栀子苷对1型糖尿病小鼠心肌损伤的保护作用和机制研究

Study on the protective effect and mechanism of geniposide on myocardial injury in type 1 diabetic mice

目的:探究栀子苷对1型糖尿病小鼠心肌损伤的保护作用及可能的机制。方法:将32只C57BL/6J小鼠随机分为4组:对照组、1型糖尿病模型组(STZ组)、25 mg/kg栀子苷给药组(GE25组)、50 mg/kg栀子苷给药组(GE50组)。测定血糖和体重水平,HE和Masson染色检测心肌纤维化,免疫组化检测LC3B、P62、Ⅰ型胶原蛋白(CollagenⅠ)和Ⅲ型胶原蛋白(CollagenⅢ)的表达。体外培养大鼠心肌细胞H9C2,将细胞分为正常组、高糖组、高糖+栀子苷组、高糖+栀子苷+AMPK抑制剂化合物C(Compound C)组。Western blot检测自噬相关蛋白LC3BⅡ/Ⅰ、P62、Beclin1、腺苷酸活化蛋白激酶(adenosine 5′-monophosphate-activated protein kinase,AMPK)、磷酸化AMPK(p-AMPK)、mTOR和p-mTOR及凋亡相关蛋白的表达。结果:与STZ组相比,栀子苷给药组(GE25组和GE50组)体重明显增加(P <0.01),血糖显著降低(P <0.01),明显减轻心肌结构异常,减少胶原沉积(P <0.01);免疫组化和Western blot结果显示,STZ组LC3BⅡ/Ⅰ、Beclin1和p-AMPK/AMPK的表达水平降低,CollagenⅠ、CollagenⅢ、P62、p-mTOR/mTOR、BAX/BCL2和Cleaved-caspase 3/Caspase 3的表达水平升高,栀子苷给药组与之趋势相反。细胞实验中,栀子苷预处理激活AMPK(p-AMPK/AMPK比值升高)和自噬活性(LC3BⅡ/Ⅰ升高,P62的表达降低),且该效应被AMPK抑制剂阻断。结论:栀子苷能改善1型糖尿病心肌病的心肌损伤,减轻心肌纤维化,其机制可能与激活AMPK介导的细胞自噬有关。

Objective:To investigate the protective effect and possible mechanism of geniposide on myocardial injury in type 1 diabetic mice. Methods:Thirty-two male C57BL/6J mice were randomly divided into 4 groups:control group,type 1 diabetes group(STZ group),geniposide 25 mg/kg administration group(GE25 group),geniposide 50 mg/kg administration group(GE50 group). The blood glucose and body weight levels of the mice were measured. HE and Masson staining were used to detect myocardial fibrosis,immunohistochemistry was used to detect the protein expression levels of LC3B,P62,CollagenⅠand Collagen Ⅲ. H9C2 cells were cultured in vitro and divided into 4 groups:negative control group(NC),high glucose group(HG),HG + geniposide group(HG+GE),and HG+GE+ Compound C group(HG+GE+CC). Western blot was conducted to detect the protein expression levels of autophagyrelated proteins LC3BⅡ/Ⅰ,P62,Beclin1,p-AMPK,AMPK,mTOR and p-mTOR and the protein expression levels of apoptosis-related proteins BAX,BCL2,Caspase3 and Cleaved-caspase3. Results:Compared with the STZ group,the geniposide treated groups(GE25 group and GE50 group)can significantly reduce blood glucose(P < 0.01),increase the body weight(P < 0.01),reduce abnormal myocardial structure and collagen deposition(P < 0.01)in mice. Immunohistochemistry and Western blot showed that the protein expression levels of LC3BⅡ/Ⅰ,Beclin1 and p-AMPK/AMPK decreased,and the protein expression levels of CollagenⅠ,Collagen Ⅲ,P62,p-mTOR/mTOR,BAX/BCL2 and Cleaved-caspase 3/Caspase 3 increased in the STZ group. The administration of geniposide 25 mg/kg or 50 mg/kg reversed these changes. In cell experiments,geniposide pretreatment activated AMPK(increased p-AMPK/AMPK ratio)and autophagy activity(increased LC3BⅡ/Ⅰ,decreased expression of P62),and this effect was blocked by AMPK inhibitor Compound C. Conclusion:Geniposide can improve myocardial injury and reduce myocardial fibrosis in type 1 diabetic mice. The mechanism may be related to the activation of AMPK-mediated autophagy.