The Mechanism Of Treatment Of AR In The Treatment Of Th1/Th2 Differentiation Induced By DCs

ObjectiveIn this experimental study,we focus on the key role of Ephedra asarum aconite decoction about imbalance of Thl/Th2 for allergic rhinitis,caused mainly by the proliferation and differentiation of CD4~+T cells and the intervention of dendritic cells,in order to analyze the mechanism of immune regulation and anti-inflammatory about Ephedra asarum aconite decoction.Methods1,Establish culture methods of dendritic cells and T cell and culture them.2,Make Preparation of the active ingredient of water extracting from Ephedra asarum aconite decoction,and detect its toxicity on dendritic cells and T cells by CCK-8.3,Detect the effect of the active ingredient of water extracting from Ephedra asarum aconite decoction on the proliferation and differentiation of CD4~+T cells by flow cytometry.4,Detect the maturation of dendritic cells after stimulating by LPS intervened by Ephedra asarum aconite decoction via flow cytometry.5,Detect the maturation of dendritic cells after stimulating by TSLP intervened by Ephedra asarum aconite decoction via flow cytometry.6,Detect the content of cytokines IL-1β,IL-6,IL-12,IFN-gamma of dendritic cells after stimulating by LPS intervened by Ephedra asarum aconite decoction via Multiple Immunoassays for Flow(AimPlex).7,Detect the content of cytokines IL-5,IL-13 of T cells interacting with dendritic cells after stimulating by TSLP intervened by Ephedra asarum aconite decoction via Multiple Immunoassays for Flow(AimPlex).8,Detect the expression of pSTAT6 and pSTAT5 of dendritic cells after stimulating by LPS intervened by Ephedra asarum aconite decoction via Western-Blot.9,Test the expression of T-bet mRNA and GATA-3 mRNA of LPS stimulaed dendritic cells after intervened by Ephedra asarum aconite decoction via qPCR.Results1,Establish the methods of culturing of dendritic cells and T cells successfully.2,By CCK-8 tests,the concentration of the active ingredient of water extracting from Ephedra asarum aconite decoction within 5mg/ml did not affect cell viability of dendritic cells,since it had the efforts to promote cell activity(P<0.01);when the concentration is above 10mg/ml,the active ingredient of water extracting from Ephedra asarum aconite decoction had inhibitory effect on DC cells activity,when the concentration was 40mg/ml,there was no difference in cell activity comparing the normal group.By CCK-8 tests,when the concentration was at 20mg/ml of the active ingredient of water extracting from Ephedra asarum aconite decoction,the cell activity T cells increased(P<0.01),and with the concentration of the drug increasing,the cell viability was enhanced.When T cells was observed under the microscope,the cell concentration in the Ephedra asarum aconite decoction group was less than that in the normal group,and the cells grew poorly in the Ephedra asarum aconite decoction group.We detected the T cell by flow cytometry,since the result from CCK-8 tests was inconsistent with what we saw under the microscope.The new result showed that when T cells treated with Ephedra asarum aconite decoction at concentration of 10mg/ml,the T cells had larger morphological changes.3,The mean intensity of fluoresce(MFI)of the blank control group,the activation group and the Ephedra asarum aconite decoction group diverged from that in the standard group(P<0.01).The MFI of the activation group was lower than that in the blank control group(P<0.01).The MFI of the Ephedra asarum aconite decoction group was under those in the blank control group,and the activation group(P<0.01).IFN-y and IL-4 contents of the blank control group were 2.4 and 8.3 respectively.The IFN-y and IL-4 contents of the activation group were 5.1 and 30.3 respectively.Furthermore,the IFN-y and IL-4 contents of the Ephedra asarum aconite decoction group were 5.0 and 20.6 respectively.4,The ratios of CD80~+DCs,MHCII~+DCs and CD83~+DCs in the LPS group(L group)were more than them in the blank control group(C group,p<0.01);the ratios in the Ephedra asarum aconite decoction group were lower than them in the LPS group(L group,P<0.01),but the proportion of CD83~+DCs was still more than it in the blank control group(C group,p<0.01).5,There were no difference of ratios of CD80~+DCs,MHCII~+DCs and CD83~+DCs among the blank control group(C group),the LPS group(L group)and the Ephedra asarum aconite decoction group(M group)6,The contents of IL-12、IFN-γ、IL-6、IL-1β in the LPS group(L group)were more than them in the blank control group(C group,p<0.01).The contents of these cytokines in the Ephedra asarum aconite decoction group(M group)were lower than them in the LPS group(L group,P<0.01),but were still more than the blank control group(C group,p<0.01).7,The contents of IL-5 and IL-13 in the