Characterizations Of Mycelium Polysaccharides From Pleurotus Geesteranus And The Mechanisms On Improving The Alcoholic Liver Disease In Mice

As a form of liver injury caused by long-term/excessive alcohol consumption,the incidence rate of alcoholic liver disease(ALD)is increasing year by year,becoming one of the common diseases that seriously threaten human health.Currently and clinically,the main therapy against ALD was pharmacotherapy,but it was limited in application and promotion owing to the easily accompanied by side-effects.Therefore,it was of great significance to clarify the pathological mechanisms of ALD and explore effective intervention strategies.Pleurotus geesteranus,an edible mushroom with delicious taste and higher nutrition,showed potential effects of antioxidant,anti-aging,hypolipidemic and immunomodulatory.However,scarce literature about the protective effects and mechanism of polysaccharides on ALD had been published.In the present work,the characterizations of P.geesteranus mycelium polysaccharides(PGPs)were conducted firstly.And the ALD mice models using Lieber-De Carli liquid diet were established to explore the hepatoprotective effects against ALD of PGPs based on the liver-intestinal axis,aiming to provide new ideas for the exploitation of natural hepatoprotective drugs.The main results were listed as follows:(1)P.geesteranus mycelium was obtained by the liquid submerged fermentation,and the biomass was 4.57±0.18g/L.After defatting,extraction,deproteinization,separation,purification and freeze-drying,the PGPs with the content of 83.23±0.38%was obtained.(2)Fourier transform infrared spectroscopy(FTIR),methylation and gas chromatography-mass spectrometry(GC-MS)analysis presented that PGPs were pyranose rings linked by?-and?-glycosidic bonds,and predominantly bonded with?4)-Glcp-(1?and Glcp-(1?.The attribution of PGPs was analyzed by multi-dimensional nuclear magnetic resonance(NMR)spectra including ~1H,¹³C,COSY,HSQC,HMBC,NOESY,TOCSY and DEPT135,and the repeat units of PGPs were preliminarily deduced and shown as follows:(?)(3)The morphological characteristics exhibited PGPs were shown obvious aggregation effects with interlocked lamellar structures under the scanning electron microscope(SEM)and aggregated conical shapes under atomic force electron microscopy(AFM).(4)The toxicity tests showed no poisoning or death,as well as normal behavioral activities and the serological parameters were observed during the test period,indicating PGPs was the safe and non-toxic natural active ingredient.(5)The ALD models in mice were established successfully,and the results demonstrated that PGPs showed potential hepatoprotective effects by effectively protecting hepatocyte structure,significantly inhibiting oxidative stress and inflammatory stress,and regulating lipid metabolism disorder.(1)PGPs could significantly inhibit alcohol-induced liver swelling,reduce liver index,and significantly reduce the serum activities of alanine transaminase(ALT)and aspartate aminotransferase(AST).(2)The liver histopathology and transmission electron microscopy(TEM)ultrastructure showed that PGPs could effectively reduce alcohol-induced hepatic lesions including lipid droplet accumulation,cell swelling,cell boundary disappearance,nuclear shrinkage,endoplasmic reticulum expansion,ribosome abscission and inflammatory cell infiltration.(3)PGPs could inhibit the disorder of alcohol metabolic enzyme system by down-regulating cytochrome P450 2E1(CYP2E1)and up-regulate alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH).(4)PGPs could effectively reduce over-accumulations of reactive oxygen species(ROS)and reduce oxidative stress damage through the nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway by promoting the nuclear translocation of Nrf2,up-regulating the antioxidant activities of enzymes such as heme oxygenase-1(HO-1),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT),and inhibiting the production of malondialdehyde(MDA).(5)PGPs could improve the disorder of lipid metabolism and reduce lipid accumulation.It could activate the signaling pathway of AMP-activated protein kinase(AMPK),inhibit expressions of sterol regulatory element binding proteins-1c(SREBP-1c)and acetyl Co A carboxylase(ACC),and up-regulate peroxisome proliferator-activated receptor-?(PPAR?)and sirtuin type 1(SIRT-1),with the results of reducing fatty acid synthesis,accelerating fatty acid oxidative decomposition,and lowing the levels of liver lipid and blood lipid including triglyceride(TG)and total cholesterol(TC).(6)PGPs showed effects on reducing liver inflammation by inhibiting the activation of myeloid differentiation factor 88(My D88)-dependent TLR4/NF-?B(Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-?B))signaling pathway,reducing the release of inflammatory factors including tumor necrosis factor-?(TNF-?)?interleukin(IL-1??IL-6,and IL-8).(6)According to the“liver-gut axis”theory,we further evaluated the protective effects of PGPs on colon in ALD mice.(1)PGPs could significantly improve the pathological changes caused by alcohol,such as thinning of colonic mucosa,severe shedding of intestinal villi,vacuolation of mitochondria and disappearance of tight junctions.(2)The western blot results showed that PGPs significantly protected intestinal barrier by up-regulating the expressions of tight junction proteins(zona occludens-1(ZO-1),Occludins,Claudin-1 and Claudin-4),and mucins(MUC1,MUC2 and MUC4).(3)The high-throughput sequencing results presented that PGPs could affect the Alpha and Beta diversity of gut microbiota,alter the gut microbial community at the Phylum and Genus levels,inhibit the expansion of intestinal LPS(lipopolysaccharide)-producing bacteria,increase the abundance of short chain fatty acids(SCFAs)-producing bacteria,and maintain the balance of intestinal flora,thereby inhibiting the intestinal leakage of LPS,and maintaining the body homeostasis.In conclusion,PGPs showed potential hepatoprotective effects against ALD by reducing oxidative stress,regulating lipid metabolism,and inhibiting the hepatic-inflammations through the Keap1/Nrf2,AMPK/SREBP and TLR4/NF-?B signaling pathways.Besides,PGPs could significantly improve the internal environment of the liver and gut by reducing the intestinal permeability,protecting the integrity of the intestinal barrier,reducing the LPS intestinal leakage,and regulating the gut microbial.All the conclusions confirmed that PGPs showed obvious hepatoprotection against ALD.