二甲双胍对HaCaT细胞增殖分化和炎症因子分泌的影响

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

目的研究二甲双胍对人角质形成细胞系HaCaT细胞的影响并分析其机制。方法采用1、2、5、10、20、50 mmol/L二甲双胍分别作用于HaCaT细胞24、48、72 h,CCK8法检测二甲双胍对HaCaT细胞存活率的影响。流式细胞仪检测0(对照组)、0.5、1、2、5、10 mmol/L二甲双胍对培养48 h的HaCaT细胞周期和凋亡的影响,Western印迹法检测HaCaT细胞增殖和分化相关蛋白角蛋白16、角蛋白17、角蛋白1、内披蛋白,凋亡相关蛋白Bax、Bcl-2及AKT/mTOR/STAT3通路蛋白表达水平的影响,ELISA检测HaCaT细胞炎症因子白细胞介素8(IL-8)、肿瘤坏死因子α(TNF-α)、IL-23分泌水平。统计学分析采用SPSS19.0软件,不同浓度组间观察指标的变化采用单因素方差分析,不同时间和不同浓度组间比较采用重复测量方差分析,两两多重比较采用LSD-t检验。结果CCK8法显示,二甲双胍对HaCaT细胞增殖有抑制作用(F=116.87,P<0.05),且随着二甲双胍浓度的增加,细胞存活率逐渐降低。0、0.5、1、2、5、10 mmol/L二甲双胍干预HaCaT细胞48 h后,G2/M期细胞比例逐渐增加(5.55%±1.03%、6.37%±0.93%、8.57%±1.18%、10.05%±0.60%、10.76%±0.87%、13.63%±1.41%,F=24.98,P<0.05),早期细胞凋亡率逐渐升高(0.78%±0.71%、19.18%±1.41%、25.67%±1.34%、28.45%±0.92%、34.97%±2.12%、40.41%±1.49%,F=296.08,P<0.05);随二甲双胍浓度增加,角蛋白1、促凋亡蛋白Bax蛋白表达呈上升趋势(F=8.86、5.38,均P<0.05),角蛋白16、角蛋白17、凋亡蛋白Bcl-2蛋白表达呈下降趋势(F=8.02、4.82、12.10,均P<0.05),但各浓度组间内披蛋白的表达差异无统计学意义(F=0.57,P >0.05);不同浓度二甲双胍干预48 h后,HaCaT细胞IL-8、TNF-α表达水平明显下降(F=33.89、14.99,均P<0.05),但IL-23表达无明显变化(F=2.12,P >0.05)。随二甲双胍浓度增加,p-AKT、p-mTOR、p-STAT3表达水平逐渐下降(F=11.38、0.35、4.38,均P<0.05),而AKT、mTOR、STAT3蛋白表达差异无统计学意义(F=0.66、0.35、4.24,均P >0.05)。结论二甲双胍可能通过调控AKT/mTOR/STAT3信号通路抑制HaCaT细胞增殖,促进HaCaT细胞分化,促进细胞凋亡及抑制炎症因子分泌。

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) , and the cell survival rates gradually decreased along with the increase in the concentrations of metformin. After 48-hour treatment with metformin at concentrations of 0, 0.5, 1, 2, 5, 10 mmol/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 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) , 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.