基于p38 MAPK通路探讨乌丹丸对子宫内膜异位症寒凝血瘀证巨噬细胞极化的影响  

Effect of Wudan Pill on Polarization of Macrophages in Endometriosis with Cold Congeal and Blood Stasis Syndrome Based on p38 MAPK Pathway

作　　者：祝洁 薛晓鸥[1] 贺稚平[1] 代勇 李军[1] 张家蔚 孙海芸[1] ZHU Jie;XUE Xiaoou;HE Zhiping;DAI Yong;LI Jun;ZHANG Jiawei;SUN Haiyun(Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China)

机构地区：[1]北京中医药大学东直门医院,北京100700

出　　处：《中国实验方剂学杂志》2022年第24期105-113,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：北京中医药大学青年教师项目(2019-jms-JYB-JS-082);北京中医药大学新奥基金“名老中医药专家经验挖掘学术研究重点项目”;王子瑜教授全国名老中医传承工作室项目。

摘　　要：目的:基于p38丝裂素活化蛋白激酶(p38 MAPK)探讨乌丹丸对子宫内膜异位症(EMT)寒凝血瘀证模型大鼠巨噬细胞M1/M2极化的影响。方法:选取雌性SD大鼠,随机数字法分为假手术组、模型组、乌丹丸高、中、低剂量组(2.4、1.2、0.6 g·kg^(-1))、中成药组和西药组,采用冰水浴联合自体子宫内膜移植法建立子宫内膜异位症寒凝血瘀证大鼠模型,连续给药4周处死取材。酶联免疫吸附测定法(ELISA)检测肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-4、转化生长因子-β(TGF-β)炎性因子的表达评估炎症;其次,实时荧光定量聚合酶链式反应(Real-time PCR)检测一氧化氮合成酶(iNOS)、TNF-α、精氨酸酶1(Arg1)、甘露糖受体(CD206)转录水平评价巨噬细胞极化;蛋白免疫印迹法(Western blot)和免疫荧光技术(IF)检测iNOS、Arg1蛋白的表达佐证巨噬细胞极化。Western blot和Real-time PCR检测p38 MAPK信号通路相关蛋白表达。结果:与假手术组比较,模型组血清TNF-α,IL-1β、TGF-β,IL-4含量均明显升高(P<0.05),异位内膜组织中iNOS、TNF-α、p-p38 MAPK、酸化细胞外信号调节激酶(p-ERK)蛋白明显升高,iNOS、TNF-α、MAPK、ERK mRNA水平明显升高,Arg1、CD206 mRNA及蛋白表达明显降低(P<0.05,P<0.01),异位内膜组织iNOS阳性细胞数明显升高,Arg1阳性细胞数明显(P<0.05,P<0.01)。与模型组比较,各给药组TNF-α,IL-1β、TGF-β,IL-4表达量均有不同程度降低,乌丹丸高、中剂量组和西药组尤为明显(P<0.05),乌丹丸高、中剂量组及中成药、西药组大鼠异位子宫内膜组织中iNOS、TNF-α、p-p38 MAPK、p-ERK蛋白明显降低,iNOS、TNF-α、MAPK、ERK mRNA水平明显降低,Arg1、CD206 mRNA及蛋白表达明显升高(P<0.05,P<0.01),乌丹丸高、中剂量组及中成药、西药组大鼠异位内膜组织中iNOS细胞阳性数量明显降低,Arg1蛋白阳性数明显升高(P<0.05,P<0.01),乌丹丸低、中、高剂量呈剂量依赖性,高剂量组与西�Objective:To investigate the effect of Wudan pill on the polarization of macrophages in the rat model of endometriosis(EMT)with cold congeal and blood stasis syndrome based on p38 mitogen-activated protein kinases(p38 MAPK).Method:Female SD rats with regular motility cycles were selected and randomly divided into sham-operated group,model group,Wudan pill high,medium,and low-dose groups(2.4,1.2,and 0.6 g·kg^(-1)),Chinese patent medicine group,and western medicine group by the random number table method.The method of ice water bath+autologous endometrial transplantation was used to establish the rat model of EMT with cold congeal and blood stasis,and the rats were executed after 4 weeks of continuous drug administration to collect materials.Expression levels of serum tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-4,and transforming growth factor-β(TGF-β)were determined by enzyme-linked immunosorbent assay(ELISA)to assess inflammation.The real-time quantitative polymerase chain reaction(Real-time PCR)was performed to determine the inducible nitric oxide synthase(iNOS),TNF-α,arginase 1(Arg1),and human mannose receptor(CD206)transcriptional levels to evaluate macrophage polarization.Western blot(WB)and immunofluorescence(IF)assays were used to determine the protein expression levels of iNOS and Arg1 to corroborate macrophage polarization.WB and Real-time PCR were used to determine the protein expression levels of the p38 MAPK pathway.Result:As compared with sham-operated group,the levels of serum TNF-α,IL-1β,TGF-β,and IL-4 of rats in the model group were significantly higher(P<0.05).In the model group,the protein levels of iNOS,TNF-α,p-p38 MAPK,and phosphorylated-extracellular signal-regulated kinase(p-ERK)in endothelial tissues were significantly higher,the mRNA levels of iNOS,TNF-α,MAPK,and ERK were significantly higher,and the mRNA and protein expression levels of Arg1 and CD206 were significantly lower(P<0.05,P<0.01).The number of iNOS positive cells in endothelial tissues was significantly

关 键 词：子宫内膜异位症 寒凝血瘀证 炎症因子 巨噬细胞极化 P38丝裂素活化蛋白激酶 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学] R2-031R285.5