补肾养精颗粒灌胃对早发性卵巢功能不全大鼠卵巢颗粒细胞凋亡的抑制作用及其机制

Inhibitory effect of Bushen Yangjing granule on apoptosis of ovarian granulosa cells in rats with premature ovarian insufficiency and its mechanism

目的观察补肾养精颗粒对早发性卵巢功能不全(POI)大鼠卵巢颗粒细胞凋亡的抑制作用,并探讨其作用机制。方法取雌性SD大鼠90只,随机分为空白组15只和模型组75只,模型组采用雷公藤多苷灌胃方法制备POI模型,空白组给予0.9%氯化纳注射液灌胃,每天1次,连续14 d。造模结束后,将模型组分为模型对照组、补佳乐组、中药低剂量组、中药中剂量组、中药高剂量组,每组各15只。补佳乐组给予0.1 mg/(kg·d)补佳乐灌胃,中药低、中、高剂量组分别给予补肾养精颗粒1.75、3.5、7.0 g/(kg·d)灌胃,空白组和模型对照组给予0.9%氯化纳注射液2 mL/d灌胃,连续给药15 d。给药结束后24 h,每组分别取6只大鼠给予孕马血清促性腺激素(PMSG)50 IU皮下注射,48 h后处死,取双侧卵巢,流式细胞定量分析法检测卵巢颗粒细胞的凋亡状态。各组剩余9只大鼠取双侧卵巢,透射电子显微镜下观察卵泡的超微化结构;取卵巢组织,采用Westernblotting法检测Hedgehog信号通路关键基因Shh、Ptch1、Gli1及凋亡相关基因Bax、Bcl-2蛋白表达。结果与空白组比较,模型对照组颗粒细胞早期凋亡率及晚期凋亡率均升高,正常细胞比例降低;与模型对照组比较,各治疗组颗粒细胞晚期凋亡率降低,正常细胞比例增加;中药高剂量组颗粒细胞早期凋亡率较其他剂量组降低;中药中、高剂量组颗粒细胞晚期凋亡率较中药低剂量、补佳乐组降低,正常细胞比例增加(P<0.05或<0.01)。与空白组比较,模型对照组见卵母细胞形态欠规则,细胞核固缩,颗粒细胞形态欠规则,见凋亡相,细胞器结构异常且数量偏少。与模型对照组比较,各治疗组细胞器形态改善或恢复正常,数量增多,卵母细胞及颗粒细胞形态改善,其中中药中、高剂量组卵母细胞形态基本接近空白组。模型大鼠卵巢组织中Shh、Ptch1、Gli1以及Bcl-2蛋白表达水平降低,Bax蛋白表达水平升高;各治疗组卵巢Shh、Ptch1、Gli1及Bcl-2蛋白表达均升高,Bax蛋白表达均降低;中药中、高剂量组Shh、Gli1蛋白表达水平较中药低剂量组、补佳乐组升高(P<0.05或<0.01)。结论中、高剂量补肾养精颗粒可明显抑制POI大鼠卵巢颗粒细胞凋亡,其机制可能与激活Hedgehog信号通路后下调促凋亡基因、上调抑凋亡基因表达有关。

Objective To observe the inhibitory effect of Bushen Yangjing granule on apoptosis of ovarian granulosa cells in rats with premature ovarian insufficiency(POI),and to explore its mechanism.Methods Ninety female SD rats were randomly divided into the blank group with 15 rats,and model group with 75 rats.The rats in the model group were given tripterygium glycosides to prepare POI models by gavage,and the rats in the blank group were given 0.9%sodium chloride injection by gavage,once a day,for 14 consecutive days.After the modeling,the model group rats were divided into five groups:model control group,Bujiale group,low-dose herbal group,medium-dose herbal group and high-dose herbal group,with 15 rats in each group.The rats in the Bujiale group were given 0.1 mg/kg Bujiale by gavage every day,the rats in the low-dose,medium-dose,and high-dose herbal groups were respectively given 1.75,3.5,and 7.0 g/kg Bushen Yangjing granule by gavage every day,and the rats in the blank group and model control group were given 2 mL of 0.9%sodium chloride injection by gavage every day,for a total of 15 days.At 24 h after the end of administration,6 rats in each group were given subcutaneous injection of pregnant mare serum gonadotropin(PMSG)50 IU,and were sacrificed 48 hours later.Quantitative flow cytometry was used to detect the apoptosis status of ovarian granulosa cells in each group.The remaining nine rats in each group were selected,we collected their bilateral ovaries,and the ultrastructural changes of follicles in each group were observed under transmission electron microscope.Western blotting was used to detect the pro⁃tein expression levels of the Hedgehog signaling pathway key genes Shh,Ptch1,Gli1,and the protein expression levels of apoptosis-related genes Bcl2-associated x(Bax),B-cell lymphoma-2(Bcl-2)in each group.Results Compared with the blank group,the early apoptosis rate and late apoptosis rate of granulosa cells in the model control group increased,and the proportion of normal cells decreased.Compared with the model control group,the late apoptosis rate of granulosa cells in each treatment group decreased,and the proportion of normal cells increased;the early apoptosis rate of granulosa cells in the high-dose herbal group was lower than that in the other dose groups;the late apoptosis rates of granulosa cells in the medium-dose herbal group and high-dose herbal group were lower than those in the low-dose herbal group and Bujia⁃le group,and the proportion of normal cells increased(P<0.05 or P<0.01).Compared with the blank group,the morphol⁃ogy of oocytes was less regular,the nucleus was pyknosis,the morphology of granulosa cells was less regular,and the or⁃ganelle structure was abnormal.Compared with the model control group,the morphology of organelles in each treatment group improved or returned to normal,the number increased,and the morphology of oocytes and granulosa cells improved.The protein expression levels of Shh,Ptch1,Gli1 and Bcl-2 in the model control group decreased,and the expression of Bax protein increased;the protein expression levels of Shh,Ptch1,Gli1 and Bcl-2 in each treatment group significantly in⁃creased,and the expression of Bax protein significantly decreased;the protein expression levels of Shh and Gli1 in the me⁃dium-dose and high-dose herbal groups were higher than those in the low-dose herbal group and the Bujiale group(P<0.05 or P<0.01).Conclusions The medium-dose and high-dose Bushen Yangjing granules can significantly inhibit apopto⁃sis of ovarian granulosa cells in POI model rats.The mechanism may be related to down-regulation of pro-apoptotic genes and up-regulation of anti-apoptotic genes after activation of Hedgehog signaling pathway.