Effects Of Aristolochic Acid â…  On The Human Umbilical Vein Endothelial-Mesenchymal Transition And Related Mechanism

Backgrounds:Aristolochic acid is one of the phenanthrene compounds belonging to the species of Aristolochia of the Aristolochiaceae,which has the ability to anti-tumor, enhance immune function and increase white blood cell count and so on.Aristolochic acid was widely used in the past.Since the 90s,more and more aristolochic acid ralated nephropathies(AAN) have been reported.The most researches focused on the injury of renal tubule endothelial cells(RTECs) and thus lead to renal tubulointerstitial fibrosis. Recently some papers concerned about the relationship between injury of microvascular endothelial cells and renal tubulointerstitial fibrosis.But there are few studies about AA's damage effects on Endothelial to Mesenchymal transition(Endo MT) and immune mediated mechanism underlying.Objective:To investigate the effects of aristolochic acid I(AAI) on the human umbilical vein endothelial cell(HUVEC) by studying the proliferation,apoptosis and Endo MT,to explore their probably immune mediated mechanisms through analysis of CD40/CD40L and transforming growth factor-β1(TGF-β1) /TGF-βⅡreceptor signals.Methods:1) HUVEC proliferation was evaluated by MTT after treated with various doses of aristolochic acid I(5,10,20,40,80μg/mL) at 96h.2) HUVEC apoptosis were detected by Flow cytometry(FCM) with markers of Annexin V and PI after 96h of culture with different concentrations(10,20,40,80μg/mL) of AAI.3) After 96h of culture with different concentrations(10,20,40μg/mL) of AAI,Endo MT was analyzed by studying expression of VEGF Receptor(Flk1,endothelial cell marker) and smooth muscle actin (α-SMA) with methods of Western Blot and FCM.4) The expressions of CD40/CD40L and TGF-β1/TGF-βⅡR signals by FCM and Western Blot were explored after 96h of cultured HUVEC with different concentrations(10,20,40μg/mL) of AAI.5) Supernatant TGF-β₁ level was measured by enzyme-linked immunosorbent assay(ELISA) at the day 1, 2,3,4,5 after cultured HUVEC with different concentrations(10,20,40μg/mL) of AAI.Results:After HUVEC were treated with AAI for 96 hours,results were achieved as followed:1) Cell proliferations were not inhibited in the groups with the AAI level of 5μg/mL,10μg/mL,20μg/mL,inhibited a bit in 40μg/mL with no statistical significance and inhibited extremely in 80μg/mL(P＜0.05).2) The apoptosis increased gradually following concentration rising of AAI.3) Down regulated Flk1 and upα-SMA expression were found,compare with control group(P＜0.05).4) The expressions of CD40/CD40L,TGF-β1/TGF-βⅡR enhanced gradually following increased the level of AAI(all P＜0.05). 5) Supematant level of TGF-β₁ increased in 40μ/ml group from day 1 to day 5 (P＜0.05),no statistical significance in both 10μg/ml and 20μg/ml group.Conclusion:In this study,we demonstrated that AAI can exert toxic effects on the HUVEC directly by inhibiting cell proliferation,inducing cell apoptosis and Endo MT which confirmed by upregulatedα-SMA and down Flk1 expression.These data suggested that AAI induced Endo MT by activation of CD40/CD40L and sequential transforming growth factor-β1(TGF-β1)/TGF-βⅡreceptor signals.This study confirmed immune pathogenesis of aristolochic acid induced vascular endothelial injury and provided potential target for the AAI related interstitial fibrosis.