三七总皂苷对心肌缺血再灌注损伤大鼠心肌ZO-1、Occludin、Cx43蛋白表达的影响

Effects of Panax Notoginseng Saponins on Myocardial ZO-1,Occludin and Cx43 in Rats with Myocardial Ischemia-Reperfusion Injury

目的探究三七总皂苷(PNS)对大鼠心肌缺血再灌注损伤(MIRI)的保护作用及机制。方法将40只SD雄性大鼠随机分为正常组、模型组、曲美他嗪组(对照组)、三七总皂苷组。正常组及模型组给予等体积生理盐水灌胃,曲美他嗪组给予5.4 mg·kg-1灌胃,三七总皂苷组给予50 mg·(kg·mL)-1腹腔注射。除正常组外,模型制备均采用冠状动脉左前降支结扎术。心电图检测ST段以评价造模情况;苏木素-伊红(HE)染色观察心肌组织损伤情况;比色法测定血清中肌酸激酶(CK)含量,酶联免疫吸附法(ELISA)测定血清中肌钙蛋白T(cTnT)、肌红蛋白(MYO)、白细胞介素1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量;蛋白免疫印迹法(Western Blot)检测闭锁小带蛋白-1(ZO-1)、紧密连接蛋白(Occludin)、缝隙连接蛋白43(Cx43)蛋白含量的表达。结果与正常组相比,模型组血清中CK、cTnT、MYO、IL-1β、IL-6、TNF-α含量显著增多(P<0.01);心肌细胞排列紊乱,存在炎性浸润,细胞核散乱无序,心肌纤维扭曲断裂;心肌组织中ZO-1、Occludin、Cx43蛋白表达均显著降低(P<0.01)。与模型组相比,三七总皂苷组与曲美他嗪组血清中cTnT、MYO含量均显著降低(P<0.01),三七总皂苷组CK含量显著降低(P<0.01),曲美他嗪组CK含量降低(P<0.05);三七总皂苷组血清中IL-1β、IL-6、TNF-α含量均显著降低(P<0.01),曲美他嗪组IL-1β、IL-6含量降低(P<0.05);三七总皂苷及曲美他嗪组心肌细胞结构完整,炎性细胞明显减少,细胞间质水肿程度降低,肌纤维排列整齐;三七总皂苷与曲美他嗪组ZO-1、Occludin、Cx43的蛋白表达均显著升高(P<0.01)。结论本实验结果表明,三七总皂苷可以降低MIRI大鼠心肌损伤标志物CK、cTnT、MYO的含量,降低血清中炎症因子IL-1β、IL-6、TNF-α含量,减轻病理损伤,可以通过调控缝隙链接蛋白Cx43、紧密连接蛋白ZO-1及Occludin的蛋白表达水平来改善血管紧密连接的结构,调节血管内皮功能,保护MIRI大鼠受损心肌。

Objective To explore the protective effect and mechanism of Panax notoginseng saponins(PNS)on myocardial ischemia-reperfusion injury(MIRI)in rats.Methods Forty SD male rats were randomly divided into normal group,model group,trimetazidine group(control group)and PNS group.The normal group and model group were given the same volume of normal saline by gavage,the trimetazidine group was given trimetazidine with the dose of 5.4 mg·kg-1 by gavage,and the PNS group was given PNS with the dose of 50 mg/(kg·mL)by intraperitoneal injection.Except for the normal group,all models were prepared by ligation of the left anterior descending coronary artery.Changes of ST segment or T wave were detected by electrocardiogram to evaluate the condition of membrane formation.Hematoxylin-eosin(HE)staining was used to observe the myocardial tissue damage.The content of creatine kinase(CK)in serum was determined by colorimetry,and troponin T(cTnT),myoglobin(MYO),interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in serum were determined by enzyme-linked immunosorbent assay(ELISA).Western Blot was used to detect the protein contents of zona occludens protein-1(ZO-1),tight junction protein(Occludin)and gap junction protein 43(Cx43).Results Compared with those of the normal group,the serum contents of CK,cTnT,MYO,IL-1β,IL-6 and TNF-αin the model group were significantly increased(P<0.01).Disordered cell arrangement,inflammatory infiltration,scattered and disordered nuclei,and twisted and broken myocardial fibers were observed,the protein expressions of ZO-1,Occludin and Cx43 in myocardial tissue were significantly decreased(P<0.01).Compared with those of the model group,the contents of cTnT and MYO in the serum of the PNS group and trimetazidine group were significantly decreased(P<0.01),the CK content of the PNS group was significantly decreased(P<0.01),and that of the trimetazidine group was significantly decreased(P<0.01).The content of CK in the trimetazidine group decreased(P<0.05),the contents of IL-1β,IL-6 and TNF-αin the serum of the PNS group were significantly decreased(P<0.01).The contents of IL-1βand IL-6 decreased in the trimetazidine group(P<0.05).The structure of myocardial cells in the groups of PNS and trimetazidine was complete,the inflammatory cells were significantly reduced,the degree of interstitial edema was reduced,and the muscle fibers were neatly arranged.In the PNS and trimetazidine groups,the protein expressions of ZO-1,Occludin and Cx43 were significantly increased(P<0.01).Conclusion The results of this experiment show that PNS can reduce the contents of myocardial injury markers CK,cTnT and MYO,reduce the contents of inflammatory factors IL-1β,IL-6 and TNF-αin serum,and alleviate the pathological damage.The protein expression levels of connexin Cx43,tight junction protein ZO-1 and Occludin can regulate the structure and function of vascular tight junctions,regulate vascular endothelial function,and protect damaged myocardium.