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Evodiamine, extracted from Chinese herb Evodia rutaecarpa, is a naturally occurring indole alkaloid. Recent studies have reported many properties of evodiamine, including anti-inflammatory, anti-obesity, anti-diabetic, anti-anxiety, anti-allergic, anti-cancer and anti-nociceptive effects. Our previous study have demonstrated that evodiamine attenuates cardiac hypertrophy and fibrosis induced by aortic banding. However, the mechanisms involved the protective effect of evodiamine on cardiac fibrosis remain unclear. The aim of this study is to investigate the effect of evodiamine on endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVEC) and myofiroblast differentiation in cardiac fibroblasts.
HUVEC-12 cells were stimulated with transforming growth factor beta 1 (TGF-β1) to induce EndMT and treated with evodiamine (5μM, 10μM) at the same time. The EndMT response in the HUVEC-12 cells was evaluated by the expression levels of endothelial and mesenchymal marker by immunofluorescence and western blot. The capacity of the transitioned endothelial cells migrating to surrounding tissue was evaluated by scratch test. Neonatal rat cardiac firoblasts were also stimulated with TGF-β1 to induce myofiroblast differentiation. After co-cultured with evodiamine (5μM, 10μM) for 24 hours, the proliferation of cardiac firoblasts in each group was evaluated by Cell Counting Kit-8. Myofiroblast differentiation level in each group was measured by immunofluorescence with α-smooth muscle actin (α-SMA). The pro-firotic proteins expression levels were evaluated by western blot. The signal pathway involved was also evaluated by western blot in both HUVEC and cardiac firoblasts.
HUVEC-12 cells stimulated with TGF-β1 exhibited significantly lower expression levels of CD31, CD34 and significantly higher levels of α-SMA and vimentin than the control cells. This phenotype was eliminated in the HUVEC-12 cells treated with both 5μM and 10μM evodiamine. Evodiamine signifiantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVEC-12 cells. Evodiamine blunted TGF-β1 induced proliferation and conversion of cardiac firoblast into myofiroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine also reduces the increased protein expression of firosis markers (Collagen I, Collagen III, connective tissue growth factor and fibronectin in neonatal rat cardiac firoblasts induced by TGF-β1. In addition, the phosphorylation of smad2 and smad3, and the nuclear translocation of smad4 in both HUVEC and cardiac firoblasts were blocked by evodiamine treatment.
Our results suggested that evodiamine prevented HUVEC from EndMT, and prevented cardiac firoblasts from proliferation and transformation into myofiroblast. These effects may be mediated by inhibition of the TGFβ/smad pathway in both HUVEC and cardiac firoblasts.