SIRT1 inhibits rheumatoid arthritis fibroblast-like synoviocyte aggressiveness and inflammatory response via suppressing NF-κB pathway

Abstract

Rheumatoid arthritis (RA) is an autoimmune disease of the joints characterized by synovial hyperplasia and chronic inflammation. Fibroblast-like synoviocytes (FLS) play a central role in RA initiation, progression, and perpetuation. Prior studies showed that sirtuin 1 (SIRT1), a deacetylase participating in a broad range of transcriptional and metabolic regulations, may impact cell proliferation and inflammatory responses. However, the role of SIRT1 in RA-FLS was unclear. Here, we explored the effects of SIRT1 on the aggressiveness and inflammatory responses of cultured RA-FLS. SIRT1 expression was significantly lower in synovial tissues and FLS from RA patients than from healthy controls. Overexpression of SIRT1 significantly inhibited RA-FLS proliferation, migration, and invasion. SIRT1 overexpression also significantly increased RA-FLS apoptosis and caspase-3 and -8 activity. Focusing on inflammatory phenotypes, we found SIRT1 significantly reduced RA-FLS secretion of TNF-α, IL-6, IL-8, and IL-1β. Mechanistic studies further revealed SIRT1 suppressed NF-κB pathway by reducing p65 protein expression, phosphorylation, and acetylation in RA-FLS. Our results suggest SIRT1 is a key regulator in RA pathogenesis by suppressing aggressive phenotypes and inflammatory response of FLS. Enhancing SIRT1 expression or function in FLS could be therapeutic beneficial for RA by inhibiting synovial hyperplasia and inflammation.

Keywords: NF-κB; fibroblast-like synoviocytes; inflammation; rheumatoid arthritis; sirtuin 1.

Figure 1. SIRT1 was down-regulated in the synovial tissues and FLS of RA patients

SIRT1 mRNA expression relative to GAPDH was measured by RT-qPCR in synovial tissues (A) and FLS (B) from healthy negative control subjects (NC, N = 6) and rheumatoid arthritis patients (RA, N = 12). Mean ± SD; **P < 0.01.

Figure 2. SIRT1 overexpression inhibited proliferation and induces apoptosis in RA-FLS

RA-FLS was transfected with pCDNA3.1 or pCDNA3.1-SIRT1 and SIRT1 mRNA (A), and protein expression (B) relative to GAPDH was measured. (C) RA-FLS proliferation was measured by a CCK-8 assay at 24, 48, and 72 h post-transfection. (D) Cell cycle analysis of transfected RA-FLS was performed by flow cytometry after DNA staining with propidium iodide and categorized into G0/G1, S, and G2/M phases. (E) Apoptosis in transfected RA-FLS was measured by flow cytometry and quantified as combined percentage of FITC+/PI- (Q4) and FITC+/PI+ (Q2) cells. (F and G) Relative activity of caspase-3 and caspase-8 was determined by colorimetric protease assays in transfected RA-FLS; N = 6, mean ± SD, *P<0.05, **P<0.01.

Figure 3. SIRT1 overexpression suppressed migration and invasion of RA-FLS

RA-FLS was transfected with pCDNA3.1 or pCDNA3.1-SIRT1. The migration of transfected RA-FLS was measured by a wound healing assay (A), and the invasion was determined by a Transwell invasion assay (B). The representative micrographs of migrated cells over scratch wounds and invaded cells across Transwell inserts were shown in (A) and (B), and quantified respectively; N = 6, mean ± SD, **P<0.01.

Figure 4. SIRT1 reduced proinflammatory cytokine production and NF-κB-related protein expression in RA-FLS

RA-FLS was transfected with pCDNA3.1 or pCDNA3.1-SIRT1. TNF-α (A), IL-6 (B), IL-8 (C), and IL-1β (D) concentration in the supernatant of transfected cells were detected by ELISAs. Protein expression of NF-κB p65, p-p65 (Ser536), and Ac-p65 (Lys310) were detected by Western blot (E) and quantified using GAPDH as loading control (F); N = 6, mean ± SD, **P < 0.01.