R)损伤后心肌细胞和血管的保护作用及潜在机制㊂方法:将60只雄性SD大鼠随机分为假手术组(Sham组)㊁I/R组㊁麝香通心滴丸组(SXTX组)和阳性组,每组15只㊂SXTX组以18.90mg/(kg㊃d)的浓度灌胃;阳性组以尼可地尔1.35mg/(kg㊃d)的浓度灌胃;Sham组和I/R组给予等体积去离子水灌胃㊂各组均灌胃1周后,行冠状动脉前降支结扎-松解手术,除Sham组外其余各组大鼠结扎左冠状动脉前降支30min后,再灌注120min,构建心肌I/R模型㊂通过心肌苏木精-伊红(HE)染色㊁心肌Masson染色观察大鼠心肌组织及血管改变;电镜观察心肌细胞和血管的超微结构改变;免疫组织化学法观察血管相关蛋白低氧诱导因子-1α(HIF-1α)和血管内皮生长因子(VEGF)表达情况㊂结果:与Sham组比较,I/R组大鼠心肌细胞坏死和血管损伤明显,心肌组织中HIF-1α表达增加;与I/R组相比,SXTX组心肌细胞和血管病理结构损伤减轻,缺血区心肌组织VEGF含量明显增加㊂结论:麝香通心滴丸预处理可保护大鼠心肌细胞和血管I/R损伤,可能与上调VEGF蛋白表达㊁促进缺血区微血管新生有关㊂关键词缺血/再灌注损伤;麝香通心滴丸;保护作用;低氧诱导因子-1α;血管内皮生长因子;实验研究d o i:10.12102/j.i s s n.1672-1349.2024.02.008Protective Effect of Shexiang Tongxin Dropping Pill Pretreatment on Myocardial Ischemia-reperfusion Injury in RatsLI Meng,LIU Yong,ZHANG Jianzhen,HAO Xuezeng,ZHANG LijingDongzhimen Hospital,Beijing University of Chinese Medicine,Beijing100700,ChinaCorresponding Author ZHANG Lijing,E-mail:****************Abstract Objective:To observe the protective effect of Shexiang Tongxin Dropping Pills on myocardial cells and blood vessels after myocardial ischemia-reperfusion(I/R)injury in rats.Methods:Sixty male SD rats were randomly divided into sham operation group (Sham group),I/R group,Shexiang Tongxin Dropping Pills group(SXTX group),and positive control group,with15rats in each group.The rats in SXTX group were given SXTX18.90mg/(kg㊃d)by gavage,and the rats in positive control group were given nicorandil1.35 mg/(kg㊃d)by gavage.The rat in Sham group and I/R group were given the same volume of deionized water by gavage.After one week of gavage,the rats left anterior descending coronary artery was ligated for30min and then reperfused for120min to build the myocardial I/R model except for the Sham group.The changes of myocardial tissue and blood vessels in rats were observed by myocardial hematoxylin-eosin(HE)staining and myocardial Masson staining.The ultrastructural changes of cardiomyocytes and blood vessels were observed by electron microscope.The expressions of hypoxia-inducible factor-1α(HIF-1α)and vascular endothelial growth factor (VEGF)were observed by immunohistochemistry.Results:Compared with Sham group,the myocardial cell necrosis and vascular injury of the rats in I/R group were obvious,and the expression of HIF-1αin the myocardial tissue pared with I/R group,the myocardial cell and vascular pathological structural damage in SXTX group alleviated,and the protein expressions of VEGF in the ischemic area significantly increased.Conclusion:Shexiang Tongxin Dropping Pills pretreatment can protect myocardial cells and vascular I/R injury in rats,which may be related to the up-regulation expression of VEGF protein and the promotion of microangiogenesis in ischemic areas.Keywords ischemia/reperfusion injury;Shexiang Tongxin Dropping Pills;protective effect;hypoxia-inducible factor-1α;vascular endothelial growth factor;experimental study急性心肌梗死(acute myocardial infarction,AMI)是心血管疾病的最危重类型之一,其发病急㊁预后差㊁病死率高,是急性心源性致死性疾病的主因㊂无论是药物溶栓㊁冠状动脉支架植入,还是冠状动脉旁路移植术,再灌注治疗都是其必然过程[1]㊂然而,心肌再灌注基金项目2021年度北京中医药大学新教师启动基金项目(No.2021-JYB-XJSJJ057);中央高校基本科研业务费专项资金资助项目(No. 2020-YJB-ZDGG-116)作者单位 1.北京中医药大学东直门医院(北京100700);2.北京中医药大学通讯作者张立晶,E-mail:****************引用信息李蒙,刘用,张健真,等.麝香通心滴丸预处理对大鼠心肌缺血再灌注损伤的保护作用机制[J].中西医结合心脑血管病杂志,2024, 22(2):250-253.