Effects And Mechanisms Of Interleukin-1β And Tumor Necrosis Factor-α On Acid Sensitive Ion Channel 1a Mediated Rat Articular Chondrocyte Apoptosis

Rheumatoid arthritis(RA)is a chronic autoimmune disease that affects around 1% of the population worldwide.It is characterized by progressive synovitis,ultimately leading to irreversible joint destruction and systemic complications.The acute-phase proinflammatory cytokines interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)demonstrate high-level expression and pleiotropic biological effects and contribute to the progression and persistence of RA.Acid hydrarthrosis is another important pathological character in RA,and a low SF pH was shown to correlate with radiological joint destruction in RA patients.Much attention has been paid to the role of acidosisinduced articular chondrocyte apoptosis in the pathogenesis of RA.Acid-sensing ion channels(ASICs),a family of extracellular H+-activated cation channels,can be transiently activated by extracellular acid and play a pivotal role in acid-induced cell injury.We previously also showed that the levels of ASIC1 a m RNA and protein expression were higher in articular cartilage from rats with adjuvant arthritis(AA)than normal rats.Furthermore,inhibition of ASIC1 a by the non-selective ASIC inhibitor amiloride attenuated articular cartilage destruction in rats with AA,suggesting that it contributed to the pathological process in RA.In recent years,studies have confirmed that ASIC1 a could mediate acid-induced articular chondrocyte apoptosis by causing [Ca2+]i overload,and cell apoptosis could be significantly attenuated by amiloride and the ASIC1a-specific blocker psalmotoxin 1(Pc TX-1).However,the associations of TNF-α and IL-1β with ASIC1 a,and their role in the mechanisms of acidosis-induced apoptosis in articular chondrocytes remain largely unknown.To further explore the underlying mechanisms of TNF-α and IL-1β on ASIC1 amediated apoptosis,rat articular chondrocytes in vitro and AA rat model in vivo were subjected to the experiments.To this end,carry out the following research: 1.Effect of IL-1β and TNF-α on ASIC1 a expression and activity.Immunofluorescence staining,Western Blot and real-time quantitative PCR(q PCR)were used to observe the effects of IL-1β and TNF-α on ASIC1 a expression in rat articular chondrocytes.Fura-3/AM probe and confocal laser scanning microscope were used to detect the intracellular calcium concentration([Ca2+]i)in chondrocytes.The effect of IL-1β and TNF-α on the extracellular acid-activated calcium ion transmembrane flux was analyzed.By constructing p GL3-ASIC1 promoter luciferase reporter gene plasmid,Dual Luciferase was used to detect the role of proinflammatory cytokines in ASIC1 promoter activity.The results showed that IL-1β and TNF-α upregulated the levels of ASIC1 a protein and m RNA expression in articular chondrocytes in a concentration-dependent manner,and also increased the acid-induced increase in [Ca2+]i level.In addition,pretreatment with IL-1β and TNF-α significantly increased ASIC1 promoter activity.The results suggest that high expressed ASIC1 a in AA rat articular chondrocytes is associated with IL-1β and TNF-α.2.The specific molecular mechanisms of IL-1β and TNF-α in regulating ASIC1 a expression and activity.Immunofluorescence double staining was used to observe the co-localization of ASIC1 a and NF-κB p65 in AA rat articular cartilage.The role of NF-κB,MAPK signaling pathway in IL-1β-and TNF-α-upregulated ASIC1 a expression and promoter activity was explored.The results showed that ASIC1 a and NF-κB p65 was highly expressed and co-localization in AA rat articular cartilage tissue.IL-1β and TNF-α could activate NF-κB p65,MAPK signaling pathway,and up-regulation of ASIC1 a expression and promoter activity induced by IL-1β and TNF-α was attenuated by inhibiting activation of those pathway.Further EMSA and CHIP experiments were performed to verify the binding of the transcription factor NF-κB p65 to the NF-κB site in the ASIC1 promoter.The results also showed that both IL-1β and TNF-α could induce NF-κB p65 recruitment to the ASIC1 gene promoter region,while PDTC(10 μM)pretreatment for 1 h significantly attenuated this binding.The results suggest that the transcription factor NF-κB is involved in up-regulation of ASIC1 a expression induced by IL-1β and TNF-α in rat articular chondrocytes by binding to the NF-κB site in the ASIC1 promoter sequence.3.Effect of IL-1β and TNF-α on ASIC1a-mediated rat chondrocyte apoptosis.The effect of pro-inflammatory cytokines,calcium ion chelator BAPTA-AM,ASIC1a-specific blocker psalmotoxin-1(Pc TX-1)and ASIC1 a short hairpin RNA(sh RNA)on ASIC1a-mediated articular cartilage cytotoxicity and apoptosis were analyzed by MTT,LDH,Hoechst 33258 staining and flow cytometry.Mitochondrial membrane potential and apoptosis related protein changes were detected by JC-1 and Western Blot,respectively.The results showed that IL-1β and TNF-α could further reduce acid-suppressed chondrocyte viability and increase acid-induced LDH release and loss of mitochondrial membrane potential,and enhance acid-induced cleaved PARP,cleaved Caspase-3/9 expression and chondrocyte apoptosis rate.This enhancement could be attenuated by ASIC1a-specific inhibitor Pc TX-1,ASIC1a-sh RNA or BAPTAAM.The results suggest that IL-1β and TNF-α enhance ASIC1a-mediated articular chondrocyte apoptosis.4.Effect of ASIC1 a activation on IL-1β and TNF-α-promoted chondrocyte matrix metabolism gene expression.The effect of acid-activated ASIC1 a on IL-1β and TNF-α-induced stromal-related gene expression in articular chondrocytes was examined by q PCR.The results showed that IL-1β and TNF-α significantly induced the expression of MMP-3,MMP-13 and ADAMTS-5 m RNA in chondrocytes,and the acidification treatment at pH 6.0 could further enhance the proinflammatory cytokine-induced MMP-3/13 and ADAMTS-5 expression.Pc TX-1 pretreatment partially decreased the level of IL-1β-induced MMP-3/13 and ADAMTS-5 m RNA.The results suggest that acid-activated ASICla enhances MMP-3/13 and ADAMTS-5 expression induced by IL-1β and TNF-α in articular chondrocytes.In summary,the results of this study suggest that IL-1β and TNF-α can promote ASIC1 a expression and activity in a NF-κB-dependent manner,thereby enhancing ASIC1a-mediated articular chondrocyte apoptosis.These studies enrich the understanding of proinflammatory factors and ASIC1 a function,and also provide novel strategy for the prevention and treatment of RA.