基于Bcl-2/Bax/Caspase-3信号通路探究参红通络方对心肌缺血再灌注损伤大鼠细胞凋亡的影响

Effect of Modified Shenhong Tongluo Prescription on Cell Apoptosis in Rats with Myocardial Ischemia-reperfusion Injury by Bcl-2/Bax/Caspase-3 Signaling Pathway

目的:探讨参红通络方加减对心肌缺血再灌注损伤(MI/RI)大鼠细胞凋亡的作用机制。方法:60只SD大鼠随机分为空白组、模型组、参红通络方剂低、中、高剂量组和辛伐他汀组。除空白组外,采用线栓结扎冠状动脉左前降支方式制备心肌缺血再灌注损伤大鼠模型(MI/RI模型),造模成功后第1天起,空白组(生理盐水1.0 mL·kg^(-1))、模型组(生理盐水1.0 mL·kg^(-1))、参红通络方剂低、中、高剂量组(1.031、2.063、4.126 g·kg^(-1)参红通络方标准浓缩液)和辛伐他汀组(辛伐他汀0.71 mg·kg^(-1)),定时灌胃给药,每日1次,连续2周。苏木素-伊红(HE)染色法观察心肌细胞病理形态学变化;酶联免疫吸附测定法(ELISA)检测血清肌酸激酶同工酶(CK-MB)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)水平变化;原位末端转移酶标记技术(TUNEL)染色法检测大鼠心肌细胞凋亡率;蛋白免疫印迹法(Western blot)检测细胞凋亡相关蛋白B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和胱天蛋白酶-3(Caspase-3)表达水平。结果:与空白组比较,模型组HE染色可见心肌细胞排列紊乱,纤维不完整,心肌细胞小灶性坏死,并伴有炎性细胞浸润;血清CK-MB、IL-6和TNF-α水平明显上升(P<0.05);心肌细胞凋亡率明显升高(P<0.05),Bax、Caspase-3蛋白表达水平则明显升高,Bcl-2明显下降(P<0.05);与模型组比较,参红通络方剂加减低、中、高剂量组可明显降低CK-MB、IL-6和TNF-α水平(P<0.05);明显下调心肌细胞凋亡率(P<0.05);Bax、Caspase-3蛋白显著降低,Bcl-2表达水平明显增加(P<0.05)。参红通络方剂加减组中Bax、Caspase-3蛋白随参红通络方给药剂量的增加而明显降低,Bcl-2表达水平随参红通络方给药剂量的增加而明显升高(P<0.05)。结论:参红通络方加减可通过减轻心肌组织病理损伤并降低心肌细胞凋亡,可能与抑制Caspase-3、Bax表达、促进Bcl-2水平表达有关。

Objective: To investigate the mechanism of modified Shenhong Tongluo prescription on cell apoptosis in rats with myocardial ischemia-reperfusion injury(MIRI). Method: Sixty Sprague-Dawley(SD) rats were randomly divided into a blank group, a model group, low-, medium-, and high-dose groups of modified Shenhong Tongluo prescription, and a simvastatin group. Except for the blank group, a rat model of MIRI was prepared by ligating the left anterior descending coronary artery. Starting from the first day after successful modeling, the blank group(1.0 mL·kg^(-1) physiological saline), model group(1.0 mL·kg^(-1) physiological saline), low-, medium-, and high-dose groups of modified Shenhong Tongluo prescription(1.031, 2.063, and 4.126 g·kg^(-1) Shenhong Tongluo prescriptiona standard concentrate), and simvastatin group(0.71 mg·kg^(-1) simvastatin) were orally administered once daily for 2 weeks. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of cardiomyocytes. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of serum creatine kinase isoenzyme(CK-MB), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α). TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis rate of rat cardiomyocytes. Western blot was used to detect the expression levels of apoptosis-related proteins B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and caspase-3. Result: Compared with the blank group, in the model group, HE staining showed disturbed arrangement of cardiomyocytes, incomplete fibers, focal necrosis of cardiomyocytes, and inflammatory cell infiltration;serum CK-MB, IL-6, and TNF-α levels were significantly increased(P<0.05);apoptosis rate of cardiomyocytes was significantly increased(P<0.01), with significantly increased expression levels of Bax and Caspase-3 proteins, and significantly decreased Bcl-2 expression(P<0.05). Compared with the model group, the low-, medium-, and high-dose groups of modified Shenhong Tongluo prescription significantly reduced CK-MB, IL-6, and TNF-αlevels(P<0.05), significantly downregulated cardiomyocyte apoptosis rate(P<0.05), significantly decreased Bax and Caspase-3 proteins, and significantly increased Bcl-2 expression levels(P<0.01). In the modified Shenhong Tongluo prescription groups, the expression levels of Bax and Caspase-3 proteins significantly decreased with increasing dosage, while the expression level of Bcl-2 significantly increased with increasing dosage of modified Shenhong Tongluo prescription(P<0.05). Conclusion: Shenhong Tongluo prescription can alleviate myocardial tissue pathological damage and reduce myocardial cell apoptosis, possibly by inhibiting Caspase-3 and Bax expression and promoting Bcl-2 expression.