Mechanism Study Of Anti-tumor Effect Of Triptolide In B16-F10 Tumor Bearing Mice Based On Treg

Background:Regulatory T (Treg) cells have the negative regulatory effect that can inhibit the function of other immune cells. It can inhibit the anti-tumor immune response by a variety of ways. In recent years, how to effectively remove and regulate Treg cells in tumor immunity has become a research hotspot. Triptolide, one of the monomer in Tripterygium wilfordii Hook. f. (Celastraceae), has the function of inhibiting lymphocyte proliferation, but its role on regulatory T cells has not been reported.Objective:Study the effect and molecular mechanism of triptolide on regulatory T cells in B 16-F10 tumor-bearing mice, which might provide a new way for improving effect of tumor immunotherapy.Methods:In vitro experiment:The cytotoxic effect of triptolide on B16-F10 cells was detected by MTT. B16-F10 cells were plated in 96-well plates at 104 cells per well. Cells were then treated with tripotolide at different concentrations for 24h,48h and 72h, respectively. Absorbance was measured at 570 nm after cells were incubated with MTT solution for 4h.In vivo experiment:50 C57 mice were randomly divided into normal group, untreated group, cyclophosphamide group, tripotolide high dose group and tripotolide low dose group. Mice were subcutaneously implanted with B16-F10 tumor cells in the right back region except normal group.24 hours later, mice in normal and untreated group were treated by intragastric administration with physiological saline, mice in cyclophosphamide group were intraperitoneally injected with cyclophosphamide for 1 week and mice in tripotolide group were treated by intragastric administration with tripotolide for 2 weeks. Fourteen days later, proportion of Treg cells in spleen were detected by flow cytometry (FCM). mRNA expression of Foxp3, IL-10 and TGF-βwere inspected by real-time PCR. Expression level of IL-10 and TGF-βin peripheral blood were measured by ELISA. Result:In vitro experiment:Triptolide inhibited B 16-F10 cells growth in a dose and time dependant manner.In vivo experiment:After being treated with triptolide for 2 weeks, the tumor inhibition rate in triptolide low dose group, triptolide high dose group and CTX group are 21.08%,33.88%and34.76%, respectively. The proportion of spleen Treg cells in untreated group increased obviously, and their level in triptolide treated group and CTX treated group decreased. RT-PCR results showed that compared with untreated group, Foxp3, IL-10 and TGF-βmRNA levels decreased significantly after intervention of triptolide (P<0.05). In addition, triptolide effectively decreased the concentrations of IL-10 and TGF-βin peripheral blood.Conclusion:Triptolide inhibits tumor growth in tumor-bearing mice, which might be through down-regulating the proportion of Treg cells and inhibit the production of some negative regulatory cytokines such as IL-10 and TGF-β...