摘要
目的:探究健脾益肾化浊方(JPYSHZ)改善多囊卵巢综合征(PCOS)卵巢功能的作用机制。方法:将70只SD雌性大鼠随机分为6组,空白组和模型组各15只,二甲双胍组(0.1 g·kg^(-1))、JPYSHZ低、中、高剂量组(1.275、2.55、5.10 g·kg^(-1))组各10只。空白组给予生理盐水(10 mL·kg^(-1))灌胃和普通饲料喂养,其余各组均采用来曲唑(1 mg·kg^(-1))灌胃联合高脂饲料喂养21 d构建PCOS模型,造模结束后,空白组和模型组给与等体积生理盐水灌胃,各药物组给予相应剂量的药物灌胃,共30 d。比较造模前与造模后大鼠体质量和空腹血糖(FPG)变化;苏木素-伊红(HE)染色观察各组大鼠卵巢组织形态学改变;酶联免疫吸附测定法(ELISA)监测大鼠血清促卵泡生成素(FSH)、促黄体生成素(LH)、睾酮(T)、抗苗勒管激素(AMH)和雌二醇(E2)水平,并计算LH/FSH;免疫组化法和蛋白免疫印迹法(Western blot)检测大鼠卵巢组织中核苷结合寡聚化域蛋白样受体3(NALP3)、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(Caspase-1)、核转录因子-κB(NF-κB)、白细胞介素(IL)-1β、IL-18和IL-6)蛋白表达水平。结果:与空白组比较,模型组大鼠卵巢中可见较多囊性扩张的大卵泡,未见优势卵泡、卵泡颗粒层减少且排列松散,黄体数量明显减少;血清T、LH、AMH和LH/FSH明显升高(P<0.05,P<0.01),FSH和E2明显降低(P<0.05,P<0.01);卵巢组织中NALP3、ASC、Caspase-1、NF-κB、IL-1β、IL-18和IL-6蛋白相对表达量均明显升高(P<0.05,P<0.01)。与模型组比较,JPYSHZ各剂量组和二甲双胍组卵巢组织中可见各级生长卵泡和黄体,囊性扩张卵泡减少,卵泡颗粒层厚度增加,可见成熟卵泡,卵母细胞局部形态完整;血清T、LH、AMH和LH/FSH明显降低(P<0.05,P<0.01),E2和FSH明显升高(P<0.05);卵巢组织中NALP3、ASC、Caspase-1、NF-κB、IL-1β、IL-18和IL-6蛋白相对表达量明显下降(P<0.05,P<0.01)。结论:JPYSHZ可通过抑制NLRP3炎症小体激活,减少卵巢组织中各炎性因子释放,调节内分泌水平,有效减轻PCOS炎症状态,发挥改善卵巢功能作用。
Objective:To explore the mechanism of Jianpi Yishen Huazhuo prescription in the improvement of ovarian function in polycystic ovary syndrome(PCOS).Method:Seventy female SD rats in SPF grade were randomly divided into 6 groups,15 in the blank group and 15 in the model group,10 in the metformin group(0.1 g·kg^(-1)·d^(-1)),and 10 in the low(1.275 g·kg^(-1)·d^(-1)),medium(2.55 g·kg^(-1)·d^(-1)),and highdose(5.10 g·kg^(-1)·d^(-1))Jianpi Yishen Huazhuo prescription groups.The blank group was given normal saline(10 mL·kg^(-1)·d^(-1))by gavage and ordinary feed,and the other groups were given letrozole(1 mg·kg^(-1)·d^(-1))by gavage combined with high-fat feed for 21 days to induce the model of PCOS.After modeling,the blank group and model group were given equal volume normal saline by gavage,and each drug group was given the corresponding dose of the drug by gavage for 30 days.The changes in body mass and fasting blood glucose(FPG)of rats before and after modeling were compared.Hematoxylin eosin(HE)staining was used to observe the morphological change in the ovaries of rats in each group.The serum levels of follicle stimulating hormone(FSH),luteinizing hormone(LH),testosterone(T),anti-Mullerian hormone(AMH),and estradiol(E2)were measured by enzyme-linked immunosorbent assay(ELISA),and the LH/FSH ratio was calculated.Immunohistochemical staining(IHC)and Western blot were used to detect the protein expression levels of nucleoside binding oligomerization domain protein like receptor 3(NALP3),apoptosis-associated speck-like protein(ASC),cysteine protease-1(Caspase-1),nuclear transcription factor-κB(NF-κB),interleukin-1β(IL-1β),interleukin-18(IL-18),and interleukin-6(IL-6)in the rat ovaries.Result:As compared with the blank group,large follicles with polycystic expansion were found in the ovaries of the model group,no dominant follicles were found,the granular layer of follicles decreased and arranged loosely,and the number of corpus luteum decreased significantly.Serum T,LH,AMH and LH/FSH increased in the model group(P<0.05,P<0.01),while FSH and E2 decreased(P<0.05,P<0.01).The relative protein expression levels of NALP3,ASC,Caspase-1,NF-κB,IL-1β,IL-18,and IL-6 increased(P<0.05,P<0.01)in the ovaries of the model group.Compared with the model group,the low,medium,and high-dose Jianpi Yishen Huazhuo prescription groups and the metformin group showed growing follicles and corpus luteum at all levels,the number of cystic expanding follicles decreased,the thickness of follicular granular layer increased,the number of follicular fluid increased,mature follicles were visible,and the local morphology of oocytes was complete.Serum T,LH,AMH,and LH/FSH in these groups decreased(P<0.05,P<0.01),while E2 and FSH increased(P<0.05).The relative protein expressions of NALP3,ASC,Caspase-1,NF-κB,IL-1β,IL-18,and IL-6 in the ovaries of these groups decreased(P<0.05,P<0.01).There was no significant difference among the treatment groups.Conclusion:By inhibiting the activation of NLRP3 inflammasome,Jianpi Yishen Huazhuo prescription reduces the release of NALP3,ASC,Caspase-1,NF-κB,IL-18,IL-1β,and IL-6 inflammatory factors in ovarian tissues,regulates endocrine level,and effectively reduces PCOS inflammatory statu,so as to play a role in improving ovarian function.
作者
陈苗
杜小利
冯亚宏
张晓静
罗嘉琦
王静
南楠
刘文钊
邢莎莎
田瑞莹
杨丽
孙苗
何瑞
陈冬梅
马会明
CHEN Miao;DU Xiaoli;FENG Yahong;ZHANG Xiaojing;LUO Jiaqi;WANG Jing;NAN Nan;LIU Wenzhao;XING Shasha;TIAN Ruiying;YANG Li;SUN Miao;HE Rui;CHEN Dongmei;MA Huiming(Ningxia Medical University,Yinchuan750004,China;Ningxia Key Laboratory of Integrated Traditional Chinese and Western Medicine,Key Laboratory of Fertility Preservation and Maintenance,Experimental Animal Center of Ningxia Medical University,Ningxia Key Laboratory of Stem Cell and Regenerative Medicine of General Hospital,Ningxia Medical University,Yinchuan750004,China;Ningxia Chinese Medicine Research Center,Yinchuan750004,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第20期61-70,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
宁夏自然科学基金项目(2020AAC03430,2022AAC03216)
国家自然科学基金项目(81660813)
宁夏科技领军人才项目(2020GKLRLX11)
宁夏药物研发创新团队项目(NXKJT2019012)。
