探索建立雷公藤多苷致早发性卵巢功能不全肾虚血瘀证的小鼠模型

Establishment of premature ovarian insufficiency kidney deficiency and blood stasis pattern mouse model with Tripterygium wilfordii polyglycoside for Bushen Culuan Decoction therapy

为研究补肾促卵方治疗早发性卵巢功能不全的作用机制,采用雷公藤多苷灌胃建立早发性卵巢功能不全肾虚血瘀证小鼠模型并进行卵巢功能和生育力评价。60只SPF级动情周期规则的雌性Blab/c小鼠随机分为空白1组、空白2组、空白生育组、模型组、模型恢复组、模型生育组,每组10只。模型组予40 mg·kg-1雷公藤多苷混悬液灌胃,空白组予等量生理盐水灌胃,每日2次,连续14 d。第15天空白1组和模型组解剖取材;其余4组终止雷公藤灌胃恢复正常喂养,空白2组和模型恢复组连续观察动情周期变化14 d后解剖取材,观察各组一般表征、动情周期、血清性激素、性腺指数、卵巢形态及卵泡计数、卵巢血管内皮生长因子(VEFG)和内皮抑制素(ES)的变化。空白生育组和模型生育组以雌雄2∶1进行合笼,记录雌鼠受孕率、产仔数、仔鼠存活数及雌雄比例。造模期间模型组和模型生育组小鼠体质量下降(P<0. 05)。与空白1组相比,模型组小鼠动情周期紊乱,卵巢及子宫指数下降,原始及生长卵泡数量减少、闭锁卵泡数量增多,VEGF表达下降、ES表达升高(P<0. 05)。与空白2组相比,模型恢复组小鼠动情周期仍紊乱,血清卵泡刺激素(FSH)升高,卵巢指数未恢复,原始及生长卵泡数量仍减少、闭锁卵泡数量仍增加,VEGF表达仍下降(P<0. 05)。与空白生育组相比,模型生育组小鼠产仔数和仔鼠存活数下降(P<0. 05)。40 mg·kg-1雷公藤多苷每日2次,连续灌胃14 d可成功建立早发性卵巢功能不全肾虚血瘀证小鼠模型,停药14 d后模型小鼠卵巢功能尚未恢复,为后续补肾促卵方的干预性实验治疗时间提供证据。

To establish a mouse model of premature ovarian insufficiency( POI) with kidney deficiency and blood stasis pattern by Tripterygium wilfordii polyglycoside( TWP) gavage,and to evaluate the ovarian function and fertility of the model,in order to find Bushen Culuan Decoction therapeutic mechanism. 60 SPF level Blab/c female mice with normal estrous cycle were randomly divided into 6 groups of 10 each: blank group 1( BG1),blank group 2( BG2),blank fertility group( BFG),model group( MG),model recovery group( MRG) and model fertility group( MFG). The mice in three model groups were treated by gastric gavage with TWP suspension 40 mg·kg-1 twice a day for 14 days,while the mice in three blank groups were treated by gastric gavage with same volume normal saline for 14 days. The mice in BG1 and MG were sacrificed and dissected on day 15. The mice in BG2,BFG,MRG and MFG were returned normal feeding from day 15 and were sacrificed and dissected on day 29. The mice in BFG and MFG were cohabited with male mice with a ratio of 2 ∶1( female ∶male) from day 15. The general situation and estrous cycles of all mice were observed every day. Serum sex hormone levels,ovarian index,uterine index,ovarian morphology,follicle count,ovarian VEGF and ES index were observed within the mice in BG1,BG2,MG and MRG. Pregnancy rate,litter size,survival number of newborn mice and male-female proportion were reported within the mice in BFG and MFG. In model establishing stage,the body weight of mice significantly decreased( P <0. 05) in MG and MFG. Compared with BG1,the mice in model group had irregular estrous cycle,decreased ovarian and uterine indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression and decreased ES expression( P <0. 05). Compared with blank group 2,the mice in model recovery group had irregular estrous cycle,increased FSH level,decreased ovarian indexes,less primordial and developing follicles,more atretic follicles,increased VEGF expression( P<0. 05). Compared with blank fertility group,the mice in model fertility group had smaller litter size and newborn mice survival count( P<0. 05). Gastric gavage with TWP 40 mg·kg-1 twice a day for 14 days is a feasible way to establish a POI kidney deficiency and blood stasis pattern mouse model. The mice ovarian functions didn’t recovery on day 14 after stopping TWP intervening,which could suggest the effectiveness of subsequent therapeutic intervention.