The Extraction Of Radix Pseudostellariae Fibrous Root Polysaccharides And Its Immunodulatory Effects

Objective:To study the optimal extraction process of Radix pseudostellariae fibrous root polysaccharides（RPFRP）and the immunomodulatory effects of RPFRP in vitro and in vivo.Methods:1.Extraction of crude polysaccharides with Radix pseudostellariae fibrous root as raw materials,extraction of crude polysaccharides by water extraction and alcohol precipitation method,the three factors of extraction time,extraction temperature and liquid-to-material ratio were selected as the investigation factors.Based on the single factor test,the Box-Behnken principle was used.The optimal extraction process of RPFRP was determined by response surface optimization method,crude polysaccharide was extracted,and protein was removed by Sevage method to obtain RPFRP for the test.2.Splenocytes of mice treated with different concentrations of RPFRP,MTT method was used to detect the proliferation of spleen cells,flow cytometry was used to detect the apoptosis of spleen cells,and Enzyme-linked immunosorbent assay（ELISA）method was used to determing contents of cytokines IL-2,IL-4,IL-6 and IFN-γ.3.96 mice were randomly divided into normal control group（CK）,model group（CY）,low,medium and high dose groups of RPFRP（50mg/kg,100 mg/kg,200 mg/kg）and Astragalus polysaccharide（APS）group（100 mg/kg）.On the 1st to 3rd days,mice in the normal control group was injected intraperitoneally with normal saline,and the other mice were injected with 80 mg/kg of cyclophosphamide.On the 4th to 17th days,mice both the normal control group and the model group were administrated with distilled water.Mice in three RPFRP groups and APS group were given the corresponding doses of RPFRP and APS by gavage.On the 18th,sampling,carbon particle clearance method was used to detect the phagocytosis of macrophages;MTT method was used to detect the mouse spleen lymphocyte proliferation index and NK cell activity;flow cytometry was used to detect the apoptosis of spleen lymphocyte apoptosis;automatic blood analyzer to detect blood routine;immunoturbidimetric method to detect serum immunoglobulin and complement content;ELISA method to detect serum cytokine contents;q RT-PCR method to detect cytokine（IL-2,IL-4,IL-6,IFN-γ）and transcription factor（T-bet,GATA-3）mRNA expression4.96 mice were grouped as in Method 3.On the 1st to 14th days,mice in the normal control group and the model group were administrated with distilled water,mice in other groups were administrated with corresponding doses of RPFRP and APS,On the 15th and 17th days,mice in normal control group were injected with normal saline,mice in other groups were injected with 80 mg/kg of cyclophosphamide.On the 18th day,take samples and perform relevant index detection according to Method 3.Results:1.The optimal conditions of polysaccharide extraction were obtained as:the extraction time is 2.95 h,the extraction temperature is 94.88℃,the liquid-to-material ratio is 32.28,the extraction rate is 3.49%,and the RPFRP sugar content after removing protein is 63.5%.2.Compared with the blank control group,RPFRP can significantly promote the proliferation of mouse spleen lymphocytes（P<0.05）,and in the concentration range of RPFRP 12.5～400μg/m L,synergistically with Con A can significantly promote the proliferation of T lymphocytes（P<0.05）.),In the concentration range of RPFRP 50～400μg/m L,synergistic LPS can significantly promote the proliferation of B lymphocytes（P<0.05）;the total apoptotic rate of the RPFRP group decreased significantly after stimulation for 24 hours（P<0.05）,the early apoptotic and late apoptotic rates were significantly reduced at a concentration range of 25～100μg/m L（P<0.05）;the total apoptotic rate of the RPFRP 50μg/m L group was significantly reduced at 48 h of stimulation（P<0.05）,the early apoptotic rate and total apoptotic rate were significantly increased at a concentration range of 200～400μg/m L（P<0.05）;RPFRP has different degrees of promotion in stimulating the secretion of cytokines IL-2,IL-4,IL-6,and IFN-γ（P>0.05 or P<0.05）.3.Compared with the normal control group,the final weight of mice in the model group,thymus index,phagocytic function of macrophages,T and B cell stimulation index,ratio of