双酚A诱发卵巢储备功能下降大鼠模型的制备及其中医证候属性

Construction of rat models of diminished ovarian reserve induced by bisphenol A and their attributes of traditional Chinese medicine syndromes

目的探讨双酚A(BPA)诱发大鼠卵巢储备功能下降(DOR)模型的最佳制备方案,并明确其中医证候属性。方法选择动情周期规律的3、6月龄雌性清洁SD大鼠各18只,不同月龄分别按随机数字表法分为低、中、高剂量组。低、中、高剂量组每日分别给予50、100、200 mg/kg BPA混悬液灌胃,均每日灌胃1次,至大鼠动情周期紊乱。观察大鼠一般情况;阴道涂片观察大鼠动情周期;取大鼠腹主动脉血,分离血清,用ELISA法检测血清促卵泡生成素(FSH)、促黄体生成素(LH)、雌二醇(E2)、抗苗勒氏管激素(AMH)、抑制素-B(INHB);取双侧卵巢,观察卵巢组织形态,测算各级卵泡比例、卵巢指数。选择观察指标变化最明显组所用的模型制备方法为最佳方法。将造模成功的18只大鼠按照随机数字表法分为对照组、右归丸组、养精种玉汤组,每组6只,分别给予蒸馏水、治疗肾阳虚的右归丸、治疗肾阴虚的养精种玉汤按1 mL/100 g体质量灌胃,连续2周。对比各组动情周期变化,肾阳虚症状,卵巢组织形态、卵巢指数、各级卵泡比例,血清激素、INHB、AMH水平。结果3、6月龄各BPA干预组大鼠活动减少,喜蜷卧扎堆。3月龄中剂量BPA组动情周期紊乱时间较其他组短;AMH水平最低,原始卵泡最少,闭锁卵泡最多,因此3月龄中剂量BPA组的模型制备方法最佳。模型组大鼠实验期间持续处于动情周期紊乱状态,养精种玉汤组大鼠有1只恢复规律动情周期,右归丸组有5只大鼠恢复规律动情周期。右归丸组大鼠规律动情周期恢复数量(5只)高于对照组(无)、养精种玉汤组(1只),肾阳虚症状明显改善。模型组大鼠卵巢苍白,体积变小,质硬;右归丸组大鼠卵巢颜色红润,体积变大,组织弹性好,表面有多个生长卵泡;养精种玉汤组大鼠卵巢表面生长卵泡少,组织弹性差。与对照组、养精种玉汤组比较,右归丸组大鼠原始卵泡比例高、闭锁卵泡比例低,AMH水平高(P均<0.05)。结论3月龄SD大鼠每日以100 mg/kg的BPA灌胃可成功制备DOR动物模型,以方测证表明该动物模型的中医证候属性为肾阳虚型。

Objective To investigate the optimal preparation method of bisphenol A(BPA)-induced diminished ovarian reserve(DOR)rat models,and to clarify the attributes of traditional Chinese medicine(TCM)syndromes.Meth⁃ods Eighteen clean female SD rats aged 3 and 6 months with regular estrous cycle were selected and were randomly divided into the low-dose,medium-dose and high-dose BPA groups.The rats in the low-dose,medium-dose and high-dose BPA groups were given 50,100,and 200 mg/kg BPA mixed suspension orally,once a day until they showed unregular estrous cycles.The general performance and vaginal smear were observed everyday.The estrous cycle of rats was observed by vaginal smear;the abdominal aortic blood of rats was collected,and serum was separated.Serum follicle-stimulating hormone(FSH),luteinizing hormone(LH),estradiol(E2),anti-Müllerian hormone(AMH),and inhibin-B(INHB)were detected by ELISA.We took bilateral ovaries,observed the morphology of ovarian tissues,and calculated the proportion of follicles at all levels and the ovarian index.We chose the model preparation method used in the group which had the most significant change of the indexes as the best method.The 18 successful model rats were randomly divided into the control group,Yougui pill group,and Yangjingzhongyu decoction group,with 6 in each.The model rats in the control group,Yougui pill group and Yangjingzhongyu decoction group were given distilled water,Yougui pill for treatment of kidny-yang deficiency,and Yangjingzhongyu decoction for kidny-yin deficiency by 1 mL/100 g body weight,for 2 consecutive weeks,respectively.The changes of estrous cycle,symptoms of kidney-yang deficiency,ovarian tissue morphology,ovarian index,proportion of follicles at all levels,serum hormone,INHB and AMH levels were compared between these two groups.Results All model rats in the BPA groups aged 3 and 6 months had reduced activity and liked to crouch and bunch.The disturbance time of estrous cycles of model rats aged 3 month in the medium-dose BPA group was significant shorter as compared with that of the other groups,and they showed decreased levels of AMH and primordial follicles and increased atretic follicles.Therefore,the preparation method in the 3-month-old rat model of the medium-dose BPA group was the best method.One rat in the Yangjingzhongyu decoction group and 5 rats in Yougui pill group resumed regular estrous cycle while all rats in the control group continued to be in disorder.The number of regular estrus cycle recovery in the Yougui pill group(5)was higher than that in the control group(0)and Yangjingzhongyu decoction group(1),while the symptom of kidneyyang deficiency improved more significantly.The ovaries of rats in the control group were pale,small and hard,while those of the Yougui pill group were bigger and ruddy.The ovaries of Yougui pill group had good elasticity and multiple growth follicles on the surface.There were fewer follicles on the ovarian surface and poor elasticity in the rats of Yangjingzhongyu decoction group.Compared with the other two groups,the Yougui pill group had higher proportion of primordium follicle,lower atresia follicle,and higher AMH level(all P<0.05).Conclusions The DOR animal models can be successfully prepared by intragastric administration of 100 mg/kg BPA in 3-month clean SD rats.The attribute of TCM syndrome of the rat models is kidney-yang deficiency by means of syndrome differentiation through formula.