Anti-inflammatory Mechanism And Effect On Ulcerative Colitis Of Polysaccharides From Hericium Erinaceus Mycelium

Ulcerative colitis(UC)is an inflammatory bowel disease,and its development is closely related with inflammation.Hericium erinaceus is a medicinal and edible fungus and applicated in the treatment of gastrointestinal diseases traditionally.Based on the protective effect of Hericium erinaceus on the gastrointestinal tract,we try to isolate and purify polysaccharides from Hericium erinaceus mycelium to evaluate the anti-inflammatory activity and explore its mechanism for treating UC.The main research is as follows:(1)Isolation,purification and identification of Hericium erinaceus polysaccharides.The crude polysaccharides(HECP)was extracted from powder of Hericium erinaceus mycelium by hot water and ethanol precipitation,and the content of total sugar was 89.1%.The single component polysaccharide of Hericium erinaceus(HEP10)was isolated by DEAE-Sepharose Fast Flow ion exchange column and Sephadex G-75 dextran gel column chromatography from HECP.The Mw of HEP10 was 9.9 kDa.HEP10 was consisted of 8 monosaccharide,including glucose(69.2%),galactose(14.7%),rhamnose(5.2%),xylose(4.7%),amino-galactose(2.0%),mannose(1.7%),fucose(1.6%),glucosamine(0.9%).The results of IR and NMR showed that HEP10 is composed of α and β glycosidic linkage of pyranose,suggesting that glycosidic linkage was(1→2)and(1→6).(2)Evaluation about the anti-inflammatory activity of HEP10 in vitro.The inflammatory model of cell was established by using 1 μg/mL LPS stimulate RAW264.7 to evalute the anti-inflammatory activity of HEP10.The result showed that HEP10 has no significant effect on cell viability at concentration of 12.5-400 μg/mL,and can inhibit nitrogen monoxide(NO)secretion at 50-200 μg/mL.Meanwhile,the secretion of tumor necrosis factor(TNF)-α and interleukin(IL)-1β was inhibited and gene transcription levels were down-regulated,but IL-6 secretion and gene transcription level did not significantly be affected when 25-200 μg/mL HEP10 acted on LPS-induced RAW264.7.Western Blot showed that 25-200 μg/mL HEP10 can significantly reduce induced nitric oxide synthase(iNOS)and cyclooxygenase(COX)-2 expression,inhibit the activation of NOD-like receptor(NLRP)3,apoptosis-associated speck-like protein containing CARD(ASC)and Caspase-1 and down-regulate the phosphorylation of NF-κB,PI3K/Akt and MAPK signaling pathway.(3)Effect of HEP10 on dextran sulfate sodium(DSS)-induced colitis in C57BL/6 mice.C57BL/6 mice were induced colitis by 2% DSS to evaluate the therapeutic effect of HEP10 on colitis.Compared with the model group,the change of body weight in the three groups of HEP10(50,100 and 200 mg/kg/d)is significantly low and the colon length were increased by23.7%,29.1% and 40.9% respectively.Meanwhile,the disease index score was improved and the ratio of mg/cm was decreased in colon of HEP10 groups.Histopathological results showed that HEP10(50,100 and 200 mg/kg/d)significantly reduces the histological severity,such as colonic mucosal ulcers,epithelium disruption and a significant immune cell infiltration.The NO level in serum and the malondialdehyde(MDA)level in colon of colitis mice were decreased,the activity of myeloperoxidase(MPO)was inhibited and the activity of total superoxide dismutase(T-SOD)was increased,which showed that HEP10 has an obviousaffect on relieving oxidative stress.qRT-PCR and Western Blot showed that HEP10(50,100 and 200 mg/kg/d)decreases the secretion of TNF-α,IL-6 and IL-1β and inhibits the expression of i NOS and COX-2 induced by inflammatory reaction.Meanwhile,HEP10 reduced the production of inflammasome NLRP3,ASC and Caspase-1 and down-regulated the phosphorylation of protein on NF-κB,PI3K/Akt and MAPK signaling pathway,which means that HEP10 can ameliorate inflammation of colitis by inhabiting the activation of signaling pathways.In summary,the experiment proved that HEP10 has anti-inflammatory activity to improve the symptoms of colitis inflammation,and its mechanism may be that HEP10 inhibits oxidative stress to improve tissue damage,down-regulates the phosphorylation level of inflammation-related signal pathway to reduce the expression of inflammatory factors,and inhibits the activation of inflammasome,thereby slows the process of colitis inflammation.