健脾养血祛风方对角质形成细胞的作用及其对紧密连接的干预机制研究

The intervention mechanism of Jianpi Yangxue Qufeng Decoction on tight junctions in keratinocytes

目的探究健脾养血祛风方对角质形成细胞(KC)的作用及其对紧密连接(tight junctions,TJ)的影响。方法CCK8检测健脾养血祛风方对人永生化角质形成细胞(HaCat)细胞活力的影响;Western blot检测不同浓度健脾养血祛风方对TJ相关蛋白occludin、claudin-1、JAM-A、Zo-1表达影响情况。ELISA检测IL-4、IL-10、INF-γ、IL-12水平。RT-qPCR检测Toll样受体4(TLR4)、MyD88、NF-κB的mRNA表达水平。结果在不影响细胞活力的前提下,健脾养血祛风方能明显增加occludin、claudin-1、JAM-A、Zo-1蛋白的表达,同时能显著抑制INF-γ、IL-12的释放,促进IL-4、IL-10的生成。此外,健脾养血祛风方还能显著抑制HaCat中TLR4、MyD88、NF-κB水平。结论健脾养血祛风方可调控HaCat中TJ相关蛋白表达,从而修复机械屏障。可能的机制是健脾养血祛风方抑制了TLR4/MyD88/NF-κB信号通路激活,进而改变了炎症因子表达。

Objective The study aims to explore the effect Jianpin Yangxue Qufeng Recipe on keratinocytes(KC)and its effect on tight on tight junctions(TJ).Methods CCK8 was used to detect the effect of Jianpi Yangxue Qufeng Compound on the activity of human immortalized keratinocytes(HaCat).The effects of Jianpi Yangxue Qufeng Recipe on TJ-related proteins,including occludin,claudin-1,JAM-A and Zo-1 were tested by western blot.The contents of IL-4,IL-10,INF-γand IL-12 were detected by ELISA.Furthermore,the mRNA expression of toll-like receptor 4(TLR4),MyD88 and NF-κB were detected by RT-qPCR.Results On the premise of not affecting cell viability,Jianpi Yangxue Qufeng Compound can significantly increase the expression of occludin,claudin-1,JAM-A,Zo-1 proteins.According to inhibit the release of INF-γ,IL-12 and promote the production of IL-4 and IL-10,significantly.In addition,invigorating spleen,nourishing blood and expelling wind can also significant inhibit TLR4,MyD88 and NF-κB levels in HaCat.Conclusion TJ-related protein expression in HaCat was regulated Jianpi Yangxue Qufeng Compound,thereby repairing the mechanical barrier.The underlying mechanism of Jianpi Yangxue Qufeng Recipe may inhibits the activation of TLR4/MyD88/NF-κB signaling pathway,thus changing the expression of inflammatory factors.