Effect Of Theophylline On H₂O₂ Exposure On Nuclear Factor-κB In C2C12 Cells

Objective To investigate the effect of theophylline on inflammatory cytokine (IL-8和TNF-a), NF-κB and NF-KB in murine myoblast C2C12 cells H2O2.Methods (1) When the C2C12 cells were differentiated into myocyte, cells were used for the follow-on experiments. To observe the appropriate concentrations of H2O2 and theophylline on cell, the H2O2 of 0.1mmol/Lκ 0.2mmol/Lκ0.3mmol/Lκ0.4mmol/Lκ0.6mmol/Land 0.8mmol/L, the theophylline of 1mol/L、0.1mol/L、0.01 mol/L、0.001 mol/L and 0.0001 mol/L, both at time points of 24h on the growth of cells were confirmed by MTT method. Then, choose the suitable concentration to interfere the cells. (2) To detect the effect of H2O2 and theophylline on NF-KB and inflammatory cytokine in C2C12 cells, the well-differentiated cells were divided into control group, H2O2 group, theophylline group, H2O2+theophylline group and PDTC+ H2O2 group. Of the H2O2 group, the cells were treated with H2O2 for 24 hours. The cells of theophylline group were treated with heophylline for 24 hours. The H2O2+theophylline group was treated with H2O2 and theophylline for 24 hours. The PDTC+H2O2 group was first treated with PDTC(20uM) for 6 hours, following by the treatment with H2O2 for 24 hours. Control group was cultured without H2O2, PDTC or theophylline, given only the culture. qRT-PCR was used to analyze the mRNA relative expression of NF-κB. Western blotting was used to analyze the NF-KB protein expression. The intranuclear activity of NF-κB was measured by EMSA. ELISA was used to measure the levels of TNF-a and IL-8 in supernatants. The SPSS 17.0 statistical software was used to analyze the data.Results (1) The effect on the growth of C2C12 cells at the H2O2 concentration of 0.4% and theophylline concentration of O.OOlmmol/L were no significant. (2) The effect of theophylline on inflammatory cytokine (IL-8和 TNF-a):In H2O2 group, the level of TNF-a and IL-8 in the supernatant was increased compared with control group and theophylline group(P<0.05). In H2O2+ theophylline group, the level of TNF-a and IL-8 in the supernatant were lower than H2O2 group (P<0.05). In PDTC+H2O2 group, the level of TNF-a and EL-8 in the supernatant were lower than H2O2 group (P<0.05). (3) The effect of theophylline on NF-B mRNA relative expression in C2C12 cell: In H2O2 group, the level of NF-κB mRNA relative expression was increased compared with control group and theophylline group (P<0.05). In H2O2+ theophylline group, the level of NF-κB mRNA relative expression was lower than H2O2 group (P<0.05). In PDTC+H2O2 group, the level of NF-κB mRNA relative expression was lower than H2O2 group (P<0.05).(4) The effect of theophylline on NF-κB protein expression in C2C12 cell:In H2O2 group, the level of NF-κB protein expression was increased compared with control group and theophylline group(P<0.05). In H2O2+ theophylline group, the level of NF-κB protein expression was lower than H2O2 group (P<0.05). In PDTC+H2O2 group, the level of NF-κB protein expression was lower than H2O2 group (P<0.05).(5) The effect of theophylline on intranuclear activity of NF-κB in C2Cl2 cell:In H2O2 group, the level of intranuclear activity of NF-κB was increased compared with control group and theophylline group(P<0.05). In H2O2+ theophylline group, the level of intranuclear activity of NF-κB was lower than H2O2 group, was higher than PDTC+H2O2 group (P<0.05). In PDTC+H2O2 group, the level of intranuclear activity of NF-κB was lower than H2O2 group (P<0.05).Conclusion The theophylline could decreased the expression of TNF-a and IL-8 by decreasing the expression of NF-κB under the condition of H2O2 exposure, inhibited the inflammation of skeletal muscle.