| Sericin,which accounts for about 25-30%of the mass of silk,is a kind of colloidal macromolecular protein with protective wrapping effect.It is secreted by the middle silk gland cells of Bombyx mori,with a relative molecular weight ranging from 13 kDa to 300 kDa.It is a water-soluble protein composed of 18 kinds of amino acids,and has various biological activities such as anti-ultraviolet,anti-oxidation,anti-tyrosinase,anti-tumor,and promoting digestion.In the traditional silkworm production and silk processing industry,silk fibroin is deeply used,and the outer layer of sericin is usually dissolved in alkaline solution through such as degumming or scouring processes,and is discarded along with such degumming wastewater.This has caused great waste of natural protein resources and environmental pollution.In this paper,silk degumming solution prepared by calcium hydroxide(Ca(OH)2)-ultrasonic degumming method was used to obtain sericin peptides and their hydrolysates by removing precipitated calcium sulfate through acid neutralization and centrifugation.This paper analyzed the degradation of sericin and simulate the metabolites of physical and chemical properties and biological activity in vitro.Through the normal liver cells of mice(Aml-12),we study the effect of sericin on the cell vitality and oxidative stress injury protection under high sugar cultivation.The treatment of sericin on T2D rats induced by STZ from the physiological and biochemical,oxidative stress,and pathological aspects of urea.We also foucus on antioxidant enzymes,sugar,lipid metabolism,PI3K/AKT signal pathway,AMPK/ACC signaling pathways.The role of sericin played in T2D rats liver damage is also discussed from repair function and mechanism of action.The research content of this paper mainly involves the following four aspects:1.In this paper,the white cocoon layer was degummized by green processing method,high molecular sericin(HS)was obtained by water boiling ultrasonic method,and low molecular sericin(LS)was get by Ca(OH)2-ultrasonic method.In this experiment,the cumulative concentrations of the above two sericins in salivary amylase,pepsin and trypsin(pH 6.8,2.0,7.4)within 10 h were investigated.The cumulative speed and amount of LS were higher than that of HS under same time,and the order of the cumulative speed and amount of sericin in simulated environment was pepsin>salivary amylase>trypsin.The metabolites of the two sericins were distributed below 15 kDa in vitro.The inhibitory activity of sericin and its simulated metabolites was positively correlated with the inhibitory activity of α-glucosidase in the concentration range of 1-10 mg/mL.The IC50 of HS and LS metabolites were 1.02±0.12 mg/mL and 0.91±0.15 mg/mL,respectively,which were 5-7 times lower than those of HS and LS.In addition,the total antioxidant capacity and hydroxyl radical scavenging ability of sericin and its metabolites were also significantly improved.The IC50 values of HS and LS were increased to 0.898±0.12 mg/mL and 0.695 ± 0.12 mg/mL.respectively,which were 2 to 3 times lower than those of HS and LS.The IC50 values of hydroxyl free radical scavenging rates of HS and LS simulated metabolites were also nearly 100%higher than those of the two metabolites.The above results fully indicated that the antioxidant and superoxide free radical scavenging ability of sericin and its glycosidase inhibitory activity were significantly enhanced after simulated metabolism.2.The protective effects of two sericins on the cell viability and oxidative stress injury of Aml-12 were investigated through the experiment that high glucose environment could cause oxidative stress of mouse normal liver cells Aml-12.The results showed that LS had a dose-effect effect on Aml-12 cell viability under high glucose environment,which was better than HS.The downregulation of reactive oxygen species(ROS)in Aml-12 cells in LS group was more significant than that in HS group.Under high glucose environment,the two sericins also had different effects on the inflammatory response of cells.LS sericin was significantly better than HS in the down-regulation of TNF-α.IL-6 and NF-κB levels in Aml-12 cells,and showed good dose effect.The levels of TNF-α were 197.06±13.1 ng/mgprot and 161.83±16.1 ng/mgprot after LS co-incubation with Aml-12 cells at 120 μg/mL and 150μg/mL,respectively.which were lower than normal levels.The down-regulation effect of LS on IL-6 and NF-κB was stronger than that of HS.The above results fully indicated that sericin could effectively reduce oxidative stress and inflammatory response of cells under high glucose environment,and eliminate the damage effect on Aml-12 of normal liver cells in mice,and the protective effect of small molecule sericin is stronger.3.In order to investigate the hypoglycemic effect of sericin oral administration on STZ-induced type II diabetes rats(T2D rats),this experiment was divided into normal group,model group,positive control group(PC),low-dose group(LLS),medium-dose group(MLS)and high-dose group(HLS),and the corresponding diets were fed quantitatively daily for four weeks.The results showed that the body weight of rats in low-dose,medium-dose and high-dose groups increased by 7.9%,11.5%and 13.9%,respectively,showing a dose-effect.After four weeks of oral administration,the blood glucose levels of rats in the three sericin low-dose,medium-dose and high-dose groups all showed a downward trend.Compared with model group,low dose,medium dose and high dose groups decreased by 46.9%,62.3%and 67.1%,respectively.Especially,the FBG level in the middle and high dose groups were almost equal to that in the normal group.In the glucose tolerance test,during 90-120 min,the blood glucose levels of the three sample groups were