TRANSLATIONAL PHYSIOLOGY
MicroRNA-141regulates the expression level of ICAM-1on endothelium to decrease myocardial ischemia-reperfusion injury
Rong Rong Liu,1*Jun Li,2*Jiu Yu Gong,1Fang Kuang,3Jia Yun Liu,4Yu Si Zhang,1Qian Li Ma,1
Chao jun Song,1Agnieszka D.Truax,5Feng Gao,2Kun Yang,1Bo Quan Jin,1and Li Hua Chen1
1Department of Immunology,Fourth Military Medical University,Xi’an,People’s Republic of China;2Department of
Physiology,Fourth Military Medical University,Xi’an,People’s Republic of China;3Department of Neurobiology,Fourth
Military Medical University,Xi’an,People’s Republic of China;4Department of Clinical Laboratory Medicine,Xijing
Hospital,Fourth Military Medical University,Xi’an,People’s Republic of China;and5Lineberger Comprehensive Cancer
Center,University of North Carolina at Chapel Hill,Chapel Hill,North Carolina
Submitted22April2015;accepted in?nal form2September2015
Liu RR,Li J,Gong JY,Kuang F,Liu JY,Zhang YS,Ma QL, Song CJ,Truax AD,Gao F,Yang K,Jin BQ,Chen LH.Mi-croRNA-141regulates the expression level of ICAM-1on endothe-lium to decrease myocardial ischemia-reperfusion injury.Am J Physiol Heart Circ Physiol309:H1303–H1313,2015.First published September14,2015;doi:10.1152/ajpheart.00290.2015.—A growing number of studies have suggested microRNAs(miRNAs)are in-volved in the modulation of myocardial ischemia-reperfusion(MI/R) injury;however,the role of endogenous miRNAs targeting endothe-lial cells(ECs)and its interaction with ICAM-1in the setting of MI/R remain poorly understood.Our microarray results showed that miR-146a,miR-146b-5p,miR-155*,miR-155,miR-497,and miR-451 were signi?cantly upregulated,whereas,miR-141and miR-564were signi?cantly downregulated in the ECs challenged with TNF-?for6 h.Real-time PCR analyses additionally validated that the expression levels of miR-146a,miR-155*,and miR-141were consistent with the microarray results.Then,ICAM-1was identi?ed as a novel target of miR-141by Target Scan software and the reporter gene system. Further functional experiments showed that elevated levels of miR-141inhibited ICAM-1expression and diminished leukocytes adhe-sion to ECs in vitro.In an in vivo murine model of MI/R injury, pretreatment with miR-141mimics through the tail vein downregu-lated the expression level of ICAM-1in heart and attenuated MI/R injury as evidenced by decreased infarct size and decline of serum cardial troponin I(cTnI)and lactate dehydrogenase(LDH)concen-tration.The cardioprotective effects of miR-141mimics may be attributed to the decreased in?ltration of CD11b?cells and F4/80?macrophages into ischemic myocardium tissue.In conclusion,our results demonstrate that miR-141,as a novel repressor of ICAM-1,is involved in the attenuation of MI/R injury via antithetical regulation of ICAM-1and in?ammatory cells in?ltration.Thus miR-141may constitute a new therapeutic target in the setting of ischemic heart disease.
miR-141;ischemic reperfusion injury;HUVEC;ICAM-1;myocardial enzyme
NEW&NOTEWORTHY
This study expand our understanding of the interaction between miRNAs and ECs.MiR-141,as an endogenous repressor of
ICAM-1,inhibited ICAM-1expression and diminished leuko-cytes adhesion to ECs to attenuate MI/R injury.MiR-141may serve as a valuable therapeutic target in the setting of ischemic heart disease.
MYOCARDIAL ISCHEMIA-REPERFUSION(MI/R)injury,sustained by ischemic myocardium following current reperfusion therapies (including thrombolysis,coronary angioplasty,and coronary bypass surgery),represents an important clinical problem with signi?cant morbidity and mortality(13).Several studies sug-gest that endothelial cells(ECs)are more sensitive to I/R injury than cardiomyocytes(CMs)and they might be a critical me-diator of I/R injury in the heart(15,24).ECs do more than provide a protective barrier between the lumen and vascular smooth muscle of blood vessels,these dynamic cells are important players in regulating blood?ow,neutrophils,plate-lets,complement activation,and myocardial function(4,6,8, 10,15,24,32).The EC surface proteins that are associated with MI/R injury are known as selectins and immunoglobulin cell adhesion molecules(CAM),which have different time courses of activation and expression(9,24).The initial inter-action between polymorphonuclear(PMNs)cells and ECs is mediated by the E-selectin expressed on the EC surface and sLe x located on the surface of PMNs.This selectin-mediated interaction results in the rolling of PMNs along the vessel wall and upregulation of?2-integrin adhesion molecules on the PMNs.As a consequence,unregulated adhesion molecules such as LFA-1(CD11/CD18)then bind to their counter recep-tors on ECs,primarily intercellular adhesion molecule-1 (ICAM-1),resulting in their?rm adhesion and transmigration through the vascular wall.Abundant evidence has shown that the expression level of adhesion molecules by coronary ECs is upregulated following MI/R(9,30).Thus understanding the recovery of ECs will allow for the development of more effective therapies that can be applied early in the I/R injury process.
miRNAs are small,noncoding RNAs that play an important role in the regulation of gene expression by binding to target messenger RNAs(3,5).Growing evidence shows that miR-NAs play a pivotal role in heart diseases(3,26,27,33)and some miRNAs are associated with CM I/R injury.Current literature describes several examples:miR-1enhances CM apoptosis by regulating the target genes Hsp60and Hsp70,
*R.R.Liu and Jun Li contributed equally to this work.
Address for reprint requests and other correspondence:L.Chen,Dept.of Immunology,Fourth Military Medical Univ.,Xi=an710032,People’s Republic of China(e-mail address:chenlh@https://www.doczj.com/doc/075684721.html,).
Am J Physiol Heart Circ Physiol309:H1303–H1313,2015.
First published September14,2015;doi:10.1152/ajpheart.00290.2015.
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whereas miR-133targets and represses caspase-9expression to decrease CM apoptosis (31).miR-320is downregulated after I/R in murine hearts,and knockdown of miR-320reduces I/R-induced CM apoptosis by suppressing Hsp20(23).miR-499is shown to protect ischemic stressed CM by suppressing calcineurin-mediated dephosphorylation of dynamin-related protein 1(28).In vivo expression of miR-24inhibited cardio-myocyte apoptosis,in part by direct repression of the proapo-ptotic protein Bim,in a mouse MI model (21).Knockdown of miR-199a during normoxia can upregulate the expression of hypoxia-inducible factor-1?(HIF-1?)and Sirtuin-1and induce hypoxia preconditioning (22).These results highlight the great Table 1.List of differentially expressed microRNA in human umbilical vein endothelial cells
Upregulated Downregulated
Gene Name Fold Change Gene Name Fold Change hsa-miR-155*13.96961939hsa-miR-5640.015906702hsa-miR-45113.07530924hsa-miR-1410.03981438hsa-miR-146b-5p 2.601147854hsa-miR-27b*0.124932883hsa-miRPlus-E1082 2.552142004hsa-miR-200b 0.228096108hsa-miR-146a 2.491157665hsa-miR-12670.242380344hsa-miR-155 2.410402118hsa-miR-142-5p 0.24466023hsa-miR-886-5p 2.303147273hsa-miR-1920.273508907hsa-miR-222* 2.188219674hsa-miR-425*0.287293756hsa-miR-497 2.159425898hsa-miR-2150.290138249hsa-miR-183 2.009146548hcmv-miR-UL1120.291378156hsa-miR-1185 1.996532142hsa-miR-12040.322018034hsa-miR-590-5p 1.972872931ebv-miR-BART1-5p 0.329702556hsa-miR-137 1.957045907hsa-miR-2120.353594045hsa-miR-31* 1.907850548hsa-miR-1940.383780752hsa-miR-1259 1.857307397hsa-miR-1430.387167053hsa-miR-218 1.813364055hsa-miR-6120.406489177hsa-miR-199a-3p/hsa-miR-199b-3p 1.756967566hsa-miRPlus-E10290.411127073hsa-miR-889 1.743078627hsa-miR-200c 0.413116511hsa-miRPlus-F1195 1.737127616hsa-miRPlus-E11730.417268718hsa-miR-29b-1* 1.703463203ebv-miR-BART6-3p 0.41832102hsa-miRPlus-E1060 1.700159605hsa-miR-187*0.427008804hsa-miRPlus-E1012 1.678456778hsa-miRPlus-E12130.438010641sv40-miR-S1-5p 1.670492948hsa-miRPlus-E12090.464965142hsa-miR-374a 1.668905068hsa-miR-7610.47355367hsa-miR-455-3p 1.645237587hsa-miR-1240.492863051hsa-miRPlus-E1202 1.645223491ebv-miR-BART19-3p 0.505740248hsa-miR-106a 1.643331491kshv-miR-K12-4-3p 0.512621434hsa-miR-605 1.639911841hsa-miR-181a-2*0.517237259hsa-miR-27a 1.639403574hsa-miR-6750.517512573hsa-miRPlus-E1100 1.634420765hsa-miR-551b 0.538937828hsa-miR-24-2* 1.633916571hsa-miR-4880.542259991hsa-miR-887 1.622060578hsa-miRPlus-E10150.54529529hsa-miR-20b 1.607681558hsa-miR-220c 0.55012168hsa-miR-1260 1.603851874ebv-miR-BHRF1-30.55079721hsa-miR-362-3p 1.587456749hsa-miR-220b 0.55570834hsa-miR-29b 1.582579067hsa-miRPlus-E10930.566834778hsa-miR-1285 1.574404184hsa-miR-186*0.57215558hsa-miR-20a 1.561641547hsa-miR-520d-5p 0.580168559hsa-miR-299-5p 1.559681001hsa-miR-200b*0.604454685hsa-miRPlus-E1139 1.558404496hsa-miRPlus-F10360.604454685hsa-miRPlus-E1108 1.557465973hsa-miR-550*0.607989508hsa-miRPlus-E1078 1.549898951ebv-miR-BART18-3p 0.613094323hsa-miR-18a 1.52974519hsa-miR-629*0.616592329hsa-miR-329 1.527043415hsa-miR-9380.633238242hsa-miR-17 1.517469095hsa-miRPlus-E11120.633655395hsa-miRPlus-E1258 1.511943882hsa-miR-490-5p 0.635339231hsa-miR-1280 1.511136713hsa-miR-518a-5p/hsa-miR-5270.636615303
hsa-miRPlus-E12360.644269066
hsa-miR-548e 0.647021916
hsa-miRPlus-F11550.647303552
hsa-miR-671-5p 0.648630518
hsa-miRPlus-C10870.653299508
kshv-miR-K12-10.654447178
ebv-miR-BART160.655150884
hsa-miR-576-5p 0.657593559
hsa-miR-9200.658270651
hsa-miR-193b*0.660937898
hsa-miRPlus-F12050.664471462
hsa-miR-13010.665142906
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importance of miRNAs and their clinical implications,but most of them target CMs instead of ECs in I/R injury.It has been reported that miR-126regulates endothelial expression of VCAM-1(12)and increased expression of miR-146a protects the myocardium from I/R injury (29).In addition,evidence shows that TNF-?-mediated induction of endothelial adhesion molecules (E-selectin and ICAM-1)can be regulated by miR-31and miR-17-3p (25).However,whether miRNAs are able to decrease endothelial adhesion molecules expression in MI/R still needs to be further examined.In our study,we are the ?rst one to demonstrate that miR-141targets and suppresses ICAM-1expression and over-expression of miR-141attenuates MI/R injury.miR-141may serve as a valuable therapeutic target in the setting of ischemic heart disease.MATERIALS AND METHODS Cell Culture Human umbilical vein endothelial cells (HUVECs)were prepared by the nature protocols (18).Cultures of HUVECs were grown on ?bronectin-coated plates (Millipore);were maintained in EGM (Lonza)supplemented with 10%fetal bovine serum (FBS;PAA),100IU of penicillin/ml,and 100?g of streptomycin/ml;and were used before the 10th passage.Acute promyelocytic leukemia (HL60)and 293T cell lines were purchased from the cell bank of the Chinese
Academy of Science (Shanghai,China)and were maintained in DMEM with 10%FBS and penicillin/streptomycin.miRNA Microarray Analysis miRNA expression in HUVECs was assessed in unstimulated
conditions as well as following 6-h stimulation with 10ng/ml TNF-?
or buffer control.Then,the total RNA was extracted using TRIzol
(Invitrogen)and an RNeasy mini kit (Qiagen,Hilden,Germany)
according to the manufacturer’s instructions.RNA quality and quan-
tity were measured using a NanoDrop spectrophotometer (ND-1000;
NanoDrop Technologies),and RNA integrity was determined using
gel electrophoresis.After the quantity of total RNA was measured,the
total RNA samples from both groups of cells were labeled using a
miRCURY Hy3/Hy5Power labeling kit (Exiqon,Vedbaek,Denmark)
and then hybridized on a miRCURY LNA Array (v.18.1,Exiqon)
using a hybridization system (Nimblegen Systems,Madison,WI).
