基于PINK1/Parkin介导的线粒体自噬与细胞凋亡探讨伸曲助育汤改善精索静脉曲张大鼠生精功能机制

Study on Mechanism of Shenqu Zhuyu Decoction in Improving Varicocele Rats Spermatogenesis Based on PINK1/Parkin Mediated Mitophagy and Apoptosis

目的基于PTEN诱导假定激酶1(PINK1)/E3泛素连接酶(Parkin)通路观察伸曲助育汤对精索静脉曲张(VC)大鼠左侧睾丸组织中线粒体自噬及细胞凋亡的影响,并探讨作用机制。方法将36只大鼠随机分为假手术组、模型组和伸曲助育汤组,每组12只,模型组和伸曲助育汤组采用左肾静脉部分结扎制备VC大鼠模型。造模成功48天后,伸曲助育汤组给予伸曲助育汤(0.7 g/100 g)灌胃,假手术组和模型组给予等量生理盐水灌胃,每天2次,连续干预60天。取各组大鼠左侧睾丸,采用HE染色观察大鼠睾丸组织形态学变化;电镜观察睾丸组织线粒体超微结构及线粒体自噬小体;Western Blot检测睾丸组织PINK1、Parkin、微管相关蛋白1轻链3B(LC3B)Ⅱ/Ⅰ、选择性自噬接头蛋白(p62)、B淋巴细胞瘤-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、裂解凋亡蛋白酶-3(Cleaved Caspase-3)的表达;采用免疫组织化学染色检测睾丸组织PINK1、Parkin的表达。结果与假手术组比较,模型组大鼠睾丸组织病理损伤严重,电镜下线粒体肿胀、形状不规则、伴有空泡化和嵴破裂,线粒体自噬小体形成增多,睾丸组织自噬相关蛋白PINK1、Parkin、LC3BⅡ/Ⅰ表达升高(P<0.01),p62表达降低(P<0.01),凋亡相关蛋白Bax、Cleaved Caspase-3表达升高(P<0.01,P<0.05),Bcl-2表达降低(P<0.05)。与模型组比较,伸曲助育汤组大鼠睾丸病理组织结构有明显改善,线粒体结构有所恢复,线粒体自噬小体减少,睾丸组织自噬相关蛋白PINK1、Parkin、LC3BⅡ/Ⅰ表达降低(P<0.01,P<0.05),p62蛋白表达升高(P<0.01),凋亡相关蛋白Bax、Cleaved Caspase-3表达降低(P<0.05),Bcl-2表达升高(P<0.05)。结论伸曲助育汤通过调控睾丸组织中PINK1/Parkin信号通路介导的线粒体自噬,减少生精细胞凋亡而改善VC大鼠睾丸生精功能。

Objective To observe the effect of the Shenqu Zhuyu Decoction(SZD)on varicocele(VC)rats mitophagy and apoptosis in left testis based on PTEN-induced putative kinase 1(PINK1)/E3 ubiquitin protein ligase(Parkin)signaling pathway,and to explore the mechanism.Methods Totally 36 male SD rats were randomly divided into sham operation group,model group and SZD group,12 rats in each group.VC rats model was established by partial ligation of the left renal vein in model group and SZD group.At 48th day after successful molding,the rats in SZD group were treated with SZD(0.7 g/100 g)by gavage,and rats in sham operation group and model group were given the equal volume of normal saline by gavage,twice a day for 60 days.The left testicle of the rats in each group were taken.HE staining was used to observe the morphology of testicles.Transmission electron microscopy was used to observe the ultrastructure of mitochondria and mitophagosomes in testicular tissue.Western Blot was used to detect protein expression of PINK1,Parkin,microtubule associated protein 1 light chain 3 beta(LC3B)Ⅱ/Ⅰ,sequestosome1(p62),B-cell lymghoma(Bcl-2),Bcl-2 associatal X protein(Bax),Cleaved Cysteine-containing aspartate-specific protease-3(Cleaved Caspase-3)in testicular tissue.The protein expressions of PINK1 and Parkin in testicular tissue were aslo detected by immunohistochemistry.Results Compared with the sham operation group,pathological changes occurred in the testis tissue of the model group,the mitochondria were swollen with irregular shapes,accompanied by vacuolization and mitochondrial crest rupture,increasing mitochondrial autophagosome formation was observed under transmission electron microscope,the expression of autophagy-related proteins PINK1,Parkin,LC3BⅡ/Ⅰincreased(P<0.01),expression of p62 protein decreased(P<0.01),the expression of apoptosisrelated proteins Bax,Cleaved Caspase-3 increased(P<0.01,P<0.05),while expression of Bcl-2 decreased(P<0.05).Compared with model group,pathological changes of testicular tissue and mitochondrial damage were recovered in SZD group,the formation of mitophagosomes decreased,the expression of autophagy-related proteins PINK1,Parkin,LC3BⅡ/Ⅰdecreased(P<0.01,P<0.05),while expression of p62 increased(P<0.01),the expression of apoptosis-related proteinsc Bax,Cleaved Caspase-3 decreased(P<0.05),while expression of Bcl-2 increased(P<0.05).Conclusion SZD could improve the spermatogenic function of VC rats by regulating PINK1/Parkin-mediated mitophagy and reducing apoptosis of spermatogenic cells.