雷帕霉素对脱氢表雄酮诱导PCOS大鼠卵巢组织的影响及作用机制探讨

The effect and mechanism of rapamycin on the ovarian tissue of rats with PCOS induced by dehydroepiandrosterone

目的探讨雷帕霉素对脱氢表雄酮(DHEA)诱导的多囊卵巢综合征(PCOS)大鼠卵巢组织的影响及作用机制。方法55只SD雌性大鼠适应性喂养1周后随机分为空白组(13只)、对照组(13只)及PCOS组(29只),其中PCOS组大鼠皮下注射DHEA诱导建立PCOS大鼠模型,对照组大鼠给予皮下注射大豆油,空白组全程不做任何处理。造模成功后分为空白组、对照组(腹腔注射等体积生理盐水),将PCOS大鼠分为模型对照组(腹腔注射等体积生理盐水)、低剂量组(腹腔注射雷帕霉素1 mg/kg)及高剂量组(腹腔注射雷帕霉素10 mg/kg),每组各8只大鼠,共干预28 d,给药结束后检测各组大鼠血糖、性激素水平,并进行HE、Masson、免疫组织化学染色,检测并比较组间mTOR、p-mTOR及p-P70S6蛋白表达,TUNEL法检测卵巢组织凋亡情况。结果PCOS大鼠动情周期出现紊乱,卵巢组织中卵泡呈现多囊样变化,PCOS大鼠体重、卵巢质量、体积、相对质量、性激素水平、纤维化阳性范围均高于对照组(P均<0.05),而低剂量组和高剂量组大鼠的上述指标均低于模型对照组(P均<0.05),且卵巢组织多囊样变化明显减轻。低剂量组与高剂量组的纤维化阳性范围较空白组及对照组增加、高剂量组较模型对照组增加(P均<0.05)。模型对照组大鼠卵巢中p-mTOR蛋白的光密度(OD)值高于其他4组(P均<0.05)、卵巢中p-P70S6蛋白的OD值与对照组相近(P>0.05),而高剂量组低于空白组、对照组及低剂量组(P均<0.05)。卵巢组织TUNEL染色结果分析,模型对照组高于空白组、对照组和高剂量组(P均<0.005),空白组低于低剂量组(P<0.005)。结论雷帕霉素使DHEA诱导的PCOS大鼠模型体重、卵巢质量减轻,卵巢体积减少,性激素水平降低,卵泡组织中多囊样改变减轻及卵巢组织细胞凋亡减少,这可能与雷帕霉素抑制mTOR信号通路的异常激活有关;但其也可能导致PCOS大鼠卵巢间质纤维化增加,大鼠体重、卵巢质量与体积低于正常水平。

Objective To evaluate the effect and unravel the mechanism of rapamycin on the ovarian tissues in rat models with polycystic ovary syndrome(PCOS)induced by dehydroepiandrosterone(DHEA).Methods After 1-week accustomed feeding,55 rats were randomly divided into the blank(n=13),control(n=13)and model groups(n=29).PCOS rat models were established by subcutaneous injection of DHEA solution.In the control group,rats were subcutaneously injected with soybean oil,while those in the blank group were not treated throughout the whole experiment.After the model was successfully established,all rats were divided into the blank group,control group(intraperitoneal injection of an equivalent volume of normal saline),the model group,low-dose group(intraperitoneal injection of 1 mg/kg rapamycin)and high-dose group(intraperitoneal injection of 10 mg/kg rapamycin)for 28 d,8 rats in each group.Blood glucose and hormone levels were detected,and HE,Masson and immunohistochemical staining was performed after corresponding interventions.The changes of the expression levels of mTOR,p-mTOR and p-P70S6 were assessed among different groups.TUNEL assay was used to detect the apoptosis of ovarian tissues among different groups.Results PCOS rats showed irregular estrous cycle,and the follicles were manifested with polycystic changes.The body weight,ovarian weight,volume,relative weight,sex hormone levels and positive area of ovarian fibrosis of PCOS rats were all higher than those in the control group(all P<0.05),whereas these parameters in the low-and high-dose groups were lower than those in the model control group(all P<0.05),and the polycystic changes of ovarian tissues were significantly mitigated.The positive area of ovarian fibrosis in the low-and high-dose groups was increased than those in the blank and control groups(all P<0.05).The positive area of ovarian fibrosis in the high-dose group was increased than that in the control group(P<0.05).No significant difference was noted in the OD value of mTOR protein among five groups(all P>0.05).The OD value of mTOR protein in the model control group was higher compared with those in the remaining four groups(all P<0.05).The OD value of mp-P70S6 protein in the model control group was similar to that in the control group(P>0.05),whereas the OD value of mp-P70S6 protein in the high-dose group was lower compared with those in the blank,control and low-dose groups(all P<0.05).TUNEL staining revealed that the apoptosis rate of ovarian tissues in the model control group was higher than those in the blank,control and high-dose groups(all P<0.005),whereas the apoptosis rate in the blank group was lower than that in the low-dose group(P<0.005).Conclusions Rapamycin can reduce body weight,decrease ovarian mass and volume,lower hormone levels,mitigate polycystic changes in follicular tissues and alleviate apoptosis of ovarian tissues in DHEA-induced PCOS rat models,which may be related to the inhibition of abnormal activation of mTOR signaling pathway by rapamycin.However,rapamycin may aggravate ovarian interstitial fibrosis in PCOS rats.The body weight,ovarian mass and volume of PCOS rats are lower than the normal levels.