Following several washing steps using a wash buffer kit (Exiqon),the
slides were scanned using an Axon GenePix 4000B microarray
scanner (Axon Instruments,Foster City,CA).GenePix pro V6.0is
used to read the raw intensity of the image.
Data analysis.For data analysis,1)the intensity of green signal
was calculated after background subtraction and four Replicated spots
of each probe on the same slide have been averaged.2)We used the
median normalization method to obtain “normalized data,”normal-
ized data ?(foreground-background)/median;the median is 50%
quantile of miRNA intensity,which is larger than 50in all samples
after background correction.3)After normalization,the statistical
signi?cance of differentially expressed miRNA was analyzed by
t -test.4)Unsupervised hierarchical clustering and correlation analysis
was performed on miRNA data.The thresholds we used to identify
up-or downregulated miRNAs were fold change ?1.5and fold
change ?0.67.
Luciferase Assay Luciferase reporter plasmid was purchased from Ambion.cDNAs encoding the entire 3=-UTR of ICAM-1(1.329kb)mRNA were cloned using speci?c primers (forward primer,5=-CTCTTCCTCG-GCCTTCCCATAT-3=;backward primer,5=-TTTGGCAGTT-GAGAAAGCTTTATTAACTA-3=)in the EcoR I and EcoR V site of the reconstructed pGL3Basic Vector (Promega,Fitchburg,WI).293T cells were transfected with 0.8?g of luciferase reporter vector containing the target site and 100nM miRNA mimics using Lipo-fectamine 2000.Assays were performed at 24h after transfection using the dual luciferase reporter assay system (Promega).Fire?y luciferase activity was normalized to Renilla luciferase activity.The recombinant 3=-UTR of ICAM-1-pGL3mutations were generated using Takara Mutant BEST Kit (Takara,Otsu,Japan)according to the manufacturer’s protocols.The following oligonucleotides were used
for mutation:3=-UTR of ICAM-1-pGL3MUT1,5=-AATCATATG-
GCCTTATTCCTCCCTTCC-3=and 5=-AGCGAATTCTATTC-CCTGGCACTCATG-3=;and 3=-UTR of ICAM-1-pGL3MUT2,5=-AATCATATGCTGGGA CTTCTCATTGGC-3=and 5=-AGC-GAATTCAGGCATAGCTTGGGCATA-3=.
HUVEC Transfection
RNA oligonucleotides were purchased from Shanghai GenePha-rma.The sequence of the antisense miR-141(referred to as miR-
141AS)from Shanghai GenePharma is as follows:GGUAGAAAUG-
GUCUGUCACAAU.All bases were modi?ed with a 2-OD.HU-VECs were transfected with 100nM miR-141miRNA mimics or with 100nM miR-141miRNA inhibitors (Dharmacon)utilizing Oligo-fectamine (Invitrogen).The effects of transfections with miR-mimics/inhibitors were assessed by quantitative (q)RT-PCR.qRT-PCR re-agents were purchased from Takara (Takara,Otsu,Japan).Real-Time PCR Assay of miRNAs
miRNAs were isolated from heart tissues or cultured cells using
the mirVanaTM miR isolation kit (Ambion)in accordance with the
manufacturer’s protocol.Quantitative real-time PCR was con-Fig.1.Micro (mi)RNA expression pro?le in human umbilical vein endo-
thelial cells (HUVECs)stimulated with TNF-?for 6h.Real-time PCR
con?rmed the upregulation of miR-146a and miR-155*and downregulation of miR-141,which were consistent with the microarray results.Bars represent the relative quantity of miRNAs expression levels in HUVECs.
Data were representative of 3independent experiments and are shown as means ?SE.*P ?0.05,***P ?0.001,****P ?0.0001.
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ducted using a 4800Real-Time PCR Machine (Bio-Rad).miRNA levels were quanti?ed by qPCR using speci?c Taqman assays for miR (Applied Biosystems)and Taqman Universal Master Mix (Applied Biosystems).Speci?c primers were obtained from Ap-
plied Biosystems [primer identi?cation numbers:000468for hsa-
miR-146a,000463for hsa-miR-141,and 001973for U6small nucleolar RNA (snRU6)].miRNA-141levels were quanti?ed with
the 2?DDct relative quanti?cation method that was normalized to the snRU6.Flow Cytometry ICAM-1,VCAM-1,and E-selectin puri?ed monoclonal anti-body were purchased from BD Phamingen.The bindings of mAbs to the ICAM-1,VCAM-1,and E-selectin molecules on the cell surface were determined by ?ow cytometry analysis.After being blocked with normal goat serum (10%),HUVECs were incubated with ICAM-1,VCAM-1,E-selectin,and control mAbs,respec-tively.After two washes in Dulbecco’s PBS (DPBS),cells were resuspended in DPBS containing a working dilution of FITC-labeled goat anti-mouse IgG (BioLegend)and were incubated at 4°C for 30min.The cells were washed and ?xed,and a minimum of 20,000-gated events/samples were collected on a FACS Calibur (Elite ESP)and analyzed using Flowjo software.HL-60Cell Binding to HUVECs
The human promyelocytic cell line HL-60cells were labeled with car-boxy?uorescein diacetate succinimidyl ester (CFSE)for 10min at 37°C and
suspended in PBS containing 1mM magnesium chloride,0.5mM calcium
chloride,and 0.1g/l glucose.HUVECs were transfected for 24h with
varying doses of mimics and inhibitors of miR-141oligonucleotides.Trans-
fected HUVECs were treated with 10ng/ml TNF-?for 6h,washed twice in PBS plus,and then incubated with 1ml of labeled HL-60(105)for 45min at 37°C.The culture wells were washed several times with PBS and the concentration of CFSE was measured by multifunctional reader (Tecan),where the wavelength of the excitation ?lter is 485nm,the wavelength of the emission ?lter is 535nm.To quantitate the precise number of adherent HL-60cells,samples were permanently ?xed,images were taken using ?uorescence microscope (Olympus,Tokyo,Japan)and then ?ve randomly selected ?10?elds were counted.
Fig.2.ICAM-1was identi?ed as a novel target of miR-141.A :miR-141is partially complementary to a region in the ICAM-13=-UTR (top ).The sequence of miR-141and its potential matching site in the ICAM-13=-UTR (bottom ).The mutated positions are shown in bold.B :luciferase assay con?rmed that ICAM-1was one of the target genes of miR-141.Fire?y luciferase activity was normalized to the average luciferase activity of cells transfected with control reporter vector.NC indicates control oligonucleotide.C :luciferase assay when the miR-141binding sites in the 3=-UTR of ICAM-1were mutated.Fire?y luciferase activity was normalized to the average luciferase activity of cells transfected with control miRNA.D :HUVECs were treated with a control oligonucleotide or miR-141mimics for 24and 48h,and the expression levels of ICAM-1,VCAM-1,and E-selection were measured by real-time PCR.Results are shown as representative of 3independent experiments.*P ?0.05,***P ?0.001.
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In Vivo Transfection of miRNAs Female BALB/c mice (10wk old)were purchased from the Laboratory Animal Center of the Fourth Military Medical University,Xi’an,China under speci?c pathogen-free conditions.Experiments followed a protocol drafted by the Animal Care Committee of the University.Entranster-in vivo reagent was purchased from Engreen Biosystem,(Beijing,China).The Entranster-in
vivo-miRNA mimics mixture was prepared according to the manufacturer’s instructions.Brie?y,1)25?g of miR141mimics or control miRNA (Ge-nePharma,Shanghai)were dissolved in 50?l of RNase-free water and mixed with 50?l 10%glucose solution;2)50?l of the Entranster-in vivo transfection reagent were mixed with 50?l 10%glucose solution;then 3)the 200-?l mixture of 1and 2was injected via the tail vein into each mouse using a microsyringe 24or 48h before the MI/R operation.Mice were randomly divided into three groups as follows:1)miR141mimics,2)control miRNA mimics,and 3)PBS treated.
Experimental Model of MI/R Injury
We used a rodent model of myocardial I/R injury that mimics the clinical scenario of myocardial infarction (14).Brie?y,female BALB/c mice (10wk old)were anesthetized by sodium pentobarbital Fig.3.Exogenous miR-141inhibits the ICAM-1expression level in HUVECs after stimulation with TNF-?.A :?uorescence-activated cell sorting (FACS)analysis showing the time-dependent upregulation of ICAM-1,VCAM-1,and E-selection expression levels in HUVECs after stimulation with TNF-?for 0,0.5,2,4,6,and 24h.B :HUVECs were treated with a control oligonucleotide or miR-141mimics for 48h and then stimulated with TNF-?for 6h,and the expression levels of ICAM-1,VCAM-1,and E-selection were measured by FACS.Results shown are representative of 3independent experiments.C :quantitative analysis for A and B .**P ?0.01.
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(30mg/kg ip)and ventilated with room air using a rodent ventilator.After left thoracotomy and exposure of the hearts,the left anterior
descending coronary artery was occluded with 6-0silk ligatures just proximal to its main branching point.The suture was tied using a shoestring knot over a 1-mm polyethylene tube (PE-10)that was left in place during the planned period of ischemia.MI was con?rmed by S-T segment elevation on the electrocardiogram.The ischemic area
was readily recognizable by a cyanotic appearance and a bulging region.The chest was compressed brie?y to expel intrapleural air and
closed,leaving one end of the coronary suture protruding from the chest.After completion of 30min of occlusion,the exteriorized suture was pulled to release the knot to perfuse the coronary artery.Before subjected to the MI/R injury,the mice were randomized to receive one of the treatments as described above.Mice were killed after 3,6,and 12h (for real-time PCR and Western blot)and 24h [for Evans/triphenyltetrazolium chloride (TTC)staining and immunohistochem-ical staining]of reperfusion.Western Blot The cellular and tissue proteins were separated by SDS-PAGE and transferred onto Hybond ECL membranes (Amersham Pharmacia,Piscataway,NJ).The ECL membranes were incubated with the appropriate primary antibody anti-ICAM-1(Santa Cruz Biotechnol-
ogy),followed by incubation with peroxidase-conjugated secondary antibodies (Cell Signaling Technology)and analysis by the ECL
system (Amersham Pharmacia,Piscataway,NJ).The signals were quanti?ed using the G:Box gel imaging system by Syngene.ELISA for Cardiac Troponin I,Creatine Kinase-MB,and Lactate Dehydrogenase Serum levels of cardiac troponin I (cTnI),creatine kinase-MB
(CK-MB),and lactate dehydrogenase (LDH)markers of myocyte necrosis,were measured by a colorimetric method,with speci?c
CK-MB,cTnI,and LDH kits (Nanjing Jiancheng Reagents),using an auto analyzer (Roche Hitachi Modular DP Systems,Mannheim,Germany)according to the manufacturer’s instructions.The recorded values are presented in international units per liter.Evans/TTC Staining
After 24h of reperfusion,the ligature around the coronary artery was retied,and the infarct size was determined by Evans blue/TTC (Sigma-Aldrich no.T8877)double staining as previously de-scribed (9).
