参附注射液通过调控自噬减轻大鼠心肌缺血再灌注损伤的作用研究

目的:探究参附注射液(SFI)通过调控自噬减轻大鼠心肌缺血再灌注损伤(MIRI)的作用及机制。方法:(1在体实验:将大鼠分为正常对照组、心肌缺血再灌注模型(I/R)组、SFI(5、10和20 mg/kg)组。大鼠采用结扎冠状动脉前降支法再灌注4 h建立心肌缺血再灌注模型。试剂盒检测心肌损伤标记物肌酸激酶(CK)、乳酸脱氢酶(LDH)、肌钙蛋白(c TnⅠ)浓度,检测心肌梗死区域百分比,HE检测心肌组织病理变化,试剂盒检测血清氧化应激分子超氧化物歧化酶(SOD)和丙二醛(MDA)水平以及血清IL-6、IL-1β和肿瘤坏死因子α(TNF-α)的浓度,Western blot检测自噬相关蛋白LC3、Beclin1、p62以及磷酸化磷脂酰肌醇3-激酶(p-PI3K)、PI3K、磷酸化蛋白激酶B(p-Akt)、Akt、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)和m TOR的表达水平。(2)离体实验:培养大鼠心肌细胞,经过25、50、100μl/ml的SFI预处理后通过缺糖缺氧/复糖复氧法(OGD/R)诱导心肌细胞损伤,MTT检测细胞活性,试剂盒检测SOD和MDA浓度,Western blot检测LC3、Beclin1和p62的表达;20 nmol/L雷帕霉素处理细胞1 h,Western blot检测p-mTOR和LC3的表达,MTT检测细胞活性。结果:①在体实验:与正常对照组比较,I/R组CK、c TnⅠ和LDH表达量升高(P<0. 01),心肌梗死面积和组织病理变化显著增加(P<0. 01),SOD活性显著降低(P<0. 01),MDA浓度显著升高(P<0. 01),TNF-α、IL-6、IL-1β表达量显著升高(P <0. 01),LC3Ⅱ/LC3Ⅰ和Beclin1表达水平增高(P<0. 01),p62表达水平降低(P<0. 01),p-PI3K/PI3K显著降低(P<0. 01);与I/R组比较,SFI(10、20 mg/kg)组CK、c TnⅠ和LDH表达量降低(P<0. 01),心肌梗死面积和组织病理变化显著减轻(P <0. 05,<0. 01),SOD活性显著升高(P <0. 05,P <0. 01),MDA浓度显著降低(P<0. 05,P<0. 01),TNF-α、IL-6、IL-1β表达量显著降低(P<0. 05,P<0. 01),LC3Ⅱ/LC3Ⅰ和Beclin1表达水平降低(P<0. 05,P<0. 01),p62表达水平升高(P<0. 05,P<0. 01),p-PI3K/PI3K、p-Akt/Akt、p-mTOR/m TOR显著升高(P<0. 05,P<0. 01)。②离体实验:与细胞对照组相比,心肌细胞缺糖缺氧复糖复氧(OGD/R)组心肌细胞活性显著降低(P<0. 01),SOD活性显著降低(P<0. 01),MDA浓度显著升高(P<0. 01),LC3Ⅱ/LC3Ⅰ、Beclin1表达水平显著升高(P<0. 01),p62表达水平显著降低(P<0. 01);与OGD/R组比较,SFI 50和100μl/ml组心肌细胞活性提高(P<0. 05,P<0. 01),SOD活性显著升高(P<0. 05,P<0. 01),MDA浓度降低(P<0. 05,P<0. 01),LC3Ⅱ/LC3Ⅰ和Beclin1表达水平降低(P<0. 05,P<0. 01),p62表达水平显著升高(P<0. 05,P<0. 01)。与未经雷帕霉素处理的SFI (100μl/ml)组比较,SFI+雷帕霉素LC3Ⅱ/LC3Ⅰ显著升高(P<0. 01),细胞活性显著降低(P<0. 01)。结论:SFI能通过调控自噬作用减轻大鼠心肌缺血再灌注损伤,作用机制与PI3K/Akt/m TOR信号通路激活有关。

Effect of Shenfu injection on alleviating myocardial ischemia-reperfusion injury in rats by regulating autophagy

