Effect of metformin on proliferation and differentiation of HaCaT cells and secretion of inflammatory factors by HaCaT cells

Objective To evaluate the effect of metformin on the human keratinocyte line HaCaT, and to explore its molecular mechanism. Methods HaCaT cells were divided into several groups to be treated with metformin at different concentrations of 1, 2, 5, 10, 20, 50 mmol/L for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8) assay was performed to evaluate the effect of metformin on the survival rate of HaCaT cells. After 48-hour treatment with metformin at concentrations of 0 (control group) , 0.5, 1, 2, 5, 10 mmol/L, flow cytometry was conducted to evaluate the effect of metformin on cell cycle and apoptosis. Western blot analysis was performed to determine the expression of cell proliferation- and differentiation-related proteins (keratin-16[K16], K17, K1, involucrin) , apoptosis-related proteins (Bax, Bcl-2) and AKT/mTOR/STAT3 pathway proteins. Enzyme-linked immunosorbent assay (ELISA) was conducted to detect the levels of interleukin (IL) -8, tumor necrosis factor (TNF) -α and IL-23 (inflammatory factors) in the culture supernatant of HaCaT cells. Statistical analysis was carried out with SPSS19.0 software using one-way analysis of variance for comparison of the above indices among the 0.5-, 1-, 2-, 5-, 10-mmol/L metformin groups and control group, repeated measures analysis of variance for comparisons among different time points or different metformin groups, least significant difference (LSD) -t test for multiple comparisons. Results CCK-8 assay showed that metformin had inhibitory effects on the proliferation of HaCaT cells (F= 116.87, P 0.05) . After 48-hour treatment with metformin at different concentrations, the levels of IL-8 and TNF-α in HaCaT cells significantly decreased (F= 33.89, 14.99 respectively, both P 0.05) . Along with the increase in the concentrations of metformin, the expression of p-AKT, p-mTOR and p-STAT3 significantly decreased (F= 11.38, 0.35, 4.38 respectively, all P 0.05) . Conclusion Metformin can inhibit the proliferation, promote the differentiation and apoptosis of HaCaT cells, and inhibit the secretion of inflammatory factors by regulating the AKT/mTOR/STAT3 signaling pathway. Key words: Metformin; Cell proliferation; Apoptosis; Cell differentiation; HaCaT cells