Immunohistochemistry
The murine hearts were rapidly excised and rinsed with PBS and ?xed in 4%paraformaldehyde overnight.After being embedded in paraf?n,5-cm sections were processed.After the endogenous perox-idase activity was inhibited,the sections were incubated with primary anti-F4/80(eBioscicence no.14–4801)or anti-CD11b (Abcam no.ab133357)antibodies at 4°C overnight.After three washings in PBS,the slices were dipped into horseradish peroxidase-conjugated sec-ondary antibody for 30min at room temperature.Antigen-antibody
complexes were incubated with DAB chromogen and observed,and
the sections were ?nally counterstained with Mayer’s hematoxylin.
The same methods were performed without the primary antibodies,as
negative controls.The numbers of CD11b ?myeloid cells and F4/80?macrophages were assessed by counting the total cell numbers in the ischemic areas in ?ve randomly chosen ?elds in each section.Statistical Analyses
The analysis was performed by SPSS and GraphPad Prism6soft-ware.Data are presented as the means ?SE.Unpaired Student’s
t -tests or ANOVA was used for statistical comparisons when appro-
priate.Values of P ?0.05were considered statistically signi?cant.RESULTS miRNA Expression Pro?le in TNF-?-Stimulated HUVECs Several studies suggest that ECs are more sensitive to I/R injury than CMs (15,24).However,miRNAs expression and regulation by ECs in the setting of MI/R remain to be eluci-dated.Hence,to determine the expression pattern of miRNAs Fig.4.miR-141suppresses neutrophil adhesion ex vivo.A :representative immuno?uorescence photomicrographs showing carboxy?uorescein diacetate succinimidyl ester (CFSE)-labeled HL-60adhesion to HUVECs.HUVECs were transfected with miR-141mimics,miR-141inhibitor,or control oligonucleotide for 24h and then treated with or without TNF-?for 6h.CFSE-labeled HL-60were added to the HUVECs,incubated at 37°C for 45min,and then washed.Scale bar ?100?m.B :number of neutrophil (CFSE-labeled HL-60)adhering to HUVECs in different groups.Bars show the mean value of 3independent experiments.Data are shown as means ?SE.**P ?0.01as indicated.
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in ECs,we used Microarray analysis.In brief,total RNA was
isolated from HUVECs that were treated for 6h with TNF-?,
fractionated by size exclusion column chromatography,la-
beled with a ?uorescent dye,and hybridized to a microarray
chip (n ?3).The threshold value we used to screen up and
down regulated miRNAs is fold change ?1.50and fold
change ?0.67.In addition,eight miRNAs,which were
reported to be involved in the development,differentiation,
and function of the immune cells,were identi?ed and
presented in italics in Table 1.The qRT-PCR results shown
in Fig.1validated our results representing miR-146a miR-
155*and miR-141.Since miR-141was the only miRNA
downregulated following TNF-?stimulation in HUVECs,
we choose miR-141as focus of our study.
ICAM-1Is a Target Gene of miR-141
Function of miRNA relies on the inhibition of the target
mRNA;therefore,we used the online prediction tool Target
scan to identify the putative target genes of miR-141.Bioin-
formatics analysis indicated that ICAM-1was one of the
potential target genes of miR-141.miR-141has sequence
similarity to a region within the 3=-UTR of the transcript for
human ICAM-1,extending between 858and 864(Fig.2A ).To
con?rm that miR-141was able to directly bind to the 3=-UTR
of ICAM-1and inhibit ICAM-1expression,a recombinant
?re?y luciferase reporter vector with a fragment of the 3=-UTR
of ICAM-1mRNA containing the putative miR-141binding
sequence was cloned and cotransfected into 293T cells with
either control miRNA mimics [neagative control (NC)],miR-
141mimics,or unrelated miRNA mimics (miR-146a),respec-
tively.As expected,only miR-141signi?cantly inhibited the
luciferase activity (Fig.2B ).To demonstrate further that the
downregulation of ICAM-1by miR-141was mediated through
the predicted binding site,two kinds of double substitution
mutations in the 3=-UTR of ICAM-1that disrupted the com-
plementation with the 5=-seed-matched sites (Fig.2A ,3=-UTR
of ICAM-1-MUT1and 3=-UTR of ICAM-1-MUT2)of miR-
141were generated,which demonstrated both mutations abol-
ished the repression effect mediated by miR-141on ICAM-1
(Fig.2C ).
Furthermore,to con?rm that exogenous miR-141is able to
decrease the mRNA level of ICAM-1in HUVECs,we trans-
fected miR-141mimics into HUVECs and detected the mRNA
level of ICAM-1.The results implied that ICAM-1expression
was inhibited by the miR-141mimic,and it has no effect on the
expression of VCAM-1and E-selection (Fig.2D ).Therefore,
the effect of miR-141is speci?c and selective with no off target
effects.
Exogenous miR-141Inhibits ICAM-1Expression on
HUVECs Following TNF-?Stimulation ICAM-1is expressed widely on nonhematopoietic and he-matopoietic cells but at a low level on normal endothelium (13).During in?ammation,adhesion molecules expressed on stimulated vascular ECs are essential for recruitment and transmigration of leukocytes to the subendothelial matrix.The released in?ammatory mediators,such as TNF-?,activate the rapid expression of ICAM-1to facilitate leukocytes adhesion and transmigration in vascular ECs (13).
Fig.5.miR-141downregulates ICAM-1expression in vivo.miR-141mimics,
miR-141inhibitor,and control oligonucleotide were transfected into BALB/c
mice using Entranster-in vivo transfection reagent through tail vein injection,mRNA level of miR-141and ICAM-1was detected by RT-PCR 48h after
transfection.A :bars show the relative miR-141mRNA level in the left ventricular tissues from different groups.B :bars show the relative ICAM-1
mRNA level in the left ventricular heart tissues from different groups.C :correlation between miR-141and ICAM-1.Bars show the relative mRNA level of miR-141and ICAM-1.Data are shown as means ?SE;n ?6per group,*P ?0.05,**P ?0.01as indicated.
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Our own data demonstrated that TNF-?stimulation induced ICAM-1,VCAM-1,and E-selectin expression in HUVECs as previously published (17)(Fig.3A ).The expression level of
ICAM-1reaches its peak after a 6-h stimulation with TNF-?.Therefore,in the following experiments we chose 6-h incuba-tion time points for maximum level of induction.To determine the effect of miR-141on ICAM-1expression,HUVECs were transfected with miR-141mimics for 48h and then stimulated with TNF-?for 6h,and then ICAM-1,VCAM-1,and E-selectin expression was measured by ?ow cytometry (Fig.3).As shown in Fig.3,B and C ,transfection of miR-141results in a signi?cant decrease in ICAM-1expression but has no effect on expression of VCAM-1and E-selectin.miR-141Suppresses Neutrophil Adhesion To explore the functional relevance of miR-141,we mea-sured the in?uence of miR-141on leukocyte adhesion to ECs.To examine that,we transfected HUVECs with miR-141,treated with TNF-?,added CFSE-labeled HL-60leukocytes,washed the mixture,and measured leukocyte binding to the ECs.TNF-?stimulation signi?cantly increased leukocyte ad-hesion to ECs,and overexpression of miR-141decreased leukocyte binding to TNF-?challenged HUVECs (Fig.4).We then tested whether endogenous miR-141regulates leukocyte adherence to ECs.HUVECs were transfected with an miR-141inhibitor (100nM),and then the leukocyte adherence to TNF-?-stimulated cells was measured as above.Our results showed that addition of inhibitor to miR-141increases leukocyte ad-herence (Fig.4).Collectively,all these data suggested that miR-141inhibits leukocyte adherence to the TNF-?challenged HUVECs.Transfection of miR-141Mimics In Vivo Attenuates MI/R Injury
To evaluate the effect of increased exogenous miR-141on MI/R-induced cellular injury.BALB/c mice (10wk old)were transfected with miR-141mimics,miR-141inhibitor and NC by Entranster in vivo transfection reagent.qRT-PCR data showed that the mRNA levels of miR-141in the myocardium of mice were signi?cantly higher compared with that of the NC-transfected mice (Fig.5A ).Meanwhile,the target gene ICAM-1was decreased as determined by qRT-PCR analysis (Fig.5B ).
Mice were subjected to 30-min I/R for 3,6,and 12h to
determine the pro?le of ICAM-1expression.Consistent with
the previous study,the ICAM-1mRNA level was upregulated
by MI/R injury,with peak at 6h following reperfusion (Fig.
6A ).Furthermore,to con?rm that exogenous miR-141could decrease the mRNA level of ICAM-1induced by MI/R,mice were transfected with miR-141for 36h before MI/R injury,and in vivo miR-141mimics transfection downregulated MI/R induced ICAM-1mRNA upregulation (Fig.6B )and protein expression (Fig.6,C and D ).
Then,serum levels of cTnI,CK-MB,and LDH were de-tected to determine the extent of the MI/R injury.The concen-trations of cTnI,CK-MB,and LDH in the blood were signif-icantly increased in wild-type animals following MI/R.How-ever,animals transfected with miR-141mimics showed
decreased serum cTnI and LDH following MI/R (Fig.7).
Infarct size was determined by Evans blue/TTC double stain-ing,and MI/R resulted in an infarct affecting ?46%of the area at ischemic risk in wild-type mice,whereas miR-141mimics pretreatment decreased the infarct size to 37%(Fig.7D ).Fig.6.miR-141downregulates ICAM-1ex-
pression induced by myocardial ischemia-
reperfusion (MI/R)injury.A :wild-type
(WT)control mice were subjected to 30min
ischemia and reperfusion for 3,6,and 12h
as indicated.ICAM-1mRNA level was up-
regulated by MI/R injury,with peak at 6h
following reperfusion.B :in vivo miR-141
mimics (miR141)transfection 48h before
MI/R injury downregulated ICAM-1mRNA
expression.C :representative immunoblots
of ICAM-1expression in ischemic heart ho-
mogenates from control oligonucleotide or
miR-141mimics transfected animals sub-
jected to MI/R (30min/6h).D :bars show
the relative ICAM-1protein in ischemic
heart tissue from control oligonucleotide or
miR-141mimics transfected animals sub-
jected to MI/R injury.Data are shown as
means ?SE;n ?6per group;**P ?0.01
as indicated.
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To investigate the possible mechanism responsible for the cardioprotective effects against MI/R injury,We assessed the
in?ammatory cells in?ltration into the ischemic and reperfused myocardium.Immunohistological analysis revealed that MI/R resulted in a large number of CD11b ?myeloid cells and F4/80?macrophages accumulation in the ischemic myocar-dium,whereas pretreatment with miR-141mimics decreased CD11b ?myeloid cells and F4/80?macrophages in?ltration following MI/R (Fig.8).DISCUSSION I/R injury contributes to pathology in a wide range of conditions,including myocardial infarction,ischemic stroke,and acute kidney injury,and is also a major challenge during organ transplantation and cardiothoracic surgery.Currently,innovative therapeutic approaches targeting I/R includes isch-emic preconditioning and postconditioning,metabolic modu-lation,therapeutic gases like nitric oxide,nucleotide,and miRNAs (7).miRNAs are small noncoding RNAs that regulate gene expression by binding to the target mRNAs,leading to translational repression or degradation.A growing number of studies have suggested miRNAs are involved in the modulation of MI/R injury.For example,miR-499administration dimin-ishes apoptosis and attenuates myocardial reperfusion injury.Inhibition of cardiomyocyte apoptosis by miR-499was as-cribed to direct targeting of a catalytic subunit of the phospha-tase calcineurin and thereby decreased activation of the mito-chondrial ?ssion program (28).In a mouse model of MI/R injury,expression of miR-24is also protective by inhibiting cardiomyocyte apoptosis and decreasing infarct size (21).In addition,one excellent study showed that miR-92a controls angiogenesis and miR-92a inhibition led to enhanced blood vessel growth and functional recovery of damaged tissue in mouse models of myocardial infarction (2).However,most of these miRNAs target cardimyocytes instead of ECs,and the
exact role of endogenous miRNAs targeting or expressed by ECs during MI/R is just emerging and remains to be deter-mined.