Objective:To explore the role and mechanism of Shenfu injection(SFI)in alleviating myocardial ischemia-reperfusion injury(MIRI)in rats by regulating autophagy.Methods:(1)In vivo experiment:rats were divided into normal control group,myocardial ischemia reperfusion model(I/R)group,Shenfu injection group SFI(5,10,20 mg/kg).Rat model of myocardial ischemia reperfusion was established by ligate the anterior descending coronary artery,the kits were used to detect the concentrations of myocardial injury markers,including creatine kinase(CK),lactate dehydrogenase(LDH),cardiac troponinⅠ(cTnⅠ),myocardial infarction percentage were measured,the pathologic changes of myocardial tissue were observed by HE staining,serum oxidative stress molecules including superoxide dismutase(SOD)and malondialdehyde(MDA),serum inflammatory factors including interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor alpha(TNF-α)were detected by kits,Western blot was used to determine the protein levels of LC3,Beclin1,p62,p-PI3K,PI3K,p-Akt,Akt,p-mTOR,mTOR in vivo.(2)In vitro experiment:rat cardiomyocytes were cultured and pretreated with 25,50,100μl/ml SFI,then cardiomyocyte injury induced by oxygen and glucose deprivation/reoxygenation,cell viability were detected by MTT;SOD and MDA were detected by kits;Western blot was used to determine the expression levels of LC3,Beclin1,p62 in vitro.Cardiomyocyte were treated with Rapamycin,then the expression of p-mTOR and LC3 were determined by Western blot;cell viability were detected by MTT.Results:(1)In vivo experiments:compared with the control group,the concentrations of CK,cTnⅠ,LDH in I/R group were significantly increased(P<0.01),the infarction area and the pathological changes of myocardial tissue were significantly more serious(P<0.01),the activity of SOD were significantly decreased,the concentrations of MDA were significantly increased(P<0.01),the concentration of TNF-α,IL-6,IL-1βwere significantly increased(P<0.01),The ratio of LC3Ⅱ/LC3Ⅰand the protein levels of Beclin1 were increased(P<0.01),the protein levels of p62 were decreased(P<0.01),the ratio of p-PI3K/PI3K were significantly decreased(P<0.01).Compared with the I/R group,the concentration of CK,cTnⅠ,LDH in SFI(10,20 mg/kg)group were decreased(P<0.01),the infarction area and the pathological changes of myocardial tissue were significantly milder(P<0.05,P<0.01),the activity of SOD were significantly in increased(P<0.05,P<0.01),the concentrations of MDA were significantly decreased(P<0.05,P<0.01),the concentration of TNF-α,IL-6,IL-1βwere significantly decreased(P<0.05,P<0.01),the ratio of LC3Ⅱ/LC3Ⅰand the protein levels of Beclin1 were decreased(P<0.05,P<0.01),the protein levels of p62 were increased(P<0.05,P<0.01),the ratio of p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR were significantly increased(P<0.05,P<0.01).(2)In cytological experiments:compared with the control group,the cell viability of oxygen and glucose deprivation/reoxygenation(OGD/R)group were significantly decreased(P<0.01),the activity of SOD were significantly decreased(P<0.01),the concentration of MDA were significantly increased(P<0.01),the ratio of LC3Ⅱ/LC3Ⅰ,protein levels of Beclin1 were significantly increased(P<0.01),the protein levels of p62 were significantly decreased(P<0.01).Compared with OGD/R group,the cell viability of SFI(50,100μl/ml)group were increased(P<0.05,P<0.01),the activity of SOD were significantly increased(P<0.05,P<0.01),the concentration of MDA were decreased,the ratio of LC3Ⅱ/LC3Ⅰand the protein levels of Beclin1 were decreased(P<0.05,P<0.01),the protein levels of p62 were significantly increased(P<0.05,P<0.01).In addition,The protein levels of p-mTOR in SFI(100μl/ml)group were inhibited by rapamycin.Compared with the SFI(100μl/ml)group without rapamycin treatment,the ratio of LC3Ⅱ/LC3Ⅰin SFI+rapamycin group were significantly increased(P<0.01),the cell viability were significantly decreased(P<0.01).Conclusion:SFI can attenuate myocardial ischemia-reperfusion injury in rats by regulating autophagy,and its mechanism is related to the activation of PI3K/Akt/mTOR signaling pathway.