Jiedu Tongluo Decoction Attenuates Myocardial Fibrosis Through Inhibition of The TGF-β1/Smad2/3 Pathway

Background: Jiedu Tongluo (JDTL) decoction is a traditional Chinese medicine (TCM) formula containing three herbal ingredients that is widely used to treat myocardial fibrosis (MF). This study aimed to investigate the molecular mechanism of action of JDTL decoction for the treatment of MF. Methods: The chemical constituents of Traditional Chinese medicine were analyzed by HPLC, Forty Wistar rats were randomly divided into normal control group, model group, Jiedu Tongluo decoction low-dose and high-dose treatment groups, with 10 rats in each group. Rat myocardial fibrosis model was induced by subcutaneous injection of 5 mg•kg-1 isoproterenol hydrochloride. 5 and 50 g•(kg.d) -1 were given intragastric administration for 7 days. Hematoxylin-eosin (HE) and Masson staining were performed for histological evaluation of myocardial tissue, MTT method detects the activity of cardiac fibroblasts, The alkaline water method is used to determine the content of hydroxyproline in myocardial tissue and myocardial fibroblasts, Immunohistochemical method was used to detect the expression of myocardial tissue transforming growth factor (TGF) β1, p smad2/3, and type III collagen (Col III) expression, Immunofluorescence to detect the expression of α-SMA/Vimentin in myocardial tissue and myocardial fibroblasts; Western blot was used to detect the protein expression levels of TGFβ1 and psmad2/3 in cardiac fibroblasts.Results: In this study, six compounds of JDTL decoction were identified by high-performance liquid chromatography (HPLC). Hematoxylin and eosin (HE) and Masson staining showed that in the isoproterenol hydrochloride (ISO)-induced MF rat model, JDTL treatment protected the myocardial structure and inhibited type III collagen (COL3) expression(p<0.05). The immunohistochemistry (IHC) results also showed that JDTL treatment significantly reduced the expression of vimentin, alpha-smooth muscle actin (α-SMA), and transforming growth factor-beta 1 (TGF-β1) as well as the phosphorylation of Smad2/3 in the rat MF model(p<0.05). Rat cardiac fibroblasts (RCFs) were used for the following assays performed in vitro: hydroxyproline detection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound-healing test, western blot, and double immunofluorescence staining; the results of these assays confirmed the inhibitory effects of JDTL decoction on the proliferation, migration, and transdifferentiation abilities of RCFs as well as the molecular mechanisms underlying these effects, including the inhibition of the TGF-β1/Smad2/3 pathway through downregulation of TGF-β1 expression and phosphorylation of Smad2/3 as well as inhibition of vimentin and α-SMA expression(p<0.05). Conclusion: JDTL decoction can prolong the process of MF through inhibition of the TGF-β1/Smad2/3 signaling pathway.