Effects Of Eugenol On Myeloid-derived Suppressor Cells And Its Molecular Mechanism

Objective:Myeloid derived suppressor cells（MDSCs）are heterogenous myeloid cells with strong inhibition of CD8⁺and CD4⁺T cells,which can inhibit the host’s anti-tumor immunity.The mechanisms of immune suppress mediated by MDSCs include ROS pathway,energy depletion,induction of Tregs and suppression of innate immunity.The immunotherapy strategies targeting MDSCs include promoting the differentiation of MDSCs,inhibiting the function of MDSCs,and reducing the number of MDSCs.The experimental group studied the effect and mechanism of eugenol on primary MDSCs,and immortalized MDSCs cell line MSC2 through in vitro experiments,so as to find a new bioactive molecule for immunotherapy and provide theoretical basis for the anti-tumor mechanism in the basic research of the active molecule.Methods:1.To investigate whether eugenol had inhibitory effect on the primary MDSCs,the subcutaneous transplantation model of CT-26 tumor cells in mice was established firstly.Then the proportion of MDSCs in the suspension of spleen cells with different concentrations of eugenol was detected by FCM.2.In the cell activity experiment,MTT method was used to detect the effect of eugenol on MSC2.At the same time,cell immunofluorescence was performed to detect the effect of eugenol on MSC2.3.To analyze the inhibitory mechanism of eugenol of different concentrations on MSC2,FCM was used to measure the quantitative detection of apoptosis function.WB was further used to detect the expression of related apoptotic proteins,including Bax,Chrochrome C,Caspase-9,Cleaved Caspase-3,etc.4.Finally,to preliminarily find the activated signal pathway and explore the anti-tumor mechanism of eugenol,the tumor subcutaneous transplantation model was established again.FCM was measured to detect whether MDSCs in related gene knockout mice(TLR4^-/-)would be affected by eugenol.5.In the cell activity experiment,MTT method was used to detect the effect of eugenol on primary abdominal macrophages,CT-26,and MC-38 and.Calculate IC₅₀ of each cell was used to compare the effect of eugenol on these cells.Results:1.FCM showed that eugenol could reduce the proportion of MDSCs in the suspension of spleen cells of tumor-bearing mice;2.MTT assay showed that the activity of MDSCs cell line MSC2 treated with eugenol at different concentrations was inhibited to varying degrees with the increase of the stimulation concentration,showing a dose-effect relationship.There was a statistical difference when eugenol concentration was 0.3mΜ,and IC₅₀ of MSC2 cell line was calculated to be 0.72mΜ.3.FCM apoptosis detection.It was found that after treated with eugenol,the proportion of Annexin V positive cells in MSC2 cells would increase,which were dose dependent.The results of WB showed that Bax,Cytochrome C,Caspase-9,and Cleaved Caspase-3 were up-regulated in the protein expression level after eugenol treatment at appropriate concentrations,while the expression level of BCL-2 was down-regulated in a time-dependent manner.4.The suspension of spleen cells of TLR4^-/-tumor-bearing were treated with eugenol.FCM revealed that the proportion of MDSCs had no significant change before and after eugenol treatment,and there was no statistical difference.5.MTT assay was used to detect the effect of eugenol on colon cancer cell lines and primary peritoneal macrophages.The results showed that eugenol had an inhibitory effect on MC-38,CT-26 only at a higher stimulation concentration,the IC₅₀ of whese were 1.92mΜand 4.02mΜ,respectively.After eugenol treatment of primary abdominal macrophages,the cell activity increased with the increase concentration of eugenol,but no inhibitory effect was observed on abdominal macrophages in the same concentration range.Conclusions:1.Eugenol Triggers myeloid-derived suppressor cell apoptosis via endogenous apoptosis pathway by binding to TLR4 receptor,thus achieving the effect of reducing the number of MDSCs.2.Within a certain concentration range,eugenol has a selective inhibitory effect on MSC2.