A wide range of pathological processes contribute to MI/R
injury,among which leukocyte-EC adhesion and EC in?am-
mation aggravate postischemic microvascular dysfunction and thus play a crucial role in reperfusion injury (1,19).There is a growing appreciation that ECs are more vulnerable to I/R injury than cardiomyocytes and they might be the critical determinants for the extent of injury and the recovery time (1,19).Hence,decreasing microvascular permeability and inhib-iting leukocyte-EC adhesion are promising therapeutic strate-gies to attenuate reperfusion injury.ICAM-1,a member of the cell adhesion molecule superfamily expressed by ECs,plays a crucial role in mediating the migration of leukocytes across the endothelium and into the myocardium.Anti-ICAM-1antibody treatment before the onset of reperfusion was demonstrated to decrease leukocytes in?ltration in the ischemic reperfused cat myocardium and thus preserved coronary vasodilatory re-sponse and reduced myocardial injury (20).Similar myocardial protection by combined antibody therapy targeting both P-selectin and ICAM-1was observed in a rat MI/R model (8).However,translation of these basic sciences into clinical prac-tice has been disappointing.Thus the endogenous regulator controlling the expression and function of ICAM-1might be more critical and clinically relevant,and the role of miRNAs
Fig.7.miR-141attenuates MI/R injury.
A –C :plasma concentration of cardiac tro-
ponin I (cTnI;A ),creatine kinase MB (CK-
MB;B ),and lactate dehydrogenase (LDH;
C )were determined after 6h of reperfusion.
D :myocardial infarction was determined by
Evans blue/triphenyltetrazolium chloride
(TTC)double-staining 24h after reperfu-
sion.Data are shown as median;n ?6–10
mice.*P ?0.05as indicated.
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and their interaction with ICAM-1in the setting of MI/R injury remains poorly understood.In the present study,the TNF-?-stimulated HUVEC was used as a model of EC in?ammation,and the pro?le of miRNA
expression by ECs was screened and analyzed by microarray.We found that miR-141expression was signi?cantly down-regulated in TNF-?stimulated HUVECs,and ICAM-1was identi?ed as the target gene of miR-141.Luciferase study con?rmed that miR-141is able to directly bind to and suppress mRNA expression of ICAM-1.TNF-?stimulation induced ICAM-1,VCAM-1,and E-selectin expression in ECs as pre-viously reported (16).More importantly,exogenous miR-141suppressed TNF-?induced ICAM-1mRNA expression in HUVECs but had no effect on the mRNA expression levels of VCAM-1and E-selectin.Further functional study demon-strated that miR-141inhibited leukocyte adherence to HU-
VECs challenged by TNF-?.Moreover,in vivo study using mouse MI/R model showed that exogenous miR-141sup-pressed MI/R-induced mRNA upregulation and protein expres-sion of ICAM-1,thereby decreasing the accumulation of CD11b ?myeloid cells and F4/80?macrophages in the isch-emic myocardium,which may account for the decreased isch-emic injury as evidenced by decreased infarct size and decline of plasma CTnI and LDH levels.Consistent with our ?ndings,a recent clinical study demonstrated that miR-320b,released from activated platelets,can be taken up by ECs and regulate ICAM-1expression in patients with myocardial infarction (11).
Taken together,for the ?rst time,we have identi?ed miR-141as an endogenous repressor of ICAM-1expression,and our data demonstrate that miR-141overexpression suppresses ischemia-induced ICAM-1expression and attenuates reperfu-sion injury.These observations expand our understanding of the interaction between miRNAs and ECs and its role in ischemic tissue injury.Thus miR-141may serve as a valuable therapeutic target in the setting of ischemic heart disease.Conclusions
Minimizing the consequences of the MI/R injury has impor-tant clinical applications and is a very important factor in predicting the outcome and time of the recovery post the cardiac surgery.ECs are uniquely situated to modulate I/R injury in the heart.They are able to tolerate long periods of
Fig.8.miR-141attenuates in?ammatory cells in?ltration to
the ischemic heart tissue following MI/R (30min/24h)
injury.A and C :representative photographs of immunohis-
tochemical staining for CD11b ?myeloid cells (A )and
F4/80?macrophages (C )in the ischemic areas 24h follow-
ing MI/R.Scale bars ?100?m.B and D :bar graph shows
the counts of CD11b ?myeloid cells (B )and F4/80?mac-
rophages (D )in the ischemic areas 24h following MI/R.
Data were obtained from 4independent experiments and
n ?4high-power ?elds per mouse.Data are shown as
means ?SE;n ?4mice per group and n ?4high-power
?elds per mouse.*P ?0.05vs NC-I/R group.
