Effect Of Calycosin Extracted From Astragalus Membranaceus On Endothelial Cells Transdifferentiation Induced By Transforming Growth Factor β1

Objective:Chronic kidney disease is often accompanied by pathological changes in renal endothelial cells, endothelial cells transdifferentiating into myofibroblasts is proposed in recent years as one of the pathological changes. Transforming growth factorβ1 (TGF-β1) has been proved to be the strongest factor to promote fibrosis, and it plays a great role in inducing epithelial-like cells transdifferentiation into fiber-like cells. Astragalus is effective to prevent renal fibrosis.Therefore,our study investigate the protective effects of calycosin extracted from astragalus membranaceus on the transdifferentiation of endothelial cells induced by transforming growth factorβ1. Further, exploring the endothelium-mesenchymal transdifferentiation in kidney fibrosis and to elucidate the mechanism of calycosin on renal fibrosis.Methods:A human umbilical endothelium derived cell line (ECV-304) from CTCC was used in this study. Cultured endothelial cells were divided randomly into 5 groups:control, transforming growth factorβ1(TGF-β1)(10μg/l),TGF-β1(10μg/l)+calycosin(0.1mg/l),TGF-β1 (10ug/l)+calycosin(1mg/1),TGF-β1(10μg/l)+calycosin(10mg/l).Morphology of endothelial cells were studied by inverted fluorescence microscope. Expression of myofibroblast marker a-SMA was detected with western blot.Results:The control group grew well in ECV-304 cells, or paving cobble stone-like, with the typical morphological characteristics of epithelial cells. Compared with the control group, the cell induced by TGF-β1 arranged fusiform, irregular, loss of cobblestone growth pattern, connections between cells reduced and cell gap became larger, showing a fibroblast-like appearance. Every dose group of astragalus calycosin can inhibit TGF-β1-induced cell morphological changes.The high-dose group were similar to normal control group, most of the cells showed a cobblestone-like growth, only a very few cells showed long spindle.Low-dose group were similar to TGF-β1 group, most cells arranged in elongated, spindle, irregular, and only a few cells showed cobblestone-like.The cells morphological of middle dose group were between high-dose group and low dose groups. The control group did not express myofibroblast marker proteins a-SMA. Compared with the control group, TGF-β1 induced high expression of a-SMA(11±0.03 vs 0.00±0.00, P<0.01). Endothelial cells cultured with astragalus calycosin and TGF-β1 significantly inhibited the expression of ofα-SMA(1.03±0.06 vs 1.11±0.03, P<0.05; 0.44±0.05 vs 1.11±0.03, P<0.0l; 0.33±0.05vs 1.11±0.03,P<0.01,respectively).The high-dose group had a stronger inhibitory effect than the low and middle dose group, the weakest inhibitory effect emerged in low dose group.Conclusions:1.TGF-β1 can induce human umbilical endothelium derived cell line vascular endothelial cells transdifferentiation into myofibroblasts.2.Astragalus calycosin can inhibit TGF-β1-induced endothelial cells (ECV-304) morphological changes, the mechanism may be blocking endothelial-to-mesenchymal transition.3.Astragalus calycosin may prevent endothelial cells-mesenchymal transdifferentiation by inhibiting TGF-β1, indirectly play a role against renal fibrosis.