人脐带间充质干细胞上调Nrf2信号改善衰老小鼠睾丸功能

Human umbilical cord mesenchymal stem cells improve testicular function in aging mice by upregulating Nrf2 signaling

目的探讨人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)移植对氧化损伤衰老小鼠睾丸功能的影响及分子机制。方法共纳入18只6~8周SPF级C57BL/C雄性小鼠,完全随机分组法分为3组,对照组:小鼠注射等量生理盐水;模型组:颈背部皮下注射D-半乳糖连续9周,造模第4周末,每只小鼠尾静脉注射生理盐水;hUCMSCs组:颈背部皮下注射D-半乳糖连续9周,造模第4周末,每只小鼠尾静脉注射hUCMSCs。9周后,称取小鼠体质量和睾丸重量,计算睾丸指数,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)法检测小鼠血清睾酮水平、睾丸组织丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)含量。肉眼观察睾丸外观,苏木精-伊红(hematoxylin-eosin,HE)染色观察睾丸组织病理学变化,实时荧光定量聚合酶链反应和Western blotting分别检测核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)信号转导相关基因和蛋白表达。结果模型组小鼠睾丸指数[(0.64±0.05)%]较对照组[(0.81±0.13)%,P=0.006]降低,睾丸体积缩小,生精小管完整性破坏,生精细胞和精子减少,间质稀疏;血清睾水平[(4.10±0.67)μg/L]、睾丸组织SOD活性[(48.87±6.40)U/mg Prot]较对照组[(5.71±0.81)μg/L,P=0.002;(78.53±9.70)U/mg Prot,P=0.001]均降低,而MDA含量[(1.11±0.19)nmol/mg Prot]较对照组[(0.77±0.07)nmol/mg Prot,P=0.001]增加。Keap1 mRNA和蛋白表达量较对照组均增高(P=0.006,P=0.043),Nrf2、SOD和NQO1 mRNA表达量较对照组减少(P=0.002,P<0.001,P=0.001),Nrf2、HO-1蛋白表达量较对照组均明显降低(P=0.011,P=0.021)。与模型组比较,hUCMSCs组小鼠睾丸指数[(0.79±0.03)%,P=0.010]增高;睾丸组织结构较为清晰、完整,生精小管各级生精细胞和精子及间质细胞较丰富;血清睾酮水平[(5.24±0.21)μg/L,P=0.028]及睾丸组织SOD活性[(79.47±14.32)U/mg Prot,P=0.001]增高,MDA含量[(0.77±0.08)nmol/mg Prot,P=0.001]降低;Nrf2、SOD和NQO1 mRNA表达量均增高(P=0.024,P=0.037,P=0.005),Keap1 mRNA表达量减少(P=0.044),Nrf2、HO-1蛋白表达量均增高(P=0.009,P=0.012),Keap1蛋白表达量降低(P=0.035)。hUCMSCs组小鼠睾丸指数、血清睾酮、SOD活性及MDA含量都与对照组差异均无统计学意义(均P>0.05)。结论hUCMSCs明显改善氧化损伤所致衰老小鼠睾丸结构和功能损伤,其作用机制与上调Nrf2信号转导及其相关下游抗氧化活性SOD、HO-1蛋白表达,减少Keap1介导的Nrf2降解有关。

Objective To investigate the effect and mechanism of human umbilical cord mesenchymal stem cells(hUCMSCs)transplantation on testicular function in aging mice with oxidative damage.Methods Totally 18 SPF grade C57BL/C male mice aged 6-8 weeks were randomly divided into 3 groups using a complete randomization method.In control group,mice were injected with an equal amount of physiological saline;in model group,mice were subcutaneous injected D-galactose into the neck and back for 9 consecutive weeks,on the 4th weekend of modeling,the mice were injected with physiological saline via the tail vein;in hUCMSCs group:mice were subcutaneous injected D-galactose into the neck and back for 9 consecutive weeks,on the 4th weekend of modeling,the mice were injected with hUCMSCs via the tail vein of each mouse.After 9 weeks,body weight and testicular weight of the three groups mice were measured and testicular index was calculated.The contents of testosterone,malondialdehyde(MDA)and superoxide dismutase(SOD)in serum and testicular tissue were detected by enzyme-linked immunosorbent assay(ELISA)method.Visual observation of testicular appearance,the histopathological changes of testis were observed by hematoxylin-eosin(HE)staining,and the expression of NF-E2-related factor 2(Nrf2)signal transduction-related genes and proteins were detected by RT-PCR and Western blotting,respectively.Results Compared with control group[(0.81±0.13)%],the testicular index of mice in model group[(0.64±0.05)%,P=0.006]was decreased.In model group,the volume of testis was reduced,the integrity of spermatogenic tubules was damaged,spermatogenic cells and sperm were reduced,and the interstitium was sparse.In model group,serum testosterone[(4.10±0.67)μg/L]and SOD[(48.87±6.40)U/mg Prot]were decreased compared with control group[(5.71±0.81)μg/L,P=0.002;(78.53±9.70)U/mg Prot,P=0.001],MDA[(1.11±0.19)nmol/mg Prot]was increased compared with control group[(0.77±0.07)nmol/mg Prot,P=0.001],Keap1 mRNA and protein expression were increased(P=0.006,P=0.043).The expression levels of Nrf2,SOD and NQO1 mRNA were significantly lower than those in control group(P=0.002,P<0.001,P=0.001),and the expression levels of Nrf2 and HO-1 protein were significantly lower than those in control group(P=0.011,P=0.021).Compared with the model group,the testicular index[(0.79±0.03)%,P=0.010]increased in hUCMSCs group,and the tissue structure of testis was clear and complete,spermatogenic cells at all levels of spermatogenic tubules,spermatogenic cells and stromal cells were abundant.Compared with the model group,the content of dihydrotestosterone[(5.24±0.21)μg/L,P=0.028]in serum and SOD[(79.47±14.32)U/mg Prot,P=0.001]in testicular tissue increased in hUCMSCs group,while the content of MDA[(0.77±0.08)nmol/mg Prot,P=0.001]decreased,Nrf2,SOD and NQO1 mRNA expression levels increased(P=0.024,P=0.037,P=0.005),Keap1 mRNA expression decreased(P=0.044),Nrf2 and HO-1 proteins expression increased(P=0.009,P=0.012),while Keap1 protein expression decreased(P=0.035).There were no statistically significant differences in testicular index,serum testosterone,SOD and MDA between hUCMSCs group and control group(all P>0.05).Conclusion hUCMSCs significantly improve testicular structure and function damage caused by oxidative damage in aging mice,and the mechanism of action is related to upregulating Nrf2 signaling and downstream antioxidant activity SOD and HO-1 protein expression,reducing Keap1 mediated Nrf2 degradation.