The Regulation Role Of Histone Acetyltransferase MOF In Inflammation Diseases

Inflammatory bowel disease (Inflammatory Bowel Disease,IBD) including ulcerative colitis (ulcerative colitis,UC) and Crohn’s disease (Crohn’s disease,CD), is a nonspecific intestinal inflammatory disease that induced by genetic factors,environmental factors and immune factors. Among them, UC involves of intestinal mucosa layer, characteristics with diffuse mucosal inflammation and ulceration. Its pathogenesis is unclear, the treatment progress slow and lack of specificity.The incidence rate in our country has risen year by year. In our country,ulcerative colitis incidence rate was 11.6/10 million, and has become the main reason of our common digestive system diseases and chronic diarrhea.The patients were usually young adults, so it caused more and more attention. Dextran sulfate sodium (DSS) is a kind of polysaccharide sulfate body.It can induce UC.The mechanism of UC may be caused by the dysregulation of intestinal flora and immune cells.Studies have shown that, inflammatory bowel disease is not only related to genetic factors,but also relate to the DNA methylation and histone modification. All these can regulate inflammation related genes.Multiple sclerosis (MS), is a kind of central nervous demyelinating autoimmune diseases.Usually occur in the youth crowd.The incidence of MS in our country is five over ten thousand.The clinical symptoms of the patients with multiple sclerosis mainly include vision loss, body action not harmonious, incontinence, etc.Due to the severe symptoms and easy to recurrence,MS has catch more and more attention.Experimental autoimmune encephalomyelitis (EAE) is a classic animal model of mimic human MS.Studies have shown that the express of CD4+ CD25+ Treg cells is very high in EAE mice neural, and regulat the development of EAE through it’s immune-suppression role.Mof also called MYST1,is a kind of histone acetyltransferase.It can acetylate the histone 4 lysine sixteenth (H4K16). Mof can also acetylate non-histone such as P53 and TIP5.Acetylation can activate gene transcription. Mof plays an irreplaceable role in the process of normal cells proliferation, differentiation,embryonic development, DNA damage repair and carcinogenesis.Studies have shown that Foxp3 could help Mof to finish acetylation, make target genes transcription. TGF-β can stimulate the Foxp3 gene activation.Thus, study the regulation for Treg cells by Mof can make us in-depth understanding the pathogenesis of autoimmune diseases.Research purposes:C57BL/6 mice were used to drink 3.5% DSS solution to establish acute UC model and the others injection of MOG35-55 peptide to establish EAE model. Through morphology, histopathology, molecular biology, cell chemistry methods to observe the changes of UC mice’s intestinal mucosa tissue compared with the wild type. Compare differences between the normal group and EAE mice during different pathogenesis periods. Then discuss the relevant role of histone acetyltransferase Mof in the pathogenesis of autoimmune disease and provide us theoretical basis for the prevention and treatment of autoimmune disease.Research methods:Select the same nest and similar weight of 6-8 weeks female C57BL/6 mice, and divided into two groups randomly. Each group have at least 3 mice,then do the following experiments:1. Select one group of mice to establish acute ulcerative colitis model,another establish EAE mouse model.2. The UC group were divided into two small groups:DSS induced 3 and 7 days.Collect the colon tissue of the same place for follow-up experiments; EAE group is divided into three periods:early onset, peak and remission, collect the spleen tissue.3. Observe of the differences between the normal group and DSS induced mice colon and EAE mice sports ability from morphology level, make sure the establish of mouse model is successful.4. Observe the severity of DSS induced 3 days and 7 days colitis using immunochemical staining.5. Immunofluorescence double staining were used to observe the expression of histone acetyltransferases Mof and Foxp3,and their position in the organization. Observe the difference of these two kinds of protein compared with the control group.6. The tissue from DSS treatment group and EAE group were extracted total cellular RNA using TRIzol. Then amplified by qPCR. Analysis the relative expression of inflammation related gene and immune factors.7. The total proteins were extracted from the two group, then using SDS-PAGE to analysis the expression of Mof and Foxp3.8. Using Co-Immunoprecipitation (Co-IP) to verification whether Mof and Foxp3 have interaction with each other.9.ChIP-qPCR experiment to analysis the target genes of Mof,so as to explore the pathogenesis of autoimmune disease and inflammation.10.Over express Mof in HCT116 cells to ensure the regulation role of Mof on other genes.Results:1. Morphological observation:colon tissue is isolated from C57BL/6 mice and observe the difference of experimental group and control group.We found the colon length of DSS induced group is shortened obviously and we can see colonic bleeding.The 7 days tissue is more obvious.The EAE experimental group mice walk slowly, tail is weak and even have double legs paralyzed.2.Using hematoxylin eosin stain to identification the pathological slices of the organization,we found DSS mice’s colon mucosa appears apoptosis, and is accompanied by severe mucosal shedding.3. Immunofluorescence staining indicated that the histone acetyltransferase Mof express in the nucleus of lamina propria cells of colon tissue and co-localizes with Foxp3.4.The mRNA levels of Mof, Foxp3,1117, Tlr3, Tlr4, Tlr5, Tlr6,Tlr7,Tlr9 were compared to the 18srRNA.The activity of Mof, Foxp3,1117 mRNA in DSS induced 7 days colon tissue was significantly higher than those in the control group. The mRNA level of Tlr4, Tlr6, Tlr9 were significantly higher than those in control group either. The mRNA level of Mof, Foxp3 and 1117 in DSS induced 3 days mouse colon have no statistically significant difference compared with the control group. The mRNA level of Mof, Foxp3 and 1117 in the EAE mice is significantly higher than control group.So does the mRNA level of Tlr4, Tlr5, Tlr7 and Tlr9.5.The results of SDS-PAGE show that the expression of Mof and Foxp3 were more than the control group in both mouse modle.6.Co-IP experiments showed that Mof interacted with Foxp3 on physiology.7.Mof can binding to the promoter of Foxp3, Tlr3, Tlr4, Tlr5, Tlr6 and RoRrt genes.8.In HCT116 cells that over express Mof, Tlr4 and Foxp3 were higher than control,so dose the mRNA levels.Conclusion:1. This experiment applied 6-8 weeks of C57BL/6 mice as materials. The application of DSS (5000D) and MOG35.55 peptide to induced colitis and EAE are simple and reliable methods. These two modles can be used to study the mechanism of human inflammatory bowel disease and Multiple sclerosis.2. This study found that, in addition to genetic factors, there is a certain relationship between the histone acetyltransferases Mof and ulcerative colitis and multiple sclerosis. Mof regulate the occurrence and development of inflammation by interacting with Foxp3.3.Inflammatory cytokines 1117 can promote the development of multiple sclerosis, but played a dual role in the UC mouse models.4. In addition, the study also found that Tlr4 and RoRrt may play a certain role in the pathogenesis of autoimmune disease. Mof may participate in the regulating of these genes expression.