| Inflammatory bowel disease(IBD),which consists of Crohn's disease(CD)and ulcerative colitis(UC),is a group of recurrent,chronic inflammatory conditions of the gastrointestinal tract.The incidence of IBD has increased yearly all over the world.The etiology and pathogenesis of IBD are still not fully clear.However,it is generally accepted that environmental factors,genetic susceptibility,gut microflora and immune responses contribute to the disease.Increased lymphocyte infiltration in inflammatory intestine tissue is an important pathologic feature of IBD.The interplay between effector T cells(Teff)and regulatory T cells(Treg)is crucial for maintaining the balance of adaptive immunity.Wogonin as a natural flavonoid from Scutellaria baicalensis Georgi(Lamiaceae)exhibits anti-tumour and anti-inflammatory activities.In this study,the effects of wogonin on intestinal inflammation were observed based on the mouse model of colitis induced by Dextran sulfate sodium salt(DSS).Then the effects of wogonin on the function of Teff/Treg cells were analyzed both in vivo and ex vivo.Finally,this study aimed to further explore the molecular mechanisms of wogonin on activity of Teff/Treg cells.Our study was divided into two parts.Part I.The effects of wogonin on DSS-induced murine colitis and the function of Teff/Treg cellObjective:To observe the effects of wogonin on the incidence of colitis induced by DSS in mice.To analyze the function of Teff/Treg cells in spleen and intestine of DSS-induced mice,and to analyze the effects of wogonin on Teff/Treg cells directly ex vivo.Methods:The mouse model of colitis was induced by DSS(2.5%)solution to observe the effects of wogonin in vivo.Different doses of wogonin or solvent were intraperitoneally injected daily,then observed the body weight,bloody stool,diarrhea of mice to calculate the disease activity index(DAI).The frequency of CD4+CD25+Foxp3+,CD4+CD25+CD127-T cells in spleen and the frequency and absolute number of CD4+CD25+CD127-T cells in intestine from DSS-induced mice were detected by flow cytometry.C57BL/6 mice were intraperitoneally injected with different doses of wogonin to observe the effects of wogonin on normal mice.Splenic CD4+ and CD8+ T cells were isolated by flow cytometry and cultured with wogonin and IL-2,and the expression of IFN-y mRNA in CD4+ and CD8+ T cells was detected by qRT-PCR.Splenic CD8+ T cells cultured with wogonin and IL-2 were mixed with murine colon cancer cell(MC-38)at indicated ratios.Cytotoxicity of CD8+ T cells against target cells was measured by an LDH releasing assay.Splenic CD4+CD25-T cells were obtained from a double-negative magnetic cell sorting,and then cultured in anti-CD3 antibody precoated wells and stimulated with medium containing anti-CD28 antibody,IL-2 and TGF-?.Different concentrations of wogonin were added into CD4+CD25+ T cells induced by TGF-? in vitro,and then the frequency of CD4+CD25+Foxp3+ T cells were detected by flow cytometry.Results:Intraperitoneal injection of wogonin exacerbated DSS-induced colitis in mice.Treatment with 50 or 100 mg/kg wogonin accelerated the loss of body weight,and intestinal stenosis,intestinal shortening as well as intestinal necrosis were more serious,particularly at 100 mg/kg.However,intraperitoneal injection of wogonin had no significant effects on C57BL/6 normal mice.The frequencies of CD4+CD25+Foxp3+ and CD4+CD25+CD127-T cells in spleen from mice with DSS-induced colitis were significantly down-regulated by wogonin(100 mg/kg)treatment,compared with those in the solvent and DSS/solvent groups.Similarly,the absolute number and frequency of CD4+CD25+CD127-T cells in total CD4+ T cells in intestine were also significantly decreased co-treated by DSS and wogonin(100 mg/kg),contrary to those of treatments of DSS/solvent or DSS/wogonin(50 mg/kg).Compared with the solvent,wogonin treatments could significantly enhance the cytotoxicity of CD8+ T cells.The expression of IFN-y mRNA in CD4+ and CD8+ T cells was significantly increased treated by wogonin in vitro.The frequency of CD4+CD25+Foxp3+ T cells was significantly suppressed by treatments of wogonin.Thus,Wogonin inhibited the induction of Tregs dose-dependently in vitro.Conclusions:Treatment with 50 or 100 mg/kg wogonin exacerbated DSS-induced colitis in mice by enhancing the function of Teff cells and inhibiting the function of Treg cells,particularly at 100 mg/kg,which proved the immune-enhancing activity of wogonin indirectly.Part ?.The molecular mechanisms of wogonin in modulating the function of Teff/Treg cellsObjective:To explore the the molecular mechanisms of wogonin in modulating the function of Teff/Treg cells.Methods:Splenic CD4+ T cells were isolated by flow cytometry and cultured with wogoninand IL-2.Splenic CD4+CD25-T cells were obtained from a double-negative magnetic cell sorting,and then cultured in anti-CD3 antibody precoated wells and stimulated with medium containing anti-CD28 antibody,IL-2 and TGF-?.Different concentrations of wogonin were added into CD4+CD25+ T cells induced by TGF-? in vitro.All cells were harvested and gently lysed in ice-cold lysis buffer respectively.Western blot was employed to analyze the expression levels of STAT3 and phosphorylated STAT3(Tyr705,Ser727),NF-?B p65 and phosphorylated NF-?B p65,Erk and phosphorylated Erk.Results:NF-?B and Erk signaling pathway were activated in Splenic CD4+ T cells.Phosphorylated NF-?B p65 was enhanced with the stimulation by wogonin treatment compared with medium group.Similarly,the expression of phosphoryl p44/42(Erk1/2)was significantly up-regulated activated with the stimulation by wogonin treatment compared with medium group,especially in 50 pg/ml wogonin group.The total STAT3 and phosphorylated STAT3-Y705 were decreased by wogonin(100 ?g/ml)treatment,contrary to those of treatments medium or wogonin(50 ?g/ml).Wogonin down-regulated the expression of phosphorylated STAT3-S727 in a dose-dependent manner.The effects of wogonin on the activation of NF-?B,Erk and STAT3 of Treg cells which were induced by IL-2,CD28 and TGF-P were examined.The phosphorylated NF-kB p65 was up-regulated by wogonin treatment at both dosages.Wogonin(100 ?g/ml)inhibited p44/42(Erkl/2)phosphorylation while wogonin at 50 ?g/ml had no significant effects on the expression of phosphorylated p44/42(Erkl/2).The total STAT3 were decreased by wogonin(100 ?g/ml)treatment.Wogonin also significantly down-regulated the phosphorylation of STAT3 on Tyr705 dose-dependently,but promoted the phosphorylation of STAT3 on Ser727 of Treg cells.Conclusions:Wogonin may promote the activation of CD4+ T cells by activating NF-?B and Erk signaling pathway,but down-regulating STAT3 phosphorylation.Wogonin inhibited the function of Treg cells by down-regulating the expression of p-Erk,p-STAT3(Y705)but up-regulating the expression of p-NF-?B p65,p-STAT3(S727). |
PDF Full Text Request