[Study on enhanced anti-hepatocellular carcinoma effect of Fuzi polysaccharide combined with lenvatinib through promoting M1 polarization of tumor-associated macrophages].

To investigate the role of Fuzi polysaccharide(FPS) in combination with lenvatinib(LEN) in regulating tumor-associated macrophage(TAM) polarization to enhance the anti-hepatocellular carcinoma(HCC) efficacy, the non-toxic concentrations of FPS and LEN on RAW264.7 cells were screened by cell counting kit-8(CCK-8). The co-culture system of RAW264.7 and Hepa1-6 cells was established, and flow cytometry was used to detect changes in M1 and M2 macrophage phenotypes. Quantitative real-time polymerase chain reaction(qRT-PCR) was performed to assess the mRNA expression levels of M1-associated factors, inducible nitric oxide synthase(iNos) and interleukin 12b(IL-12b), as well as M2-associated factors arginase 1(Arg-1) and IL-10. The effects of macrophages treated with FPS and LEN on HCC cell proliferation, migration, invasion, and apoptosis were evaluated by 5-ethynyl-2'-deoxyuridine(EdU) staining, wound healing assay, Transwell invasion assay, and Annexin V/PI double-staining flow cytometry. A C57BL/6 subcutaneous HCC xenograft mouse model was established to observe the tumor growth, body weight, spleen index, and tumor-infiltrating macrophage polarization. CCK-8 assay showed that 50-200 mg·L~(-1) FPS and 1-5 μmol·L~(-1) LEN had no inhibitory effects on RAW264.7 macrophages. Flow cytometry and qRT-PCR results demonstrated that 200 mg·L~(-1) FPS + LEN significantly increased the proportion of M1 macrophages(P<0.01) and the M1/M2 ratio(P<0.01), upregulated the expressions of M1-associated genes iNos and IL-12b(P<0.01), reduced the proportion of M2 macrophages(P<0.01), and inhibited the expressions of M2-associated genes Arg-1 and IL-10(P<0.01). Functional assays revealed that macrophages of the FPS + LEN group inhibited Hepa1-6 proliferation(P<0.01), promoted apoptosis(P<0.01), and reduced migration and invasion(P<0.01). In vivo experiments showed that compared to those of the LEN group, the tumor volume was significantly reduced(P<0.05), and the spleen index was significantly increased(P<0.05) in the FPS + LEN group. The body weight was not decreased, and the proportion of M1 macrophages and the M1/M2 ratio in the tumors were significantly increased in the FPS + LEN group(P<0.01). In conclusion, FPS combined with LEN enhances the anti-HCC effect by promoting the M1 polarization of TAMs.