Hypoglycemic Effects Of Peptides From On Sea Cucumber(Holothuria Nobilis) On Type Ⅱ Diabetic Rats And The Related Mechanisms

Food derived bioactive peptides have gradually become a research hotspot in the development of health care products with specific efficacy due to their wide sources,simple preparation process,easy digestion and absorption and diverse activities.Diabetes mellitus has become the third largest chronic non communicable disease after cancer and cardiovascular disease.Diabetes can lead to a variety of complications,which seriously affect the quality of life of patients.So far,there are few studies on bioactive peptides/hydrolysates with hypoglycemic effect,and the exploration of activity evaluation methods in vitro and the mechanisms of peptides on hypoglycemic peptides are still in the initial stage.This paper focuses on the preparation of bioactive peptides from the hydrolysates of aquatic fish,which can improve insulin resistance and hypoglycemic effect.The hypoglycemic activity of sea cucumber and its molecular mechanism were studied by T2 DM animal model.Then the bioactive peptides were identified and synthesized,and the effects of the synthesized bioactive peptides on improving insulin resistance,anti-inflammation and anti-oxidation effect were investigated through Hep G2 cells and MES 13 cells.The main research contents and results of this paper are as follows:(1)Papain,protamex,neutral,alkaline,bromelain and flavourzyme protease were used to screen the hydrolysates of sea cucumber.The results showed that papain hydrolysates had the strongest antioxidant activity and insulin resistance ameliorating effect.Papain and other five kinds of protease were compounded,the combination of papain and protamex hydrolysate had the highest glucose uptake and the strongest effects on improving insulin resistance.On the basis of the above enzyme addition method,the total amount of enzyme added and hydrolysis time were further explored,and the preparation technology of sea cucumber was screened as follows: 1:1 combination of papain and protamex,enzyme dosage was 1%,enzymolysis time was 4h,temperature was55 °C and p H was 7.(2)In this study,anti-diabetic activity and potential mechanisms of sea cucumber hydrolysates(SCH)were investigated in a rat model of Type II diabetes induced by STZ combined with high-fat diet.Results showed that SCH ameliorated the body weight loss,water intake,fasting blood glucose level,glycated hemoglobin,lipid metabolism,liver and kidney function.In addition,UPLC-q TOF-MS/MS was used for identifying SCH peptides.The results showed that 242 peptides were identified from SCH,which were rich in hydrophobic and aliphatic amino acids.The hypoglycemic and hypolipidemic effects of SCH peptide might be attributed to the presence of a large number of hydrophobic amino acids and aliphatic amino acids,as well as a large number of hypolipidemic peptides.30 peptides were screened as potential bioactive peptides by analyzing the intensity of mass spectra combined with Peptide Ranker.(3)SCH exerted potent and effective hypoglycemic,hypolipidemic and insulin-sensitizing effects in T2 DM rats.The potential molecular mechanism investigations indicated that SCH activated PI3K/Akt signaling pathway,further regulating the expression of the GLUTs and p-GSK-3β protein,thus promoting glycogen storage and improving insulin sensitivity.Through further analysis of242 identified peptides,it is concluded the anti-diabetic effect of SCH is associated with the low MW peptides,which contain abundant hydrophobic amino acids,aliphatic amino acids,and some specific amino acids with insulin-sensitizing effect,such as Pro,Phe,Leu/Ile and Ala.(4)In order to identify and verify the peptides in SCH,the insulin resistance model of Hep G2 cells(IR-Hep G2)induced by high glucose and palmitic acid(PA)was used to evaluate the effect of drugs on promoting glucose uptake and improving insulin resistance,and further to clarify its protective mechanism and related signaling pathways.The results showed that SPA showed the highest glucose uptake.Moreover,SPA increased the expression of GLUT2,p-GSK-3β,GS,IRS1,PI3 K,p-Akt and decreased the expression of GSK-3β,p-GS,p-IRS1 in IR-Hep G2 cells.The results indicated that SPA could improve insulin resistance,glucose transport and glycogen synthesis by activating PI3K/Akt signaling pathway,thus improving glucose metabolism of insulin resistant IR-Hep G2 cells.(5)The purpose of the present study was to investigate the renoprotective effects of sea cucumber hydrolysates(SCH)against diabetic nephropathy induced by STZ in rats and further to explore the underlying anti-inflammatory and antioxidant mechanisms of SCH on T2 DM rats.Results showed that SCH significantly alleviated the micro-albumin urine(U-MA),and SCH supplementation alleviated oxidative stress by increasing the activities of SOD and GSH-px and reducing MDA accumulation.Moreover,SCH treatment attenuated levels of inflammation factors such as IL-1β,TGF-β and TNF-α.Histological observation also indicated that SCH treatment notably reversed renal damage and protected the kidneys from hyperglycemia-mediated oxidative and inflammation injury.The potential molecular mechanism investigations indicated that SCH ameliorated oxidative stress and Inflammation by triggering Nrf2/NQO1 and suppressing TLR4/NF-κB signaling pathway,indicating that SCH may possess anti-diabetic nephropathy effects.(6)In order to explore the renal protective mechanism and related signaling pathway of peptide,the anti-inflammatory and antioxidant protective effects of the drug on rat mesangial cells were evaluated by high glucose induced MES 13 cells injury model.Molecular docking technology was used to explore the antioxidant and anti-inflammatory mechanisms of Nrf2 and NF-κB pathway.The results showed that WWGP and APGY had strong antioxidant activity,which might be related to their hydrophobic(Tyr,Pro and Ala)and aromatic(Tyr,Trp and Phe)amino acids.Besides,WWGP and APGY alleviated a series of oxidative stress by promoting nuclear translocation of Nrf2.Molecular docking analysis revealed that these peptides such as WWGP and APGY might directly bind to Keap1 and thus modulate Akt/Nrf2 pathway.On the other hand,the strong anti-inflammatory effects of ALGP and WWGP might be related to their hydrophobic(Pro,Ala and Trp),aromatic(Tyr,Trp and Phe)amino acid and specific amino acids with anti-inflammatory effect,such as Gly.The underlying molecular mechanism suggests that ALGP and WWGP alleviated a series of inflammatory responses by preventing nuclear translocation of NF-κB.Molecular docking analysis revealed that these peptides such as WWGP and APGY might directly bind to TLR4,interfere with the interaction of IκBα-NF-κB,inhibit the nuclear translocation of NF-κB,and thus modulate TLR4/NF-κB pathway.