我们探讨了孤儿核受体4A1(NR4A1)在转化生长因子-β1(TGF-β1)介导的心肌纤维化中的参与及其对细胞孢子酮B(Csn-B)的反应。我们通过给予高脂饮食和低剂量链脲佐菌素注射开发了糖尿病心肌病小鼠模型。我们的分析涉及监测血糖和血脂水平的变化,心脏功能和结构,以及促纤维化因子,如α平滑肌肌动蛋白(α-SMA),胶原蛋白I,胶原蛋白III,TGF-β1,结缔组织生长因子,和纤连蛋白.这些评估是使用生化技术进行的,多普勒超声,组织病理学,和实时定量聚合酶链反应。从乳鼠中提取心脏成纤维细胞(CFs),并在高糖培养基中培养以体外模拟糖尿病诱导的心肌纤维化。然后将这些CFs与TGF-β1或Csn-B进行共培养实验。使用细胞计数试剂盒8(CCK-8)测定和Transwell测定来评估CFs的增殖和迁移,分别。采用蛋白质印迹和免疫荧光测定来评估NR4A1敲低或Csn-B给药后用TGF-β1处理的CFs中NR4A1,p-NR4A1和α-SMA的表达水平,分别。在糖尿病心脏组织中,p-NR4A1的表达显著升高。此外,在高葡萄糖暴露后,CF表现出增强的增殖能力和增加的p-NR4A1表达。有趣的是,NR4A1敲低导致用TGF-β1治疗后CFs中纤维化相关蛋白的表达显着增加。此外,我们的观察显示,Csn-B治疗后,p-NR4A1水平显著降低,纤维化相关蛋白表达降低.在用Csn-B治疗的糖尿病小鼠中,我们注意到NR4A1磷酸化减少和心肌纤维化减轻.我们得出结论,在小鼠模型中,Csn-B通过激活NR4A1在抑制糖尿病诱导的心肌纤维化中起关键作用。
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.