| Objective:To observe the effect of traditional Chinese medicine Xingbi Gel on the expression of the Fyn-STAT5 pathway in nasal mucosa fibroblasts of allergic rhinitis(AR)guinea pigs,in order to search for the therapeutic targets of Xingbi Gel on AR,further illuminate its therapeutic mechanism,and provide experimental basis for improving clinical efficacy on AR.Methods:The guinea pigs were modeled to AR by ovalbumin sensitization,then isolated and cultured the nasal mucosa fibroblasts of AR guinea pigs in vitro,identified the fibroblasts by vimentin immunocytochemical staining.The nasal mucosa fibroblasts were intervened with different concentration of transforming growth factor-beta 1(TGF-?1)for 6h,12h and 24h,and methyl thiazolyl tetrazolium(MTT)assay was examined to estimate cell proliferation after TGF-?1 intervention,so as to select the optimal condition of TGF-?1 intervention in the fibroblasts.Guinea pigs nasal mucosa fibroblasts were randomly divided into normal group(A group),model group(B group),TGF-?1 intervention group(C group),Xingbi Gel intervention group(D group)and Rhinocort intervention group(E group),the C group,D group and E group were intervened with TGF-?1,Xingbi extract and Rhinocort respectively.The mRNA levels of protein tyrosine kinase Fyn,interleukin-10(IL-10)and stem cell factor(SCF)in fibroblasts were measured by real-time PCR,while the protein levels of the Fyn-STAT5 pathway in fibroblasts were detected by Western blot.Results:1.The immunocytochemical staining indicated that the vimentin was positive in F3 generation guinea pigs nasal mucosa fibroblasts,and its growth curve appeared as a typical"S-shaped curve".2.MTT assay showed that the cell viability was increased most obviously when intervened with TGF-?1 25ng/ml,which was increased to 129.356 ± 4.354%after intervened for 6h,increased to 134.810 ± 8.047%after intervened for 12h,and increased to]43.824 ±4.773%after intervened for 24h(compared with intervened with 25ng/ml for 24h,P<0.01 for 6h,P<0.05 for 12h).3.Real-time PCR suggested that,?the mRNA levels of Fyn and SCF in B group were significantly higher than A group(P<0.01),while the IL-10 in B group was significantly lower than A group(P<0.01);?the mRNA levels of Fyn and SCF in C group,D group and E group were remarkably decreased,while the IL-10 in that groups were remarkably increased after intervened for 12h and 24h respectively(compared with B group at the same intervention time,all groups P<0.01);the mRNA levels of Fyn and SCF in C group,D group and E group,as well as the IL-10 in C group were not significantly different from B group after intervened for 48h(compared with B group at the same intervention time,these groups P>0.05),while the the mRNA levels of IL-10 in D group and E group were remarkably increased(compared with B group,P<0.01);?the mRNA levels of Fyn and SCF in D group were higher than C group after intervened for 12h(P<0.05),were not significantly different from C group after intervened for 24h(P>0.05),and after intervened for 48h,the Fyn in D group was lower than C group(P<0.05),the SCF in D group was not significantly different from C group(P>0.05);the mRNA levels of IL-10 in D group were remarkably higher than C group after intervened for 12h and 24h(P<0.01),but remarkably lower than C group after intervened for 48h(P<0.01);? compared at the same intervention time,the mRNA of all genes in D group were no significant difference from E group(P>0.05).4.Western blot indicated that,?the protein levels of Fyn in B group were significantly higher than A group(P<0.01),while the signal transduction and activator of transcription 5(STAT5)in B group was significantly lower than A group(P<0.01);?the protein levels of Fyn in C group,D group and E group were remarkably decreased,while the STAT5 in these groups were remarkably increased after intervened for 12h and 24h respectively(compared with B group at the same intervention time,both Fyn and STAT5 in C group P<0.01,in D group and E group P<0.05 for 12h,and P<0.01 for 24h);the protein levels of Fyn and STAT5 in C group,D group and E group were not significantly different from B group after intervened for 48h(compared with B group,these groups P>0.05);?the protein levels of Fyn in D group were not significantly different from C group after intervened for 12h,24h and 48h(P>0.05),while the STAT5 in D group were lower than C group after intervened for 12h(P<0.05),but not significantly different from C group after intervened for 24h and 48h(P>0.05);?compared at the same intervention time,both of the two protein in D group were no significant difference from E group(P>0.05).Conclusions:1.The cells isolated and cultured had typical biological characteristics of fibroblasts.2.Fyn,STAT5 and SCF were associated with the development of AR.Fyn is involved in the activation of mast cells by activating Fc?RI,and SCF makes STAT5 phosphorylate by mediating the activation of JAK2.On the one hand,the activated STAT5 can combine Fyn into an immune complex to activate the Fyn-STAT5 pathway,then promotes the degranulation of mast cells and participates in the immune response of AR.On the other hand,its own transcription factors may inhibit B cell renewal to prevent IgE from continuing to synthesize.IL-10 has a negative regulatory effect in the immune response of AR.3.TGF-?1 can intervene in AR fibroblasts by inhibiting the activation of Fyn-STAT5 pathway,then inhibit the immune response of AR in vitro.4.Xingbi Gel may reduce the expression of Fyn and SCF,prevent Fc?RI receptor cross-linking,inhibit the proliferation and degranulation of mast cells,reduce the release of histamine and various inflammatory cytokines,and up-regulate STAT5 to antagonize IgE synthesizing,correct the immune imbalance of TH1/TH2,thus inhibiting the Fyn-STAT5 pathway,and reducing the immune response of AR fibroblasts.Furthermore,Xingbi Gel can significantly increase IL-10,inhibit the production of various pro-inflammatory factors,so as to relieve airway inflammation and regulate immune function of organism. |
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