| Background : Recently,the isolation and extraction of effective anti-tumor components from natural medicinal herbs become a new strategy for tumor therapy and attract great attentions.Astragalus is pouplar in natural medicinal herbs,and its main effective chemical components include astragalus polysaccharides(APS),astragalus saponin,astragalus flavonoids and so on.Astragalus polysaccharides have variety pharmacological effects of active substances,including anti-tumor,immune regulation,treatment of cardiovascular diseases and diabetes.Honey-processing Astragalus polysaccharides,(HP-APS)are astragalus polysaccharides extracted after honey roasting,its biological activity and pharmacodynamics of HP-APS improved compare with APS.The anti-tumor effects of Astragalus polysaccharides can be achieved by immunological regulation.The immunogenic death of tumor cells is might be a key mechanism of immunomodulatory drugs.Therefore,this study mainly focuses on exploring the effects of HP-APS and APS on the immunogenicity of tumor cells and their correlation with antitumor immunity.Objective: To investigate the antitumor activity of Astragalus Polysaccharides and honey-processing Astragalus Polysaccharides in honey and its influence on the immunogenicity of tumor cells.Methods:1.Extraction of Astragalus Polysaccharides by water extraction and ethanol precipitation.Astragalus Polysaccharides and honey roasted astragalus polysaccharides were then purified by gel chromatography.phenol colorimetry and they content determined by phenol colorimetry.2.CCK8 assay was used to detect the effects of HP-APS and APS on the proliferation of human lung cancer cell A549,mouse colon cancer cell MC38,and mouse melanoma cell B163.Annexin V and 7AAD staining were used to detect the effects of HP-APS and APS on the apoptosis of A549,MC38 and B16 cancer cell lines,and PI staining was used to study the influence of HP-APS and APS on the cell cycle of A549 cell line.4.Using flow cytometry to detect the immunogenicity of HP-APS and APS by testing for the expression of the CRT(calreticulin),MHC-I,CD80,CD86 on the A549 cells,and MC38,B16 cell line.5.Enzyme linked immunosorbent assay(ELISA)was used to determine the effects of HP-APS and APS on the release of immunogenic death signaling molecule in A549,MC38 cells---adenosine triphosphate(ATP).6.Flow cytometry was used to detect the effects of HP-APS and APS on the expression of surface markers in mouse cell line DC2.4 and mouse primary DC cells.7.Construct the B16 melanoma xenograft model on C57/B6 mice,and study the effects of HP-APS and APS on melanoma growth.Results1.The contents of Astragalus Polysaccharides and honey roasted astragalus polysaccharides were 67.22±0.58% and 69.81±0.76%,respectively.2.HP-APS and APS showed a cytotoxic effect on A549,MC38 and B16 cells at a concentration of 5mg/mL-10mg/mL,and was in a concentration dependent manner.When the concentration of HP-APS was increased to 10mg/mL,the maximum inhibitory rates of A549,MC38 and B16 were 48.9±0.98%,42.60±0.89%,48.40±1.35% respectively.3.HP-APS can promote the apoptosis of A549,MC38 and B16 in the concentration range of 5mg/m-10mg/m L.When the concentration of HP-APS was 10mg/m L,the apoptotic rate of each cell line was 18.8%,9.82% and 12.9%,respectively.HP-APS content of A549 cells in the G0 / G1 phase was: 60.3 ± 3.48%.4.HP-APS could promote the expression of CRT,MHC-I and HSP70 molecules on the surface of A549 cells.When the concentration of HP-APS was 10mg/mL,the average fluorescence intensity of CRT,MHC-I and HSP70 on the surface of A549 cells was 388.80±9.60,4734.66±58.58,494.66±18.58 respectively,which was significantly different from that of the blank control group(P<0.05).HP-APS can also promote the expression of MHC-I,CRT and CD86 on the surface of MC38 and B16 cells,and this difference is of statistical significance compared with the blank group.5.HP-APS can promote the expression of CD80,CD86 and MHC-I on the surface of DC2.4 cells in mice.When the concentration is 10mg/mL,the corresponding fluorescence expression intensity is 805.00±10.53,1433.02±23.02,128.00±6.53.respectively.When the concentration of HP-APS was 10mg/mL,compared with the blank group,the expression of CD11 c,CD80 and MHC-I on the surface of bone marrow DC cells could be significantly increased,and the corresponding fluorescence intensity was 22628.00±116.32,11390.33±123.55,118.00±4.32,respectively.6.The release of HP-APS in A549,MC38,B16 was determined by ELISA.The release of ATP was linearly related with the concentration ranging from 5mg/m L to 10mg/m L.When the concentration was 10mg/mL,the release of ATP was 3834.38±24.59,3021.53±96.80,2276.53±43.84,respectively.7.To detect the effect of HP-APS in vivo by melanoma xenograft mouse models,and observe the tumor volume and tumor weight and tumor growth rate of the experimental group was significantly slower.ConclusionHP-APS and APS can induce the immunogenic death of tumor cells.The possible mechanism may be its effect on enhancing the expression of ATP,HSP70 and CRT to induce the immunogenic death of A549 cells,improving the expression of MHC-I and CD86,CD80 to induce the apoptosis of MC38 and B16 cells,and blocking the A549 cells in the G0/G1 phase.It may also promote the mouse bone marrow DC cell maturation to further activate the antigen presenting capabilities of antigen-presenting cells,and finally improving the anti-tumor effect. |
PDF Full Text Request