Study On The Effects Of Autophagy In The Vascular Endothelial Cell Injury Induced By Nicotine

Objective:All the researches based on the primary human umbilical vein endothelial cells(HUVEC). To definite the influence aboute function of autophagy in the human umbilical vein endothelial cells by nicotine stimulated and to investigate the effects of autophagy in the vascular endothelial cell injury induced by nicotine and its possible mechanism.Methods:Human umbilical vein endothelial cells cultured with different concentration of nicotine for 6 h, detecting the protein expression of cell autophagy marker protein LC3Ⅱ and autophagy related protein Beclin 1, p62/SQSTM1 and LAMP2 by protein immunoblot technology---Western Blot. Human umbilical vein endothelial cells cultured with 6×10-8 M, 6×10-6M nicotine in different time(0h, 1.5h, 3h, 6h, 12h), detecting the protein expression level of LC3 Ⅱ, p62/SQSTM1 and LAMP 2 by Western Blot. Using flow cytometry to detect human umbilical vein endothelial cells apoptosis rate induced by 6×10-8 M, 6×10-6M nicotine. Knockout AMPKα by the technology of gene silence,then detect LC3 Ⅱ and AMPKα protein expression by Western Blot and detect cell apoptosis rate by flow cytometry.Results:1. The expression of LC3 Ⅱ protein in HUVECs is concentration-dependent with different concentration of nicotine. LC3 Ⅱ protein expression increased significantly(P<0.01)when cells stimulated by nicotine which the concentration is 6×10-6M. However,the changes between the expression of Beclin-1 and LC3 Ⅱ are not completely consistent: Beclin-1 elevated compared with control group(P<0.01) when cells stimulated in a low concentration of nicotine(6×10-9M, 6×10-8M). Beclin-1 reduced compared with the control group(P<0.01) when cells stimulated in a high concentrations(6×10-7M, 6×10-6M). The changes of LAMP- 2 protein expression are same with the changes of Beclin-1. It is interesting to note that the changes of SQSTM1/p62 protein expression is completely opposite with the changes of Beclin-1 and LAMP- 2 protein expression.2. HUVEC is stimulated by nicotine(6×10-8M, 6×10-6M) in different time. The expression of LC3 Ⅱ protein increased significantly(P<0.01) in 6 h. At the same time,LAMP- 2 expression decreased obviously(P<0.01)and SQSTM1/p62 protein expression increased significantly(P<0.01)stimulated by 6×10-6M nicotine. However, the changes of LAMP- 2 and p62/SQSTM1 protein expression when cells stimulated by nicotine which the concentration is 6×10-8M.3. Compared with control group, the apoptosis rate of 6×10-8M nicotine treatment group is not statistically significant(P?0.05). 6×10-6 M nicotine treatment group compared with control group, the apoptosis rate increased significantly(P<0.001).6×10-6 M nicotine treatment group compared with 6×10-8M nicotine treatment group, the apoptosis rate increased significantly(P<0.001).4. AMPKα can be phosphorylated by nicotine. 6×10-8M nicotine+AMPKα siRNA(AMPKsiRNA)experimental group compared with the 6×10-8M(consiRNA) nicotine group, the expression of LC3 Ⅱ protein decreased significantly(P<0.001) and the apoptosis rate increased significantly(P<0.001).Conclusion:1. Nicotine can affect human umbilical vein endothelial cell autophagy in a concentration-dependent and time-dependent way.2. Autophagy level enhanced can adersely affect nicotine induced apoptosis inendothelial cells. On the contrary, autophagy dysfunction is disadvantage of endothelial cell survival.3. Autophagy mediated by AMPK plays a key role in the apoptosis induced by nicotine in endothelial cells.