Research On Immunomodulatory Activity Of Polysaccharides From Paederia Scandens

Objective:To extract and isolate polysaccharide from Paederia scandens,study and evaluate the immunomodulatory activity of Paederia scandens polysaccharide on macrophages and immunosuppressed mice,and the acute toxicity of PSP.It provides a certain theoretical basis for the further research,development and application of Paederia scandens polysaccharide.Methods:(1)Explore the method of making immunosuppressive mouse model and determine the positive control drug.Immunosuppressive mice model was established by cyclophosphamide(CY)induction.Model group,Jinshuibao group and levamisole group were intraperitoneally injected with CY 50 mg/kg on the 3rd,5th,7th and 9th day of the experiment to make immunosuppressive mice,continuous administration for 10 days.At the end of the experiment,the changes of body mass,thymus index,spleen index,the number of Peyer’s Patches(PPs)and cytokine content of mice in each group were compared to evaluate whether the modeling method is feasible and which drug is more suitable as positive control substance in vivo experiment.(2)The preliminary experiment was carried out on the immunoregulatory activity of Paederia scandens polysaccharide.PSP was extracted by water extraction and alcohol precipitation.To study the immunomodulatory activity of PSP in vitro,MTT reagent was used to detect the changes of cell proliferation rate after PSP with different concentrations acted on Raw264.7 macrophages;Neutral red phagocytosis test was used to investigate the changes of phagocytosis rate of Raw264.7 cells after PSP with different concentrations was treated;ELASA was used to detect the changes of cytokine expression level in Raw264.7 cells treated with PSP with different concentrations.To study the immunoregulatory activity of PSP in vivo,the experimental method refers to the experiment of screening positive control substance in(1).In addition,the phenotype and proportion of CD3+,CD19+and CD4+lymphocytes in PPs cells were detected by flow cytometry.(3)The immunoregulatory activity of polysaccharide from Paederia scandens was tested.PSP was extracted from Paederia scandens dispensing granules by water extraction and alcohol precipitation,and its immunomodulatory activity was studied in vivo and in vitro according to the experimental method in(2).(4)Paederia scandens dispensing granules was used to extract the PSP to carry on the acute toxicity experiment.The experiment was divided into control group and PSP experimental group.The PSP experimental group was gavaged once every 5 hours and twice a day according to the maximum volume of mice:40 mL.kg-1 and the maximum dissolved concentration of PSP:300mg.mL-1,while the control group was gavaged with pure water in the same way.During the experiment,all aspects of mice’s performance were closely observed and recorded.At the end of the experiment,the animals were killed and dissected to observe whether the internal organs changed,and the main organs were weighed and histopathologically observed.Results:(1)The positive control drug screening experiment of PSP in vivo showed that the immunosuppressive mouse model was successfully established by intraperitoneal injection of cyclophosphamide.Compared with Jinshuibao group,the weight gain,thymus index,spleen index,the number of Peyer’s Patches and the amount of IL-4 and IL-8 secreted by intestinal tract of immunosuppressive mice in levamisole group were higher.Therefore,levamisole is more suitable as a positive control substance for in vivo experiments in mice.(2)The results of Preliminary study on immunoregulatory activity of PSP showed that PSP could promote the proliferation of macrophages and phagocytosis of neutral red in vitro,and could promote the secretion of cytokines such as IL-6,IL-2,IL-8,TNF-α,NO and IFN-γ by Raw264.7 macrophages in different degrees.In vivo experiments,PSP could restore the body weight,thymus index,spleen index,the number of Peyer’s Patches,the proportion of CD3+,CD19+and CD4+lymphocytes in Peyer’s Patches,and the contents of NOS and H2O2 in serum of immunosuppressive mice induced by cyclophosphamide.(3)The results of immunoregulatory activity of PSP showed that PSP could promote the proliferation of macrophages and the phagocytosis of neutral red and FITC-dextran 40,and promote the secretion of IL-6,TNF-α,NO,NOS and H2O2 factors by Raw264.7 macrophages.PSP could restore the body mass,thymus index,spleen index,the proportion of CD3+,CD 19+and CD8+lymphocytes in Peyer’s Patches,the content of IL-10 and IgG in serum,and the content of sIgA and IL-12p70 in supernatant of intestinal contents of immunosuppressed mice induced by cyclophosphamide.(4)The results of acute toxicity test of PSP showed that:compared with the blank control group,the general state,death and histopathology of PSP treated groups of female and male mice were not significantly abnormal;there were no significant differences in food consumption,body weight change,body weight gain and organ index(thymus index,spleen index,liver index and kidney index)between the two groups(P>0.05).Conclusion:In this study,polysaccharide from Paederia scandens was obtained by water extraction and alcohol precipitation,and its immunomodulatory activity was studied in vivo and in vitro.Preliminary experiments showed that PSP could promote the proliferation,phagocytosis and secretion of related cytokines of Raw264.7 macrophages,and could resist immunosuppression caused by CY.The formal experiments further confirmed the results of the pre experiment.Acute toxicity test showed that the toxicity of PSP was very low.The above experiments provide a certain theoretical basis for further research,development and application of PSP.