竹节参总皂苷减轻高脂饮食诱导的小鼠睾丸支持细胞连接功能损伤

Protective effects of total saponins from Panax japonicus against high-fat diet-induced testicular Sertoli cell junction damage in mice

目的探究竹节参总皂苷(TSPJ)对高脂饮食诱导的小鼠睾丸支持细胞功能损伤的保护作用及机制。方法将40只C57BL/6J雄性小鼠随机分为正常饮食组、高脂饮食组、TSPJ低剂量(25 mg/kg)组和TSPJ高剂量(75 mg/kg)组,10只/组。正常饮食组给予普通饲料喂养,其余组均给予高脂饲料喂养,TSPJ连续灌胃给药5月,小鼠称重处死,取出睾丸和附睾组织称重并计算其指数;取附睾组织进行精液分析;HE染色观察小鼠睾丸组织形态变化,测量统计生精上皮厚度与生精小管直径;Western blot检测睾丸支持细胞紧密连接功能蛋白ZO-1、Occludin和Claudin11以及外质特化功能蛋白N-cadherin、E-cadherin和β-catenin的表达水平;免疫荧光法检测睾丸组织中ZO-1和β-catenin蛋白定位及表达;免疫荧光双标法检测睾丸支持细胞中LC3B、p-AKT和p-mTOR表达变化。结果与高脂饮食组相比,TSPJ能明显减轻高脂饮食小鼠体质量(P<0.001),增加睾丸和附睾指数(P<0.05,P<0.01),提高精子浓度与精子活率(P<0.05,P<0.01),改善睾丸组织形态异常,增加生精上皮厚度(P<0.05,P<0.001),但对生精小管直径无明显影响。Western blot结果显示,TSPJ能增加ZO-1、Occludin、N-cadherin、E-cadherin和β-catenin的蛋白表达水平(P<0.05,P<0.01,P<0.001),但对Claudin11的表达无显著性影响;免疫荧光结果显示,TSPJ能增加睾丸组织中ZO-1和β-catenin蛋白的表达(P<0.001),下调睾丸支持细胞中LC3B的表达,上调p-AKT和p-mTOR蛋白表达。结论TSPJ能减轻高脂饮食诱导的睾丸支持细胞连接功能损伤,改善小鼠生精功能减退,其机制可能与激活支持细胞AKT/mTOR信号通路抑制自噬有关。

Objective To investigate the protective effects of total saponins from Panax japonicus(TSPJ)against high-fat diet induced testicular Sertoli cell junction damage in mice.Methods Forty male C57BL/6J mice were randomized into normal diet group,high-fat diet group,and low-dose(25 mg/kg)and high-dose(75 mg/kg)TSPJ treatment groups(n=10).The mice in the normal diet group were fed a normal diet,while the mice in the other groups were fed a high-fat diet.After TSPJ treatment via intragastric administration for 5 months,the testes and epididymis of the mice were collected for measurement of weight,testicular and epididymal indices and sperm parameters.HE staining was used for histological evaluation of the testicular tissues and measurement of seminiferous tubule diameter and seminiferous epithelium height.The expression levels of ZO-1,occludin,claudin11,N-cadherin,E-cadherin andβ-catenin in Sertoli cells were detected with Western blot,and the localization and expression levels of ZO-1 andβ-catenin in the testicular tissues were detected with immunofluorescence assay.The protein expressions of LC3B,p-AKT and p-mTOR in testicular Sertoli cells were detected using double immunofluorescence assay.Results Treatment with TSPJ significantly improved high-fat diet-induced testicular dysfunction by reducing body weight(P<0.001),increasing testicular and epididymal indices(P<0.05),and improving sperm concentration and sperm viability(P<0.05).TSPJ ameliorated testicular pathologies and increased seminiferous epithelium height of the mice with high-fat diet feeding(P<0.05)without affecting the seminiferous tubule diameter.TSPJ significantly increased the expression levels of ZO-1,occludin,N-cadherin,E-cadherin andβ-catenin(P<0.05)but did not affect claudin11 expression in the testicular tissues.Immunofluorescence assay showed that TSPJ significantly increased ZO-1 andβ-catenin expression in the testicular tissues(P<0.001),downregulated LC3B expression and upregulated p-AKT and p-mTOR expressions in testicular Sertoli cells.Conclusion TSPJ alleviates high-fat diet-induced damages of testicular Sertoli cell junctions and spermatogenesis possibly by activating the AKT/mTOR signaling pathway and inhibiting autophagy of testicular Sertoli cells.