Study On The Modulation Of Lipid Metabolism By Grifola Frondosa And Its Polysaccharides

Hyperlipidemia,a form of dyslipidemia is a chronic metabolic disease that poses a substantial danger to human health by causing thrombosis,coronary heart disease,and other cardiovascular diseases.Current therapeutic options involve the usage of chemical synthetic medicines,however,long-term use of hypolipidemic drugs may cause several debilitating side effects,including rhabdomyolysis,hepatotoxicity,and new-onset diabetes.The development of cheaper novel treatments for hyperlipidemia that entail fewer adverse effects therefore,is a pressing need.Edible medicinal fungi exhibit multiple beneficial activities and have a long history of application in disease prevention and treatment.The mushroom Grifola frondosa(GF)grows primarily in Asia,Europe,and eastern North America,and is widely consumed in China and several other Asian countries on account of its anti-hyperlipidemic,anti-diabetic,anti-hypertensive,anti-inflammatory,anti-oxidative,and immunomodulatory properties.While current research about GF focuses on liquid fermentation of the strain,analysis of nutritional components,separation and purification of constituent polysaccharides,and assessment of biological activities,no systemic research on anti-hyperlipidemic effects that are exerted by GF or its purified polysaccharide components,via modulation of inflammation is known.The analysis of the main components of GF as part of our study revealed that it comprises 44.30%total sugar,22.50%reducing sugar,15.90%crude protein,10.80%crude fiber,5.60%total ash,4.80%crude fat,0.90%total saponins,0.76%mannitol,0.30%total phenol,0.25%total triterpenoids,0.21%total sterol,0.21%total flavonoids,and0.03%total alkaloids.These findings indicate that while the major components of GF are carbohydrates and proteins,it contains lower quantities of fat.Of the 11 vitamin types that were detected,vitamins B₃,B_1,and C were the most abundant.Additionally,nine minerals,including potassium,magnesium,iron,calcium,sodium,zinc,copper,manganese and selenium were identified,of which potassium(2.2×10⁴ mg/kg)was present in the highest amount.The concentrations of heavy metals including lead,cadmium,and mercury were within the acceptable limits of contaminants in food as defined by the state criteria(GB2762-2017),which ensures the edible security of GF.The second aspect of our study involved investigation of the effects of GF on lipid metabolism in high fat diet(HFD)fed mice.Administration of GF was found to significantly control weight gain and blood glucose,as well as restore related organ indices in these mice.Additionally,it regulated the levels of factors related to lipid metabolism and insulin resistance,restored the morphology of adipose and pancreatic tissues,and improved dyslipidemia and insulin resistance apart from alleviating hepatic steatosis,and regulating levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),and associated hepatic inflammatory factors,thereby demonstrating hepato-protective properties.An analysis of the intestinal microflora of HFD fed mice revealed a significant reduction in the abundance of 20 bacteria,including Streptococcus,Staphylococcus,and Mycobacterium along with an increase in the relative abundance of 6 bacteria,including Ruminococcus post GF administration.GF was further found to regulate the heme biosynthesis superpathway that is associated with inflammation according to the Meta Cyc database,which may partially account for its hypolipidemic effects.Lipidomic analysis revealed regulation of 11 metabolites levels by GF,including that of ceramide(Cer),which is relevant in the context of inflammation and insulin resistance in HFD fed mice.Enzyme linked immunosorbent assay(ELISA)results in conjunction with western blot analysis,revealed decreased serum Cer levels in HFD fed mice,that was accompanied by suppression of toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-?B)and nucleotide-binding domain,leucine-rich-containing family,pyrin domain containing 3(NLRP3)signaling pathways that are mediated by ceramide,as a consequence of GF administration.The optimal extraction of crude polysaccharides from GF(GFP),with a yield rate of4.4%,by single factor experiments was obtained under the following conditions:extraction temperature:80°C,extraction time:120 min,and solid-liquid ratio:1:40(g/m L).Extracts from two consecutive extractions were combined,concentrated,deproteinized with Sevag reagent,and precipitated with 80%alcohol.The GFP obtained was purified using a diethylaminoethyl cellulose anion exchange column to obtain GFP2with a yield rate of 19.4%(based on GFP).GFP2 was further purified using an Ez Load26/60 Chromdex 200 column to obtain GFPA with a yield rate of 7.6%(based on GFP2).The structure and physicochemical properties of GFPA were subsequently analyzed.The lack of absorbance peaks at 260 nm and 280 nm indicated the absence of any obvious nucleic acid and protein component in GFPA.The number average molecular weight and the weight average molecular weight of GFPA were 4188.9 k Da and 5570.2 k Da respectively,and the polydispersity index was 1.33,which indicated a relatively homogeneous molecular weight.GFPA was determined to have a molecular configuration of irregular coils,thus suggesting the existence of branches,and was found to consist of glucose,galactose,and fucose with a molar ratio of 99.73:0.17:0.10.The Fourier-transform infrared spectrum of GFPA showed peaks characteristic of polysaccharides,such as O-H stretching vibration,C-H stretching vibration,C=O stretching vibrations,C-O stretching vibrations,and O-H variable angle vibration.The major glycosidic residues in GFPA were found to be?-D-Glcp-(1?,?4)-?-D-Glcp-(1?and?4,6)-?-D-Glcp-(1?.Nuclear magnetic resonance was employed to decipher the structure,glycosidic bond compositions,and main connection modes of GFPA.Further,the presence of an irregular branched network structure on the surface of GFPA was confirmed by scanning electron microscopy.Finally,the regulatory effect of GFPA on lipid metabolism via modulation of inflammation was studied in HFD fed mice.The results showed that GFPA administration efficiently reversed body weight gain and blood glucose concentration in these mice.The purified polysaccharide counteracted hyperlipidemia by normalizing triglyceride,total cholesterol,low-density lipoprotein cholesterol,and high-density lipoprotein cholesterol levels in serum and liver,while simultaneously decreasing adipocyte size and ameliorating inflammatory infiltration of adipose tissues.GFPA also alleviated hepatic steatosis in these mice by decreasing serum and hepatic AST and ALT levels,hepatic fat accumulation,and inflammatory infiltration.Further,GFPA was found to regulate lipid metabolism via downregulation of ceramide levels in HFD fed mice.While ELISA based assays demonstrated a decrease in the serum and hepatic levels of Cer,TLR4,myeloid differential factor 88,tumor necrosis factor receptor-associated factor 6,tumor necrosis factor-?,interleukin(IL)-6,and IL-1?,western blotting analysis confirmed inhibition of the TLR4/NF-?B signaling pathway by GFPA.The above-mentioned findings led us to conclude that:(1)GF contains multifarious nutritive components.The fungus has high carbohydrate and protein content,of which total sugar(44.30%)is the most abundant,and low fat content;(2)GF regulates lipid metabolism via inhibition of chronic inflammation and insulin resistance that is mediated by ceramide in HFD fed mice;(3)GF contains a macromolecular polysaccharide with a branched structure that primarily contains the glycosidic residues?-D-Glcp-(1?,?4)-?-D-Glcp-(1?and?4,6)-?-D-Glcp-(1?,which was successfully purified and termed GFPA;(4)GFPA regulated lipid metabolism via suppression of the TLR4/NF-?B inflammatory pathway that is mediated by ceramide in HFD fed mice.Our study will serve as the experimental basis for the utilization of GF and its polysaccharide constituents,for the development of therapeutic agents to treat hyperlipemia.