苓桂术甘汤调控Wnt/β-catenin通路抑制心肌成纤维细胞纤维化  

Mechanism of Linggui Zhugan Decoction in inhibiting myocardial fibrosis by regulating Wnt/β-catenin pathway

作　　者：丁芮 李向阳 王翔[1] 王靓[1] 周鹏[1] 黄金玲[1] DING Rui;LI Xiang-yang;WANG Xiang;WANG Liang;ZHOU Peng;HUANG Jin-ling(Anhui University of Chinese Medicine,Hefei 230012,China)

机构地区：[1]安徽中医药大学,安徽合肥230012

出　　处：《中国中药杂志》2024年第15期4178-4187,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(30973707,81373533,81973844)。

摘　　要：该研究旨在通过观察苓桂术甘汤(Linggui Zhugan Decoction,LGZGD)含药血清对心肌成纤维细胞(cardiac fibroblasts,CFs)纤维化及Wnt/β-连环蛋白(β-catenin)信号通路蛋白分子表达的调控机制。制备空白血清和LGZGD含药血清,胰酶-胶原酶依次消化并结合差速贴壁法分离培养原代CFs,采用免疫荧光标记鉴定原代CFs。设正常对照组,模型组,20%空白血清组,5%、10%、20%LGZGD含药血清组。分别用20%空白血清,5%、10%、20%LGZGD含药血清预处理12 h,除正常对照组,其余5组均加入过氧化氢(hydrogen peroxide,H_(2)O_(2))刺激细胞,建立原代CFs纤维化模型。采用划痕愈合实验观察细胞迁移能力,ELISA法检测Ⅰ型胶原(collagenⅠ,ColⅠ)、Ⅲ型胶原(collagenⅢ,ColⅢ)含量,Western blot法检测α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Wnt1、糖原合酶激酶3β(glycogen synthase kinase 3 beta,GSK-3β)、磷酸化糖原合酶激酶3β(phospho synthase kinase 3 beta,p-GSK-3β)、β-catenin及细胞核β-catenin的蛋白表达,RT-qPCR检测β-catenin、基质金属蛋白酶9(matrix metalloprotein 9,MMP9)的基因表达,免疫荧光技术检测关键蛋白α-SMA、β-catenin的表达及定位。制备Wnt1过表达CFs,采用H_(2)O_(2)处理CFs。设正常对照组、模型组、20%LGZGD含药血清组、空载质粒+20%LGZGD含药血清组、Wnt1过表达+20%LGZGD含药血清组。采用ELISA法检测ColⅠ、ColⅢ的含量与其比值,Western blot法检测α-SMA和Wnt1、GSK-3β、p-GSK-3β、β-catenin及细胞核β-catenin的蛋白表达,RT-qPCR检测β-catenin、MMP9的基因表达。免疫荧光染色显示,心肌成纤维细胞Vimentin表达阳性,呈绿色,胞核为蓝色,纯度大于90%,鉴定是原代CFs。结果显示,与正常对照组相比,模型组CFs愈合率增强,ColⅠ、ColⅢ含量升高,ColⅠ/ColⅢ比值增大,α-SMA、Wnt1、p-GSK-3β、β-catenin、核β-catenin蛋白表达上调,GSK-3β蛋白表达降低,β-catenin、MMP9 mRNA表达显著升高,β-catenin、�This study aimed to investigate the regulatory mechanism of Linggui Zhugan Decoction(LGZGD)-medicated serum on the fibrosis of cardiac fibroblasts(CFs)and the protein expression of the Wnt/β-catenin signaling pathway.Blank serum and LGZGD-medicated serum were prepared,and primary CFs were isolated and cultured using trypsin-collagenase digestion and differential adhesion method.Immunofluorescence labeling was used to identify primary CFs.Cells were divided into normal control group,model group,20%blank serum group,and 5%,10%,and 20%LGZGD-medicated serum groups.Except for the normal control group,all other groups were stimulated with hydrogen peroxide(H_(2)O_(2))after pretreatment with 20%blank serum or 5%,10%,20%LGZGD-medicated serum for 12 hours to establish a model of fibrosis in primary CFs.Scratch healing assay was used to observe cell migration ability.ELISA was used to detect the content of collagen typeⅠ(ColⅠ)and typeⅢ(ColⅢ).Western blot was used to detect the protein expression ofα-smooth muscle actin(α-SMA),Wnt1,glycogen synthase kinase 3β(GSK-3β),phosphorylated GSK-3β(p-GSK-3β),β-catenin,and nuclearβ-catenin.RT-qPCR was used to detect the gene expression ofβ-catenin and matrix metalloproteinase 9(MMP9),and immunofluorescence technique was used to detect the expression and localization of key proteinsα-SMA andβ-catenin.CFs with Wnt1 overexpression were prepared and treated with H_(2)O_(2).The following groups were set up:normal control group,model group,20%LGZGD-medicated serum group,empty plasmid+20%LGZGD-medicated serum group,and Wnt1 overexpression+20%LGZGD-medicated serum group.ELISA was used to detect the content and ratio of ColⅠand ColⅢ.Western blot was used to detect the protein expression ofα-SMA,Wnt1,GSK-3β,p-GSK-3β,β-catenin,and nuclearβ-catenin.RT-qPCR was used to detect the gene expression ofβ-catenin and MMP9.Immunofluorescence staining showed that CFs expressed Vimentin positively,appearing green,with blue nuclei and purity greater than 90%,which were identifi

关 键 词：苓桂术甘汤 慢性心力衰竭 心肌纤维化 心肌成纤维细胞 Wnt/β-连环蛋白信号通路 

分 类 号：R285.5[医药卫生—中药学]