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自噬在大鼠睾丸扭转致缺血再灌注损伤中的作用机制
胡志1,徐律2,孙伟2,付桥2,张炜2,陈一衍2,褚浩2*
0
(1. 武汉大学附属同仁医院(武汉市第三医院)光谷院区泌尿外科, 武汉 430014;
2. 武汉大学附属同仁医院(武汉市第三医院)泌尿外科, 武汉 430000
*通信作者)
摘要:
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。
关键词:  睾丸扭转  缺血再灌注损伤  自噬  氧化性应激  生殖细胞  细胞凋亡
DOI:10.16781/j.CN31-2187/R.20230051
投稿时间:2023-02-15修订日期:2023-07-10
基金项目:武汉市卫生健康委员会面上项目重点项目(WX20A16).
Role of autophagy in ischemia/reperfusion injury caused by testicular torsion in rats
HU Zhi1,XU Lü2,SUN Wei2,FU Qiao2,ZHANG Wei2,CHEN Yiyan2,CHU Hao2*
(1. Department of Urology, Guanggu Branch of Tongren Hospital of Wuhan University (Wuhan Third Hospital), Wuhan 430014, Hubei, China;
2. Department of Urology, Tongren Hospital of Wuhan University (Wuhan Third Hospital), Wuhan 430000, Hubei, China
*Corresponding author)
Abstract:
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.
Key words:  testicular torsion  ischemia/reperfusion injury  autophagy  oxidative stress  germ cells  apoptosis