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ischemia and upon reperfusion activate a variety of blood elements including neutrophils,platelets,and complement.Because of their proximity to cardiomyocytes,they are very important in modulating cardiomyocyte function and injury.Further focus on ECs may present a deeper understanding of I/R injury and provide new opportunities for its treatment or prevention.Translational studies using miRNA-based thera-pies in I/R injury are needed.Our studies make a great contribution to that ?eld by identifying a novel miR-141that as we have shown using in vitro as well in vivo approach is involved in attenuation of MI/R-induced cardiac injury and dysfunction via antithetical regulation of ICAM-1.Our data demonstrate that miR-141has a great potential to be a new therapeutic target for ischemic heart disease.GRANTS This work was supported by Grant 2013ZX1004609and the National Natural Science Foundation of China (91442108and 81000088).DISCLOSURES No con?icts of interest,?nancial or otherwise,are declared by the author(s).AUTHOR CONTRIBUTIONS Author contributions:R.R.L.,F.G.,and L.H.C.conception and design of research;R.R.L.,J.L.,J.Y.G.,F.K.,J.Y.L.,Y.S.Z.,Q.L.M.,C.J.S.,K.Y.,and B.Q.J.performed experiments;R.R.L.,J.L.,and J.Y.G.analyzed data;R.R.L.,J.L.,and J.Y.G.interpreted results of experiments;R.R.L.and J.L.prepared ?gures;R.R.L.and J.L.drafted manuscript;R.R.L.,J.L.,A.D.T.,and L.H.C.edited and revised manuscript;R.R.L.,A.D.T.,and L.H.C.approved ?nal version of manuscript.REFERENCES 1.Bekkers SC,Yazdani SK,Virmani R,Waltenberger J.Microvascular obstruction:underlying pathophysiology and clinical diagnosis.J Am Coll Cardiol 55:1649–1660.2.Bonauer A,Carmona G,Iwasaki M,Mione M,Koyanagi M,Fischer A,Burch?eld J,Fox H,Doebele C,Ohtani K,Chavakis E,Potente M,Tjwa M,Urbich C,Zeiher AM,Dimmeler S.MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice.Science 324:1710–1713,2009.3.Braunwald E.The war against heart failure:the Lancet https://www.doczj.com/doc/075684721.html,ncet 385:812–824,2014.4.Brutsaert DL.Cardiac endothelial-myocardial signaling:its role in car-diac growth,contractile performance,and rhythmicity.Physiol Rev 83:59–115,2003.5.Bushati N,Cohen SM.microRNA functions.Annu Rev Cell Dev Viol 23:175–205,2007.6.Davidson SM,Duchen MR.Endothelial mitochondria:contributing to vascular function and disease.Circ Res 100:1128–1141,2007.7.Eltzschig HK,Eckle T.Ischemia and reperfusion–from mechanism to translation.Nat Med 17:1391–1401.8.Fukushima S,Coppen SR,Varela-Carver A,Yamahara K,Sarath-chandra P,Smolenski RT,Yacoub MH,Suzuki K.A novel strategy for myocardial protection by combined antibody therapy inhibiting both P-selectin and intercellular adhesion molecule-1via retrograde intracoro-nary route.Circulation 114:I251–256,2006.9.Gao F,Yue TL,Shi DW,Christopher TA,Lopez BL,Ohlstein EH,Barone FC,Ma XL.p38MAPK inhibition reduces myocardial reperfu-sion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation.Cardiovasc Res 53:414–422,2002.10.Gawaz M.Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium.Cardiovasc Res 61:498–511,2004.11.Gidlof O,van der Brug M,Ohman J,Gilje P,Olde B,Wahlestedt C,Erlinge D.Platelets activated during myocardial infarction release func-tional miRNA,which can be taken up by endothelial cells and regulate ICAM1expression.Blood 121:3908–3917,S3901–3926.12.Harris TA,Yamakuchi M,Ferlito M,Mendell JT,Lowenstein CJ.MicroRNA-126regulates endothelial expression of vascular cell adhesion molecule 1.Proc Natl Acad Sci USA 105:1516–1521,2008.13.He Q,Pu J,Yuan A,Lau WB,Gao E,Koch WJ,Ma XL,He B.Activation of liver-X-receptor alpha but not liver-X-receptor beta protects against myocardial ischemia/reperfusion injury.Circ Heart Fail 7:1032–1041,2014.14.Hua F,Ha T,Ma J,Li Y,Kelley J,Gao X,Browder IW,Kao RL,Williams DL,Li C.Protection against myocardial ischemia/reperfusion
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围术期心肌缺血的监测和治疗 围术期心肌缺血是手术治疗中的严重并发症之一，心肌缺血可引起心脏功能的显著变化，并诱发一系列严重事件，如心肌梗死、心律失常、肺水肿，甚至死亡。对围术期心肌缺血的评估、预防和有效的诊疗，是减少心脏意外和并发症的关键，有助于病人近期康复和远期的预后。 一、心肌缺血的代谢与生理机制 冠脉供血量不能满足心肌对能量的需要时即发生心肌缺血。因此心肌缺血既可发生在冠脉供血量明显减少时，也可发生在心肌对能量的需要明显增加时。心肌缺血时不仅有心肌组织缺氧,并且不能把具有潜在毒性的代谢产物移走,因而同时有乳酸,二氧化碳和氢离子的堆积。此外，血流恢复可能进一步加重损伤的程度（再灌注）。 正常情况下，心肌完全依赖碳于有氧代谢，其细胞内氧与ATP 量很少，脂肪酸是心肌氧化磷酸化作用的主要供能方式，其它底物包括葡萄糖、氨基酸、丙酮酸和乳酸。一旦发生心肌缺血，心肌迅速从有氧代谢转为无氧代谢，从而产生大量乳酸。 冠脉堵塞后，在不到1分钟内即有K＋从缺血细胞外移，细胞外K＋浓度升高。心肌细胞内K＋的丢失导致心肌细胞膜极化的改变和心电图ST段的异常，并成为心肌缺血早期室性心律失常的基础。 钙稳态是维持正常心功能的关键因素，钙稳态失调是心肌细胞损伤的重要发病因素。缺血心肌细胞内钙离子的增加是缺血心肌发生挛缩的原因。 心肌缺血时上述代谢变化导致进行性膜功能改变和离子稳态失调，早期膜功能变化的特点是离子泵和离子通道一个个相继发生障碍，最早是钾离子从缺血心肌细胞外流，此现象出现在Na＋－K＋－ATP酶功能障碍以前，当ATP减少到一定程度时，Na＋－K＋－ATP酶功能发生明显障碍，于是Cl－和水在细胞内大量积聚，K＋进一步丢失，细胞丧失了调节自身容积的能力，于是发生细胞内水肿。随着缺血加重，离子泵转运失调，大量钙离子进入细胞内并激活磷脂酸和脂肪酶，而使细胞膜结构损坏及细胞解体，出现不可逆变化。 在力学方面方面，急性心肌缺血可影响心脏的收缩与舒张功能。舒张功能障碍往往早于收缩功能的变化。心肌缺血对心室顺应性的即刻影响与缺血的病因学有关。氧供下降开始时伴有心室顺应性增加，而氧需增加与心室顺应性即刻显著下降有关（即心室变成僵硬）。心室需要较高的充盈压（LVEDP），以维持一定的每搏量。此时病人可能表现出室壁运动异常，心律失常和传导阻滞。如果冠脉血流下降80%，则可引起心室收缩无力；冠脉血流下降95%，则出现心室动力障碍。心肌缺血严重时，LVEDP升高可引起肺水肿。缺血心肌可呈不可逆性损伤（梗死）或立即恢复，同时还有其它的生理途径，短暂性严重心肌缺血后，心肌收缩功能可逐渐恢复即心肌顿抑；而慢性严重缺血可引起心脏收缩作功下降如慢性室壁运动异常即心肌冬眠。 稳定性缺血综合征可能是在冠状动脉固定斑块的基础上发生氧需增加。而一般认为，不稳定性缺血综合征是斑块破裂伴局部栓塞与局部血管反应，结果使处于临界的冠状血管氧供间断性降低。CAD或高血压病人内皮细胞功能受损，从而导致血管收缩加剧。左室肥厚病人在这
冠心病健康管理方案 冠心病是冠状动脉粥样硬化性心脏病的简称，中医称为胸痹。冠状动脉供应心脏自身血液，冠状动脉发生严重粥样硬化或痉挛，使冠状动脉狭窄或闭塞，导致心肌缺血缺氧或梗死的一种心脏病。冠心病的主要临床表现是心肌缺血、缺氧而导致的心绞痛、心律失常、严重者可发生心肌梗死危及生命。冠心病已成为当今危害我国人民健康和生命的主要疾病。对于冠心病的诊断，目前国际上没有统一的标准，冠状动脉造影检查是其一项“金标准”，但行此有创检查者还是少数，更多的冠心病的诊断需要依据病史、临床症状、辅助检查结果进行综合判断，这也给冠心病的健康管理提出更高要求。 1冠心病的一级管理 1.1一级管理对象对有心血管病危险因素存在，但尚未确诊冠心病人群采取预防措施，控制或减少心血管疾病危险因素，并维持稳定，以减少冠心病的发病率、死亡率与致残率［。目前，已经确认的冠心病危险因素达300种以上。主要因素分为不可变因素（如年龄、性别、心血管疾病家族史等）和可变因素（如高血压、高血脂、吸烟、缺乏体育锻炼、饮食等）。对可变因素的干预重点在于改善血管内皮功能，防止血管内皮溃疡面形成，阻止脆弱的动脉粥样硬化斑块形成血栓，减慢动脉粥样硬化的进展。而干预方式除了药物治疗以外，更多的危险因素需要生活方
式和行为干预。冠心病的一级管理即危险因素的管理。 1.2一级管理措施 1.2.1戒烟临床研究显示，吸烟能增加患者心血管疾病死亡率50％，心血管死亡的风险与吸烟量直接相关。吸烟还与血栓形成、斑块不稳定及心律失常相关。对于所有冠心病高危人群及患者，均需详细询问吸烟史。对于这个危险因素的控制多用于发病前的预防性措施，对高危人群及患者宣传吸烟的危害，协助其完全戒烟并且避免被动吸烟。已有一些行为及药物治疗措施，如尼古丁替代治疗等，可以协助其戒烟。 1.2.2适量运动运动应尽可能与多种危险因素的干预结合起来，成为冠心病高危人群及患者综合治疗的一部分。目前有资料显示，运动锻炼能减轻患者症状、改善运动耐量，减轻同位素显像的缺血程度及动态心电图上的ST段压低［6］。以症状限制性有氧运动为主，运动方式有步行、慢跑、骑自行车、游泳等。每次20~30 min，逐渐延长至40~60 min左右，每周4~5次，以能耐受、感觉舒适为宜，运动过程中自测心率以达到（170-年龄）次/min为宜。 1.2.3减轻体重按照中国肥胖防治指南定义，肥胖指体重指数（BMI）≥28 kg/m2；腹形肥胖指男性腰围≥90 cm，女性≥80 cm。肥胖多伴随其他促发冠心病的危险因素，包括高血压、胰岛素抵抗、HDL-C降低和TG升高等。与肥胖相关的冠心病危险的增加多由上述危险因素导致［8］。减轻体重（控制饮食、活动和
