自噬调控对小鼠精原细胞缺氧/复氧损伤的影响研究

Effect of autophagy regulation on hypoxia/reoxygenation injury of mouse spermatogonia

目的研究自噬调控对小鼠精原细胞缺氧/复氧损伤(H/RI)的影响,探讨自噬对小鼠睾丸组织缺血再灌注损伤(IRI)的影响。方法以小鼠精原细胞系GC1 spg为研究对象构建H/RI模型,雷帕霉素(RAPA)和3-甲基腺嘌呤(3-MA)分别作为自噬激动剂和抑制剂,设置对照组、模型组(H/RI)、自噬激动剂干预组(H/RI+自噬激动剂)、自噬抑制剂干预组(H/RI+自噬抑制剂)。用噻唑蓝(MTT)法检测各组细胞增殖能力,流式细胞术检测各组细胞活性氧(ROS)释放水平和凋亡水平,实时荧光定量PCR(qPCR)检测各组细胞自噬相关基因Beclin1和凋亡相关基因Bcl-2、Bax mRNA的表达水平,Western blot检测各组细胞自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ、Beclin1、p62和凋亡相关蛋白Bcl-2、Bax、caspase-3的表达水平。结果与对照组比较,模型组增殖能力、Beclin1和Bcl-2 mRNA表达水平明显下降(P<0.01),p62和Bcl-2蛋白表达水平明显下降(P<0.01),ROS水平、细胞凋亡率、Bax mRNA相对表达水平明显上升(P<0.01),Beclin1、Bax、caspase-3蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ蛋白比值明显上升(P<0.01)。与模型组比较,自噬激动剂干预组细胞增殖能力、Beclin1和Bcl-2 mRNA表达水平明显上升(P<0.01),Beclin1、Bcl-2蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ蛋白比值均明显上升(P<0.01),ROS水平、细胞凋亡率、Bax mRNA表达水平明显下降(P<0.01),p62、Bax、caspase-3蛋白表达水平明显下降(P<0.01)。与模型组比较,自噬抑制剂干预组细胞增殖能力、Beclin1和Bcl-2 mRNA表达水平明显下降(P<0.01),Beclin1、Bcl-2蛋白表达水平和LC3-Ⅱ/LC3-Ⅰ蛋白比值明显下降(均P<0.01),ROS水平、细胞凋亡率、Bax mRNA表达水平明显上升(P<0.01),p62、Bax、caspase-3蛋白表达水平明显上升(P<0.01)。结论增强自噬能抑制小鼠精原细胞凋亡,修复H/RI,为治疗睾丸组织IRI提供了理论基础。

Objective To study the effect of autophagy regulation on hypoxia/reoxygenation injury(H/RI)in mouse spermatogonia,and to explore the effect of autophagy on ischemia-reperfusion injury(IRI)in mouse testicular tissue.Methods The mouse spermatogonial cell line GC1 spg was used as the research object to construct the H/RI model.Rapamycin(RAPA)and 3-methyladenine(3-MA)were used as autophagy agonists and inhibitors.The control group,the model group,the autophagy agonist intervention group(H/RI+autophagy agonist intervention),and the autophagy inhibitor intervention group(H/RI+autophagy inhibitor intervention)were set up.The cells proliferation ability of each group was detected by methyl thiazol tetrazolium(MTT)method.The release level of reactive oxygen species(ROS)and apoptosis level of each group were detected by flow cytometry.The expression levels of autophagy-related gene Beclin1 and apoptosis-related genes Bcl-2 and Bax mRNA in each group were detected by real-time fluorescence quantitative PCR(qPCR).The expression levels of autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ,Beclin1,p62 and apoptosis-related proteins Bcl-2,Bax,caspase-3 in each group were detected by Western blot.Results Compared with the control group,the proliferation abiliy,the expression levels of Beclin1 and Bcl-2 mRNA in the model group were significantly decreased(P<0.01),the relative expression levels of p62 and Bcl-2 proteins were significantly decreased(P<0.01).The ROS level,apoptosis rate and the mRNA expression level of Bax were significantly increased(P<0.01),and the protein expresion levels of Beclin1,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰwere significantly increased(P<0.01).Compared with the model group,the cell proliferation a-bility,the expression levels of Beclin1 and Bcl-2 mRNA in the autophagy agonist intervention group were the protein ratio of significantly increased(P<0.01),the protein expression levels of Beclin1 and Bcl-2 and the protein ration of LC3-Ⅱ/LC3-Ⅰwere significantly increased(P<0.01).The ROS level,apoptosis rate and the expression level of Bax mRNA were significantly decreased(P<0.01),and the protein expression levels of p62,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰwere significantly decreased(P<0.01).Com-pared with the model group,the cell proliferation ability,the mRNA expression levels of Beclin1 and Bcl-2 in the autophagy inhibitor intervention group were significantly decreased(P<0.01),the protein expression lev-els of Beclin1 and Bcl-2 protein were significantly decreased(P<0.01).The ROS level,apoptosis rate and the mRNA expression level of Bax were significantly increased(P<0.01),and the protein expression levels of p62,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰwere significantly iecreased(P<0.01).Conclusion Enhanced au-tophagy can inhibit apoptosis of spermatogonia and repair H/RI in mice,which provides a theoretical basis for the treatment of testicular tissue IRI。