白藜芦醇对银屑病细胞生长的影响及其作用机制的探讨

Effect and mechanism of resveratrol on proliferation of psoriatic cells

摘要:

目的 探讨白藜芦醇对银屑病细胞生长的影响及其作用机制。方法 不同浓度的角质细胞生长因子（KGF）诱导人永生角质形成细胞（HaCaT），显微镜观察及四甲基偶氮唑盐比色（MTT）法检测HaCaT细胞增殖。选出最佳浓度作用HaCaT细胞复制银屑病模型，采用不同浓度白藜芦醇作用HaCaT细胞，计算半抑制浓度（IC50），根据IC50选出后续实验白藜芦醇的浓度；将银屑病模型细胞分为阴性对照组（30 μg/L生理盐水），2.5 μmol/L、5.0 μmol/L、7.5 μmol/L白藜芦醇组，阳性对照组（5.0 μg/mL维A酸）及空白对照组（未加任何药物），MTT法检测各组银屑病模型细胞的增殖，流式细胞术检测各组银屑病模型细胞的凋亡率，Western blotting检测各组银屑病模型细胞Caspase-3、p-ERK1/2、p-P38蛋白的表达。结果 不同KGF浓度组HaCaT细胞0 h、12 h、24 h、48 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点的OD值有差异（P <0.05）；②不同浓度组的OD值有差异（P <0.05），40 ng/mL KGF组的OD值高于其他浓度组；③不同浓度组OD值的变化趋势有差异（P <0.05），40 ng/mL KGF组的OD值从24 h开始明显高于其他浓度组。0 μmol/L、0.5 μmol/L、1.0 μmol/L、2.0 μmol/L、4.0 μmol/L白藜芦醇作用HaCaT细胞24 h后的OD值比较，差异有统计学意义（P <0.05），白藜芦醇浓度升高OD值降低，白藜芦醇可抑制HaCaT细胞增殖，IC50值约5.3 μmol/L。空白对照组、阴性对照组、2.5 μmol/L白藜芦醇组、5.0 μmol/L白藜芦醇组、7.5 μmol/L白藜芦醇组、阳性对照组银屑病模型细胞24 h、48 h、72 h的OD值比较，采用重复测量设计的方差分析，结果 ①不同时间点的OD值有差异（P <0.05）；②各组OD值有差异（P <0.05），③各组OD值的变化趋势有差异（P <0.05），从48 h开始，5.0 μmol/L、7.5 μmol/L白藜芦醇对银屑病模型细胞具有一定的抑制增殖作用。空白对照组、阴性对照组、2.5 μmol/L白藜芦醇组、5.0 μmol/L白藜芦醇组、7.5 μmol/L白藜芦醇组、阳性对照组银屑病模型细胞的凋亡率比较，差异有统计学意义（P <0.05），5.0 μmol/L、7.5 μmol/L的白藜芦醇对银屑病模型细胞有促凋亡作用。5.0 μmol/L白藜芦醇组、7.5 μmol/L白藜芦醇组、阳性对照组与阴性对照组和空白对照组比较，Caspase-3蛋白相对表达量升高（P <0.05），p-ERK1/2、p-P38蛋白相对表达量降低（P <0.05）。结论 白藜芦醇可能是通过抑制ERK/MAPK和/或P38/MAPK通路发挥作用进而抑制银屑病细胞增殖并促进其凋亡，具体作用机制还有待更深入研究。

Abstract:

Objective To investigate the effect of resveratrol on the growth of psoriatic cells and its mechanism.Methods Human immortal keratinocytes (HaCaT) cells were induced by different concentrations of keratinocyte growth factor (KGF), and the proliferation of HaCaT cells was detected by microscopic observation and tetramethylazol salt colorimetric (MTT) assay to select the optimal concentration to stimulate HaCaT cells to establish a psoriasis cell model. HaCaT cells were stimulated by different concentrations of resveratrol, the effect of resveratrol on the proliferation of HaCaT cells was detected by MTT assay to calculate the IC50, and the subsequent experimental resveratrol stimulation concentration was selected according to the IC50. The psoriasis cell model was divided into blank, negative, positive and resveratrol low, medium, and high concentration groups, the proliferation of psoriasis cell model- HaCaT cells was detected by MTT assay, the apoptosis of psoriasis cell model- HaCaT cells was detected by flow cytometry, and the expression of caspase-3, P-ERK1/2, and P-p38 protein in HaCaT cells of each group was detected by Western blotting.Results The OD values of HaCaT cells at 0 h, 12 h, 24 h, and 48 h in different KGF concentration groups were compared by repeated measurement design analysis of variance, which showed that: (1) The OD values at different time points were different (P < 0.05); (2) The OD value of 40 ng/ml KGF group was higher than that of other concentration groups; (3) The change trend of OD value in different concentration groups was different (P < 0.05). The OD value of 40 ng/ml KGF group was significantly higher than that of other concentration groups from 24 hours. The OD values of HaCaT cells treated with 0 μmol/L, 0.5 μmol/L, 1.0 μmol/L, 2.0 μmol/L, 4.0 μmol/L resveratrol after 24h were statistically significant (P < 0.05). After 24 h, 48 h, and 72 h, resveratrol can inhibit the proliferation of HaCaT cells, and the IC50 value is about 5.3 μmol/L. The OD values of blank control group, negative control group, 2.5 μmol/L, 5.0 μmol/L, 7.5 μmol/L resveratrol group, and positive control group were analysis of variance with repeated measurement design; The results were as follows: (1) The OD values at different time points were different (P < 0.05); (2) The OD value of each group was different (P < 0.05); (3) The change trend of OD value of each group was different (P < 0.05); Starting from 48 hours, 5.0 μmol/L and 7.5 μmol/L resveratrol can inhibit the proliferation of psoriatic model cells. There were significant differences in the apoptosis rate of blank control group, negative control group, 2.5 μmol/L, 5.0 μmol/L, 7.5 μmol/L resveratrol group, and positive control group (P < 0.05), which showed 5.0 μmol/L and 7.5 μmol/L resveratrol promoted the apoptosis of psoriatic model cells. Compared with the negative control group and the blank control group, the relative expression of caspase-3 protein in 5.0 μmol/L, 7.5 μmol/L resveratrol group, and positive control group increased (P < 0.05), and the relative expression of p-ERK1/2 and p-p38 protein decreased (P < 0.05).Conclusions Resveratrol may inhibit the proliferation and promote the apoptosis of psoriatic cells by inhibiting ERK/MAPK and/or p38/MAPK pathway. The specific mechanism needs to be further studied.