Amygdalin Promotes The Activity Of T Cells To Suppress The Progression Of HBV-related Hepatocellular Carcinoma Via JAK2/STAT3 Signaling Pathway

Objective:This study intends to elucidate the mechanism of action of amygdalin,a main component of TCM prescription Bielong Ruangan Decoction,in HBV-related HCC and cellular immune response during HCC progression.Methods:Extract T lymphocytes from peripheral blood of HBV-related HCC patients and healthy controls,to determine the optimal use concentration of amygdalin;MTT detect peripheral blood T cells(HBV-T)and amygdalin-treated HBV-T cells in peripheral blood T cells(NC-T)and HBV-related HCC patients in healthy people.ELISA test the content of IFN-?,TNF-?,IL-2 and IL-10 in all three groups of cell culture medium;Flow cytometry detect the ratio of CD4~+and CD8~+T cell subsets to CD3~+T cells in all groups,the phosphorylation level of STAT3 in CD4+and CD8~+T cell subsets;The expressions of STAT3,p-STAT3,JAK2 and p-JAK2 in each group were detected by QPCR and western blot.HBV-T,amygdalin-treated HBV-T cells and HBV-associated HepG2.2.15 direct co-culture model,followed by ELISA to detect IFN-?,TNF-?,IL-2 and IL-10;Construction of NC-T cells,HBV-T,amygdalin-treated HBV-T cells and HepG2.2.15 non-contact co-culture model,MTT assay for cell proliferation,flow cytometry for apoptosis,transwell for cell migration and invasion.Results:The optimal treatment concentration of amygdalin was selected at 10 mg/mL;Compared with NC-T cells,HBV-T cell activity is weakened,while amygdalin can enhance HBV-T cell activity;The contents of IFN-?,TNF-?and IL-2 in HBV-T group were lower,IL-10 content was higher,and after treatment with amygdalin for 48h,IFN-?,TNF-?,IL-2 levels were significantly up-regulated,IL-10 was reduced;The number of CD4~+subpopulations was down-regulated in HBV-T cells,the CD8~+subpopulation was up-regulated,and the ratio of CD4~+/CD8~+was decreased.Amygdalin enhanced the number of CD4~+subpopulations in HBV-T cells,decreased the number of CD8~+subpopulations,and up-regulated CD4~+/CD8~+ratio.The expression levels of STAT3 and JAK2 mRNA in HBV-T cells were significantly higher,they were down-regulated after amygdalin intervention.The cytokines of HBV-T cells released under co-culture conditions such as IFN-?,TNF-?,IL-2 are still lower,IL-10 is higher,and bitter almonds after glucoside intervention,the levels of IFN-?,TNF-?and IL-2 released by HBV-T cells were up-regulated,while IL-10 was down-regulated to level close to NC-T cells.The effects of HBV-T cells on target cells HepG2.2.15 were significantly attenuated,but enhanced after amygdalin intervention.Conclusions:Amygdalin can increase the activity of HBV-T cells.Amygdalin can regulate CD4~+/CD8~+T cells through JAK2-STAT3 pathway,attenuating the immunosuppressive effects of HBV-T cells,enhancing cell interference and killing of cytokines,and enhancing the viability and invasion of HBV-associated HCC cells.And the inhibition of migration and the promotion of apoptosis.