Protective Role Of Hericiumerinaceus Polysaeeharides On Gastrointestinal Mucosal Barrier Function

ObjectiveFour classical grastric moucal lesion rat models were set up respectively by alcohol, indomatacin, acetic acid and water immersion stress methods.And then relative indicators were measured in order to study the protective effect and mechanism of Hericiumerinaceus Polysaeeharides (HEP) against gastric mucosal lesion, guiding the effective adding amount in fromular accordingly.By developing acute inflammation model of Caco-2cells and co-cultured Caco-2/RAW264.7system induced by LPS,we assessed the protective role of HEP on intestinal mucosal barrier function through in vitro experiment system.Methods1. Protective effect and mechanism of HEP aginst gastric mucosal lesion.Make sure the experimental dosages with ulcer area in grastric moucal lesion model induced by anchol.SD rats were divided into normal group, model control group, positive group and HEP groups. The minimun experimental dosage (HEP1) was set five times as the human body’s lowest dosage (0.2g/d), the maximum one (HEP4) was set as twenty times as the human body’s highest dosage (0.4g/d), and two gradients (HEP2, HEP3) were set between them.Dose Settings:HEP117mg/kg, HEP268mg/kg, HEP3102mg/kg, HEP4136mg/kg. For further study, another three models were adopted to observe the protective effect and mechanism of HEP against gastric mucosal lesion which were stimulated respectively with indomatacin, acetic acid and water immersion stress.Male SD rats, weighting from180to220g, were used and divided into normal group, model control group, positive group, HEP dose groups. Drugs were administered for a total of10days, the control group given amount of distilled water.(1) one hour after the last dilivery, except normal group, rats were administered indomethacin80mg/kg, normal group given equal amount of water. Afer7hour, Rats were sacrificed by cervical dislocation.Stomaches were taken out and the ulcer area were recored by Image J software.Concentrations of prostaglandin E2(PGE2) and epidermal growth factor (EGF) in gastric mucosa tissue homogenate supernatant were measured by enzyme-linked immunoassay. (2) Before administration, chronic gastritis rat model was prepared with30%acetic acid by grastric serosal injection. Twety-four hours after the last delivery, Rats were sacrificed by cervical dislocation.Stomaches were taken out and the ulcer area were recored by Image J software. The concentrations of basic fibroblast growth factor (bFGF) in gastric mucosa tissue homogenate supernatant was measured by enzyme-linked immunoassay and the expression level of transforming growth factor-a mRNA(TGF-a) was evaluated with RT-PCR method.(3) Half hour after the last dilivery, except normal group, rats were fixed into the metal mesh cages, vertically immsed into the water (temperature is20℃) for6h, then rats were anesthetized with chloral hydrate by intraperitoneal injection.open anterior wall and pull part of stomach out,scratch the body and antrum of stomach in a0.5cm transverse incision, grastric moucal blood flow(GMBF) in these two points were determinated with the laser Doppler perfusion monitors, and the mean values were used for evaluation.Gastric mucosa ulcer area also measured later.2. Protective effect of HEP aginst acute inflammation in Caco-2cell model.Caco-2cells were recovered and cultured on Transwell plate with DMEM solution (pH7.4, containing10%FBS,2mmol·I-1L-glutamine,10mmol·l-1hepes, Neaa, penicillin&streptomycin),in condition of37℃and5%CO2for24h incubation. The adherent cells approached confluence and were passaged after4-6days.Then micro-morphological observation, transmembrane potential detection, Papp measurment and alkaline phosphatase activity test were used to evaluate the cell. The acute infla-mmation response of caco-2and co-cultured caco-2/RAW264.7cells were stimulated by lipopolysaccharide,drug intervention for24h,then downside liquid were collected, and productive inflammatory cytokines were measured by Elisa.(1) Caco-2cells were cultured for15days; LPS50pg/ml and HEP500ug/ml were added into upside solution successively. The contents of IL-6, IL-8, IL-10, IL-12, TNF-a and IFN-y in productive liquid were determined.(2) Caco-2cells were cultured for15days, another species cell RAW264.7were inoculated under the transwell plate, co-culture for24h.LPS50pg/ml and HEP500ug/ml were added into upside solution successively. The contents of IL-6, IL-8, IL-10, IL-12, TNF-a and IFN-y in productive liquid were determined.Results1. Protective effect and mechanism of HEP aginst gastric mucosal lesion.(1) Dose-screening test results show that17mg/kg,68mg/kg and136mg/kg of HEP all had an inhibitory action on acute gastric mucosa injury.17mg/kg and68mg/kg were chosed as low and high dose of HEP in the following experiments.(2) Gastric mucosal ulcer area induced by acetic acid was reduced, and concentrations of PGE2and EGF in gastric mucosa tissue homogenate supernatant were increased both in low and high dosage of HEP groups.(3)Gastric mucosal ulcer area induced by indomatacin was reduced.The concentrations of bFGF and the expression level of TGF-α Mrna in gastric mucosa tissue homogenate supernatant were increased both in low and high dosage of HEP groups.(4) Gastric mucosal ulcer area induced by water immersion stress was reduced, and GMBF were increased both in low and high dosage of HEP groups.2. Protective effect of HEP aginst acute inflammation in Caco-2cell model.(1) IL-6, IL-8, IL-10, IL-12, TNF-a and IFN-y secretion were significantly increased in actue inflammation cell model of caco-2and co-culture Caco-2/RAW264.7system induced by LPS.(2) HEP acted though approaches of inhibiting IL-6、IL-12secretions, promoting IL-10secretion and adjusting others cytokines such as IFN-γ,in order to relieve the inflammatory response of cell model and protect intestinal mucosal barrier.ConclusionHericium polysaccharide (HEP) acted as a protective and improving role in four grastric moucal lesion rat models using absolute enthanol, indomatacin, acetic acid and water immersion stress methods respectively. The mechanism study found that GMBF, content and secretion of PGE2, EGF and bFGF were enhanced as well as the expression level of TGF-a in low and high dosage of HEP groups.In conlusion, HEP could acts effectively against grastric moucal lesion through those approaches above.HEP has a benign effect against the actue inflammatory response in caco-2and co-culture caco-2/RAW264.7cells stimulated with LPS. HEP acted by inhibiting inflammatory cytokines secretion and promoting the anti-inflammatory cytokines secretion, suggesting that the mechanism related to its physiological activity of immune regulation.