基于p38-MAPK通路探讨真武汤对心力衰竭心肌细胞结构重构-电重构的影响  

Exploring Effect of Zhenwu Decoction(真武汤)on Structural Remodeling-Electrical Remodeling of Cardiomyocytes in Heart Failure Based on p38-MAPK Pathway

作　　者：王晓琳 张倩 田鑫 陈智勇 车驰 徐丽丽 刘智明 陈智军[3] 王兴 廖金花 廖东华[3] 李林[3] WANG Xiaolin;ZHANG Qian;TIAN Xin;CHEN Zhiyong;CHE Chi;XU Lili;LIU Zhiming;CHEN Zhijun;WANG Xing;LIAO Jinhua;LIAO Donghua;LI Lin(Graduate School of Jiangxi University of Chinese Medicine,Nanchang 330000,Jiangxi,China;The First People's Hospital of Xiushui County,Jiujiang 332000,Jiangxi,China;Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330000,Jiangxi,China;Hunan Provincial Hospital of Traditional Chinese Medicine,Zhuzhou 412000,Hunan,China;Guangzhou Zengcheng District Traditional Chinese Medicine Hospital,Guangzhou 510000,Guangdong,China)

机构地区：[1]江西中医药大学研究生院,江西南昌330000 [2]修水县第一人民医院,江西九江332000 [3]江西中医药大学附属医院,江西南昌330000 [4]湖南省直中医医院,湖南株洲412000 [5]广州市增城区中医医院,广东广州510000

出　　处：《中华中医药学刊》2024年第4期130-137,I0032-I0034,共11页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81960820);全国中医药创新骨干人才培养项目(国中医药人教函〔2019〕128号);江西省自然科学基金项目(20202BABL206141);江西省中医药青年骨干人才项目(〔2020〕59号);江西中医药大学研究生创新专项(JZYC21S19)。

摘　　要：目的真武汤在治疗慢性心力衰竭(Heart Failure,HF)过程中能够改善心律失常,旨在从p38-MAPK通路调控肥大心肌结构重构和电重构角度探讨真武汤治疗HF相关心律失常的作用机制。方法用AngⅡ诱导H9c2心肌细胞构建心肌肥大模型,实验共分6组:正常组(N),模型组(M)和真武汤低剂量组(D)、中剂量组(Z)、高剂量组(G)及抑制剂组(S)。正常组(N)用含有胎牛血清的高糖培养基培养,模型组(M)用含有10^(-6) mol/L浓度的血管紧张素Ⅱ(Angio-tensinⅡ,AngⅡ)培养基处理,低(D)、中(Z)、高(G)剂量组分别予2%、4%、8%体积分数的真武汤含药血清及AngⅡ培养基共同处理,抑制剂组(S)用含有p38MAPK抑制剂SB203580的培养基处理。处理48 h后,镜下观察各组心肌细胞的生长情况,通过酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay,ELISA)法测心肌肥大标志物ANP的含量,用β-半乳糖苷酶染色法检测细胞衰老情况,采用Western blot法检测各组心肌细胞缝隙连接蛋白43(Connexin 43,Cx43)、p38丝裂原激活的蛋白激酶(p38 Mitogen-ctivated Protein Kinase,p38-MAPK)、基质金属蛋白酶2(Matrix Metalloproteinase 2,MMP2)、基金金属蛋白酶9(Matrix Metalloproteinase 9,MMP9)、金属蛋白酶组织抑制剂(Tissue Inhibitor of Metalloprotei-nase1,TIMP1)、p53蛋白的表达情况,采用实时荧光定量PCR(Quantitative Real-time PCR,qPCR)定量分析各组心肌细胞ANP、Cx43、p-Cx43、p38-MAPK、MMP2、MMP9、TIMP1的mRNA转录水平,免疫荧光检测Cx43蛋白的表达和分布情况。结果高剂量的真武汤能够减轻肥大心肌细胞的表面积,中低剂量无明显效果;真武汤能够降低心肌肥大标志物ANP及其mRNA的水平,且其效果与剂量呈正相关;真武汤能改善肥大细胞的衰老情况,效果与抑制剂相似;真武汤能够降低肥大心肌细胞Cx43、p38-MAPK、MMP2、MMP9、p53、p-Cx43蛋白的表达水平及Cx43、p38-MAPK、MMP2、MMP9的mRNA转录水平,且能够增加TIMP1的表达�Objective Zhenwu Decoction(真武汤)can improve arrhythmia in the treatment of chronic heart failure(HF).The purpose of this study is to explore the mechanism of Zhenwu Decoction in the treatment of HF-related arrhythmias from the point of view of p38-MAPK pathway regulating hypertrophic myocardial structural remodeling and electrical remodeling.Methods H9c2 cardiomyocytes were induced by Angiotensin Ⅱ(AngⅡ)to establish the cardiac hypertrophy model.The experiment was divided into six groups:normal group(N),model group(M),Zhenwu Decoction low dose group(D),middle dose group(Z),high dose group(G)and inhibitor group(S).The normal group(N)was cultured in high glucose medium containing fetal bovine serum,and the model group(M)was treated with AngⅡ medium containing 10^(-6)mol/L concentration.The low(D),middle(Z)and high(G)dose groups were treated with 2%,4% and 8% volume fraction of Zhenwu Decoction containing serum and AngⅡ culture medium,respectively,and the inhibitor group(S)was treated with medium containing p38MAPK inhibitor SB203580.After 48 hours of treatment,the growth of cardiomyocytes in each group was observed under microscope,the content of cardiac hypertrophy marker Atrial Natriuretic Peptide(ANP)was measured by Enzyme Linked Immunosorbent Assay(ELISA)method,the senescence of cardiomyocytes was detected by β-galactosidase staining,the expressions of Connexin 43(Cx43),p38 Mitogen-ctivated Protein Kinase(p38-MAPK),Matrix Metalloproteinase 2(MMP2),Matrix Metalloproteinase 9(MMP9),Tissue Inhibitor of Metalloproteinase1(TIMP1)and p53 protein in cardiomyocytes were detected by Westernblot method,and the mRNA transcription levels of ANP,Cx43,p-Cx43,p38-MAPK,MMP2,MMP9 and TIMP1 were quantitatively analyzed by Quantitative Real-time PCR(qPCR).The expression and distribution of Cx43 protein were detected by immunofluorescence.Results High dose of Zhenwu Decoction could reduce the surface area of hypertrophic cardiomyocytes,but the middle and low doses had no obvious effect.Zhenwu Decoction could reduce t

关 键 词：真武汤 心力衰竭 结构重构 电重构 CX43 P38-MAPK 缝隙连接 基质金属蛋白酶 

分 类 号：R289.5[医药卫生—方剂学]