Jianpi Huayu Decoction Attenuates Immunosuppression In H₂₂Hepatocellular Carcinoma-bearing Mice By Regulating Myeloid-derived Suppressor Cells

Background The long-term and effective reversal of tumor-induced immunosuppression is still the difficulty of antitumor therapy.Previous studies found that Jianpi Huayu Decoction(JHD)can reduce the immune-suppression induced by hepatocellular carcinoma,but the underlying mechanism is not clear.Combined with the research progress in related literature,it is speculated that JHD may reduce immunosuppression by regulating myeloid-derived suppressor cells(MDSCs).Objective To investigate the mechanism of JHD to weaken the immunosuppression status of subcutaneous H22 hepatocellular carcinoma mice,and the regulatory effect of JHD on MDSCs derived from hepatocellular carcinoma mice.Methods1?In vivo experiment:The BALB/c mouse model of subcutaneous H22 liver cancer was established and randomly divided into the JHD group(n=5)and the PBS group(n=5).JHD(24.96g/kg/day)and PBS were given gavage treatment for 12 days,and the tumor volume was measured daily.Immunofluorescent markers were combined with flow cytometry to detect the proportion of macrophages,dendritic cells,MDSCs,Treg,Th17,Th1,Th2,CTL,and granulocytes in the spleen and/or bone marrow.Tissue structure of spleen was observed by H&E staining.2?In vitro experiment:MDSCs derived from hepatocellular carcinoma mice were separated by immunomagnetic beads and treated with JHD or oxaliplatin.Cell activity was detected by CCK-8;Annexin V-FITC/PI assay was used to detect apoptotic cell;Immunofluorescent markers were combined with flow cytometry to detect cell differentiation.Additionally,MDSCs were induced in vitro and stimulated by JHD.Intracellular ROS were detected by flow cytometry.In vitro proliferation inhibition assay of CD4+T cells was performed to detect proliferation inhibition of MDSCs treated with JHD.ResultsIn vivo experiment:1?Jianpi Huayu Decoction significantly delay the growth of subcutaneous H22 tumors and inhibited the proliferation and activity of H22 hepatocellular carcinoma cells in vivo and in vitro.2?Jianpi Huayu Decoction significantly reduce spleen weight and spleen tissue structure damage.3?Jianpi Huayu Decoction significantly increase the proportion of CD11b+Gr-1+cells in spleen,the proportion of CD11b+Ly6G+cells in bone marrow and the proportion of CD11b+Ly6C+ cells in spleen.4?Jianpi Huayu Decoction significantly increase the proportion of dendritic cells in the spleen.5?Jianpi Huayu Decoction did not significantly affect the proportion of CD11b-Gr-1+ granulocytes in the spleen and bone marrow.6?Jianpi Huayu Decoction significantly increase the proportion of CD8+IFN-y+cells,and significantly reduce the proportion of Treg and Th17 cells in the spleen.In vitro experiment:1?When the concentration of Jianpi Huayu Decoction is less than 250?g/ml,it increase the activity of MDSCs,and when the concentration is more than 250?g/ml,it inhibit the activity of MDSCs.2?After 24 hours of stimulation,the early apoptotic cells in Jianpi Huayu Decoction group(250?g/ml,500?g/ml)and oxaliplatin group increase significantly;The late apoptotic cells in the 500?g/ml Jianpi Huayu Decoction group and oxaliplatin group significantly increase compared with the control group.After 36 hours of stimulation,Jianpi Huayu Decoction(250?g/ml,500?g/ml)significantly reduced the early and late apoptosis of MDSCs.3?After 36 hours of stimulation,Jianpi Huayu Decoction significantly increase the proportion of F4/80+cells and decrease the proportion of CD 11c,CD80,MHCII+cells;After 48 hours of stimulation,Jianpi Huayu Decoction significantly increase the proportion of cells expressing F4/80,CD11c,CD80,MHCII molecules.4?Jianpi Huayu Decoction significantly reduce the ROS expression of MDSCs.5?Jianpi Huayu Decoction significantly reduce the proliferation inhibition of MDSCs on CD4+T cells.Conclusions1?Jianpi Huayu Decoction inhibit the growth and proliferation of H22 hepatocellular carcinoma cells in vivo and in vitro.2?Jianpi Huayu Decoction attenuate the immunosuppression status of H22 hepatocellular carcinoma mice by regulating the apoptosis,differentiation and immunosuppressive ability of MDSCs.SignificanceThis study further elucidates the mechanism of Jianpi Huayu Decoction to attenuate immunosuppression of hepatocellular carcinoma,and provides a research basis for finding drug molecules that target and regulate MDSCs.