基于MAPKs信号通路探讨补肾助孕方改善BN大鼠黄体功能的作用机制  

Mechanism of Bushen Zhuyun Prescription on Improving Luteal Function of Brown Norway Rats Based on MAPKs Signaling Pathway

作　　者：唐星冉 周惠芳 冯华 尚玉洁 袁艺祯 代雅欣 丁银银 TANG Xing-ran;ZHOU Hui-fang;FENG Hua;SHANG Yu-jie;YUAN Yi-zhen;DAI Ya-xin;DING Yin-yin(Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210046,China;Jiangsu Vocational College of Medicine,Yancheng 224008,China;Xishan Traditional Chinese Medicine Hospital,Wuxi 214194,China)

机构地区：[1]南京中医药大学附属医院,南京210046 [2]江苏医药职业学院,江苏盐城224008 [3]无锡市锡山区中医医院,江苏无锡214194

出　　处：《中国实验方剂学杂志》2022年第12期78-85,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(81973898);江苏省社会发展重大项目(BE2021726);江苏省中医院高峰学术人才项目(y2021rc11);研究生科研创新计划项目(KYCX21_1669)。

摘　　要：目的:探讨补肾助孕方降低黄体功能不全(LPD)自然模型-棕色挪威(BN)大鼠卵巢凋亡水平的可能作用机制。方法:使用随机数表法将50只SPF级雌性BN大鼠分为模型组、地屈孕酮组(0.002 g·kg^(-1))、补肾助孕方低、中、高剂量组(4.5、9、18 g·kg^(-1)),给予相应药物剂量灌胃,每天1次,给药3个动情周期。使用蛋白免疫印迹法(Western blot)检测各组大鼠c-Jun氨基酸末端激酶(JNK)、磷酸化(p)-JNK、细胞外信号调节激酶(ERK)、p-ERK、p38丝裂原活化蛋白激酶(p38 MAPK)、p-p38MAPK、B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)、Bcl-2的蛋白表达水平,实时荧光定量聚合酶链式反应(Real-time PCR)检测各组大鼠JNK、ERK、p38 MAPK、Bax、Bcl-2 mRNA表达水平,酶联免疫吸附测定法(ELISA)检测各组大鼠血清孕酮(P)和雌二醇(E;)水平,苏木素-伊红(HE)染色观察卵巢形态。结果:与模型组比较,补肾助孕各剂量组使用1个周期后,大鼠动情周期恢复情况差异有统计学意义(P<0.05);补肾助孕方低剂量组卵巢组织形态有所改善,中、高剂量组卵巢组织各级卵泡结构清晰,囊状卵泡及闭锁卵泡减少,颗粒细胞层数较多,黄体数增多、境界清晰、体积较大,以新鲜黄体为主;补肾助孕方低、中、高剂量组大鼠卵巢组织Bcl-2 mRNA表达水平上调,Bax mRNA表达水平下调(P<0.05,P<0.01);补肾助孕方高剂量组大鼠卵巢组织JNK、p38 MAPK mRNA水平显著下调(P<0.01);补肾助孕方低、中剂量组大鼠卵巢组织ERK mRNA表达水平上调(P<0.05)。与模型组比较,补肾助孕方中、高剂量组Bcl-2蛋白表达水平均显著上调(P<0.01),补肾助孕方各剂量组Bax蛋白表达水平显著下调(P<0.01);补肾助孕方各剂量组大鼠卵巢组织中JNK、ERK、p38 MAPK蛋白表达水平差异无统计学意义;补肾助孕方中、高剂量组大鼠卵巢组织中p-ERK蛋白表达水平显著升高(P<0.01),p-JNK、p-p38 MAPK蛋白表达水平均显著降低(P<0.01)。补肾助Objective:To investigate the possible mechanism of Bushen Zhuyun prescription(BSZYP)to reduce the level of ovarian apoptosis in Brown Norway(BN)rats with luteal phase deficiency(LPD).Method:Fifty SPF female BN rats were randomly divided into a model group,a dydrogesterone group(0.002 g·kg^(-1)),and a low(4.5 g·kg^(-1)),a medium(9 g·kg^(-1)),and a high-dose(18 g·kg^(-1))BSZYP groups,with ten rats in each group.The rats were administrated with corresponding drugs by gavage,once a day for three estrus cycle.Western blot was used to detected the protein expression levels of c-Jun NH2 terminal kinase(JNK),extracellular signal-regulated kinase(ERK),phosphorylated-ERK(p-ERK),phosphorylated-JNK(p-JNK),p38 mitogen-activated protein kinase(p38 MAPK),phosphorylated-p38 MAPK(p-p38 MAPK),B-cell lymphoma(Bcl-2),and Bcl-2 associated X protein(Bax)in ovary.Real-time quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression levels of ERK,JNK,p38 MAPK,Bax,and Bcl-2 in ovary.Enzyme-linked immunosorbent assay(ELISA)was used to detect the serum progesterone(P)and estradiol(E;)levels.Hematoxylin-eosin(HE)staining was used to observe the ovarian tissue morphology.Result:Compared with the model group,the recovery of estrus cycle of rats in all BSZYP groups had statistical significance after 1-circle administration(P<0.05).The ovarian tissue morphology in the low-dose BSZYP group was improved,and that in the medium and high-dose BSZYP groups was significantly improved with clear follicle,less vesicular follicle and atretic follicle,and more granular layers.The number of luteum,especially the fresh luteum,in the medium and high-dose groups was increased with smooth edge and large volume.The mRNA expression level of Bcl-2 was increased in all-dose BSZYP groups,while the mRNA expression level of Bax was significantly decreased in all-dose BSZYP group(P<0.05,P<0.01).The mRNA expression levels of JNK and p38 MAPK were significantly decreased in the high-dose BSZYP group(P<0.01),and the mRNA expression lev

关 键 词：补肾助孕方 丝裂原活化蛋白激酶(MAPKs) 黄体功能不全(LPD) 凋亡 卵巢 

分 类 号：R2-0[医药卫生—中医学] R22