人参皂苷Rg1联合人参皂苷Rg3对雄性生殖功能损伤模型小鼠生殖功能的改善作用

Improvement effect of ginsenoside Rg1 combined with ginsenoside Rg3 on reproductive function in male model mice with reproductive function injury

目的:探讨人参皂苷Rg1联合人参皂苷Rg3对邻苯二甲酸二丁酯(DBP)诱导的雄性生殖功能损伤模型小鼠生殖功能的改善作用,阐明人参皂苷Rg1和人参皂苷Rg3对生殖功能保护作用的相关机制,为其深入开发提供实验依据。方法:28只清洁级雄性C57BL/6小鼠随机分为模型组(400 mg·kg^-1 DBP)、人参皂苷Rg1+DBP组(20 mg·kg^-1人参皂苷Rg1,400 mg·kg^-1 DBP)、人参皂苷Rg3+DBP组(20 mg·kg^-1人参皂苷Rg3,400 mg·kg^-1 DBP)和联合组(20 mg·kg^-1人参皂苷Rg1,20 mg·kg^-1人参皂苷Rg3,400 mg·kg^-1 DBP),每组7只。DBP溶于玉米油于每天上午灌胃给药,人参皂苷Rg1和人参皂苷Rg3溶于生理盐水于每天下午皮下注射给药,每天各给药1次,连续给药35 d。采用WLJY-9000型精子质量检测系统检测各组小鼠的精子密度、精子活力和总畸形率,HE染色观察小鼠睾丸组织病理形态表现,采用酶联免疫吸附实验(ELISA)检测小鼠血清中睾酮(T)、卵泡刺激素(FSH)和黄体生成素(LH)水平,采用免疫荧光法检测睾丸组织缝隙连接蛋白43(Cx43)蛋白表达水平。结果:与模型组比较,联合组小鼠睾丸脏器系数明显升高(P<0.05),人参皂苷Rg1+DBP组、人参皂苷Rg3+DBP组和联合组小鼠精子密度和精子活力明显升高(P<0.01);与模型组比较,人参皂苷Rg1+DBP组、人参皂苷Rg3+DBP组和联合组小鼠血清中T和LH水平明显升高(P<0.01),FSH水平明显降低(P<0.01),小鼠睾丸组织中Cx43蛋白表达水平明显升高(P<0.05)。HE染色,模型组小鼠睾丸组织生精上皮较薄,管腔中生精细胞严重脱落;与模型组比较,人参皂苷Rg1+DBP组和人参皂苷Rg3+DBP组小鼠睾丸组织生精上皮变厚,管腔中脱落细胞较少;联合组小鼠睾丸组织生精上皮结构完整,各级生精细胞排列规则,腔内精子丰富。析因分析,人参皂苷Rg1联合人参皂苷Rg3可致小鼠精子活力(F=6.704,P=0.016)、血清中T水平(F=4.912,P=0.036)和睾丸组织中Cx43蛋白表达水平(F=6.937,P=0.018)升高。结论:人参皂苷Rg1和人参皂苷Rg3单独使用及人参皂苷Rg1与人参皂苷Rg3联合使用,可提高精子密度和精子活力,使小鼠血清中T和LH水平升高,FSH水平降低,减轻小鼠生殖功能损伤,其作用机制可能与上调Cx43表达水平发挥生精保护作用有关,可抑制DBP所致的生殖功能损伤,且人参皂苷Rg1与Rg3联合使用表现为协同作用。

Objective:To investigate the improvement effect of ginsenoside Rg1 combined with ginsenoside Rg3 on the reproductive function in the dinbutyl phthalate(DBP)-induced reproductive function injury model mice,to elucidate the mechanism of ginsenoside Rg1 combined with ginsenoside Rg3 in the protection of reproductive function,and to provide the experimental basis for their further development.Methods:Twenty-eight clean grade male C57BL/6 mice were randomly divided into model group(400 mg·kg^-1 DBP),ginsenoside Rg1+DBP group(20 mg·kg^-1 ginsenoside Rg1+400 mg·kg^-1 DBP),ginsenoside Rg3+DBP group(20 mg·kg^-1 ginsenoside Rg3+400 mg·kg^-1 DBP)and combination group(20 mg·kg^-1 ginsenoside Rg1+20 mg·kg^-1 ginsenoside Rg3+400 mg·kg^-1 DBP);there were 7 mice in each group.DBP dissolved in corn oil was given to the mice by gavage every morning,ginsenoside Rg1 and ginsenoside Rg3 were dissolved in normal saline and injected subcutaneously in the mice every afternoon,and DBP,ginsenoside Rg1 and ginsenoside Rg3 were administered once a day for 35 consecutive days.The sperm density,sperm viability and total malformation rate of the mice in each group were detected by Wljy-9000 sperm quality detection system.HE staining was used to observe the patholmorphology of testis tissue of the mice.The levels of serum testosterone(T),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)were detected by enzyme-linked immunosorbent assay(ELISA).The expression level of Cx43 protein in testis tissue was detected by immunofluorescence assay.Results:Compared with model group,the testicular organ coefficient of the mice in combination group was significantly increased(P<0.05),and the sperm densities and the sperm viabilities of the mice in ginsenoside Rg1+DBP group,ginsenoside Rg3+DBP group and combination group were significantly increased(P<0.01).Compared with model group,the levels of serum T and LH of the mice ginsenoside Rg1+DBP group,ginsenoside Rg3+DBP group and combination group were significantly increased(P<0.01)and the FSH levels were significantly decreased in(P<0.01);the expression levels of Cx43 protein were significantly increased(P<0.05).The HE staining results showed that the spermatogenic epithelium of the testis tissue of the mice in model group was relatively thin,and the spermatogenic cells in the duct cavity were seriously exfoliated;compared with model group,the spermatogenic epithelium of the testis tissue of the mice in ginsenoside Rg1+DBP group and ginsenoside Rg3+DBP group became thicker,and fewer exfoliated cells were found in the lumen;the spermatogenic epithelial structure of the testis tissue in combination group was complete,with regular spermatogenic cells at all levels and abundant intracellular sperm.The result of factorial analysis showed that there was an interaction between ginsenoside Rg1 and ginsenoside Rg3,which resulted in the increase of sperm viability(F=6.704,P=0.016),the serum T level(F=4.912,P=0.036)and the ex pression level of Cx43 protein in testis tissue(F=6.937,P=0.018).Conclusion:Ginsenoside Rg1 and ginsenoside Rg3 used alone or combined can improve the sperm density and the sperm viability,increase the serum T and LH levels,decrease the FSH level in the mice,and reduce the reproductive function injury in the mice,and its mechanism might be related to the protective role in spermatogenesis by up-regulating the expression level of Cx43.Ginsenoside Rg1 combined with ginsenoside Rg3 has a synergistic effect.