Structural Characterization And Mechanism On Improving Dyslipidemia Of Mycelia Polysaccharides From Ganoderma Lucidum

As an edible and medicinal fungi preciously,Ganoderma lucidum has been employed in traditional Chinese medicine for thousands of years,and its medicinal value has been widely recognized.Polysaccharides have been considered to be the important bioactive constituents in G.lucidum which showed various biological activities such as anti-obesity,anti-inflammation,antioxidation,antitumor and immunomodulatory effects.As a kind of typical metabolic disease with the increased incidence year by year,hyperlipidaemia is regarded as the critical cause of cardiovascular diseases.Therefore,it is of great importance to promote the prevention and treatment of hyperlipidemia.In this study,t hree fractions were isolated and purified from the crude polysaccharides of G.lucidum mycelia,and GLP was selected for further experiments based on the evaluation of lipid-lowering activities in vitro.The structure of GLP was characterized to explore the structural basis which is beneficial for exerting its bioactivities.The effects and mechanisms of GLP intervention on hyperlipidemia were revealed using a hyperlipidemia mice model induced by high-fat diet,aiming to provide new ideas for the development of new drugs on hyperlipidemia and the further utilization of natural active substances.The main results were listed as follows.（1）The biomass of G.lucidum mycelia was 2.35±0.27 g/L which was obtaind through liquid-submerged fermentation.After hot water extraction,the crude polysaccharides of G.lucidum mycelia were obtained,and then three polysaccharide fractions including GLP,A-GLP and S-GLP were purified by DEAE-52 cellulose anion-exchange column chromatography.The carbohydrates contents of GLP,A-GLP and S-GLP were 96.10%,92.24%and 85.37%,respectively.（2）The in vitro hypolipidemic analysis showed that GLP,A-GLP and S-GLP could bind with sodium glycocholate and sodium taurocholate,adsorb cholesterol and inhibit the activity of cholesterol esterase,which indicated that they all possessed lipid-lowering abilities in vitro.However,GLP had the best lipid-lowering effects comprehensively,and was used in the subsequent experiments in consequence.（3）The UV scanning spectrum showed that GLP did not contain nucleic acid and protein.The scanning electron microscopy（SEM）showed that GLP displayed an irregular flaky structure with some flecks and holes in its unsmooth surface.The results of ion chromatography（IC）indicated that GLP was mianly composed of fucose,galactose,glucose,xylose,mannose,ribose and glucuronic acid with mass percentages of 14.70%,64.21%,17.51%,0.16%,2.21%,0.74%and 0.46%,respectively,and galactose was the main component in the backbone of GLP.The GPC-RI-MALS was used to measure the absolute molecular weight of GLP,and the results showed that GLP was a homogeneous polysaccharide with the Mw of 12.53 k Da,Mn of 11.16 k Da and polydispersity index of 1.12.Fourier transform infrared spectrometry（FT-IR）and methylation analyses manifested that GLP,with pyranose rings linked byα-type andβ-type glycosidic bonds,consisted of 11 kinds of sugar residues which 6-Gal（p）accounted for the highest proportion.Finally,by means of one-dimensional(¹H and ¹³C)and two-dimensional（COSY,NOESY,HSQC and HMBC）NMR spectra,the signal attributions of GLP were revealed,and the possible repeat units of GLP were predicted and displayed as follows.（?）（4）The acute toxicity analysis demonstated that GLP-treated mice did not exhibit any toxic responses,behavioral changes and deaths during the 14 d trial period.Besides,the body weights and serological parameters of GLP-treated mice showed no obvious difference when compared with the mice in control group.The abo ve results suggested that GLP was practically non-toxic and could be used for further in vivo animal experiments.（5）High-fat diet was employed to eatablish the hyperlipidemia mice model,and GLP intervention therapy was performed to evaluate its protective effects.The detailed results were showed as follows.（1）GLP administration significantly decreased the body weight gains,alleviated liver tissue swelling and epididymal fat accumulation but without significant effects on anenterotrophy induced by high-fat diet.（2）GLP could lower the activities of alanine aminotransferase（ALT）and aspartate aminotransferase（AST）in serum,and effectively reverse the abnormal secretion of leptin（LEP）and adiponectin（ADPN）,thus mitigating the liver injury and energy metabolism disorder of mice.