Effects Of Metformin On Proliferation,Differentiation Of HaCaT Cells And Its Mechanisms

Objective:Psoriasis is a common,chronic,immune-mediated,inflammatory skin disease.Which not only harms skin,but also affects many systems of the body.Metformin,an insulin sensitizer,is widely used in the treatment of type 2 diabetes.Recent studies have shown that metformin could inhibit the growth of tumor such as breast cancer,pancreatic cancer,ovarian cancer and so on.And its mechanisms may be because that metformin can inhibit the excessive proliferation of cells and promot cell apoptosis.Both of psoriasis and malignant tumors are characterized by hyperproliferation.Hyperproliferation,disdifferentiation of keratinocytes and infiltration of inflammatory cells are the significant characteristics of psoriasis.So could metformin treat psoriasis by inhibiting excessive proliferation of keratinocytes and promoting apoptosis of keratinocytes?The effect of metformin on keratinocytes remains unclear.This study aims to explore the effects of metformin on the proliferation and differentiation of keratinocytes and its possible molecular mechanism so as to provide the possibility for the application of metformin in the treatment of psoriasis in future.Methods:HaCaT cells were cultured in vitro and treated with metformin at different concentrations cultured for 24,48 and 72 hours respectively.The morphological changes of HaCaT cells were observed by inverted microscope.The cell survival rate was evaluated by CCK-8 assay after 24,48,72h.Flow cytometry was conducted to detect the cell cycle and apoptosis after a 48h treatment.Western blot analysis was performed to determine the expression of cell proliferation and differentiation related protein K16,K17,K1,Involucrin and apoptosis related protein Bax,Bcl-2 and the expression of p-AKT,p-mTOR p-STAT3 protein.ELISA was conducted to detect the effect of different concentrations of metformin on cytokines secreted by HaCaT cells.Results:(1)Effects of metformin at different concentrations on proliferation,differentiation and apoptosis of HaCaT cells? When HaCaT cells were cultured normally for 48 hours,The cells are regular,transparent,well-circumscribed,closely arrange under the inverted microscope.While when HaCaT cells were cultured with 25,50 and 100 mmol/L metformin for 48 hours,the cell morphology became irregular,loose,the number of cells are decreased,and this change becomes more obviously with the increasing of drug concentration.? CCK8 assay showed that metformin inhibited the proliferation of HaCaT cells.Repeated variance analysis showed that 1,2,5,10,20,50 mmol/L metformin inhibited the survival rate of HaCaT cells(F concentration=116.87,P<0.05).And the results showed the higher the concentration,the stronger the effect of inhibition.The survival rate of HaCaT cells in each group changed over time(Ftime=9.01,P<0.05).While 48 and 72 h cells were lower than 24h(P<0.05).There was no significant difference between 48 and 72 hours(P>0.05).There is an interaction between drug concentration and the cell cultured time(F=8.12,P<0.05).? Flow cytometry showed that the percentage of G2/M phase cells increased gradually with the increasing of metformin concentration after the 48 hours intervention of 0,0.5,1,2,5,10 mmol/L metformin on HaCaT cells,the percentage of G2/M phase cells were(5.55 ± 1.03)%,(6.37±0.93)%,(8.57 ± 1.18)%,(10.05± 0.60)%,(10.76±0.87)%,(13.63± 1.41)%.The difference is of statistically significant(F=24.98,P<0.05);the percentage of early apoptotic cells were(0.78 ± 0.71)%,(19.18 ±1.41)%,(25.67±1.34)%,(28.45±0.92)%,(34.97±2.12)%,(40.41±1.49)%.The difference is of statistically significant(F=296.08,P<0.05).? Western blot showed that the expression of K1 and Bax protein increased with the increasing of metformin concentration after a 48 hours intervention of 0,0.5,1,2,5,10 mmol/L metformin on HaCaT cells,while the expression of K16,K17 and Bcl-2 protein decreased(P<0.05),but the expression of Involucrin is not of significant difference(P>0.05).(2)Effect of Metformin at Different Concentrations on the Secretion of Inflammatory Cytokines ELISA results showed that metformin could significantly reduce the secretion of IL-8 and TNF-a of HaCaT cells after a 48 hours intervention(F=33.89,14.99,P<0.05).(3)Effects of Metformin at Different Concentrations on AKT/mTOR/STAT3 Signaling Pathway Western blot showed that the expression levels of p-AKT,p-mTOR and p-STAT3 decreased with the increasing of metformin concentration after a 48 hours intervention of HaCaT cells compared to control group(F=11.38,0.35,4.38,P<0.05),while the expression levels of AKT,mTOR and STAT3 in each group at different concentrations are not of significant difference(F=0.66,0.35,4.24,P>0.05).Conclusions:? Metformin can inhibit the proliferation of HaCaT cells in vitro,induce G2/M arrest and early apoptosis and promote the differentiation of HaCaT cells;? Metformin can inhibit the secretion of IL-8 and TNF-a of HaCaT cells;? Metformin can inhibit AKT,mTOR and STAT3 proteins phosphotyrosine of HaCaT cells,The mechanism of the effect of metformin on keratinocytes may be related to AKT/mTOR/STAT3 signaling pathway.