Objective:To explore the effect of Xiaozhen Zhiyang Spray on keratinocyte proliferation and differentiation and the effect on macrophage infiltration.Methods:MTT method was used to observe the different concentrations of Xiaozhen Zhiyang spray on skin keratinocytes HaCaT growth effect;Wound healing and Transwell experiment was used to examine the effect of Xiaozhen Zhiyang Spray on HaCaT and macrophage RAW264.7 exercise ability;Western blotting experiment was used to investigate the effect of Xiaozhen Zhiyang Spray on the proteinic expression of PI3K,AKT,tPA and CD147 in HaCaT cells and MMP-2,MMP-9 in RAW264.7 cells.RT-PCR was used to investigate the effects of Xiaozhen Zhiyang spray on MMP-2 and MMP-9 mRNA levels in RAW264.7 cells.Results:Xiaozhen Zhiyang Spray can dose dependently promote the proliferation of HaCaT cells and inhibit its differentiation.In wound healing and transwell experiment,the migration number and invasive ability of HaCaT and macrophage RAW264.7 decreased with Xiaozhen Zhiyang Spray concentration increased.Xiaozhen Zhiyang Spray can also dose dependently inhibit the expression of proliferation protein PI3K and AKT,and decrease the expression of differentiation protein tPA and CD147.In addition,Xiaozhen Zhiyang spray can significantly inhibit the mRNA and protein expression of MMP2 and MMP9 in RAW264.7.Conclusion:Xiaozhen Zhiyang spray can promote keratinocyte proliferation and reduce the infiltration of macrophage,which is one of the important mechanisms for the treatment of EGFRI associated skin rashes.%目的:探究消疹止痒喷剂对皮肤角质细胞增殖分化及巨噬细胞浸润的影响及其作用机制.方法:MTT法观察不同浓度消疹止痒喷剂对皮肤角质细胞HaCaT生长能力的影响;划痕实验(Wound healing)及Transwell实验考查消疹止痒喷剂对HaCaT及巨噬细胞RAW264.7运动能力的影响,采用western blotting实验考察消疹止痒喷剂对HaCaT细胞的PI3K、AKT、tPA、CD147及RAW264.7细胞中基质金属蛋白酶MMP-2(Matrix metalloproteinases-2,MMP-2)、MMP-9蛋白表达的影响,采用RT-PCR考察消疹止痒喷剂对RAW264.7细胞的MMP-2、MMP-9 mRNA水平的影响.结果:消疹止痒喷剂可以剂量依赖性地促进HaCaT细胞的增殖,抑制其分化;Wound healing实验和Transwell小室迁移实验中,随着消疹止瘁喷剂浓度提高,HaCaT及巨噬细胞RAW264.7迁移数目呈递减趋势;随着消疹止瘁喷剂浓度的提高,HaCaT及巨噬细胞RAW264.7侵袭能力呈递减趋势;消疹止瘁喷剂可以剂量依赖性抑制HaCaT细胞PI3K和AKT增殖蛋白表达,同时降低分化蛋白tPA和CD147的表达;此外消疹止痒喷剂可以显著抑制RAW264.7 MMP2和MMP9的mRNA和蛋白水平表达.结论:消疹止痒喷剂促进皮肤角质细胞增殖并抑制巨噬细胞浸润是其治疗EGFRI相关性皮疹的重要机制之一.
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