西洋参茎叶总皂苷对缺血/再灌注大鼠心肌线粒体膜电位及细胞凋亡的影响

 目的：研究西洋参茎叶总皂苷 (PQS) 对大鼠缺血/再灌注 (I/R) 损伤后心肌细胞凋亡的影响，并从线粒体膜电位 (ΔΨm) 及线粒体凋亡通路探讨其可能的机制。方法：健康雄性SD大鼠90只，随机分为假手术(sham)组、模型(I/R)组、PQS (200 mg·kg-1·d-1, 灌胃6周)+I/R组、环孢霉素A (CsA；10 mg·kg-1，再灌前10 min腹腔注射)组、CsA+I/R组和PQS+CsA+I/R组，各组n=15。除sham组和CsA组大鼠开胸后冠状动脉左前降支（LAD）下穿线不结扎外，其余各组大鼠常规麻醉后，结扎LAD 30 min，再灌注120 min复制I/R模型。生化分析仪测血清乳酸脱氢酶（LDH）含量，氯化三苯基四氮唑（TTC）和伊文思蓝双染法测心梗面积，原位缺口末端标记法（TUNEL）检测各组大鼠心肌细胞凋亡情况，Western blotting检测细胞凋亡相关蛋白的表达。采用JC-1作为荧光探针，用激光共聚焦显微镜和荧光酶标仪测定ΔΨm水平。结果：与sham组比较，I/R组血清LDH活性、心梗面积和心肌细胞凋亡率均显著升高（P<0.05）；与I/R组相比，PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组血清LDH活性、心梗面积和心肌细胞凋亡率均显著降低（P<0.05）。Western blotting结果显示，与sham组相比，I/R组心肌Bcl-2蛋白表达量降低，Bax、胞浆cytochrome C和cleaved caspase-3升高（均P<0.05）；与I/R组相比，PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组心肌Bcl-2蛋白表达量升高，Bax、胞浆cytochrome C及心肌cleaved caspase-3蛋白表达量均降低（P<0.05）。激光共聚焦显微镜观察结果示，I/R组线粒体JC-1染色后红色荧光强度减弱，荧光酶标仪测相对荧光单位（RFU） 较sham组降低（P<0.05）；PQS+I/R组、CsA+I/R组和PQS+CsA+I/R组RFU均较I/R组升高（P<0.05）。结论：PQS显著降低大鼠I/R后心肌细胞损伤，减少细胞凋亡，其机制与维持再灌注期ΔΨm稳定、抑制线粒体凋亡通路的激活有关。

Effects of Panax quinquefolium saponin on mitochondrial membrane potential and cardiomyocyte apoptosis in ischemia/reperfusion rats

AIM：To investigate whether mitochondrial membrane potential (ΔΨm) and the mitochondrial apoptotic pathway are involved in the protective mechanism of Panax quinquefolium saponin (PQS) against cardiomyocyte apoptosis after ischemia/reperfusion (I/R) injury in rat myocardium. METHODS：Ninety healthy male SD rats were randomly divided into sham group, I/R group, PQS (200 mg·kg-1·d-1) +I/R group, cyclosporine A (CsA) group, CsA (10 mg·kg-1) +I/R group and PQS +CsA +I/R group. The model of myocardial I/R injury in vivo was established by ligating the left anterior descending artery (LAD) for 30 min followed by 120 min of reperfusion in the rats. The serum activity of lactate dehydrogenase (LDH) was measured by automatic chemistry analyzer. The myocardial infarct size was measured by Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis was detected by in situ TDT-mediated dUTP nick end labeling (TUNEL). The protein levels of Bcl-2, Bax, cleaved caspase-3 and cytosolic cytochrome C were determined by Western blotting. ΔΨm was measured by laser scanning confocal microscopy and fluorescence microplate reader. RESULTS：Compared with I/R group, the serum content of LDH，the infarction size in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group and the myocardial apoptotic index were  decreased. Compared with I/R group, the fluorescence intensity of mitochondria after JC-1 staining was enhanced in PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, and the relative fluorescence units (RFU) of ΔΨm were improved in those 3 groups. In PQS+I/R group, CsA+I/R group and PQS+CsA+I/R group, the protein expression of Bcl-2 was increased, and that of Bax was decreased compared with I/R group. Moreover, in those 3 groups, the protein levels of cleaved-caspase-3  and cytosolic cytochrome C were decreased compared to I/R group, respectively. CONCLUSION：PQS attenuates myocardial injury and cardiomyocyte apoptosis during I/R, and the protective mechanisms of PQS were associated with the modulation of ΔΨm and the inhibition of mitochondrial apoptosis pathway.