过程本身可诱导心肌细胞损伤㊁坏死进一步加重,这一过程即为缺血再灌注(ischemia reperfusion,I/R)损伤[2]㊂因此,预防和治疗再灌注损伤尤为关键㊂麝香通心滴丸由‘太平惠民和剂局方“中至宝丹衍生而来,具有芳香益气通脉㊁活血化瘀止痛的功效㊂前期研究发现麝香通心滴丸对经皮冠状动脉介入治疗(PCI)术后慢血流病人的有效性及安全性较好[3]㊂关于麝香通心滴丸对I/R后心肌细胞和血管的保护作用及作用机制尚未明确㊂本研究通过构建I/R损伤大鼠模型,观察麝香通心滴丸预处理对大鼠心肌I/R损伤后心肌细胞和血管的保护作用,以及对低氧诱导因子-1α(HIF-1α)㊁血管内皮生长因子(VEGF)表达的影响,进而探讨其保护I/R的潜在作用机制,为麝香通心滴丸防治I/R的临床应用提供实验依据㊂1材料与方法1.1实验材料1.1.1实验动物无特定病原体(SPF)级雄性SD大鼠60只,9~ 10周龄,体质量(250ʃ10)g,购自北京华阜康生物科技股份有限公司㊂实验操作通过北京中医药大学东直门医院动物伦理的要求,伦理号(No):21-49㊂1.1.2实验药物麝香通心滴丸,规格:35mg,批准文号:国药准字Z20080018,由内蒙古康恩贝药业有限公司提供,以去离子水配成[18.90mg/(kg㊃d)]溶液,超声溶解,高压灭菌后于-20ħ贮存备用㊂尼可地尔片,规格:每片5 mg,批准文号:国药准字H20160540,由Nipro Pharma Corporation Kagamiishi Plant生产,使用去离子水配制成1.35mg/(kg㊃d)的混悬液保存备用㊂1.1.3实验试剂及仪器实验试剂:苏木素染液(武汉市皮诺飞生物科技有限公司生产),HIF-1α抗体(28b)(Santa,sc-13515-200μg/mL),VEGF Rabbit Polyclonal Antibody(proteintech, 19003-1-AP-150μL),过氧化氢溶液(国药集团化学试剂有限公司生产,10011218),牛血清白蛋白(BSA) (Solarbio,A8020),包埋液(环氧树脂Epon812),二抗[辣根过氧化物酶(HRP)标记山羊抗兔/鼠通用二抗] (DAKO,K5007),组化试剂盒二氨基联苯胺(DAB)显色剂(DAKO,K5007)㊂实验仪器:超薄切片机(徕卡ULTRACUT R),光学显微镜(CIC,XSP-C204),透射电镜(日本日立H-7650),正置光学显微镜(日本尼康),成像系统(日本尼康)㊂1.2实验方法1.2.1动物造模对SD大鼠进行冠状动脉前降支结扎松解手术构建心肌I/R模型㊂将SD大鼠以4%戊巴比妥钠(0.4mL/100g)腹腔麻醉注射,之后气管插管,接动物呼吸机和心电图机,于左侧第三肋与第四肋间切开皮肤,钝性分离肌层,打开胸腔,充分暴露心脏,用带线缝合针在左心耳与肺动脉圆锥之间,于左心耳根部下方约2mm处进针,将左前降支连同少量的心肌组织一起结扎,先缺血30min㊁再灌注120min制备大鼠心肌I/R模型㊂抬高的ST段下降1/2以上为大鼠心肌I/R 模型造模成功㊂1.2.2分组及给药适应性喂养1周,采用随机数字表法将60只SD 大鼠随机分为4组:假手术组(Sham组)㊁I/R组㊁麝香通心滴丸组(SXTX组)和阳性组,每组15只㊂所有用药剂量按照人与大鼠体表面积换算,SXTX组按成人用量的2倍,给予麝香通心滴丸18.90mg/(kg㊃d)灌胃,阳性组以尼可地尔1.35mg/(kg㊃d)灌胃,Sham 组和I/R组给予等体积去离子水灌胃㊂每日1次,连续灌胃1周,末次灌胃1h后构建大鼠I/R模型㊂1.3观察指标1.3.1心肌苏木精-伊红(HE)染色实验结束后取出心脏,立即将10%多聚甲醛溶液50mL注入离体心脏主动脉内,5min后将左心室前壁心肌组织中间层切成条状,放在上述固定液中固定48h 后,蒸馏水清洗㊁梯度乙醇脱水,后制作病理切片,切片厚度为4μm,脱蜡㊁复水后行HE染色,封固,光镜下观察大鼠心肌组织及血管改变㊂1.3.2心肌Masson染色取出甲醛中固定24h后的心肌组织,按乙醇浓度由低至高脱水,石蜡包埋,切片,脱蜡后蒸馏水水洗㊂Weiger氏铁苏木素染色5~10min后流水充分水洗,再用1%的盐酸乙醇分化,再用丽春红酸性品红染液染色5~10min后用蒸馏水水洗,加1%磷钼酸水溶液5min,后苯胺蓝液复染5min,随后1%冰醋酸即刻洗涤后用95%乙醇多次脱水,后置于二甲苯透明,中性树胶封片,光镜下观察心脏组织纤维化情况㊂1.3.3电镜观察心肌血管内皮细胞超微结构留取再灌注末左心室梗死周边缺血区心肌体积约1mm3的标本,放置于4%戊二醛内固定,0.1mol/L二甲膦酸钠缓冲液漂洗,1%四氧化锇固定,然后梯度丙酮脱水,使用环氧树脂进行包埋㊁聚合,制作超薄切片,最后用醋酸双氧铀㊁柠檬酸铅染色,在透射电镜下观察并拍照㊂1.3.4免疫组织化学法检测血管相关蛋白HIF-1α㊁VEGF的表达情况取10%甲醛固定梗死周边缺血区心肌组织,并进行常规石蜡包埋及心肌切片(5μm),石蜡切片脱蜡至水,抗原修复,阻断内源性过氧化物酶,3%牛血清白蛋白室温封闭30min,加一抗(HIF-1α1ʒ50稀释,VEGF 1ʒ200稀释),4ħ孵育过夜,加HRP标记山羊抗兔二抗孵育30min,DAB显色,复染细胞核,脱水封片,光镜下分别观察HIF-1α㊁VEGF蛋白的表达情况㊂1.4统计学处理采用SPSS20.0统计软件进行数据分析㊂正态分布的定量资料以均数ʃ标准差(xʃs)表示,组间比较采用单因素方差分析,方差齐性采用Student-Newman-Keuls检验,方差不齐用Tamhane's T2检验㊂以P<0.05为差异有统计学意义㊂2结果2.1HE染色结果Sham组心肌组织结构正常㊁完整,心肌细胞分布均匀㊁有序,轮廓清晰可见,胞核与胞质边缘分明,血管管腔完整㊂I/R组心肌组织染色着色较浅,有细胞轮廓不清及破碎现象,组织排列异常紊乱㊁呈无序状,并伴有炎性细胞浸润,坏死区充血出血㊂阳性组心肌细胞部分相互连接,排列整齐,心肌细胞形态正常,心肌纤维中少量心肌纤维变性坏死及脂肪变性,伴少量炎性细胞浸润㊂与I/R组比较,SXTX组心肌组织排列较为整齐,着色相对更均匀,有炎性细胞浸润,血管管腔完整度较好㊂详见图1㊂图1各组大鼠心肌组织病理变化比较(HE染色,ˑ400)2.2Masson染色结果Sham组Masson染色心肌纤维无纤维化,仅在血管壁周边有着色㊂I/R组显示紫红色的心肌组织明显减少,存在大量呈蓝色的胶原纤维,血管破碎,部分血管呈管腔狭窄和闭合㊂与I/R组相比,阳性组㊁SXTX 组Masson染色显示以紫红色心肌组织为主,蓝色胶原纤维增生沉积现象显著减轻㊂详见图2㊂图2各组大鼠心肌组织病理变化比较(Masson染色,ˑ400)2.3透射电镜观察心肌血管内皮细胞超微结构Sham组微血管基底膜未见增厚,内弹性膜未见变窄,管腔规整,内皮细胞排列整齐,内皮细胞胞质和核仁结构清楚,周围心肌细胞形态完整,未见脂质沉积;I/R组内膜下结缔组织肿胀,内弹性膜局部变窄,管腔严重变形,内皮细胞胞质肿胀,可见胞质碎片,核膜部分缺失,周围心肌细胞出现明显损伤,细胞破碎严重,大量的脂质沉积;与I/R组相比,SXTX组微血管基底膜轻度增厚,内弹性膜局部变窄,管腔稍有变形,内皮细胞排列较整齐,可见内皮细胞质和核仁结构,周围心肌细胞形态基本完整,少量脂质沉积㊂详见图3㊂2.4免疫组织化学法检测心肌中HIF-1α㊁VEGF的蛋白表达与Sham组比较,I/R组心肌组织中HIF-1α表达增加,提示在缺血㊁缺氧状态下HIF-1α蛋白反应性增加㊂与I/R组比较,SXTX组心肌组织HIF-1α表达下降,VEGF蛋白表达增加,提示麝香通心滴丸可促进VEGF蛋白的生成,进而促进缺血区血管形成,改善心肌组织乏氧环境㊂详见图4㊁图5㊂图3各组大鼠心肌细胞及血管超微结构变化比较图4麝香通心滴丸对HIF-1α蛋白表达的影响(ˑ100)图5麝香通心滴丸对VEGF蛋白表达的影响(ˑ100)3讨论时间就是心肌 ,由于心肌组织细胞对缺氧㊁缺血的敏感性高,尤其是其具有不可再生的特性,因此,在AMI的救治过程中,及时再灌注治疗是挽救濒死心肌细胞的关键㊂然而缺血再灌注治疗必然会造成再灌注损伤,是AMI不可避免的风险事件㊂保护心肌I/R损伤可防止心肌梗死后不良重塑和更差的临床结果㊂研究发现持续的心肌I/R可诱导各种形式的心肌细胞死亡和冠状动脉微血管损伤[4]㊂麝香通心滴丸由人工麝香㊁蟾酥㊁人工牛黄㊁冰片㊁人参茎叶㊁丹参㊁熊胆粉组成,具有芳香益气通脉㊁活血化瘀止痛之功效㊂多项研究表明麝香通心滴丸可减轻心肌缺血损伤,可能通过升高内源性一氧化氮水平,抑制炎症反应,改善冠状动脉微循环等[5-7]㊂本研究亦发现麝香通心滴丸可保护心肌I/R引起的心肌细胞和血管损伤㊂王伟等[8]对麝香通心滴丸组方入血成分进行网络药理学研究,发现VEGF/血管内皮细胞生长因子受体2(VEGFR2)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(AKT)通路可能为麝香通心滴丸治疗冠心病的信号通路㊂本研究结果同样发现麝香通心滴丸可促进缺血区心肌组织VEGF蛋白的生成,并提示可能与HIF-1α介导的信号通路有关㊂HIF-1α是一种关键的氧敏感转录因子,心肌组织细胞缺血缺氧时,诱导HIF-1α表达增加,进而调控多种基因表达,发挥机体对低氧或缺血的保护性适应[9]㊂VEGF与血管生成密切相关㊂在心肌I/R损伤中,增加血管生成可有效改善病灶缺氧状态,从而保护心脏[10]㊂本研究发现心肌I/R损伤时HIF-1α的表达增加以适应乏氧环境,麝香通心滴丸可能通过促进VEGF蛋白的生成,促进缺血心肌血管新生,改善乏氧环境,参与大鼠心肌I/R心肌保护的过程㊂综上所述,麝香通心滴丸可有效保护大鼠心肌I/R 的细胞和血管损伤,减轻心肌组织病理损伤,其作用机制可能与上调心肌组织VEGF蛋白的表达及促进缺血区微血管新生有关,从而达到保护心脏㊁抗心肌I/R损伤的作用㊂参考文献:[1]ZHU S S,XU T D,LUO Y Y,et al.Luteolin enhances sarcoplasmicreticulum Ca2+-ATPase activity through p38MAPK signalingthus improving rat cardiac function after ischemia/reperfusion[J].Cellular Physiology and Biochemistry,2017,41(3):999-1010.[2]LAHNWONG S,PALEE S,APAIJAI N,et al.Acute dapagliflozinadministration exerts cardioprotective effects in rats with cardiacischemia/reperfusion injury[J].Cardiovascular Diabetology,2020,19(1):91.[3]韩松洁,张晓雨,张立晶,等.麝香通心滴丸对PCI术后患者慢血流的临床证据评价[J].世界科学技术-中医药现代化,2018,20(10):1772-1777.[4]HEUSCH G.Myocardial ischaemia-reperfusion injury and cardioprotectionin perspective[J].Nature Reviews Cardiology,2020,17(12):773-789.[5]张艳达,施珊岚,厉娜,等.麝香通心滴丸改善心肌梗死小鼠微循环障碍的作用机制[J].中西医结合心脑血管病杂志,2017,15(23):2969-2972.[6]LIU H H,ZHAO J J,PAN S L,et al.Shexiang Tongxin Dropping Pill(麝香通心滴丸)protects against sodium laurate-inducedcoronary microcirculatory dysfunction in rats[J].J Tradit ChinMed,2021,41(1):89-97.[7]王华伟,金丹丹,潘海华.麝香通心滴丸通过p-Akt/p-GSK3β/GSK3β通路对心肌梗死小鼠的心肌保护作用研究[J].中国中医药科技,2020,27(3):352-355.[8]王伟,刘星雨,尚云龙,等.基于血清药物化学与网络药理学探究麝香通心滴丸治疗冠心病的机制[J].中成药,2020,42(10):2768-2777.[9]ZHENG J,CHEN P E,ZHONG J F,et al.HIF-1αin myocardialischemia-reperfusion injury(review)[J].Molecular MedicineReports,2021,23(5):352.[10]LI J,ZHOU W J,CHEN W,et al.Mechanism of the hypoxiainducible factor1/hypoxic response element pathway in ratmyocardial ischemia/diazoxide post conditioning[J].MolecularMedicine Reports,2020,21(3):1527-1536.(收稿日期:2022-03-31)(本文编辑王丽)。

◇基础研究◇摘要目的：基于代谢组学探索黄芪甘草汤防治三氧化二砷诱导QT 间期延长的保护作用及其机制。

方法：建立三氧化二砷诱导大鼠QT 间期延长模型。

检测大鼠心电图、血常规、代谢组学差异代谢物，并结合网络药理学收集关键靶点，通过功能注释、通路富集分析筛选黄芪甘草汤保护作用的可能候选基因与通路，并进行体外实验验证。

结果：黄芪甘草汤对三氧化二砷诱导的SD 大鼠QT 间期具有显著的缓解作用（P <0.05）；GO 富集分析发现黄芪甘草汤和三氧化二砷诱导QT 间期延长的关键靶点主要涉及炎症应答、活性氧、氧化应激等生物过程，内细胞囊泡、褶皱、内细胞囊泡膜等细胞组分，SMAD 结合、R-SMAD 结合、信号受体激活剂的活性等分子功能；KEGG 通路分析发现其主要富集于PI3K-Akt 信号通路、脂质和动脉硬化、FOXO 信号通路、TNF 信号通路、HIF-1等信号通路。

通过体外H9c2细胞模型，验证了黄芪甘草汤能够逆转SIRT1、FOXO1蛋白表达。

结论：黄芪甘草汤可能通过调控SIRT1/FOXO1信号通路，从而改善三氧化二砷诱导QT 间期延长，减轻三氧化二砷心脏毒性。

关键词黄芪甘草汤；三氧化二砷；QT 间期；代谢组学中图分类号：R965.2文献标志码：A文章编号：1009-2501(2024)02-0130-09doi ：10.12092/j.issn.1009-2501.2024.02.002三氧化二砷（ATO ）是中药砒霜的主要活性成分，常用于治疗急性早幼粒细胞白血病，也用于多种癌症的治疗［1-2］。

然而，研究发现有54.2%的患者在使用ATO 的过程中出现QT 间期延长的副作用［3］。

针对ATO 心脏毒性的临床管理建议都不能解决根本问题，而且对于抗心律失常药物的使用和用量在临床上一直存在争议［4］。

总之，目前ATO 引起心脏毒性原因尚未完全明确，且缺乏有效防治药物，仍需探明关键发病机制并找到有效防治药物。

