蟾毒灵通过抑制TAM介导的STAT3磷酸化降低结直肠癌细胞PD-L1表达OA

Objective:To investigate the regulatory effect of bufalin(BU)on tumor-associated macrophage(TAM)-mediated programmed death-ligand 1(PD-L1)expression in colorectal cancer(CRC)cells and to elucidate the underlying molecular mechanism.Methods:An in vitro co-culture system was established using HCT116 cells and THP-1-derived macrophages.THP-1 cells were differentiated into M0 macrophages by PMA treatment and further stimulated with HCT116-conditioned medium(CM)to generate TAM-like cells.Flow cytometry was used to detect CD11b and CD206 expressions and assess macrophage polarization level.The changes in the expression levels of TGF-β,IL-10,STAT3/p-STAT3,and PD-L1 were detected by RT-qPCR and WB assay.A STAT3 knockdown cell line(HCT116sh-STAT3),constructed by lentiviral transduction,was combined with BU treatment to verify the role of the STAT3/PD-L1 signaling pathway.Results:HCT116-derived CM induced macrophage polarization toward the M2 phenotype,as evidenced by an increased proportion of CD11b⁺CD206⁺ cells(P<0.01)and elevated expression of TGF-β and IL-10(both P<0.001).TAM-conditioned medium significantly promoted STAT3 phosphorylation(P<0.001)and upregulated PD-L1 expression at both mRNA and protein levels in HCT116 cells(P<0.001 or P<0.01).BU treatment markedly suppressed TAM-mediated STAT3 phosphorylation and concomitantly reduced PD-L1 expression(both P<0.01).STAT3 knockdown decreased PD-L1 expression,showing a similar trend to BU treatment.Conclusion:BU downregulates PD-L1 expression in CRC cells by inhibiting TAM-mediated STAT3 signal activation,suggesting its potential application value in regulating the tumor immune microenvironment.