Study On The Hypolipidemic Efficacy Of Inonotus Hispidus And Its Purified Polysaccharides

Hyperlipidemia(HLP)is a chronic metabolic disorder characterized by elevated circulating levels of lipids or lipoproteins in the body.Abnormal lipids in the body can lead to the development of atherosclerosis and cardiovascular disease.Dyslipidemia is mainly caused by high-fat diet and other diseases in life.Drugs such as statins,bile acid chelators,betulinic acid and niacin are commonly used to treat HLP,but can cause serious side effects or adverse reactions such as muscle disease,liver damage,epigastric distress and reversible liver dysfunction.Therefore,there is an urgent need to develop a strategy with few side effects and good therapeutic efficacy to treat HLP.Edible fungi are rich in nutrients,such as polysaccharides,proteins,and vitamins,and their long-term consumption will improve human health.Extracts or powders of edible fungi are often used for prevention,relief,treatment or providing a balanced diet in patients with several diseases.In addition,edible fungal polysaccharides have the ability to repair the intestinal barrier,improve the structure and composition of the intestinal microbiota,and are degraded by the enzymes produced by microorganisms into small molecules that can be easily absorbed,thus improving the body’s metabolism.Inonotus hispidus(Bull.:Fr.)P.Kurst(IH),also known as Inonotus hispidus,belongs to the genus Inonotus of the subphylum Ciliophora and is an edible fungus.Studies by domestic and foreign scholars have found that IH has a variety of bioactive components,such as polysaccharides,polyphenols,terpenoids,steroids,etc.,and has initially shown multiple pharmacological activities such as hypolipidemic,hypoglycemic,antiinflammatory,antioxidant,antitumor,antibacterial and antiviral.Although IH substrates are rich in polysaccharides,the establishment of isolation and purification methods,structural characterization and hypolipidemic efficacy in vivo of polysaccharides have not been reported systematically.Therefore,in this study,we used a high-fat diet(HFD)-induced HLP mouse model to clarify the hypolipidemic efficacy and related mechanisms of IH and its isolated and purified polysaccharide IHP3,providing experimental and theoretical support for the full exploitation of IH resources.However,the effective active ingredients and pharmacological mechanisms of this edible fungi still need to be further investigated to fully explore and utilize this valuable resource.Therefore,in this study,we used the HFD-induced HLP mouse model to clarify the hypolipidemic efficacy mechanism of IH and its purified polysaccharides IHP3 to provide experimental and theoretical support for the full exploitation of IH resources.Thus,the specific studies are as follows.1.Study on the hypolipidemic effect of IH on HLP mice.Firstly,after 8 weeks of HFD feeding to establish HLP mouse model,then 8 weeks of IH administration treatment significantly inhibited the weight gain and fasting blood glucose level in HLP mice,reversed the pathological state of liver and three kinds of fat(groin,epididymis and perirenal),and significantly decreased the expression levels of low density lipoprotein cholesterol(LDL-C),total cholesterol(TC),triglyceride(TG)and leptin(LEP)and increased the expression level of high-density lipoprotein cholesterol(HDLC)in serum.Meanwhile,IH reduced liver injury and thus achieved hepatoprotection by regulating the expression levels of liver and serum markers of liver injury such as alanine aminotransferase(ALT),aspartate aminotransferase(AST),inflammatory factors such as interleukin(IL)-1β,IL-6,tumor necrosis factor α(TNF-α),plasminogen activator inhibitor-1(PAI-1)and monocyte chemoattractant protein-1(MCP-1),and oxidative stress-related factors such as reactive oxygen species(ROS)and malondialdehyde(MDA).In addition,the combination of intestinal flora analysis and serum lipidomics revealed that IH significantly modulated the composition of intestinal microbiota,improved the relative abundance of microbiota at different taxonomic levels,and modulated serum lipid levels,respectively.Finally,Western blot results confirmed that IH could regulate the nuclear factor erythroid-2-related factor 2/ Nuclear factor kappa-B(Nrf2/NF-κB)signaling pathway to mediate inflammation and oxidative stress,which in turn regulated the intestinal flora and improved the body’s lipid levels,achieving the hypolipidemic effect.2.Isolation,purification and structural characterization of polysaccharides from IH substrates.The IH polysaccharide was extracted by the extraction temperature of 90 ℃,extraction time of 3 h,extraction material-liquid ratio of 1:30,and extraction times of 2 times.The crude polysaccharide IHP was obtained by deproteinization,dialysis,concentration,alcohol deposition and lyophilization of the extracts.The polysaccharide yield of IHP was 0.81%,and the total sugar content of IHP was 51.30%.Then,the crude polysaccharide IHP was purified using anion exchange chromatography column DEAE Bestarose FF for two separations to obtain IHP1.The purified polysaccharide IHP2 was obtained by molecular exclusion purification of IHP1 using a gel filtration