Effect of metformin on proliferation and differentiation of HaCaT cells and secretion of inflammatory factors by HaCaT cells / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the effect of mefformin on the human keratinocyte line HaCaT,and to explore its molecular mechanism.Methods HaCaT cells were divided into several groups to be treated with mefformin 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 mefformin had inhibitory effects on the proliferation of HaCaT cells (F =116.87,P ＜ 0.05),and the cell survival rates gradually decreased along with the increase in the concentrations of mefformin.After 48-hour treatment with mefformin at concentrations of 0,0.5,1,2,5,10mmol/L,the proportion of HaCaT cells in G2/M phase gradually increased (5.55％ ± 1.03％,6.37％ ±0.93％,8.57％ ± 1.18％,10.05％ ± 0.60％,10.76％ ± 0.87％,13.63％ ± 1.41％,respectively,F =24.98,P ＜0.05),and the early apoptosis rate also gradually increased (0.78％ ± 0.71％,19.18％ ± 1.41％,25.67％ ±1.34％,28.45％ ± 0.92％,34.97％ ± 2.12％,40.41％ ± 1.49％,respectively,F =296.08,P ＜ 0.05).Along with the increase in the concentrations of metformin,there were increasing trends in the expression of K1 and the pro-apoptotic protein Bax (F =8.86,5.38 respectively,both P ＜ 0.05),while there were decreasing trends in the expression of K16,K17 and the apoptotic protein Bcl-2 (F =8.02,4.82,12.10 respectively,all P ＜ 0.05).There was no significant difference in the expression of involucrin among different metformin groups (F =0.57,P ＞ 0.05).After 48-hour treatment with mefformin 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),while there was no significant change in the IL-23 level (F =2.12,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),but there were no significant changes in the protein expression of AKT,mTOR and STAT3 (F =0.66,0.35,4.24 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.