Mechanism Of Immunosuppressant In The Treatment Of Multiple Sclerosis By Interfering With Gut Microbiota

Background Multiple sclerosis(MS)is a central nervous system(CNS)disease characterized by highly disabling white matter inflammatory demyelination,accompanied by immune response and axonal damage,which has a high incidence in young adults and is difficult to cure.In China,the incidence of multiple sclerosis has been on the rise in recent years,especially with the deepening of clinical understanding of the disease,the number of patients diagnosed in recent years has increased significantly.As multiple sclerosis is a chronic and progressive disease and difficult to cure,it severely affects the quality of life of the patients and also increases the economic burden of the family and society.There are many studies on the pathogenesis of multiple sclerosis,but the specific reasons are not completely clear,which may be related to autoimmune response,viral infection,individual heredity,living environment of patients and other factors.Immunosuppressive drugs or immunomodulatory drugs are used in clinic to treat the clinical symptoms.Neuronal axonal damage of multiple sclerosis is caused by the inflammation which is induced by immune cells.In different stages of the disease,nerve damage presents different ways.The inflammatory response caused by tissue damage and infection is the body's defense and repair process produced by different immune cells and inflammatory factors.However,an overactive inflammatory response can lead to injury and disease and demyelination of nerve cells in the central nervous system.Therefore,when the immune response is activated,moderate control of the level of inflammatory response can prevent its harmful effect.Rapamycin induces autophagy by inhibiting the m TOR signaling pathway,non-specifically inhibits the overactivation of immune cells,and reduces the inflammatory response of nerve tissues.MCC950(cp-456773)can reduce the expression of inflammatory factors by inhibiting the construction of NLRP3 inflammatory bodies.Therefore,both rapamycin and MCC950 are expected to reduce the damage of nerve cells and relieve the clinical symptoms of multiple sclerosis by inhibiting the transitional activation of immune cells.Since multiple sclerosis is usually treated with systemic administration,and more than 70% of the body's immune cells are stored in lymphoid tissue of the intestine,the effect of immunosuppressants will also affect the activity of the immune system in the intestine.Intestinal lymphatic tissue and mucosal layer constitute the first line of defense of the body immunity,with mucosal barrier as the main place for invasion and colonization of foreign pathogens and antigens.Central and the intestinal immune system and gut microbiota,all levels of the nervous system and related endocrine system form interaction network with each other,forming the brain gut enteric microbiota axis(BGMA).The nervous system at all levels regulates the intestinal tract so that two-way regulation is formed between the intestinal flora and the brain through the brain-gut axis,so as to promote the activation of effector cells in the intestinal tract and release of signal molecules,and regulate the growth,composition,adhesion and colonization of the gut microbiota in the intestinal cavity.The digestive tract is the largest immune tissue in the body.In the pathological state,when the intestinal mucosal barrier is damaged,the stability of the intestinal microecology is destroyed,which can lead to the imbalance of gut microbiota,and can affect the immune cell activity of brain and intestinal tissues through the brain gut axis to promote theprogress of neurological diseases.At the same time of the onset of multiple sclerosis,the central nervous system is damaged due to the breakdown of the immune balance in vivo,and the nerve impulse conduction at various levels which regulated by it is also damaged or inhibited to varying degrees,resulting in limb paralysis and other obvious clinical symptoms.The immune system in the intestinal tract is affected,resulting in changes in the permeability of intestinal mucosal tissue and the mode of microbial attachment,exacerbating the course of the disease.Therefore,this study proposed a hypothesis: the treatment with immunosuppressive agents such as rapamycin and MCC950 maybe inhibit the excessive activation of immune cells in brain and intestinal tissue to reduce its damage to the myelin sheath structure of nerve cells,restore the immune barrier effect of intestinal mucosa and the microecological balance in the intestinal tract.Therefore,the action of immunosuppressive agents helps to improve the therapeutic effect of the disease and slow down the progress of the disease.Objectives In this study,the mouse model of experimental autoimmune encephalomyelitis(EAE)was prepared by polypeptide immunization.Based on the brain-gut axis theory,the mice were treated with rapamycin and MCC950 respectively/in combination.The pathogenesis of multiple sclerosis and the treatment strategies of immunosuppressive agents were systematically discussed from three aspects: brain,intestinal immune barrier and gut microbiota.The therapeutic effects and mechanisms of rapamycin and MCC950 in different stages of disease were investigated by detecting the proliferation and mode of action of immune responsibline-related cells in the brain and intestinal mucosa,the changes in the diversity and abundance of intestinal flora,and the relationship between them and the manifestations of multiple sclerosis.By verifying whether there is an association between brain diseases and gut microbiota,it provides new ideas for studying the pathogenesis and treatment of multiple sclerosis.Methods In this study,using MOG35-55 which is one of the major antigenic determinant of the myelin oligodendrocytes glycoprotein(MOG)as antigen to prepare experimental autoimmune encephalomyelitis animal model by subcutaneous injection in C57BL/6 mice.Early treatment with rapamycin and MCC950 was initiated on day 3 of immunization.1.The drug treatment group received