| Background and objectives:Autoimmune diseases?ADs?are characterized by the abnormal response of immune system on normal body parts.The etiology of ADs is remaining elusive.The pathogenesis of ADs in Traditional Chinese medicine theory is attributed to“zheng xu Xie sheng”,while Contemporary Medicine consider ADs are caused by the interaction between genetic and environmental factors,and gut dysbiosis is currently considered the most important environment factor due to its critical role in initiating excesses immune response as well as autoimmune response.Herein,in combination with the theory of Traditional Chinese medicine and Contemporary Medicine,a novel scientific hypothesis that“gut dysbiosis causes the‘ying wei shi tiao'and‘zheng xu and xie sheng'contributes to the pathogenesis of ADs”was firstly put forward in this thesis.Based on this hypothesis,targeting regulate the intestinal microecosystem is a reliable treatment strategy for ADs in line with both the theory of Western medicine and Traditional Chinese medicine.Inulin is a fructan composed by fructose with?-2,1linkage and has been reported can benefits intestinal microecological.Therefore,we proposed a scientific hypothesis that“inulin can benefits ADs through modulating microecology”,and takes the rheumatoid arthritis mouse model?CIA?and multiple sclerosis mouse model?EAE?as research objects to clarify the therapeutic effect of inulin and to verify the role played by microecosystem.In order to lay a foundation for the clinical practice of inulin for ADs,and provide a basis for the ADs drug development targeting intestinal microecology,as well as enrich the scientific connotation of TCM theory of ADs,and provide clues for the biological mechanism of how hard-to-absorb polysaccharides exert their pharmacological effects in vivo.Methods:1.The extraction,separation,purification and structure characterization of inulin from three herbs:Crude polysaccharides from three kinds of Chinese herbal medicines?Artemisia japonica,Aucklandia lappa,Codonopsis pilosula?were obtained by water extraction and alcohol precipitation;neutral polysaccharides were separated by DEAE-Sepharose Fast Flow ion exchange chromatography;Superdex-75gel chromatography was used to purify inulin;HPGPC was used to determine molecular weight and homogeneity;GC-MS was used to detect monosaccharide composition;IR was used to detect the characteristic peaks of polysaccharides;methylation analysis was used to determine the linkage types of polysaccharides;NMR was used to elucidate structural characteristics.2.The in vitro immunological activity of inulin with different molecular weight:LPS stimulates macrophage inflammation,CCK-8 detects cytotoxicity of inulin,Griess detects NO secretion,ELISA detects inflammatory factor secretion,Con A induces lymphocyte proliferation,CCK-8 detects cell proliferation,Elisa detects inflammatory factor secretion.3.The treatment of inulin on EAE mice through modulating the intestinal microecosystem:EAE was induced by immunization of MOG35-55,CFA and PTX.To confirm the efficacy of inulin on EAE and its protection effect on the pathological changes of the spinal cord,inulin was oral administrated in EAE mice and the clinical scores were assessed and recorded daily,H&E and LFB staining were used to evaluate the inflammatory demyelination lesions in the spinal cord,immunohistochemistry was used to detect the infiltration of immune cells in the spinal cord,RT-PCR was used to detect the expression of inflammatory and chemokine genes in the spinal cord.To clarify the modulation of inulin on intestinal microecology,16Sr DNA sequencing was used to detect the changes of gut microbiota,gas chromatography was used to detect the short-chain fatty acids in feces.To clarify the regulation of inulin on intestinal barrier and immune response,HE staining and immunohistochemistry were used to observe the changes of colonic tissue morphology and tight junction,flow cytometry was used to analyze the proportion of CD103+CD11c+DC cells,M1 and M2 macrophages and Ig A+B220+B cells in MLN,RT-PCR was used to detect the expression of inflammatory genes in colonic tissue.To clarify the influence of inulin on systemic immune response,Elisa assay was used to detect the levels of cytokines in serum and cell supernatant,flow cytometry was used to analyze the changes of immune cell subsets in spleen,3H-Tdr