| Objective:Gastric cancer is one of the cancers with high morbidity and mortality in the world.Most patients are found in the middle and late stage,with poor prognosis,and there are some problems such as tumor recurrence,metastasis,toxicity and side effects in surgery,radiotherapy and chemotherapy and other treatments.Therefore,there is an urgent need to find a better treatment.In recent years,tumor immunotherapy has made great progress in clinic,anti-tumor by regulating immunity,which brings great innovation to the treatment of gastric cancer,and gastric cancer immune microenvironment is one of the research hotspots.Tumor-associated macrophage（TAMs）and Natural killer cell（NK）are important immunosuppressive cells and immune effector cells in immune microenvironment.TAMs can inhibit the proliferation and function of NK cells.By regulating these two kinds of cells,relieving immunosuppression and restoring immune function is very important for the treatment and prognosis of gastric cancer.Based on this clinical background,this study selected three components of Sanwubai Powder:Crotonin,Platycodin D,Peimine and their compatibility,to explore whether monomer and compatibility can regulate TAMs,whether monomer and compatibility can relieve the immunosuppression of NK cells by regulating TAMs,improve the immune state of microenvironment and promote the treatment of gastric cancer,and further explore the mechanism involved.Methods:（1）THP-1 cells were induced into TAMs cells by IL-4+IL-13+PMA+complete medium containing 5%serum in vitro,and the phenotype was identified.（2）The safe concentration ranges of Crotonin,Platycodin D and Peimine on AGS cells,TAMs cells and NK cells in vitro were screened by CCK8 method.（3）Screening the threshold concentration range,optimal concentration and subthreshold concentration of Crotonin,Platycodin D and Peimine in regulating TAMs by flow cytometry and ELISA.（4）Orthogonal experiment was designed,Crotonin,Platycodin D,Peimine and blank were used as four factors,and the subthreshold concentration of each monomer,subthreshold concentration of 1 beat 2 and 0 as three levels were used for orthogonal compatibility.Flow cytometry and ELISA were used to screen the best compatibility combination for regulating TAMs.（5）In Transwell system,TAMs was inoculated into the upper chamber,and AGS cells and NK cells were co-cultured in the lower chamber to simulate the immune microenvironment of gastric cancer,and drug intervention was carried out.（6）The cytotoxicity of NK cells was detected by lactate dehydrogenase（LDH）release assay.（7）The contents of MMP9,sMICA and sMICB in the culture supernatant were detected by ELISA.（8）The expression of cell membrane molecules NKG2D and MICA/B was quantitatively detected by flow cytometry.Results:（1）After THP-1 induction,the expressions of TAMs phenotypic markers CD206 and secretory factors IL-10 and TGF β were significantly up-regulated as compared with the control group（P<0.05）;（2）.AGS cells were treated with different concentrations of monomers,and verified by TAMs and NK cells,the concentration range of cell survival rate was as follows:Crotonin:10-5,10-6,10-7,10-8,10-9mol/L.Platycodin D:10-5,10-6,10-7,10-8,10-9 mol/L,Peimine:10-4,10-5,10-6,10-7,10-8mol/L.（3）In terms of decreasing CD206 level,crotonin at 10-1,10-6,10-7,10-8,10-9mol/L,Platycodin D at 10-5,10-6,10-8mol/L and Peimine at 10-1,10-6,10-8mol/L showed significant difference（P<0.05）.In terms of decreasing IL-10 and TGF β,crotonin at 10-1,10-6,10-1,10-8,10-9mol/L,Platycodin D at 10-5,10-6,10-7,10-8mol/L and Peimine at 10-4,10-5,10-6,10-8mol/L showed significant difference（P<0.05）.The optimal concentrations of Crotonin,Platycodin D and Peimine to regulate TAMs were 10-5,10-5 and 10-4mol/L respectively,and the subthreshold concentrations were 10-10,10-9 and 10-7mol/L respectively.（4）In terms of reducing the levels of CD206,IL-10 and TGF β,the best compatibility was 10-10mol/L Crotonin+10-9mol/L Platycodin D+10-7mol/L Peimine.The influence degree of each factor on CD206 and TGF β was Crotonin>Platycodin D>Peimine,and the influence degree on IL-10 was Crotonin>Peimine>Platycodin D.（5）In immune microenvironment,the compatibility group could significantly enhance the cytotoxicity of NK cells,but each single group had no effect.（6）The results of flow cytometry showed that compared with the model group,there was a significant difference in the increase of NKG2D level between the peimine group and the compatibility group,and there was a significant difference in the decrease of MICA/B level between the crotonin group and the compatibility group.（7）The results of ELISA showed that the sMICA/B of the compatibility group decreased significantly（P<0.05）.Although the MMP9 index of each group was not significant,it had a downward trend,and the decrease of the compatibility group was the most obvious.Conclusion:（1）THP-1 cells can be successfully induced into TAMs;（2）Crotonin,Platycodin D,Peimine and their compatibility can regulate the phenotype of TAMs,decrease the proportion of CD206+TAMs cells and decrease the content of secretory factors IL-10 and TGF β.（3）In immune microenvironment,the compatibility group can enhance the cytotoxicity of NK cells,but each monomer group has no effect on it.（4）The compatibility group can regulate the immune state in the microenvironment,which is related to the regulation of NKG2D/NKG2DL. |
PDF Full Text Request