基于铁死亡探讨参苓白术散对高尿酸血症小鼠睾丸生精功能的改善作用

Investigate the effect of Shenling Baizhu Powder on the spermatogenic function of the testes in hyperuricemic mice based on ferroptosis

目的基于铁死亡探讨参苓白术散改善高尿酸血症小鼠睾丸生精功能的作用。方法60只BALB/c小鼠按随机数字表平均分为正常组,模型组,参苓白术散高、中、低剂量组(灌胃,20.14、10.07、5.04 g/kg)及3-氨基-4-环己基氨基苯甲酸乙酯(Fer-1)组(尾静脉注射,0.8 mg/kg)。除正常组外,其他各组小鼠通过连续7 d腹腔注射氧嗪酸钾悬液[600 mg/(kg·d)],复制高尿酸血症模型。造模成功后,各组分别给予相应药物,连续14 d。生化法检测血清尿酸,睾丸组织Fe2+、还原型谷胱甘肽(GSH)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;测定附睾、睾丸指数;HE染色法评估睾丸生精功能;动物精子全自动分析仪检测精子质量;加强普鲁士蓝染色法检测睾丸组织铁沉积;免疫组化法检测睾丸组织酰基辅酶A合成酶长链家族成员4(ACSL4)、谷胱甘肽过氧化物酶4(GPX4)蛋白表达;蛋白免疫印迹检测睾丸组织核因子E2相关因子2(Nrf2)/血红素加氧酶1(HO-1)/GPX4通路相关蛋白表达水平。结果与正常组比较,模型组小鼠血清尿酸、睾丸组织MDA、Fe2+含量升高,GSH含量、SOD活性降低,附睾及睾丸指数、睾丸生精功能、精子密度和活动率降低,睾丸组织铁沉积、ACSL4蛋白表达升高,Kelch样ECH相关蛋白1(Keap1)、细胞质Nrf2蛋白表达量升高,细胞核Nrf2、HO-1、GPX4、溶质载体家族7成员11(SLC7A11)蛋白表达均降低(P<0.01)。与模型组比较,参苓白术散各剂量组及Fer-1组以上指标均有不同程度改善(P<0.05,P<0.01)。结论参苓白术散可通过Nrf2/HO-1/GPX4信号通路抑制睾丸细胞铁死亡,改善高尿酸血症生精功能障碍小鼠的睾丸生精功能。

Objective We aimed to investigate the effect of ferroptosis on Shenling Baizhu Powder,a compound prescription of Chinese herbal medicine,in improving testicular spermatogenic function in hyperuricemic mice with spermatogenic dysfunction.Methods Sixty BALB/c mice were randomly divided into normal group,model group,Shenling Baizhu Powder high-,medium-,and low-dose groups(20.14,10.07,5.04 g/kg,by gavage),and ferrostatin-1(Fer-1)group(0.8 mg/kg,by tail vein injection),with 10 mice each group.Except for the normal group,the other groups were intraperitoneally injected with potassium oxonate suspension[600 mg/(kg·d)]for 7 days to establish the hyperuricemic model,and then the corresponding intervention was given for consecutive 14 days.Content of serum uric acid(UA),testicular Fe2+,reduced glutathione(GSH),malondialdehyde(MDA)and superoxide dismutase(SOD)activity were detected by biochemical method.Epididymal and testicular indices were measured.The spermatogenic function of testes was evaluated by eosin-hematoxylin staining.Sperm quality was detected by an automatic animal sperm analyzer.Prussian blue staining was used to detect iron deposition in testicular tissue.Immunohistochemistry was used to detect the related protein expressions of acyl-coenzyme A synthetase long-chain family member 4(ACSL4)and glutathione peroxidase 4(GPX4)in testicular tissue.Western blotting was used to detect the related protein expression levels of nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/GPX4 signaling pathway in testicular tissue.Results Compared with the normal group,the contents of serum UA,MDA,and Fe2+in the testis tissue of the model group were increased,the GSH content and SOD activity were decreased,the epididymal and testicular index,testicular spermatogenic function,sperm density and activity rate were decreased,and the iron deposition and ACSL4 protein expression in the testis tissue were increased.The expressions of kelch-like ECH-associated protein-1(Keap1)and Nrf2 were increased.The expressions of nuclear Nrf2,HO-1,GPX4,and recombinant solute carrier family 7 member 11(SLC7A11)protein were decreased(P<0.01).Compared with the model group,the above indexes in the Shenling Baizhu Powder groups and the Fer-1 group were improved to varying degrees(P<0.05,P<0.01).Conclusion Shenling Baizhu Powder can inhibit the ferroptosis of testicular cells through the Nrf2/HO-1/GPX4 signaling pathway,and improve the testicular spermatogenic function of mice with hyperuricemia spermatogenic dysfunction.