| Multiple sclerosis(MS),an autoimmune disease affecting the central nervous system(CNS),is characterized by chronic inflammatory infiltration of the nervous system with varying degrees of demyelination.It is one of the main causes of cripple and paralysis in middle-aged and young people.Clinically,relapse-remitting multiple sclerosis(RRMS)is the most common type,characterized by relapse-remitting process,long course of disease,and acute aggravation of neurological symptoms.Etiology and pathogenesis of MS are unclear,it is generally accepted that environmental factors,autoimmune reaction,virus infection,diet,vitamin D deficiency and other factors caused the increase of CD4+T cells and subsequent cytokines,which activated the intrinsic glial cells of the CNS,and caused different degrees of inflammatory infiltration and demyelination of brain and spinal cord tissues.For the treatment,disease-modifying treatments(DMTs)and other symptomatic treatments are the first-line drugs,such as interferon-β(IFN-β),but there are also many problems,such as the decline of clinical efficacy over time and high treatment cost.Matrine,a quinolazine alkaloid component which is extracted from the root of Sophora flavescens,has antibacterial and anti-inflammatory effects in clinical practice,and is commonly used by the hepatitis B,C and various causes of leukopenia.More and more studies have confirmed that matrine can inhibit the process of oxidative stress and inflammation,inhibit hippocampal neuronal apoptosis,and has been increasingly applied in the treatment of nervous system diseases such as autoimmune disease.Matrine has significant immunomodulatory effects,but its effect on MS has not been thoroughly studied.Thus,we began with a combination of myelin Oligodendrocyte glycoprotein(MOG)35-55 and mycobacterium tuberculosis,to establish the classical animal model of MS,the experimental autoimmune encephalomyelitis(EAE)mice.And matrine with different doses was injected respectively to investigate the efficacy of matrine on EAE mice.Then,according to the effect of matrine on T cells,16S rRNA technology was further used to detect intestinal flora in cecal contents of mice in each group.Analysis of the difference in intestinal flora and further prediction of the function were performed.Based on the results of 16S rRNA technology,concentrations of the short-chain fatty acids(SCFAs)in cecal contents of mice were detected by targeted metabolomics.Next,we further explored the effect of matrine on the intestinal immune mechanism of EAE mice.Immunofluorescence staining was used to determine the intestinal barrier function and the difference of T cell composition in the colon of mice.Primary culture of mouse spleen or lymph node cells,and mouse immortalized cell line CT26,were used to investigate the molecular mechanism of matrine action.Finally,pseudo-sterile mice were established by intragastric administration of non-absorbable antibiotics,and then pseudo-sterile EAE mice were established by antigen immunization prepared by MOG35-55 and Mycobacterium tuberculosis.Cecal contents of EAE mice treated with matrine were transplanted into pseudo-sterile EAE mice,to analyze matrine effects under the condition of relative depletion of intestinal flora of mice.And these results further verified the regulation of matrine on intestinal microenvironment of EAE mice.Part Ⅰ:A preliminary study on the effect of matrine on multiple sclerosis animal model,EAE miceAimTo investigate the therapeutic effect of matrine on EAE mice.Methods1.By the method of subcutaneous injection of immune antigen to establish animal models of MS,EAE mice.Mice were divided into normal group,EAE group and matrine-treated group.The treatment group was given different doses of matrine injection and the EAE group was given the same volume of normal saline at the same time.Researchers observed and recorded the weight and the nerve function score after immunization every day;2.The mice in each group were euthanized on the 23rd day after immunization,and blood was collected from eyeballs.After heart perfusion with normal saline,brain and spinal cord tissues were collected.HE staining was used to analyze the infiltration of inflammatory cells in the spinal cord tissues,and LFB staining was to analyze the demyelination.Cytometric Bead Array(CBA)was used to determine the Th1/Th2/Th17 cytokine concentrations in mice plasma.RT-PCR was used to detect the mRNA relative expression of Th1/Th17/Treg cell related transcription factors and cytokines.The distribution of Th1/Th17/Treg cells was analyzed by immunofluorescence double staining(CD4+IFN-γ+,CD4+IL-17A+,CD4+Foxp3+)in brain tissues.3.Statistical analysis was carried