Angelicin Alleviates Post-Trauma Osteoarthritis Progression by Regulating Macrophage Polarization via STAT3 Signaling Pathway

Abstract

Post-trauma osteoarthritis (PTOA) is the most common articular disease characterized by degeneration and destruction of articular cartilage (Bultink and Lems, Curr. Rheumatol Rep., 2013, 15, 328). Inflammatory response of local joint tissue induced by trauma is the most critical factor accelerating osteoarthritis (OA) progression (Sharma et al., 2019; Osteoarthritis. Cartilage, 28, 658-668). M1/M2 macrophages polarization and repolarization participates in local inflammation, which plays a major role in the progression of OA (Zhang et al., 2018; Ann. Rheum. Dis., 77, 1524-1534). The regulating effect of macrophage polarization has been reported as a potential therapy to alleviate OA progression. Synovitis induced by polarized macrophages could profoundly affect the chondrocyte and cartilage matrix (Zhang et al., 2018; Ann. Rheum. Dis., 77, 1524-1534). Generally, anti-inflammatory medications widely used in clinical practice have serious side effects. Therefore, we focus on exploring a new therapeutic strategy with fewer side effects to alleviate the synovitis. Angelicin (ANG) is traditional medicine used in various folk medicine. Previous studies have revealed that angelicin has an inhibitory effect on inflammation (Wei et al., 2016; Inflammation, 39, 1876-1882), tumor growth (Li et al., 2016; Oncology reports, 36, 3,504-3,512; Wang et al., 2017; Molecular Medicine Reports, 16, 5441-5449), DNA damage (Li et al., 2019; Exp. Ther. Med., 18, 1899-1906), and virus proliferation (Li et al., 2018; Front. Cell. Infect. Microbiol., 8, 178). But its specific effects on influencing the process of OA were rarely reported. In this study, the molecular mechanism of angelicin in vivo and in vitro was clearly investigated. Results showed that angelicin could regulate the M1/M2 ratio and function and alleviate the development of PTOA in the meanwhile. Bone marrow monocytes were isolated and induced by macrophage colony-stimulating factor (M-CSF), lipopolysaccharide (LPS) and interferon (IFN)-γ for M1 polarization and interleukin (IL)-4/IL-13 for M2 polarization. Subsequently, repolarization intervention was performed. The results indicate that angelicin can repolarize M1 toward M2 macrophages by upregulating the expression of CD9. Besides, angelicin can also protect and maintain M2 polarization in the presence of LPS/IFN-γ, and subsequently downregulate the expression of inflammatory mediators such as IL-1β and TNF-α. Mechanistically, angelicin can activate the p-STAT3/STAT3 pathway by conducting CD9/gp130 to repolarize toward M2 macrophages. These results suggest angelicin can alleviate the progression of OA by regulating M1/M2 polarization via the STAT3/p-STAT3 pathway. Therefore, angelicin may have a promising application and potential therapeutic value in OA clinical treatment.

Keywords: angelicin; inflammation; macrophage; osteoarthritis; polarization.

FIGURE 1

Evaluation of angelicin toxicity in bone marrow macrophages (BMMs). (A) Chemical structure of angelicin. (B, C) Effect of angelicin on the cell viability of BMM. The cytotoxic effects of angelicin were detected at the different concentrations for 24 and 48 h using CCK-8 assay. (D, E) Representative image of apoptosis rate detected by flow cytometry of each group. The values presented are the mean ± SD of five independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 (ANOVA).

FIGURE 2

Angelicin protectsM1 polarization and M2 macrophage polarization in vitro. (A) Schematic diagram of bone marrow macrophages (BMMs) isolation and macrophage M1/M2 polarization and repolarization strategy. (B) Representative morphology images in each group of three independent experiments. Bar represents 10 μm. (C, D) Immunostaining of enzyme-inducible nitric oxide synthase (iNOS) and CD206 (red) in isolated BMMs in different groups with quantification of iNOS⁺ and CD206⁺ cell numbers. (E, F) Western blot assay and qRT-PCR assay of Arg-1, iNOS and β-actin in BMMs. (G) qRT-PCR assay of IL-1β and TNF-α expression in BMMs. (H) ELISA for IL-1β and TNF-α concentration from cellular supernatant. The data in figures represents the mean ± SD. Significant differences are indicated as **p < 0.01. Scale bar represents 30 μm. DAPI, 4′,6-diamidino-2-phenylindole; IL, interleukin; LPS/IFN-γ, lipopolysaccharide/interferon-γ; ANG, angelicin.

FIGURE 3

Angelicin could regulate M1/M2 polarization via STAT3 signal pathway. (A) Western blot analysis of CD9, gp130, STAT3, p-STAT3, and β-actin in BMMs treated with LPS, IL-4/IL-13, LPS + Angelicin, LPS + IL-4/IL-13 + Angelicin or LPS + IL-4/IL-13. (B) Western blot analysis of iNOS, Arg-1, STAT3, p-STAT3, and β-actin in BMMs treated with LPS, LPS + Angelicin, LPS + IL-4/IL-13 + Angelicin + AG490. (C) Western blot analysis of iNOS, Arg-1, STAT3, p-STAT3, and β-actin in M2 macrophages treated with LPS, LPS + Angelicin, LPS + IL-4/IL-13 + Angelicin + AG490. (D, E) Flow cytometry analysis of iNOS and CD206 expression of BMMs. (F) qRT-PCR assay of IL-1β and TNF-α expression in BMMs. (G) ELISA for IL-1β and TNF-α concentration from cellular supernatant.

FIGURE 4

Angelicin alleviates PTOA development by regulating M1/M2 polarization. (A) Experiment design of treatment on LPS-induced mice. (B, C) Flow cytometry analysis of iNOS⁺F4/80⁺ and CD206⁺F4/80⁺macrophage in total bone marrow cells. (D, E) Immunostaining of iNOS and CD206 (red) in DMM model. Bar represents 200 μm. (F) Representative Safranin-O staining and H&E staining of cartilage and articular tissues from the different experimental groups (scale bar = 50 µm). (G) Diagram showing the OARSI scores of the articular cartilage.

FIGURE 5

Schematic illustration of the potential protective effects of angelicin in osteoarthritis development. Angelicin increased the phosphorylation of STAT3 and activated M2 macrophage polarization with the presence of LPS and IFN-γ.