心肌缺血再灌注损伤的发生机制和防治研究进展 1 9 6 0年J e n n i n g s等，第一次提出心肌缺血再灌注损伤的概念，证实再灌注会引起心肌超微结构不可逆坏死，并逐渐引起医学界的高度重视。缺血心肌恢复再灌注后，病情反而恶化，引起超微结构、功能、代谢及电生理方面发生进一步的损伤，是由于在缺血损伤的基础上再次引起的损伤，因此称为缺血．再灌注损伤( i s e h e m i a — r e p e r f u s i o n i n j u r y ，I R I ) k 2 J 。临床上表现为闭塞的冠状动脉再通、梗死区血液灌流重建后一段时间内，有的病例发生血压骤降、心功能不全、心律失常甚至猝死等一系列病情反而恶化的现象。因此， I R I 的发生机制与防治越来越引起人们的关注，并一直试图寻找能对 I R I 产生确切保护作用的药物。现就 I R I的发生机制和防治的研究进展作一综述。 1 . 心肌缺血再灌注损伤的发生机制 目前，缺血再灌注损伤发生的机制尚未完全阐明，研究表明自由基、钙超载、心肌纤维能量代谢障碍、中性粒细胞、血管内皮细胞、细胞黏附分子与细胞凋亡等均可能参与缺血再灌注损伤。 1 ． 1 氧自由基( F R) 生成正常细胞内有自由基清除剂超氧化物歧化酶( S O D)，使氧自由基转变为过氧化氢，后者又通过触酶及谷胱甘肽过氧化物酶的作用还原为水和分子氧，故小量氧自由基不造成损伤。再灌注时产生的大量氧自由基不能被清除，其中包括非脂质氧自由基和脂质氧自由基，如超氧阴离子、羟自由基、过氧化氢等。缺血再灌注后，它可与各种细胞成分，如膜磷脂、蛋白质、核酸等发生反应，造成细胞结构损伤和功能代谢障碍。C a s t e d o 等在动物实验中发现，再灌注后细胞内膜脂质过氧化增强，形成多种生物活性物质，如血栓素、前列腺素等，促进再灌注损伤。 1 9 8 6年,M u r r y等，首次在犬缺血／再灌注模型实验中发现反复短暂缺血发作可使心肌在随后持续性缺血中得到保护，从而提出了缺血预适应( I P C) 心脏保护的概念，为缺血心肌的保护及其机制探讨开辟了崭新的领域。自由基可能参与了预适应保护的触发机制。Z h o n g等证明预适应过程中产生的低浓度自由基对延迟心肌缺血再灌注损伤有保护作用。冉擘力等从细胞水平证明早期产生的氧自由基能诱导延迟保护作用产生，其机制可能是通过早期氧化反应一方面改变S O D形态结构而提高酶的活性，诱导延迟相S O D合成增加，另一方面诱导热休克蛋白信使核糖核酸转录和持续合成，保护心肌细胞对抗细胞外氧自由基的损伤；氧化氮合酶( N O S ) 产生的一氧化氮能有效对抗氧自由基的损害，延迟期心肌 N O S活性增加。延迟保护作用增强，其机制可能是氧自由基诱导了后期 N O S信使核糖核酸转录和合成增加，因为在缺血等应激状态下，氧化氮能够调控心脏基因的表达。总之，热休克蛋白、抗氧化酶和 N O S等不是孤立地对抗氧自由基损伤，而是有机地结合起来发挥作用。 1 ． 2 钙超载。生理状态下，胞浆内钙浓度约为 l 0-7 m o l ／ L ，而细胞外及胞浆内的钙储存系统( 如内质网和线粒体) 中钙浓度为1 0 -3m o l ／L 。正常状态下，细胞通过一系列转运机制可以保持这种巨大的浓度梯度，以维持细胞内低钙状态。但是再灌注后，钙离子向线粒体转移，导致线粒体功能障碍；钙离子浓度升高，可激活多种酶( 如激活膜磷脂酶 A ， )同时促使心
创伤病人麻醉的注意事项 据统计，目前在世界范围内创伤已成为年轻人伤残和死亡的首要原因。因创伤而需要急诊手术的病人，病情严重程度很不一致，麻醉处理的难度也各不相同，处理得当与否直接关系到治疗效果。严重外伤和复合伤病人需要立即进行麻醉和手术，更有些病人在急诊室即要求麻醉人员处理各种紧急情况: 呼吸、循环、镇痛和麻醉方面。为此首先要了解严重创伤的特点和病理生理变化，其次是掌握紧急气道和循环处理措施，最后是选择合适的麻醉方法和药物，以及预防和治疗术中和术后的并发症。 创伤病人的麻醉科根据创伤部位、手术性质和病人情况选用局麻区域阻滞或全麻。一般说来，不能绝对的肯定某一麻醉药或麻醉技术较其他药物或方法优越，麻醉方法的选择取决于: ①病人的健康情况; ②创伤范围和手术方法; ③对某些麻醉药物是否存在禁忌; ④麻醉医师的经验和水平。 麻醉前用药 休克、低血容量和意识障碍的病人可免用镇静、镇痛药。但不宜省略抗胆碱药。有些外伤病人可能十分烦躁，需术前使用镇静、镇痛药，以免影响意识和瞳孔的观察。长骨骨折和腹部创伤疼痛剧烈。对一般情况稳定者，吗啡 10mg，阿托品 0． 5mg 静脉注射，可有效的减少分泌物和防止诱导期某些药物引起的心动过缓。 麻醉方法的选择 ( 1) 区域阻滞：对一些创伤范围小，失血少的病人，区域阻滞有一定优点，如降低交感神经张力，减轻应激反应，减少术中出血和术后深静脉血栓形成，患者在手术期间保持清醒状态，有利于神经和意识的判断积极有助于术后的阵痛等。原则上对于循环不稳定、有意识障碍、呼吸困难或凝血功能差的病人，忌用区域阻滞。 ( 2) 全身麻醉: ①麻醉诱导。对于严重创伤的病人，麻醉药物和治疗指数非常低。同样的病人，如果是受伤，其所谓的安全诱导剂量也会造成致命性的危险，对于病情稳定的创伤患者麻醉诱导与一般选择性手术患者无明显区别，而对低血容量的多发伤患者则要警惕。 ②麻醉维持。低血容量病人用阿片类药和肌松药维持麻醉。因吗啡和哌替啶均具有组胺释放作用，故常选用芬太尼。芬太尼对心血管 功能差得病人能提供良好镇痛作用，对血流动力学影响较小。但因有轻度扩张周围静脉作用，开始应用剂量要小。近年来，对术中知晓问题进一步重视，可用地西泮、咪达唑仑或异丙酚预防术中知晓。吸入麻醉剂一般用于全麻维持，氧化亚氮有加重气胸或颅脑积气的危险且其
心肌缺血再灌注损伤介绍和实验设计 Ⅰ.心肌缺血再灌注损伤： 它是指缺血心肌组织恢复血流灌注时，导致再灌注区心肌细胞及局部血管网显著的病理生理变化，这些变化共同作用可促使进一步的组织损伤。那这里的关键词就是缺血心肌组织。那为什么会产生缺血的心肌组织呢？这就与临床上的疾病有关了。一些心脏疾病，比如急性心肌梗死、冠心病等他们会使心脏发生缺血的症状，其基本的生理过程就是心肌缺血。 Ⅱ.心肌缺血的危害： 心肌缺血：指单位时间内的冠脉血流量减少，供给组织的氧量也减少，缺血必定存在缺氧表明缺血缺氧。心肌缺血比单纯性心肌缺氧无血流障碍要严重，因为前者除了缺氧的影响之外，缺血组织也不能获得足够的营养物质又不能及时清除各种代谢产物带来的有害影响。 一、心肌缺血的原因主要分为两种情况：1是冠脉血流量的绝对不足。这种情况是由自身疾病产生的，主要包括冠状动脉阻塞，冠状动脉痉挛。2是冠脉血流量的相对不足：包括供氧降低或耗氧增加，比如高原高空或通风不良的矿井吸入氧减少；肺通气或换气功能障碍，可致血氧含量降低红细胞数量和血红蛋白含量减少等。 二、缺血对心肌的危害主要包括以下几个方面：1是心肌收缩能力降低。2是导致心肌舒张功能降低。3是心肌组织的血流动力学发生改变，比如说血流的阻力增加等。4是心肌电生理的变化，比如说静息点位降低，传导速度减慢；室颤阈降低等。5是导致心肌形态学的改变。当然还有其他的危害，在这里就不一一列举了。 由于心肌缺血存在这么多的危害，临床上针对这一疾病采取了再灌注治疗方法，但随之而来的又是另外一个临床问题：缺血再灌注损伤。 下面具体介绍一下心肌缺血再灌注损伤。心肌缺血再灌注损伤英文缩写为MIRI，最早由詹宁斯等于1960年提出，发现其临床表现为再灌注心律失常、心肌顿抑、心肌能量代谢障碍等现象。随后又有学者在临床手术中也证实了这一观点，发现在冠脉搭桥术完成后，心肌坏死进一步加重的现象。接着布朗沃尔德教
心肌缺血预处理 第一节预处理的概述 一．预处理的概念 缺血是造成心肌细胞代谢障碍和功能异常的重要原因，严重时可引起细胞坏死。多年来人们一直认为，短暂缺血会引起心肌可逆性损伤，并使心肌难以承受再次缺血，反复多次的缺血发作可造成累积性心肌损伤甚至心肌梗死。1986年Murry等报道，短暂夹闭狗冠状动脉左旋支5 min，再灌注5 min，重复4次后，持续夹闭左旋支40 min，再灌注3 h，可使心肌梗死面积比单纯夹闭左旋支40 min，再灌注3 h组减少75％，而局部血流量并无明显变化，从而首次提出了缺血预处理(ischemic preconditioning，I-Pre-C)的概念：反复短暂缺血-再灌注可以激发自身的适应性反应，使心肌对随后发生的持续性缺血的耐受力提高，对随后长时间的缺血再灌注损伤产生明显保护作用的一种适应性机制。这一概念的提出不但更新了以往的认识，而且为缺血心肌的保护尤其是激发机体内源性抗损伤机制开辟了新思路，迅速成为心血管领域的一个研究热点。 二．预处理的特点 （一）有限记忆性若预处理与长时间缺血的间隔时间从10min延长至1～2个小时，心肌细胞将不再“记忆”它曾被预处理过，故保护作用将随之消失。 （二）双时相性预处理的保护作用在时间上呈现2个不连续的时相变化。 1．早期保护作用(early protection) 早期保护作用是短暂缺血后即刻出现的保护作用，又称经典保护反应，是延迟阶段保护作用的基础。它发生迅速，一般在预处理后2小时内发生，保护作用明显但持续时间较短，随再灌注时间延长而消失。一般而言，首次预处理后1-5 min即可显现保护效应，其持续时问因动物种属而异，兔30-60 min，猪、大鼠约60 min，狗90-120 min。早期保护作用的意义主要在于延迟了缺血心肌发生坏死的时间。例如，正常狗心肌缺血20 min 即可发生不可逆损伤，但经预处理后，需缺血40 min才出现坏死表现，这为挽救缺血心肌赢得了宝贵时间。 2．延迟保护作用(delayed protection) 指在预处理后24 h出现的保护作用。没有初始短暂阶段的保护作用，不可能发生延迟阶段的耐受。1992年Yamashita
病例讨论 山东大学齐鲁医院（青岛）麻醉科周金锋
病例简介 ?患者，男，52岁，76kg，身高177cm，术前诊断“上颌骨骨样骨瘤”。拟全身麻醉下行上颌骨肿瘤切除术。 ?既往史：患者自诉偶有劳累时发作性胸闷，发作不规律，数分钟后可自行缓解，病程超过一年，具体不详，未进一步诊治，近3月内未再发； ?心功能I级，日常活动未受限； ?否认高血压、糖尿病、肾病、肝病等病史； ?心电图、胸片、心超、肺功能等辅助检查及血常规、凝血谱、血生化、两便常规等实验室检查均正常； ?麻醉术前评估ASAI-II级。
？ ?是否需要进一步完善术前检查？?术中监测？ ?有无特殊注意事项？
?入室后常规监护，II导联心电监护提示窦性心律，心率78次/min；血压149/91mmHg，血氧饱和度98%。 ?开放外周静脉通路，抗生素静滴后，予以乳酸林格氏液静滴维持。 ?麻醉诱导：丙泊酚（静安）120mg（约1.5mg/Kg）、舒芬太尼40ug、顺式阿曲库铵15mg（约0.2mg/Kg）缓慢静注，4min后气管插管控制通气。 ?插管后VT500ml，f 12bpm，气道压力33-35cmH2O，PetCO247mmHg,听诊双肺上叶哮鸣音，以右上肺为著，未闻及湿罗音。液体通畅，但输液肢体略肿胀，SPO2最低降至65%。心率102次/min，测血压为76/36mmHg。
？ ?怎么了？ ?首先要处理的问题？?药物？
?5min内心率渐升高至152次/min，袖带血压测不出； ?立即纯氧通气，给予沙丁胺醇气雾剂（万托林）气管内喷雾3次，去甲肾上腺素5ug iv，胺碘酮150mg （稀释至20ml，40ml/h）快速泵注；?同时再次确定导管无扭曲或阻塞，位置无误，但听诊发现双肺底有明显湿罗音； ?约10min后心率降至121次/min，血压 77/35mmHg。测体温36.8℃； ?快速建立有创压力监测，测ABP约75-80/35-40mmHg，CVP 11mmHg。
