益肾疏肝汤对卵巢储备功能减退模型大鼠卵巢颗粒细胞凋亡的影响

Effects of Yishen Shugan Decoction on Apoptosis of Ovarian Granulosa Cells in Rats with Diminished Ovarian Reserve

目的观察益肾疏肝汤对雷公藤多苷所致卵巢储备功能减退(DOR)模型大鼠卵巢颗粒细胞凋亡的影响,探讨其改善DOR的作用机制。方法32只SD大鼠按随机数字表法分为空白组8只和造模组24只,空白组不予处理,造模组予雷公藤多苷片混悬液50 mg/kg灌胃14 d制备DOR模型,成模后将造模组再随机分为模型组、中药低剂量组和中药高剂量组,每组8只。中药低、高剂量组分别予益肾疏肝汤16.38、32.76 g/kg灌胃,空白组和模型组予等体积生理盐水,连续15 d。观察大鼠动情周期变化,计算卵巢指数和子宫指数,HE染色观察卵巢组织形态,ELISA检测血清抗米勒管激素(AMH)、促卵泡素(FSH)、雌二醇(E2)含量,TUNEL染色检测卵巢颗粒细胞凋亡情况,Western blot检测卵巢组织Bax、Caspase-3、Bcl-2蛋白表达。结果与空白组比较,模型组大鼠动情周期紊乱,卵巢指数和子宫指数明显降低(P<0.01),血清AMH、E2含量明显减少(P<0.01),FSH含量明显增加(P<0.01);各级卵泡及成熟卵泡数目明显减少,闭锁卵泡数目增多,卵巢颗粒细胞凋亡率明显增加(P<0.01);卵巢组织Bax、Caspase-3蛋白表达明显升高(P<0.01),Bcl-2蛋白表达及Bcl-2/Bax比值明显降低(P<0.01)。与模型组比较,中药低、高剂量组大鼠动情周期趋于正常,卵巢指数和子宫指数明显升高(P<0.05),血清AMH、E2含量明显增加(P<0.01),FSH含量明显减少(P<0.01);各级卵泡及成熟卵泡数目增加,闭锁卵泡数目减少,卵巢颗粒细胞凋亡率明显减少(P<0.05);卵巢组织Bax、Caspase-3蛋白表达明显降低(P<0.05,P<0.01),Bcl-2蛋白表达及Bcl-2/Bax比值明显升高(P<0.05,P<0.01)。结论益肾疏肝汤能改善DOR模型大鼠病理状态,提高卵巢储备功能,其机制可能与抑制卵巢颗粒细胞凋亡,上调Bcl-2、下调Bax及Caspase-3蛋白表达有关。

Objective To observe the effects of Yishen Shugan Decoction on apoptosis of ovarian granulosa cells in rats with diminished ovarian reserve(DOR)induced by tripterygium wilfordii polyglycoside;To discuss its mechanism of action for improving DOR.Methods Totally 32 SD rats were divided into blank group(8 rats)and modeling group(24 rats)according to random number table method.The blank group was not treated,the modeling group was given tripterygium wilfordii polyglycoside tablet suspension 50 mg/kg by gavage for 14 days to establish DOR model.After modeling,the modeling group was randomly divided into model group,TCM low-and highdosage groups,with 8 rats in each group.TCM low-,and high-dosage groups were given 16.38 and 32.76 g/kg of Yishen Shugan Decoction respectively,and the blank group and model group were given the same volume of normal saline for 15 days.The estrus cycle was observed,the ovarian and uterine indexes were counted,the morphology of ovarian tissue was observed by HE staining,ELISA was used to detect serum anti-Müllerian hormone(AMH),follicle-stimulating hormone(FSH),and estradiol(E2)contents,the apoptosis of ovarian granulosa cells was detected by TUNEL staining,Western blot was used to detect the protein expression of Bax,Caspase-3 and Bcl-2 in ovarian tissue.Results Compared with the blank group,the estrous cycle of the rats in model group was disordered,the ovarian index and uterine index significantly decreased(P<0.01),the contents of AMH and E2in serum significantly reduced(P<0.01),the content of FSH significantly increased(P<0.01);the number of follicles at each level and mature follicles reduced significantly,the number of atretic follicles increased,and the apoptosis rate of ovarian granulosa cells increased significantly(P<0.01);the expressions Bax and Caspase-3 protein in ovarian tissue increased significantly(P<0.01),the expression of Bcl-2 protein and the ratio of Bcl-2/Bax decreased significantly(P<0.01).Compared with the model group,the estrous cycle of the rats in TCM low-and high-dosage groups tended to be normal,and the ovarian index and uterine index significantly increased(P<0.05),the contents of AMH and E2in serum significantly increased(P<0.01),the content of FSH significantly reduced(P<0.01);the number of follicles at each level and mature follicles increased,the number of atretic follicles reduced,and the apoptosis rate of ovarian granulosa cells significantly decreased(P<0.05);the expressions of Bax and Caspase-3 protein in ovarian tissue significantly decreased(P<0.05,P<0.01),and the expression of Bcl-2 protein and the ratio of Bcl-2/Bax significantly increased(P<0.05,P<0.01).Conclusion Yishen Shugan Decoction can improve the pathological state and ovarian reserve function of DOR rats,and the mechanism may be related to the inhibition of ovarian granulosa cell apoptosis,up-regulation of Bcl-2,down-regulation of Bax and Caspase-3 protein expression.