| The grubs are the larvae of the Korean black Scarab which belongs to the Golden Turtle Family.They were first recorded in the "Shen Nong Herbal Classic",and later included in the "Compendium of Materia Medica" and "the Synopsis of The Golden Chamber".Studies have shown that grub extract has the effects of inhibiting bacterial growth,inhibiting tumors,protecting liver and dissolving thrombus.Grubs peptides,one of the components extracted from grubs,has some pharmacological activities such as anti-tumor,anti-oxidation and improving immunity,but little research has been done on improving immunity.In this study,Grubs polypeptides were extracted from grubs,purified and identified by protease hydrolysis,ultrafiltration,chromatography and electrophoresis.The regulatory effects of immunosuppression and protective effects of Grubs polypeptides were investigated by immunosuppressive mice model and immune liver injury model.Expected research results will provide experimental basis for grubs to be developed immunomodulatory drugs.1 The first experiment was to extract,purify and identify the Grub polypeptide and to lay a material foundation for the investigation of its immunoregulatory function.After the dried grubs were crushed,the grub ponders were hydrolyzed by papain and purified by ultrafiltration and ion chromatography.The molecular size of the grub peptides was identified by Tricine-SDS-PAGE.The scavenging ability of Grub polypeptide to benzopicrin acyl radical was analyzed by spectrophotometry.The results showed that the concentration of polypeptide extracted from grubs was 37.01mg/mL,the extraction rate was 18.66%,the molecular size was less than 10 KDa,and the scavenging activity of acyl radical was 42.84%.The results indicated that small molecule grub polypeptide was obtained by enzymatic hydrolysis,separation and purification,and it had good ability to scavenge acyl free radicals.2 The second experiment was to investigate the immunomodulatory effects of grub polypeptide on the immunosuppressive mice induced by cyclophosphamide.Mouse were randomly divided into control group,model group and grub polypeptide group with different doses.The mice in the grub polypeptide group were given the grub polypeptide,and the mice in the control group and the model group were given the same amount of saline once a day for 20 consecutive days.Three days before the end of the experiment,all mice except the control group were given CTX intraperitoneally for 3consecutive days.After the last 24 hours of administration,the mice were weighed and blood was collected to prepare serum.The cells from abdominal cavity,spleen and bone marrow by aseptic operation were prepared and cultured,then the live,spleen and thymus were taken from the mice and weighted.By means of weighing,spectrophotometry,blood cell analyzer and flow cytometry,organ index,white blood cell count,delayed hypersensitivity,lymphocyte proliferation and secretion factor,the phagocytosis rate of peritoneal macrophages and serum hemolysin content of mice were analyzed.Compared with the control group,there was significantly a decrease in the model group,in the index of liver,spleen and thymus in the model group,and in the number of white blood cells and the content of serum hemolysin,and in the proliferation and NO secretion of lymphocytes,and in the phagocytosis rate of macrophages,and in the degree of ear swelling in mice.Compared with the model group,therewas significantly an increase in the visceral index of mice,and in the number of white blood cells and the content of serum hemolysin,and in the proliferation of lymphocyte and the content of NO secretion those value near the control group,and in the phagocytosis ability of macrophages and the swelling degree of mice ears alleviated in grub peptide group.The results showed that Grub polypeptide could significantly enhance the immune function of immunosuppressed mice,indicating that Grub polypeptide could improve the specific and non-specific immune function of mice.3 The third experiment was to investigate the protective effect of Grub polypeptide on immune liver injury mice induced by ConA.Mouse were randomly divided into control group,model group and Grub polypeptide group with different doses.The mice in the grub polypeptide group were given the grub polypeptide,and the mice in the control group and the model group were given the same amount of saline once a day for 20 consecutive days for 10 consecutive days.ConA was injected into the tail vein of all mice except the normal group eight hours before the end of the experiment.24 hours after the last administration,the mice were weighed and blood was collected to prepare serum.The liver and spleen were taken from open the abdominal cavity and weighed.The one part of the liver was taken to prepare homogenate,and the other part of the liver was fixed with polyformaldehyde and prepared into tissue sections.The organ indexes of mice were analyzed by weighing.HE staining,spectrophotometry,ELISA,PR-PCR were used to detect the liver function index,peroxide content,antioxidant enzyme activity,inflammatory factor protein and gene expression level.The liver tissue structure was observed by microscopy.Compared with the control group,in the model group the liver and spleen index increased significantly.In mouse liver tissue the liver lobules were blurred,the hepatic veins were severely deformed,and there was a lot of blood stasis in the hepatic sinuses by microscopic observation.There was significantly an increase in the serum levels of hepatic enzymes and the contents of peroxide MDA in the liver tissue.The activities of antioxidant enzymes decreased significantly.Inflammatory factors protein level and their gene expression were significantly increased.Compared with the model group,the visceral index of mice in grub polypeptide group was significantly lower.In grub polypeptide group the hepatocytes were arranged neatly without obvious necrosis and degeneration.The contents of ALT and AST in serum were significantly lower.The activities of antioxidant enzymes in liver were significantly increased,and the contents of peroxides were significantly decreased.The levels of inflammatory factor protein and gene expression in serum were significantly lower.Results showed that grub polypeptide significantly reduced the liver function of mice with immune liver injury,significantly increased the activity of antioxidant enzymes,decreased the production of peroxide and inflammatory factors.The results indicated that grub polypeptide had a certain degree anti-inflammatory and anti-oxidative effects,and had certain protective function to mice with immune liver injury.To sum up,this study obtains low molecular weight grub polypeptide by extraction and purification,and found that it can improve the specific and non-specific immune function of immunosuppressive mice,and has certain protective function on immune liver injury mice. |
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