OHSS模型大鼠卵巢组织中自噬、凋亡和氧化应激相关蛋白的表达及意义

Expression and significance of autophagy,apoptosis and oxidative stress related proteins in ovarian tissue of rat OHSS model

目的探讨卵巢过度刺激综合征(OHSS)模型大鼠卵巢中自噬、氧化应激、细胞凋亡作用并探讨相关调节机制。方法将22日龄雌性SD大鼠20只随机分为对照组、OHSS组,每组各10只。在第22~23天每天给对照组大鼠注射等体积0.9%生理盐水,第24天注射孕马血清促性腺激素(PMSG),第26天注射人绒毛膜促性腺激素(HCG);在第22~25天每天给OHSS组大鼠连续腹腔注射PMSG,在第26天注射HCG。连续记录生长体重变化,ELISA法检测血清卵泡刺激素(FSH)、黄体生成素(LH)水平,全自动化学发光免疫分析法检测雌二醇(E 2)、孕酮(P)、睾酮(T)水平,HE染色观察卵巢组织学改变,Western blot、ELISA或IHC检测卵巢组织自噬相关蛋白(Atg3、Beclin1、LC3、p62、P-ERK1/2及ERK1/2)、凋亡相关蛋白(Bax、Bcl-2、p53、Cleaved-caspase3)、氧化与抗氧化指标(Nrf2、ROS、SOD)表达水平。结果与对照组比较,OHSS组大鼠卵巢体积、卵巢/体重比值显著增加(P<0.001),卵巢组织总卵泡数(P<0.05)和黄体数显著增多(P<0.001),而初级卵泡、窦状卵泡、排卵前卵泡、闭锁卵泡数目均显著减少(P<0.05)。与对照组比较,OHSS大鼠卵巢组织中自噬相关蛋白(Atg3、Beclin1、LC3、pERK1/2)表达水平(P<0.05)和ROS浓度显著增加(P<0.05),而抗氧化能力显著下降(P<0.01);另外,促凋亡相关蛋白(Bax、p53、Cleaved-caspase3)表达显著下调(P<0.05),而抗凋亡蛋白(Bcl-2)显著增加(P<0.01)。结论卵巢过度刺激导致卵巢中氧化与抗氧化状态失衡,诱导自噬水平升高并减少卵巢内细胞凋亡,进而促进黄体组织增多、激素水平释放增加。

Objective:To investigate the role and related mechanism of autophagy,oxidative stress,apoptosis in ovarian of rat with hyperstimulation syndrome(OHSS)model.Methods:A total of 20 female SD rats aged 22 days were randomly divided into control group and OHSS group with 10 rats in each group.The rats in the control group were injected with equal volume of 0.9%normal saline every day from 22^(nd) to 23^(rd) days,PMSG was injected on the 24^(th) day,and HCG was injected on the 26^(th) day.On the 22^(nd)-25^(th) day,the rats were intraperitoneally injected with PMSG continuously,and HCG on the 26^(th) day in OHSS group.The changes of body weight were recorded continuously.Serum levels of FSH and LH were detected by ELISA,the levels of estradiol(E_(2)),progesterone(P)and testosterone(T)were detected by automatic chemiluminescence immunoassay,and the histological changes of ovary were observed by HE staining.The autophagy related proteins(Atg3,Beclin1,LC3,P62,P-ERK1/2 and ERK1/2),apoptosis related proteins(Bax,Bcl-2,p53,Cleaved caspase3),oxidation and antioxidant indexes(Nrf2,ROS and SOD)were detected by Western blot,ELISA or IHC.Results:The ovarian volume and ovarian/body weight ratio of rats in the OHSS group were significantly increased(P<0.0001).The total numbers of follicles in ovarian tissue(P<0.05)and corpus luteum(P<0.001)were significantly increased,while the numbers of primary follicle,antral follicle,preovulatory follicle and atretic follicle were significantly decreased(P<0.05).Compared with the control group,the expression levels of autophagy related proteins(Atg3,Beclin1,LC3,P-ERK1/2&ERK1/2)and the ROS concentration in ovarian tissues of OHSS rats were significantly increased(P<0.05),while the antioxidant capacity was significantly decreased(P<0.01).In addition,the expressions of pro-apoptotic related proteins(Bax,p53,Cleaved caspase3)were down-regulated(P<0.05),while the expressions of anti-apoptotic proteins(Bcl-2)were significantly increased(P<0.01).Conclusions:Hyper-stimulation of ovary leads to an imbalance between oxidation and antioxidant status in the ovary,induces an increase in autophagy and reduces apoptosis in the ovary,thereby promoting an increase in corpus luteum tissue and an increase in hormones release.