Studies On Structural Characterization And Hypoglycemic Mechanism Of Enteromorpha Prolifera Oligossaccharide Based On Proteomics

Enteromorpha prolifera belongs to the family of ulva and the Phylum of Chlorophyta.In this paper,based on the analysis and identification of the structure of E.prolifera oligosaccharide(EPO),combined with the results of animal physiology and pathology experiments,by the Tandem Mass Tag(TMT)-based quantitative proteomics,to obtain differential target proteins to analyze the target proteins involved biological function and signal pathway mechanism.It provided a theoretical and experimental basis for the development of nutrient functional foods transformed by green alga Enteromorpha prolifera oligosaccharides improved blood-sugar metabolism in vivo.The main results are as follows:(1)One E.prolifera oligosaccharide named EPO-1,that a water-soluble heteropolysaccharide was isolated and purified from Enteromorpha prolifera polysaccharide degradation by DEAE-52 and Bio-Gel P-2 column chromatography.Fourier transform infrared spectroscopy(FT-IR),high performance liquid chromatography(HPLC),multi-angle laser light scattering(MALLS),and nuclear magnetic resonance(NMR)spectroscopy were used to characterize the structure of EPO-1.EPO-1 mainly consisted of D-mannose,L-rhamnose,D-glucuronic acid,D-galacturonic acid,D-glucose,and D-galactose with the molar ratios of 0.61:12.53:30.59:3.26:1.73:21.69.EPO-1showed the average molecular weight(M_W)and number molar masses(Mn),which were 4.28 k Da and 2.47 k Da,respectively.It contained six types of linkage units as?2)-?-D-Glcp A-(1?,?3,6)-?-D-Manp-(1?,?4)-?-D-Glcp-(1?,?6)-?-D-Galp-(1?,?-L-Rhap-(1?,and?4)-?-D-Galp A-(1?.(2)The T2DM rats were established to study the effect of interventional treatment of T2DM rats at low-dose with 150 mg/kg EPO(EPOL)and high-dose with 300 mg/kg EPO(EPOH)intragastrically for28 days.At the beginning of the experimental,compared with the normal group,fasting blood glucose(FBG),glycated hemoglobin(HBAC),and oral glucose tolerance test(OGTT)levels were significantly increased in the model group throughout the experimental(P<0.05).Nevertheless,after 4 weeks of treatments with metformin hydrochloride,EPOL,and EPOH,the levels of FBG and OGTT decreased significantly in diabetic rats(P<0.05),implying that EPO ameliorated glucose metabolism in T2DM rats.In particular,oral administration of EPO at 150 mg/kg group has the best effect on improving the OGTT levels in diabetic rats.Histopathological evidence showed that EPO reduced oxidative damage stress in tissue,thereby preventing diabetic complications in liver,pancreas and jejunum tissues.The phosphoinositide 3-kinases(PI3K)/protein kinase B(Akt)were detected by real-time polymerase chain reaction(RT-q PCR).Compared with the model group,EPOL treatment significantly increased the m RNA levels of PI3K and Akt in the jejunum(P<0.05).It showed that both EPOL and EPOH treatments significantly increased the expression of PI3K and Akt using western blotting(WB).Mitogen-activated protein kinase kinase(MEK)and Extracellular signal-regulated kinases(ERK 1/2)genes that are associated with the maintenance of intestinal endothelial cells homeostasis.Compared with model group,EPOL treatment significantly up-regulated the m RNA transcription of MEK in jejunum tissues.Only EPOL treatment group significantly up-regulated the protein expressions of MEK and ERK 1/2proteins.Overall,the results showed that the EPOL dose group influenced T2DM-related indicators led to improving the intestinal barrier stabilization and glucose metabolism.(3)TMT proteomics was used to quantify the differential proteins in jejunum tissue of T2DM rats to study hypoglycemic and action mechanisms after intragastric administration of EPO to T2DM rats.In this proteomics experiment,the total number for 6810 of the proteins were detected and 5977 quantitative information were obtained,in which the protein change multiples were 1.2 times,and the P-value less than0.05 was the threshold.Among the quantified proteins,71 proteins were up-regulated and 77 proteins were down-regulated in the EPOL/Model group comparison group.In the Model/Normal group comparison group,69 protein expressions were up-regulated and 196 protein expressions were down-regulated.Based on the(EPOL/Model)comparison group,compared with the model group,bioinformatics analyzed different differential proteins(DEPs)after EPOL treatment.The cellular component of GO analysis revealed that those proteins,mainly were extracellular matrix protein.KEGG pathway analyzes mainly including ECM-receptor interaction,protein digestion and absorption,arachidonic acid metabolism,pentose and glucuronate interconversions and PPAR signaling pathway.Then,by analyzing the interaction network of DEPs(EPOL/Model,Model/Normal)found that Col1a1,Ttr,Gc,and C3 were an important part of the functional network intersection,of which corresponding protein expression trends are complementary.Obviously,Ttr and Col1a1 proteins were chosen the path with the largest number of nodes.Finally,the genes of Col1a1 and Ttr were selected.Correlation analysis of the Normal,Model and EPOL groups based on the heatmap analysis,to evaluated the relationships between the biochemical parameters related to glucose metabolism and differentially expressed proteins.The heatmap showed strong inverse correlation between the serum levels of alanine transaminase(ALT),total cholesterol(TC),aspartate aminotransferase(AST),low-density lipoprotein cholesterol(LDL-C),OGTT,HBAC,triglyceride(TG),FBG and Col1a1,Ttr.The obviously positive relationships between the serum levels of high-density lipoprotein cholesterol(HDL-C),the gene expression of PI3K,Akt,MEK,ERK 1/2 and Col1a1,Ttr in the jejunum.Further study confirmed that the m RNA and protein expression levels of Col1a1 were consistented with bioinformatics screening results.Overall,combining the above bioinformatics analysis results with validation results showed that Col1a1could be used as a potential biomarker for T2DM symptoms.