CD3⁺,CD4⁺,CD8⁺,CD4⁺/CD8⁺in spleen lymphocyte,white blood cell count,and lymphocyte count,lymphocyte percentage in peripheral blood,serum immunoglobulin IgA,IgG,IgM content,IFN-γcontent,IL-2 mRNA relative expression and T-bet/GATA-3 were significantly reduced（P<0.05）,The spleen index and the apoptosis rate of spleen cells were significantly increased（P<0.05）.Compared with the CY model group,there was no significant difference in the final weight of the RPFRP groups（P>0.05）;the thymus indexs of the RPFRP groups were significantly increased（P<0.05）;the spleen indexs were significantly reduced（P<0.05）;and significant increase in phagocytic indexαin the middle-dose RPFRP group（P<0.05）,T and B lymphocyte stimulation indexs（SI）were significantly increased in the low and medium dose groups of RPFRP and APS group（P<0.05）,and the killing activity of NK cells in the medium dose group of RPFRP was significantly increased（P<0.05）;The ratios of CD3⁺,CD4⁺,and CD8⁺increased significantly in the RPFRP group,and CD4⁺/CD8⁺increased significantly both in low and high dose RPFRP groups（P<0.05）.The total apoptosis rate in RPFRP groups decreased significantly（P<0.05）.The number of white blood cells and lymphocytes increased significantly at medium dose（P<0.05）,and the contents of IgA,IgG,IgM,C₃,C₄,IL-2,and IFN-γin RPFRP groups increased significantly（P<0.05）.The IL-4 content in low dose and medium group were significantly increased（P<0.05）;the cytokines IL-2,IFN-γ,T-bet/GATA-3 mRNA expression level in three RPFRP groups were significantly increased（P<0.05）.4.Compared with the normal control group,spleen index,thymus index,T,B cell stimulation index,NK cell killing activity,CD3⁺,CD4⁺,CD8⁺,CD4⁺/CD8⁺,white blood cell count,lymphocyte count,lymphocyte percentage,serum immunoglobulin IgA,IgG,IgM and complement C₃content,the relative expression levels of IL-2,IL-4,IFN-γ,T-bet/GATA-3mRNA in model group were significantly reduced（P<0.05）,and the early,late and total apoptosis rates were significantly increased（P<0.05）.Compared with the CY model group,the thymus indexs in three RPFRP groups and APS group were increased significantly（P<0.05）.The spleen index in RPFRP low and high dose group were increased significantly（P<0.05）;T lymphocyte stimulation index SI increased significantly in RPFRP medium and high dose groups and APS group（P<0.05）,and B lymphocyte stimulation index SI in the low and medium dose groups and APS group significantly increased（P<0.05）,NK cell killing activity in RPFRP medium dose group and APS group increased significantly（P<0.05）;RPFRP high dose group CD3⁺,CD4⁺,CD8⁺,and CD4⁺/CD8⁺significantly increased（P<0.05）The early apoptosis,late apoptosis and total apoptosis rate of RPFRP groups decreased significantly（P<0.05）,and the number of white blood cells and lymphocytes increased significantly in low,mediumRPFRP dose,and APS groups（P<0.05）.The content of IgG,C₃,and C₄ in the dose group increased significantly（P<0.05）,the content of IL-4 in the low and medium dose groups increased significantly（P<0.05）,and the content of IFN-γin the medium and high dose groups increased significantly（P<0.05）;the expression of cytokine IL-2 mRNA in the low-dose RPFRP group was significantly increased（P<0.05）.IFN-γmRNA expression in the high dose group of cytokines was significantly increased（P<0.05）,each dose group T-bet/GATA-3 mRNA expression was significantly increased（P<0.05）.Conclusions:1.Using the response surface optimization method,the RPFRP extraction rate is 3.49%,and the sugar content after purification is 63.5%.2.In vitro experiments show that RPFRP can promote the proliferation of mouse splenic lymphocytes,reduce apoptosis,promote cytokine secretion,and have an immune promoting effect on mouse splenic lymphocytes.3.The in vivo test results show that RPFRP can correct the swelling or atrophy of the immune organs of mice caused by cyclophosphamide and the imbalance of T lymphocyte subsets,improve the killing activity of NK cells,reduce apoptosis of spleen cells and increase white blood cells And the number of lymphocytes,regulate the secretion of immunoglobulins,complement cytokines and the expression of cytokines,transcription factors,etc.so as to have a certain immune repair and immune protection effect on immunosuppressed mice.