栀子苷通过抑制VPO1/ERK1/2信号通路减轻糖尿病心肌病大鼠的心肌细胞凋亡

Gardenoside alleviates diabetic cardiomyopathy via inhibiting VPO1/ERK1/2 signaling pathway to improve myocardial apoptosis in rats

目的:探讨栀子苷(geniposide,GE)改善大鼠糖尿病心肌病的作用及其具体机制。方法:24只成年雄性SD大鼠,随机分为3组:正常组(Control组,8只)、糖尿病心肌病组(DCM组,8只)、糖尿病心肌病组+栀子苷组(DCM+GE组,8只),采用高脂饲料联合链脲佐菌素(STZ)构建糖尿病心肌病大鼠模型,应用次氯酸(HOCl)诱导H9C2损伤模型。干预12周后,HE和Masson染色观察心脏组织病理学改变;TUNEL法检测大鼠心肌细胞凋亡水平;免疫组化检测及Western blot检测法检测VPO1/ERK1/2信号通路及凋亡相关蛋白表达水平。给予次氯酸刺激心肌细胞后,Western blot检测法检测ERK1/2、p-ERK1/2、Bcl-2、Bax蛋白表达水平的改变。给予ERK1/2酶抑制剂U0126后,用Western blot检测法再次检测ERK1/2、p-ERK1/2、Bcl-2、Bax蛋白表达水平的改变。结果:HE及Masson染色显示,与Control组相比,DCM组出现心肌纤维排列紊乱、心肌胶原含量明显增多,而DCM+GE组心肌损伤情况明显改善(P<0.05)。与Control组相比,DCM组心肌细胞凋亡水平及Bax/Bcl-2比值明显增加,而DCM+GE组心肌凋亡情况得到明显好转(P<0.05)。免疫组化和Western blot结果显示,在DCM组中VPO1、p-ERK1/2蛋白表达水平升高,而DCM+GE组中VPO1、p-ERK1/2蛋白表达水平得到了抑制(P<0.05)。进一步对H9C2细胞行HOCl干预发现,与Control组相比,次氯酸组出现p-ERK1/2蛋白表达水平及Bax/Bcl-2比值增加,而加入ERK1/2酶抑制剂U0126后p-ERK1/2蛋白表达水平及Bax/Bcl-2比值下降(P<0.05)。结论:栀子苷通过抑制VPO1/ERK1/2信号通路抑制心肌细胞凋亡从而改善糖尿病引起的心肌损伤。

AIM:To investigate the effect of geniposide(GE)on diabetic cardiomyopathy and its mechanism.METHODS:Twenty-four adult male SD rats were randomly divided into three groups:Control group(n=8),DCM group(n=8)and DCM+GE group(n=8).The diabetic cardiomyopathy model was established by high fat diet combined with streptozotocin(STZ),and H9C2 injury model was induced by hypochlorite(HOCl).After 12 weeks of intervention,the histopathological changes of heart were observed by HE and Masson staining,the level of cardiomyocyte apoptosis was detected by TUNEL staining,and the expression levels of VPO1/ERK1/2 signal pathway and apoptosis-related proteins were detected by immunohistochemistry and Western blot.The changes of ERK1/2,p-ERK1/2,Bcl-2 and Bax protein expression in cardiomyocytes were detected by Western blot after HOCl stimulation.After administration of ERK1/2 inhibitor U0126,the protein expression levels of ERK1/2,p-ERK1/2,Bcl-2 and Bax were detected again by Western blot assay.RESULTS:Compared with the control group,the arrangement of myocardial fibers was disordered and the content of myocardial collagen was significantly increased in DCM group by HE and Masson staining,while the myocardial injury was significantly improved in DCM+GE group(P<0.05).Compared with the control group,the apoptotic rates of cardiomyocytes and the ratio of Bax/Bcl-2 in DCM group were remarkably increased,while the myocardial apoptosis in DCM+GE group was significantly improved(P<0.05).The results of immunohistochemistry and Western blot showed that the expression of VPO1 and p-ERK1/2 was increased in DCM group,while the expression level of VPO1 and p-ERK1/2 was inhibited in DCM+GE group(P<0.05).When H9C2 cells were stimulated with HOCl,the expression of p-ERK1/2 and the ratio of Bax/Bcl-2 were both increased as compared with the control group,while the expression level of p-ERK1/2 and the ratio of Bax/Bcl-2 were decreased after the addition of ERK1/2 inhibitor U0126(P<0.05).CONCLUSION:Geniposide alleviates the diabete-induced myocardial injury by suppressing cardiomyocyte apoptosis via inhibiting VPO1/ERK1/2 signal pathway.