TSLP group(T group)were more than them in the blank control group(C group,p<0.01).The contents of these cytokines in the Ephedra asarum aconite decoction group(M group)had little difference comparing the TSLP group(T group,P<0.01),but were still more than the blank control group(C group,p<0.01).8,The expression of STAT6 of dendritic cells in LPS group(L group)was less than the expression in the blank control group(C group,p<0.05).The expression of STAT6 of dendritic cells in the Ephedra asarum aconite decoction group(M group)was lower than them in the blank control group and the LPS group(P<0.01,P<0.05).The expression of pSTAT6 of dendritic cells in LPS group(L group)was less than the expression in the blank control group(p<0.01).The expression of pSTAT6 of dendritic cells in the Ephedra asarum aconite decoction group(M group)was more than it in the LPS group(P<0.01),and making no difference comparing the blank control group(C group).The ratio of pSTAT6/STAT6 of dendritic cells in LPS group(L group)was less than the ratio in the blank control group(C group,p<0.05).The ratio of pSTAT6/STAT6 of dendritic cells in the Ephedra asarum aconite decoction group(M group)was higher than it both in the blank control group and the LPS group(P<0.05,P<0.01).There were no difference of the expression of STAT5 of dendritic cells among the blank control group(C group),the LPS group(L group)and the Ephedra asarum aconite decoction group(M group).The expression of pSTAT5 of dendritic cells in LPS group(L group)was less than the expression in the blank control group(p<0.01).The expression of pSTAT5 of dendritic cells in the Ephedra asarum aconite decoction group(M group)was more than it in the blank control group(C group,P<0.01),and making no difference comparing the LPS group(L group).The ratio of pSTAT5/STAT5 of dendritic cells in LPS group(L group)was less than the ratio in the blank control group(C group,p<0.05).The ratio of pSTAT5/STAT5 of dendritic cells in the Ephedra asarum aconite decoction group(M group)was lower than it in the blank control group(C group,P<0.05),and making no difference comparing the LPS group(L group).9,The expression of T-bet mRNA of dendritic cells in the LPS group(L group)was more than it in the blank control group(C group,P<0.05).The expression of T-bet mRNA of dendritic cells in the Ephedra asarum aconite decoction group(M group)was both less than it in the blank control group and the the LPS group(p<0.01).There was no difference of the expression GATA-3 mRNA of dendritic cells between the blank control group(C group)and the LPS group(L group).The expression of GARA-3 mRNA of dendritic cells in the Ephedra asarum aconite decoction group(M group)was both more than it in the blank control group and the the LPS group(p<0.01,p<0.05).Conclusions1,In this study,we successfully established primary cultures methods of dendritic cells and T cells.2,The toxicity of the clinical common dose of Ephedra asarum aconite decoction on dendritic cells and T cells is very low.3,Ephedra asarum aconite decoction can promote the proliferation of activated T cells in vitro and reduce the secretion of IL-4.It is vital that the decoction can play a role in allergic rhinitis for promoting the body’s immune function by regulating the differentiation of Th1/Th2.4,Ephedra asarum aconite decoction can inhibit the expression of mature molecules on dendritic cells after LPS stimulation,which inhibits antigen presentation of dendritic cells and can play an anti-inflammatory role.5,The effect of the maturation of dendritic cells stimulated by TSLP was not obvious,and the Ephedra asarum aconite decoction had no significant effect on the state of dendritic cells after TSLP stimulation.6,Ephedra asarum aconite decoction can reduce the secretion about inflammatory factors of IL-12,IFN-gamma,IL-6,IL-1β of dendritic cells after LPS stimulation,which making the Ephedra asarum aconite decoction has obvious on effect anti-inflammatory.7,Ephedra asarum aconite decoction can not reduce the secretion about inflammatory factors of IL-5 and IL-13 of T cells interacting with dendritic cells after TSLP stimulation,which making the Ephedra asarum aconite decoction has no obvious on effect Th2 type inflammation induced by TSLP-dendritic cells.8,Ephedra asarum aconite decoction can increase the expression of STAT6 of dendritic cells after LPS stimulation,and has the potential to regulate the Thl imbalance.However,the STAT5 of dendritic cells was not the target of regulation of type Thl inflammation,since Ephedra asarum aconite decoction could not reverse the decrease of pSTAT5 after LPS stimulation.9,Ephedra asarum aconite decoction can inhibit the expression of T-bet mRNA in LPS stimulated dendritic cells,and further regulate the skewing of Thl cells by STAT6-GATA-3 pathway.