2021年第47卷第1期(总第421期)125㊀DOI:10.13995/ki.11-1802/ts.024501引用格式:孔庆敏,朱慧越,田培郡,等.嗜酸乳杆菌La28对丙戊酸暴露引起的子代大鼠外周炎症和肝损伤的缓解作用[J].食品与发酵工业,2021,47(1):125-131.KONG Qingmin,ZHU Huiyue,TIAN Peijun,et al.Alleviation effects on peripheral in-flammation and liver damage by Lactobacillus acidophilus La28in offspring rats induced by valproic acid exposure [J].Food and Fermentation Industries,2021,47(1):125-131.嗜酸乳杆菌La28对丙戊酸暴露引起的子代大鼠外周炎症和肝损伤的缓解作用孔庆敏,朱慧越,田培郡,赵建新,张灏,陈卫,王刚∗(江南大学食品学院,江苏无锡,214122)摘㊀要㊀为评价嗜酸乳杆菌La28对丙戊酸(valproic acid ,VPA )暴露引起的子代大鼠外周炎症和肝损伤的作用,同时明确其对VPA 大鼠肠道菌群的调节作用㊂在大鼠孕期12.5d 腹腔注射VPA 和生理盐水,断奶后,随机选取雄性子代分成对照组㊁VPA 组㊁La28组和利培酮(MED )组(n =5),La28组每天灌胃菌液(109CFU /mL ),对照㊁VPA 和MED 组灌胃等量生理盐水和药物,干预期28d ㊂干预结束后,评价外周炎症㊁肝功能和肠道菌群的改善情况㊂与VPA 组大鼠相比,嗜酸乳杆菌La28干预能显著地改善VPA 大鼠的外周炎症和肝功能,其中对IL-6㊁TNF-α㊁ALT 和GGT 的作用效果更为显著㊂此外,嗜酸乳杆菌La28可能通过上调Allobaculumhe 和Mucispirillum 的相对丰度,增加厚壁菌门丰度,降低拟杆菌门丰度从而改善VPA 暴露引起子代大鼠的神经炎症和肝功能㊂关键词㊀嗜酸乳杆菌La28;丙戊酸;外周炎症;肝功能;肠道菌群第一作者:博士研究生(王刚副教授为通讯作者,E-mail:wanggang@)基金项目:国家自然科学基金项目(31972052);中央高校基本科研业务费专项资金项目(JUSRP22006)收稿日期:2020-05-20,改回日期:2020-07-15㊀㊀丙戊酸(valproic acid,VPA)是目前治疗癫痫最常用的药物之一[1],而肝毒性是其应用过程中最严重的并发症之一,一种类型是可逆性肝功能不全,另一种类型的肝损害是不可逆性的肝中毒甚至死亡[2-3],实验表明,孕期服用VPA 的含量与胎儿致畸程度㊁认知及社交障碍正相关,并且对子代的免疫系统和肝脏功能也同样具有一定的影响[4]㊂结合RO-DIER 等[5]在1996年首次通过大鼠子宫暴露VPA 得到子代自闭症(autism spectrum disorder,ASD)大鼠,因其所得ASD 大鼠与临床症状相似,孕期高浓度VPA 干预已成为获取ASD 模型动物的常用造模方法㊂ASD 患者常伴随胃肠道功能紊乱,与ASD 严重程度呈正相关,多表现为肠漏㊁便秘㊁腹泻㊁食道反流㊁腹痛和胀气等症状[6]㊂当肠道微生态系统失衡,肠道黏膜屏障受损时,肠道微生物代谢产物包括内毒素㊁氨㊁吲哚㊁酚类㊁短链脂肪酸㊁假性神经递质前体㊁炎症介质等透过肠道屏障进入肠系膜淋巴组织,过度激活机体免疫系统,引起异常免疫反应,导致肝细胞凋亡㊁坏死㊂此外,来自肠道中的各种毒素需要由肝脏清除,同时肝脏还能清除肠源性细菌㊁真菌等,更加重了肝功能受损[7]㊂已有研究表明益生菌可以通过对肠道微生态系统的维护,降低内毒素和血氨等浓度,缓解肝脏的损伤,改善肝脏代谢功能[8]㊂本研究通过孕期高浓度VPA 暴露获得ASD 大鼠,考察从断乳后到性成熟嗜酸乳杆菌La28干预对孕期VPA 暴露引起的子代大鼠外周炎症和肝功能的作用,为筛选出更适合ASD 患儿发育过程的益生菌提供参考㊂1㊀材料与方法1.1㊀实验材料1.1.1㊀实验菌种及培养基嗜酸乳杆菌(Lactobacillus acidophilus )La28来自于菌种保藏中心,菌株分离自健康人肠道㊂Wistar 大鼠(雄性5只,初始体质量280~290g;雌性10只,初始体质量220~250g),北京维通利华实验动物技术有限公司㊂本研究经江南大学实验动物伦理委员会批准(JN.NO.20180915S0601230[183]),并按照欧洲共同体关于实验动物的准则(Directive 982010/63/EU)进行操作㊂培养基:MRS 培养基+0.05%L -半胱氨酸盐酸盐㊂126㊀2021Vol.47No.1(Total 421)1.1.2㊀主要试剂IFN-γ㊁TNF-α㊁IL-1β㊁IL-6和IL-10Elisa 试剂盒,R&D Systems 公司;利培酮,西安杨森制药有限公司;粪便提取试剂盒(FastDNA Spin Kit for Feces),MP Biomedicals,Santa Ana,CA;L -半胱氨酸盐酸盐,国药集团化学试剂有限公司㊂1.1.3㊀主要仪器与设备BS-480全自动生化分析仪,深圳迈瑞生物医疗电子股份有限公司;AW500SG 大型厌氧工作站,英国Electrotek 有限公司;SW-CJ-1CV 型微生物操作超净工作台,苏州安泰空气技术有限公司;大容量冷冻离心机,美国Beckman 公司;高速冷冻离心机,Eppen-dorf 公司;UV-1800紫外可见分光光度合计,岛津仪器有限公司;高温高压灭菌锅,日本SANYO 公司㊂1.2㊀灌胃用菌制备将冻存在保护剂内的嗜酸乳杆菌La28于液体培养基活化3次,活化后的菌液按体积分数为2%接种到液体培养基中,37ħ条件下厌氧培养18h,8000ˑg 离心20min,以无菌生理盐水清洗菌体2遍后,用10%脱脂乳作为保护剂(采用倾注法测得菌落总数为109CFU /mL),冻存在-80ħ备用,灌胃前,6000ˑg 离心3min,用生理盐水重悬㊂1.3㊀动物实验大鼠饲养于江南大学实验动物中心,室温(25ʃ2)ħ,相对湿度50%~55%,光照时间12h,黑暗时间12h㊂适应1周后,于下午17ʒ00合笼,雌雄数量比例2ʒ1㊂第2天早上8ʒ00前对每只雌鼠进行阴道涂片检查,观察到精子则记录为妊娠第0.5天,雌鼠受孕后分笼饲养,分为实验组(n =5)和对照组(n =5)㊂孕期12.5d,孕鼠腹腔注射600mg /kg 的VPA (用生理盐水配制丙戊酸钠溶液至质量浓度250mg /mL)[9],而在对照组腹腔注射等量的生理盐水㊂实验组出生的雄性大鼠被分配到模型组,而对照组出生的雄性大鼠在出生后第21天被分配到健康组㊂从4周龄到8周龄,每天同一时间,嗜酸乳杆菌La28组(La28,n =5)灌胃菌液,模型组(VPA,n =5)和空白组(对照,n =5)灌胃等量生理盐水,药物对照组(MED,n =5)灌胃等量生理盐水配制的利培酮㊂8周龄的最后一天,乙醚麻醉,取血后处死,凝血后于1500ˑg 离心15min,取血清,冻存在-80ħ备用㊂1.4㊀外周炎症评价采用全自动生化分析仪检测3组血清中IgA㊁IgG㊁IgM 和C-反应蛋白(C-reactive protein,CRP);用Elisa 试剂盒检测IFN-γ㊁TNF-α㊁IL-1β㊁IL-6和IL-10㊂1.5㊀肝功能评价采用全自动生化分析仪检测3组血清中的谷丙转氨酶(alanine aminotransferase,ALT)㊁谷草转氨酶(aspartate aminotransferase,AST)㊁碱性磷酸酶(alka-line