肾衰方干预PINK1/Parkin介导的线粒体自噬治疗CKD心肌损伤的作用机制研究

Mechanism of Shenshuai recipe in PINK1/Parkin-mediated mitochondrial autophagy for treatment of CKD myocardial injury

目的探究PINK1/Parkin介导线粒体自噬在慢性肾病(chronic kidney disease,CKD)心肌损伤中的作用及肾衰方干预CKD心肌损伤大鼠的作用机制。方法将48只大鼠随机分成6组,分别为假手术组,模型组,贝那普利组,肾衰方低、中、高剂量组,采用5/6肾切除建立CKD心肌损伤模型,模型建立给予相应的干预2周,然后处死大鼠,取材进行相应的检测:用ELISA法测定大鼠的肌酸激酶同工酶(CK-MB)、高敏肌钙蛋白(hs-cTnI);对心脏组织进行HE染色后,光镜下观察组织变化;透射电镜观察自噬小体和自噬溶酶体;RT-PCR测定PINK1、Parkin、P62、LC3B mRNA水平;Western Blot测定肾切大鼠PINK1、Parkin、P62、LC3B蛋白水平。结果与假手术组相比,模型组CK-MB、hs-cTnI明显提升(P<0.01),与模型组相比,贝那普利组,肾衰方低、中、高剂量组均有明显降低(P<0.05);与假手术组相比,模型组肾切大鼠心肌组织明显紊乱、间质增生、血管壁增厚,呈玻璃样改变,贝那普利组和肾衰方低、中、高剂量组尚未见明显异常;与模型组相比,肾衰方低、中、高剂量组中电镜的自噬小体及自噬溶酶体的数量明显增多(P<0.01);与假手术组相比,模型组PINK1、Parkin、P62、LC3B mRNA明显下降(P<0.05),与模型组相比,贝那普利组和肾衰方低、中、高剂量组PINK1、Parkin、P62、LC3B mRNA明显升高(P<0.05);与假手术组相比,模型组大鼠PINK1、Parkin、P62、LC3B蛋白明显降低(P<0.01),与模型组相比,贝那普利组和肾衰方低、中、高剂量组的PINK1、Parkin、P62、LC3B蛋白明显升高(P<0.01)。结论5/6肾切CKD心肌损伤大鼠心肌细胞PINK1/Parkin线粒体自噬通路受到了抑制,肾衰方可以通过激活PINK1/Parkin线粒体自噬通路,增强线粒体自噬,发挥心肌细胞保护作用,其中肾衰方高剂量组线粒体自噬强度相关指标最为显著。

Objective To investigate the role of the PINK1/Parkin mitochondrial autophagy pathway in myocardial injury of chronic kidney disease,and the mechanism of Shenshuai recipe in the treatment of 5/6 nephrectomy CKD myocardial injury in rats.Methods Overall,48 nephrectomized rats were randomly divided into six groups:sham operated,model,benazepril,and low,medium,and high dose Shenshuai recipe groups.Rats were treated for 2 weeks and then sacrificed.Heart tissue was collected,stained with HE,and observed under a light microscope.Autophagosomes and autophagic lysosomes were observed by transmission electron microscopy.PINK1,Parkin,P62,and LC3B mRNA levels were measured by RT-PCR.PINK1,Parkin,P62,and LC3B protein levels in nephrectomized rats were measured by Western Blot.Results Compared with the sham-operated group,CK-MB and hs-cTnI were significantly increased in the model group(P<0.01).Compared with the model group,benazepril group,low,medium,and high dose Shenshuai recipe groups were significantly decreased(P<0.05).Compared with the sham operated group,myocardial tissue in the model group after nephrectomy was obviously disordered and displayed interstitial hyperplasia,vascular wall thickening,and glassy changes.There were no obvious abnormalities in Benazepril and low,medium,and high dose Shenshuai Recipe groups.Compared with the model group,the numbers of autophagosomes and autophagic lysosomes were increased significantly in low,middle,and high dose Shenshuai recipe groups(P<0.01).Compared with the sham operated group,PINK1,Parkin,P62,and LC3B mRNA levels were significantly decreased in the model group(P<0.05).Compared with the model group,PINK1,Parkin,P62,and LC3B mRNA levels were significantly increased in benazepril and high,medium,and low dose Shenshuai recipe groups(P<0.05).Compared with the sham operated group,PINK1,Parkin,P62,and LC3B protein levels were significantly decreased in the model group(P<0.01).Compared with the model group,PINK1,Parkin,P62,and LC3B protein levels were significantly increased in Benazepril and low-,medium-,and high-dose Shenshuai recipe groups(P<0.01).Conclusions The PINK1/Parkin mitochondrial autophagy pathway in myocardial cells of rats with myocardial injury caused by 5/6 nephrectomy was inhibited.Shenshuai recipe enhanced mitochondrial autophagy by activating the PINK1/Parkin mitochondrial autophagy pathway,and played a protective role in myocardial cells.Among the groups,the highest dose of Shenshuai recipe had the most significant effects on mitochondrial autophagy intensity.