OBJECTIVE To investigate the effect of Huangqi-Ezhu-Chonglou combination on macrophage polarization and its mechanism of inhibiting colorectal cancer (CRC) cells proliferation and migration.
METHODS THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and interleukin-4 (IL-4) to establish M2 macrophage polarization model. The experiment was divided into M0 group (PMA treatment), M2 group (PMA+IL-4 treatment), and M2+ Huangqi-Ezhu-Chonglou combination group (PMA+IL-4+Huangqi-Ezhu-Chonglou combination treatment).The effect of Huangqi-Ezhu-Chonglou combination freeze-dried powder on the viability of macrophage was detected by CCK-8 method. The expression of macrophage polarization markers, glutaminase (GLS) mRNA and protein was detected by qPCR and Western blot. The levels of interleukin-10 (IL-10), transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) in cell supernatant were detected by ELISA. CCK-8 method and Transwell assays were used to detect the proliferation and migration of HCT116 cells intervened by the supernatant of macrophage culture treated with Huangqi-Ezhu-Chonglou combination, namely conditioned medium (CM).
RESULTS Compared with the M0 group, the expression levels of IL-10, mannose receptor (CD206), arginase 1 (ARG1), and GLS mRNA and protein in the M2 group were significantly increased (P < 0.01, P < 0.001), the levels of IL-10 and TGF-β secreted by macrophages were significantly increased (P < 0.01, P < 0.001); compared with the M2 group, the M2+ Huangqi-Ezhu-Chonglou combination group had significantly reduced IL-10, CD206, ARG1, and GLS mRNA and protein expression (P < 0.05, P < 0.01), the mRNA and protein levels of TNF-α and inducible nitric oxide synthase (iNOS) were significantly increased (P < 0.05, P < 0.01, P < 0.001), the interleukin-1β (Interleukin-1β, IL-1β) mRNA expression significantly increased (P < 0. 01), and the contents of IL-10 and TGF-β in the cell supernatant significantly decreased (P < 0.05, P < 0.01), while TNF-α content significantly increased (P < 0.01). CCK-8 and Transwell results showed that compared with the M0-CM group, the M2-CM promoted the proliferation and migration of HCT116 cells (P < 0.01, P < 0.001), the M2+ Huangqi-Ezhu-Chonglou-CM group significantly inhibited HCT116 cell proliferation and reduced cell migration compared to the M2-CM group (P < 0.01, P < 0.001).
CONCLUSION Huangqi-Ezhu-Chonglou combination can inhibit colorectal cancer cells proliferation and migration by regulating macrophage polarization, and its mechanism may be related to the changes in the expression of GLS, a key enzyme in glutamine metabolism.
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