积雪草苷通过Notch1/Hes1信号上调自噬减轻糖尿病心肌病心肌损伤的作用及机制

Asiaticoside attenuates myocardial injury in diabetic cardiomyopathy by enhancing autophagy through Notch1/Hes1 signaling

目的 探讨积雪草苷(ASI)激活Notch1/Hes1信号调控自噬改善糖尿病心肌病(DCM)心肌损伤的作用及机制。方法 将100只8周龄雄性C57/BL成年小鼠随机分为对照组(sham组)、DCM组、ASI处理DCM组(ASI+DCM组)以及ASI联合Notch1抑制剂DAPT处理DCM组(ASI+DAPT+DCM组)。对照组以普通饲料喂养,DCM组、ASI+DCM组以及ASI+DAPT+DCM组以高脂饲料喂养。1月后DCM组、ASI+DCM组以及ASI+DAPT+DCM组禁食12 h后进行链脲佐菌素(STZ)腹腔注射,对照组腹腔注射同等体积生理盐水,STZ注射后72 h,测尾静脉空腹血糖(fasting blood glucose, FBG),FBG>16.7 mmol/L为造模成功。造模成功后,ASI+DCM组腹腔注射ASI 10 mg/(kg·d),ASI+DAPT+DCM组同时腹腔注射ASI 10 mg/(kg·d)以及DAPT(10 mg/kg)。自实验开始5月后,检测小鼠血清乳酸脱氢酶(LDH)、结缔组织生长因子(CTGF)水平、左室射血分数(LVEF)等心功能指标,Masson染色检测心肌纤维化程度,免疫印迹检测Beclin1、Atg5、LC3Ⅱ/Ⅰ及Notch1和Hes1的表达。结果 与sham组比较,DCM组LVEF值降低(P<0.05),心肌纤维化程度增加(P<0.05),血清LDH、CTGF水平升高(P<0.05),LC3Ⅱ/Ⅰ比值、Beclin1和Atg5表达降低(P<0.05),而Notch1和Hes1表达增高(P<0.05)。与DCM组比较,ASI+DCM组LVEF值增加(P<0.05),心肌纤维化程度改善(P<0.05),血清LDH、CTGF水平降低(P<0.05),LC3Ⅱ/Ⅰ比值、Beclin1和Atg5表达升高(P<0.05),而Notch1和Hes1表达降低(P<0.05)。与ASI+DCM组比较,ASI+DAPT+DCM组LVEF值降低(P<0.05),心肌纤维化程度增加(P<0.05),血清LDH、CTGF水平升高(P<0.05),LC3Ⅱ/Ⅰ比值、Beclin1和Atg5表达降低(P<0.05),而Notch1和Hes1表达增高(P<0.05)。结论 ASI通过抑制Notch1/Hes1信号增强自噬减轻DCM引起的心肌损伤。

Objective To investigate the effect of asiaticoside(ASI) on myocardial damage in diabetic cardiomyopathy(DCM) by re-gulating autophagy via Notch1/Hes1 signaling and its mechanism. Methods A total of 100 male C57/BL adult mice(8 weeks old) were randomly divided into sham group, DCM group, ASI+DCM group and ASI+DAPT+DCM group. The mice in sham group were fed with normal diet. The mice in DCM group, ASI+DCM group and ASI+DAPT+DCM group were fed with high-fat diet until the end of the experiment. At one month after the feeding, the mice in DCM group, ASI+DCM group and ASI+DAPT+DCM group were intraperitoneally injected with streptozocin(STZ) after fasting for 12 h. At 72 h after STZ, if the fasting blood glucose(FBG) was greater than 16.7 mmol/L, the model was successfully established, and then the therapeutic drug intervention was carried out. ASI[10 mg/(kg·d)] was dissolved in 0.9% sodium chloride solution. The mice were injected intraperitoneally with ASI [10 mg/(kg·d)] in ASI+DCM group, and ASI[10 mg/(kg·d)] and DAPT(10 mg/kg) in ASI+DAPT+DCM group. The mice in sham group and DCM group were intraperitoneally injected with the same volume of normal saline. The mortality of mice in each group was recorded and calculated after the experiment. At 5 month after the beginning of the experiment, the levels of serum LDH and CTGF were detected. The cardiac function indexes were detected by ultrasound. The degree of cardiac fibrosis was detected by Masson staining. The expression of autophagy related protein LC3Ⅱ/Ⅰ was detected by immunofluorescent staining. The expression levels of Beclin1, Atg5, LC3Ⅱ/Ⅰ, Notch1 and Hes1 were detected by Western blot. Results Compared with sham group, LVEF was decreased, the degree of myocardial fibrosis and the levels of serum LDH and CTGF were increased(P<0.05). The LC3Ⅱ/Ⅰ ratio, the expression levels of Beclin1 and Atg5 were decreased(P<0.05), while the expression levels of Notch1 and Hes1 were increased in DCM group(P<0.05). Compared with DCM group, LVEF was increased, while the degree of myocardial fibrosis was decreased in ASI+DCM group(all P<0.05);the serum levels of LDH and CTGF were decreased(P<0.05);LC3Ⅱ/Ⅰ ratio and the expression levels of Beclin1 and Atg5 were increased, while the expression levels of Notch1 and Hes1 were decreased in ASI+DCM group(P<0.05). Compared with ASI+DCM group, LVEF was decreased(P<0.05), while the level of myocardial fibrosis was increased in ASI+DAPT+DCM group(P<0.05);the levels of LDH and CTGF in serum were increased(P<0.05);LC3Ⅱ/Ⅰ ratio and the expression levels of Beclin1 and Atg5 were decreased(P<0.05), while the expression levels of Notch1 and Hes1 were increased(P<0.05). Conclusion ASI can enhance the autophagy by inhibiting Notch1/Hes1 signal and reduce the myocardial injury caused by DCM.