消银解毒方颗粒对Jurkat T细胞内JAK1/STAT3信号通路作用机制的研究

Effect of Xiaoyin Jiedu Fang on JAK1/STAT3 signaling pathway in Jurkat T cells

目的探讨消银解毒方颗粒对人急性T淋巴细胞白血病细胞株(Jurkat T)细胞内酪氨酸激酶1(JAK1)/信号转导和转录激活因子3(STAT3)信号通路的调控作用。方法以植物血凝素(PHA)和白细胞介素-6(IL-6)共同诱导Jurkat T淋巴细胞建立血热证银屑病T细胞异常活化病理细胞模型,将模型细胞接种于24孔板,制备消银解毒方颗粒、凉血方颗粒(消银解毒方颗粒拆方)、解毒方颗粒(消银解毒方颗粒拆方)、阿维A(阳性对照)、蒸馏水(阴性对照)的药理血清,择取最佳浓度药理血清及JAK/STAT信号通路阻断剂Filgotinb(GLPG0634)作用于模型细胞48 h,同时设空白细胞组。收集各组细胞,采用蛋白质印迹(Western blot)、实时定量聚合酶链式反应(real-time PCR)法检测JAK1、STAT3、糖蛋白(GP)130的蛋白表达与基因转录水平。结果模型组的JAK1、STAT3、GP130的蛋白和mRNA表达较空白组明显增高(P<0.01)。与模型组相比,各实验组磷酸化酪氨酸溶酶(p JAK)1/JAK1、磷酸化信号转号和转录激活因子(p STAT)3/STAT3、GP130蛋白表达量均明显降低(P<0.01);与模型组相比,各实验组JAK1、STAT3、GP130的mRNA表达量均降低(P<0.05或P<0.01)。结论消银解毒方颗粒能够通过抑制JAK1/STAT3信号通路的开放进而抑制T细胞异常活化,以发挥其治疗银屑病的作用。

Objective To study the effect of Xiaoyin Jiedu Fang, a Chinese medicine compound formula- ted by TCM principle of cooling blood with detoxifieation, on nonreceptor tyrosine kinase JAK1/ signal transducer and activator of transcription 3 （ STAT3 ） signaling pathway in human acute leukemia Jurkat T cells. Methods The abnormal activation of T cells of psoriasis with TCM pattern of blood-heat was in- duced by stimulating Jurkat T cells with phytohaemagglutinin （PHA） and IL-6. The medicated serum contained Xiaoyin Jiedu Fang granule （XYJDF） , Liangxue Fang （LXF） or Jiedu Fang granule （JDF） which was a disassembled prescription of XYJDF, acitretin （ positive drug） and distilled water （ negative control） were prepared. The model cells were inoculated into 24-hole cell culture plate and were cultured with the prepared medicated serum and Filgotinb（ GLPG063d）, an inhibitor of JAK/STAT signaling path- way for 48 hours. Jurkat T cells with no treatment was served as the blank group. Protein and mRNA ex- pression of JAK1, STAT3, glucoprotein（GP）130 were detected by using Western blot and real-time PCR. Results The protein and mRNA expression of JAKI ,STA33 and GP130 in the model group were significantly higher than those of the blank group （P 〈 0.01 ）. Compared with the model group, the protein expression of pJAK1/JAK1, pSTAT3/STAT3 and GP130 in all medicated group were decreased significantly （P 〈 0.01 ）, and the mRNA expression of JAK1, STAT3 and GP130 declined （P 〈0.01 or P 〈 0.05）. Conclusion The effect of XYJDF against psoriasis could be via the way of inhibiting JAKI/ STAT3 signaling pathway leading to prevent abnormal activation of T cells.