补肾化痰祛瘀方对铜负荷肝豆状核变性模型小鼠卵泡发育障碍的影响

Effects of Bushen Huatan Quyu Fang on follicular dysgenesis in copper-loaded mice models with hepatolenticular degeneration

目的探讨补肾化痰祛瘀方对铜负荷小鼠卵泡发育障碍的影响及其作用机制。方法将雌性C57小鼠随机分为6组,即正常组、铜负荷模型组、二巯丁二酸组、肝豆灵组、补肾化痰祛瘀方组、补肾化痰祛瘀方+二巯丁二酸组,每组20只。采用高铜复制铜负荷小鼠模型,即以100 mg/kg硫酸铜(CuSO4·5H2O)每天上午同一时间灌胃1次,正常组用等量的生理盐水灌胃,共28 d。在给铜的第15天开始除正常组和模型组灌胃生理盐水外,其他4组每日分别灌胃给药,二巯丁二酸组0.16 g/kg、肝豆灵组1.2 g/kg、补肾化痰祛瘀方组1.5 g/kg、补肾化痰祛瘀方+二巯丁二酸组,共14 d至给铜的第28天结束。运用促性腺激素促排方法检测小鼠排卵数,苏木精-伊红染色观察各级卵泡发育情况,电镜观察小鼠卵丘复合物超微结构,采用免疫荧光染色法检测卵丘复合物颗粒细胞Ki67的表达水平以观察细胞增殖情况。结果与正常组比较,各组小鼠排卵数、正常次级卵泡和窦卵泡数、正常线粒体数目和颗粒细胞Ki67表达水平显著减少(P<0.05);异常线粒体数目显著增加(P<0.05);补肾化痰祛瘀方组上述指标较模型组和肝豆灵组改善明显(P<0.05),补肾化痰祛瘀方+二巯丁二酸组也优于二巯丁二酸组(P<0.05)。结论补肾化痰祛瘀方能增加铜负荷小鼠排卵数,促进铜负荷小鼠卵泡的发育,改善其线粒体超微结构,提高其卵丘复合物颗粒细胞的增殖能力。

Objective To investigate the effect of Bushen Huatan Quyu Fang(kidney-tonifying,phlegm-resolving,and blood-stasis-eliminating formula,BSHTQYF)on follicular dysgenesis in copper-loaded mice and elucidate its underlying mechanism.Methods Female C57 mice were randomly divided into 6 groups:1)normal group,2)copper-loaded model group,3)dimercaptosuccinic acid(DMSA)group,4)Gandouling(Cure for Hepatolenticular Degeneration)(GDL)group,5)BSHTQYF group and 6)BSHTQYF+DMSA group,with 20 mice in each group.To establish copper-loaded models,mice were administered with copper sulfate(CuSO4·5 H2O)solution(100 mg/kg)by gavage once a day for 28 days while normal saline was given to the normal group.Since the 15th day of copper administration,the DMSA group was treated with DMSA(0.16 g/kg),the GDL group with GDL(1.2 g/kg),the BSHTQYF group with BSHTQYF(1.5 g/kg)and the BSHTQYF+DMSA group with DMSA and BSHTQYF by gavage once a day for 14 days while the normal group and the model group were given normal saline.HCG was injected to induce ovulation and the number of eggs released was counted.Follicular development was observed with hematoxylin-eosin staining.Ultrastructure of mouse cumulus complex was observed with electron microscopy.The expression of cumulus complex granulosa cell Ki67 was observed with immunofluorescence.Results Compared with the normal group,the number of eggs released,the number of normal secondary follicles and antral follicles,the number of normal mitochondria and the expression of Ki67 in granulosa cells in the copper-loaded mice of all the other groups were significantly decreased(P<0.05).Meanwhile,the number of abnormal mitochondria increased significantly in the copper-loaded mice,compared with the normal group(P<0.05).The above parameters were significantly improved in the BSHTQYF group,compared with the model group and the GDL group(P<0.05),and those in the BSHTQYF+DMSA group were significantly better than those in the DMSA group(P<0.05).Conclusion BSHTQYF can increase the number of eggs released by copper-loaded mice,promote the development of follicles in copper-loaded mice,improve the ultrastructure of mitochondria,and enhance the proliferation of cumulus complex granulosa cells.