TRPA1基因敲除HEK293细胞

Pain is often debilitating, and current treatments are neither universally efficacious nor without risks. Transient receptor potential (TRP) ion channels offer alternative targets for pain relief, but little is known about the regulation or identities of endogenous TRP ligands that affect inflammation and pain. Here, transcriptomic and targeted lipidomic analysis of damaged tissue from the mouse spinal nerve ligation (SNL)-induced chronic pain model revealed a time-dependent increase in mRNA and a concurrent accumulation of 8,9-epoxyeicosatrienoic acid (EET) and 19,20-EpDPA post injury. Production of 8,9-EET and 19,20-EpDPA by human/mouse CYP1B1 was confirmed in vitro, and 8,9-EET and 19,20-EpDPA selectively and dose-dependently sensitized and activated TRPA1 in overexpressing HEK-293 cells and -expressing/AITC-responsive cultured mouse peptidergic dorsal root ganglia (DRG) neurons. TRPA1 activation by 8,9-EET and 19,20-EpDPA was attenuated by the antagonist A967079, and mouse TRPA1 was more responsive to 8,9-EET and 19,20-EpDPA than human TRPA1. This latter effect mapped to residues Y933, G939, and S921 of TRPA1. Intra-plantar injection of 19,20-EpDPA induced acute mechanical, but not thermal hypersensitivity in mice, which was also blocked by A967079. Similarly, -knockout mice displayed a reduced chronic pain phenotype following SNL injury. These data suggest that manipulation of the CYP1B1-oxylipin-TRPA1 axis might have therapeutic benefit.

Relapsing-remitting multiple sclerosis (RRMS) is characterized by increased oxidative compound production and neuroinflammation, accompanied by neuropathic pain and anxiety. Activation of nicotinamide adenine dinucleotide phosphate oxidase (Nox) generates oxidative stress by-products that induce nociception and anxiety-like behaviours by transient receptor potential ankyrin 1 (TRPA1) activation in the relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) model. Nox activation stimulates myeloperoxidase (MPO), producing advanced oxidation protein products (AOPPs). AOPPs are oxidative stress biomarkers and agonists of the receptor for advanced glycation end products (RAGE). Elevated plasma AOPP levels in multiple sclerosis are associated with disability progression, suggesting that the AOPPs/MPO/Nox pathway contributes to TRPA1 activation. This study investigated AOPP-mediated TRPA1 activation in a RR-EAE mouse model using pharmacological interventions and TRPA1 gene deletion. We tested AOPP-induced intracellular calcium influx in HEK cells transfected with TRPA1 and in dorsal root ganglion (DRG) neurons isolated from wild-type (Trpa1+/+) and TRPA1-deficient (Trpa1-/-) mice. In vivo, Trpa1+/+ and Trpa1-/- female C57BL/6J mice received intrathecal (i.t.) AOPPs (0.1-30 nmol/site) with or without the TRPA1 antagonists (HC030031 and A967079, 10 nmol/site i.t.), or the RAGE antagonist (FPS-ZM1, 10 nmol/site, i.t.). In the RR-EAE model, we assessed the effect of non-specific Nox inhibition on the AOPPs pathway activity using apocynin (APO; 100 mg/kg, intragastric) administered for 15 days, evaluating nociception and anxiety-like behaviour. Following APO treatment, we measured AOPP levels, MPO/Nox activity, neuroinflammatory biomarkers and demyelination in the spinal cord and brain. We evaluated the effects of anti-AOPP antibody (5 µl/site, i.t.) in blocking RR-EAE- and AOPP-induced nociception and anxiety-like behaviour to selectively inhibit AOPP action. AOPPs selectively induced calcium influx in TRPA1-transfected HEK cells and DRG neurons from Trpa1+/+ mice. AOPP administration triggered nociception, which was blocked by TRPA1 antagonists but not by the RAGE antagonist. Trpa1+/+ mice showed AOPP-induced nociception and phosphorylated extracellular signal-regulated kinase (p-Erk) signalling in the spinal cord dorsal horn, while Trpa1-/- mice exhibited no nociceptive behaviour or p-Erk signalling. APO treatment reduced AOPP levels, MPO/Nox activation, nociception, anxiety-like behaviour, neuroinflammation, spinal cord and brain demyelination in the RR-EAE model. Acute anti-AOPP injections exerted antinociceptive and anxiolytic-like effects in RR-EAE- and AOPP-induced nociception by lowering spinal cord and brain AOPP levels. Our findings highlight AOPPs as a TRPA1 agonist and as contributors to nociception, anxiety-like behaviour and neuroinflammation in RR-EAE, suggesting that targeting AOPPs may offer a novel approach for managing these symptoms. Therefore, targeting the AOPP formation pathway may provide a novel therapeutic strategy for RRMS patients.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.