Abstract
Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG2), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.
Highlights
Gouty arthritis is the most painful arthritis caused by an inflammatory reaction that arises in response to the deposition of uric acid in the form of monosodium urate (MSU) crystals in articular joints and bursal tissues of individuals with hyperuricemia, provoking robust inflammation and unbearable pain [1,2]
MSU crystals that have been engulfed by macrophages interacts with pathogen-recognition receptors, Toll-like receptors (TLR) 2/4 and CD 14 likely triggers the MyD88/TRIF pathway that leads to nuclear factor-κB (NF-κB) activation and formation of a protein complex called NLRP3 inflammasome, resulting in the activation of caspase-1 and processing and secretion of IL-1β, a pro-inflammatory cytokine
The primary antibodies against NF-κBp65 (Cell Signaling Technology; Cat No 4764), COX-2 (Cell Signaling Technology; Cat No 12282), TNF-α (Cell Signaling Technology; Cat No 11948), βactin (Cell Signaling Technology, Beverly, MA, USA; Cat No 8457) and Lamin A were purchased from Abcam, (Cambridge, UK; Cat No ab26300)
Summary
Gouty arthritis is the most painful arthritis caused by an inflammatory reaction that arises in response to the deposition of uric acid in the form of monosodium urate (MSU) crystals in articular joints and bursal tissues of individuals with hyperuricemia, provoking robust inflammation and unbearable pain [1,2]. A preponderance of literature suggests that MSU crystals can be recognized as an endogenous adjuvant and pro-inflammatory signals analogous to a motif, called danger associated molecular pattern (DAMP) by innate phagocytes including dendritic cells, macrophages and neutrophils. These DAMPs that are similar to pathogen-associated molecular pattern can drive systemic inflammatory immune responses in the absence of infectious triggers [4]. A study by Martin et al [8] reported that resident macrophage depletion significantly inhibited neutrophil infiltration at the inflamed joints and abrogated the production of proinflammatory cytokines, including IL-1β, suggesting that resident macrophages are key in initiating the inflammatory cascade. There is an urgent need to develop new safe anti-inflammatory treatments with maximum efficacy for gouty arthritis therapy
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