phosphatase,ALP )和γ-谷氨酰转移酶(gamma-glutamyl transferase,GGT)㊂1.6㊀肠道菌群分析VPA 组大鼠(n =5)㊁对照组大鼠(n =5)㊁MED 组(n =5)和La28组(n =5)大鼠在出生后第56天收集粪便样本,使用粪便DNA 提取试剂盒提取DNA㊂利用Illumina MiSeq 平台对V3~V4区域进行扩增和测序[10]㊂1.7㊀统计与分析实验数据均以平均值ʃ均值标准误差(mean ʃSD)表示㊂每个实验至少重复3次㊂采用One-Way ANOVA 以及Fisher s LSD 检验进行数据统计分析,P <0.05时具有显著性差异㊂采用GraphPad Prism 7和R 作图,在Glaxay 平台进行LEfSe 软件分析,设置LDA Score 的默认筛选值为2㊂2㊀结果与分析2.1㊀孕鼠VPA 暴露对子代的影响孕期暴露的VPA 可通过外周血循环和胎盘屏障均可直接进入子代体内,利用其神经毒性参与子代神经管闭合,从而引发先天性的神经系统发育性疾病,此外,还会引起子代唇裂或腭裂㊁先天性心脏病㊁肢体缺陷㊁泌尿生殖器缺陷及肝脏脂质代谢障碍等疾病[9]㊂如图1所示(对照和VPA 组大鼠均为5日龄),孕期子宫暴露600mg /kg VPA 的子代大鼠出现了全部尾部畸形和部分手部畸形,尾部的畸形多表现为弯曲形状,且与健康组比较短,手部的畸形表现为缺指,表明VPA 在孕鼠子宫中成功暴露[11]㊂此外,SCHNEIDER 等[12]发现孕期VPA 暴露所得的自闭雄性小鼠的行为表现与ASD 患者的临床表现更为相似,因此本文选择雄性子代进一步实验㊂2.2㊀嗜酸乳杆菌La28对孕期VPA 暴露引起的子代大鼠外周炎症的作用2.2.1㊀嗜酸乳杆菌La28对血清细胞因子的作用细胞因子通过促炎和抗炎途径调节免疫反应㊂外周细胞因子可跨越血脑屏障参与中枢神经活动㊂外周和神经炎症在ASD 进程中发挥重要作用,ASD2021年第47卷第1期(总第421期)127㊀图1㊀VPA 暴露对子代大鼠的致畸作用Fig.1㊀The teratogenic effect on offspring rats by VPA exposure患者外周促炎因子水平上调,抑炎因子水平下调,同时伴随着持续的神经炎症[13]㊂由图2可知,从断奶到性成熟期,孕期VPA 暴露的子代大鼠血清中促炎因子IL-1β㊁IL-6㊁IFN-γ和TNF-α水平显著升高,抑炎因子IL-10水平显著下降㊂利培酮可以显著下调大鼠的血清中IL-1β和TNF-α水平,但对IFN-γ水平没有明显的下调作用,甚至MED 组比VPA 组稍高,此外,利培酮可以辅助下调IL-6水平,上调IL-10水平㊂嗜酸乳杆菌La28可显著下调大鼠血清中IL-6和TNF-α水平,同时IFN-γ和IL-1β水平均有所下降,IL-10水平上调,因此,嗜酸乳杆菌La28可以辅助降低促炎因子浓度上调抑炎因子水平,从而调节早期VPA 暴露引起的血清促炎和抑炎因子水平的异常㊂已有研究明确益生菌在免疫调节中的作用,主要通过提高上皮细胞的完整性,调节上皮细胞分泌细胞因子提高免疫应答[14]㊂另外还可以辅助抑制NF-κB 激活[15]和调节Toll 样受体的表达[16],降低促炎细胞因子的表达㊂图2㊀嗜酸乳杆菌La28对血清细胞因子IL-1β㊁IL-6㊁IL-10㊁IFN-γ和TNF-α水平的影响Fig.2㊀Effects of L.acidophilus La28on the level of IL-1β,IL-6,IL-10,IFN-γand TNF-α2.2.2㊀嗜酸乳杆菌La28对血清CRP 的作用CRP 是由肝脏合成的一种五聚体蛋白,是体内急性相反应标志物[17]㊂已有研究表明,外周血CRP 水平升高与抑郁症㊁躁郁症㊁精神分裂症等神经系统类疾病有关[18-19]㊂此外,动物研究已经报道,外周血CRP 水平反映的慢性炎症状态与小鼠的ASD 样行为相关[20-21]㊂且已有报道益生菌可参与下调CRP 水平[22],本研究中孕期VPA 暴露的子代大鼠血清中CRP 水平显著上调(图3)㊂利培酮可下调大鼠的血清CRP 水平,从断奶到性成熟期灌胃嗜酸乳杆菌La28的大鼠血清中CRP 水平下调,虽然MED 组和嗜酸乳杆菌La28组均可下调CRP 水平,但两组与VPA组均没有显著性差异,因此,嗜酸乳杆菌La28可以缓解早期VPA 暴露引起的血清免疫因子CRP 水平异常㊂128㊀2021Vol.47No.1(Total 421)图3㊀嗜酸乳杆菌La28对血清CRP 水平的影响Fig.3㊀Effects of L.acidophilus La28on the level of CRP2.2.3㊀嗜酸乳杆菌La28对血清免疫球蛋白的作用研究发现ASD 患者的IgG 亚型不全,IgA 的血清含量低[23],其中血清型IgA 存在于血清中,血清IgA 具有某些IgG 和IgM 的免疫功能,特异性IgA 能中和血液中的抗原,同时也出现替代性补体免疫系统[24]㊂另外,有研究表明有些ASD 患者的IgA 含量极低,甚至几乎没有,而分泌型IgA 具有保护肠壁的黏膜组织的功能,因而ASD 患者的肠黏膜较容易受损[23],这与本实验结果一致㊂由图4可知,孕期VPA 暴露的子代大鼠血清中IgG 和IgA 水平显著上调,IgM 无显著性差异㊂利培酮可显著下调大鼠的血清IgG 水平,对IgA 和IgM 没有明显调节作用㊂从断奶到性成熟,嗜酸乳杆菌La28的干预可下调大鼠血清中IgG 水平,但与VPA 组没有显著性差异,与MED 组同样对IgA 和IgM 没有明显调节作用㊂嗜酸乳杆菌La28对早期VPA 暴露引起的免疫球蛋白IgG 水平异常有明显作用,且这与已有研究表明益生菌干预机体IgA 的水平没有显著调控效果结果一致[25-26]㊂图4㊀嗜酸乳杆菌La28对血清IgG㊁IgM 和IgA 水平的影响Fig.4㊀Effects of L.acidophilus La28on the level of IgG,IgM and IgA2.3㊀嗜酸乳杆菌La28孕期VPA 暴露的子代大鼠肝功能的作用如图5所示,孕期VPA 暴露的子代大鼠ALT㊁AST㊁ALP 和GGT 水平显著上调㊂利培酮可下调大鼠ALT㊁AST㊁ALT 和GGT 水平,提升肝功能,从断奶到性成熟期,嗜酸乳杆菌La28的干预可下调大鼠ALT ㊁AST ㊁ALP 和GGT 水平,且可显著调节ALT 和GGT 水平㊂因此,嗜酸乳杆菌La28可以显著缓解早期VPA 暴露引起的肝损伤㊂研究表明,益生菌主要通过改善肠道环境,调控肠道微生物组成结构和短链脂肪酸等代谢产物水平来维持肠道屏障功能,加强肠道的黏膜屏障作用,减少肠源性内毒性通过门静脉系统㊁内循环系统进入肝脏,进而改善肝脏病变程度[27-28]㊂图5㊀嗜酸乳杆菌La28对ALP㊁ALT㊁AST 和GGT 水平的影响Fig.5㊀Effects of L.acidophilus La28on the level of ALP,ALT,AST and GGT2.4㊀孕期VPA 暴露的子代大鼠外周炎症和肝功能指标的相关性分析已有研究表明,外周炎症和肝功能密切相关,来自肠道中的各种毒素通过门静脉系统作用于肝脏,引发炎症反应,导致肝功能受损[29-30]㊂因而,对本实验中的外周炎症指标和肝功能指标进行相关性分析(图6),发现IFN-γ㊁TNF-α㊁IL-1β㊁IL-6㊁CRP 和IgG 与肝功能指标均呈正相关,其中IL-6与ALT(r =0.700,P =0.024)㊁AST(r =0.702,P =0.024)㊁ALP(r =0.723,P =0.018)和GGT (r =0.790,P =0.007)4个指标都显著正相关,IL-1β与ALP (r =0.729,P =0.017)呈显著正相关㊂IL-10与ALT(r =2021年第47卷第1期(总第421期)129㊀-0.773,P =0.009)㊁AST(r =-0.745,P =0.013)和GGT(r =-0.705,P =0.023)4个指标都显著负相关㊂嗜酸乳杆菌La28可以显著降低外周IL-6的水平,同时也显著降低了ALT 和GGT 的表达水平,因而推测嗜酸乳杆菌La28可能是通过调节VPA 大鼠肠道环境降低外周IL-6的水平从而达到改善肝功能效果㊂图6㊀外周炎症与肝功能指标相关性分析Fig.6㊀Correlation analysis between peripheral inflammationand liver function indicators2.5㊀嗜酸乳杆菌La28对孕期VPA 暴露的子代大鼠神经炎症和肝功能的综合作用效果利用主成分分析对上述所有指标进行综合分析,由图7可知,嗜酸乳杆菌La28对VPA 暴露引起的子代大鼠的外周炎症和肝功能的综合缓解能力较好㊂图7㊀外周炎症与肝功能指标综合主成分分析Fig.7㊀Principal component analysis of peripheral inflammationand liver function indicators2.6㊀嗜酸乳杆菌La28对VPA 暴露的子代大鼠肠道菌群的影响越来越多的证据表明肠道菌群失衡与ASD 的严重程度有关[31],基于肠道菌群失调在ASD 中的作用,利用益生菌将肠道菌群重塑为健康状态是近几年提出的作为ASD 的潜在治疗方法㊂因此,基于16S rRNA 扩增测序手段,进一步研究了嗜酸乳杆菌La28对孕期VPA 暴露的子代大鼠的肠道微生物的影响㊂已有发现,在孤独症儿童的粪便样本中,厚壁菌门的丰度低于健康儿童,厚壁菌门与拟杆菌门比值(F /B)升高[32-33]㊂这与本实验研究结果一致,如图8-a 所示,孕期VPA 暴露的子代大鼠F /B 显著上调,嗜酸乳杆菌La28和利培酮干预可以降低F /B水平,表明嗜酸乳杆菌La28菌群组成上能够发挥调节作用㊂利用LEfSe 分析确定了各组的优势菌群(图8-b),在属水平上,孕期VPA 暴露的子代大鼠的优势菌属为Roseburia ;利培酮干预后优势菌属为Pseudomonas 和Bifidobacterium ;嗜酸乳杆菌La28干预后的优势菌属为Anaerostipes ㊂进一步对各组肠道菌群的属水平进行分析,从图8-c 可知,与孕期VPA 暴露的子代大鼠的肠道菌群比,嗜酸乳杆菌La28可以显著提高Mucispirillum ㊁Allobaculumhe ㊁Desulfo-vibrio ㊁Lactobacillus ㊁Odoribacter 和Bifidobacterium 丰度(P <0.05),其中对照组与嗜酸乳杆菌La28组的Allobaculumhe 丰度没有差异,且均高于VPA 组和MED 组,嗜酸乳杆菌La28组的Mucispirillum 显著性高于其他3组㊂说明嗜酸乳杆菌La28通过上调Allobaculumhe 和Mucispirillum 的相对丰度,增加厚壁菌门丰度,降低拟杆菌门丰度来调节肠道菌群从而改善VPA 暴露引起子代大鼠的神经炎症和肝功能㊂3㊀结论经嗜酸乳杆菌La28干预后,对VPA 暴露引起子代大鼠的神经炎症和肝功能具有有效的改善作用㊂其中,嗜酸乳杆菌La28对抑制促炎因子IL-6和TNF-α的表达和调节肝代谢酶ALT 和GGT 的表达效果更佳,且在肠道菌群调节上效果更为显著,为筛选出更适合ASD 患儿发育过程的益生菌提供一定参考㊂参考文献[1]㊀VAJDA F J E,O BRIEN T J,GRAHAM J E,et al.Valproate-asso-ciated foetal malformations-rates of occurrence,risks in attemptedavoidance [J ].Acta Neurologica Scandinavica,2018,139(1):42-48.[2]㊀ABDELKADER N F,ELYAMANY M,GAD A M,et al.Ellagic acidattenuates liver toxicity induced by valproic acid in rats[J].Journalof Pharmacological Sciences,2020,143(1):23-29.[3]㊀BASSETT J T,RODRIGUEZ B,MULLIGAN L,et al.Acute liverfailure in a military recruit treated with valproic acid and harboring a130㊀2021Vol.47No.1(Total 421)a-厚壁菌门与拟杆菌门比值;b-属水平各组优势菌群;c-门水平和属水平丰度差异g-属水平;p-门水平;f-科水平;o-目水平;k-界水平图8㊀嗜酸乳杆菌La28对肠道菌群的影响Fig.8㊀Effects of L.acidophilus La28on gut microbiotapreviously unrecognized polg-1mutation [J ].Epilepsy &behavior reports,2019,12:100342.[4]㊀ZHAO G,GAO J Q,LIANG S,et al.Study of the serum levels of pol-yunsaturated fatty acids and the expression of related liver metabolic enzymes in a rat valproate-induced autism model [J].InternationalJournal of Developmental Neuroscience,2015,44:14-21.[5]㊀RODIER P M,INGRAM J L,TISDALE B,et al.Embryological originfor autism:Developmental anomalies of the cranial nerve motor nu-clei [J ].the Journal of Comparative Neurology,1996,370(2):247-261.[6]㊀HOLINGUE C,NEWILL C,LEE L,et al.Gastrointestinal symptomsin autism spectrum disorder:A review of the literature on ascertain-ment and prevalence[J].Autism Research,2018,11(1):24-36.[7]㊀BRANISTE V,ALASMAKH M,KOWAL C,et al.The gut microbiotainfluences blood-brain barrier permeability in mice [J ].Science Translational Medicine,2014,6(263).DOI:10.1126/scitranslmed.3009759.[8]㊀TRIPATHI A,DEBELIUS J W,BRENNER D A,et al.Publisher cor-rection:The gut-liver axis and the intersection with the microbiome[J].Nature Reviews Gastroenterology &Hepatology,2018,15(7):785-785.