凉血解毒汤对人表皮角质形成细胞CCL20表达及分泌影响

Effect of Liangxue Jiedu(LXJD)Prescription on IL-17-induced CCL20 Expression and Secretion in Human Keratinocytes

目的:观察凉血解毒汤对人表皮角质形成细胞(Ha Ca T细胞)CC趋化因子配体20(CCL20)表达及分泌的影响,并探讨其作用机制。方法:用IL-17(100 ng·m L-1)刺激Ha Ca T细胞,并加入不同浓度凉血解毒汤(8、2、0.5μg·m L-1),干预体外培养Ha Ca T细胞模型。采用MTT法检测凉血解毒汤剂对Ha Ca T细胞活性的影响;ELISA检测细胞上清CCL20蛋白的含量,Western blot检测细胞内CCL20相关蛋白(p-IκBα、p-P65)的变化,Realtime PCR检测细胞样本中CCL20相关基因(Piasy、Trim32 m RNA)转录水平的变化情况。结果:IL-17能够诱导Ha Ca T细胞CCL20的表达及分泌。加入凉血解毒汤水煎剂粉末后,CCL20在分泌、蛋白、m RNA表达水平均明显下降(P<0.05),NF-κB通路相关蛋白p-P65、p-IκBα磷酸化水平显著下降(P<0.01),但对CCL20相关调节分子Piasy、Trim32 m RNA的表达无显著影响(P>0.05)。结论:凉血解毒汤通过调节NF-κB通路磷酸化抑制Ha Ca T细胞分泌及表达CCL20可能是其发挥治疗银屑病作用机制之一。

Objective：To observe the effect of Liangxue Jiedu（LXJD）Prescription on IL-17-induced CC chemokine ligand 20（CCL20）expression and secretion in Human Keratinocytes（Ha Ca T cells）and to explore its mechanism. Methods：Ha Ca T cells were stimulated with IL-17（100 ng·m L-1）and incubated with different concentrations of LXJD Prescription（8,2,0.5 μg·m L-1）. The effects of LXJD Prescripition on viability of Ha Ca T cells were observed by MTT method. ELISA was used to detect CCL20 protein content in the cell supernatant. Western blot was used to detect the changes of intracellular CCL20 related protein（p-IκBα,p-P65）. Real-time PCR was used to detect the expression of intracellular CCL20 m RNA,Piasy m RNA and Trim32 m RNA. Results：IL-17 led to the upregulation of CCL20 expression in Ha Ca T cells. After incubation with LXJD Prescripition,the expression of CCL20 were down-regulated at secretion（P〈0.05）,m RNA（P〈0.05）and protein（P〈0.05）levels significantly. The relative protein expression of NF-κB pathway on the phosphorylation level was significantly decreased（P〈0.01）. But the expression of related molecules Piasy m RNA and Trim32 m RNA had no significant effect. Conclusion：LXJD Prescription can lead to the down-regulation of IL-17-induced CCL20 expression and secretion in Ha Ca T cells by activating NF-κB pathway,which is one of the mechanisms for the treatment of psoriasis.