Study On The Therapeutic Effect Of Inonotus Hispidus Polysaccharides On Ulcerative Colitis

Inonotus hispidus is a valuable medicinal fungus.In this paper,the process of hot water extraction of I.hispidus polysaccharide was optimized.In vitro LPS-induced inflammation model of macrophage RAW264.7 was established to evaluate the intervention mechanism of I.hispidus polysaccharide homogeneous fraction INP-A2 on cellular inflammation;in vivo DSS-induced UC model of C57BL/6 mice was established to evaluate the effect of INP-A2 on the prevention and treatment of ulcerative colitis in mice.Specific research findings are as follows:（1）The extraction of I.hispidus polysaccharide was carried out by hot water extraction method.The effect of the five main levels of material-to-liquid ratio,extraction temperature and extraction time on the polysaccharide yield was investigated by single-factor experiments.The optimum extraction process of I.hispidus was 1:18 g·m L^-1,80.5℃and 1.9 h.The yield of polysaccharide was 0.612±0.057%.（2）The total polysaccharides of I.hispidus substrates were extracted from the crude polysaccharides by Sevag method,protein removal,adsorption on macroporous resin and salt removal,and separated by ion exchange chromatography and gel filtration chromatography to obtain a homogeneous polysaccharide fraction of molecular weight,INP-A2,with a sugar content of 97.8%and a molecular weight of 13.6 k Da.The structure was analysed by FT-IR,methylation,GC-MS and NMR,and the monosaccharide composition consisted mainly of Gal,Glc,Man and Glc A with a molar ratio of 30.4:25.1:17.1:8.9.FT-IR showed a typical absorption peak for polysaccharides,with a C-O-C stretching vibrational absorption peak near 1018 cm-1,a pyran ring conformation,and a+27°dextrose,and methylation and NMR patterns showed three structural domains in INP-A2:dextran,mannan and galactan.Gal has a side chain attached at the O-2 position with 31.6%branching,Glc is mainly present as 1,3-Glcp,1,6-Glcp,1,3,6-Glcp and a small amount of T-Glcp,and Man is present as 1,3-Manp and T-Manp linkages.（3）A classical cellular inflammation model was established for LPS-induced macrophage RAW264.7,and the concentrations of INP-A2 and LPS administration were investigated by MTT method;cell morphology was observed;the secretion levels of NO,IL-6 and TNF-αwere detected by ELISA;the relative expression of IL-6,IL-1βand TNF-αm RNA were detected by fluorescence quantitative PCR;protein immunoblotting was performed to detect p38MAPK/NF-κB and AKT signaling pathway related protein expression.The results were following:the optimal concentration for cell administration was 125-500μg-m L-1,and the optimal modeling concentration for LPS was 500 ng-m L-1;INP-A2 improved the state of polarization and branching of cells in the LPS model group;INP-A2 significantly decreased the secretion of inflammatory cytokines IL-6,TNF-αand inflammatory mediator NO;in RT-PCR results,the INP-A2 significantly down-regulated the m RNA expression levels of TNF-α,IL-6 and IL-1β;in the protein immunoblotting results,INP-A2 could effectively reduce the expression levels of p-p38,p-p65,p-Jnk1/2,p-Erk1/2,p-AKT and IκBαprotein phosphorylation in p38MAPK/NF-κB and AKT signaling pathways.（4）The DSS-induced C57BL/6 mouse UC model was selected to detect daily body weight,DAI score,spleen index,colon tissue length and weight measurements;H.E staining of colon histopathological sections and pathological damage scores;ELISA to detect the secretion levels of inflammatory factors IL-6,IL-1βand TNF-αin colon tissues,and protein immunoblotting to determine the relative expression of key proteins in the p38MAPK/NF-κB and AKT signaling pathways was determined by protein immunoblotting.Compared with the DSS group,the mice in the high-dose INP-A2 administration group showed a slower trend of weight loss,lower DAI score,and normalized colon length and quality;HE staining showed that the INP-A2 group had good crypt status and no obvious inflammatory infiltration;ELISA showed that lower INP-A2could reduce the secretion levels of IL-6,IL-1βand TNF-αin colon tissues;Western-Blot results showed that a high dose of 200 mg-kg-1 INP-A2 significantly inhibited the phosphorylation of p38MAPK/NF-κB,AKT signalling pathway key protein IκB,p-p65,p-p38,p-Jnk,p-Erk and p-AKT proteins.In summary,the homogenized I.hispidus polysaccharide can provide good therapeutic effects on macrophages and ulcerative colitis in mice,providing some scientific basis for the future development of I.hispidus polysaccharide into an effective and safe drug for the treatment of colitis.