Background:Liver kinase B1 (LKB1) is known as a tumor suppressor gene and also inhibits reactive oxygen species (ROS) levels. Intracellular ROS are catalyzed by the enzyme complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). We previously reported that NOX4 induced the migration and invasion of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA). Although LKB1 is expected to alleviate synovial inflammation through ROS regulation, the role of LKB1 in RA has not been examined.Objectives:To explore whether LKB1 affects RA inflammation, we transfected LKB1 siRNA and analyzed related gene expressions in RA FLS.Methods:Synovial tissues were obtained from RA patients who were undergoing synovectomy or joint replacement. The isolated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and maintained in a 5% CO2 incubator at 37 °C. FLS were used for experiments after four to six passages. Cells were transfected with lipofectamine transfection reagent and LKB1 siRNA duplex targeting constructs. After incubation for 24 h, downregulation of target expression was evaluated by real-time PCR and western blot analysis.Results:RA FLS was transfected with LKB1 siRNA and 90% of LKB1 mRNA expression was decreased. LKB1 knock-down also caused the decreased expression of mechanistic target of rapamycin (mTOR; 0.38 fold) and serine/threonine kinase (AKT) 2 (0.40 fold), which are downstream targets of LKB1. NOX4 was significantly increased (4.94 fold) by LKB1 inhibition. On the other side, the down regulated NOX4 induced significantly elevated LKB1 mRNA expression in RA FLS. When the expressions of proinflammatory cytokines were examined, IL-1β, IL-6, TNF-α were highly increased by LKB1 deficiency. FLS migration-related chemokines, IL-8 and MMP-3 were also enhanced compared to control.Conclusion:There was a negative correlation between NOX4 and LKB1 in RA FLS. As LKB1 deficiency induced the expression of proinflammatory cytokines and migration related chemokines, LKB1 could play a critical role in RA pathogenesis.References:[1]Bartok B, Firestein GS. Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010;233(1):233–55.[2]Mateen S, Moin S, Khan AQ, Zafar A, Fatima N. Increased reactive oxygen species formation and oxidative stress in rheumatoid arthritis. PLoS One. 2016;11(4):e0152925.Disclosure of Interests:None declared.
Cryptotanshinone induces reactive oxygen species‑mediated apoptosis in human rheumatoid arthritis fibroblast‑like synoviocytes