intraperitoneal injection of rapamycin and/or MCC950 every other day,while the control group and EAE group receivedintraperitoneal injection of PBS buffer every other day instead of drug treatment.The neurological function scores of mice were observed and body mass was weighed the next day after immunization.2.Mice were anesthetized and put to death on the 8th,16 th and 24 th day after immunization,and tissue samples of spleen,cecum,large intestine and brain were collected to determine the disease course and drug treatment effect through histopathological examination.m TOR/NLRP3 related signaling pathways and expression changes of CD4,CD8,GFAP and other related immune markers were detected respectively to explore the mechanism of treatment with rapamycin and MCC950.3.Stool samples were collected from each group of mice,and genomic DNA was extracted.The variable region 4 of 16 S r RNA of gut microbiota in each group was amplified by PCR through universal primer amplification.The amplified products,which were detected by electrophoresis and recovered after purification,were used in Illumina-Misq high-throughput sequencing platform for sequencing.Cluster analysis was performed using QIIME software and compared in Silva database to detect changes in the specise and composition of gut microbiota in the control group and the rapamycin and/or MCC950 treatment group.4.Calculate the Alpha diversity index of gut microbiota through bioinformatics analysis,and compare the abundance and diversity of microorganisms in each group;The Beta diversity was compared and the diversity of species among different samples was analyzed.LEf Se difference analysis,evolutionary tree distribution analysis of each sample species,phylogenetic and functional prediction of species were used to explore the correlation between the inhibition of immune cell activity induced by rapamycin and/or MCC950 treatment and changes in gut microbiota in the course of disease progression.Results 1.C57BL/6 mice immunized with MOG35-55 began to develop disease successively around 7d after immunization,and showed obvious disease symptoms,and gradually developed tail weakness and limb paralysis.The mice gradually recovered after the peak of the disease,but generally lost weight.The onset peak of the mice treated by rapamycin and MCC950 respectively/combined was delayed and the self-healing period was advanced,but the mice in the combined treatment group lost weight significantly.2.As an immunosuppressant,rapamycin can induce autophagy by inhibiting m TOR pathway to alleviate the clinical symptoms of EAE mice.MCC950 can inhibit the damage of IL-1? and other inflammatory factors on brain tissues of EAE mice by inhibiting the assembly of NLRP3 inflamosome.By detecting the brain tissues in the early stage,peak period and recovery period of mice respectively,after the treatment with rapamycin and MCC950,the onset period of mice was shortened,and the disease symptoms were alleviated while the expressions of CD4,CD8 and GFAP were decreased.3.Inflammatory cell infiltration was observed in the brain tissue and intestinal mucosa of the model mice,and the intestinal wall was thickened and the spleen was enlarged,all of which were caused by autoimmune response.The increase of CD4+T and CD8+T cells in brain and intestinal mucosa were detected in the EAE model,which participated in the immune regulation of intestinal mucosa 4.After the mice were immunized and treated with drugs,stool samples of each group were collected and microbial DNA was extracted.Samples were sequenced on Illumina-Misq high throughpot sequencing platform,using the QIIME software for data processing and cluster analysis.Analysis and comparison were performed in the Silva database to calculate the composition and proportions of gut microbiota.Compared with the control group,after immunizd by MOG35-55,total OTUs of EAE group was reduced,while the othor groups theated by rapamycin or MCC950,the OTUs were increase.The total increase of OTUs in the combined treatment group was significantly higher than that in the single treatment group.5.The main bacteria of stool samples in the control group were Bacteroidetes(63.31%),Firmicutes(24.11%)and Proteobacteria(9.1%).Compared with the control group,the proportion of Bacteroidetes in the model group decreased significantly,while the proportion of Firmicutes and Proteobacteria increased.6.Alpha diversity and Beta diversity of gut microbiota in mouse stool samples were analyzed respectively.Through Alpha diversity analysis,there was no significant difference in the species diversity between EAE group and the control group.The abundance of microbiota in the control group was higher than that in EAE group,while the abundance of species in the drug treated groups was in between.7.Through the Beta diversity analysis,species composition of the control group and rapamycin treated group were more different than other groups.The main bacteria in control group was Bacteroidia,etc.The mark bacteria in EAE group was Bacteroides,while in rapamycin treated group it was Firmicutes in the treatment group.In MCC950 treated group,the mark bacteria was Christensenellaceae.In the combined treatd group,the markers were Akkermansia.Conclusion 1.In the early treatment of EAE mice,the used of rapamycin and MCC950 can effectively slow down the disease progression,and can relieve the clinical symptoms.Rapamycin and MCC950 respectively by inhibiting m TOR signaling pathways and NLRP3 assembly,suppression of brain tissue and intestinal immune inflammatory reaction,so as to relieve the progress of the disease,and can inhibit the excessive activation of immune cells in brain and intestinal tissue.2.During the preparation of the EAE mouse model,immune cells in the brain and intestinal mucosa were activated,resulting in changes in the intestinal microenvironment and a decrease in the abundance of gut microbiota.The early treatment of the immunosuppressants rapamycin and MCC950 restored the composition of gut microbiota by reducing the activity of immune cells in the tissues and repairing the intestinal mucosal immune barrier.