The ROS/GRK2/HIF-1α/NLRP3 Pathway Mediates Pyroptosis of Fibroblast-Like Synoviocytes and the Regulation of Monomer Derivatives of Paeoniflorin

Abstract

Hypoxia is an important factor in the development of synovitis in rheumatoid arthritis (RA). The previous study of the research group found that monomeric derivatives of paeoniflorin (MDP) can alleviate joint inflammation in adjuvant-induced arthritis (AA) rats by inhibiting macrophage pyroptosis. This study revealed increased levels of hypoxia-inducible factor- (HIF-) 1α and N-terminal p30 fragment of GSDMD (GSDMD-N) in fibroblast-like synoviocytes (FLS) of RA patients and AA rats, while MDP significantly inhibited their expression. Subsequently, FLS were exposed to a hypoxic environment or treated with cobalt ion in vitro. Western blot and immunofluorescence analysis showed increased expression of G protein-coupled receptor kinase 2 (GRK2), HIF-1α, nucleotide-binding oligomerization segment-like receptor family 3 (NLRP3), ASC, caspase-1, cleaved-caspase-1, and GSDMD-N. Electron microscopy revealed FLS pyroptosis after exposure in hypoxia. Next, corresponding shRNAs were transferred into FLS to knock down hypoxia-inducible factor- (HIF-) 1α, and in turn, NLRP3 and western blot results confirmed the same. The enhanced level of GSDMD was reversed under hypoxia by inhibiting NLRP3 expression. Knockdown and overexpression of GRK2 in FLS revealed GRK2 to be a positive regulator of HIF-1α. Levels of GRK2 and HIF-1α were inhibited by eliminating excess reactive oxygen species (ROS). Furthermore, MDP reduced FLS pyroptosis through targeted inhibition of GRK2 phosphorylation. According to these findings, hypoxia induces FLS pyroptosis through the ROS/GRK2/HIF-1α/NLRP3 pathway, while MDP regulates this pathway to reduce FLS pyroptosis.

Pyroptosis and Its Role in Autoimmune Disease: A Potential Therapeutic Target

“…Intriguingly, according to recent studies, in addition to traditional IL-1β inhibitors, NLRP3 or P2X7R blockers also reduce the release of cartilage destruction factor IL-1β, and alleviate joint inflammation by controlling macrophage or FLS pyroptosis ( 151 – 153 ), which increases the potential therapeutic targets of RA.…”

Pyroptosis in inflammatory bone diseases: Molecular insights and targeting strategies

“…Recently, one study showed hypoxia excessed the level of ROS, which in turn caused the phosphorylation of G protein-coupled receptor kinase 2 (GRK2) serine 670 (S670), promoting the HIF-1α to initiate the NLRP3 inflammasome and consequent pyroptosis. 119 As a result, the hypoxia-activated ROS/GRK2/HIF-1α/NLRP3 axis may make RA inflammation worse.…”

“…Later, Wang et al 118 showed that IL‐6 activated the NLRP3 inflammasome through the axis of the cathepsin B (CTSB)/ATP pathway, and S100A9 (a calcium‐binding protein) may engage in this process. Recently, one study showed hypoxia excessed the level of ROS, which in turn caused the phosphorylation of G protein‐coupled receptor kinase 2 (GRK2) serine 670 (S670), promoting the HIF‐1α to initiate the NLRP3 inflammasome and consequent pyroptosis 119 . As a result, the hypoxia‐activated ROS/GRK2/HIF‐1α/NLRP3 axis may make RA inflammation worse.…”