chromatography column Sephacryl TM S-400.Finally,IHP2 was purified using a gel filtration chromatography column Finedex 200 pg to obtain the purified polysaccharide IHP3 with relatively concentrated and homogeneous molecular weight.High performance liquid chromatography(HPLC),gel permeation chromatography(GPC),fourier transform infrared spectroscopy(FT-IR),ultraviolet analysis(UV),nuclear magnetic resonance(NMR)and scanning electron microscope(SEM)were used to characterize the monosaccharide composition,molecular weight analysis of polysaccharides,the type of glycosidic bonding between monosaccharides,and the branching structure and molecular conformation of polysaccharides of IHP3,respectively.The results showed that the IHP3 was almost free of nucleic acid and protein impurities,contained O-H,C=O,and C-O stretching vibration absorption peaks,and was composed of monosaccharides such as Gal,Man,Fuc,Glc,Glc-UA,Rha,and Man-UA,with monosaccharide molar ratios of 48.84:21.85:17.88:5.93:5.46:0.03:0.01,which indicates that IHP3 is mainly composed of Gal,Fuc,and Man.The GPC results showed that IHP3 has a uniform single peak with a molecular weight range of 10.00 k Da-27.48 k Da,accounting for 99.50%.The molecular weight(Mw)of IHP3 is 19.16 k Da and the polydispersity index(Mw/Mn)is 1.02,which indicates that IHP3 has a small molecular weight and good homogeneity.Combined with the results of NMR analysis showed that the purified polysaccharide IHP3 was inferred to be a heteropolysaccharide,and the main chain structure was mainly composed of →6)-α-D-Galp-(1→ and →2,6)-α-D-Galp-(1→,with a branched structure at the O-2 position of the →2,6)-α-D-Galp-(1→ sugar residue.The SEM results showed that IHP3 consisted of irregular lamellar and striated composition of mixed structure with different sizes and shapes,smooth surface morphology,and showed amorphous structure.3.Study on the hypolipidemic effect of IHP3 in HLP mice.Firstly,after 8 weeks of HFD feeding to establish HLP mouse model,then 4 weeks of IHP3 administration treatment significantly inhibited the weight gain and fasting blood glucose level in HLP mice,reversed the pathological state of liver and four kinds of fat(groin,epididymis,perirenal and scapular),and significantly reduced the expression levels of LDL-C,TC and TG in serum and increase the expression levels of HDL-C expression levels.Meanwhile,IHP3 inhibited liver injury by suppressing AST and ALT expression levels in the liver and serum of HLP mice.In addition,the results of intestinal flora metabolite analysis showed that IHP3 administration significantly up-regulated four metabolites such as 12(13)-epoxy-9z-octadecenoic acid and linolenic acid,and down-regulated eight metabolites such as gamma-rhamnocholic acid,ceramide(d18:1/18:1(9Z)),Dribulose-1-phosphate,and 12-keto-deoxycholic acid.Among them,linolenic acid showed positive correlation with 12(13)-epoxy-9z-octadecenoic acid,while ceramide(d18:1/18:1(9Z))showed positive correlation with linolenic acid and 12(13)-epoxy-9zoctadecenoic acid.Liver proteomic analysis showed that 22 proteins were significantly up-regulated and 12 proteins were down-regulated in liver protein expression of mice after IHP3 administration.The GO enrichment analysis of the above differential proteins concluded that IHP3 is involved in biological processes such as acute phase response,acute inflammatory response,and complement activation in the liver of HLP mice.Meanwhile,it was screened with Acetyl-Cco Aa carboxylase1(ACC1),a key enzyme for lipid synthesis,and haptoglobin(Hp),orosomucoid 1(ORM1)and serum amyloid a1(Saa1),acute inflammatory response proteins.In addition,IHP3 promoted white adipose(WAT)browning and increased brown adipose(BAT)activity by inhibiting the phosphorylation of PPARγ,which effectively improved the thermogenesis in HLP mice.Finally,the results of ELISA,IHC and Western blot showed that IHP3 could regulate IL-17 signaling pathway to improve the inflammatory response,and then regulate the lipid level in HLP mice to achieve the effect of hypolipidemia.In conclusion,this study used the HFD-induced HLP mice model to clarified the hypolipidemic efficacy of IH substrates was associated with intestinal microbial composition,oxidative stress and inflammatory response;it was clarified that the Mw of purified polysaccharide IHP3 is 19.155 k Da and its structural backbone structure is mainly composed of →[6)-α-D-Galp-(1→6)-α-D-Galp-(1]n→ and →[6)-α-D-Galp-(1→2,6)-α-D-Galp-(1]n→,with a branched structure present at the O-2 position of the→2,6)-α-D-Galp-(1→ sugar residue.The branched structures may include α-D-Manp-(1→,α-D-Manp-(1→6)-β-D-Manp-(1→,α-L-Fucp-(1→6)-β-D-Manp-(1→,α-DManp-(1→2)-α-L-Fucp-(1→6)-β-D-Manp-(1→.In addition,it was further clarified that IHP3 suppressed the inflammatory response,inhibited PPARγ phosphorylation,promoted WAT browning and activated BAT activity in HLP mice by regulating IL-17 signaling pathway,and regulate the expression of intestinal microbial metabolism thereby alleviating the symptoms of hyperlipidemia.Therefore,this study provides a theoretical basis for the therapeutic mechanism and the development of related drugs of IH and its purified polysaccharide on HLP.