龙胆草对HaCaT细胞增殖和凋亡及表皮生长因子受体磷酸化的影响

Effect of Chinese gentian on the proliferation of, apoptosis and phosphorylation of epidermal growth factor receptor in HaCaT cells

目的 探讨龙胆草提取物对表皮生长因子（EGF）刺激后HaCaT细胞增殖和凋亡及表皮生长因子受体（EGFR）磷酸化的影响.方法 取0～ 50g/L龙胆草提取物作用于EGF刺激后的HaCaT细胞24 h,噻唑蓝法检测细胞增殖率,流式细胞仪检测细胞凋亡率,Western印迹法检测磷酸化EGFR.结果 龙胆草提取物浓度依赖性地抑制EGF刺激后HaCaT细胞的增殖（r=-0.991,P＜0.01）,促进EGF刺激后HaCaT细胞的凋亡（r=0.996,P＜ 0.05）.同时,龙胆草提取物使EGF刺激后的HaCaT细胞EGFR的磷酸化水平降低,该作用随着药物浓度的增加而增加.结论 龙胆草可能通过降低EGFR的磷酸化,阻断相关细胞内信号通路来抑制角质形成细胞的过度增殖并促进细胞的凋亡.

Objective To evaluate the effect of Chinese gentian extracts on the proliferation of,apoptosis and phosphorylation of epidermal growth factor receptor （EGFR） in HaCaT cells induced by epidermal growth factor （EGF）.Methods Methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferation of HaCaT cells pretreated with EGF of 20 μg/L for 24 hours followed by 24 hours of treatment with various concentrations of Chinese gentian extracts.Flow cytometry was carried out to detect apoptosis in HaCaT cells pretreated with EGF of 20 μg/L for 24 hours followed by 4 hours of treatment with different concentrations of Chinese gentian extracts.Western blot was conducted to measure the level of phosphorylated EGFR in HaCaT cells treated with different concentrations of Chinese gentian extracts for 24 hours followed by treatment with EGF of 20 μg/L for 10 minutes.Results Chinese gentian extracts inhibited the proliferation （r =-0.991,P ＜ 0.01）,but promoted the apoptosis （r =0.996,P ＜ 0.05） of HaCaT cells induced by EGF in a dose-dependent manner.At the same time,the extracts suppressed the phosphorylation of EGFR in HaCaT cells induced by EGF,and the suppressing effect increased with the rise in the concentration of the extracts.Conclusions Chinese gentian may inhibit the proliferation,but promote the apoptosis of keratinocytes by decreasing EGFR phosphorylation and blocking relevant intracellular signaling pathways.