[9]㊀TANOSHIMA M,KOBAYASHI T,TANOSHIMA R,et al.Risks ofcongenital malformations in offspring exposed to valproic acid in ute-ro:A systematic review and cumulative meta-analysis [J].Clinical Pharmacology &Therapeutics,2015,98(4):417-441.[10]㊀YANS,YANG B,ZHAO J C,et al.A ropy exopolysaccharide pro-ducing strain Bifidobacterium longum subsp.longum YS108R alle-viates DSS-induced colitis by maintenance of the mucosal barrier and gut microbiota modulation [J ].Food &Function,2019,10(3):1595-1608.㊀[11]㊀FOLEY A G,GANNON S,ROMBACH-MULLAN N,et al.Classihistone deacetylase inhibition ameliorates social cognition and cell adhesion molecule plasticity deficits in a rodent model of autism spectrum disorder [J ].Neuropharmacology,2012,63(4):750-760.㊀[12]㊀SCHNEIDER T,ROMAN A,AGNIESZKA B K,et al.Gender-spe-cific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid [J].Psycho-neuroendocrinology,2008,33(6):728-740.[13]㊀DOENYAS C.Gut microbiota,inflammation,and probiotics on neu-ral development in autism spectrum disorder [J ].Neuroscience,2018,374:271-286.㊀[14]㊀FERNANDES BS,STEINER J,BERNSTEIN H G,et al.C-reactiveprotein is increased in schizophrenia but is not altered by antipsy-chotics:Meta-analysis and implications[J].Molecular Psychiatry,2016,21(4):554-564.[15]㊀KEMGANG T S,KAPILA S,SHANMUGAM V P,et al.Cross-talkbetween probiotic lactobacilli and host immune system[J].Journalof Applied Microbiology,2014,117(2):303-319. [16]㊀RAISON C L,PIKALOV A,SIU C,et al.C-reactive protein and re-sponse to lurasidone in patients with bipolar depression[J].BrainBehavior and Immunity,2018,73:717-724.[17]㊀FELGER J C,HAROON E,PATEL T A,et al.What does plasmacrp tell us about peripheral and central inflammation in depression?[J].Molecular Psychiatry,2020,25(6):1301-1311. [18]㊀CURRAIS A,FARROKHI C,DARGUSCH R,et al.Dietary glyce-mic index modulates the behavioral and biochemical abnormalitiesassociated with autism spectrum disorder[J].Molecular Psychiatry,2016,21(3):426-436.[19]㊀GUPTA S,SAMRA D,AGRAWAL S.Adaptive and innate immuneresponses in autism:Rationale for therapeutic use of intravenous im-munoglobulin[J].Journal of Clinical Immunology,2010,30:S90-S96.[20]㊀MILOSEVIC I,VUJOVIC A,BARAC A,et al.Gut-liver axis,gutmicrobiota,and its modulation in the management of liver diseases:A review of the literature[J].International Journal of MolecularSciences,2019,20(2).DOI:10.3390/ijms20020395. [21]㊀BAJAJ J S.Alcohol,liver disease and the gut microbiota[J].NatureReviews Gastroenterology&Hepatology,2019,16(4):235-246.[22]㊀CHAIDEZ V,HANSEN R L,HERTZ-PICCIOTTO I.Gastrointesti-nal problems in children with autism,developmental delays or typi-cal development[J].Journal of Autism and Developmental Disor-ders,2014,44(5):1117-1127.[23]㊀LIU F T,LI J,WU F,et al.Altered composition and function of in-testinal microbiota in autism spectrum disorders:A systematic re-view[J].Translational psychiatry,2019,9(1):43.㊀[24]㊀SHAABAN S Y,EL G Y G,MEHANNA N S,et al.The role of probiot-ics in children with autism spectrum disorder:A prospective,open-la-bel study[J].Nutritional Neuroscience,2018,21(9):676-681. [25]㊀ZHANG H,YE H C,JIN Z,ET AL.Prospective study of probioticsupplementation results in immune stimulation and improvement ofupper respiratory infection rate[J].Synthetic and Systems Biotech-nology,2018,3(2):113-120.