基于自噬探讨丹蒌片减轻心肌缺血再灌注损伤的机制研究

Study on the Mechanism of Danlou Tablet Alleviates Myocardial Ischemia Reperfusion Injury via Regulation of Autophagy

目的观察丹蒌片对心肌缺血再灌注损伤的保护作用,并探讨其具体作用机制。方法40只8周龄雄性C57BL/6J小鼠,随机分为4组:假手术组、模型组、丹蒌片组、3-甲基腺嘌呤(3MA)组,每组10只。丹蒌片组给予丹蒌片超微粉混悬液3 g/(kg·d)连续灌胃5天,假手术组和模型给予同体积生理盐水连续灌胃5天,3MA组分别于第1天及造模前15 min给予3MA 15 mg/kg腹腔注射。治疗5天后采用结扎左冠状动脉前降支的方法制备小鼠心肌缺血再灌注模型,假手术组仅缝线下穿不结扎。心脏超声评价心功能,TUNEL染色法评价心肌凋亡水平,TTC染色法评价心肌灌流水平,免疫印迹法检测自噬相关蛋白LC3B、BNIP3表达。结果与假手术组比较,模型组小鼠左室射血分数(LVEF)、左室短轴缩短率(LVFS)下降(P<0.01),心梗面积比、心肌细胞凋亡率以及心肌LC3B、BNIP3蛋白表达升高(P<0.01)。与模型组比较,丹蒌片组和3MA组LVEF、LVFS升高(P<0.05),心梗面积比以及心肌LC3B、BNIP3蛋白表达下降(P<0.05,P<0.01),丹蒌片组心肌细胞凋亡率下降(P<0.05)。结论丹蒌片能够减少缺血再灌注后心肌坏死面积,提高心功能,从而保护缺血心肌,其机制可能与调节心肌细胞自噬及减少细胞凋亡有关。

Objective To investigate the effect of Danlou Tablet on myocardia in mice with ischemia reperfusion injury(IRI) and underlying mechanism. Methods Totally 40 male C57 BL/6 J mice of 8 weeks old were randomly divided into 4 groups: sham group, vehicle group, Danlou Tablet group and 3-methyladenine(3 MA) group,10 in each group. Mice in Danlou Tablet group were gavaged with suspension of Danlou Tablet ultrafine powder 3 g·kg-1·d-1 for 5 consecutive days. Meanwhile, sham group and vehicle group were given the equal volume of normal saline. In 3 MA group, mice were treated with 3 MA solution(15 mg/kg) via intraperitoneal injection on the first day and 15 minutes before the modeling. The mice model of myocardial IRI was induced by left coronary artery ligation after 5 days of treatment. Sham group underwent the same procedure without ligation of the left coronary artery. Cardiac function was evaluated by echocardiography system. The myocardial apoptosis was investigated by TUNEL staining. TTC staining was used to detect myocardial infarct size. The protein expression of LC3 B and BNIP3 were analyzed by immunoblot assay. Results Compared with sham group, the left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS) decreased in vehicle group(P<0.01), the proportion of positive nucleus, myocardial infarct size and the expression of LC3 B and BNIP3 protein increased(P<0.05). Compared with vehicle group, the LVEF and LVFS in Danlou Tablet and 3 MA group increased(P<0.05), myocardial infarct size and the expression of LC3 B and BNIP3 protein in Danlou Tablet group and 3 MA group decreased(P<0.05, P<0.01), the proportion of positive nucleus in Danlou Tablet group was reduced(P<0.05). Conclusion Danlou Tablet could alleviate myocardial infarction area and improve cardiac function for the mice with IRI via regulation of autophagy and reducing cardiomyocytes apoptosis, thereby protecting ischemic myocardium.