The Structural Characteristics And Antitumor Immunoregulatory Mechanism Of Polysaccharides From Polygala Tenuifolia Willd.

The root of Polygala tenuifolia Willd.,is a raw material of health food for treating anti-depressant,anti-tumor,anti-inflammatory,anti-forgetfulness,etc.Besides,the polysaccharide has attracted great attention of researchers in the fields of food and biomedicine because of its diverse structural characteristics and biological activities.Polysaccharide is an important chemical component of Polygala tenuifolia Willd.,however the structural feature and antitumor immunomodulatory activity are still rarely reported.In terms of biological activity of Polygala tenuifolia Willd.polysaccharide,the most reported is about the neuroprotective effects.Therefore,the purpose of this paper was to extract and characterize the structure of polysaccharides from Polygala tenuifolia Willd.,and then to identify their antitumor immunoregulatory activity in vivo and in vitro.Firstly,the polysaccharides of Polygala tenuifolia Willd.（PTP-80 and PTP-120）were obtained through hot water extraction and ethanol precipitation,and column chromatography methods.The research on its physics-chemistry properties indicated that the carbohydrate contents of PTP-80 and PTP-120 were measured as 94.85±4.83%and93.74±6.91%,respectively,and with little uronic acid,proteins and sulfates.The results of scanning electron microscope and high-performance gel liquid chromatography revealed that the PTP-80 was a homogeneous macromolecular polysaccharide with porous and rough surface structure,while PTP-120 was a homogeneous macromolecular polysaccharide with smooth flat flakes surface structure and good malleability,and the molecular weights were 815 k Da and 234 k Da,respectively.Combined with the results of monosaccharide composition,fourier transform infrared spectroscopy,methylation analysis,1D and 2D nuclear magnetic resonance spectroscopy,we found that PTP-80 and PTP-120were composed of rhamnose,arabinose,xylose,mannose,glucose and galactose.And we could also infer that the backbone of PTP-80 was composed by repeated→6)-β-Galp-（1→,→3,6）-β-Galp-（1→and→3）-α-Araf-（1→,and then branches of the region were linked by→2,5）-α-Araf-（1→,→3,5）-α-Araf-(1→,α-Araf-（1→,→3,6）-α-Manp-（1→,→3）-α-Araf-(1→,α-Xylp-(1→andβ-Rhap-（1→.At the same time,we could deduce that therepeatingunitofPTP-120was[→3）-α-Araf-（1→3）-α-Araf-（1→5）-α-Araf-（1→5）-α-Araf-（1→3）-α-Araf-(1→]_n,and the side chain could be deduced asα-Araf-（1→6）-β-Galp-（1→6）-β-Glcp-（1→6）-α-Manp-（1→,which was attached to the C3 of→3,5）-α-Araf-(1→.Secondly,in this paper,the peritoneal macrophages of S180-bearing mice were used as the object to investigate the immunoregulatory mechanism.The expression level of NO in the 200 mg/kg PTP-80 and PTP-120 groups significantly rose to 15.66μg/m L and 18.68μg/m L,respectively,indicated that the macrophages were activated.MTT and Elisa results suggested that PTP-80 and PTP-120 remarkably enhanced the phagocytic function of abdominal macrophage,and increased the secretions of TNF-α,IL-6,IL-1βthan the model group.Besides,the western blot result turned out that the experiment groups had significantly increased in the expressions of P-p38,P-ERK,P-JNK compared with the model group.Consequently,PTP-80 and PTP-120 mediated MAPK pathway might be activated,and then exert immune activity to suppress the proliferation of malignant cells.Thirdly,this paper further explored the other immune indexes of S180-bearing mice.The results suggested that PTP-80 and PTP-120 could inhibit the proliferation of malignant tumors in mice.Meanwhile,PTP-80 and PTP-120 could effectively protect the immune organs of the S180 bearing mice and activate the immune cells,including significantly improve the killing activity of NK cells,enhance the proliferative capacity of spleen lymphocytes and increase the CD3⁺,CD4⁺,CD8⁺T cells and CD19⁺B cells proportions in peripheral blood.In addition,the results of solid tumor cells tests revealed that PTP-80 and PTP-120 could change the morphology of solid tumor cells and arrest the cell cycle in G0/G1 phase,and then induce the cell apoptosis.Western blot method proved that the levels of Bcl-2,Bax,Cytochrome C,Caspase 9 and Caspase 3 proteins in the solid tumor cells have significantly changed compared with the model group,implied that PTP-80 and PTP-120 have significant anti-tumor immunoregulation effect in S180 tumor-bearing mice model,and its mechanism was to activate immune cells to enhance the immune function of the body,and induce apoptosis of cells in tumor tissue.Finally,this paper established the MGC-803 cells model to directly explore the antitumor immunomodulatory effect of PTP-80 and PTP-120.Here,we could intuitively see that the cell state changed markedly with the PTP-80 and PTP-120 concentration increasing under the inverted fluorescence microscope,such as cell blebbing,loss of adhesion and cellular volume.The CCK-8 results showed that the growth of MGC-803cells treated with PTP-80 and PTP-120 for 24 h was inhibited,the IC₅₀value could be further calculated,which was 104.19μg/m L and 152.97μg/m L,respectively.Meanwhile,the translocation of phosphatidylserine,the accumulation of reactive oxygen,the reduction of mitochondrial membrane potential and the arrest of cell cycle in S pahse proved that PTP-80 and PTP-120 could induce MGC-803 cells apoptosis in a dose-dependent manner.Western Blot result revealed that the ratio of Bax and Bcl-2 increased,which promoted the release of Cytochrome C,and it further maximized the expression of activated-Caspase 9/3,implied that PTP-80 and PTP-120 could induce cell apoptosis through the mitochondrial pathway in vitro,which may serve as a theoretical basis for subsequent cancer research in clinical trials.