Effect Of Cigarette Smoke Exposure On Nuclear Factor-?b And Histone Deacetylase-2 In C2c12 Cells

Objective To investigate the effect of CSE on inflammatory cytokine(IL-8?TNF-?),NF-?B and HDAC2 in murine myoblast C2C12 cells.Methods(1)When the C2C12 cells were differentiated into myocyte,cells were used for the follow-on experiments.To observe the appropriate concentrations of CSE on cell,the CSE of 0.1%,0.2%and 0.4%at time points of 24h,48h and 72h on the growth of cells were confirmed by MTT method.And the suitable concentration was selected to interfere the cells.(2)To investigate the effects of CSE on the release of inflammatory mediator of C2C12 cells,cells were divide as follow:control group,0.1%CSE group(the cells were incubated with 0.1%CSE for 24 hours),0.2%CSE group(the cells were incubated with 0.2%CSE for 24 hours).The levels of IL-8 and TNF-a in the supernatant were determined by sandwich ELISA.(3)To detect the effect of CSE on HDAC2 and inflammatory cytokine in C1C12 cells,the well-differentiated cells were divided into control group,CSE group,HDAC2-siRNA group and HDAC2-siRNA+CSE group.Of the CSE group,the cells were treated with CSE for 24 hours.The cells of HDAC2-siRNA group were transfected with short silence HDAC2-siRNA sequences plasmid gene into the cells by liposome for 24 hours to interfere the expression of HDAC2 protein.The HDAC2-siRNA+CSE group was first treated as HDAC2-siRNA group,following by the treatment with CSE for 24 hours.Control group was cultured without CSE or HDAC2-siRNA,given only the culture.qRT-PCR was used to analysis the mRNA expression of HDAC2.Western blotting was used to analyse the HDAC2 protein expression.The activity of HDAC2 was measured by colorimetry.ELISA was used to measured the levels of TNF-a and IL-8 in supernatants.(4)To detect the effect of CSE on the expression NF-?B in C2C12 cells and the release of IL-8 and TNF-a levels in supernatants,the cells were divided into control group,CSE group,PDTC group and PDTC+CSE.CSE group was treated with CSE for 24 hours.PDTC+CSE group was preincubated with NF-?B inhibitor PDTC for 24 hours before the CSE was added to the culture dishes.PDTC group was treated with PDTC for 24 hours.Western blotting was used to analyze the expression of NF-?B protein.ELISA was used to measured the levels of TNF-a and IL-8 in supernatants.(5)To explore the interaction of HDAC2 and NF-?B p65,co-immunoprecipitation was used to determined the HDAC2/NF-?B p65 compound.Western blot was uesd to detect the expression of acetyl-NF-KB p65.The SPSS 16.0 statistical software was used to analyze the data.Results(1)The effect on the growth of C2C12 cells at the CSE concentration of 0.1%and 0.2%was no significant(2)After treatment with CSE for 24 hours,the level of TNF-a and IL-8 in the supernatant was CSE does-dependent increased(P<0.05).(3)When the cells were treated with CSE for 24 hours,the expression and activity of HDAC2 in the cells was lower than cells in control group,the level of TNF-a and IL-8 was incressed(P<0.05).When the cells were transfected with HDAC2-siRNA followed by CSE stimulation,the expression and activity of HDAC2 was decreased compared with control group and CSE group,and the increase of TNF-a and IL-8 was more obvious.As a negative control,the expression and activity of HDAC2 in the HDAC2-siRNA group were decreased compared with control group(P<0.05).(4)Treatment with CSE for 24 h increased NF-?B proteins expression,and the supernatant levels of IL-8 and TNF-? were significantly increased(P<0.05).When preincubated with PDTC for 24 h before the addition of CSE,the expression of NF-?B induced by CSE was inhibited,the IL-8 and TNF-?level was lower than the CSE group.As a negative control,PDTC inhibited NF-?B expression.(5)Co-mmunoprecipitation confirmed that HDAC2/NF-?B p65 compound was increased in the CSE-exposed C2C12 cells with a CSE dose-dependence.Western blotting result showed an increased expression of acetyl-NF-?B p65with a CSE dose-dependence.Conclusion(1)The C2C12 cells increased the expression of TNF-a and IL-8 by decreasing the expression and activity of HDAC2 and increasing the expression of NF-?B under the condition of CSE exposure.(2)The CSE induced the expression of HDAC2/NF-?B p65 compound and acetyl-NF-?B p65,showing a interaction of HDAC2 and NF-?B p65.