栝楼薤白半夏汤对心肌缺血再灌注损伤大鼠自噬及PINK1/Parkin通路作用研究

Effects of Gualou Xiebai Banxia Decoction on autophagy and PINK1/parkin pathway in rats with myocardial ischemia-reperfusion injury

目的:观察栝楼薤白半夏汤对心肌缺血再灌注损伤(MI/RI)大鼠心肌自噬的影响,并研究PINK1/Parkin通路在其中可能发挥的作用。方法:将60只SD大鼠按随机数字表法分为假手术组、MI/RI模型组、3-MA(3-Methyladenine,3-甲基腺嘌呤)抑制剂组、栝楼薤白半夏汤高剂量组、栝楼薤白半夏汤低剂量组,采用可逆性冠脉左前降支结扎缺血30 min、再灌注2 h复制MI/RI模型,透射电镜下观察心肌组织超微结构,全自动生化仪检测血清中肌酸激酶同工酶MB(CK-MB)、乳酸脱氢酶(LDH)含量,Western Blot法测定心肌组织自噬相关蛋白Beclin-1、选择性自噬接头蛋白P62、线粒体融合蛋白2(Mfn2)、PTEN诱导假定激酶1(Pink1)、Parkin蛋白的表达。结果:假手术组大鼠心肌肌丝致密排列,线粒体结构正常;MI/RI模型组大鼠心肌肌丝断裂,心肌线粒体增大变形,自噬体形成;3-MA抑制剂组、栝楼薤白半夏汤高、低剂量组心肌肌丝及线粒体结构基本保持相对完整,自噬体较少。与假手术组比较,MI/RI模型组大鼠血清CK-MB、LDH含量及心肌组织Beclin-1、P62、Pink1、Parkin蛋白表达均显著升高而Mfn2蛋白表达显著降低;与MI/RI模型组比较,3-MA抑制剂组、栝楼薤白半夏汤高、低剂量组大鼠血清CK-MB、LDH含量及心肌Beclin-1、P62、Pink1、Parkin蛋白表达显著下降而Mfn2蛋白表达显著上升。结论:MI/RI可激活Pink1/Parkin线粒体自噬通路,导致过度自噬而加重心肌损伤,栝楼薤白半夏汤可通过抑制Pink1/Parkin通路活化,减轻线粒体自噬而发挥心肌保护作用。

Objective:To study the effect of Gualou Xiebai Banxia decoction(GXBD)on myocardial autophagy in rats with myocardial ischemia reperfusion injury(MI/RI)and the possible role of PINK1/Parkin pathway in this disease.Methods:60 SD rats were randomly divided into sham-operated group,MI/RI model group,3-methyladenine(3-MA)group,GXBD high-dose group and GXBD low-dose group.The MI/RI model was reproduced by ligation of left descending artery for 30 min and followed by releasing the ligation for 2 hours in rats.Ultrastructure of myocardial tissue was observed under the transmission electron microscope.Serum contents of creatine kinase-MB(CK-MB)and lactate dehydrogenase(LDH)were measured by automatic biochemical analyzer.The protein levels of the autophagy-related protein Beclin-1,the selective autophagy-adaptor protein p62,mitofusin 2(Mfn2),the PTEN-inducible kinase 1(Pink1)and Parkin were analyzed by Western Blot.Results:The rats of sham-operated group showed that the cardiac myofilament was well in alignment and mitochondrial structure was normal.In the MI/RI model group,it was observed that the myocardial myofilaments were broken,the myocardial mitochondria were enlarged and deformed,and the autophagosomes formed finally.In contrast,the myocardial myofilaments and mitochondria structure remained relatively intact and there were fewer autophagosomes in the 3-MA inhibitor group,high-dose and low-dose groups of GXBD.Compared with sham-operated group,the contents of CK-MB,LDH and the protein expression of Beclin-1,P62,Pink1 and Parkin were all significantly increased,while the expression of Mfn2 was evidently decreased in MI/RI model group.Compared with MI/RI model group,the contents of CK-MB,LDH and the protein expression of Beclin-1,P62,Pink1 and Parkin were all obviously decreased,while the expression of Mfn2 was increased in the 3-MA inhibitor group,high-dose and low-dose groups of GXBD.Conclusions:MI/RI can activate the Pink1/Parkin pathway,which lead to excessive mitochondrial autophagy and aggravation of myocardial damage.GXBD may exert myocardial protection by inhibiting Pink1/Parkin pathway activation and reducing mitochondrial autophagy.