积雪草苷对小鼠糖尿病心肌病的干预作用及机制探讨

Intervention effect of asiaticoside on diabetic cardiomyopathy mice and the mechanism

目的观察积雪草苷(ASI)对小鼠糖尿病心肌病(DCM)的干预作用,并探讨其机制。方法将60只8周龄雄性C57/BL成年小鼠随机分为对照组(Sham组)、DCM组、ASI干预组(ASI+DCM组)、ASI与Notch1抑制剂DAPT联合干预组(ASI+DAPT+DCM组)。Sham组以普通饲料喂养,余组高脂饲料喂养,并以注射链脲佐菌素联合高脂饮食建立糖尿病心肌病模型;ASI+DCM组腹腔注射ASI 10 mg/(kg·d),ASI+DAPT+DCM组腹腔注射ASI 10 mg/(kg·d)和DAPT 10 mg/(kg·d),DCM组给予相同体积生理盐水,共给药5个月。超声检查测算左心室射血分数(LVEF)、左心室短轴缩短率(FS)、左心室舒张末期容积(LVEDV)、左心室收缩末期容积(LVESV)、左心室舒张期前壁厚度(LVAWd)等心功能指标;检测小鼠血清肌酸激酶同Ⅰ酶(CK-MB)、结缔组织生长因子(CTGF)水平;HE染色、Masson染色观察心肌结构及纤维化情况;Tunel染色法观察心肌细胞凋亡情况;RT-PCR法检测心肌组织中的Bax、Caspase-3 mRNA,Western blotting法检测Notch1、Hes1、Bax、Caspase-3蛋白。结果与DCM组、ASI+DAPT+DCM组相比,ASI+DCM组肌纤维排列整齐,纤维化程度改善。DCM组LVEF、FS、LVEDV及心肌组织中Bcl-2、Notch1、Hes1蛋白表达低于Sham组,LVAWd、血清CK-MB、CTGF水平及心肌组织Bax、Caspase-3 mRNA和蛋白表达高于Sham组(P均<0.05)。ASI+DCM组LVEF、FS、LVEDV高于DCM组,LVAWd低于DCM组(P均<0.05);ASI+DAPT+DCM组LVEF低于ASI+DCM组,LVEDV高于ASI+DCM组(P均<0.05)。ASI+DCM组血清CK-MB、CTGF水平及心肌组织中Bax、Caspase-3 mRNA和蛋白表达低于DCM组、ASI+DAPT+DCM组,心肌组织中Bcl-2、Notch1、Hes1蛋白表达高于DCM组、ASI+DAPT+DCM组(P均<0.05)。DCM组、ASI+DCM组、ASI+DAPT+DCM组细胞凋亡率高于Sham组,ASI+DCM组细胞凋亡率低于DCM组、ASI+DAPT+DCM组(P均<0.05)。结论ASI可减轻糖尿病心肌病小鼠心肌损伤,减轻心肌纤维化,作用机制可能与抑制Notch1/Hes1信号通路、减少心肌细胞凋亡有关。

Objective To observe the intervention effect of asiaticoside(ASI)on diabetic cardiomyopathy(DCM)mice and to investigate the mechanism.Methods Totally 60 male C57/BL mice were randomly divided into four groups:Sham group,DCM group,ASI+DCM group,and ASI+DAPT+DCM group.The mice in the Sham group were fed with normal diet and the mice in the other groups were fed with high-fat diet.DCM models were established by injection of streptozotocin combined with high-fat diet.ASI(10 mg/kg·d)was given in the ASI+DCM group.ASI(10 mg/kg·d)and DAPT(10 mg/kg·d)were given in the ASI+DAPT+DCM group.The mice in the DCM group were given the same volume of physiological saline.The total time of the experiment was five months.The cardiac function indexes such as left ventricular ejection fraction(LVEF),left ventricular fractional shortening(FS),left ventricular end-diastolic volume(LVEDV),left ventricular end-systolic volume(LVESV),and left ventricular anterior wall diameter(LVAWd)were detected by ultrasound.The levels of serum creatine kinase isoenzyme(CK-MB)and connective tissue growth factor(CTGF)were detected.The myocardial structure and fibrosis in each group were detected by HE staining and Masson staining.The myocardial apoptosis was detected by TUNEL staining.The mRNA expression levels of Bax and Caspase-3 in the myocardial tissues were detected by RT-PCR.The protein expression levels of Notch1,Hes1,Bax,and Caspase-3 were detected by Western blotting.Results Compared with the DCM group and the ASI+DAPT+DCM group,the muscle fibers in the ASI+DCM group were arranged neatly and the degree of fibrosis was improved.LVEF,FS,LVEDV and Bcl-2,Notch1,Hes1 protein expression levels in the myocardial tissues were lower in the DCM group than in the Sham group,while LVAWd,serum CK-MB,CTGF level and Bax,Caspase-3 mRNA and protein expression levels in the myocardial tissues were higher than those of the Sham group(all P<0.05).LVEF,FS,and LVEDV in the ASI+DCM group were higher than those in the DCM group,and LVAWd was lower than that of the DCM group(all P<0.05);LVEF in the ASI+DAPT+DCM group was lower than that of the ASI+DCM group,but LVEDV was higher(both P<0.05).The levels of serum CK-MB and CTGF and the mRNA and protein expression levels of Bax and Caspase-3 in the myocardial tissues were lower in the ASI+DCM group than in the DCM group and ASI+DAPT+DCM group,while the protein expression levels of Bcl-2,Notch1 and Hes1 in the myocardial tissues were higher than those of the DCM group and ASI+DAPT+DCM group(all P<0.05).The apoptosis rates of the DCM group,ASI+DCM group,ASI+DAPT+DCM group were higher than that of the Sham group,and the apoptosis rate of ASI+DCM group was lower than those of the DCM group and ASI+DAPT+DCM group(all P<0.05).Conclusion ASI can attenuate myocardial injury and reduce myocardial fibrosis in DCM mice,which may be related to the inhibition of Notch1/Hes1 signaling pathway and reduction of cardiomyocyte apoptosis.