out by Graphpad 7.0,and the results were expressed as mean ±mean standard error.Comparison of body weight and neurofunctional score between groups was performed by ANOVA of repeated measurement design variables,and comparison of mean between groups was performed by t-test.Results1.Matrine improved the weight loss during the onset of EAE mice,reduced the severity of neurological function,and slowed down the disease progression of EAE mice;2.HE staining results showed that matrine alleviated the inflammatory infiltration levels in the spinal cord of EAE mice.LFB staining showed that matrine reduced the demyelination in EAE mice.CBA results showed that matrine decreased the concentrations of pro-inflammatory cytokines such as IFN-γ and IL-17A,and increased the concentrations of anti-inflammatory cytokines such as IL-10.RT-PCR showed that matrine decreased the mRNA relative expression levels of Th1/Th17 cell transcription factors and cytokines,and increased the mRNA relative expression levels of Treg cell transcription factors and cytokines.Double immunofluorescence staining of brain tissue showed that matrine decreased the number of Th1/Th17 cells but increased the number of Treg cells.ConclusionMatrine improved the weight loss,alleviated the severity of neurological function,slowed disease progression in EAE mice,reduced inflammatory infiltration and demyelination of spinal cord tissues,and also reduced the number of Th1/Th17 cells and increased the number of Treg cells in CNS of EAE mice.These results indicated that matrine had a significant therapeutic effect on EAE mice.Part Ⅱ:Matrine exerted anti-inflammatory effects by regulating the composition of intestinal flora and promoting the production of short-chain fatty acids in EAE miceAimTo investigate the effect mechanism of matrine on intestinal microflora and metabolites in EAE mice.Methods1.Cecum contents of all mice were collected,namely in Normal group,EAE+NS group,EAE+MAT-L group and EAE+MAT-H group,and 16S rRNA technology was conducted to analyze the effects of matrine on intestinal micro flora of EAE mice.2.Further functional prediction analysis was performed of the different flora by comparing different database;3.Based on the results of functional prediction analysis,the intestinal SCFAs concentrations of mice were further detected.4.Correlation analysis was conducted for the different bacterial communities and the concentrations of SCFAs among each group.Results1.α diversity analysis showed that matrine increased the number,species richness and evenness of intestinal flora in EAE mice;β diversity analysis showed that matrine changed the composition and increased the diversity of intestinal flora in EAE mice.especially in Firmicutes,Bacteroidetes,such as lactobacillus,Clostridium,bacteroidetes,etc.2.Functional analysis showed that the differential flora induced by matrine was related to pathways of glucose metabolism,lipid metabolism and amino acid metabolism.The results showed that the samples of the model group and the matrine-treated group differed mainly in the following pathways:NAD biosynthesisⅡ(from tryptophan),lipid ⅣA biosynthesis,Kdo transfer to lipid ⅣA Ⅲ,reduc tive acetyl coenzyme A pathway,superpathway of hexitol degradation(bacteria),superpathway of glucose and xylose degradation,superpathway of pyrimidine deoxyribonucleotides de novo biosynthesis,teichoic acid(poly-glycerol)biosynthesis,superpathway of tetrahydrofolate biosynthesis and so on.In particular,the comparative analysis results of EAE+NS and EAE+MAT-H showed that there were differences in superpathway of fatty acid biosynthesis initiation and in hexitol fermentation to lactate,formate,ethanol and acetate.3.Further detection of intestinal SCFAs concentrations in four groups showed that matrine increased the concentrations of acetic acid,propionic acid and butyric acid in EAE mice,especially in the EAE+MAT-H group,butyric acid concentrations were significantly increased.4.By correlation analysis of the results of 16S rRNA and metabonomics,it showed that in the phylum level,differences of the acetic acid concentration were closely relative to synergistetes but negatively correlated with deferribacteres.Differences of the propionic acid concentration was positively relative to verrucomicrobia,synergistetes,bacteroidetes but negatively correlated with Firmicutes.Differences of the butyric acid concentration was positively correlated with synergistetes,but negatively correlated with deferribacteres and epsilonbacteraeota,At the classification level of genus,the difference of acetic acid concentration was positively correlated with alistipes,bacteroides and firmicutes unclassified and negatively correlated