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Fexofenadine Protects Against Intervertebral Disc Degeneration Through TNF Signaling.

ABSTRACT: Objective: Fexofenadine (FFD) is an antihistamine drug with an anti-inflammatory effect. The intervertebral disc (IVD) degeneration process is involved in inflammation in which tumor necrosis factor-α (TNF-α) plays an important role. This study aims to investigate the role of FFD in the pathological process of IVD degeneration. Methods: Safranin O staining was used for the measurement of cartilageous tissue in the disc. Hematoxylin-Eosin (H&E) staining was used to determine the disc construction. A rat needle puncture model was taken advantage of to examine the role of FFD in disc degeneration in vivo. Western Blotting assay, immunochemistry, and immunoflurence staining were used for the determination of inflammatory molecules. ELISA assay was performed to detect the release of inflammatory cytokines. A real-time PCR assay was analyzed to determine the transcriptional expressions of molecules. Results: Elevated TNF-α resulted in inflammatory disc degeneration, while FFD protected against TNF-α-induced IVD degeneration. Mechanism study found FFD exhibited a disc protective effect through at least two pathways. (a) FFD inhibited TNF-α-mediated extracellular matrix (ECM) degradation and (b) FFD rescued TNF-α induced inflammation in disc degeneration. Furthermore, the present study found that FFD suppressed TNF-α mediated disc degeneration via the cPLA2/NF-κB signaling pathway. Conclusions: FFD provided another alternative for treating disc degeneration through a novel mechanism. Additionally, FFD may also be a potential target for the treatment of other inflammatory-related diseases, including IVD degeneration.

SUBMITTER: Liu K

PROVIDER: S-EPMC8421647 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIntervertebral disc degeneration (IVDD) is a leading cause of low back pain (LBP). The pathological process of IVDD is associated with inflammatory reactions and extracellular matrix (ECM) disorders. Digoxin is widely used for treating heart failure, and it has been reported to have anti-inflammatory effects.ObjectiveThis study is to investigate the role of digoxin in the pathogenesis of intervertebral disc degeneration as well as the involved molecular mechanism, particularly the potential target protein.MethodsWe exploited a rat needle model to investigate digoxin's role in intervertebral disc degeneration in vivo. Safranin O staining was used to measure cartilaginous tissue in the intervertebral disc. The morphological changes of intervertebral discs in animal models were determined by Hematoxylin-Eosin (H&E) staining and the pathological score. Primary nucleus pulposus cells (NP cells) from intervertebral discs of patients and murine were used in the present study. Western-Blotting assay, Real-time PCR assay, immunofluorescence staining, and immunochemistry were used to detect the role of digoxin in anti-TNF-α-induced inflammatory effects in vitro. Transfection of siRNA was used to regulate low-density lipoprotein receptor-related protein 4 (LRP4) expression in NP cells to investigate the potential protein target of digoxin.ResultsDigoxin protected against intervertebral disc degeneration in rat needle models. Digoxin was found to exert its disc-protective effects through at least three different pathways by a) suppressing TNF-α-induced inflammation, b) attenuating ECM destruction, c) significantly promoting ECM anabolism. Additionally, LRP4 was found to be the downstream molecule of digoxin in NP cells for anti-inflammation and regulation of ECM metabolism. The knockdown of LRP4 downregulated the protective effect of digoxin in NP cells.ConclusionThese findings suggest that digoxin may be a potential therapeutic agent for intervertebral disc degeneration through anti-catabolism and pro-anabolism. Digoxin might also work as an alternative for other inflammation-related diseases.

Project description:Background contextLow back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear.PurposeTo study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling.Study designThe expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro.MethodsHuman disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells.ResultsIn vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM).ConclusionOur results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Project description:ObjectiveTumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis.MethodsIn vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine's anti-TNF activity on cPLA2.ResultsSerial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine's anti-TNF activity.ConclusionCollectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

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Fexofenadine Protects Against Intervertebral Disc Degeneration Through TNF Signaling.

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