incorporation assay was used to evaluate the lymphocyte auto-reactive proliferation,RT-PCR was used to determine the gene expression of transcription factors and cytokines.To clarify the causal relationship between intestinal microecology and the efficacy of inulin,intraperitoneal injection of inulin on EAE mice was used to determine the efficacy through parenteral routes,fecal microbiota transplantation was used to determine the efficacy of fecal from inulin-treated normal mice on EAE mice,and high-fat diet interference was used to evaluated the efficacy of inulin on EAE mice when the benefits effect on microecosystem was blocked by HFD.4.The treatment of inulin on CIA mice through modulating the intestinal microecosystem:CIA was induced by bovine type?collagen and immunologic adjuvant.To confirm the efficacy of inulin on CIA mice and its protection effect on the pathological changes of joints,inulin was oral administrated in CIA mice and the clinical scores were assessed and recorded daily,H&E and toluidine blue staining were used to evaluate the inflammatory cells infiltration and cartilage damage in the joints.To clarify the modulation of inulin on intestinal microecology,16Sr DNA sequencing was used to investigate the changes of gut microbiota.To clarify the regulation of inulin on intestinal barrier and inflammatory response,HE staining and immunohistochemistry were used to observe the changes of colonic tissue morphology and tight junction,RT-PCR was used to detect the expression of inflammatory genes in colonic tissue.To clarify the influence of inulin on systemic immune response,Elisa assay was used to detect the levels of cytokines in serum and cell supernatant,flow cytometry was used to analyze the changes of immune cell subsets in spleen,RT-PCR was used to determine the gene expression of transcription factors and cytokines.To clarify the causal relationship between intestinal microecology and the efficacy of inulin,fecal microbiota transplantation was used.Results:1.Three inulin with different molecular weihght were obtained from Artemisia Japonica,Aucklandia lappa,and Codonopsis pilosula:The yield of crude polysaccharides of Artemisia Japonica was 3.86%,a neutral polysaccharide?MWP?was isolated and purified by using DEAE-Sepharose Fast Flow column and superdex-75 column.MWP has a molecular weight of 3.1 k Da as calculated by High Performance Gel Permeation Chromatography?HPGPC?and is comprised of fructose and glucose analyzed by GC/MS,IR result showed that MWP had characteristic absorption peaks of polysaccharides and fructose-beta-glycoside bonds,methylation analysis indicated the linkage type was 1,2-linkage,NMR results showed the H-1 of glucose and fructose both correspond with the C-2 of fructose,these results indicated MWP is a typical inulin-type fructan.The yield of crude polysaccharides of Aucklandia lappa and Codonopsis pilosula were 16.8%and 4.1%,respectively.Two homogeneous polysaccahrides were isolated and purified from and named as AWP and CWP,respectively,HPGPC results indicated the molecular weight of AWP and CWP were 4.8KDa and 2.2KDa,NMR results indicated both there two polysaccharides are inulin type fructan.2.Three inulin have no immunosupressees activity:AWP?CWP and MWP have no cytotoxicity on RAW264.7 cells,and have no inhibition effect on NO secretion of RAW264.7 with LPS stimulation;AWP and CWP but not MWP promoted the proliferation of lymphacytes,indicated AWP and CWP have immune enhancement effect.3.Inulin colud benefits EAE mice through modulating intestinal microecosystem:EAE was successfully induced on C57BL/6 mice,inulin with different molecular weights have therapeutic effect on EAE,and the efficacy between three inulin has no significant difference,but MWP is a slightly better that CWP and AWP.The follow-up results showed that the efficacy of inulin?MWP?had no obvious dose dependence,and 200 mg/kg showed better efficacy,this dose also reduce the incidence of EAE,ameliorate myelitis demyelinating lesions,and reduce the infiltration of immune cells and the expression of inflammatory genes in spinal cords;Compared with normal mice,the intestinal microecological of EAE mice is unbalanced,and the intestinal morphology had changed in company with colon inflammation,which indicated that there were pathogenic manifestations of Yingwei imbalance in EAE mice.However,Inulin treatment