DOI ：10.3877/cma.j.issn.1674-0793.2010.04.022 基金项目：国家自然基金资助（30872446） 作者单位：510080n 广州，中山大学附属第一医院麻醉科 近年来，利用机体自身抗损伤机制和耐受性从而提高机体自身保护能力的观点日益受到人们关注。自1986年Murry 等[1]提出心肌缺血预处理（ischemic]preconditioning ，IPC ）的概念后，后续研究表明缺血预处理是机体的一种内源性保护机制。Przyklenk 等[2]首次在犬心脏缺血模型中发现，当局部冠状动脉接受缺血预处理后，可使远离该区域的心肌组织产生缺血耐受，从而产生保护作用。据此学者们提出了缺血预处理有脏器交叉保护效应的假设，后续的研究结果支持了此假设。如Gho 等[3]证实小肠或肾脏缺血预处理可诱导心脏缺血耐受；Oxman 等[4]发现给予大鼠下肢10nmin 的短暂缺血，可对随后的心肌缺血产生保护作用。据此，学者们 提出远程缺血预处理（ remotenischemicnpreconditioning ，RIPC ）或器官间预处理（inter-organnpreconditioning ）的概念：对远离缺血部位的器官或组织行短暂缺血预处理，可对缺血部位产生保护作用。随后发现肢体、胃肠道、肠系膜或肾脏的短暂性缺血预处理可以减轻长时间心肌缺血/再灌注（ischemia/reperfusion ，I/R ）所致的心肌损伤、心律失常和代谢紊乱等。本文就肠远程缺血预处理对心肌保护作用的研究进展作一综述。 一、肠远程缺血预处理的研究 自RIPC 的概念提出后，近年来以大鼠为研究对象的实验证明肠RIPC 能从组织水平减少心肌I/R 所致 心肌梗死的面积，见表1。有研究[3，5-9]表明单次循环肠系膜上动脉夹闭 （mesentericnarterynocclusion ，MAO ）15nmin 介导的RIPC 以及随后预处理小肠的再灌注减少了心肌梗死面积。有研究表明，由多次循环MAO 介导的RIPC 同样减少心肌梗死面积[10，11]；Pateln 等[9]发现单次循环RIPC 比多次循环RIPC 更为有效。Wang 等[12]还证实即使在诱导心肌缺血24nh 后其仍有延迟相心肌保护作用。这些研究共同为MAO 介导的RIPC 效应提供了证据。研究者通常把组织学作为观察终点，而并未对髓过氧化物酶（MPO ）活性或心肌肌酸激酶水平等其 ·讲座与综述· 肠远程缺血预处理在心肌缺血/再灌注 损伤中的研究进展 温仕宏姚溪刘克玄 研究者 缺血预处理的位置诱导缺血位置模型终末点器官保护作用可能机制Gho 等[3] 肠系膜和肾心肌缺血大鼠梗死面积减少梗死面积神经、体液因素Schoemaker 等[5] 肠系膜心肌缺血大鼠梗死面积减少梗死面积缓激肽介导和神经通路Liem 等[8] 肠系膜心肌缺血大鼠梗死面积减少梗死面积增加间质的腺苷水平；神经刺激；心肌腺苷受体的激活Patel 等[9] 肠系膜心肌缺血大鼠梗死面积减少梗死面积内源性阿片类物质Wolfrum 等[6] 肠系膜心肌缺血大鼠梗死面积减少梗死面积通过体液缓激肽途径和神经通路激活心肌PKC Wolfrum 等[7] 肠系膜心肌缺血大鼠梗死面积减少梗死面积降钙素基因相关肽Tangn 等[10] 肠系膜心肌缺血大鼠梗死面积减少梗死面积辣椒素敏感性感觉神经Xiao 等[11] 肠系膜心肌缺血大鼠梗死面积减少梗死面积辣椒素敏感性感觉神经和NOS Wang 等[12] 肠系膜心肌缺血大鼠梗死面积减少梗死面积iNOS 的作用Petrishcev 等[13] 心肌和肠系膜心肌缺血大鼠梗死面积减少梗死面积非NO 机制Vlasov 等[14] nn nn 肠系膜心脏和肠大鼠梗死面积没有心肌保护作用，只产生小肠特殊适应不是远程预处理而是直接预处理产生的NO nn nn Liem 等[15] 肠系膜心肌缺血大鼠梗死面积减少梗死面积肠系膜缺血腺苷依赖性途径Huda 等[16]肠系膜心肌缺血 大鼠梗死面积减少梗死面积基因表达的改变表1 短暂性肠系膜缺血介导RIPC 的相关研究
宣威市人民医院 心脏起搏器植入诊疗应急预案 一、总则 1.各项手术前必须做好充分的准备，包括设备的运行情况、物品的准备、抢救药品的准备 等。 2.术者应于术前充分考虑患者手术的风险性，制定出详实的手术方案，做好应急的特殊物 品的准备和心理准备。 3.一旦出现紧急意外情况按照如下流程进行抢救工作： ⑴第一时间，术者为主要负责人（有特殊安排者除外）指挥现场人员进行抢救，抢救效 果好的，待患者病情稳定后，据情况继续手术或终止； ⑵如在场人员自己处理有困难，应立即提出紧急会诊要求，邀请相关科室人员协助抢救， 同时通知医务处； ⑶如有必要，应报告给主管院长、院长； ⑷及时与患者家属沟通，通报患者病情和抢救情况，以征得家属理解； ⑸抢救过程中要有专人负责记录医嘱和患者的病情变化情况，当时记录不全的抢救结束 后立即补记。 ⑹处理完后召开讨论会，分析出现紧急情况的原因、抢救是否及时、存在的问题，从中 得出教训、以及改进方案等，并详细记录备案。 4、本应急预案适用于心内科开展的冠脉介入手术、起搏器安置与及心内电生理检查与射频 消融术、先心病介入封堵术等心脏病介入手术。 二、实施细则
1、介入诊疗术中可能因导管钢丝等器械引起血管或心肌穿孔导致心包积液，需要立即行心包穿刺和引流。 2、介入器械、药物干扰心脏敏感区域可能引起或加重心律失常，甚至出现危及生命的室速、室颤或心脏停搏），情况危急时需要药物、电复律或起搏器植入。 3、病情和病变性质决定介入术的策略，有时于介入术中临时改变计划，会向家属交待。 4、介入术中心脏、血管内血栓形成或脱落，以及器械的断裂或脱落可能造成血管阻塞，引起心肌梗死、中风、肺栓塞或其他部位缺血，情况危急时会考虑中断手术。 5、部分介入术需联合应用抑制血液凝固的药物，可能诱发或加重出血，包括伤口、消化道、腹膜后、胸腔、眼底、颅内出血等，严重出血时需要输血、压迫、腔镜、外科手术等处理。 6、患者可能对消毒剂、对比剂或其他药物、材料过敏，出现皮疹、头晕、呼吸困难、休克、溶血等，予相应处理，情况不能控制应终止手术。 7、急诊介入治疗过程会出现再灌注综合征如：血压下降、室性心律失常、心动过缓等，可予多巴胺、利多卡因、阿托品等静推。 血压升高应急预案 【抢救流程】 严密监测患者血压等生命体征 ↓ 给予患者心理安慰，消除其紧张情绪，必要时予镇静 ↓ 尽可能排除引起患者血压升高的因素（焦虑、紧张等）
心肌缺血再灌注损伤机制的研究进展 摘要急性心肌梗死是临床常见急症重症，及时、有效的恢复心肌的血液灌注,挽救“濒死”的心肌是抢救成功的关键，因此探索缺血再灌注损伤的机制，减轻或防止再灌注损伤的发生，是临床的重要课题。本文综述了心肌缺血再灌注损伤发生机制研究领域的最新进展。 关键词心肌缺血再灌注；氧自由基；钙超载；中性白细胞；血管内皮细胞；一氧化氮；细胞黏附因子；细胞凋亡 急性心肌梗死（AMI）是临床常见急症重症，及时、有效的恢复心肌的血液灌注,挽救“濒死”的心肌是抢救成功的关键。探索心肌再灌注损伤(MRI)的机制，减轻或防止再灌注损伤的发生，是临床的重要课题。至今为止MRI的机制还没有完全清楚，目前主要认为与氧自由基、钙超载、活化的中性白细胞、心肌纤维能量代谢障碍、血管内皮细胞、一氧化氮、细胞黏附因子和细胞凋亡等都可能参与MRI的发病过程。[1、2] 1氧自由基与心肌缺血再灌注损伤 生理情况下，细胞内存在的抗氧化物质可以及时清除自由基，对机体并无有害影响。当组织细胞缺血、缺氧时，由于活性氧生成过多或机体抗氧化能力不足，可引起氧化应激反应，造成膜流动性与钙离子通透性增加,破坏膜结构完整性,钙跨膜内流与超负荷导致细胞损伤甚至死亡。氧化应激是缺血组织再灌注的特征之一。而且应用自由基清除剂辅酶Q10[3]可以减轻缺血再灌区细胞的损伤。 2钙超载与心肌缺血再灌注损伤 近年研究表明,细胞内Ca2+超载在心肌缺血再灌注损伤发病机制中起中心作用。钙超载可以造成线粒体功能障碍，激活磷脂酶类，使细胞膜及细胞器膜结构受到损伤。还可激活蛋白酶，促进细胞膜和结构蛋白的分解，同时促进氧自由基的生成。激活某些ATP酶和核酶，加速ATP消耗，引起染色体损伤。Ca2+超载还可引起再灌注心律失常。心肌缺血再灌注损伤的始动环节是能量代谢障碍,而直接损伤原因则是自由基,其结果导致细胞内钙超载,并形成恶性循环。钙超载
气管插管全麻技术风险评估及应急预案 气管插管全麻技术主要有以下并发症：呼吸道阻塞、通气量不足、低氧血症、低血压、高血压、窦性心动过缓或过速、心肌缺血、误吸、恶性高热、术后瞻妄。针对以上各并发症的发生可能，特制定以下处置方案： 1、呼吸道阻塞 （1）上呼吸道阻塞：阻塞部位在喉头以上 原因：机械阻塞的原因有舌后坠、口腔内分泌物及异物阻塞、喉头水肿等。性能性原因有喉痉挛。 预防及处理： ①全身麻醉下发生的呼吸道阻塞，其阻塞症状可不明显，因此应密切观察，麻醉恢 复期的护理更为重要。 ②舌后坠时可将头后仰，托起下颌或置入口咽通气道。 ③吸除口咽部分泌物，将病人头转向一侧，有利于分泌物的流出。 ④喉头水肿多发生于婴幼儿及气管导管插进困难者，遇此情况，可预防性注射氢化 可的松0.5—1.0mg/kg：术后发生喉头水肿者除吸氧、激素治疗外，严重者尚需行气管切开。 ⑤轻度喉痉挛者可加压给氧，严重者可经环甲膜穿刺置进粗针头行加压给氧，多数 均可缓解。对上述处理无效或严重喉痉挛下刺激后喉头；采用硫喷妥钠麻醉或行尿道、宫颈扩张等手术时，应给予阿托品0.3-0.5mg，预防喉头副交感神经张力增高。 （2）下呼吸道阻塞：阻塞部位在喉头以下者。 原因：机械性阻塞最常见原因是气管导管扭折、导管斜面过长而紧贴在气管壁上、粘痰或呕吐物误吸堵塞气管及支气管。性能型原因：由于支气管痉挛，多见于浅麻
醉时、支气管内异物、炎症刺激、肌松药的组胺开释作用以及支气管哮喘者。 预防及处理： ①仔细挑选气管导管，过软或分歧格者应丢弃。 ②经常听诊肺部，及时清除呼吸道内的分泌物。 ③维持适当麻醉深度，预防及解除支气管痉挛的诱因。保持麻醉深度及氧合（通气） 适当是缓解支气管痉挛的重要措施，必要时可静注氨茶碱0.25mg或氢化可的松100mg。 2.通气量不足: (1)原因： ①麻醉药对呼吸中枢的抑制；肌松药对呼吸肌的麻痹而辅助呼吸及控制呼吸又不充分者。 ②吸进麻醉药残存0.1MAC时仍可抑制缺氧—通气反应，致麻醉恢复期通气不足。 ③麻醉恢复期肌松药的残存作用。 ④术中过度通气2小时可消耗近3L的co2储备，术后机体则需降低通气量以补充所消耗的co2，故通气不足，且可导致低氧血症 ⑤术中所有的麻醉性镇痛药常为术后呼吸抑制的重要原因，尤以高龄、肥胖者为然。 ⑥麻醉期间发生通气不足时，主要表现为O2潴留；而恢复期发生通气不足，除O2潴留外，还可发生底氧血症，而后者的威胁尤甚。 （2）预防及处理： ①辅助呼吸或控制呼吸应适当，避免通气不足或长时间过度通气。 ②加强围术期病人的呼吸功能检测，尤其对高龄、肥胖等“高危”病人。 ③严格把握拔除气管导管的指征，即呼唤病人可睁眼、抬头、握拳、用力吸气压力可达2.9kPa（-30cmH20）、TOF中T4∕和T1＞75
大鼠心肌缺血/再灌注损伤 【实验目的】 1.复制大鼠在体与离体心肌缺血/再灌注损伤模型； 2.观察缺血/再灌注过程中心功能的变化 【实验动物】成年Wistar 大鼠（体重200－300g） 【仪器药品】 电子天平，肾形盘，动物呼吸机，BL-420F记录装置，眼科开睑器，微血管钳，组织镊，眼科镊，组织剪，眼科剪，眼科止血钳，止血钳，动脉夹，眼科缝合针，1号及00缝合线。Langedroff灌流装置。 20%乌拉坦，1ml注射器，5ml 注射器，纱布块 实验1 在体模型 【实验步骤】 1.实验采用体重200-300ｇ健康雄性Wistar大鼠，20%乌拉坦腹腔注射麻醉（0.5ml/100g）； 2.颈胸部备皮及手术，分离气管及右侧颈总动脉 3.气管插管连接呼吸机（呼吸肌参数：潮气量9ml，呼吸比=3:2，呼吸频率55~60） 4.经右侧颈总动脉逆行插管至左心室, 再经BL-420F软件输入计算机,（一通道描记心 电，（右上黄、右下黑、左下红）二通道描记心室内压，三通道描记微分）持续监测心脏左心室内压力及心电的变化情况 5. 沿胸骨左侧剪开2，3肋骨，开睑器开胸暴露心脏；寻找冠状动脉左前降支，穿线备 用; 6.采用结扎5min后再放开5min两次,造成缺血预处置；采用结扎30mim再放开30min 复制缺血/再灌注模型； 思考题： 1.如何判定缺血模型复制成功 2.如何判定有再灌注损伤发生
实验2 离体模型 【实验步骤】 (1) 大鼠称重，腹腔注射20%乌拉坦（0.5ml/100g）麻醉，仰卧固定于鼠板，上腹部及前胸部剪毛。 (2) 舌下/阴茎背静脉注入1%肝素（0.05ml/100g）后，切开胸腹部皮肤，用剪刀横行剪开腹腔，向上剪断隔膜，沿两侧肋骨向上平行剪开，翻起前胸壁，把心脏及胸膈周围的结缔组织拨到一侧，充分暴露心脏。 (3) 用镊子提起心脏根部，暴露出主动脉和肺动脉，在距主动脉起始部0.5cm处用手术剪切断血管，迅速取出心脏至于4℃生理盐水平皿中使之停搏。 (4) 经主动脉将心脏悬挂在灌流装置上，用丝线结扎固定，打开灌流液行逆向灌流，待心脏恢复自主跳动，小心减去心脏周围附着组织。 (5)用眼科剪剪去左心耳，通过左心耳经房室瓣插入左心室一乳胶球囊，球囊连接一个内充生理盐水的导管，导管经三通管和换能器与BL-420F连接。 (6) 在BL-420F仪的监测下，通过向球囊内注入一定量的生理盐水是左心室的舒张末压调整在0~10mmHg之间。 (7)连接心电导线，心尖、右心耳和地线，一通道设置记录， (8) 预灌流10~20分钟，观察心率，二通道记录心室内压、三通道取微分记录±dp/dtmax 等心动指标，同时描记ECG，待上述各指标平衡后开始以下实验。 心肌缺血-再灌注损伤 (1) 心脏用正常灌流液预灌流15分钟后完全停灌40分钟，然后恢复灌流20分钟，观察心脏在正常，停灌初期和再灌期的心功能变化。 (2) 分别收集正常灌流时，再灌流后3分钟时的心脏冠脉流出液1ml，测定其中乳酸脱氢酶的活性。 思考题： 1.如何判定缺血模型复制成功 2.如何判定有再灌注损伤发生
