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. 2022 Aug 31:13:905376.
doi: 10.3389/fphar.2022.905376. eCollection 2022.

Myricitrin inhibits fibroblast-like synoviocyte-mediated rheumatoid synovial inflammation and joint destruction by targeting AIM2

Chuyu Shen  1 , Meilin Xu  1 , Siqi Xu  1 , Shuoyang Zhang  1 , Wei Lin  1 , Hao Li  1 , Shan Zeng  2 , Qian Qiu  1 , Liuqin Liang  1 , Youjun Xiao  1 , Hanshi Xu  1
Affiliations

Myricitrin inhibits fibroblast-like synoviocyte-mediated rheumatoid synovial inflammation and joint destruction by targeting AIM2

Chuyu Shen et al. Front Pharmacol. .

Abstract

Objective: To explore the effect and underlying mechanism of Myricitrin (Myr) in regulating fibroblast-like synoviocyte (FLS)-mediated synovitis and joint destruction in RA. Methods: FLSs were isolated from synovial tissues from patients with RA. Gene expression was measured using quantitative RT-qPCR. Protein expression was detected by immunohistochemistry or Western blot. Cell apoptosis was performed by an Annexin-PI staining assay. EdU incorporation was used to assess the proliferation of RA FLS. Transwell assay was used to characterize the cell migration and invasion ability of RA FLS. The potential target of Myr was identified by RNA sequencing analysis. The in vivo effect of Myr was assessed in a collagen-induced arthritis (CIA) model. Results: Myr treatment inhibited the lamellipodia formation, migration, and invasion, but not the apoptosis and proliferation, of RA FLSs. Myr also reduced the expression of CCL2, IL-6, IL-8, MMP-1, MMP-3, and MMP-13 induced by TNF-α. The RNA-seq results indicated that AIM2 may be a target gene of Myr in RA FLSs. Furthermore, compared to healthy controls, AIM2 expression showed higher levels in synovial tissues and FLSs from RA patients. AIM2 knockdown also inhibited RA FLS migration, invasion, cytokine, and MMP expression. In addition, either Myr treatment or AIM2 knockdown reduced the phosphorylation of AKT induced by TNF-α stimulation. Importantly, Myr administration relieved arthritis symptoms and inhibited AIM2 expression in the synovium of CIA mice. Conclusion: Our results indicate that Myr exerts an anti-inflammatory and anti-invasion effect in RA FLSs and provide evidence of the therapeutic potential of Myr for RA.

