Korean J Physiol Pharmacol.  2025 Jan;29(1):45-56. 10.4196/kjpp.24.079.

Retinoic acid ameliorates rheumatoid arthritis by attenuating inflammation and modulating macrophage polarization through MKP-1/MAPK signaling pathway

• Xin M
• Jin H
• Guo X
• Zhao L
• Li X
• Xu D
• Zheng L
• Liu L

• KMID: 2563499
• DOI: http://doi.org/10.4196/kjpp.24.079

Abstract

Macrophages are innate immune cells connected with the development of inflammation. Retinoic acid has previously been proved to have anti-inflammatory and anti-arthritic properties. However, the exact mechanism through which retinoic acid modulates arthritis remains unclear. This study aimed to investigate whether retinoic acid ameliorates rheumatoid arthritis by modulating macrophage polarization. This study used retinoic acid to treat mice with adjuvant arthritis and evaluated anti-inflammatory effects by arthritis score, thermal nociceptive sensitization test, histopathologic examination and immunofluorescence assays. In addition, its specific anti-arthritic mechanism was investigated by flow cytometry, cell transfection and inflammatory signaling pathway assays in RAW264.7 macrophages in vitro. Retinoic acid significantly relieved joint pain and attenuated inflammatory cell infiltration in mice. Furthermore, this treatment modulated peritoneal macrophage polarization, increased levels of arginase 1, as well as decreased inducible nitric oxide synthase expression. In vitro, we verified that retinoic acid promotes macrophage transition from the M1 to M2 type by upregulating mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1) expression and inhibiting P38, JNK and ERK phosphorylation in lipopolysaccharide-stimulated RAW264.7 cells. Notably, the therapeutic effects of retinoic acid were inhibited by MKP-1 knockdown. Retinoic acid exerts a significant therapeutic effect on adjuvant arthritis in mice by regulating macrophage polarization through the MKP-1/MAPK pathway, and play an important role in the treatment of rheumatic diseases.

Keyword

Adjuvant arthritis; Dual specificity phosphatase 1; Inflammation; Macrophage activation; Tretinoin

Figure

• 

  Fig. 1 Retinoic acid alleviates arthritis. (A) Anti-arthritis treatment schedule. (B) Left hind paw of mice in each group on day 30. (C, D) Overall assessment of arthritis and foot and metatarsal swelling were performed in each group from day 0 to day 28 and assessed at 7-day intervals. (E) Paw withdrawal latency was assessed in the different groups. (F) Spleens were harvested, and splenic indices were determined on day 30 after immunization. Statistical analysis was performed by using two-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD (n = 6). CFA, complete freund’s adjuvant; MTX, methotrexate; RA, rheumatoid arthritis. ##p < 0.01 and ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 vs. model group.

• 

  Fig. 2 Retinoic acid ameliorates histopathology in mice with arthritis. (A) Histopathologic changes were evaluated using H&E and Safranin-O staining (10×, scale bar = 200 μm). a (synovial proliferation), b (cartilage erosion), c (pannus formation), d (inflammatory cell infiltration). The red arrow indicates that the joint space has narrowed. (B) Representative micrographs of matrix metallopeptidase 13 (MMP13) protein expression in IHC (20×, scale bar = 100 μm; 40×, scale bar = 50 μm). (C) The score of histological changes of ankle joints in different groups were assessed. (D) MMP13-positive areas of ankle cartilage. Data were analyzed using one-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD. Results are represented by at least three independent experiments. MTX, methotrexate; RA, rheumatoid arthritis. #p < 0.05, ###p < 0.001 vs. control group; *p < 0.05 and **p < 0.01 vs. model group.

• 

  Fig. 3 Retinoic acid reduces the secretion of pro-inflammatory factors. (A–C) Tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) levels were measured. Data were analyzed using one-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD (n = 6). MTX, methotrexate; RA, rheumatoid arthritis. ##p < 0.01 and ###p < 0.001 vs. control group; **p < 0.01 vs. model group.

• 

  Fig. 4 Retinoic acid affects macrophage polarization and function in mice with adjuvant arthritis. (A) Quantification of inducible nitric oxide synthase (iNOS) expression in peritoneal macrophages by immunofluorescence (100× magnification). (B) Quantification of arginase 1 (Arg-1) expression in peritoneal macrophages using immunofluorescence (100× magnification). (C, F) Representative images showing macrophage migration in various groups (200× magnification). (D) Quantitative analysis of iNOS levels. (E) Quantitative analysis of Arg-1 levels. Statistical analysis were performed by one-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD. Results are represented by at least three independent experiments. MTX, methotrexate; RA, rheumatoid arthritis. ###p < 0.001 vs. control group; *p < 0.05 and **p < 0.01 vs. model group.

• 

  Fig. 5 Retinoic acid inhibited lipopolysaccharide (LPS)-induced M1 polarization in RAW264 .7 macrophages. (A) Removal of dead cells and debris. (B) The gate strategy of iNOS+ macrophages (M1) and CD206+ macrophages (M2). (C–E) The proportion of M1 and M2 macrophages in each group was analyzed by flow cytometry. (F) Protein levels of inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPAR-γ) were analyzed by Western blotting. (G) Quantitative analysis of iNOS, Arg-1, COX-2, and PPAR-γ protein levels. Statistical analysis were performed by using one-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD. Results are represented by at least three independent experiments. RA, rheumatoid arthritis. ##p < 0.01 and ###p < 0.001 vs. control; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group.

• 

  Fig. 6 Retinoic acid inhibits the MAPK signaling by upregulating MKP-1. (A) Detection of protein levels of mitogen-activated protein kinase (MAPK) phosphatase 1 (MKP-1), MAPK pathway in macrophages using Western blotting. (B–E) Quantitative analysis of MKP-1, p-JNK/JNK, p-ERK/ERK, p-P38/ P38 expression levels. Statistical analysis were performed by using one-way ANOVA and Tukey’s post-hoc test. Data are expressed as mean ± SD. Results are represented by at least three independent experiments. JNK, c-Jun amino-terminal kinase; ERK, extracellular signal-regulated kinase; RA, rheumatoid arthritis. ###p < 0.001 vs. control; **p < 0.01 vs. model group.

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