Effects And Mechanism Analysis Of Tumor Necrosis Factor α On The Myoblast Line C2C12

Objective: Our study aims to examine the role of NF-κB in the inhibition of myogenic differentiation caused by TNF-α and to determine whether IGF-1 could counteract TNF-α’s negative effect on myotube formation.Methods:(1) The C2C12 myoblasts were differentated into myotubes in DMEM containing2% horse serum in the presence or absence of various treatment such as tumor necrosisfactor-α(TNF-α), insulin-like growth factor(IGF-1) and / or the nf-kappa B inhibitors pyrrolidine dithiocarbamate salts(PDTC);(2) Cell apoptosis were performed by flow cytometry;(3) The expression of Akt was assessed by Western blotting;(4) Dual-luciferase reporter assay(DLR) was used to to examine activation of NF-kB promoter.Results: TNF-α’s inhibitory effect on differentiation can be observed as early as 24 hours after treatment. On day 6, the differentiation of cells was completely inhibited,accompanied by reduced protein levels of myogenin, p-Akt and total Akt, indicating that IGF-1 signaling was impaired by TNF-α treatment.In order to define the role of NF-kB in myogenic differentiation, we added PDTC together with TNF-α to C2C12 cells cultured in differentiation medium. The results showed that PDTC treatment prevented TNF-α induced inhibition of C2C12 differentiation, which suggests that TNF-α inhibits C2C12 differentiqation via activation of NF-kB, The involvement of NF-kB in TNF-α’s action was validated by dual-luciferase reporter assay(DLR).Conclusion: Our results demonstrate that myotube formation was completely inhibited by TNF-α when added to the differentiating C2C12 cells. The inhibitory effectof TNF-α on differentiation was accompanied by activation NF-kB and down regulation of myogenin and Akt. Importantly, TNF-α induced inhibition of myogenic differentiation was prevented by IGF-1. The results support the using IGF-1 as a potential treatmenet for muscle wasting.