The Influence Of Alcoholism On Hypoxia Ingury Of SK-N-SH Cells Induced By Cobalt Chloride

Objective:To investigate the effect of alcoholism on which cobalt chloride induced oxygen deficit in human neuroblastoma SK-N-SH cells.Methods:SK-N-SH cells are divided into four groups:blank group,cobalt chloride group,low concentration alcohol group and high concentration alcohol group.The blank group did not make any processing.The cobalt chloride group is treated by 250?mol/L cobalt chloride of medium for 6h.The alcohol groups are pretreated by alcohol at dose of 300mg/L?900mg/L for 1 hours,and add cobalt chloride at dose of 250?mol/L for 6 hours;The cells which using in the experiment are in logarithmic growth period.Cells are c?ltured in 37? incubator with 5%CO2.The cell morphology of SK-N-SH cells are observed by microscope.Cell viability is determined by MTT assay.Apoptotic cells are detected by Hoechst staining.At the same time,the expression of HIF-1?,Bcl-2 and Bax protein are estimated by Western blot.Results:(1)The microscope observes the 4 groups of SK-N-SH cell morphology which shows that the cobalt chloride group and alcohol group have shorter nerve morphology and dead cells;the alcohol groups have shorter nerve morphology and more dead cells and the high concentration alcohol group has shorter nerve morphology and more dead cells.(2)MTT assay shows that the survival rate of SK-N-SH cells is significantly decreased in cobalt chloride group(P<0.001)(compared with the blank group).Compared with cobalt chloride group,the survival rate of the low and high concentration alcohol group is obviously decreased(P<0.01 or P<0.001).Compared with the low concentration alcohol group,the survival rate of the high concentration alcohol group is significantly decreased(P<0.001).(3)Hoechst method showes that nuclear condensation of SK-N-SH cells in the group of cobalt chloride,strong blue fluorescence presented at the microscope and the proportion of apoptosis increased obviously.And the alcohol groups have more blue fluorescence and the proportion of apoptosis increased obviously.The number of strong blue fluorescence increased and the proportion of apoptosis increased obviously in the high concentration alcohol group.(4)Western blot displays that the expression of Bax and HIF-1? protein in the group of cobalt chloride and alcohol is significantly increased(P<0.001);and the expression of Bcl-2 protein in the chloride group is decreased(P<0.001),the expression of Bcl-2 protein in the low and high group is decreased(P<0.001 ? P<0.01)(compared with the control group)?The expression of Bax and HIF-la protein in alcohol group is significantly increased(P<0.001),while the expression of Bcl-2 protein is decreased(P<0.001)(compared with the cobalt chloride group).The expression of Bax and HIF-la protein in the high concentration alcohol group(900mg/L)is significantly increased(P<0.001),and the expression of Bcl-2 protein is decreased(P<0.001)(compared with the low concentration alcohol group).Conclusion:1.Alcoholism can reduce the survival rate of SK-N-SH cells caused by hypoxia and increase the damaged of hypoxic cells.2.Alcoholism increases the apoptosis of SK-N-SH cells induced by cobalt chloride and its mechanism which related to the up-regulation of HIF-1??Bax expression and down-regulation of Bcl-2 expression.