Antibacterial peptides inhibit MC3T3-E1 cells apoptosis induced by TNF-α through p38 MAPK pathway

BACKGROUND: Antimicrobial peptides (AMP), as a small molecular polypeptide with a broad antibacterial spectrum and high efficiency, have attracted more and more attention. Few pieces of research on the effect of the antimicrobial peptide on osteoblast under inflammatory conditions have so far been reported. The main aim of this work was to investigate the antiapoptosis effect of the antimicrobial peptide on MC3T3-E1 cells induced by TNF-α and its related mechanism. METHODS: Rat MC3T3-E1 cells were co-cultured with different concentrations of antibacterial peptide DP7 and TNF-α.MTS assay, cell scratch test, alkaline phosphatase activity, and alizarin red staining assay were used to determine osteoblast viability in this experiment. Annexin V-FITC/PI double staining cells and flow cytometry were used to analyze apoptosis and Western blot assay detection to show mitogen-activated protein kinase (MAPK) protein expression in rat MC3T3-E1 cells. Then, Realtime polymerase chain reaction (PCR) was used to examine the caspase-3 gene expression. Also, ELISA detection was used to clarify the anti-apoptotic effect of the p38 MAPK inhibitor, SB203580, on cells’ apoptosis. RESULTS: Antimicrobial peptide could promote the proliferation, migration, and osteogenic ability of MC3T3-E1 cells induced by TNF-α, but inhibit cell apoptosis rate (P<0.05), and the effect was concentration-dependent. Western blot results showed after TNF-αtreatment, the expression of p-p38 MAPK in the MC3T3-E1 cells increased after TNF-α and antimicrobial peptide cotreatment, TNF-α induced p-p38 MAPK phosphorylation was inhibited, and the difference was statistically significant (P<0.05). Realtime PCR results showed that the gene expression of caspase-3 mRNA was up-regulated after TNF-α treatment, while their expression was down-regulated after cultured with TNF-α and antimicrobial peptide. Elisa's analysis showed that cell apoptosis increased after TNF-α treatment alone, and cell apoptosis was reduced to the normal levels when combined with antimicrobial peptide, and cell apoptosis induced by TNF-α was partially abolished when combined with SB203580. CONCLUSIONS: Antimicrobial peptide DP7 could inhibit MC3T3-E1 cells apoptosis induced by TNF-α, and the effect was concentration-dependent. The antiapoptosis activation of the antimicrobial peptide on MC3TE-E1 cells may be related to the inhibition of the p38 MAPK pathway.