Preliminary Study On The Amelioration Activity Of Polysaccharides Of Pycnoporus Sanguineu Against Dextran Sulphate Sodium-induced Inflammatory Bowel Disease

Objective:Inflammatory bowel disease(IBD)is a chronic immune disease with unknown etiology.It has been a consensus that IBD is caused by environmental factors acting on genetic susceptibility,finally resulting in topical autoimmune inflammation activated by the intestinal immune system and non-immune system with the participation of gut microbiota.Indeed,the autoimmunity of IBD is closely related to T lymphocyte immune,especially that the imbalance of the subsets of helper T cell(Th)in intestinal mucosa is involved.Therefore,it is important to study the regulation of Th subsets and autophagy in the pathogenesis of IBD,which also makes great contribution for immunotherapy against IBD.Pycnoporus sanguineus(L.)Murrill is a kind of edible fungus.Folk medicine and existing research in foreign countries have shown that P.sanguineus has the activities of antitumor,improving immunity and anti-inflammation.And,it has been used to treat arthritis,sore throat,ulcer,gingival pain and so on.Based on the immune regulation and anti-inflammatory effects of existing studies,the present study explored the mechanism of the amelioration activity of polysaccharide of P.sanguineus(PPS)in murine colitis model induced by dextran sulphate sodium(DSS).Methods1.The protein content of PPS was determined by butyleyanoacrylate(BC A)assay,and that of polysaccharide was determined by phenol-sulfuric acid method.The relative molecular weight of PPS was analyzed by gel permeation chromatography(GPC),and the monosaccharide composition of PPS was analyzed by gas chromatography-mass spectrometer(GC-MS).2.60 male BALB/c mice were randomly divided into 6 groups(10 mice in each group),including Normal(N)group,Model(M)group,5-ASA group,and the PPS groups.From day 1 to 14,PPS groups were orally treated with PPS in doses of 100,200,400 mg/kg,(namely PL,PM,PH groups).From day 8 to 14,5-ASA group was treated with 5-ASA(400 mg/kg,p.o.),while Normal group and Model group were given distilled water.To induce colitis,Model group,5-ASA group,and the PPS groups were free access to 3%(weight/volume)DSS solution from day 8 to 14,meanwhile Normal group was drunk with distilled water.3.During the experiment,body weight,feces characteristics and motility were recorded daily,and datas of the 7 days of DSS-challenge were used to evaluate disease activity index(DAI).On the 15th day,all mice were sacrificed and colons(from anus to cecum)were harvested for macroscopic score(MS).4.Part of the colons was fixed in 4%formaldehyde(pH～7.2)and embedded in paraffin.Some sections were subjected to Haematoxylin and Eosin(H&E)staining to analyze tissue damage,and the other were subjected to immunohistochemistry(IHC)to analyze the expression of proliferating cell nuclear antigen(PCNA).The activity of myeloperoxidase(MPO)was detected by enzyme-linked immunosorbent assay(ELISA),and IL-12p40,IL-15,IL-10,IL-17,MCP-1β and MIP-1β were detected by Luminex.In addition,lipopolysaccharide(LPS)content in serum was detected by ELISA.5.Western blot(WB)was used to detect the expression of junction proteins(ZO-1 and E-Cadherin)and autophagy-related proteins(ULK1,p62,Beclin-1,LC3 I and LC3 II)in colon tissue.6.Part of the fresh colon was analyzed by flowcytometry analysis(FCM)immediately for the expression of IFN-y,IL-4,IL-17 and Foxp3,to evaluate ratios of Th1 cells,Th2 cells,Th17 cells,and regulatory cells(Treg)in colons.Results1.PPS is a heteropolysaccharide that is composed of ribose,rhamnose,arabinose,xylose,mannose,glucose,galactose.The range of relative molecular mass of PPS is 5000～6.7×105 Da.In addition,polysaccharide content of PPS is 74%,and its protein contentis 25%.2.Mice of M group showed evident colitis features during the DSS challenge,including body weight loss,loose stool,and hemafecia,and colons of this group were shorter than those of N group,resulting in a remarkable increased DAI and MS.By contrast,body weight of mice in PPS groups went up,the colitis features were ameliorated,and their colons were prolonged.DAI and MS were decreased.3.By H&E staining,colons of M group displayed evident structure lesion.In PPS groups,it was found that colons of them showed fewer crypt branching in addition to the remission in epithelium,submucous layer and muscular layers.PCNA expression was evidently suppressed in colons of M group,while it was apparently recovered in those of PPS group.4.Western blotting revealed that the two crucial proteins of epithelium junction,ZO-1 and E-Cadherin,were significantly decreased in colons of M group,whereas they were obviously up-regulated in those of PPS groups.In addition,LC3 I,LC3II,ULK1,Beclin-1,and p62 were all dramatically reduced in the colons of M group,and the ratio of LC3 Ⅱ/Ⅰwas increased.PPS treatment significantly promoted the expressions of LC3 Ⅰ,LC3 Ⅱ,Beclin-1,and p62,but decreased the ratio of LC3Ⅱ/Ⅰ.Moreover,serum LPS content of PPS groups was much lower than that of Model group.5.MPO activity in the colons of M group were extremely higher than that of N group,MPO activities of PPS groups(100,200,400mg/kg)displayed decreases.Cytokine assays found that IL-12p40,IL-15,MCP-1β and MIP-1β in M group were significantly increased,but PPS treatment reduced IL-10,IL-15,IL-17 and IL-12p40.6.Compared with those of N group,DSS challenge induced obvious increase of Th cells in the colons of M group,as well as its subsets,including Th1,Th2,Th17 and Treg.PPS displayed various impacts on the Th cells.It was found that PPS treatment(200 and 400mg/kg)was able to reduce both proportions of the pro-inflammatory subsets(Th2 and Th17),and the anti-inflammatory Treg.Nevertheless,PPS remarkably raised Th1 in the colons.ConclusionsPPS displayed evidently remission in IBD,which would be attributed to its mucosal healing by up-regulating the expression of Zonula occludens-1(ZO-1)and E-cadherin within the colon as well as the restrain on excessive autophagy.PPS can suppresss reduce the infiltration of Th cells and its differentiation towards the pro-inflammatory subsets Th2 cells,Th17 cells and Treg in the lesions,and inhibited the secretion of inflammatory cytokines,thus maintaining the immune homeostasis.In conclusion,PPS,an active constituent of Pycnoporus sanguineus,has several benefitical anti-inflammatory effects in the DSS-induced model,including epithelium barrier repair,autophagy suppression,and autoimmune inhibition,highlighting a promising potential in the treatment for IBD.