Immunomodulatory Effects And Mechanisms Of Jinyin Qingre Oral Liquid Mediated By TLR Signaling Pathway

Jinyin Qingre Oral Liquid main components include honeysuckle,forsythia,rhubarb,astragalus,licorice,capillaris,Gardenia and red sage root;which is a Chinese materia medica compound formulations,and developed by hubei university of traditional Chinese medicine and shiyan taihe hospital.This prescription has been proved to have the functions of clearing away heat and toxic materials,promoting blood circulation and cooling blood,has remarkable curative effect on treating influenza,and can enhance the immune function of the body,after been studied about prescription optimization,toxicity,stability,pharmacodynamics,clinical efficacy and in vitro experiments in the early stage.In modern Chinese medicine theory and practice,it is proved that Chinese medicine can achieve certain therapeutic effects by affecting the immune function of the body.At the same time,a large number of studies have shown that Chinese medicine exerts immunoregulatory effects through the TLR signaling pathway.The research group found through the experiment of Jinyin Qingre Oral Liquid on anti-Hep2 cell pathological changes caused by influenza A(H1N1)virus that the test drug can significantly increase the levels of antiviral factors IFN-la and TNF-a of Hep2 cells,and reduce the levels of inflammatory factors such as interleukin IL-la and IL-6.Therefore,in order to explore the effect of Jinyin Qingre Oral Liquid on immune cells and its mechanism,this study uses humanPeripheral blood mononuclear cell(PBMC)to study the effect of Jinyin Qingre Oral Liquid on TLRs signaling pathway in immune cells and its effect on genes and proteins related to signaling pathway,and to explore the role of TLRs signaling pathway in the immune regulation of Jinyin Qingre Oral Liquid.Objective Exploring whether Jinyin Qingre Oral Liquid(test drug)can play a role through TLR signaling pathway,and study which TLR signaling pathways of Jinyin Qingre Oral Liquid play a role,further explore the immunomodulatory mechanism of Jinyin Qingre Oral Liquid.Methods 1.Establishment of TLR1-10 and GAPDH standard curves PBMC was incubated with LPS,total RNA of PBMC was extracted as template,Primer 5.0 primer software was used to design TLR1-10 and GAPDH primers,PCR method was used to obtain TLR1-10 and GAPDH amplification products,TLR1-10 and GAPDH standards were prepared,different real-time fluorescence quantitative PCR standard curves were established respectively,PCR reaction conditions were optimized,and amplification efficiency and specificity of the method were detected.2.Effect of Jinyin Qingre Oral Liquid on TLRs mRNA in PBMC PBMC was incubated with different concentrations of test drug,the cell activity was detected by MTT colorimetric method,and the maximum non-toxic concentration(TC0)of test drug to cells was calculated.A blank control group and a drug test group were set up,and the expression of TLR1-10 mRNA was detected by Real-time PCR.3.Effect of Jinyin Qingre Oral Liquid on TLR2 Signal Pathway RT-PCR and Western Blot were used to detect the expression of related signal molecules TLR2,MyD88,IRAK4,TRAF6,IRF3 mRNA and protein in PBMC of blank control group and test group.ELISA was used to detect the secretion levels of cytokines IL-la,IL-6,TNF-a,IFN-a.The inhibitor group,the blank control group,the drug test group and the agonist group were set up respectively,to extract nuclear protein and plasma protein.The Nuclear translocation of IRF3 and NF-kB p65 was detected by Western Blot method,and the gray analysis was carried out.Results 1.the standard curves of TLR1-10 and GAPDH have been established:the linear relationship between Ct value of each gene and concentration of standard substance is good,and the correlation coefficient R2 is more than 0.995;The amplification efficiency is basically more than 90%.The lower detection limit is 1×103 copies/ul,proved its high sensitivity.No primer dimer or non-specific amplification products appear,and the specificity of these primers can be reflected well.2.Jinyin Qingre Oral Liquid significantly up-regulates TLR1,2 and 6 mRNA expression in PBMC:MTT method was used to detect the survival rate of PBMC,and TC0 was 2.015 mg mL-1.RT-PCR results showed that the mRNA levels of TLR5,TLR7,and TLR8 mRNA the test group were decreased by 53%、21%、15%respectively,while TLR1 was increased by 4.11 times,TLR2 by 6.32 times,TLR6 by 5.58 times and TLR9 by 1.53 times(p<0.05).3.Jinyin Qingre Oral Liquid Activates MyD88-Dependent TLR Signal Pathway:RT-PCR results showed that the mRNA expressions of TLR2,MyD88,and TRAF6 were up-regulated 5.93 times,1.77 times,2.95 times in the test group.Western-blot analysis showed that the proteins expression of MyD88 and TRAF6 were up-regulated by 1.26 and 1.24 times compared with the blank control group.The results of ELISA showed that the expression of IL-la and IL-6 in the test group was down-regulated 55%and 61%compared with that in the blank control group.The secretion levels of TNF-a and IFN-a were up-regulated 4.3 times and 2.9 times,with statistically significant difference(p<0.05).4.TLR2 inhibitor can block the immune activation effect of Jinyin Qingre Oral Liquid on PBMC:IRF3 and NF-κB Via nuclear translocation after treatment with Jinyin Qingre Oral Liquid on PBMC,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 without any treatment.In contrast,TLR2 agonist pretreatment can further up-regulate the nuclear translocation of IRF3 and NF-κB mediated by Jinyin Qingre Oral Liquid.Conclusion 1.Jinyin Qingre Oral Liquid significantly up-regulates TLR1,2 and 6 mRNA expression in PBMC,suggesting that it may play an immune regulatory role mainly through TLR1,TLR2 and TLR6 signaling pathways;2.Jinyin Qingre Oral Liquid can up-regulate the antiviral natural immunity of PBMC;3.Jinyin Qingre Oral Liquid can activate MyD88-dependent TLR signaling pathway,and TLR2 inhibitor can interdict translocation of IRF3 and NF-κB mediated by Jinyin Qingre Oral Liquid,indicating that TLR2 signaling pathway plays a key role in immune regulation function of Jinyin Qingre Oral Liquid.