摘要
目的:探讨归肾丸加味通过调控丝裂原活化蛋白激酶(MAPK)/核转录因子-κB(NF-κB)通路抑制多囊卵巢综合征(PCOS)大鼠炎症的作用机制。方法:按照随机数字表法将60只SPF级雌性SD大鼠随机分为正常组10只及PCOS模型组50只。正常组予以常规喂养,模型组采用来曲唑(1 mg·kg^(-1)·d^(-1))灌胃21 d建立PCOS模型。将建模成功的大鼠再次随机分为模型组,达英-35组(0.2 g·kg^(-1)·d^(-1))和归肾丸加味高、中、低剂量组(16.04、8.02、4.01 g·kg^(-1)·d^(-1)),正常组和模型组予以等体积生理盐水,持续灌胃28 d。治疗结束后,采用革兰氏染色观察各组大鼠阴道涂片观察动情周期;酶联免疫吸附测定法(ELISA)检测血浆中卵泡刺激素(FSH)、雌二醇(E_(2))、黄体生成素(LH)、睾酮(T)、孕酮(PROG)、血浆及卵巢组织中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)水平,并计算LH/FSH;苏木素-伊红(HE)染色观察卵巢组织形态学变化;蛋白免疫印迹法(Western blot)检测大鼠卵巢组织中细胞外调节蛋白激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38 MAPK、NF-κB p65、NF-κB抑制蛋白α(IκBα)、磷酸化(p)-JNK、p-ERK、p-p38 MAPK、p-NF-κB p65、p-IκBα的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测大鼠卵巢组织中ERK、JNK、p38 MAPK、NF-κB p65、IκBαmRNA表达。结果:与正常组比较,模型组大鼠处于动情间期,卵巢囊泡数量明显增多,颗粒细胞及黄体生成均明显减少(P<0.05),血浆FSH、E_(2)表达降低,LH、T及LH/FSH明显升高(P<0.05);与模型组比较,归肾丸加味高、中、低剂量组均能有效改善PCOS大鼠的发情周期,增加颗粒细胞及黄体的生成,减少囊泡数量,增加FSH、E_(2)的表达,降低LH、T及LH/FSH(P<0.05,P<0.01),且呈剂量依赖性,以归肾丸加味高剂量组疗效最佳,故后续机制实验在正常组、模型组和归肾丸加味高剂量组中进行。与模型组比较,归肾丸加味高剂量组能明显降低血浆及卵巢组织中IL-1β、TNF-α水平,升高IL-10表达(P<0.05,P<0.01),且能明显降低PCOS大鼠p-ERK、p-JNK、p-p38 MAPK、p-NF-κB p65、p-IκBα蛋白表达,增加IκBα蛋白表达(P<0.01);同时,归肾丸加味高剂量组明显降低ERK、JNK、p38 MAPK、NF-κB p65 mRNA表达(P<0.05,P<0.01)。结论:归肾丸加味能有效改善来曲唑诱导的PCOS大鼠的卵巢功能,并对其具有抗炎作用,其机制可能与抑制MAPK/NF-κB通路有关。
Objective:To explore the mechanism by which modified Guishenwan alleviates inflammation in the rat model of polycystic ovary syndrome(PCOS)by regulating the mitogen-activated protein kinase(MAPK)/nuclear factor kappa B(NF-κB)pathway.Methods:According to the random number table method,60 SPF female SD rats were randomized into a normal group(n=10)and a modeling group(n=50).The normal group received routine feeding,while the modeling group was administrated with letrozole(1 mg·kg^(-1)·d^(-1))by gavage for 21 days for the modeling of PCOS.The successfully modeled rats were randomized into model,diane-35(0.2 g·kg^(-1)·d^(-1)),high-(16.04 g·kg^(-1)·d^(-1)),medium-(8.02 g·kg^(-1)·d^(-1)),low-(4.01 g·kg^(-1)·d^(-1))dose modified Guishenwan groups.The drug intervention groups were administrated with modified Guishenwan at corresponding doses by gavage,and the normal group and model group were given equal volumes of normal saline.All the groups were continuously treated for 28 days.After treatment,Gram staining of vaginal smears was employed to observe the estrous cycle in each group.Enzyme-linked immunosorbent assay was employed to determine the levels of follicle-stimulating hormone(FSH),estradiol(E_(2)),luteinizing hormone(LH),testosterone(T),and progesterone(PROG)in the plasma,as well as interleukin-1 beta(IL-1β),tumor necrosis factor-alpha(TNF-α),and interleukin-10(IL-10)in the plasma and ovarian tissue.The LH/FSH ratio was calculated.The morphological changes in the ovarian tissue were observed by hematoxylin-eosin(HE)staining.Western blot was employed to determine the protein levels of extracellular-regulated protein kinase(ERK),c-Jun N-terminal kinase(JNK),p38 MAPK,NF-κB p65,IκBα,p-JNK,p-ERK,p-p38 MAPK,p-NF-κB p65,and p-IκBαin the ovarian tissue.Real-time quantitative polymerase chain reaction was used to determine the mRNA levels of ERK,JNK,p38 MAPK,NF-κB p65,and IκBαin the ovarian tissue.Results:Compared with the normal group,the model group was in the estrus phase,with an increase in the number of ovarian vesicles and decreases in granulosa cells and corpus luteum formation(P<0.05),and lowered levels of FSH and E_(2) and elevated levels of LH,T,and LH/FSH in the plasma(P<0.05).Compared with the model group,high-,medium-,and lowdose modified Guishenwan recovered the estrous cycle,increased the generation of granulosa cells and corpus luteum,reduced the number of vesicles,elevated the levels of FSH and E_(2),and lowered the levels LH,T,and LH/FSH(P<0.05,P<0.01)in a dosedependent manner.High-dose modified Guishenwan demonstrated the best therapeutic effect.Therefore,subsequent experiments for exploring the treatment mechanism were conducted in the normal group,model group,and high-dose modified Guishenwan group.The results showed that compared with the model group,high-dose modified Guishenwan lowered the levels of IL-1β,TNF-α,and IL-10 and elevated the level of IL-10 in the plasma and ovarian tissue(P<0.05,P<0.01),down-regulated the protein levels of p-ERK,p-JNK,p-p38 MAPK,p-NF-κB p65,and p-IκBα,while up-regulating the protein level of IκBα(P<0.01).At the same time,the mRNA levels of ERK,JNK,p38 MAPK,and NF-κB p65 in the high-dose modified Guishenwan group were downregulated(P<0.05,P<0.01).Conclusion:Modified Guishenwan can improve the ovarian function in rat model of PCOS induced by letrozole and has anti-inflammatory effects,which may be related to inhibition of the MAPK/NF-κB pathway.
作者
田珈瑜
秦文熠
杨涓
荣晓凤
TIAN Jiayu;QIN Wenyi;YANG Juan;RONG Xiaofeng(The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第5期86-94,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金面上项目(81973653)。
