Formononetin, an isoflavone compound, has been extensively researched due to its various biological activities, including a potent protective effect on the cardiovascular system. However, the impact of formononetin on cardiac fibrosis has not been investigated. In this study, C57BL/6 mice were used to establish cardiac fibrosis animal models by subcutaneous injecting of isoproterenol (ISO) and formononetin was orally administrated. The results showed that formononetin reversed ISO-induced heart stiffness revealed by early-to-atrial wave ratio (E/A ratio). Masson staining, western blot, immunohistochemistry and real-time PCR exhibited that the cardiac fibrosis and fibrosis-related proteins (collage III, fibronectin, TGF-β1, α-SMA, and vimentin) and genes (Col1a1, Col3a1, Acta2 and Tgfb1) induced by ISO were significantly suppressed by formononetin. Furthermore, by combining metabolomics and network pharmacology, we found three important targets (ALDH2, HADH, and MAOB), which are associated with mitochondrial function, were involved in the beneficial effect of formononetin. Further validation revealed that these three genes were more abundance in cardiomyocyte than in cardiac fibroblast. The mRNA expression of ALDH2 and HADH were decreased, while MOAB was increased in cardiomyocyte upon ISO treatment and these phenomena were reversed by formononetin. In addition, we investigated mitochondrial membrane potential and ROS production in cardiomyocytes, the results showed that formononetin effectively improved mitochondrial dysfunction induced by ISO. In summary, we demonstrated that formononetin via regulating the expressions of ALDH2, HADH, and MAOB in cardiomyocyte to improve mitochondrial dysfunction and alleviate β-adrenergic activation cardiac fibrosis.

Cardiac fibrosis is a prevalent characteristic of various cardiovascular diseases and poses a significant global health challenge. Recent research has established a robust correlation between gut microbiota and cardiovascular diseases. Hesperidin has been shown to possess cardioprotective properties to some extent. Furthermore, studies suggest that hesperidin may enhance overall health by regulating intestinal flora. However, there is a lack of reports regarding the effects of hesperidin on cardiac fibrosis. This study aimed to investigate the mechanisms by which hesperidin ameliorates cardiac fibrosis through the regulation of gut microbiota and associated metabolites. Cardiac fibrosis was induced in C57BL/6 mice via subcutaneous injection of isoproterenol (5 mg/kg per day) for a duration of 7 days. Echocardiography was used to assess cardiac function, while Masson staining, western blot analysis, and real-time polymerase chain reaction were used to evaluate fibrosis-related indicators. Changes in gut microbiota were analyzed through 16S ribosomal RNA gene sequencing. Our findings indicate that hesperidin significantly mitigates cardiac fibrosis in mice. These beneficial effects are associated with improvements in the dysbiosis of intestinal microbiota observed in fibrotic mouse models. The involvement of gut microbiota in cardiac fibrosis was further corroborated by administering hesperidin therapy to mice depleted of gut microbiota. To our knowledge, this study provides the first evidence that the modulation of gut microbiota by hesperidin contributes to improved outcomes in cardiac fibrosis. The use of traditional Chinese medicine to modulate gut microbiota presents a promising strategy for the treatment of cardiac fibrosis. SIGNIFICANCE STATEMENT: The work is extremely interesting because it acts on a frontier of science that relates the influence of the intestinal microbiota with human physiological systems and associated pathologies. This study provides the first evidence that the modulation of gut microbiota by hesperidin contributes to improved outcomes in cardiac fibrosis.

Background: Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug‐eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti‐proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis.Methods: C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin–Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real‐time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking.Results: The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS‐induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC‐ and CuB‐treated neointima was downregulated.Conclusions: CuECs, especially CuB, exert an anti‐proliferation effect in VSMCs and may be potential drugs to prevent restenosis.