Characterization And Mechanism On Renovating Diabetic Nephropathic Of Mycelia Polysaccharides From Coprinus Comatus

Coprinus comatus(C.comatus),which belonged to the family of Coprinusaceae and genus of Coprinus,was a fungus riching in various nutrients,not only a kind of high-quality food,but also a kind of folk medicine.As one of the most important physiologically active substances,the polysaccharides played vital roles in maintaining the biological effects of C.comatus.Previous literatures had been mainly focused on the polysaccharides from fruiting-bodies,however,scare report on the isolation,purification,structural characteristics,hypoglycemic activity and the ability to relieve diabetic nephropathy of the polysaccharides from the mycelia of C.comatus had been published.Thus,in the present work,two components,CMP and N-CMP,were isolated and purified form the crude C.comatus mycelia polysaccharides(C-CMP).The in vitro antioxidant analysis and simulated digestion experiments of the two components were evaluated,providing the evidence for chosing the potential candidate of CMP for further animal experiments.Meanwhile,the CMP was characterized by physicochemical analysis and the in vitro hypoglycemic abilities was evaluated by inhibiting?-amylase and?-glucosidase activities,respectively.Additionally,the protective effects and potential mechanisms of CMP on lowering blood glucose and blood lipids,improving energy metabolism disorders,and protecting the hyperglycemia-induced kidney damages have been investigated.The present results could provide evidencs in developing and utilizating of polysaccharides from edible fungus on the treatment of diabetes and its complications,theoretically.The main results were listed as follows:(1)The biomass of C.comatus mycelia with 2.23±0.43g/L were obtaind by liquid submerged fermentation technology.After extracting by hot water and defatting,two polysaccharide fractions,CMP and N-CMP,were islated and purified from C-CMP by DEAE-52 chromatography with the yields of 29.4%and 15.8%,repspectively.Furthermore,the gel permeation chromatography(GPC)analysis showed that the weight average molecular weights of CMP and N-CMP were 495.86kDa and 1.12kDa,respectively.(2)The in vitro antioxidant and in vitro digestion experiments showed that the half scavenging concentration(IC₅₀)of CMP to hydroxyl radicals and DPPH free radicals reached 0.981mg/mL and 1.092mg/mL,respectively,which showed better effects than N-CMP.Besides,the results of digestion experiments indicated that simulated gastric juice and intestinal juice have non-significant effects on CMP.The molecular weight(Mw)of CMP was decreased from 491.48kDa to 423.09kDa and the reducing sugar content was increased from 0.395mg/g to 0.877mg/g.However,the N-CMP cannot exist stably during the tertiary digestion process,and its Mw was reduced from 1.12kDa to 171Da,which was close to the monosaccharides level,while the content of reducing sugars increased from 0.449mg/g to2.480mg/g.The results indicated that the CMP has stronger stability than N-CMP and the potential to enter the body to exert biological effects.(3)Ion chromatography(IC)results showed that the CMP was composed of fucose,ribose,arabinose,xylose,mannose,galactose and glucose.The galactose accounted the highest proportion,which was the main component in the polysaccharide chain.The methylation and infrared measurement(FT-IR)analyses indicated that CMP,with the?-type glycosidic bonds and pyranose rings in the main chain,was mainly consisted of?6)-Galp-(1?,?2,6)?Manp(1?,and-Galp-(1?,and the total proportion of the three residues was more than 78%.The atomic force microscope(AFM)results showed that CMP existed by multi-branched network structures in the solution,with the main chain length ranging from 50nm to 200nm,the single chain thickness ranging from 2nm to 6nm,and the width of the single chain about 16nm.(4)The in vitro hypoglycemic analysis showed that CMP could significantly inhibit the?-amylase and?-glucosidase activities.The IC₅₀of CMP on?-amylase and?-glucosidase reached 6.84mg/m L and 2.26mg/m L,respectively.The Lineweaver-Burk curve analysis showed that CMP exerted inhibitory activities on?-amylase and?-glucosidase in a mixed inhibition type,indicating that CMP had hypoglycemic activity,and could be used in the further research.(5)The model of diabetic nephropathy mice was established by streptozotocin(STZ)injection combining with high-fat diet.After the 60-days continuous intervention of CMP,we found that CMP could significantly improve insulin resistance and energy metabolism,inhibit renal dysfunction,reduce renal oxidative stress and inflammation,repair renal podocyte damage,and delay the process of renal fibrosis in diabetic nephropathy mice.The detailed results were listed as follows:(1)CMP could improve the symptoms of insulin resistance by reducing the levels of fasting blood glucose(FBG),fasting insulin(FINS),glycated serum protein(GSP)and homeostasis model assessment of insulin resistance(HOMA-IR)in diabetic nephropathy mice.(2)CMP could reduce the contents of total cholesterol(TC),triacyl glycerols(TG),and low-density lipoprotein cholesterol(LDL-C)in serum,inhibit the accumulation of blood lipids,and lowed blood lipids.(3)CMP could up-regulate the phosphorylation levels of adenosine monophosphate activated protein kinase?(AMPK?),decrease the contents of serum leptin(LEP),and increase the levels of adiponectin(ADPN),correcting the energy metabolism disorder in diabetic mice.(4)CMP could promote the m RNA expressions of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)through the Nrf2/Keap1 pathway,enhance the endogenous antioxidant enzyme activity,reduce the level of malondialdehyde(MDA),and relieve the kidney damage caused by reactive oxygen species(ROS).(5)CMP could reduce the release of tumor necrosis factor-?(TNF-?),interleukin-1b(IL-1b)and interleukin-6(IL-6)by inhibiting the activation of TLR4/NF-(?)B signaling pathway,thus improving the inflammatory stress in the kidney.(6)CMP could promote the expression of PTEN protein in a dose-dependent manner,and negatively regulate the PI3K/AKT signaling pathway.Meanwhile,CMP could also reduce the accumulation of the key protein of?-catenin in the Wnt/?-catenin signaling pathway in the cytoplasm,reducing high glucose environmental damage to renal podocytes.(7)CMP could prevent or reverse the renal fibrosis by inhibiting the transduction of TGF-?1/Samd3 signaling pathway and reducing fibrosis related factors such as cystatin C(Cysc),transforming growth factor?1(TGF-?1)and tissue inhibitors of metalloproteinase 1(TIMP-1).The results suggested that polysaccharides form C.comatus could be used as functional foods/drugs on preventing diabetic nephropathy.