Autophagy/Cathepsins Play A Role In Protecting Endothelial Cells Against Ischemia/reperfusion-mediated Injury Directed By Curcumin

Aim To investigate the effect of autophagy/cathepsins activation mechanisms on protecting HUVEC against ischemia /reperfusion (I/R) injury directed by curcumin.Methods Establish hypoxia/reoxgenation(H/R) model on HUVEC. Using MTT colorimetric assay to observe the injury degree of hypoxia and reoxygenation at the diferent time. Preconditioning with different concentration of Cur, the survival rate of HUVEC subjected to H/R was assessed by MTT colorimetric assay. Pretreated with Cur, the expression of LC3,cathepsin B,cathepsin L,Bax and Bcl-2 was observed by fluorescent staining and Western Blot in HUVEC during H/R process. The level of Bax and Bcl-2 was tested by Western Blot during H/R when 3-methyladenine(3- MA), an inhibitor of autophagy was added.Results In the hypoxia phase , the expression of LC3,Beclin 1,cathepsin B and the ratio of Bax /Bcl-2 increased, and the nuclear translocation of cathepsin L was induced; and in the H/R phase ,these proteins just refered further enhanced;Inhibition of autophagy by 3-MA significantly increased the ratio of Bax/Bcl-2 which was induced by H/R. Cur(1.25～5μmol·L﹣1) played a protective role during H/R in HUVEC in a dose-dependent manner . When Cur (5μM) pretreated , LC3 further strengthened , at the same time , the up-regulation of cathepsin B,the ratio of Bax /Bcl-2 and the nuclei-location of cathepsin L was inhibited partly by Cur.Conclusions Autophagy was induced by hypoxia and further enhanced by reoxgenation in HUVEC, in vitro. Apoptosis was enhanced by 3-MA suggesting that autophagy is protective against hypoxia and H/R in HUVEC. Cathepsin B and cathepsin L protein up-ragulated and the apoptosis increased. Cur can raised the survival rate of HUVEC in the process of H/R. Cur increased the autophagy activity , down-regulated cathepsin B,L and inhibited the ratio of Bax /Bcl-2 to protect the endothelial cells.