Astragaloside IV inhibits the progression of liver cancer by modulating macrophage polarization through the TLR4/NF-κB/STAT3 signaling pathway

Aim: The purpose of the present research was to investigate the effect and mechanism of Astragaloside IV (AS-IV) on liver cancer progression in vivo and in vitro. Since M1 macrophages play an essential role in suppressing tumors, while M2 macrophages can accelerate the incidence and progression of tumors by promoting angiogenesis, increasing tumor cell invasion and inhibiting tumor immune response, the effect and mechanism of AS-IV on macrophage polarization and their role in the development of HCC was explored.

Methods: The effects of different concentrations of AS-IV (0, 50, 80, 100, 120, and 150 μM) on the capacity of hepatocellular carcinoma (HCC) cells to proliferate, migrate, and invade were detected. THP-1 cells were subjected to incubation in PMA for the purpose of stimulating differentiation into M0 macrophages. These macrophages were treated using LPS, IFN-γ, and PMA to produce M1 macrophages or treated using PMA, IL-13, and IL-4 to produce M2 macrophages. HCC cells and M1 or M2 macrophages were co-cultured for 48 hours, then the cell proliferation and migration were measured. The MTT assay was employed to determine cell viability. The capability of the cells to migrate and invade was investigated utilizing the Transwell assay and the wound healing assay. The expression of the M2 macrophage CD206 in macrophages treated with AS-IV was evaluated by flow cytometry. The expression of p-signal transducer and activator of transcription 3 (STAT3), phosphorylated (p)-NF-κB, and toll-like receptor 4 (TLR4) in macrophages was measured after treatment with AS-IV and M2 induction. To verify the function of the TLR4/NF-κB/STAT3 signaling pathway, TLR4 expression was knocked down in M2 macrophages, then the proliferation and migration and the M2 macrophage markers of HCC cells were measured. The effect of AS-IV on HCC in vivo was confirmed by a subcutaneous tumor mouse model. AS-IV was 2 was administered by gavage (0, 40, 80, and 100 mg/kg) for every 3 days. The tumor volume and weight were recorded.

Results: AS-IV suppressed the capacities of HCC cells to proliferate, migrate, and invade in a dose-dependent way. M2 macrophages could promote the proliferative, migratory, and invasive ability of Huh-7 cells, which were suppressed by AS-IV. AS-IV directly attenuated the expression of M2 macrophage markers, indicating that AS-IV can inhibit macrophage M2 polarization. M2 macrophages stimulated the expression of p-STAT3, p-NF-κB, and TLR4, while AS-IV decreased the expression compared to the M2 group, indicating that AS-IV can regulate the TLR4/NF-κB/STAT3 signaling pathway. TLR4 small interfering RNA (siRNA/si) inhibited the proliferation of Huh-7 cells. The tumor volume, as well as weight of mice, was significantly reduced by AS-IV, indicating the antitumor impact of AS-IV in vivo.

Conclusion: AS-IV can inhibit the proliferative, invasive, and migratory ability of liver cancer through the suppression of the M2 polarization of macrophages, and the mechanism may involve the TLR4/NF-κB/STAT3 signaling pathway. The present study indicates that AS-IV could be an alternative drug to treat liver cancer, and the polarization of macrophages may be a novel treatment target for HCC.