| 1.Background and objectiveInflammatory bowel disease(IBD),comprising ulcerative colitis(UC)and Crohn's disease(CD),is an idiopathic inflammatory condition characterized by an autoimmune disorder.Since IBD is a refractory disease with high morbidity,new alternative medical strategies for IBD are still desperately needed.Dihydroartemisinin(DHA)was extracted from Artemisia annua L.by Chinese scientists in 1970s.Besides anti-malaria,DHA was also found to posses prominent immunological modulation activity and ameliorate autoimmune disease,like lupus erythematosus,rheumatoid arthritis and multiple sclerosis.However,the effect and mechanism of DHA on IBD are still at the early stage.Among chemically induced IBD mice models,oxazolone and 2,4,6-trinitro-benzene sulfonic acid induced mice model resemble human UC and CD with histological features and could be used for Th9 and Thl/Th17 study separately.The aim of this study was to explore the potential effect of DHA for treating OXA-and TNBS-induced IBD mice model,so as to lay the foundation for further research on its exploration of new therapeutic applications.2.Research methods and contents2.1 Efficacy study of DHA in OXA-and TNBS-induced IBD mice modelThe model mice received a single intracolonic application of OXA or TNBS after presensitization.At 8,24,and 48 h after infusion,the mice were treated with 4,8,or 16 mg/kg DHA or 1 mg/kg dexamethasone(DXM)per day intraperitoneally a total of three times.In the NC and model groups,the mice were administered the same volume of solvent.The development and progress of disease were assessed at Oh,8h,24h,48h and 72h by measuring performance status,body weight index,diarrhea and bloody stool,mortality and DAI score.All mice were sacrificed by cervical dislocation at 72 h after infusion.Colon or intetine tissue were removed and cleaned rapidly,measured weight and length,fixed with 4%paraformaldehyde and embedded in paraffin,then analyzed macroscopically and histologically.Tissue sections(3-?m-thick)were stained with H&E and Sirius-Red,and the degree of inflammation was observed under a microscope and scored.The percentages of the positive collagen fiber areas in the colon or intestine were calculated using Image-pro plus 6.0.2.2 Efficacy study of DHA for regulating CD4+T subsets in IBD mice modelFor OXA mice model,the mRNA level of Th9 related transcription factor PU.1 and cytokine IL9,Th22 related transcription factor AHR and cytokine IL22,Treg related transcription factor Foxp3 and cytokine IL10 in colon tissue of OXA induced colitis were measured by Real-Time PCR;the ratio of Th9,Th22 and Treg cells in spleen of OXA induced colitis were measured by flow cytometry;the protein level of PU.1,AHR,Foxp3 in colon lamina propria lymphocyte of OXA induced colitis were measured by capillary electrophoresis Western blotting.For TNBS mice model,the mRNA level of Thl related transcription factor T-bet and cytokine IFNy,Th17 related transcription factor RORyt,Treg related transcription factor Foxp3 and cytokine IL10 in intestine tissue of TNBS induced colitis in mice were measured by Real-Time PCR;the ratio of Thl,Th17 and Treg cells in spleen of TNBS induced colitis were measured by flow cytometry;T-bet,RORyt,Foxp3 in intestine lamina propria lymphocyte of TNBS induced colitis were measured by capillary electrophoresis Western blotting.2.3 Efficacy study of DHA in the inhibition of activated CD4+T lymphocyte based on heme oxygease-1 inductionCD4+T cells purified by MACS beads were stimulated with plate-bound anti-CD3e and anti-CD28.Then cell survival rate was calculated after DMSO,DHA 0.lmg/ml,0.2mg/ml,0.4mg/ml,0.8mg/ml and 1.6mg/ml cultured for 72 h.CD4+T cells purified by MACS beads were stimulated with plate-bound anti-CD3e and anti-CD28.Then cell survival rate was calculated after DMSO,DHA 0.8mg/ml,SnPP(HO-1 inhibitor)30nmol/ml and DHA+SnPP cultured for 72 h.Cells were stained with Annexin V-FITC and propidium iodide(PI),then analyzed by flow cytometry to calculate apoptotic rate.The concentrations of HO-1 in the cell culture media were measured by ELISA.For OXA mice model,colon lamina propria lymphocyte was extracted and calculated,ratio of apoptotic cell were measured by TUNEL staining of colon tissue.For TNBS mice model,intestine lamina propria lymphocyte was extracted and calculated,ratio of apoptotic cell were measured by TUNEL staining of intestine tissue.2.4 Efficacy study of DHA in OXA-and TNBS-incuced IBD mice model based on heme oxygenase-1 inductionFor OXA and TNBS mice model,mice were divided randomly into NC?Model?DHA16mg/kg/d?SnPP30umol/kg/d?DHA+SnPP groups.The mice were treated with DHA at 8h,24h and 48h after infusion intraperitoneally a total of three times.The mice were treated with SnPP at Oh,24h and 48h after infusion subcutaneously into the posterior neck tissue a total of three times.In the NC and Model groups,the mice were treated with the same volume of