“…Nrf2/HO-1 axis inhibits NF-κB pathway to attenuate pyroptosis 88 SDF-1 activates AMPK signal to suppress NLRP3 inflammasome and subsequent pyroptosis 89 A3AR inhibits the ROS/NLPR3/GSDMD axis to relieve pyroptosis-mediated symptoms in OA 90 Osteomyelitis NLRP3 inflammasome is activated to cause bone loss 93 Periodontitis Hypoxia with P. gingivalis-LPS synergistically activate NLRP3 to induce pyroptosis 101 MARK4 induces NLRP3-mediated pyroptosis 102 The Nrf2/HO-1 axis inhibits LPS-induced ROS to prevent NLRP3-mediated pyroptosis 98 NLRP6 inflammasome activates caspase-1 to induce pyroptosis of HGFs 103 Caspase-4/GSDMD pathway mediates noncanonical pyroptosis 104,105 Butyrate activates caspase-3/GSDME axis to induce pyroptosis 106 LPS-induced Dec1 upregulates NF-κB via the PI3K/AKT pathway to promote pyroptosis, while LPS-induced Dec2 attenuates the phosphorylation of NF-κB to inhibit pyroptosis [108][109][110][111] RA TNF-α plus CRT induces NLRP3 inflammasome-mediated pyroptosis 116 ACPAs induces NLRP3 inflammasome-mediated pyroptosis via the Akt/NF-κB axis 117 S100A9 may participate in the IL-6-induced pyroptosis via activating the CTSB/ATP pathway 118 Hypoxia induces NLRP3 inflammasome-mediated pyroptosis via the ROS/GRK2/HIF-1α axis 119 TNF-α plus hypoxia induce caspase-3/GSDME-mediated pyroptosis 120 ASIC1a promotes the influx of Ca 2+ , activating the calpain/calcineurin axis to induce NLRP3 inflammasome-mediated pyroptosis 122,123 CaSR promotes the influx of Ca 2 to induce NLRP3 inflammasome-mediated pyroptosis 124 Abbreviations: ACPAs, anti-citrullinated protein antibodies; AP, apical periodontitis; ASIC1a, acid-sensitive ion channel 1a; ATP, adenosine triphosphate; A3AR, adenosine A3 receptor; CaSR, calcium-sensing receptor; CRT, calreticulin; CTSB, cathepsin B; Dec1, embryo-chondrocyte expressed genes 1; FLS, fibroblast-like synoviocyte; Gout, gouty arthritis; GRK2, G protein-coupled receptor kinase 2; GSDMD, gasdermin D; HGFs, human gingival fibroblasts; HIF-1α, hypoxia-inducible factor-1α; HO-1, heme oxygenase-1; IL, interleukin; LPS, lipopolysaccharide; MARK4, microtubule affinity regulating kinase 4; NF-κB, nuclear factor kappa-B; NLRP3, nucleotide-binding oligomerization domain receptor family PYD domain containing 3; NOX, NAD(P)H oxidases; Nrf2, nuclear factor erythroid-2-related factor 2; OA, osteoarthritis; P. gingivalis, Porphyromonas gingivalis; ROS, reactive oxygen species; SDF-1, stromal cell-derived factor-1; TNF-α, tumor necrosis factor-α; USP7, ubiquitin-specific protease 7.…”

“…Direct inhibitors of the NLRP3 inflammasome include CY-09, 128 glyburide, 93 icariin, 82 MCC950, 21,98,99 monomeric derivatives of paeoniflorin (MDP), 119 metformin, 129 cranberry proanthocyanidins (PACs), 130 and resveratrol. 75 Indirect NLRP3 inhibitors include A3AR activator (CF101), 90 ASIC-1a inhibitors (amiloride, psalmotoxin-1), 122 GRK2 phosphorylation inhibitor (MDP), 119 glycogen synthase kinase-3β (GSK-3β) inhibitor (GSKI), 131 NF-κB inhibitors (Baeckein E, loganin, morroniside, PDTC, punicalagin), 21,[132][133][134][135] Nrf2/HO-1 activators (eldecalcitol, licochalcone A), 88,98 and ROS inhibitors (enamel matrix derivative, NAC, quercetin). 98,99,136,137 5.2 | MicroRNAs (miRNAs) targeting strategies Epigenetic processes, including DNA methylation, histone modifications, and miRNAs, which causing the heritable silence of genes without alteration to their coding sequence.…”