| Objective:To explore the effect of IL-22 on the activation and poliferation of HSC-T6 s induced by acetaldehyde,and further investigate whether this inhibition via antioxidant anix Nrf2-keap1-ARE.Methods:(1)HSC-T6 cells culturing: HSC-T6 s were cultured in DMEM/H containing 10% FBS and 1% streptomycin-penicillin at 37°C in a humidified incubator with 5% CO2;(2)Establishment of the in-vitro model of alcoholic liver fibrosis: HSC-T6 s were divided into the blank control group and the acetaldehyde-stimulated groups.In the blank control group HSC-T6 cells were incubated with an conventional DMEM/H medium whereas in the acetaldehyde-stimulated groups,HSC-T6 s were incubated with various concentration of acetaldehyde(25,50,100,200,400?mol/L).After 24,48 h of reaction,MTT assay was employed to detect the proliferation of HSC-T6 s in order to choose the best concentration and action time;(3)Influence of IL-22 on the activation and proliferation of HSC-T6 cells induced by acetaldehyde: HSC-T6 cells in logarithmic growth phase were divided into five groups,namely the blank control group(cells were cultured in the conventional DMEM/H medium for 48h),the model group(200?mol/L acetaldehyde was added into the DMEM/H for 48h)and high,medium,low-dose of IL-22 intervention groups(cells were co-incubated with 200?mol/L acetaldehyde and 50ng/ml,20ng/ml,10ng/ml of IL-22 for 24 h after pretreatment with 200?mol/L acetaldehyde for 24h).Cell proliferation rate was detected by MTT assay,the cell cycle was analyzed by Flow Cytometric analysis,the expression of ?-SMA protein were detected by western blotting and ICC;(4)Mechanism exploration of IL-22: the groups as former,the expression of Nrf2 protein were detected by western blotting and ICC,the levels of MDA and GSH were measured by spectrophotometric method.SPSS 17.0 was used for all statistical analysis and data were expressed as meanąSD values(X ąS),the difference was statistically significant with P<0.05.A one-way analysis of variance was used for comparison of means between any two groups.Results:(1)HSC-T6 s grew very well and the characteristics of the cells was stable after subcultured;(2)MTT assay showed that OD values were obviously increased when incubated with different concentrations of acetaldehyde,and it was most obvious in 200?M after adding for 48h;(3)Flow Cytometric analysis showed that when stimulated with acetaldehyde,HSC-T6 s in the G0/G1 phases were decreased,while increased in the S phase in comparision with the control group.When intervened with different concentrations of IL-22,the progression of HSC-T6 cells cycle was arrested from G1 to S phase.Western blotting and ICC showed that the expression of ?-SMA was significantly increased in the acetaldehyde-stimulated group,after intervened with different concentrations of IL-22,HSC-T6 s activity and proliferation rate was decreased in a dose-depedent manner(P<0.05).(4)Western blotting demonstrated that the expression of total Nrf2 had no significant change.In the model group,the expression of nuclear Nrf2 was higher than in the control group.When intervened with gradient concentrations of IL-22,the expression of nuclear Nrf2 was significant higher than in the model group,the difference has statistically significant(P<0.05).ICC also showed that in the IL-22 intervention groups,the rate of positive Nrf2 nuclear staining was higher than in the model group,whereas there was nearly no positive nuclear staining in the control group.The level of MDA and GSH in the model group was significantly enhanced compared with the control group,while intervened with IL-22,the MDA level was attenuated but remained higher than the control group,the content of GSH was further elevated(P<0.05),the difference was statically significant(P<0.05).The higher the IL-22 concentration,the more obvious the effect.Conclusion:(1)Acetaldehyde can promote activation and proliferation of HSC-T6 s,we successfully set up an in-vitro model of rat alcoholic liver fibrosis HSC-T6 s.(2)IL-22 effectively inhibits acetaldehyde-induced HSC-T6 s activation and proliferation and the effect may be partly related to the promoted intranuclear transfer of Nrf2 and the enhanced activity of antioxidant axis Nrf2-keap1-ARE. |
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