Therapeutic Effect Of Ethanolic Extract From The Green Cocoon Sericin Layer Of Silkworm Bombyx Mori On Type 2 Diabetic Mice And Its Hypoglycemic Mechanism

Sericin is a kind of colloidal spherical protein secreted from the middle silk gland of the silkworm Bombyx mori,which acts as an adhesive joining two fibroin filaments in order to form a solid ellipsoidal cocoon shell.In recent years,sericin has been proved to have many biological activities,such as anati-ultraviolet,anti-inflammatory,anti-oxidation,inhibition of tyrosinase and free radical scavenging.Sericin was prepared from new green cocoon variety(caoyuan?shenyun)which contains abundant flavonoids by a novel(Ca(OH)₂)degumming method.Then the ethanol extracts from sericin(EE)was obtained.Firstly,we explored the composition and activity of EE in vitro,and the protective effect of L02 cells on oxidative stress in high glucose culture.On this basis,we further studied the therapeutic effect of EE on type 2 diabetic mice induced by the combination of a high-fat diet(HFD)and streptozotocin(STZ).Furthermore,the related mechanism was also studied from the level of physiological and biochemical,oxidative stress,inflammatory responses,insulin signaling pathways and glucose and lipid metabolism.The research of this paper mainly involved the following four aspects:(1)The experimental results of the basic composition analysis showed that the cocoon shell of the green cocoon is composed of 69%silk fibroin fibre,22.39%sericin,and 8.61%ethanol components.And,the EE contains 27.06%of amino acids.The antioxidant amino acids and essential amino acids accounted for 34.11%and 27.07%of the total amino acids,respectively.Rutin,astragalin,kaempferol,quercetin,kaempferol,kaempferol-5-O-glucoside,quercetin-3-O-galactopyranoside,quercetin-3-O-rutinoside,quercetin-3-O-glucoside,quercetin-4',7-2-O-glucoside were identified by UHPLC-QTOF-MS used R program package and internal MS2 data.The total flavonoid aglycones were 33.42±0.08 mg/g measured by an innnovaive hydrolysis-assited-HPLC methods.In vitro assay,the free radical scavenging abilities of DPPH,ABTS and hydroxyl radical were measured to evaluate the antioxidant ability of EE.And,IC₅₀ values of scavenging abilities were 296.95±13.24?g/m L,94.31±9.13?g/m L and 9.21±0.15mg/m L,respectively.This mean EE have good antioxidant ability in vitro.The IC₅₀ values of inhibitory activities of?-amylase and?-glucosidase were 37.57±6.45?g/m L and 212.69±22.94?g/m L.EE can improve blood glucose and body damage in type 2 diabetes though scavenging superoxide derived free radicals,reducing oxidative stress and inhibiting carbohydrate hydrolases.(2)Previously,sericin has been proved to have the activities of reducing blood sugar and preventing diabetes syndrome.However,these experiments were all used in the white commercial cocoon shell.A new green cocoon variety which contains abundant flavonoids was used in this paper.The protective effect of EE on oxidative stress was investigated using L02 cells cultured with high glucose in vitro.We found that EE can reduce the level of ROS and oxidative stress in the cells induced by high glucose.EE could effectively decrease the content of 8-Ohd G,NF?B and TNF?in cells,and had a good does effect.This indicated that EE can significantly reduce the damage to cells by oxidative stress and inflammatory reaction.(3)After 4 weeks of HFD feeding,ICR mice were fasted for 12 hours,and each mouse was injected once with STZ(100 mg/kg.BW).Seven days after the injection,mice were fasted for 10hours and fasting blood glucose(FBG)was determined;mice with more than 11.1 mmol/L of FBG were considered to be diabetic and included in the study.The diabetic mice were random Ly divided into 4 groups:the diabetic model group and three EE-gavaged diabetic groups with doses of 150mg/kg,250 mg/kg and 350 mg/kg.In sixth week,1.5 g/kg glucose were gavaged to measure glucose tolerance.The rate of glucose reduction was 71.73%,18.02%,34.41%,58.65%and 66.34%in the normal group,the diabetic model group and the three EE-treated group.The decrease rate of the high dose group was 3.68 times than the model group with good dose response.And,the measurement of insulin tolerance also indicated that EE treatment could relieve the abnormality of glucose tolerance and insulin resistance in type 2 diabetes.The administration was given daily for 7weeks and the weight and FBG were measured weekly.At last,blood,livers,kidney and pancreas were used for further investigation.Our results showed that the FBG level of mice in diabetic model group was consistently high,which was between 19.0-29.0 mmol/L.Moreover,serum insulin levels of this group were abnormally elevated.EE treatment significantly reduced FBG and fasting serum insulin levels in diabetic mice in a dose-dependent manner,and the rate of glucose reduction was 25.16%,34.97%and 39.38%.EE can obviously alleviate hyperglycaemia and insulin resistance in diabetic mice.EE treatment also significantly reduced the abnormal glycosylated hemoglobin level and serum liver function enzyme activity in diabetic mice,and had a good regulatory effect on blood lipids.The results also showed that EE treatment increased the level of antioxidant enzymes in the liver of diabetic mice.In the 350 mg/Kg high dose group,the level of T-SOD and GSH-Px reached to 89.40±16.33 U/mgprot and 1477.15±49.69 U/mgprot,which was almost close to the normal group.The level of MDA decreased to 57.89%of the model group.All this indicated that EE could protect the body from oxidative stress.We found that fat accumulation,inflammatory infiltration and edema area significantly reduced in the EE-treated group.The EE treatment had repair effect on pancreatic injury resulting in the distribution of islet?cell distribution in mice more uniform and rich.At the same time,EE could improve the level of antioxidant enzymes in the kidney and reduce the level of TNF-?,MCP-1 and p-P38?which related to diabetic nephropathy.In order to further explore the mechanism of hypoglycemic effect of EE,we investigated the effects of EE on the insulin signaling pathway,glucose metabolism,lipid metabolism and inflammatory response in the liver of type 2 diabetic mice by Western blot in detail.After EE treatment,the level of NF?B,IL-6 and TNF-?decreased to 68.82%,53.12%and 51.01%of the model group in the 250 mg/Kg group,and decreased to 49.90%,45.74%,45.75%in the 350mg/Kg group.This suggested that EE could reduce the effect of inflammatory factors on the phosphorylation of insulin receptor substrates in diabetic mice and improve insulin resistance.The levels of insulin receptor(IR)and insulin receptor substrate(IRS)protein in the liver of type 2diabetes model group were 50%and 16.95%of the normal group.The treatment of EE can increase the expression of IR and IRS close to the normal level.The EE treatment could increase glycogen synthesis to inhibit the effect of gluconeogenesis though phosphorylating GSK3?and enhancing the activity of GS by activating the damaged PI3K/AKT pathway.Glucose is oxidized and activated to convert glucose into glycogen by activating GLUT4 transposition to enhance glucose uptake.The activated AMPK could promotes glycolysis and the production of lactic acid.Inhibition of gluconeogenesis by down regulation of PEPCK and G6Pase.EE treatment could also increase the lipid metabolism in the liver by improving the level of PPAR?.In this experiment,a green cocoon containing high active ingredients was firstly used to get the ethanol extracts which composed of oligopeptide and flavonoids with strong antioxidant activity.EE can improve the blood sugar level of type 2 diabetes by enhancing the body's antioxidant capacity,reducing inflammatory reaction and insulin resistance,regulating glycolysis and gluconeogenesis.It can also play a preventive role in diabetic nephropathy and renal fibrosis.This study has profound significance for the comprehensive utilization and sustainable development of sericulture industry.