摘要
目的:观察针刺干预对卵巢早衰大鼠卵巢功能及卵巢组织自噬相关蛋白的影响。方法:将雌性Wistar大鼠随机分为空白组、模型组、药物组、核心腧穴组和非经非穴组,除空白组外的大鼠均采用腹腔注射环磷酰胺的方法构建卵巢早衰大鼠模型。药物组大鼠给予己烯雌酚灌胃,核心腧穴组大鼠针刺关元穴和双侧三阴交穴,非经非穴组大鼠针刺关元穴和双侧三阴交穴旁开5 mm非经非穴处,空白组和模型组大鼠给予非干预因素相同处理。14天后取材,大鼠称重,计算卵巢、子宫脏器指数。采用HE染色观察卵巢和子宫形态并统计各级卵泡、黄体数。采用酶联免疫吸附法检测血清雌二醇(E_(2))、促卵泡刺激素(FSH)、促黄体生成素(LH)、抗缪勒管激素(AMH)水平,Western blot法检测卵巢组织PTEN、AKT、p-AKT、LC3、Beclin1蛋白的表达水平。采用RT-qPCR法检测卵巢组织PTEN、AKT、LC3、Beclin1的mRNA相对表达量。结果:与空白组比较,模型组大鼠体质量和卵巢、子宫脏器指数下降(P<0.05),各级生长卵泡和黄体数量减少,闭锁卵泡数量增加(P<0.05),血清AMH和E_(2)水平降低、FSH和LH水平升高(P<0.05),卵巢组织PTEN、LC3、Beclin1蛋白和mRNA的表达水平上调,p-AKT/AKT水平下调(P<0.05)。与模型组比较,核心腧穴组大鼠体质量和卵巢、子宫脏器指数上升(P<0.05),原始卵泡数量增加、闭锁卵泡数量减少(P<0.05),血清AMH和E2水平升高、FSH和LH水平降低(P<0.05),卵巢组织PTEN、LC3、Beclin1蛋白和mRNA的表达水平下调(P<0.05),p-AKT/AKT水平上调(P<0.05)。与非经非穴组比较,核心腧穴组大鼠体质量和卵巢、子宫脏器指数上升(P<0.05),原始卵泡数量增加、闭锁卵泡数量减少(P<0.05),血清AMH和E_(2)水平升高、LH水平降低(P<0.05),卵巢组织Beclin1蛋白表达水平下调(P<0.05)。结论:针刺“关元、三阴交”核心腧穴组合可调节大鼠血清激素水平,恢复卵巢功能,并通过影响PTEN/AKT信号通路降低卵巢早衰大鼠卵巢组织LC3和Beclin1蛋白及mRNA在卵巢组织中的表达,抑制卵巢颗粒细胞的过度自噬损伤,减少卵泡闭锁。
Objective:To observe the effects of acupuncture intervention on ovarian function and autophagy-related proteins in ovarian tissue of rats with premature ovarian failure.Methods:Female Wistar rats were randomly divided into a blank group,a model group,a medication group,a core acupoints group,and a non-meridian and non-acupoint group.All rats except for the blank group were injected intraperitoneally with cyclophosphamide to establish a rat model of premature ovarian failure.Rats in the medication group were given diethylstilbestrol by gavage;the core acupoints group was treated with acupuncture at the Guanyuan(CV4)point and bilateral Sanyinjiao(SP6)points;the non-meridian and non-acupoint group was given acupuncture at locations that were neither on the meridians nor acupoints and 5 mm away from the Guanyuan(CV4)point and bilateral Sanyinjiao(SP6)points;the blank group and the model group were given the same treatment without any intervention factors.After 14 days,samples were taken,rats were weighed,and ovarian and uterine organ indicators were calculated.HE staining was used to observe the morphology of the ovaries and uterus,and the follicles and corpus luteum were counted at all levels.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of serum estradiol(E_(2)),follicle-stimulating hormone(FSH),luteinizing hormone(LH),and anti-Müllerian hormone(AMH).Western blot was used to detect the expression levels of PTEN,AKT,p-AKT,LC3,and Beclin1 proteins in ovarian tissue.The RT-qPCR was used to detect the relative mRNA expression levels of PTEN,AKT,LC3,and Beclin1 in ovarian tissue.Results:Compared with the blank group,the body mass,and ovarian and uterine organ indicators in the model group were decreased(P<0.05);the number of growing follicles and corpus luteum was decreased,and the number of blocked follicles was increased(P<0.05);the serum AMH and E_(2) levels were declined,and FSH and LH levels were increased(P<0.05);the protein and mRNA expression levels of PTEN,LC3,and Beclin1 in ovarian tissue were upregulated,and the p-AKT/AKT level was downregulated(P<0.05).Compared with the model group,the core acupoints group showed an increase in body mass and ovarian and uterine organ indicators(P<0.05),an increase in the number of primordial follicles,and a decrease in the number of blocked follicles(P<0.05);the serum AMH and E_(2) levels in the core acupoints group were enhanced,and FSH and LH levels were declined(P<0.05);the protein and mRNA expression levels of PTEN,LC3,and Beclin1 in ovarian tissue in the core acupoints group were downregulated,and the p-AKT/AKT level was upregulated(P<0.05).Compared with the non-meridian and non-acupoint group,the core acupoints group showed an increase in body mass and ovarian and uterine organ indicators(P<0.05),an increase in the number of primordial follicles and a decrease in the number of blocked follicles(P<0.05),an increase in serum AMH and E_(2) levels,a decrease in LH levels(P<0.05),and a downregulation of Beclin1 protein expression in ovarian tissue(P<0.05).Conclusion:Acupuncture at the core acupoints of the Guanyuan(CV4)point and bilateral Sanyinjiao(SP6)points can regulate serum hormone levels in rats,restore ovarian function,and reduce the expression of LC3 and Beclin1 proteins and mRNA in ovarian tissue of rats with premature ovarian failure by affecting the PTEN/AKT signaling pathway.It also inhibits excessive autophagy damage of ovarian granulosa cells and reduces follicular atresia.
作者
崔文哲
朱柯璇
李冉
董静雯
程怡心
张晶
CUI Wenzhe;ZHU Kexuan;LI Ran;DONG Jingwen;CHENG Yixin;ZHANG Jing(School of Acupuncture,Moxibustion and Tuina,Shandong University of Traditional Chinese Medicine,Jinan Shandong 250355,China)
出处
《新中医》
2025年第3期116-124,共9页
New Chinese Medicine
基金
山东省自然科学基金重点项目(ZR2020KH027)。
关键词
卵巢早衰
针刺
PTEN/AKT信号通路
自噬
关元
三阴交
Premature ovarian failure
Acupuncture
PTEN/AKT signaling pathway
Autophagy
Guanyuan(CV4)point
Sanyinjiao(SP6)point
