Ginsenoside Rh2 Enhances Anti-tumor Immunity By Regulating Fatty Acid Metabolism

Objective:Ginsenoside Rh2(G-Rh2),an important bioactive constituent of Chinese medicine ginseng,exhibits rich pharmacological activities,especially anti-tumor effect.The research aims to explore the therapeutic effect of G-Rh2 on non small cells lung cancer(NSCLC),the synergistic treatment effect of G-Rh2 and cyclophosphamide(CY),and the regulation of G-Rh2 on liver metabolism and immunity.Methods:(1)We established NSCLC subcutaneous xenograft model of C57/BL6J mice and immunodeficient nude mice.C57/BL6J model mice were treated with CY,G-Rh2or CY combined with G-Rh2,nude mice were treated with G-Rh2.Living imaging of mice and tumor volume were used to evaluate tumor size.Hematoxylin and eosin(H&E)of liver was applied to detect pathological injury.Biochemical kits were used to test the levels of Alanine transaminase(ALT),Aspartate transaminase(AST),creatinine(CRE)and urea nitrogen(BUN)to evaluate the function of liver and kidney.Flow cytometry(FCM)analysis was used to detect the proliferation and function of immune cells,and the expression of immunity related molecular.Mixed lymphocyte reaction(MLR)was applied to detect the stimulation ability of dendritic cells(DCs)on the proliferation of T cells.ELISA kits were used to measure cytokines levels in serum of mice.(2)C57/BL6J mice were injected intraperitoneally with methotrexate for two weeks or treated with the whole body irradiation by 4Gy X-ray once to establish the immunosuppressive model.Mice were treated with thymopentin which can enhance immune function or G-Rh2,and whose weight was recorded every day.To evaluate the influence of G-Rh2 on immune function,FCM was applied to test the proliferation,differentiation and function of immune cells,and expression levels of immune related molecules.The concentration of cytokines in the serum was detected by ELISA kits.(3)To evaluate the effect of G-Rh2 on lipid metabolism,the biochemical kits were used to detect the levels of fatty acid(FA),high density lipoprotein(HDL),low density lipoprotein(LDL),total cholesterol(TC)and total triglycerides(TG)in serum of mice.Quantitative real-time polymerase chain reaction(qRT-PCR)and western blot were used to detect the mRNA and protein expression levels of phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),sterol regulatory element binding protein 1(SREBP-1)and fatty acid synthetase(FASN)in mice liver,mouse normal liver cells AML12,mouse dendritic cells DC2.4 and mouse lung cancer cells LLC.To verify whether G-Rh2 regulated the expressions of SREBP-1 and FASN through the PI3K-AKT signaling pathway,we treated AML12,DC2.4 and LLC with the inhibitor of PI3K-AKT signaling pathway(LY294002)and the agonist of the pathway(740Y-P),and then we detected the expressions of SREBP-1 and FASN.The immunofluorescence assay was applied to detect SREBP-1 expression inside and outside nuclear of AML12 and DC2.4.We treated AML12,DC2.4 and LLC with Fatostin,the inhibitor of SREBP-1,to detect whether G-Rh2 affected FASN expression by inhibiting SREBP-1,thereby regulated the synthesis of fatty acid(FA).Chromatin immunoprecipitation(ChIP)assay was applied to detect the interaction of SREBP-1 and FASN.In addition,we established liver specific FASN knockout FASN~fl/fll/fl mice by transfecting Cre plasmid into FASN~fl/fll/fl mice via hydrodynamic tail vein injection.After treated with G-Rh2 for two weeks,we used FCM to test the immune function of mice to verify the role of FASN on the immune regulation of G-Rh2.Results:(1)G-Rh2 significantly inhibited tumor growth,reduce the aggragation of cancer cells in tumor site,and enhanced the anti-tumor effect of CY when combined with it.In vitro exprements showed that G-Rh2 inhibited the proliferation of A549 cells,but exhibited no influence on the proliferation of human normal hepatocytes L02.The results suggested that G-Rh2 had anti-tumor effect and had no damage on normal cells.Additionally,G-Rh2 maintained the stable increase of mice weight,the rate of increase was similar to normal mice.When combined with CY,G-Rh2 alleviated the effect of CY on mice weight.G-Rh2 had no damage on liver,decresed the secration of ALT,AST,CRE and BUN in serum to protect liver and kidney function of mice.(2)G-Rh2 significantly up-regulated the proportions of CD8~+T cells in thymus and lymph nodes,CD4~+T cells in thymus,natural killing cells(NK)cells in spleen and decreased the number of regulatory T cells(Tregs)in spleen of NSCLC mice.G-Rh2 treatment up-regulated the expressions of CD86 and major histocompatibility complex(MHCII)on DCs to promote the DCs maturation,down-regulated programmed death molecular ligands 1(PD-L1)expression and enhanced the ability of DCs to stimulate the proliferation of T cells to boost immune function.(3)For immunodeficient mice,G-Rh2 alleviated weight loss caused by radiotherapy and methotrexate,increased the numbers of CD4~+T and CD8~+T cells in immune organs,up-regulated the expression of T cell receptor(TCR),decreased bone marrow-derived immunosuppressive cells(MDSC),up-regulated MHCII expression on DCs,down-regulated PD-L1 expression on DCs.Besides,G-Rh2 promoted the seccration of interleukin(IL-2)and tumor necrosis factor?(TNF-?).G-Rh2 revised the immunodeficient of mice,enhanced immune function,alleviated damage of radiotherapy and methotrexate.(4)G-Rh2 treatment inhibited the expressions of FA synthesis related genes,promoted the expressions of FA decomposition related genes in liver,suppressed FA synthesis,increased ketones level,and decreased glucocorticoid secretion of NSCLC mice.G-Rh2 down-regulated the expressions of PI3K-AKT signaling pathway,SREBP1,FASN and disturbed the interaction of SREBP-1 and FASN which indicated G-Rh2 disturbed transcriptional regulation of SREBP-1 on FASN to down-regulated FASN expression.For liver specific FASN knockout mice,G-Rh2 failed to enhance the immune function which suggested the immune regulation of G-Rh2 was correlated with FASN.Conclusion:(1)G-Rh2 exhibits a synergistic anti-tumor effect with CY,not only significantly enhances the anti-tumor effect,but also improves immunodeficiency caused by CY.Besides,G-Rh2 alleviates damage of liver and kidney and weight loss resulted from CY.And G-Rh2 enhances immune function of immunodeficient mice.(2)G-Rh2 regulates FA metabolism to enhance immunity.G-Rh2 inhibites FA synthesis,promotes FA decomposition,induces ketones production,and reduces glucocorticoid level.When FASN is deficient in liver,G-Rh2 fails to boost immunity.(3)G-Rh2 inhibites the PI3K-AKT signaling pathway,inhibites nuclear translocation of SREBP-1,decreses the expression of SREBP-1 in nuclear,disturdes the transcriptional regulation of SREBP-1 on FASN,down-regulates FASN espression.