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

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. 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. 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. 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.

Min L ，Wang H ，Qi H 《American Journal of Translational Research》

Astragaloside IV antagonizes M2 phenotype macrophage polarization-evoked ovarian cancer cell malignant progression by suppressing the HMGB1-TLR4 axis.

Macrophages are the most abundant cells in tumor stroma and their polarization within tumor microenvironment exert the key roles in tumorigenesis. Astragaloside IV is a natural extract from traditional Chinese herbal Radix Astragali, and fulfills pleiotropic function in several cancers. Nevertheless, its function in ovarian cancer microenvironment remains elusive. In the present research, astragaloside IV exhibited little cytotoxicity within a certain dose range in THP-1 cells. Moreover, astragaloside IV suppressed the ratio of CD14+CD206+ cells in IL-4/IL-13-treated THP-1 macrophages and transcripts of M2 macrophage markers (including CD206, CCL24, PPARγ, Arg-1, IL-10), indicating the inhibitory effects of astragaloside IV on IL-4/IL-13-induced macrophage M2 polarization. Intriguingly, astragaloside IV antagonized M2 macrophages coculture-evoked cell proliferation, invasion and migration in ovarian cancer cells. During this process, administration with astragaloside IV restrained the high expression of high-mobility group box1 (HMGB1) and TLR4 in macrophages co-cultured with ovarian cancer cells, concomitant with decreases in release of M2 marker TGF-β, MMP-9 and IL-10. Moreover, targeting the HMGB1 signaling reversed M2 macrophages-induced ovarian cancer cell proliferation, invasion and migration. Noticeably, exogenous HMGB1 overturned the inhibitory efficacy of astragaloside IV against macrophage M2 polarization-evoked malignant potential in ovarian cancer cells. Together, these findings suggest that astragaloside IV may protect against M2 macrophages-evoked malignancy in ovarian cancer cells by suppressing the HMGB1-TLR4 signaling. Therefore, astragaloside may alleviate the progression of ovarian cancer by regulating macrophage M2 polarization within tumor microenvironment, implying a promising therapeutic strategy against ovarian cancer.

Wang X ，Gao S ，Song L ，Liu M ，Sun Z ，Liu J ... -  《-》

Polygonatum Polysaccharide Regulates Macrophage Polarization and Improves LPS-Induced Acute Lung Injury through TLR4-MAPK/NF-κB Pathway.

To investigate the effects of polygonatum sibiricum polysaccharides (PSPs) on the polarization of macrophages to M1 and M2 phenotypes and their potential mechanism. PSPs samples were prepared through water extraction and alcohol precipitation assay. The properties of PSPs were identified and analyzed by high-performance liquid chromatography, FT-IR, and NMR assay. Then, the effects of PSPs on mouse macrophage RAW264.7 viability were measured by CCK-8 assay. The cells were randomly divided into the control group, PSPs group, LPS group, and LPS + PSPs group. M1 phenotype polarization of RAW264.7 cells was induced by LPS treatment. The effects of various treatments on expression of M2 phenotype CD206, activation of TLR4-MAPK/NF-κB signal pathway, and translocation of NF-κB into the nucleus were determined by ELISA, western blot, and immunofluorescence assay, respectively. TLR4 inhibitor, TAK-242, and MAPK inhibitor, BIRB 796, were used to verify the effects of PSPs on the TLR4-MAPK/NF-κB pathway. The mice model of acute lung injury (ALI) was established and randomly divided into control group, PSPs group, LPS group, and LPS + PSPs group. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected to measure protein, inflammatory cells, neutrophil and macrophage cells number, and the levels of IL-6 and TNF-α in BALF. Flow cytometry and western blot assay measured the phenotypic changes of macrophages and the activation of the TLR4-MAPK/NF-κB signaling pathway. The concentrations of PSPs lower than 100 μg/mL showed no toxicity to RAW264.7 cells. PSPs treatment could significantly reverse the reduction of CD206 protein expression (P < 0.05) and the increase of the expression of inflammatory factor TNF-α, IL-1β, and IL-6 (all P < 0.05), TLR4-MAPK/NF-κB signaling pathway activation (all P < 0.05), and NF-κB translocation into the nucleus induced by LPS. The effect of inhibitors TAK-242 and BIRB 796 was consistent with that of PSPs. In the mice model of ALI, PSPs treatment could reduce the total protein levels of BALF and the number of inflammatory cells level, reverse the number changes of neutrophils and macrophages, and downregulate the proinflammatory factors IL-6 and TNF-α caused by LPS (all P < 0.05). In addition, PSPs treatment could also significantly reverse the increase in the number of iNOS expressing macrophages in alveolar lavage fluid induced by LPS (P < 0.05). In contrast, CD206-expressed cells decreased (P < 0.05). PSPs could also reverse LPS-induced TLR4-MAPK/NF-κB signal pathway protein activation (all P < 0.05). PSPs could suppress TLR4-MAPK/NF-κB activation induced by LPS, inhibit M1 phenotypic polarization of macrophages, and promote M2 phenotypic polarization, thus playing an anti-inflammatory role.

Zhou W ，Hong J ，Liu T ，Li M ，Jin H ，Wang X ... -  《-》

IL-6/STAT3 pathway intermediates M1/M2 macrophage polarization during the development of hepatocellular carcinoma.

Human cancers, including hepatocellular carcinoma (HCC), are characterized by a high degree of drug resistance in chemotherapy. However, the underlying molecular mechanism remains unknown. To the role of interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in the regulation of macrophage polarization, M1-type and M2-type macrophages were separately induced using lipopolysaccharide and interleukin-4 (IL-4), and we found that the IL-6/STAT3 signaling pathway was inhibited in M1-type macrophages but activated in M2-type macrophages. After anti-IL-6-treated macrophages were separately induced by lipopolysaccharide and IL-4, we found that the inhibition of IL-6/STAT3 signaling pathway turned macrophages into M1-type. Co-culture with M1-type macrophages reduced HCC cell viability, proliferation, invasion, migration, drug resistance, but increased apoptosis. Co-culture with M2-type macrophages yielded reciprocal results. The inhibition of IL-6/STAT3 signaling pathway mediated by anti-IL6 was shown to significantly enhance the effects of M1-type macrophages on HCC cells and rescue HCC cells from co-culture with M2-type macrophages. Tumor xenografts of co-cultured HCC cells were established in nude mice and the results showed that the inhibition of IL-6/STAT3 signaling pathway mediated by anti-IL6 was found to reduce tumor formation of HCC cells co-cultured with M1- or M2-type macrophages and lung metastases. The current study reveals a novel mechanism of IL-6/STAT3 signaling pathway in the regulation of macrophage polarization, thus contributing to HCC metastasis and drug resistance in chemotherapy.

Yin Z ，Ma T ，Lin Y ，Lu X ，Zhang C ，Chen S ，Jian Z ... -  《-》

Astragaloside IV inhibits lung cancer progression and metastasis by modulating macrophage polarization through AMPK signaling.

Xu F ，Cui WQ ，Wei Y ，Cui J ，Qiu J ，Hu LL ，Gong WY ，Dong JC ，Liu BJ ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》