缺血性心脏病是导致人类死亡的主要原因，在治疗上，早期成功恢复心肌再灌注是改善临床转归的最有效方法。但缺血心肌恢复血流的过程可造成损伤，这一现象称为心肌缺血/再灌注损伤(myocardial ischemia/reperfusion injury,MI/RI)［1 2］。而氧自由基（oxygen free radical,OFR）也是心血管疾病时诱导心肌细胞死亡的重要因素之一［3］。在正常生理条件下，细胞内存在抗氧化物质可以及时清除OFR，使自由基的生成与降解处于动态平衡，对机体无害，而在心肌缺血再灌注损伤情况下，由于OFR生成过多或机体抗氧化能力不足，引发氧化应激反应，介导心肌损伤［4 5］。本研究重点阐述OFR与心肌缺血再灌注损伤之间的关系。 1 OFR合成、清除及生物学作用 自由基（free radical）是指具有一个不配对电子的原子和原子团的总称。由氧诱发的自由基称为OFR，主要包括超氧阴离子(O-2)、过氧化氢(H2O2)和羟自由基(OH)［6］。H2O2本身并非自由基而是一种活性氧(reactive oxygen species,ROS)，但它与OFR的产生有密切关系，易接收一个电子生成羟自由基（OH）。正常情况下OH不能形成，因为OH的形成要求O-2及H2O2同时存在。当O-2及H2O2在组织中过剩, O-2及H2O2在金属离子及金属离子复合物的催化下发生Haber Weiss反应，生成氧化性更强的OH。OH是十分不稳定的氧化物，几乎与细胞内所有的有机物反应，破坏核酸、蛋白质、氨基酸和脂类化合物，从而损害细胞功能［7］。在生理情况下，氧通常是通过细胞色素氧化酶系统接收4个电子还原生成H2O，同时释放能量，但也有1%～2%的氧接收1个电子生成O-2，或再接收1个电子生成H2O2。O-2寿命极短，可通过连锁反应产生OH，H2O2能直接或间接促进细胞膜脂质过氧化。 自由基反应的扩展较广，但生物体内存在一套完整的抗氧化酶和抗氧化剂系统，可以及时清除它们，所以对机体无害。抗氧化酶包括超氧化物歧化酶（SOD)、谷胱甘肽过氧化物酶（GSH PX)和过氧化氢酶(CA T)。它们存在于胞浆和线粒体中，其重要意义在于降低H2O2浓度，保护细胞不受强毒性OFR OH的损伤。抗氧化剂包括存在于细胞质的维生素E 和维生素A；细胞外液中的半胱氨酸、抗坏血酸、谷胱甘肽；存在胞浆中的还原型谷胱甘肽(GSH)和还原型病理辅酶Ⅱ(NADPH)等。在OFR清除系统功能降低或丧失,生成系统活性增强，一旦恢复组织血液供应和氧供,OFR便大量产生与急剧堆积，从而造成心肌细胞急性或慢性损伤［8］。特异靶向抑制NADPH氧化酶可以减弱心血管氧化应激［9］。 2 OFR在心肌缺血再灌注损伤中的作用及地位 目前关于心肌缺血再灌注损伤的发病机制有许多假设和报道，主要与心肌再灌注时与OFR损伤、细胞内Ca2+超载、心肌细胞能量代谢障碍［10］、微血管损伤和粒细胞浸润以及心肌细胞的凋亡等作用有关。MI/RI时OFR合成增多主要与线粒体单电子还原、黄嘌呤氧化酶形成增多、儿茶酚胺自氧化增强、细胞内钙超载以及中性粒细胞呼吸暴发等有关［11］。由于OFR产生过多以及抗氧化酶类活性下降，引发链式脂质过氧化反应，损伤细胞膜、细胞器乃至细胞核酸，导致细胞坏死凋亡。应用外源性OFR清除剂及抗氧化剂则能降低组织中OFR浓度，促进心功能恢复，表明OFR在心肌缺血再灌注损伤中起着重要作用。 3 OFR与脂质生物膜
? 文献综述 ? 63 心肌缺血再灌注损伤（myocardial ischemic reperfusion in j ury ，MIRI ）指心肌缺血恢复血流供应后，造成代谢功能障碍及结构损伤加重的现象[1]。MIRI 是临床上常见的疾病，其病理过程与冠状动脉血管形成术，冠状动脉重建术，心脏移植等术后并发症密切相关[2]。MIRI 涉及的机制复杂，尚有待更深入的研究阐述。近年来，由于电生理学、基因组学和蛋白组学等技术的应用，对MIRI 机制的研究也获得了一定的进步，其主要机制概述如下：1 氧自由基与MIRI 自由基（free radical ），又称游离基，指在外层电子轨道上具有不配对的单个电子、原子、原子团或分子的总称[3] 。由机体内氧诱发化学性质活泼的自由基称为氧自由基，包括羟自由基和超氧阴离子。生理状态下自由基存在较少，在细胞缺血时，其氧自由基清除能力下降[4]。当组织恢复血液供应时，触发氧自由基“爆增”并累积，攻击自身和周围细胞，造成损伤[5]。自由基损伤细胞膜，致其结构破坏造成心肌酶溢漏；自由基氧化破坏机体蛋白，改变蛋白酶表面结构使功能受损；自由基诱导遗传物质DNA 、RNA 断键或破损，影响核酸正常功能[6]。自由基可导致心律失常，心肌损伤，细胞凋亡等事件[7]。2 炎症反应与MIRI MIRI 发生时心脏组织内皮结构受损触发功能障碍，而中性粒细胞趋集、黏附血管内皮是炎症“级联”反应的诱发阶段[8] 。激活的中性粒细胞合成释放肿瘤坏死因子、IL-1、IL-6 等炎症介质，介导其他炎症细胞共同攻击心肌组织[9] 。此外，白细胞浸润在MIRI 中涉及的主要机制为，MIRI 使细胞膜受损和膜磷脂降解，具有很强趋化作用的白三烯等代谢产物增多，使更多白细胞循环浸润，对心肌细胞造成多次损伤。MIRI 时，心肌缺血细胞生成大量的促炎介质如补体C 5a 、LPS 、IL-8等，激活并诱导心肌细胞多种黏附如ICAM-1，ICAM-2等分子表达[10]。膜表面的黏附分子作为受体和配体介导白细胞与内皮细胞、心肌细胞的黏附，并为炎性浸润提供物质基础。3 钙超载与MIRI 由于细胞内钙浓度显著升高并造成心脏功能代谢障碍的现象称为钙超载（Ca 2+ 超载）[11] 。生理条件下，钙浓度稳态维持着正常心功能。当心肌缺血时，钠泵功能障碍，Na + 与Ca 2+ 的交换紊乱，使细胞内Ca 2+大量积累，触发线粒体功能障碍、钙泵障碍等[12]。Ca 2+超载与细胞损伤有相关性。其可引起：①减少线粒体ATP 生成。②激活钙依赖性降解酶，损伤细胞结构。③诱导自由基生成，损害心肌细胞。④促使 Ca 2+与CaM 结合，影响细胞内信号转导。⑤引起心律失常。 4 能量代谢障与MIRI MIRI 发生时，心肌细胞依赖无氧代谢途径供能，但其生成ATP 的能力有限。而ATP 的明显不足会触发一系列代谢的异常和紊乱：①依赖性ATP 的细胞膜泵活性下降，膜电位改变。②Ca 2+内流增加，激活膜磷酶导致缺血性肌挛缩，并产生氧自由基进一步损害细胞。③酸中毒，破坏细胞的生存环境。④严重阻碍ATP 的生成[13]。研究表明，能量代谢障碍可造成有关基因及蛋白表达的异常，同时细胞内的ATP 含量是触发细胞凋亡促进因素之一。5 细胞凋亡与MIRI 细胞凋亡，又称程序性细胞死亡，指由促凋亡因素触发细胞内死亡程序而发生的细胞死亡过程[14]。细胞凋亡调控着机体中细胞稳态，并摒除体内有害的细胞、无功能的细胞、突变的细胞以及受损的细胞。而过度活跃的细胞凋亡进程会加重MIRI 病情。MIRI 中的细胞凋亡的机制涉及的凋亡途径多种途径，以多方式、多水平的交叉联系，构成复杂的信号通路网络。线粒体途径、细胞因子信号转导途径、JAK-STAT 途径、LOX-1通路、MAPKs 通路等均可介导心肌MIRI 发生发展，造成的心肌细胞凋亡。提示抗凋亡作用或特异性对抗有关信号通路是治疗MIRI 的有效措施之一。6 小 结 综上所述，心肌缺血再灌注损伤（MIRI ）的发生机制涉及多因素的复杂过程，需要广大科研攻关者更全面、更深入的科学研究，积极寻求更有效的防治措施，为MIRI 造福。近年来，随着科学技术的不断发展，在基因调控、细胞凋亡、信号转导等角度的深层次研究也在逐步开展，期待对MIRI 机制研究取得重要的突破。 参考文献 [1] 赵亚玲,敖虎山.心肌缺血再灌注损伤的研究进展[J].中国循环杂 志,2011,26(5):396-398. [2] C astedo E,Segovia J,Escudero C,et a1.Ischemia-reperfusion in j ury during experimental heart transplantation. Evaluation of trimetazidine's cytoprotective effect[J].Rev Esp Cardiol. 2005,58(8):941-950. [3] C hen AF,Chen DD,Daiber A,et a1.Free radical biology of the cardiovascular system[J].Clin Sci (Lond),2012,123(2):73-91.[4] V al ko M,Leibf r itz D,Moncol J,et a1.Free radicals and antioxidants in normal physiological functions and human disease [J].Int J Biochem Cell Biol, 2007,39(1):44-84. [5] D r?ge W.Free radicals in the physiological control of cell function[J].Physiol Rev, 2002,82(1):47-95. [6] 林灼锋,李校坤,孟娟.活性氧自由基对心肌细胞损伤效应研究[J]. 心肌缺血再灌注损伤的机制研究进展 邓海英* 赖为国 （钦州市第二人民医院药剂科，广西 钦州 535099） 【摘要】冠心病严重危害人类的生命健康，主要临床表现为心绞痛或心肌梗死。心肌缺血后再获取血液供应，常会出现心律失常、梗死面积扩大、心功能低下等心肌细胞损伤现象，即心肌缺血再灌注损伤（MIRI ）。国内外研究表明MIRI 发生机制较为复杂，目前认为与再灌注后机体氧自由基攻击，炎症反应浸润，Ca 2+超载，能量代谢障碍、细胞凋亡进程等有关。现对MIRI 的机制及治疗的研究进展综述如下。本文通过归纳并总结有关MIRI 研究进展的国内外文献，对MIRI 的机制做出综述。【关键词】心肌缺血再灌注；损伤；机制 中图分类号：R542.2 文献标识码：A 文章编号：1671-8194（2013）01-0063-02 *通讯作者:E-mail: denghaiying2012@https://www.doczj.com/doc/075684721.html,
心肌缺血再灌注损伤采用缺血预处理和后处理的相关作用和机制研 究 目的探究缺血预处理和后处理在心肌缺血再灌注损伤时的作用及其机制。方法选取100只雄性大鼠，将其平均分为对照组、缺血再灌注组、缺血再灌注预处理组、缺血再灌注后处理组、缺血再灌注预处理和后处理组，测定血清中乳酸脱氢酶、肌酸激酶含量，估算心肌梗死的面积大小，同时检测丙二醛含量和组织髓过氧化物酶的活性。结果血清乳酸脱氢酶和肌酸激酶含量在缺血再灌注组中明显升高；丙二醛在缺血再灌注组明显升高而在缺血预处理和后处理组中含量较低；组织髓过氧化物酶在缺血再灌注组明显降低而在缺血预处理和后处理组中含量显著升高。结论缺血再灌注预处理和后处理对心肌均有保护作用，但预处理和后处理并不能协同保护，这说明预处理和后处理组之间的信号传导机制可能相同。 标签：心肌缺血再灌注；缺血预处理；缺血后处理；含量 心肌缺血损伤是由于心肌缺氧及营养成分导致心肌细胞的暂时性功能缺损或坏死[1]，而缺血再灌注造成的损伤则是由于氧和受损心肌细胞或者坏死心肌细胞的反应导致氧自由基对心肌存在损伤作用[2]。主要表现心律失常、心室收缩力下降等不良后果，给人们的生命安全带来巨大威胁。曾有报道显示，缺血预处理可以使冠状动脉在多次短暂缺血后增加心肌对之后一段时间内缺血的耐受性，它是一种内源性的保护机制[3]。而缺血后处理是指当心肌再灌注发生时，出现多次短暂的停灌、复灌，同样具有对心脏的保护作用[4]。为探究缺血预处理和后处理在心肌缺血再灌注损伤时的作用及其机制，笔者采用回顾性分析的方法，选取100只雄性大鼠，将其平均分为对照组、缺血再灌注组、缺血再灌注预处理组、缺血再灌注后处理组、缺血再灌注预处理和后处理组，现总结报道如下。 1 资料与方法 1.1 一般资料选择雄性大鼠100只（Wister大鼠），体重为（275±25）g，将其平均分成5组，分别为对照组、缺血再灌注组、缺血再灌注预处理组、缺血再灌注后处理组、缺血再灌注预处理和后处理组，编号为1～5。每组鼠的体重、年龄、身体情况均无显著差异。 1.2 药品及器材20%乌拉坦、注射器、气管插管装置、动物呼吸机、心电监护仪、手术刀、止血钳、手术剪、缝合线、弯针、1%TTC磷酸缓冲液。 1.3方法用20%的乌拉坦对大鼠进行腹腔麻醉（6mL/Kg），将麻醉好的大鼠背部固定，对大鼠进行气管插管并连接于动物呼吸机上，之后连接心电监护仪，密切监视心电图变化[5]。用手术剪剪开大鼠胸腔暴露心脏，之后小心剪开心脏包膜，在做信儿和肺动脉圆锥的中间，用穿有缝合线的弯针结扎左冠状动脉前降支，將一带有凹槽的乳胶管放置于结扎线和左冠状动脉前降支之间，使之心肌缺