七氟醚后处理对缺血再灌注大鼠心肌内质网应激反应的影响及可能机制  

Effects of sevoflurane postconditioning on myocardial endoplasmic reticulum stress response in a rat model of ischemia-reperfusion and underlying molecualr mechanism

作　　者：秦佳月 朱志鹏 周红梅 路建 吴城 QIN Jia-yue;ZHU Zhi-peng;ZHOU Hong-mei;LU Jian;WU Cheng(Department of Anesthesiology,The Second Affiliated Hospital of Jiaxing University,Jiaxing 314000,China)

机构地区：[1]浙江省嘉兴市第二医院麻醉科,嘉兴314000

出　　处：《浙江中西医结合杂志》2022年第11期1001-1007,共7页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

基　　金：浙江省医药卫生科技计划项目(No.2019ZD053)。

摘　　要：目的明确七氟醚后处理对缺血再灌注大鼠心肌损伤的保护作用,初步阐明其对心肌内质网应激反应的影响及可能靶点。方法取SPF级雄性SD大鼠55只,建模取25只,每组5只,分为对照组(sham)和缺血再灌注组(I/R),每组5只。I/R组分别于缺血30 min之后进行再灌注2、4、6和8 h,制造在体心脏左冠状动脉前降支结扎缺血再灌注动物模型,获取心肌损伤和内质网应激反应最理想模型参数。实验取大鼠30只,随机数字表法分为sham组、I/R组、七氟醚后处理组(sevoP,分为2.5%、3.0%亚组)、4-苯基丁酸(4-PBA)复合七氟醚组(4-PBA+2.5%、3.0%sevoP组)。sevoP组在行微创左冠状动脉前降支结扎后,即刻吸入相应浓度的七氟醚15 min后洗脱,松开结扎线恢复血流至额定时间,观测心肌HE形态,血清的乳酸脱氢酶(LDH)、肌钙蛋白(cTnI),以及组织的GRP78、CHOP mRNA和蛋白表达,sham组只放线不结扎。利用体外H9C2心肌细胞缺氧/复氧损伤模型(3H/3R),除正常对照组和H/R组外,sevoP组复氧前以2.5%七氟醚处理缺氧箱内H9C2细胞30 min,另两组H9C2细胞分别以小干扰肌浆网Ca^(2+)-ATP酶(siSERCA)和阴性对照(ncSERCA)处理48 h后进行sevoP同样的操作,检测上清液中的上述指标,以及细胞内质网应激蛋白指标。结果与sham组比较,I/R组大鼠心肌缺血0.5 h再灌注2 h后血清LDH[(5.62±0.68)U/L比(3.44±0.54)U/L,P<0.05]、cTnI[(5.64±1.01)ng/mL比(2.72±0.61)ng/mL,P<0.05]浓度的升高,GRP78蛋白表达到缺血0.5 h再灌注6 h后才增高明显[(2.36±0.49)比(0.38±0.18),P<0.05]。与Sham组比较,I/R组大鼠肺组织GRP78、CHOP的mRNA显著高表达(22.22±8.22比1.02±0.27,14.84±2.49比1.01±0.22,P均<0.05),蛋白表达也同样增加(0.59±0.11比2.06±0.36,0.44±0.12比1.97±0.42,P均<0.05)。七氟醚后处理能够有效抑制其高表达,4-PBA能够进一步减弱七氟醚的抑制作用。H9C2细胞的H/R损伤和内质网应激可以引起GRP78和CHOP的4倍高表达和细胞上清液�Objective To assess the protective effect of sevoflurane postconditioning on ischemic re-perfusion injury of myocardium in a rat model and the possible targets of the endoplasmic reticulum stress response.Methods A total of 55 Sprague-Dawley male rats in specific pathogen-free(SPF)condition were purchased.First25 rats were randomly divided into control(sham,n=5)and ischemic re-perfusion(I/R)groups(n=20)and the latter group of rats was re-perfused at 2 h,4 h,6 h and 8 h after 30 min after ischemia,respectively(n=5 per group).The model was established by ligation of the front left coronary artery.The optimal model parameters for myocardial injury and endoplasmic stress response were obtained.Next,the remaining 30 rats were randomly divided into sham,I/R,I/R+sevoflurane postconditioning low or high(sevoP;2.5%and 3%),I/R+4-phenybutyric acid(4-PBA)+sevoflurane groups(I/R+4-PBA+2.5%or 3%sevoP;n=5 per group).SevoP group of rats were given inhalation of the corresponding sevoflurane dose for 15 min and then loosening the ligation line to restore blood flow.The heart tissues were taken from rats for H&E staining and serum lactic acid dehydrogenase LDH(U/L)and troponin cTnI and tissue GRP78 and CHOP mRNA and protein levels were assessed.In addition,an in vitro hypoxia reoxygenation model(3H/3R)was also established by exposure of H9C2 cells to a hypoxia tank and then treated with 2.5%sevoflurane before reoxygenation in the sevoP group or with pre-incubation with small interference Ca^(2+)-ATP enzyme(siSERCA)and negative control(ncSERCA)for 48 h.The above-mentioned serum parameters and expression of GRP78 and CHOP were detected.Results Compared with the sham group of rats,serum LDH and cTnI levels were significantly increased after 2 h re-perfusion in I/R rats(3.44±0.54 vs.5.62±0.68;2.72±0.61 vs.5.64±1.01;all P<0.05,respectively),while expression of GRP78 protein was also significantly induced after 0.5 h ischemia and 6 h re-perfusion(0.38±0.18 vs.2.36±0.49;P<0.05).Compared to the sham group,levels of GRP78 and CHOP mR

关 键 词：大鼠 七氟醚 内质网应激 缺血再灌注 H9C2细胞 细胞低氧 

分 类 号：R614[医药卫生—麻醉学]