姜黄素激活哺乳动物西罗莫司靶蛋白信号抑制自噬减轻小鼠心肌梗死损伤的作用

Effect of mammalian target of sirolimus on autophagy in reducing myocardial infarction injury of C57 mice by curcumin

目的研究姜黄素(curcumin,Cur)减轻小鼠心肌梗死(myocardial infarction,MI)损伤的作用以及哺乳动物西罗莫司靶蛋白(mammalian target of sirolimus,mTOR)调控的自噬在其中的作用。方法将40只8周龄C57BL/6小鼠随机分为假手术组(Sham)、单纯姜黄素(500 mg·kg^(-1)·d^(-1))处理组(Cur)、心肌梗死组(MI)和姜黄素(500 mg·kg^(-1)·d^(-1))干预心肌梗死组(MI+Cur)。结扎冠状动脉左前降支建立心肌梗死模型,用苏木精-伊红(HE)染色、Masson染色观察心肌组织形态学变化及纤维化程度,检测各组血清中乳酸脱氢酶(LDH)、肌酸激酶同工酶(CK-MB)的水平,原位末端转移酶标记技术(TUNEL)染色检测各组细胞凋亡水平,用蛋白质印迹(Western blot)法检测心肌组织中磷酸化的西罗莫司靶蛋白(p-mTOR)、p62、Beclin1、自噬蛋白微管相关蛋白1轻链3B(LC3B)的表达,以免疫荧光染色法检测心肌组织LC3B的表达。结果Sham组、MI组和MI+Cur组射血分数(EF)分别为(66.33±7.09)%,(35.00±5.00)%,(50.00±5.00)%,差异有统计学意义(P<0.05),MI+Cur组心功能明显改善。与Sham组相比,Cur组各项指标均无明显变化;MI组心肌纤维断裂明显排列紊乱,纤维化程度增加。Sham组、MI组和MI+Cur组LDH分别为(384.20±20.80),(1010.90±70.27),(648.10±86.96)U·L^(-1),CK-MB分别为(406.83±101.55),(1767.93±321.04),(934.20±277.22)U·L^(-1),差异均有统计学意义(均P<0.05)。与MI组比较,MI+Cur组TUNEL阳性细胞明显减少,LC3B和Beclin1表达显著降低,而p-mTOR和p62的表达显著升高,免疫荧光结果显示LC3B表达减少(均P<0.05)。结论姜黄素通过激活mTOR信号抑制自噬减轻小鼠心肌梗死损伤。

Objective To explore the role of curcumin in regulating autophagy through mammalian target of sirolimus(mTOR)signaling pathway in reducing myocardial infarction injury in mice.Methods Forty 8-week-old C57BL/6 mice were randomly divided into sham operation group(sham),single curcumin group(Cur,500 mg·kg^(-1)·d^(-1)),myocardial infarction group(MI)and curcumin(500 mg·kg^(-1)·d^(-1))intervention in myocardial infarction group(MI+Cur).The myocardial infarction model was established by ligating the left anterior descending branch of coronary artery.The myocardial morphology and fibrosis degree were observed by hematoxylin-eosin(HE)and Masson staining.Lactate dehydrogenase(LDH)and creatine kinase-MB(CK-MB)in serum of each group was detected.Apoptosis levels in each group were detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)staining.The expression of phosphorylated mammalian target of sirolimus(p-mTOR),p62,Beclin1,and autophagy protein microtuble associated protein light chain 3B(LC3B)were detected by Western blot,and the expression of LC3B was detected by immunofluorescence staining.Results The ejection fraction(EF)in sham group,MI group and MI+Cur group were(66.33±7.09)%,(35.00±5.00)%,(50.00±5.00)%,the difference were significant(P<0.05).Compared with sham group,the indexes of Cur group had no significant change.In MI group,the myocardial fibers were broken and disordered,and the degree of fibrosis increased.The LDH in sham group,MI group and MI+Cur group were(384.20±20.80),(1010.90±70.27),(648.10±86.96)U·L^(-1),the CK-MB were(406.83±101.55),(1767.93±321.04),(1767.93±321.04)U·L^(-1),the difference were all significant(all P<0.05).Compared with MI group,the TUNEL positive cells in MI+Cur group were significantly decreased,the expressions of LC3B and Beclin1 were significantly decreased,and the expressions of p-mTOR and p62 were significantly increased.Immunofluorescence results showed that the expression of LC3B was decreased(all P<0.05).Conclusion Curcumin can reduce the myocardial damage in mice by regulating mTOR signal-mediated autophagy.