The Mechanism Of Metformin Alleviated Ulcerative Colitis In A Mouse Model

Ulcerative colitis(UC)is a subtype inflammatory bowel disease of unknown etiology,but is commonly characterized by bloody diarrhea,a damaged epithelial barrier,immune response disorder and relevant gene mutations in the intestinal immune system.B cells play important roles in antibody production,antigen presentation,and cytokine production,all of which contribute to immune responses.IgA is the most abundant antibody secreted into the gastrointestinal tract and provides initial immune protection.A major function of IgA is to exclude pathogenic microbes by restricting their access to the epithelial surfaces.B220~+B cells and CD138~+plasma cells have also been observed to secrete both IL-10 to inhibit inflammation.IL-35 is preferentially secreted by Foxp3~+Treg cells.Regulatory B cells and DCs also produce IL-35.Intestinal IL-35-producing B cells have been found in patients with Crohn’s disease.IgG is the most abundant immunoglobulin in serum.Although intestinal IgG confers protection against infection,activation of complement or binding to Fc receptors may aggravate intestinal inflammation.Metformin is an AMP-activated protein kinase(AMPK)activator mainly used for the treatment of type II diabetes.Previous studies have found that metformin interactions with the microbiota promoted the production of amine and short chain fatty acids in patients with type II diabetes.Our data and previous studies have shown amine can alleviate the inflammatory damage in mice colon tissue in previous study.We aimed to explore whether metformin can play a protective role in UC and identify the anti-inflammatory mechanism of metformin treatment.ObjectiveThis study aimed to investigate the effect of metformin on improving colitis symptoms in C57BL/6 mice and explore the mechanism of metformin in alleviating ulcerative colitis.Methods1.UC was induced in mice using 3%dextran sodium sulfate(DSS).Control,DSS,and DSS+metformin(DSS+MET)groups were established.Bodyweight loss,stool characteristics,rectal bleeding,colon length,and the pathological index of the colon tissue were recorded daily to evaluate colon inflammatory damage in three groups.2.The levels of cytokines and immunoglobulins in the serum and colon were determined using quantitative polymerase chain reaction(q-PCR)or enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to detect the differences in the ratio of CD45~+IL-10~+,B220~+IL-10~+,CD138~+IL-10~+,CD45~+P35~+EBI3~+,B220~+P35~+EBI3~+,CD138~+IgA~+,CD138~+IgG~+,and CD138~+IgM~+cells among the control,DSS,and DSS+MET groups.3.Anti-CD20 antibody was used to clear B cells with the exception of plasma cells in mice,and purified rat IgG2b,isotype Control was injected as a control.On the7th day,the clearance efficiency of CD19~+B220~+B cells in the spleen and mesenteric lymph nodes was determined by flow cytometry.Six groups of mice were established as follows:isotype Control,isotype+DSS,isotype+DSS+MET,anti-CD20 Control,anti-CD20+DSS and anti-CD20+DSS+MET.The bodyweight loss,stool characteristics and rectal bleeding were recorded daily.The concentrations of IL-10,IgA,IgG,and IgM in the colon tissue were detected by ELISA.Flow cytometry was used to detect intestinal epithelial and lamina propria ratio of CD138~+IgA~+cells in different groups.4.CD19~+B cells were isolated from the spleens of C57BL/6 mice in vitro.q-PCR and ELISA were used to detect the effects of MET on the secretion of IL-10,IL-35,IgA,IgG1,IgG2a,and IgG2b in B cells.5.Total RNA was extracted from the colon tissues of the Control,DSS,and DSS+MET groups.RNA sequencing(RNA-seq)was performed by PE150 to detect differences in gene expression between the three groups.Genes were enriched using the gene ontology(GO)or Kyoto Encyclopedia of Genes and Genomes(KEGG)databases to investigate significant differences in three groups.q-PCR was used to verify the RNA-seq results.Results1.MET improved the survival of mice in a DSS-induced UC model.MET relieved the bodyweight loss,diarrhea,rectal bleeding,rectal strictures and pathological damage caused by DSS.Pathological analysis showed that MET reduced the infiltration of inflammatory cells into the lamina propria and improved goblet cell loss.2.Anti-inflammatory cytokine,IL-10 and IL-35 were upregulated after treatment with MET.IgA concentrations in colon tissues were significantly higher than the DSS group.Flow cytometry showed that the ratios of intestinal epithelial and lamina propria CD45~+IL-10~+and CD138~+IgA~+cells were significantly higher in the DSS+MET group than the DSS group.3.When C57BL/6 mice were injected with anti-CD20 antibody,the ratio of CD19~+B220~+cells in the spleen and mesenteric lymph nodes of the mice was significantly lower.The DAI score of the anti-CD20+DSS group was lower than that of the isotype+DSS group.Anti-CD20+DSS+MET group got the lowest DAI score,whereas the isotype+DSS group had the highest score.ELISA showed that the IgG concentration in the isotype+DSS group was higher than the anti-CD20+DSS+MET group,indicating that IgG may exacerbate the symptoms of UC and MET could alleviate colitis by downregulating IgG levels.4.MET upregulated the m RNA levels of IL-10,IL-35,IgA,IgG1 and downregulated the level of IgG2a in B cells in vitro.ELISA showed that MET increased the levels of IL-10 protein and inhibited the expression of IgG2a and IgG2b proteins secreted by B cells.5.Compared with the control group,upregulated genes were enriched in the classical pathway of complement activation item and TNF signaling pathway in the DSS group,whereas downregulated genes were also enriched in above items or pathway after administration of MET.Compared to the DSS group,the upregulated genes were enriched in the defense response to bacteria item and arginine biosynthesis pathway after MET treatmet.q-PCR showed that the level of the Notch3 gene in the DSS group was significantly lower than the Control and DSS+MET group,suggesting that metformin may play an anti-inflammatory role by upregulating Notch3 gene.ConclusionThe study showed that metformin can improve the survival rate,bodyweight loss,diarrhea,rectal bleeding and pathological damage caused by DSS in a mouse model by increasing IgA and decreasing IgG level in conlon.Metformin inhibited B cell secretion of pro-inflammatory IgG2a and IgG2b in vitro.RNA-seq analysis suggested that arginine synthesis after metformin administration may be related to the promotion of IgA secretion.Metformin may inhibit the classical complement pathway and TNF signaling pathway by reducing IgG expression.