modulated the intestinal microecology which include regulated the proportion of Bacteroidetes and Firmicutes and decreased Odoribacter,as well as increased the concentrations of SCFAs;in addition,Inulin protected the intestinal mucosal barrier?alleviate the pathological changes of colon?and induce intestinal immune tolerance?increased the proportion of CD11C+CD103+DC cells and M2macrophages,reduced the M1 macrophage?,and reduced the expression of inflammatory genes;inulin regulated the systemic immune response by lowering the level of serum inflammatory factors,decreasing lymphocyte autoreactive proliferation,reducing the proportion of antigen presenting cells,and regulating the balance between Th17 cells and Treg cells?reduced gene expression and levels in cell supernatant of IL-17,while promoted gene expression and levels in cell supernatant of IL-10?;Furthermore,there is a causal relationship between the regulation of inulin on intestinal microecology and its therapeutic effect on EAE mice,firstly,intraperitoneal injection of inulin has no beneficial effect on EAE development,secondly,fecal microbiota transplantation from inulin-treated normal mice to EAE mice alleviated the severity,thirdly,the treatment effects of inulin on EAE mice is counteracted after feed by high-fat diet.Finally,SCFAs played an important role in the efficacy of inulin,acetate,propionate and butyrate exhibited immunoregulatory activity in vitro,and butyrate showed best inhibition effect on LPS stimulated RAW264.7 cells,in addition,butyrate could inhibit lymphocyte proliferation induced by Con A dose-dependent,furthermore,the lymphocyte auto-reactive proliferation assay indicated that butyrate could significantly inhibit the proliferation induced by MOG35-55,but inulin had no effect,FACS analysis showed that butyrate increased the proportion of Treg cells and inhibited the proportion of Th17 cells in lymphocytes.Interestingly,the immunoregulatory activity of butyrate depended on its histone deacetylation inhibitory activity,when histone acetyltransferase inhibitor MG149 was added,the immunoregulatory effect of butyrate disappeared.4.Inulin colud benefits CIA mice through modulating intestinal microecosystem:Inulin treatment can significantly reduce the clinical score of CIA mice,alleviated the foot swollen,reduce inflammatory cell infiltration and cartilage damage in joints;compared with normal mice,CIA mice exhibited unbalanced intestinal microecology and changed intestinal morphology,while inulin treatment regulated intestinal microecology,include increased proportion of Bacteroidetes,reduced proportion of Firmicutes,increased proportion of S24-7 and Lactobacillus and reduced proportion of odoribacter bacteria;inulin administration protected the intestinal mucosal barrier?alleviating colonic pathological changes?and reduced the expression of inflammatory genes;inulin inhibited systemic immune response by lowering the level of serum inflammatory factors,reducing the proportion of spleen antigen presenting cells,and regulating the balance between Th17 cells and Treg cells?reduced gene expression of IL-17 and increased gene expression of IL-10?.Fecal microbiota transplantation benefits CIA indicated that there was a causal relationship between the regulation of inulin on intestinal microecology and its therapeutic effect.Conclusion:Both EAE mice and CIA mice showed intestinal microecological imbalance?structural shifts of the gut microflora and changed metabolites?,intestinal mucosal barrier damage,and intestinal inflammation.These two autoimmune diseases have the characteristics of"Ying wei shi tiao"and"Zheng-xu-xie-sheng",which provide experimental basis for the hypothesis put forward in this thesis.Inulin can effectively improve intestinal microecology,restore intestinal mucosal barrier and regulate intestinal immune response,further regulate the systemic immune response?mainly regulating the balance of Th17 and Treg cells?,thus playing a therapeutic role on EAE and CIA mice,and there is a causal relationship between the modulation of intestinal microecology of inulin and its therapeutic effects.These results demonstrated the scientific hypothesis that“inulin can treat ADs by regulating intestinal microecology”,and suggested that inulin has the potential to be used as a therapeutic drug for ADs. |
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