with mucispirillum and lactobacillus.The difference of propionic acid concentration was positively correlated with alistipes,bacteroides,firmicutes unclassified,Akkermansia and negatively correlated with lactobacillus and streptococcus.ConclusionMatrine played an anti-inflammatory role in EAE mice by regulating the intestinal flora,increasing the number,richness,evenness and diversity,increasing the microbes producing SCFAs,and further promoting the release of acetic acid,propionic acid and butyric acid.Part III:Matrine promoted IL-10 production and restored intestinal barrier function in EAE mice by regulating SCFA receptor GPR109AAimTo explore the specific mechanism of matrine on intestinal immune function of EAE mice.Methods1.WGA-FITC immunofluorescence staining was performed on the colon tissues of mice in each group on the 23rd day after immunization to determine the effect of matrine on the colonic mucosa;2.Immunofluorescence staining on colon tissues of EAE mice on day 23 after immunization was performed to determine the effect of matrine on colon CD4+T cells,such as Th17(CD4+IL-17A+),Treg(CD4+FOXP3+)and CD4+IL-10+.3.Total lymphocytes were further extracted from the lymph nodes or spleens of naive C57BL/6 mice and stimulated with MOG35-55(10μg/mL)for 24h,then co-cultured with matrine monomer(10,50 or 100μM).The group with medium was as control.Forty-eight hours later,the supernatant was collected respectively to detect the secretion levels of IFN-γ,IL-17A and IL-10 by ELISA.4.Next step was to explore the specific relationship between matrine and IL-10.The single cells from spleens or lymph nodes of naive C57BL/6 mice were co-cultured with IL-10 neutralizing antibody to compare the therapeutic effect of matrine in the absence of IL-10.Co-cultured with Isotype was as control as control.Forty-eight hours later,cells were collected respectively to detect Thl(CD4+IFN-γ+)and Th17(CD4+IL-17A+)cells by flow cytometry.5.Whether there are similarities on EAE mice between matrine and butyric acid,single cells were isolated from spleens or lymph nodes of mice in the EAE+NS group.And then co-cultured with butyric acid(1.0mM,2.5mM,5mM)or matrine(100μM),and PBS co-cultured group was as control.Forty-eight hours later,the concentrations of IFN-γ,IL-17A and IL-10 in supernatant were detected respectively by ELISA,and the IL-10-producing cells were detected by flow cytometry.6.Mouse immortalized cell line,CT26 was further transfected with the targeted small interfering RNA(si-RNA)to knock down the expression of GPR109A,butyric acid and matrine monomer or alone was co-cultured.Cells were collected after 48h,and WB was used to determine the protein expression levels of inflammatory factors,IL-17A,IFN-γ and TNF-α,RT-PCR was used to detect the mRNA expression levels of tight junction proteins,ZO-1,Claudin-land IL-10,Helios.Results1.Matrine restored the integrity of intestinal mucosal barrier in EAE mice.Compared with Normal group,the intestinal fluorescence intensity of WGA-FITC in EAE+NS group was weaker and less continuous,and the integrity of intestinal tract was significantly damaged.However,the fluorescence intensity in the matrine-treated group was relatively increased,and the fluorescence intensity was enhanced with the increase of matrine concentration.Further comparison among groups,there was statistical significance,p<0.05.2.Matrine reduced the number of Th17 cells but increased the number of FOXP3+Treg cells and CD4+IL-10+cells in the colon tissue of EAE mice.In the matrine-treated group,the fluorescence intensity of CD4+IL-17A+was significantly weaker than that of the EAE+NS group,and decreased with the increase of matrine concentration.The average fluorescence intensity in each group was statistically significant,p<0.001.In the matrine-treated group,the fluorescence intensity of CD4+FOXP3+ or CD4+IL-10+was significantly stronger than that of the EAE+NS group.The average fluorescence intensity of each group was further calculated.There was no statistical significance between EAE+NS and EAE+MAT-L group,p>0.05,but there was significant statistical significance between EAE+NS and EAE+MAT-H group,p<0.001.3.Matrine decreased the secretion of IFN-γ and IL-17A in peripheral immune organs,but increased IL-10.Compared with MOG group,100μM of matrine monomer significantly decreased the secretion of IFN-γ and IL-17A,but increased the secretion of IL-10,there was significant statistical significance,p<0.001.4.Matrine relied on IL-10 to regulate Thl and Thl7 cells thus exerting anti-inflammatory effects.Compared with Isotype group,matrine monomer could significantly reduce the number of Thl and Thl7 cells,there was significant statistical