The Preliminary Study On The Immunomodulation Mechanism And Immunomodulatory Substances Of Smilax Glabra Roxb.

ObjectiveSmilax glabra Roxb.(SGR)is sweet and pale in taste,belongs to spleen,stomach,and liver meridians.It is commonly used for treating immune-related diseases such as chronic nephritis,eczema and psoriasis,indicated that SGR can affect immunomodulatory.Therefore,in this paper,effect of SGR on immune was studied by immune-inhibiting and immune-stimulating model,and the relevant mechanism and the substance was investigated for its further development.Methods1.Effect of SGR extraction on cyclosporine-A immunosuppressive model:96 mice were divided into normal control group,model control groups levamisole positive group,and low(4.45g·Kg-1),medium(9.1g·Kg-1)and high(13.65g·Kg-1)dose of SGR extraction groups.Except the normal group,all mice in the other groups were continuously intraperitoneally injected with cyclosporine-A solution for five days.After the model was established,mice in treated groups were intragastric administration by corresponding drugs,the normal and model group mice were given saline for five days;the spleen of mice were taken and weighed,then were pulverized into cell serum;the amount of CD3+CD4+ cell and CD3+CD8+ cell in mice spleens were investigated by flow cytometry.The mice spleen were embedded and sliced for HE-pathological section;the levels of IL-6,IL-2 and IFN-? protein in mice spleens were determined by enzyme-linked immunosorbent assay(ELISA)at 570 nm wavelength.2.Immuneregulating effect of astilbin on mice with blood-stasis psoriasis model:first the spleen deficiency and blood stasis model was established in mice.Except for the normal group mice,other mice were go on a diet and companied with ice swimming for 15 days,thereafter model mice were divided into model control,methotrexate group,low(30.7mg·Kg-1),middle(61.4mg·Kg-1),and high(92.lmg·Kg-1)astilbin groups,and propranolol emulsion was applied on the back skin to prepare blood-stasis psoriasis model for 6 days;the corresponding therapeutic drug was given to the treated groups and the normal and model control group mice with normal saline;the PASI score was measured in the dorsal skin lesions of mice;the weight of spleen and thymus were wei.ghted,and the index was calculated,Treg/Thl/Thl7 lymphocyte numbers in the spleen and lymph nodes were determined by flow cytometer;the Baker score was performed on the skin pathological sections of mice;the mice skin inflammatory level of IL-6/IL-17/IFN-y was determined by enzyme-linked immunosorbent assay(ELISA).3.Effect of polysaccharides from SGR extraction on the cyclophosphamide immunosuppressive model:mice were divided into normal control,model control and polysaccharides low(0.3595g·Kg-1),medium(0.7189g·Kg-1)and high(1.0784 g·Kg-1)dose groups,levamisole group,besides the normal control group,mice in other groups were intraperitoneal given 40 mg·Kg-1 cyclophosphamide solution for 10 days,mice in drug treated group were given the corresponding drugs at the meantime.Thereafter mice were sacrificed,spleen and thymus were weighted and the number of CD3+ and CD19+ cells in the spleen was determined by flow cytometer.The cytotoxicity test was used to investigate mice spleen lymphocytes proliferation after stimulated by conA.The effect of polysaccharides from SGR on T cells of mice spleen was determined by immunofluorescence assay.Results1.Effect of SGR water extract on cyclosporine immunosuppressive model:after intraperitoneal injection of cyclosporine,mice in model group gain weight slowly,spleen enlargement,spleen index increased,and thymus index decreased,indicated the model was successful.The Levamisole and the middle-dose group of SGR significantly reduced the mice spleen index;levamisole increased the thymus index,however all three doses of smilax glabra roxb have marginal effect on thymus index.SGR increased the CD4+T lymphocytes in the spleen significantly,while have no effect on the CD8+T cells.The pathological section showed that the white pulp in spleen was shriveled,SGR significantly released the symptoms significantly.SGR decreased IFN-? and had no effect on IL-6 and IL-2.2.The immune-regulating effect of astilbin on blood-stasis psoriasis model:the mice back skin was red and thick,some mice had erythema,and there was pain when touched lightly.The middle and high dose of astilbin released the skin lesions significantly.The PAS I scores in the model group were increased,and astilbin dose-dependently reduced the PASI score;high-dose group of astilbin decreased the Baker score significantly;the number of Treg,Th17 and Thl in the mice lymph nodes was significantly increased after modeling,and low,medium and high doses group of astilbin decreased the Treg and Thl number of significantly.However,astilbin had marginal effect on the spleen T-regulated cells;IL-17A,IL-6 and IFN-? in lesions were significantly increased;astilbin decreased the contents of IL-17A,IL-6 and IFN-?significantly,and shown a more strong effect on IL-17A and IFN-?.3.Effect of SGR polysaccharides on the cyclophosphamide immunosuppressive model:the proliferation of spleen lymphocytes in the model group was significantly reduced,and SGR polysaccharides dose-dependently reversed it;the number of spleen T and B lymphocytes and CD4+T lymphocytes in the model group was decreased;SGR polysaccharide increased the number of the CD3+labeled T lymphocytesand CD4+T lymphocytes significantly,but had no effect on the CD19+labeled B lymphocytes.ConelusionThe results of this study indicate that SGR can promote T cell-mediated immunity,and polysaccharide of SGR is the effective part;astilbin,the main flavonoid component of SGR is the imnunesuppressive agent and could used for psoriasis.Astilbin and polysaccharide together play the imnunomodulatory effect of SGR.