（3）GLP markedly decreased the levels of total cholesterol（TC）,triacylglycerol（TG）,low-density lipoprotein cholesterol（LDL-C）,total bile acid（TBA）,free fatty acid（FFA）and apolipoprotein B（Apo B）,while increased high-density lipoprotein cholesterol（HDL-C）,apolipoprotein A1（Apo A1）,hepatic lipase（HL）and lipoprotein lipase（LPL）in serum.Meanwhile,the deposition of TC and TG in liver was alleviated.The results showed that GLP could relieve lipid metabolism disorder by reducing lipid accumulation in the body.（4）GLP could recover the abnormal expressions of lipid metabolism regulatory proteins in liver.By promoting the phosphorylation of adenosine monophosphate activated protein kinaseα（AMPKα）,GLP further inhibited the expressions of sterol regulatory element-binding proteins 1C（SREBP1C）,fatty acid synthase（FAS）and acetyl-Co A carboxylase（ACC）.Furthermore,peroxisome proliferator-activated receptorα（PPARα）was upregulated,while stearoyl-Co A desaturase 1（SCD1）and proprotein convertase subtilisin/kexin type 9（PCSK9）were downregulated,jointly inhibiting the synthesis and desaturation of fatty acid,and accelerating the oxidative decomposition of fatty acid,thereby relieving liver steatosis and regulating cholesterol homeostasis.（5）GLP promoted the expressions of antioxidant enzyme including superoxide dismutase（SOD）,glutathione peroxidase（GSH-Px）,catalase（CAT）and heme oxygenase-1（HO-1）by activating the Nrf2 pathway.At the same time,the generations of malondialdehyde（MDA）and accumulations of reactive oxygen species（ROS）were alleviated to resist the oxidative stress damage.（6）GLP was capable of improving hepatic inflammation via modulating the release of inflammatory cytokines such as tumor necrosis factor-α（TNF-α）,interleukin-1β（IL-1β）,interleukin-6（IL-6）and interleukin-10（IL-10）through negatively regulating TLR4/NF-κB signaling pathway.（7）GLP promoted reverse cholesterol transport by mediating the LXRα-ABCA1/ABCG1pathway.The expressions of cholesterol 7α-hydroxylase（CYP7A1）and mitochondrial steroid27-hydroxylase（CYP27A1）responsible for bile acids production were increased,accompanied by inhibition of colonic FXR-FGF15 pathway,which indicated that GLP could stimulate the conversion of cholesterol into bile acids,thus lowering the cholest erol levels.（8）GLP could significantly improve the impaired colon morphology and recover the intestinal barrier damage by enhancing the expressions of tight junction proteins including Occludin,Claudin-1 and ZO-1.Consequently,the intestinal leakage of LPS was alleviated and the levels of inflammatory cytokines（TNF-α,IL-1βand IL-6）in serum were reduced by inhibiting colonic NF-κB pathway.（9）The 16S r RNA sequencing results showed that GLP intervention had no significant effects on Alpha diversity of gut microbiota,but significantly affected its Beta diversity.The community composition analysis at phylum and genus level indicated that GLP could reduce the ratio of Firmicutes to Bacteroidetes,increase the abundance of beneficial bacteria and inhibit the expansion of harmful bacteria related to inflammation and obesity,thereby changing the composition and structure of intestinal flora.There were 7,7 and 10 key biomarker strains screened by LEf Se analysis in the NC,MC and HGLP groups,respectively,suggesting that these strains might play important roles in maintaining the homeostasis of lipid metabolism,or the occurrence and development of hyperlipidemia.Functional prediction analysis showed that high-fat diet increased the abundance of cardiovascular disease and lipid metabolism,and 18 markedly different pathways were identified.However,GLP significantly increased the abundance of energy metabolism,and cell growth and death pathways.The above results indicated that GLP could improve intestinal flora disorder and maintain intestinal homeostasis.（10）The results of non-targeted metabolomics based on liquid chromatography-mass spectrometry（LC-MS）suggested that GLP had obvious influences on fecal metabolites profile which was involved in 44 differential metabolites in high-fat-diet mice.Amino acid metabolism and lipid metabolism pathway were significantly regulated by GLP,indicating that GLP had a certain regulative effects on the alterations of fecal metabolites in hyperlipidemia mice.In addition,correlation analysis indicated that there was close relationships between intestinal flora,fecal metabolites and hyperlipidemia related parameters.In summary,GLP significantly regulated lipid metabolism associated proteins,eased oxidative stress and inflammation response,promoted reverse cholesterol transport,modulated bile acids metabolism and restored the damaged intestinal barrier function in hyperlipidemia mice.Furthermore,GLP could effectively improve intestinal microbial community disorders and changes of fecal metabolites resulted from high-fat diet.The above findings suggested that GLP showed potential lipid-lowering effects,which may possibly used as a dietary supplement or adjuvant therapy medication for hyperlipidemia.