芪参益气滴丸对2型糖尿病大鼠心肌细胞线粒体自噬的调节机制研究

Regulating mechanism of Qishen YiqiDripping Pills on mitochondrial autophagy in type 2 diabetic rats

目的观察芪参益气滴丸对心肌缺血再灌注损伤2型糖尿病大鼠心肌的保护作用及线粒体自噬磷酸甘油酸变位酶5(PGAM5)/Fun14结构相关蛋白1(FUNDC1)信号通路的影响。方法雄性SD大鼠48只,分为空白对照组、假手术组、心肌缺血再灌注损伤(MIRI)1组、MIRI 2组、抑制剂组和芪参益气滴丸组。除空白对照组和MIRI 1组外,其余大鼠通过高脂饮食联合链脲佐菌素腹腔注射,建立2型糖尿病模型。芪参益气滴丸组灌胃芪参益气滴丸450 mg/kg,1次/d,抑制剂组除给予芪参益气滴丸外同时腹腔注射三甲基腺嘌呤(3-MA)100 mmol/L,1次/d,其余4组灌胃等体积生理盐水。灌胃1周后,除空白对照组、假手术组外,其余4组采用左冠状动脉前降支结扎30 min、再灌注2 h的方法构建MIRI模型,再灌注2 h后取材。计算大鼠死亡率,检测心肌酶肌酸激酶(CK)、乳酸脱氢酶(LDH)和天门冬氨酸氨基转移酶(AST)水平和心肌组织超氧化物歧化酶(SOD)、丙二醛(MDA)水平,实时荧光定量PCR测定心肌组织PGAM5/FUNDC1通路节点蛋白表达水平的变化。结果与MIRI 1组比较,MIRI 2组大鼠血清AST、LDH、CK和MDA水平升高(均P<0.05),SOD水平降低(P<0.05),心肌组织中FUNDC1、PGAM5、B细胞淋巴瘤-xL(Bcl-xL)、蛋白轻链3(LC3)、自噬相关蛋白5(ATG5)、Beclin-1水平降低(均P<0.05),P62水平升高(P<0.05),含半胱氨酸的天冬氨酸蛋白水解酶-9(Caspase-9)水平有增高的趋势,但差异无统计学意义(P>0.05);与MIRI 2组及抑制剂组比较,芪参益气组大鼠血清AST、LDH、CK和MDA水平降低(均P<0.05),SOD水平升高(P<0.05),心肌组织FUNDC1、PGAM5、Bcl-xL、LC3、ATG5、Beclin-1水平升高(均P<0.05),而P62、Caspase-9水平降低(均P<0.05)。结论高血糖加重了MIRI,芪参益气滴丸通过调节PGAM5/FUNDC1通路介导线粒体自噬减轻MIRI,对糖尿病大鼠心肌发挥保护作用。

Objective To observe the protective effect of Qishen Yiqi Dripping Pills on myocardial ischemia-reperfusion injury in type 2 diabetic rats and its effects on mitochondrial autophagy phosphoglycerate mutase family member 5(PGAM5)/Fun14 domain-containing protein 1(FUNDC1)signaling pathway.Methods 48 male SD rats were divided into a blank control group,sham operation group,No.1 myocardial ischemia reperfusion injury(MIRI)group,No.2 MIRI group,inhibitor group,and Qishen Yiqi group.In addition to the blank control group and the No.1 MIRI group,the other 32 rats were fed with a high-fat diet combined with intraperitoneal injection of streptozotocin to establish animal models of diabetes.Then,the rats in the Qishen Yiqi group were ig Qishen Yiqi Gropping Pills 450 mg/kg,once daily.The rats in the inhibitor group were given Qishen Yiqi Gropping Pills and trimethylamine(3-MA)by intraperitoneal injection 100 mmol/L,once daily.And the rats in the other four groups were ig normal saline.One week after intragastric administration,except for the blank control group and the sham operation group,the rats in the other four groups were used to establish the animal model of myocardial ischemia-reperfusion injury by ligating the anterior descending branch of the left coronary artery for 30 min and reperfusion for 2 h.Then,the materials were taken after reperfusion for 2 h.Finally,the mortality of rats was calculated,the changes in creatine kinase(CK),lactate dehydrogenase(LDH),aspartate aminotransferase(AST),and the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in myocardial tissue were detected,and the expression level of PGAM5/FUNDC1 pathway node protein in myocardial tissue was measured by real-time fluorescence quantitative PCR.Results Compared with the No.1 MIRI group,serum indicators of the AST,LDH,CK,and MDA levels in the No.2 MIRI model group increased(all P<0.05),while the level of SOD decreased(P<0.05).Compared with the No.1 MIRI group,myocardial tissue indicators of FUNDC1,PGAM5,B cell lymphoma-xL(Bcl-xL),light chain 3(LC3),autophagy associated protein 5(ATG5),and Beclin-1 level decreased(all P<0.05),the level of P62 increased(P<0.05),while the level of cysteinyl aspartate specific proteinase-9(Caspase-9)increased,but he difference is not statistically significant(P>0.05).Compared with the No.2 MIRI group and the inhibitor group,serum indicators of the AST,LDH,CK,and MDA levels in the Qishen Yiqi group decreased(all P<0.05),and the level of SOD increased(P<0.05).Compared with the No.2 MIRI group and the inhibitor group,myocardial tissue indicators of FUNDC1,PGAM5,Bcl-xL,LC3,ATG5,and Beclin-1 levels increased(all P<0.05),while the levels of P62 and Caspase-9 decreased(all P<0.05).Conclusions High blood sugar levels can aggravate MIRI.Qishen Yiqi Dripping Pills can regulate mitochondrial autophagy through the PGAM5/FUNDC1 pathway and alleviate myocardial ischemia-reperfusion injury.MIRI plays a protective role in the myocardium of diabetic rats.