[26]㊀SAPUTRO I D,PUTRA O N,PEBRIANTON H,et al.Effects ofprobiotic administration on IGA and IL-6level in severe burn pa-tients:A randomized trial[J].Annals of Burns and Fire Disasters,2019,32(1):70-76.[27]㊀MANCINI A,CAMPAGNA F,AMODIO P,et al.Gut:liver:brainaxis:The microbial challenge in the hepatic encephalopathy[J].Food&Function,2018,9(3):1373-1388.[28]㊀YE J,LV L,WU W,et al.Butyrate protects mice against methio-nine-choline-deficient diet-induced non-alcoholic steatohepatitis byimproving gut barrier function,attenuating inflammation and reduc-ing endotoxin levels[J].Frontiers In microbiology,2018,9(21).DOI:10.3389/fmicb.2018.01967.[29]㊀MILOSEVIC I,VUJOVIC A,BARAC A,et al.Gut-Liver axis,gutmicrobiota,and its modulation in the management of liver diseases:A review of the literature[J].International Journal of MolecularSciences,2019,20(2):395-399.[30]㊀ALCOHOL B J.Liver disease and the gut microbiota[J].NatureReviews Gastroenterology&Hepatology,2019,16(4):235-246.[31]㊀CHAIDEZ V,HANSEN R L,HERTZ-PICCIOTTO I.Gastrointesti-nal problems in children with autism,developmental delays or typi-cal development[J].Journal of Autism and Developmental Disor-ders,2014,44(5):1117-1127.[32]㊀LIU F,LI J,WU F,et al.Altered composition and function of intes-tinal microbiota in autism spectrum disorders:A systematic review[J].Translational Psychiatry,2019,9(1):43.[33]㊀SHAABAN S Y,GENDY Y G,MEHANNA N S,et al.The role ofprobiotics in children with autism spectrum disorder:A prospective,open-label study[J].Nutritional Neuroscience,2017,21(9):676-681.Alleviation effects on peripheral inflammation and liver damage by Lactobacillus acidophilus La28in offspring rats induced by valproic acid exposureKONG Qingmin,ZHU Huiyue,TIAN Peijun,ZHAO Jianxin,ZHANG Hao,CHEN Wei,WANG Gang∗(School of Food Science and Technology,Jiangnan University,Wuxi214122,China)ABSTRACT㊀To evaluate the effects of Lactobacillus acidophilus La28on relieving peripheral inflammation and improving liver function in rats induced by valproic acid,Wistar rats were intraperitoneally injected with VPA or normal saline on day12.5after pregnancy and the male offspring were randomly grouped as the control group,VPA group,La28group and risperidone(MED)group(n=5).La28group was treated with L.acidophilus La28(109CFU/mL)by gavage for28days.The control,VPA and MED groups were treated with equiva-lent saline and risperidone by gavage.After the intervention,peripheral inflammation,liver function and gut microbiota were evaluated. Compared with rats in VPA group,L.acidophilus La28significantly improved peripheral inflammation and liver function in rats.The effects on IL-6,TNF-α,alanine aminotransferase(ALT)andγ-glutamyl transpeptidase(GGT)were more significant.In addition,L.ac-idophilus La28might improve the peripheral inflammation and liver function through regulating the relative abundance of Allobaculumhe and Mucispirillum,increasing Firmicutes abundance,and decreasing Bacteroides abundance.Key words㊀Lactobacillus acidophilus La28;valproic acid;peripheral inflammation;liver function;gut microbiota2021年第47卷第1期(总第421期)131㊀。