| BackgraoundAt present,patients with type 2 diabetes are increasing year by year,and become the third non contagious diseases following cardiovascular disease and tumor.Type 2 diabetes[1]showed high blood glucose,is a kind of complex metabolic disease caused by multiple factors,previous studies have shown that type 2 diabetes causes chronic inflammation,and islet autoimmunity,may also cause serious complications of diabetic nephropathy,diabetic retinopathy,heart cerebrovascular disease.One of the main causes of type 2 diabetes is insulin resistance?Insulin Resistance?.The reason is that the body's sensitivity to insulin decreases,resulting in the imbalance of glucose metabolism[2].Therefore,the study of hypoglycemic drugs has become an urgent task for researchers.Galectin-3?Galectin-3,Gal-3?,which belongs to the carbohydrate binding protein family,as a good biomarker factor for diabetic nephropathy,can provide the basis for the prevention and treatment of judgment and prognosis of diabetes,recent studies have shown that the formation of Galectin-3 and insulin resistance are directly linked,it directly hindered the connection of insulin and insulin receptors leading to the downstream pathway that the glucose transporter?Glut-4?can make glucose output arrhythmia resulting in elevated blood glucose concentration caused by insulin resistance from cytoplasmic translocation to the cell membrane,a conclusion can be drawn that Galectin-3 may affect the expression by insulin receptor signaling pathway and inflammatory genes to induce insulin resistance,therefore,will be a new target as Galectin-3 to develope new drug for diabetes treatment has great prospects.Bovine lactoferrin?Bovine lactoferricin,LfcinB?,has various biological effects of monomer protein hydrolysis in milk.Previous studies have found that it has significant antibacterial and antitumor activity,but recent studies have found that the antibacterial activity of LfcinB amino acid sequence of the LfcinB center RRWQWR has been reported as ACE inhibitors play a hypotensive action of peptides may also have potential hypoglycemic activity and insulin resistance and even have some connection,but the specific mechanism by now has no relevant reports.Based on the above research background,the purpose of this experiment is to study the effect of mimetic peptide designed based on cationic lactoferrin peptide LfcinB64-9?RRWQWR?on glucose metabolism of human hepatoma cells and mouse adipocytes in insulin resistance state,and to explore its mechanism.In the former part of the research on peptide library of APD2 according to the amino acid structure of antibacterial peptide sequence pattern requirements,advantage of site and its chemical modification,to carry out virtual combinatorial design hypoglycemic peptides,and through quantitative structure-activity relationship analysis of the implementation a series of high-throughput chemical modification on LfcinB screening,which may have lower blood glucose and to ameliorate the condition of insulin resistance.To this end,a number of novel lactoferrin peptide derivatives use molecular docking to the target protein structure Galectin-3 were to serching for the potential peptides who have the good affinity with Galectin-3.The preliminary study of the in vitro hypoglycemic activity of the synthetic peptide and the mechanism of its hypoglycemic activity were studied.The effects of mimetic peptide on adipocyte 3T3L1,insulin target cell HepG2 insulin resistance model were studied.It provides new ideas and experimental basis for the search for new hypoglycemic peptides.ObjectiveScreening of target Galectin-3 LFcinB peptide derivatives tested on glucose consumption and adipose proliferation activities in hepatocellular carcinoma cells and adipose cell under the condition of insulin resistance,to observe whether it has beneficial effects on glucose metabolism,if there was explore what kind of mechanisms behind these effects.Method?1?Downloading the Galectin-3 protein receptor from the PDB library.Molecular docking of 7 LFcinB mimetic peptides screened out in the early stage of the laboratory were used to screen out the best Galectin-3 fitting candidate peptide.?2?The polypeptides were synthesized by solid phase,and the structure and purity of the synthetic results were identified.?3?High concentration insulin was used for establishing HepG2 insulin resistance cell model.And dexamethasone,insulin and isobutyl methyl xanthine were used to induce insulin resistance 3T3L1 cell model.?4?GOD-POD method was used to determine glucose consumption in cell models.We observed and screened the effects of mimetic peptides on glucose consumption of insulin resistance cells,and made further screening for LFcinB peptide derivatives for subsequent experiments.?5?Oil red-O staining method was used to determine the effect of the selected peptide derivatives on the proliferation and differentiation of adipocytes,so as to observe the effect of LFcinB peptide derivatives on body weight gain.?6?The biological safety of LFcinB peptide derivatives was screened by CCK8 assay.?7?The effect of peptide and control group on the expression of Galecin-3,IRS-1,pIRS-1,pAKT and pGSK-3 beta,Glut-4 protein was detected by Western-Blot method.?8?The direct effect of LFcinB peptide derivatives on Glut-4 transposition to the membrane of insulin resistant 3T3L1 cells was observed by fluorescence immunomicroscopy.Result?1?Using molecular docking method to screened out the Galectin-3 as a target protein receptor for docking method,we synthesized R1,2,3,5,7 5 peptide analogs including template peptides according to the synthetic cost.?2?With 1?mol/L insulin for HepG2 cells induced by 24h successfully established the model of insulin resistance in HepG2/IR cells by GOD-POD method,and it will last for48h.Test the glucose consumption experiment showed that the synthesized peptide R7RWR-EtBn at the concentration of 100?mol/L on glucose consumption has significant effect at the concentration of 1?mol/L in 12h.The next step of the experiment was to select the most significant peptide R7 of the LFcinB derivative to improve the effect of glucose consumption.?3?R7 peptide RWR-EtBn can inhibit the proliferation and differentiation of 3T3L1cells into mature fat cells,and in a concentration dependent inhibition,indicating R7peptide has potential fat reduction activity.?4?CCK8 cell proliferation inhibition test showed that Peptide R7 had almost no toxic effect on adipocyte 3T3L1,and the biological safety was higher than that of R1 template peptide and Rosiglitazone.?5?Western blot assay showed that the peptide R7 and R1 template peptide can produce inhibition effect of the expression of diabetic biomarker factor Galectin-3 in HepG2 cell model in a concentration depentent way,but the R7 is more significant than R1template and Rosiglitazone.R7 may through the insulin receptor signaling pathway to improve insulin resistance and lower the glucose level and regulate the glucose metabolism.?6?Immunofluorescence assay showed that the R7 peptide had a decrease effect in the protein expression of Galectin-3 and a promotion in the translocation of glucose transporter to the cell membrane.ConclusionsThe experimental design LFcinB peptides derivativies showed great promoting decrease effect of glucose consumption in established in vitro model of insulin resistance human hepatoma cells HepG2 at 100?mol/L level,R7 peptide can significantly promote the glucose consumption,also on the proliferation and differentiation of 3T3L1 into mature fat cells in a concentration dependent inhibition,which means peptide R7 may have influnce on the weight gain,combined with the above function,preliminary judgment can be draw that screened R7 peptide based on the molecular docking with Galectin-3 had effect on insulin resistance and glucose metabolism process,and the effect is better than that of rosiglitazone and R1 template peptide.And the protein expression of the biomarker factor Galectin-3 of diabetic nephropathy was reduced with R7 in a concentration dependent manner.The mechanism of LFcinB peptide mimetic peptide R7 is probably through activating the IRS-1-pAKT-pGsk-3?-Glut-4 insulin receptor pathway,and ultimately promoting Glut-4 translocation to the cell surface. |
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