金茵清热口服液对人PBMC中TLR2信号通路的调节机制研究

Immunomodulatory effects of Jinyin Qingre oral liquid on TLR2 signaling pathway of PBMC in human

目的:研究金茵清热口服液(JYQR)对人外周血单个核细胞(PBMC)中Toll样2受体(TLR2)信号通路免疫调节作用机制。方法:体外分离、培养人PBMC,设空白对照组和JYQR组,JYQR组加入JYQR刺激PBMC,与空白组经37℃,5%CO2培养24 h后,分别提取各组PBMC中总RNA、总蛋白、细胞上清液,RT-PCR和Western Blot法检测空白对照组和JYQR组PBMC中相关信号分子TLR2、MyD88、IRAK4、TRAF6、IRF3 mRNA和蛋白的表达情况,ELISA法检测细胞因子IL-1α、IL-6、TNF-α、IFN-α的分泌水平;分别设空白对照组:不作处理;JYQR组:JYQR 2.015 mg·mL^-1;TLR2激动剂组:Pam3CSK4 300 ng·mL^-1;TLR2抑制剂组:OxPAPC 30μg·mL^-1+JYQR 2.015 mg·mL^-14组,提取核蛋白和浆蛋白,Western Blot法检测IRF3和NF-κB p65的核转位情况。结果:与空白对照组相比,JYQR组中TLR2、IRAK4、TRAF6、IRF3 mRNA表达水平分别上调5.93倍、1.77倍、2.95倍、1.43倍(*P<0.05,**P<0.01);MyD88、TRAF6蛋白的表达分别上调1.26倍、1.24倍(*P<0.05),IRAK4和IRF3的相对表达水平差异无统计学意义;细胞因子IL-1α、IL-6的表达下调55%、61%,TNF-α、IFN-α的分泌水平上调4.3倍、2.9倍(*P<0.05,**P<0.01)。JYQR预处理PBMC后,IRF3及NF-κB核转位增加,但是经过TLR2抑制剂预处理后,IRF3和NF-κB的核转位减弱;相反,TLR2激动剂预处理可以进一步上调IRF3和NF-κB的核转位。结论:金茵清热口服液对人PBMC中TLR2信号通路的作用机制可能是通过MyD88依赖型信号通路激活中、下游的IRAK4、TRAF6、IRF3以及NF-κB调控终端效应因子发挥免疫调节作用。

OBJECTIVE To study the immunomodulatory mechanism of Jinyin Qingre oral liquid(JYQR) on Toll-like 2 receptor(TLR2) signaling pathway in human peripheral blood mononuclear cells(PBMC).METHODS Human PBMCs were isolated and cultured in vitro. The JYQR group and the blank control group were set up. The PBMCs in test drug group were stimulated by JYQR and incubated with the blank control group at 37 ℃, 5% CO2 for 24 h. Total RNA, protein and supernatant were extracted from PBMCs of each group, respectively. RT-PCR and Western Blot were used to detect the expression of related signaling molecules TLR2, MyD88, IRAK4, TRAF6, IRF3 mRNA and protein in PBMCs of the blank control group and the JYQR group. ELISA was used to detect the secretion level of cytokines IL-1α、IL-6、TNF-α、IFN-α. The control group: no treated, JYQR group: JYQR 2.015 mg·mL^-1, TLR2 agonist group: Pam3 CSK4 300 ng·mL^-1 and TLR2 Inhibitor group: OxPAPC 30 μg·mL^-1+ JYQR 2.015 mg·mL^-1), were set up to extract nuclear protein and cytoplasmic protein, respectively. Western Blot was used to detect nuclear translocation of IRF3 and NF-κB p65.RESULTS The results showed that compared with blank control group, the mRNA expression levels of TLR2, IRAK4, TRAF6 and IRF3 in the JYQR group were up-regulated by 5.93, 1.77, 2.95 and 1.43 times, respectively(*P<0.05,**P<0.01);The expression of MyD88 and TRAF6 proteins were up-regulated by 1.26 and 1.24 times, respectively(*P<0.05). There was no significant difference in the relative expression levels of IRAK4 and IRF3. The expression of cytokines IL-1α and IL-6 were down-regulated by 55% and 61%, and the secretion levels of TNF-α and IFN-α were up-regulated by 4.3 and 2.9 times(*P<0.05,**P<0.01). IRF3 and NF-κB Via nuclear translocation after treatment with JYQR on PBMCs, but after pretreatment with TLR2 inhibitor, the nuclear translocation of IRF3 and NF-κB is significantly inhibited, even lower than that of blank control group. In contrast, TLR2 agonist pretreatment can further up-regulate the nuclear translocation of IRF3 and NF-κB mediated by JYQR.CONCLUSION The immunomodulatory mechanism of JYQR on TLR2 signaling pathway in PBMCs may be through MyD88-dependent signaling pathway to activate IRAK4, TRAF6, IRF3 and NF-κB regulatory terminal effect factors in the middle and downstream to exert immune regulation.