基于PI3K/Akt信号通路探讨哈蟆油对卵泡发育作用的影响  

Effect of Ranae Oviductus on Follicular Development Based on PI3K/Akt Signaling Pathway

作　　者：赵宏宇[1] 王玉[1] 孙雪缘 宋莲莲[1] 刘新宇[1] 邸琳[1] ZHAO Hong-yu;WANG Yu;SUN Xue-yuan;SONG Lian-lian;LIU Xin-yu;DI Lin(Jilin Academy of Chinese Medicine Science,Changchun 130012,China)

机构地区：[1]吉林省中医药科学院,长春130012

出　　处：《中国实验方剂学杂志》2021年第21期121-130,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：吉林省健康卫生技术创新项目(2019J086);吉林省中医药科技项目(2020103)。

摘　　要：目的:探讨哈蟆油对大鼠卵巢卵泡发育、卵巢磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路及怀胎功能的影响,以及哈蟆油雌激素样作用机制。方法:雌性Wistar大鼠70只,随机分为正常组、戊酸雌二醇+黄体酮组(1 mg·kg^(-1)+40 mg·kg^(-1)),克罗米芬(10 mg·kg^(-1))组,哈蟆油高、低剂量(400,200 mg·kg^(-1))组,连续灌胃8周。灌胃7周时,所有大鼠测定发情周期。灌胃8周时,每组取4只大鼠进行产仔实验,其余大鼠在测定出发情期当天采血,测定血清雌二醇(E2),孕酮(P),睾酮(T),促卵泡生成素(FSH),促黄体生成素(LH)含量,摘取子宫、卵巢,称量重量计算器官指数,一侧卵巢制成病理切片,进行不同发育阶段卵泡及黄体计数,另一侧卵巢进行实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Westen blot)检测PI3K/Akt信号通路mRNA及蛋白变化。雌性昆明种小鼠40只,随机分为正常组、哈蟆油14 d组(400 mg·kg^(-1)),哈蟆油28 d组(400 mg·kg^(-1)),哈蟆油56 d组(400 mg·kg^(-1))。哈蟆油各组小鼠分别在灌胃14,28,56 d后与雄性小鼠合笼饲养,18 d后观察两侧子宫内胎鼠数目及发育不良胎数目。结果:8周灌胃哈蟆油后,大鼠子宫指数明显降低(P<0.05),血清LH明显降低(P<0.05),卵巢中黄体数明显减少(P<0.01),初级卵泡数明显减少(P<0.05),卵泡闭锁率显著上升(P<0.01),卵巢皮质层中出现短期内较多黄体或间质腺退化为间质的结构,卵巢PI3K和Akt mRNA表达水平显著升高(P<0.01),哺乳动物雷帕霉素靶蛋白(mTOR)mRNA和人第10号染色体缺失的磷酸酶(PTEN)mRNA表达水平显著降低(P<0.01);Akt蛋白磷酸化水平有降低趋势,差异无统计学意义;大鼠产仔数明显减少(P<0.05)。小鼠不同时长灌胃哈蟆油后,受孕率不同程度下降,服用14 d受孕率最低仅30%。灌胃哈蟆油28 d,左右子宫怀胎差值明显增大(P<0.05)。结论:哈蟆油长期灌胃后表现出与长期使用克罗米芬类似�Objective:To investigate the effect of Ranae Oviductus(RO)on ovarian follicular development,phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,and pregnancy function in rats,and the estrogen-like mechanism of OR.Method:Seventy female Wistar rats were randomly divided into a normal group,a progynova+luteohormone group(1 mg·kg^(-1)+40 mg·kg^(-1)),a clomiphene group(10 mg·kg^(-1)),and high-dose(400 mg·kg^(-1))and low-dose(200 mg·kg^(-1))RO groups.Rats were administered correspondingly by gavage for eight weeks.After seven weeks of intragastric administration,the estrus cycle of all rats was measured.After eight weeks of intragastric administration,four rats from each group were selected to give birth.For other rats,blood was collected on the day of estrus,and the serum levels of estradiol(E2),progesterone(P),testosterone(T),follicle-stimulating hormone(FSH),and luteotropic hormone(LH)were detected.Uterus and ovaries were extracted and weighed to calculate organ index.One ovary was made into pathological sections,and the follicles at different developmental stages and corpus luteum were counted.Realtime fluorescence-based quantitative polymerase chain reaction(Real-time PCR)and Western blot were performed on the other ovary to detect mRNA and protein changes in the PI3K/Akt signaling pathway.Forty female Kunming mice were randomly divided into a normal group and RO groups(400 mg·kg^(-1))with 14 days,28 days,and 56 days of intervention.Mice in the RO groups were raised with male mice in cages after intragastric administration of OR for 14,28,and 56 days,respectively.After 18 days,the number of intrauterine fetuses on both sides and the number of stunted fetuses were counted.Result:After eight weeks of intragastric administration of OR,the rats showed decreased uterine index(P<0.05),declining serum LH(P<0.05),reduced luteum(P<0.01),dwindled primary follicles(P<0.05),and increased rate of follicle atresia(P<0.01).Additionally,more luteal or interstitial glands degenerated into interstitial

关 键 词：哈蟆油 卵巢 卵泡 磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号通路 性激素 

分 类 号：R2-0[医药卫生—中医学] R289