The Study Of Immunomodulatory Effects Of Ganoderma Lucidum Polysaccharides In The Experimental Ulcerative Colitis In Mice

Objective:Inflammatory bowel disease(IBD)is a chronic disorder caused by dysregulated immune responses to intestinal microorganisms that occurs within all or part of intestinal tract.IBD consists of two major phenotypes,ulcerative colitis(UC)and Crohn's disease(CD).Although the complex etiology of both CD and UC has not been definitively elucidated,imbalanced cytokine production and T cell dysfunction are responsible for the pathogenesis of IBD.Particularly,increased secretion of pro-inflammatory cytokines is considered as the key factor in the pathophysiology of IBD.Ganoderma lucidum(G.lucidum)have anti-tumor,regulate immunity,anti-aging and many other effects,polysaccharides is considered to be one of the most important bioactive components.Ganoderma lucidum polysaccharides(GLP)have,with the above effects,anti-oxidation,anti-virus,anti-radiation effects.However,there is no direct study regarding the efficacy of GLP on colitis and the mechanism of immune regulation.In the present study,we investigated the protective role and the immunomodulatory effects of GLP on DSS-induced colitis and explore the underlying mechanisms of these effects.Methods:Male C57BL/6 mice aged 6-8 weeks weighing 20�1g were randomly distributed into two groups including experimental group(GLP+DSS group)and control group(DSS group)and containing ten animals each.The experimental group mice were administered with GLP(100 mg/kg body weight;dissolved in 0.2ml water)by intragastric gavage every day for two weeks prior to the DSS treatment.The control group was administered water(0.2 mL).Two group mice were orally administered with2.5%DSS dissolved in drinking water with GLP or water treatment for 6 days.The mice were killed on day 7 after induction of colitis.A total of 28d,detect the following changes:?The colon tissue was removed and cut into pieces for histological analyses.Survival rate,loss of body weight,shortened colon length and DAI score were detected;?Neutrophils,macrophages,??T cells,NK,NKT,B cells,T cells,CD4~+CD44~+T cells,CD4~+CD25~+T cells were detected by flow cytometry Surface staining;Th17 cells and Th1 cells were detected by flow cytometry intracellular staining.?The levels of TNF-?,IL-1?,IL-6,IL-23,IL-17A,IL-10,IFN-?and IL-4were detected by ELISA.?The mRNA expression of transcription factors(ROR-?t,Foxp3,T-bet and GATA-3)in colon tissue was detected by real-time PCR.Results:1.All mice orally administered with 2.5%DSS for 6 days showed symptoms of colitis,including diarrhea,loss of body weight,and rectal bleeding,which demonstrated that the model of colitis mouse model was successful.Pretreatment with GLP prevented the development of DSS-induced acute colitis,reflecting better survival,loss of body weight,shortened colon length,histological changes of colon tissues and DAI score.Compared with model mice,GLP treated mice exhibited obviously enhanced survival rates,lower loss of body weight,decreased DAI score initially on day 7,less colon shortening and decreased histological scores.Thus,these results suggested that GLP-treatment had beneficial effect on acute colitis of DSS induced in mice.2.Flow cytometry results showed:GLP could suppress Th17 mediated cell response in the colonic lamina propria of C57BL/6 mice with DSS-induced acute colitis.In the GLP-treated acute colitis group,the percentage of B220~+CD3~-cells in LPL significantly increased in GLP-treated mice,while CD3~+B220~-cells in LPL significantly decreased compared with control mice.The percentages and absolute numbers of CD4~+CD44~+in LPL were significantly lower in the GLP-treated acute colitis group than in the control group.The percentage of NK cell(NK1.1~+CD3~-)and NKT cells(NK1.1~+CD3~+)in LPL of GLP-treated mice significantly reduced compared to untreated mice,while the absolute numbers of only NKT cells(NK1.1~+CD3~+)of LPL in the GLP-treated mice significantly decreased compared with controls.There was a significant decrease in the percentage and absolute number of IL-17A-producing CD4~+T-LPLs in GLP-treated mice compared with model mice.These results suggested that the effect of GLP may promote the proliferation of B cells,regulate Th1,Th17 andTh2 cell responses and inhibit the proliferation NK cell and NKT cells in LPL of colon in DSS-induced acute colitis mice.3.ELISA results showed:Measurement of cytokine concentrations in the culture supernatants of unstimulated LPL manifested that the levels of TNF-?,IL-1?and IL-6were significantly decreased in the GLP-treated acute colitis group compared with the control group.The level of IL-17A with anti-CD3 and anti-CD28 mAbs stimulation and without any stimulation was significantly reduced in the GLP-treated acute colitis group compared with the control group.The levels of IL-4 were significantly decreased with anti-CD3 and anti-CD28 mAbs stimulation.4.Real-time PCR results showed:GLP treatment led to a reduction in the expression of ROR-?t.However,it did not lead to any change in the expression of Foxp3,T-bet and GATA-3.These results suggested that GLP inhibits th17 cell response by inhibiting its differentiation.Conclution:1.GLP could alleviate DSS-induced acute colitis in C57BL/6 mice.2.GLP could suppress Th17 mediated cell response in the colonic lamina propria of C57BL/6 mice with DSS-induced acute colitis.3.GLP could significantly reduce the expression of ROR-?t in the colonic tissue of C57BL/6 mice with DSS-induced acute colitis to surpess Th17 cell differentiation.