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Myocardial fibrosis is the main morphological change of ventricular remodelling caused by cardiovascular diseases, mainly manifested due to the excessive production of collagen proteins. SRY-related high mobility group-box gene 9 (SOX9) is a new target regulating myocardial fibrosis. Bellidifolin (BEL), the active component of G. acuta, can prevent heart damage. However, it is unclear whether BEL can regulate SOX9 to alleviate myocardial fibrosis. The mice were subjected to isoproterenol (ISO) to establish myocardial fibrosis, and human myocardial fibroblasts (HCFs) were activated by TGF-β1 in the present study. The pathological changes of cardiac tissue were observed by HE staining. Masson staining was applied to reveal the collagen deposition in the heart. The measurement for expression of fibrosis-related proteins, SOX9, and TGF-β1 signalling molecules adopted Western blot and immunohistochemistry. The effects of BEL on HCFs, activity were detected by CCK-8. The result showed that BEL did not affect cell viability. And, the data indicated that BEL inhibited the elevations in α-SMA, Collagen I, and Collagen III by decreasing SOX9 expression. Additionally, SOX9 suppression by siRNA downregulated the TGF-β1 expression and prevented Smad3 phosphorylation, as supported by reducing the expression of α-SMA, Collagen I, and Collagen III. In vivo study verified that BEL ameliorated myocardial fibrosis by inhibiting SOX9. Therefore, BEL inhibited SOX9 to block TGF-β1 signalling activation to ameliorate myocardial fibrosis. [ABSTRACT FROM AUTHOR]

Ethnopharmacological Relevance. GanDouLing (GDL) is a Chinese medicinal herb produced by the preparation center of Anhui Hospital of TCM for preventing and treating Wilson's disease (WD), an ATP7B mutation-inherited disease that affects copper transport and is characterized by liver and nervous system manifestations with variable and often unpredictable manifestations. However, the "multicomponent" and "multitarget" characteristics of TCM make it challenging to clarify the potential therapeutic mechanisms of GDL for WD. Aim of the Study. This study aimed at the systematic encoding of WD potential target for GDL and experimental verification for the relevant core targets, providing a deeper insight into the understanding of the mechanisms of GDL protection underlying WD with liver injury. Material and Methods. Following the strategy of the network pharmacology, we, firstly, predicted the active components of GDL and putative targets for WD. By employing clusterProfiler, the enrichment of functional and pathway terms was analyzed. Further, the protein-protein-interaction network was analyzed by STRING. Lastly, after establishing the toxic-milk mouse (TX) model with GDL treating, Hematoxylin and Eosin stain (HE) and western blotting (WB) for apoptosis biomarker were experimented. Results. Firstly, 324 active compounds have been identified in the GDL formula. Meanwhile, we identified 1496 human genes which are related to WD or liver cirrhosis. Functional and pathway enrichment analysis indicated that NOD-like receptor signaling pathway, bile secretion, calcium signaling pathway, steroid hormone biosynthesis, T cell receptor signaling pathway, apoptosis, MAPK signaling pathway, and so forth can be obviously regulated by GDL. Further, in a mouse model of WD, in vivo experiments showed that GDL treatment can not only reduce the pathological symptoms of the liver but also reduce the apoptosis of hepatocytes. Conclusions. In this study, systemic pharmacological methods were proposed and the mechanism of GDL combined therapy for WD was explored. This method can be used as a reference for the study of other mechanisms of traditional Chinese medicine. [ABSTRACT FROM AUTHOR]