Effects Of Ethanol With The Change Of NAD~+,NADH,nitric Oxide And Mitochondrial Function In Rat Hepatoma H4-ⅡE Cells

Objective:To investigate the survival rate and the content of NAD+,NADH,NAD+/NADH and nitric oxide by alcohol intervention in rat hepatoma H4-IIE cells,research the molecular mechanism of ethanol induced to hepatocyte injury..Methods:To recoveried the rat H4-IIE cells,and cultured them,when the experiment needed,to cryopreservation and subculture.Seted control group and ethanol intervention group,The intervention group seted the concentration gradient of 25 mM,50 mM,75 mM,100 mM,and the time which used the ethanol treatment were seted to 12h,24h and 36h,the survival rate of each group were measured by MTT assay.The content of NAD+,NADH,and NAD+/NADH in each group were measured by WST-8 assay.The nitric oxide produced in each group was measured by chemical assay.The experimental data of each group were represented were expreeesd as X±S The analysis of variance of multi-level and single factor was used to compare between groups.When P<0.05,it was different in considered statistically significant.Results:1.MTT results showed that ethanol inhibited the activity of H4-IIE cells,and when the concentration of ethanol was increased,cell activity was decreased it compared to the control group(P<0.05),and when the treatment time of ethanol prolonged,the cell activity was decreased it compared to the group of 12h group,(P<0.05).2.The NADH of each intervention group was higher than the control group.The concentration increased with the increase of ethanol treatment after 12h of ethanol treatment,but when ethanol treatment after 24h and 36h,with the concentration of ethanol was increased,the concentration of NADH were increaseed in beginning,then reduced,cell activity was decreased,it compared to the control group was different in considered statistically significant(P<0.05).In the group of 25mM ethanol treatment group,when the time of ethanol treatment prolonged,the concentration of NADH was increaseed.In the group of 100mM,when the time of ethanol treatment prolonged,the concentration of NADH was reduced,it compared to the group of 12h group was different in considered statistically significant(P<0.05).About the concentration of NAD+,by the ethanol treatment after 12h and 24h,were increaseed in beginning,then reduced.In the ethanol treatment after 36h,were reduced when the time of ethanol treatment prolonged,it compared to the control group was different in considered statistically significant(P<0.05).NAD+/NADH were reduced when the concentration of ethanol increaseed,it compared to the control group was different in considered statistically significant(P<0.05),and when the time of ethanol treatment prolonged,NAD+/NADH were reduced,too.It compared to the group of 12h group was different in considered statistically significant(P<0.05).3.The concentration of nitric oxide in each intervention group were higher than the control group.In the time of ethanol treatment after 12h and 24h group,when the concentration of ethanol increased,the concentration of nitric oxide increased,and in the time of ethanol treatment after 36h,with the concentration of ethanol increased,the concentration of nitric oxide increased increaseed in beginning,then reduced,it compared to the control group was different in considered statistically significant(P<0.05).By the treatment to the concentration of ethanol 25 mM,the concentration of nitric oxide increased with the increase of ethanol treatment time.By the treatment to the concentration of ethanol 50 mM and 75 mM,the concentration of nitric oxide increased in beginning,then reduced.By the treatment to the concentration of ethanol 100 mM,the concentration of nitric oxide reduced with the time increaseed of ethanol treatment,it compared to the group of 12h group was different in considered statistically significant(P<0.05).Conclusions:1.The metabolite of ethanol can damage the cells,the concentration of ethanol increased and the time of ethanol treatment increased,the cytotoxic effect reinforcement;2.Ethanol can induce the NAD+/NADH decrease and nitric oxide product increased of cells,maybe oxidative stress of mitochondria,depressing the fuction of respiratory chain and change the protein of mitochondria,lead to dysfunction of mitochondria,damage the cells.