自噬在大鼠睾丸扭转致缺血再灌注损伤中的作用机制

Role of autophagy in ischemia/reperfusion injury caused by testicular torsion in rats

目的探讨自噬在大鼠睾丸扭转所致缺血再灌注损伤(I/RI)中的作用机制。方法将40只健康雄性SD大鼠随机分为假手术组、I/RI组、I/RI+雷帕霉素(RAPA)组和I/RI+3-甲基腺嘌呤(3-MA)组,每组10只。后3组大鼠建立左侧睾丸扭转(720°,1 h)复位模型,I/RI+RAPA组和I/RI+3-MA组大鼠造模前分别给予自噬激动剂RAPA和自噬抑制剂3-MA干预。于睾丸复位12 h后取各组大鼠左侧睾丸和附睾,收集精子进行精子质量分析,采用H-E染色观察睾丸组织病理变化,采用试剂盒检测睾丸组织中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、总抗氧化能力(T-AOC),采用TUNEL染色检测睾丸组织细胞凋亡水平,通过蛋白质印迹法检测睾丸组织中自噬相关蛋白(Beclin1、p62)和凋亡相关蛋白(Bcl2、Bax、cleaved caspase 3)表达水平。结果与假手术组比较,I/RI组大鼠精子质量下降,睾丸曲细精管排列松散,生殖细胞减少、脱落,睾丸组织SOD活性、T-AOC降低(均P<0.05),MDA含量升高(P<0.05),p62和Bcl2蛋白表达水平降低(均P<0.05),Beclin1、Bax和cleaved caspase 3蛋白表达水平升高(均P<0.05),凋亡生殖细胞明显增多。与I/RI组比较,I/RI+RAPA组大鼠精子质量提高,睾丸曲细精管排列正常,睾丸组织SOD活性、T-AOC升高(均P<0.05),MDA含量降低(P<0.05),Beclin1和Bcl2蛋白表达水平升高(均P<0.05),p62、Bax和cleaved caspase 3蛋白表达水平降低(均P<0.05),凋亡生殖细胞减少;I/RI+3-MA组大鼠精子质量下降,睾丸曲细精管排列松散,睾丸组织SOD活性、T-AOC降低(均P<0.05),MDA含量升高(P<0.05),Beclin1和Bcl2蛋白表达水平降低(均P<0.05),p62、Bax和cleaved caspase 3蛋白表达水平升高(均P<0.05),凋亡生殖细胞增多。结论激活自噬可改善I/RI大鼠精子质量和睾丸组织病理损伤,缓解氧化应激,抑制生殖细胞凋亡。

Objective To investigate the role of autophagy in ischemia/reperfusion injury(I/RI)caused by testicular torsion in rats.Methods Forty healthy male SD rats were randomly assigned(1∶1∶1)to sham group,model(I/RI)group,I/RI+rapamycin(RAPA)group,or I/RI+3-methyladenine(3-MA)group.Left testicular torsion(720°,1 h)reduction rat models were established in the latter 3 groups.The rats in the I/RI+RAPA group and I/RI+3-MA group were intervened with autophagy agonist RAPA and autophagy inhibitor 3-MA before modeling,respectively.At 12 h after testicular reduction,the left testis and epididymis of rats in each group were collected for sperm quality analysis.The histopathological changes of testicular tissue were observed by hematoxylin-eosin(H-E)staining.The levels of superoxide dismutase(SOD),malondialdehyde(MDA),and total antioxidant capacity(T-AOC)in testicular tissue were measured by kits.The level of apoptosis in testicular tissue was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay(TUNEL).The expression levels of autophagy-related proteins(Beclin1 and p62)and apoptosis-related proteins(Bcl2,Bax,and cleaved caspase 3)in testicular tissue were detected by Western blotting.Results Compared with the rats in the sham group,the rats in the I/RI group had poorer sperm quality,loosened testicular varicocele arrangement,fewer and shed germ cells,significantly lower SOD activity,T-AOC level,and p62 and Bcl2 expression levels(all P<0.05),significantly higher MDA content and expression levels of Beclin1,Bax,and cleaved caspase 3(all P<0.05),and more apoptotic germ cells.Compared with the rats in the I/RI group,the rats in the I/RI+RAPA group had better sperm quality,normalized testicular varicocele arrangement,significantly higher SOD activity,T-AOC level,Beclin1 and Bcl2 expression levels(all P<0.05),significantly lower MDA content,p62,Bax and cleaved caspase 3 expression levels(all P<0.05),and fewer apoptotic germ cells;the rats in the I/RI+3-MA group had poorer sperm quality,loosened testicular varicocele arrangement,significantly lower SOD activity,T-AOC level,Beclin1 and Bcl2 expression levels(all P<0.05),significantly higher MDA content,p62,Bax and cleaved caspase 3 expression levels(all P<0.05),and more apoptotic germ cells.Conclusion Activation of autophagy can improve the sperm quality and testicular histopathological damage in I/RI rats,alleviate oxidative stress,and inhibit germ cell apoptosis.