significantly lower than that of the model group(P<0.05),and the serum insulin contents of the three dose groups were significantly lower than that of the model group after four weeks of sericin supplementation.Serum insulin levels in the middle and high dose groups decreased by about 35%and tended to normal insulin levels.Insulin tolerance(ISI)test showed that the ISI value of the low,medium and high dose sericin group increased with the increase of the concentration,and the glycolated serum protein level of the model group decreased by 0.1,0.2 and 0.3 mmol/mL,respectively,showing a certain dose effect.After four weeks of oral sericin,the ratio of aspartate aminotransferase/alanine aminotransferase in low-dose group was 3,medium-dose and high-dose groups were both 2.5,which was only 50%of that in the model group.The albumin level was reduced to the same level as that in the normal group.The levels of total bilirubin and alkaline phosphatase were significantly lower than those in model group.The total cholesterol level of rats in medium and high dose groups decreased by about 25%.Compared with the model group,the triglyceride levels in the three low-dose,medium-dose and high-dose sericin groups were reduced by 27.3%,28%and 34.7%,respectively.The triglyceride level of HLS group was close to that of the normal group.The LDL levels in three dose groups than the model group.The lactate dehydrogenase and free fatty acid level was significantly lower than model group(P<0.05).Pancreatic tissue biopsies and insulin immunofluorescence staining results show that pancreas is complete,islet cells distribution were better uniformed and more abundant,small molecule sericin on pancreas damage has obvious repair action in three dose groups.These results indicated that oral administration of sericin peptides and their hydrolysates can effectively reduce blood glucose levels and insulin levels,improve serum liver function enzymes,and reduce blood lipid levels,thus exerting hypoglycemic effects in T2D rats.4 In order to explore the repair effect of sericin on liver injury in T2D rats,liver physiological and biochemical indexes and histopathology were detected in each group,and the effects of sericin on glucose metabolism,PI3K/Akt,AMPK/ACC signaling pathway and inflammatory response in T2D rats were futher investigatcd.After four weeks of oral sericin treatment,the liver organ coefficient of each sericin sample group gradually decreased.The level of 8-hydroxydeoxyguanosine decreased significantly(P<0.01),showing a dose-dependent effect.The liver glycogen level in the three dose groups significantly increased and showed a dose-positive trend.The liver glycogen level in the low-dose group was twice as high as that in the model group,and the liver glycogen levels in the medium-dose and high-dose groups were also close to those in the normal rats,suggesting that sericin supplementation may down-regulate the blood glucose level in type Ⅱ diabetic rats by increasing the liver glycogen synthesis.After four weeks of oral sericin,the activity of glutathione peroxidase in medium and high dose groups was increased by 53.8%and 58.2%,respectively,and almost returned to the level of normal rats.The content of malondialdehyde in the sericin sample groups was negatively correlated with the sericin concentration,while the total antioxidant capacity in the low-dose,medium-dose and high-dose groups was 218%,221%and 280%of that in the model group,with obvious dose-effect.The results of liver histopathological examination showed that the morphology of liver tissues in the three sericin groups were restored to a certain extent,especially in the medium and high dose groups,the abnormal liver pathology was improved obviously,the liver cells were arranged neatly,the structure of liver lobules was complete,and there were no obvious changes within vacuoles and inflammatory infiltration.Oral sericin activated the impaired PI3K/Akt/AMPK pathway,inactivated phosphorylation,increased GS activity,down-regulated G6Pase and PCK levels,glycogen synthesis and inhibited gluconeogenesis were enhanced.GLUT4 translocation was activated to accelerate glucose utilization.p-AMPK increased the expression level of enzymes related to glycolysis and accelerating the occurrence of glycolysis.p-AMPK also inhibited ACC production,thereby fat synthesis was inhibited.In addition,the protein expressions of related inflammation factor NF-κB,IL-6 and TNF-α of also means inflammation were reduced in the body.The physicochemical properties and hypoglycemic effects of HS and LS obtained from cocoon were investigated for the first time.The mechanism of oral administration of LS on liver injury repair was also discussed.With calcium hydroxide-ultrasonic degumming method of small molecule by sericin and high temperature ultrasonic macromolecular sericin comparison experiment,the simulation of the metabolites of alpha glycosidase enzyme inhibition and anti-oxidation ability were greatly enhanced.Sericin could protected Aml-12 from oxidative stress and inflammatory response to alleviate and environmental damage.Oral administration of small molecular sericin for four weeks can reduce blood glucose level,improve insulin resistance,protect liver and reduce pancreatic injury by promoting antioxidant capacity,enhancing insulin sensitivity and balancing glycolysis and gluconeogenesis in T2D rats.The results of this study had important reference significance for the sustainable development and utilization of silk resources,especially for sericin waste.It could reduce environmental pollution,and provide a new idea for the adjuvant treatment of diabetes and the development of hypoglycemic functional food. |
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