Orphan nuclear receptor 4 A1 involvement in transforming growth factor beta1-induced myocardial fibrosis in diabetic mice.

Abstract

We explored the involvement of orphan nuclear receptor 4 A1 (NR4A1) in myocardial fibrosis mediated by transforming growth factor-beta1 (TGF-β1) and its response to cytosporone B (Csn-B). We developed a diabetic cardiomyopathy mouse model by administering a high-fat diet in conjunction with a low-dose streptozotocin injection. Our analysis involved monitoring alterations in blood glucose and lipid levels, cardiac function and structure, as well as profibrotic factors such as α smooth muscle actin (α-SMA), collagen I, collagen III, TGF-β1, connective tissue growth factor, and fibronectin. These assessments were conducted using biochemical techniques, Doppler ultrasound, histopathology, and real-time quantitative polymerase chain reaction. Cardiac fibroblasts (CFs) were extracted from suckling mice and cultivated in a high-glucose medium to simulate diabetes-induced myocardial fibrosis in vitro. These CFs were then subjected to coculture experiments with TGF-β1 or Csn-B. The proliferation and migration of CFs were assessed using cell counting kit 8 (CCK-8) assays and Transwell assays, respectively. Western blotting and immunofluorescence assays were employed to evaluate the expression levels of NR4A1, p-NR4A1, and α-SMA in CFs treated with TGF-β1 after NR4A1 knockdown or Csn-B administration, respectively. In diabetic heart tissue, the expression of p-NR4A1 was notably elevated. Furthermore, CFs exhibited enhanced proliferative capabilities and increased p-NR4A1 expression following high glucose exposure. Interestingly, NR4A1 knockdown resulted in a significant increase in the expression of fibrosis-related proteins in CFs following treatment with TGF-β1. Moreover, our observations revealed a marked decrease in p-NR4A1 levels and a reduction in the expression of fibrosis-related proteins after Csn-B treatment. In diabetic mice treated with Csn-B, we noted diminished NR4A1 phosphorylation and a mitigation of myocardial fibrosis. We concluded that in the mouse model, Csn-B played a pivotal role in inhibiting diabetes-induced myocardial fibrosis by activating NR4A1.