探讨活血养阴法调控Bcl-2/Bax通路改善精索静脉曲张大鼠生精功能的作用机制

Exploring the mechanism of regulating Bcl-2/Bax pathway by the therapy of promoting blood circulation and nourishing yin to improve spermatogenesis in rats with varicocele

目的基于B淋巴细胞瘤-2(Bcl-2)/Bcl-2相关X蛋白(Bax)通路探讨活血养阴法改善精索静脉曲张大鼠生精功能的作用机制。方法选取8周龄SD雄性大鼠30只,按随机数字表法分为假手术组、模型组、左卡尼汀组(1.0 g/kg)、活血养阴法低剂量组(5.2 g/kg)、活血养阴法中剂量组(10.4 g/kg)及活血养阴法高剂量组(20.8 g/kg),每组5只。除假手术组外,其余大鼠均通过经典的Turner法复制精索静脉曲张模型。术后4周,假手术组及模型组大鼠灌胃生理盐水,各给药组灌胃相应药物,每日1次,连续4周。灌胃结束后,使用计算机辅助精子分析仪检测精子质量,HE染色法观察睾丸组织病理学形态并通过Johnsen评分评价睾丸生精功能,TUNEL法检测睾丸细胞凋亡情况,实时荧光PCR法检测睾丸组织Bcl-2及Bax mRNA表达,免疫组织化学法检测细胞色素C蛋白表达。结果与假手术组比较,模型组大鼠的精子总数、精子活动率及(a+b)级精子率均降低(P<0.05);睾丸组织中生精小管排列紊乱,有明显空泡样改变,Johnsen评分下降(P<0.05);睾丸组织细胞凋亡率增加(P<0.05);睾丸组织Bax mRNA表达上升(P<0.05);细胞色素C蛋白阳性表达增加(P<0.05)。与模型组比较,活血养阴法中剂量组大鼠的精子总数,低、中剂量组精子活动率,以及低剂量组(a+b)级精子率均上升(P<0.05);大鼠睾丸病理学结构有不同程度的改善,活血养阴法中剂量组Johnsen评分增加(P<0.05);左卡尼汀组与活血养阴法各剂量组睾丸细胞凋亡率均下降(P<0.05);活血养阴法低、高剂量组Bcl-2 mRNA表达上升(P<0.05),左卡尼汀组与活血养阴法中剂量组Bax mRNA表达下降(P<0.05);活血养阴法中剂量组细胞色素C蛋白阳性表达下降(P<0.05)。结论活血养阴法可提高精索静脉曲张大鼠的精子质量,修复损伤的睾丸组织结构,改善生精功能,其作用机制可能与激活Bcl-2/Bax凋亡通路、抑制细胞凋亡有关。

Objective To explore the mechanism of the therapy of promoting blood circulation and nourishing yin(PBCNY)in improving spermatogenesis in rats with varicocele based on Bcl-2/Bax pathway.Methods Thirty 8-week-old male SD rats were randomly divided into six groups(five rats per group):sham,model,levocarnitine(1.0 g/kg),and PBCNY low-dose(5.2 g/kg),PBCNY mediumdose(10.4 g/kg),and PBCNY high-dose(20.8 g/kg)groups.Apart from the sham group,all the groups were built a varicocele model by the classical Turner method.After 4 weeks,the sham group and model group were gavaged with normal saline,and the remaining groups were given with levocarnitine or different doses of PBCNY decoction once a day for 4 weeks.At the end of gavage,the sperm quality of rats in each group was detected by a computer-assisted sperm analyzer,and HE staining was used to observe the testicular tissue morphology of rats in each group and performed with Johnsen score.TUNEL method was used to detect apoptosis,RT-qPCR was used to detect Bcl-2 and Bax mRNA expression,and immunohistochemical method was used to detect cytochrome C protein expression.Results Compared to the sham group,sperm total count,motility rate and percentage of sperm with grade(a+b)motility were lower in the model group of rats(P<0.05).HE staining showed disturbed arrangement of seminiferous tubules in the testicular tissue with significant changes and decreased Johnsen score(P<0.05).The result of TUNEL assay showed that the apoptosis rate was increased in the model group(P<0.05).Bax mRNA expression was increased in testicular tissue(P<0.05).The result of immunohistochemistry showed that the positive expression of cytochrome C was increased in the model group(P<0.05).Compared with the model group,the rat sperm total count in the PBCNY medium-dose group,motility rate in the PBCNY low-and medium-dose groups,and percentage of sperm with grade(a+b)motility in the PBCNY low-dose group increased(P<0.05);the pathological structure of rat testis had different degrees of improvement,and Johnsen score increased in the PBCNY medium-dose group(P<0.05);the apoptosis rate of testicular cells decreased in the levocarnitine group and all doses of PBCNY groups(P<0.05);the expression of Bcl-2 mRNA increased in the PBCNY low-dose and high-dose groups(P<0.05),and the expression of Bax mRNA decreased in the levocarnitine group and the PBCNY mediumdose group(P<0.05);the positive expression of cytochrome C decreased in the PBCNY medium-dose group(P<0.05).Conclusion The therapy of PBCNY can improve sperm quality,repair damaged testicular tissue structure and improve spermatogenic function in rats with varicocele,and its mechanism of action may be related to the activation of Bcl-2/Bax pathway and inhibition of cell apoptosis.