基于TLR4/NF-κB p65信号通路研究左归丸防治产前应激子代大鼠卵巢储备功能下降的分子机制

Zuoguiwan Prevent Decreased Ovarian Reserve in Prenatal Stressed OffspringRats via TLR4/NF-κB p65 Signaling Pathway

目的:观察左归丸对产前应激(PS)模型大鼠子代雌性大鼠卵巢储备功能的影响,基于Toll样受体4/核转录因子-κB p65(TLR4/NF-κB p65)信号通路探讨其作用机制。方法:制备32只孕鼠,随机分为正常组、模型组、左归丸组(18.9 mg·kg^(-1))、维生素E组(1.44 mg·kg^(-1))4组,每组8只,除正常组以外,其余3组在确定妊娠的第11天开始通过慢性不可预测轻度应激法(CUMS)复制PS孕鼠模型,造模的同时进行相应药物的灌胃治疗,直至生产。以蔗糖偏好实验(SPT)、旷场实验(OFT)、血清皮质酮(CORT)含量评估PS孕鼠模型。通过监测发情周期、观察卵巢形态学变化、酶联免疫吸附测定法(ELISA)检测75日龄子代大鼠血清雌二醇(E2)、促黄体生成素(LH)、卵泡刺激素(FSH)、抗苗勒管激素(AMH)水平评价卵巢储备功能;计算卵巢指数和子宫指数;ELISA检测血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)含量;苏木素-伊红(HE)染色观察出生当天和75日龄子代卵巢组织形态的改变;免疫荧光法(IF)检测75日龄子代大鼠卵巢中NF-κB p65的入核情况;蛋白免疫印迹法(Western blot)检测卵巢组织TLR4、NF-κB p65等相关蛋白的表达。结果:与正常组比较,出生当天模型组子代仔鼠原始卵泡数大幅度显著减少(P<0.01),75日龄模型组子代大鼠动情周期紊乱,卵巢指数、子宫指数显著降低(P<0.01),黄体数减少,闭锁卵泡显著增多(P<0.01),性成熟模型组子代大鼠血清AMH、E2水平显著降低(P<0.01),LH、FSH水平升高(P<0.01),IL-1β、TNF-α水平明显升高(P<0.05,P<0.01),TLR4、NF-κB p65、髓分化因子88(MyD88)、磷酸化NF-κB抑制因子α(p-IκBα)蛋白表达显著升高(P<0.01);与模型组比较,出生当天左归丸组,维生素E组仔鼠原始卵泡数显著增多(P<0.01),75日龄左归丸组、维生素E组大鼠动情周期恢复,卵巢指数明显升高(P<0.05,P<0.01),子宫指数均明显升高(P<0.05),黄体数均显著增加(P<0.01),闭锁卵泡数均显著减少(P<0.01),血清AMH、E2水平均明显升高(P<0.05,P<0.01),LH、FSH水平明显降低(P<0.05,P<0.01),IL-1β、TNF-α水平均明显降低(P<0.05,P<0.01),TLR4、NF-κB p65、MyD88、p-IκBα蛋白表达均明显降低(P<0.05,P<0.01)。结论:左归丸能有效改善先天肾虚子代大鼠卵巢储备功能,作用机制可能与TLR4/NF-κB p65信号通路有关。

Objective:To observe the effect of Zuoguiwan on ovarian reserve in the female offspring rat model of prenatal stress(PS)and explore the mechanism based on Toll-like receptor 4/nuclear factor-κB p65(TLR4/NF-κB p65)signaling pathway.Method:Thirty-two pregnant rats were prepared and randomized into four groups(n=8):control,model,Zuoguiwan(18.9 mg·kg^(-1)),and vitamin E(1.44 mg·kg^(-1)).Except the control group,the other three groups were subjected to chronic unpredictable mild stress(CUMS)from day 11 of pregnancy,and the modeling was accompanied by gavage with corresponding drugs until delivery.The PS model was evaluated by the sucrose preference test,open field test,and serum corticosterone(CORT)level.The estrous cycle was monitored and the morphological changes in the ovarian tissue were observed.The serum levels of estradiol(E2),luteinizing hormone(LH),follicle-stimulating hormone(FSH),and anti-Mullerian hormone(AMH)in the 75-day-old offspring rats were measured by enzyme-linked immunosorbent assay(ELISA)to evaluate the ovarian reserve.The ovary and uterus indices were calculated.The serum levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were measured by enzyme-linked immunosorbent assay(ELISA).The morphology of the ovarian tissue in the offspring on the day of birth and day 75 after birth was observed by hematoxylin-eosin staining.The transport of NF-κB p65 to the nucleus in the ovaries of the 75-day-old offspring was detected by the immunofluorescence(IF)assay.The expression of TLR4,NF-κB p65 and other related proteins in the ovarian tissue was determined by Western blot.Result:Compared with the control group,the model group showed reduced primordial follicles in the offspring on the day of birth(P<0.01)as well as disturbed estrous cycle,decreased ovary index and uterus index(P<0.01),reduced corpus luteum,increased atretic follicles(P<0.01),lowered serum levels of AMH and E2(P<0.01),elevated serum levels of LH,FSH,IL-1β,and TNF-α(P<0.05,P<0.01),and up-regulated protein levels of TLR4,NF-κB p65,recombinant myeloid differentiation factor 88(MyD88),and phosphorylated NF-κB inhibitor(p-IκBα)(P<0.01)in the 75-day-old offspring rats.Compared with the model group,Zuoguiwan and vitamin E increased the primordial follicles in the offspring on the day of birth(P<0.01).Moreover,they resumed the estrous cycle,increased the ovary and uterine indices(P<0.05,P<0.01)and corpus luteum(P<0.01),reduced atretic follicles(P<0.01),elevated the serum levels of AMH and E2(P<0.05,P<0.01),lowered the serum levels of LH,FSH,IL-1β,and TNF-α(P<0.05,P<0.01),and down-regulated the expression of TLR4,NF-κB p65,MyD88,and p-IκB-α(P<0.05,P<0.01)in the 75-day-old offspring.Conclusion:Zuoguiwan can improve the ovarian reserve in the offspring rat model of congenital kidney deficiency by regulating the TLR4/NF-κB p65 signaling pathway.