Role Of Autophagy On Ursolic Acid-mediated Suppression Of Proliferation In Vascular Endothelial Cells

Purpose: Ursolic acid (UA)extracted from Chinese herbs has beenshowen a inhibiting role in proliferation of Human Umbilical VeinEndothelial Cells(HUVECs),but the specific mechanisms underlying thisprocess remain unclear.In this study,we investigated the role of autophagyin the suppressive effect of UA on the proliferation of human umbilical veinendothelial cells (HUVECs).Methods： HUVECs were cultured in vitro and treated with variousconcentrations of UA and3-methyladenine (Autophagyinhibitor,3-MA)+UA respectively. The effects of UA on proliferation aswell as3-MA+UA on survival of HUVECs were determined by MTTmethod. The change of ultrastructure in HUVECs was detected usingTransmission Electron Microscope(TEM). The expressions ofautophagy-associated proteins in HUVECs treated with UA weredetermined by fluorescnt staining of microtubule-associated protein1lightchain3(LC3)and flow cytometry.Autophagy was characterized bymonodansylcadaverin (MDC) staining with fluorescent microscope. Theprotein levels of autophagy-associated protein Beclin-1and LC3were determined by Western blot. The transcription levels of ofautophagy-associated gene Beclin-1and LC3were determined by RT-PCR.Cell apoptotic ratio was measured by flow cytometry analysis.Results： UA showed a suppressive effect on the proliferation ofHUVECs in a dose-dependent manner. Autophagic vacuoles increased in theHUVECs after treatment with UA. The fluorescent density of LC3positiveHUVECs increased significantly as compared with those in controlgroup.The protein levels and transcription levels of LC3and Beclin-1inHUVECs were significantly increased following treatment with UA,whichwas also in a time-dependent manner. Treatment with3-MA+UAsignificantly enhanced the proliferation inhibition and exacerbated theapoptosis of HUVECs.Conclusion： UA inhibited the the proliferation and induced theautophagy of HUVECs,in which autophagy plays a protective role. Theinhibition of autopbagy significantly promoted the death of HUVECsinduced by UA.