Abstract
IntroductionSecretory phospholipase A2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). The objective of this study was to understand the inhibitory mechanism of phospholipase inhibitor from python (PIP)-18 peptide in cultured synovial fibroblasts (SF), and to evaluate its therapeutic potential in a human tumor necrosis factor (hTNF)-driven transgenic mouse (Tg197) model of arthritis.MethodsGene and protein expression of sPLA2-IIA, MMP-1, MMP-2, MMP-3, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 were analyzed by real time PCR and ELISA respectively, in interleukin (IL)-1β stimulated rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts cells treated with or without inhibitors of sPLA2 (PIP-18, LY315920) or MMPs (MMP Inhibitor II). Phosphorylation status of mitogen-activated protein kinase (MAPK) proteins was examined by cell-based ELISA. The effect of PIP-18 was compared with that of celecoxib, methotrexate, infliximab and antiflamin-2 in Tg197 mice after ip administration (thrice weekly for 5 weeks) at two doses (10, 30 mg/kg), and histologic analysis of ankle joints. Serum sPLA2 and cytokines (tumor necrosis factor (TNF)α, IL-6) were measured by Escherichia coli (E coli) assay and ELISA, respectively.ResultsPIP-18 inhibited sPLA2-IIA production and enzymatic activity, and suppressed production of MMPs in IL-1β-induced RA and OA SF cells. Treatment with PIP-18 blocked IL-1β-induced p38 MAPK phosphorylation and resulted in attenuation of sPLA2-IIA and MMP mRNA transcription in RA SF cells. The disease modifying effect of PIP-18 was evidenced by significant abrogation of synovitis, cartilage degradation and bone erosion in hTNF Tg197 mice.ConclusionsOur results demonstrate the benefit that can be gained from using sPLA2 inhibitory peptide for RA treatment, and validate PIP-18 as a potential therapeutic in a clinically relevant animal model of human arthritis.
Highlights
Introduction Secretory phospholipaseA2 and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA)
Our results demonstrate the benefit that can be gained from using secretory phospholipase A2 (sPLA2) inhibitory peptide for RA treatment, and validate phospholipase inhibitor from python (PIP)-18 as a potential therapeutic in a clinically relevant animal model of human arthritis
Gene and protein expression analyses, along with nuclear magnetic resonance and molecular modelingbased investigations, we have demonstrated that a linear 18residue peptide PIP-18 potently inhibits IL-1β-induced secretions of sPLA2 and matrix metalloproteinases (MMPs; 1, 2, 3, and 9) in RA synovial fibroblasts (SF), at protein and mRNA levels [11]
Summary
Introduction Secretory phospholipaseA2 (sPLA2) and matrix metalloproteinase (MMP) inhibitors are potent modulators of inflammation with therapeutic potential, but have limited efficacy in rheumatoid arthritis (RA). Among the vast family of PLA2 enzymes, which includes three cellular (cPLA2) isoforms and 10 secretory PLA2 (sPLA2) isoforms (IB, IIA, IIC, IID, IIE, IIF, III, V, X, and XII), group IIA secretory phospholipase (sPLA2-IIA) is proinflammatory in vivo [5]. It is an attractive target in RA because it releases arachidonic acid from cell membranes under some conditions, enhances cytokine induction of prostaglandin (PGE) production, and is associated with enhanced release of IL-6 [6]. Inhibition of sPLA2 may logically block the formation of a wide variety of secondary inflammatory mediators
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