solvent.The development and progress of disease were assessed at Oh,8h,24h,48h and 72h by measuring performance status,body weight index,mortality and DAI score.All mice were sacrificed by cervical dislocation at 72 h after infusion.Colon or intestine tissue were removed and cleaned rapidly,fixed with 4%paraformaldehyde and embedded in paraffin,then analyzed macroscopically and histologically.Tissue sections(3-?m-thick)were stained with H&E and Sirius-Red,and the degree of inflammation was observed under a microscope and scored.The percentages of the positive collagen fiber areas in the colon or intestine were calculated using Image-pro plus 6.0.2.5 Efficacy study of DHA for regulating CD4+T subsets in IBD mice model based on heme oxygenase-1For OXA mice model,the ratio of Th9,Th22 and Treg in mice spleen were measured by flow cytometry;the protein level of Th9 related transcription factor PU.1,Th22 related transcription factor AHR,Treg related transcription factor Foxp3 in colon lamina propria lymphocyte were measured by capillary electrophoresis Western blotting.For TNBS mice model,the ratio of Thl,Th17 and Treg in mice spleen were measured by flow cytometry;the protein level of Thl related transcription factor T-bet,Thl7 related transcription factor RORyt,Treg related transcription factor Foxp3 in intestine lamina propria lymphocyte were measured by capillary electrophoresis Western blotting.2.6 Statistical analysisGraphpad Prism 7 software was used to analyze the data,all values are expressed as means ± standard errors of the means.Data were compared among groups using one-way ANOVA,repeated measured parameters in different time point were analyzed by repeated measures analysis of variance.Values of P<0.05 were considered statistically significant.3.Research results3.1 Efficacy study of DHA in OXA-and TNBS-induced IBD mice model1.Model verification:After intracolonic application of OXA or TNBS,model mice showed poor appetite,little drinking-water,lack of movement,slow response,diarrhea,loose bloody stools,decreasing weight.The colon or intestine mucosa appeared ulcerated,edematous,hyperemic and hemorrhagic.The histological slides showed tissue structure destruction,accompanied by increased lymphocytes infiltration and positive collagen fiber areas.2.For OXA mice model,in comparison with NC group,OXA model group showed significantly decreased weight index,increased DAI score,shortened and lightened colon;colon tissue H&E staining sections showed significantly increased lymphocytes infiltration;colon tissue Sirius-Red staining sections showed significantly increased positive collagen fiber areas.In comparison with OXA model group,mice in DHA groups and DXM group showed improved performance status and other symptom parameters,less mortality,increased weight index and decreased DAI score;colon tissue H&E staining sections showed less lymphocytes infiltration;colon tissue Sirius-Red staining sections showed less positive collagen fiber areas.3.For TNBS mice model,in comparison with NC group,TNBS model group showed significantly decreased weight index,increased DAI score,and a decreased trend of intestine weight;intestine tissue H&E staining sections showed significantly increased lymphocytes infiltration;intestine tissue Sirius-Red staining sections showed significantly increased positive collagen fiber areas.In comparison with TNBS model group,mice in DHA groups and DXM group showed improved performance status and other symptom parameters,less mortality,increased weight index and decreased DAI score;intestine tissue H&E staining sections showed less lymphocytes infiltration;intestine tissue Sirius-Red staining sections showed less positive collagen fiber areas.3.2 Efficacy study of DHA for regulating CD4+T subsets in IBD mice model1.For OXA model,quantitative RT-PCR results showed that the mRNA expression levels of the transcription factor PU.1 and cytokine IL9,IL22 were significantly increased,the transcription factor Foxp3 was significantly decreased in colon tissue of mice in OXA model group compared with NC group,there was a rising trend of AHR expression in OXA model mice despite no statistical difference,quantitative RT-PCR results also showed that DHA reduced the mRNA expression levels of PU.1,AHR,IL9 and IL22,and increased the level of Foxp3 and IL10;flow cytometry results showed that the ratio of Treg significantly decreased and Th9,Th22 significantly increased in mice spleen of OXA model groups compared with NC group,DHA decreased the ratio of Th9 and Th22,and increased the ratio of Treg cells;capillary electrophoresis Western blotting results showed that the protein expression level of PU.1 significantly increased,Foxp3 significantly decreased in colon lamina propria lymphocytes of OXA model mice compared with NC group,there was a rising trend of AHR expression in OXA model mice despite no statistical difference,DHA decreased the protein expression