significance,p<0.001,while the effects of matrine were weakened or even disappeared after the addition of IL-10 neutralizing antibody,compared with Isotype+MAT group,there was statistically significant,p<0.05.5.The anti-inflammatory effects of matrine were similar to that of butyric acid in the regulation of EAE mice immune cells,especially T cells.Both matrine and butyric acid(5mM)could reduce IFN-γ and IL-17A secretion and increase IL-10 secretion,with statistically significant difference,p<0.001.The number of IL-10-producing cells was also increased,which was statistically significant compared with PBS group,p<0.01.6.Matrine could regulate the protein expression of IFN-γ and IL-17A,and promote the relative mRNA expression of tight junction proteins ZO-1,Claudin-1 and IL-10 and Helios by the short chain fatty acid receptor,GPR109A.The expressions of IFN-γ and IL-17A were decreased by matrine and butyric acid,while the expressions were not significantly decreased with the presence of si-GPR109A,with statistically significant difference,p<0.001.Similarly,the mRNA relative expressions of tight junction protein ZO-1,Claudin-1 and IL-10,Helios were significantly increased by matrine and butyric acid,and the comparison was statistically significant,p<0.001.However,there was no significant change after si-GPR109A was added,and the comparison was also no statistically significant,p>0.05.ConclusionMatrine restored the integrity of intestinal mucosal barrier in EAE mice,reduced the number of Th 17 cells,increased the number of FOXP3+Treg cell and IL-10 producing cells,reduced the secretion of IFN-γ and IL-17A in peripheral immune organs,increased the secretion of IL-10,and depended on IL-10 to regulate the number of Th1 and Th17 cells.Similar to butyrate acid,matrine could regulate the protein expression of IFN-γ and IL-17A,and promote the relative mRNA expression of tight junction proteins ZO-1,Claudin-1,IL-10 and Helios by short chain fatty acid receptor,GPR109A.Part Ⅳ:Study on the therapeutic effect of bacterial community transplantation from the matrine-treated mice on multiple sclerosis animal model.AimTo verify the therapeutic effect of bacterial community transplantation from the matrine-treated mice on multiple sclerosis animal model.Methods1.Pseudo-aseptic EAE mouse model was established by continuous intragastric administration of non-absorbable antibiotics.Cecal contents of matrine-treated EAE mice were collected,suspended,and then the solution was prepared.On the 7th day after immunization,pseudo-aseptic EAE mice were given bacterial community transplantation by intragastric administration.And normal group,matrine-treated group and pseudo-aseptic EAE mice model group were as control.Neurological function scores and body weight changes of mice in each group were observed and recorded every day.2.On the 23rd day after immunization,all mice were euthanatized and performed cardiac perfusion.Spinal cord tissues were collected for HE staining,LFB staining and MBP+DAPI immunofluorescence staining to analyze inflammatory infiltration and demyelination.3.On the 23rd day after immunization,spleen or lymph nodes of mice in each group were collected,and the number of Th1(CD4+IFN-γ+),Th2(CD4+IL-4+),Th17(CD4+IL-17A+),Treg(CD4+CD25+)cells was detected by flow cytometry,and the percentage of each type of cells was counted.4.On the 23rd day of immunization,the mice were euthanized,and blood was collected from the eyeballs.Plasma cytokines concentrations of IFN-γ,IL-17A and IL-10 were detected by CBA method.Results1.The intestinal flora transplantation from matrine-treated EAE mice reduced the clinical severity and improved the weight loss of pseudo-aseptic EAE mice.2.The intestinal flora transplantation from matrine-treated EAE mice reduced inflammatory infiltration and demyelination of the spinal cord of pseudo-aseptic EAE mice.3.The intestinal flora transplantation from matrine-treated EAE mice reduced the number of Th2 and Th17 cells and increased the number of Treg cells.4.The intestinal flora transplantation from matrine-treated EAE mice reduced the concentration of IL-17 A and increased the concentration of IL-10 in peripheral blood of mice.ConclusionBacterial community transplantation from the matrine-treated mice reduced clinical severity,improved weight loss,reduced inflammatory infiltration and demyelination of spinal cord tissue,reduced the number of Th2 and Th17 cells,and increased Treg cells,reduced the concentration of IL-17A and increased the concentration of IL-10 in peripheral blood of mice.The regulation of matrine on intestinal microenvironment in multiple sclerosis was further verified. |
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