Keywords: AIM2; aggression; fibroblast-like synoviocytes; inflammation; myricitrin; rheumatoid arthritis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Effect of Myr on the viability, proliferation and apoptosis of RA FLSs. The cells were treated with Myr at different concentrations (0–800 μM) for 24 h. (A)The CCK-8 assay was used to detect the cell viability. (B,C) Effect of Myr on the apoptosis of RA FLSs. Cell apoptosis was detected by a flow cytometry-based Annexin V-APC/PI assay. Representative images are shown. (D,E) Effect of Myr on the proliferation of RA FLSs. Cell proliferation was assessed by the EdU assay. Representative images are shown (original magnification, ×100). The data are expressed as the mean ± SD from at least 3 independent experiments. * p < 0.05 versus DMSO group.
FIGURE 2
FIGURE 2
Myr inhibits the migration, invasion, and TNF-α–induced expression of proinflammatory cytokines and MMPs of RA FLSs. The cells were pretreated with DMSO or Myr (100, 200, and 400 μM) for 24 h (A–D) The migration and invasion of RA FLSs were evaluated by a Boyden chamber assay. Transwell inserts coated with a Matrigel basement membrane matrix were used to detect the invasion of RA FLSs. The relative migration or invasion rate was calculated by counting migrated or invaded cells and then normalized to that in DMSO group. Representative images (original magnification, ×100) are shown. Graphs show the relative migration (B) and invasion (D) rates. (E) Myr impaired lamellipodia formation of RA FLSs. Arrows indicate lamellipodia formation. Representative images are shown (original magnification, ×400). (F,G) The cells were preincubated with DMSO, DMSO with TNF-α (10 ng/ml) or Myr (100, 200, and 400 μM) with TNF-α for 24 h. RT-qPCR was used to measure the expression of proinflammatory cytokines (F) and MMPs (G) in RA FLSs. Data (B,D,F,G) show the mean ± SD of samples from at least 3 independent experiments. * p < 0.05, ** p < 0.01 versus DMSO; # p < 0.05, ## p < 0.01 versus TNF-α+DMSO.
FIGURE 3
FIGURE 3
AIM2 is identified as a target of Myr. (A) Volcano plot indicated up-regulated and down-regulated gene (Padj<0.05 and |log2FC| >1) by RNA sequencing in Myr-treated versus DMSO-treated RA FLSs from 3 patients. (B) Heatmap of the top 20 differentially expressed genes (DEGs). (C) Bubble plot of the top 13 enriched KEGG pathways of DEGs. (D,E) Myr downregulated the gene and protein expression of AIM2 by using RT-qPCR (D) and Western blot (E). Data are presented as the mean ± SD from at least 3 independent experiments. (F) Immunohistochemistry staining was used to measured AIM2 expression in synovial tissues from RA patients and HC subjects. Original magnification, ×200. ** p < 0.01 versus HC.
FIGURE 4
FIGURE 4
AIM2 knockdown regulated RA FLS migration and invasion. RA FLSs were transfected with siRNAs against AIM2 (siAIM2-1, siAIM2-3) or control siRNA (siC). (A,B) Effect of AIM2 knockdown on the migration and invasion of RA FLSs. RA FLS migration (A) and invasion (B) were measured with a Boyden chamber assay. The relative migration and invasion rates were calculated by counting migrated or invaded cells and then normalized to that in control group. Representative images (original magnification, ×100) are shown. Data show the mean ± SD of samples from at least 3 independent experiments. (C) Effect of AIM2 knockdown on lamellipodia formation of RA FLSs. White arrows indicate lamellipodia formation. (D,E) AIM2 knockdown downregulated TNF-α–induced expression of proinflammatory cytokines and MMPs. RT-qPCR was used to detect the expression of cytokines (D) and MMPs (E). Data show the mean ± SD from at least 3 independent experiments. * p < 0.05, ** p < 0.01 versus siC; # p < 0.05, ## p < 0.01 versus TNF-α + siC.
FIGURE 5
FIGURE 5
Effect of Myr treatment or AIM2 knockdown on the activation of the AKT pathway in RA FLSs. Protein expression was detected using Western blot analysis. (A) RA FLSs were transfected with siRNAs for AIM2 (siAMI2-1, siAIM2-3) or control siRNA (siC) for 72 h and then stimulated with TNF-α (10 ng/ml) for 30 min (B) RA FLSs were treated with Myr (400 μM) or DMSO for 24 h, and then stimulated with TNF-α (10 ng/ml) for 30 min. Data (A,B) show the mean ± SD from at least 3 independent experiments. * p < 0.05 versus siC or DMSO, # p < 0.05, ## p < 0.01 versus siC + TNF-α or DMSO + TNF-α.
FIGURE 6
FIGURE 6
Myr reduces the severity of arthritis in mice with CIA. CIA mice were gavaged with Myr (100 mg/kg/d, n = 5) or 1%DMSO (as a model control, n = 5) daily for 14 days, normal mice were presented as normal control (normal). (A,B) Myr attenuated clinical symptom in CIA mice. The values in (B) are the mean ± SD of clinical scores in normal mice or mice treated with Myr or DMSO. (C,D) H&E and Safranin O-Fast green staining was used to evaluate synovial hyperplasia, inflammation, cartilage destruction, and bone erosion. Original magnification, ×100. The graph (D) indicates the mean ± SD scores for synovial hyperplasia, inflammation, cartilage destruction, and bone erosion. (E) Immunohistochemical staining was used to measure the expression of AIM2 in synovial tissues from mice. Representative images were from normal mice, DMSO-treated mice, and Myr-treated mice (original magnification, × 200). (F) Effect of Myr on Body weight in mice. Data were shown as the mean ± SD. (G) Effect of Myr on the levels of serum glucose (SG), serum creatinine (SCr), and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mice. Data were shown as the mean ± SD. (H) Representative images show the pathological changes of the liver and kidney in mice. Graph indicates the mean ± SD from 5 mice. Original magnification, ×200. * p < 0.05, ** p < 0.01 versus Normal, # p < 0.05, ## p < 0.01 versus DMSO.
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