| ObjectiveTo investigate the antioxidant of silk fibroin(SF). We hydrolyzed silk fibroin with enzyme and designed orthogonal experiment to optimize the hydrolysis process. A stable and reliable hydrolysis process was selected. Then study on the antioxidant activity of hydrolysate by vitro cell experiment.Methods1. The optimization of hydrolytic process to prepare antioxidant silk fibroin protein.Papain, pancreatin, pepsin, trypsin, neutral protease were used to hydrolyze antioxidant silk fibroin. We detected amino nitrogen content by neutral formalin titration to observe hydrolysis process. Pyrogallol autoxidation method was used to measured superoxide radicals(O2-·) clearance rate. Screened hydrolysis process and designed orthogonal experiment to optimize hydrolysis process with index of O2-· clearance rate.Hydrolyzate molecular weight distribution was detected by polyacrylamide gel electrophoresis(SDS-PAGE) assay. Identify ability of hydrolyzate to scavenge other free radical. Hydroxyl radicals(·OH) clearance rate was detected by salicylic acid chromogenic assay. And suppression of H2O2-induced oxidative hemolysis in erythrocytes.2.The effect of silk fibroin on H2O2-induced A549 and Hep G2 cells injury.The appropriate concentration of H2O2 to induce cells: A549 and Hep G2 cells were treated with 50, 100, 200, 400, 600, 800μmol/L H2O2 for 24 h, cell viability was tested via MTT assay and the level of malondialdehyde(MDA) was detected via thiobarbituric acid(TBA) assay. Select concentration of H2O2 which injury cells viability about 50%.The concentrations of SF treated cells were 10, 20, 30, 50mg/m L. Cells were treated with an antioxidant N-acetylcysteine(NAC) in positive control group. The effect of SF on cell viability: Cells were divided into blank group, pre-treatment groups(SF, NAC pretreatment cells for 24 h and then cells were cultured for another 24h), and post-treatment group(cells were cultured 24 h, then SF, NAC incubated cells for 24h). Effect of SF oninjured cells: Cells were divided into blank group, H2O2 group, pretreatment group(10, 20,30 and 50mg/m L SF pretreated for 24 h, cells exposed to H2O2 for another 24h) and post-treatment group(H2O2-induced cells 24 h and treated with 10, 20, 30 and 50mg/m L SF for another 24h). Detection of cell viability and MDA content, WST-8 kit was used to detect the activity of superoxide dismutase(SOD). Catalase enzyme activity(CAT) was detected via chromogenic reaction method. Intracellular total antioxidant capacity(T-AOC)was detected via ABTS assay.3. Amelioration effect of silk fibroin on insulin resistance induced by high glucose and insulin in Hep G2 cells.Establishment of insulin resistance model : The cells were divided into blank group,30mmol/L glucose group, 30mmol/L glucose +(10-5, 5×10-6, 10-6, 5×10-7, 10-7, 5×10-8, 10-8)mol/L insulin groups. We detected cell glucose consumption values via glucose oxidase-peroxidase assay to defined the insulin concentrations, time required for modeling,duration time of this model. GPO-PAP assay used to detect triglyceride(TG) content,chromogenic reaction method was used to detect the glycogen accumulation values. We evaluated whether the model was established successfully by glucose consumption values,triglyceride(TG) content and the glycogen accumulation values.The effect of SF on insulin resistance cells: Cells were divided into blank group,insulin resistance group, SF group, metformin group, SF + metformin group.Concentration of SF was 50mg/m L and concentration of metformin was 0.01mg/m L, the cells treatment time is 24 h. Indexes of glucose and lipid metabolism were glucose consumption values, TG content, glycogen accumulation values. Indexes of oxidation-antioxidant were MDA content, SOD vitality, CAT vitality, T-AOC ability, the flow cytometry was used to detect the level of reactive oxygen species(ROS).Inflammatory cytokines tumor necrosis factor(TNF-α) and human interleukin-6(IL-6)content were detected via Elisa assay.Results1. The optimization of hydrolytic process to prepared antioxidant silk fibroin protein.(1) O2-· clearance rate of pancreatin hydrolyzate was maximum, so we used pancreatin hydrolysis process to prepare antioxidant silk fibroin. The optimal conditions of SF hydrolysis in this study was: concentration of enzyme was 6%, reaction time was160 min, substrate concentration was 20mg/m L, p H value was 8, temperature was 38℃.Under this hydrolysis process the superoxide radical scavenging rate of hydrolyzate was(72.73±0.41%).(2) The molecular weight of hydrolyzate were below 10 k Da, the hydrolysate radical scavenging rate was(47.24±0.78%) and inhibition rate of H2O2-induced erythrocyte hemolysis was(24.30±0.98%).2. The effect of silk fibroin on H2O2-induced A549 and Hep G2 cells injury.(1) We selected 600μmol/L H2O2 injury A549 cells and 800 μmol/L H2O2 injury Hep G2 cells. Then cell viability decreased significantly(p<0.05) and level of MDA in cells increased significantly(p<0.05).(2) 10, 20, 30, 50mg/m L SF and NAC were non-toxic to cells. Compared with the H2O2 group, the activity of the cells increased and the content of MDA decreased(p<0.05) in the pre-treatment groups and post-treatment groups, and the effect of post-treatment group was better than the pretreatment group(p<0.05).(3) The levels of SOD, CAT and T-AOC were decreased in H2O2 group(p<0.05).SOD, CAT and T-AOC(p<0.05) were increased by post-treating with SF(p<0.05).3. Amelioration effect of silk fibroin on insulin resistance induced by high glucose and insulin in Hep G2 cells.(1) 30mmol/L glucose+10-6mol/L insulin-induced Hep G2 cells for 48 h to establish insulin resistance model. Glucose consumption values decreased to(1.18±0.21mmol/L)(p<0.05), TG content increased to(1.01±0.01mmol/g Pro)(p<0.05), glycogen accumulation values decreased to(7.78±0.19μg/mg Pro)(p<0.05). Glucose consumption values of model with no significant change within 60 h.(2) SF ameliorated glucose and lipid metabolism by increasing glucose consumption,decrease TG levels and increase the amount of glycogen accumulation(p<0.05).Compared with the single administration group, results of ameliorate glucose and lipid metabolism were more significant in SF and metformin combination group(p<0.05).(3) The levels of ROS and MDA increased in insulin resistance cells(p<0.05),inflammatory cytokines TNF-α and IL-6 were increased(p<0.05), antioxidant enzymes SOD, CAT activity and T-AOC ability were decreased(p<0.05). The levels of ROS, MDA,TNF-α and IL-6 in insulin resistance cells were decreased after treated with SF(p<0.05),and also the antioxidant enzymes SOD, CAT activity and T-AOC ability were increased(p<0.05). Compared with the single administration group, the results of restorebalance of oxidation-antioxidant system and decrease inflammatory factors were more significant in SF and metformin combination group(p<0.05).ConclusionThe silk fibroin obtained from optimization of hydrolysis process had a strong ability of scavenging free radical, the molecular weight of silk fibroin peptide are below 10 k Da.Silk fibroin restore balance of oxidation-antioxidant system, ameliorate cells injury and insulin resistance caused by oxidative stress. |
PDF Full Text Request