levels of PU.1 and AHR,increased Foxp3 in the colon lamina propria lymphocytes.2.For TNBS model,quantitative RT-PCR results showed that the mRNA expression levels of the transcription factor RORyt was significantly increased in intestine tissue of mice in TNBS model group compared with mice in NC group,there was a rising trend of T-bet and IFNy,a decreasing trend of Foxp3 expression in TNBS model mice despite no statistical difference,quantitative RT-PCR results also showed that DHA reduced the mRNA expression levels of T-bet?RORyt and IFN?,and increased the level of Foxp3 in the TNBS colitis model mice;flow cytometry results showed that the ratio of Treg significantly decreased and Th9,Th22 significantly increased in mice spleen of TNBS model groups compared with NC group,DHA decreased the ratio of Th1 and Th17,and increased the ratio of Treg cells;capillary electrophoresis Western blotting results showed that the protein expression level of T-bet and RORyt significantly increased,Foxp3 significantly decreased in intestine lamina propria lymphocytes of TNBS model mice compared with NC group,DHA decreased the protein expression levels of T-bet and RORyt,increased Foxp3 in the intestine lamina propria lymphocytes of the TNBS colitis.3.3 Efficacy study of DHA in the inhibition of activated CD4+T lymphocyte based on heme oxygease-1 induction1.DHA(0.1-1.6 mg/ml)markedly reduced the numbers of surviving CD4+T cells with anti CD3/CD28 stimulation.Notably,the suppression effect of DHA(0.8 mg/ml)was partially reversed by treatment with the HO-1 inhibitor SnPP(30 nM).Consistently,ELISA results showed that the HO-1 protein level in the cell culture supernatant was increased and decreased by DHA and SnPP respectively,and the HO-1 upregulation could be significantly neutralized by SnPP.DHA induced the apoptosis of activated CD4+T cells according to the Annexin V/PI staining assay,which was also partly reversed by SnPP.2.For OXA model,DHA significantly decreased the number of colon lamina propria lymphocytes,and TUNEL staining results showed that DHA increased the ratio of apoptotic cells in colon tissue of OXA model mice,SnPP significantly neutralized the effect of DHA.3.For TNBS model,DHA significantly decreased the number of intestine lamina propria lymphocytes,and TUNEL staining results showed that DHA increased the ratio of apoptotic cells in intestine tissue of TNBS model mice,SnPP significantly neutralized the effect of DHA.3.4 Efficacy study of DHA in OXA-and TNBS-incuced IBD mice model based on heme oxygenase-1 induction1.For OXA model,mice in SnPP+DHA group showed greater weight loss,more serious symptoms,and higher mortality than those in DHA group;H&E staining of histopathological sections of the colon showed greater lymphocyte infiltration in the SnPP+DHA group than DHA treated group;in addition,the collagen fibrosis area in the colon was larger in the SnPP+DHA group according to Sirius-Red staining.2.For TNBS model,mice in SnPP+DHA group showed greater weight loss,more serious symptoms,and higher mortality than those in DHA group;H&E staining of histopathological sections of the intestine showed greater lymphocyte infiltration in the SnPP+DHA group than DHA treated group;in addition,the collagen fibrosis area in the intestine was larger in the SnPP+DHA group according to Sirius-Red staining.3.5 Efficacy study of DHA for regulating CD4+T subsets in IBD mice model based on heme oxygenase-11.For OXA model,according to flow cytometry results,there was a greater proportion of Th9 cells and lower proportion of Treg cells in the SnPP+DHA group compared to those of the DHA-treated-group,although there was a trend of an increase in Th22 cells in the SnPP+DHA group,the difference was not statistically significant;similarly,the DHA-induced decrease in the PU.1 and AHR protein expression in LPMCs of OXA colitis mice was weakened,when HO-1 was inhibited,the up-regulated trend of Foxp3 expression in the DHA group was also diminished upon treatment with SnPP.2.For TNBS model,according to flow cytometry results,there was a greater proportion of Thl and Th17 cells and lower proportion of Treg cells in the SnPP+DHA group compared to those of the DHA-treated-group;similarly,the DHA-induced decrease in the T-bet and RORyt protein expression in LPMCs of TNBS colitis mice was weakened,when HO-1 was inhibited,the up-regulated trend of Foxp3 expression in the DHA group was also diminished upon treatment with SnPP.Conclusion1.DHA ameliorated OXA-and TNBS-induced colitis in a dose-dependent manner in vivo;2.DHA regulated CD4+T subsets in OXA-and TNBS-induced colitis;3.DHA suppressed activated CD4+T cell subsets through inducing apoptosis based on HO-1 induction;4.DHA ameliorated OXA-and TNBS-induced colitis based on HO-1 induction;5.DHA regulated the Th/Treg balance in OXA-and TNBS-induced colitis based on HO-1 induction. |
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