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

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 (H₂O₂) 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 type Ⅰ (Col Ⅰ) and type Ⅲ (Col Ⅲ). Western blot was used to detect the protein expression of α-smooth muscle actin (α-SMA), Wnt1, glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β), β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and matrix metalloproteinase 9 (MMP9), and immunofluorescence technique was used to detect the expression and localization of key proteins α-SMA and β-catenin. CFs with Wnt1 overexpression were prepared and treated with H₂O₂. 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 Col Ⅰ and Col Ⅲ. Western blot was used to detect the protein expression of α-SMA, Wnt1, GSK-3β, p-GSK-3β, β-catenin, and nuclear β-catenin. RT-qPCR was used to detect the gene expression of β-catenin and MMP9. Immunofluorescence staining showed that CFs expressed Vimentin positively, appearing green, with blue nuclei and purity greater than 90%, which were identified as primary CFs. RESULTS: : showed that compared with the normal control group, CFs in the model group had enhanced healing rate, increased content of Col Ⅰ and Col Ⅲ, increased ratio of Col Ⅰ/Col Ⅲ, upregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, decreased GSK-3β expression, elevated mRNA expression of β-catenin and MMP9, and enhanced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the model group, 5%, 10%, 20% LGZGD-medicated serum significantly inhibited cell migration ability, reduced the content of Col Ⅰ and Col Ⅲ, decreased ratio of Col Ⅰ/Col Ⅲ, downregulated protein expression of α-SMA, Wnt1, p-GSK-3β, β-catenin, nuclear β-catenin, increased GSK-3β expression, decreased mRNA expression of β-catenin and MMP9, and reduced fluorescence intensity and expression of β-catenin and α-SMA. Compared with the empty plasmid+20% LGZGD-medicated serum group, the effect of LGZGD-medicated serum was significantly reversed after overexpression of Wnt1. LGZGD can reduce excessive deposition of collagen fibers, inhibit excessive proliferation of fibroblasts, and improve the process of myocardial fibrosis. The improvement of myocardial fibrosis by LGZGD is related to the regulation of the Wnt/β-catenin pathway, reduction of collagen deposition, and protection of myocardial cells.