CYP1B1-derived epoxides modulate the TRPA1 channel in chronic pain

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 Cyp1b1 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 Trpa1-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, Cyp1b1-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.

Bioflavonoid exerts analgesic and antiinflammatory effects via transient receptor potential 1 channel in a rat model / Bioflavonoide exerce efeitos analgésicos e antiinflamatórios via canal receptor do potencial transitório 1 em um modelo de rato

Abstract Background Pain is an uncomfortable sensation in the body. Kaempferol is a flavonoid with antinociceptive effects. Transient receptor potential (TRP) channels have been characterized in the sensory system. Objective This study evaluated the central antinociceptive effect of Kaempferol and possible mechanisms of action of transient receptor potential cation channel subfamily V member 1 (TRPV1). Methods Capsaicin as a TRPV agonist (5 μg/μL, intracerebroventricular [ICV]) and capsazepine as its antagonist (10 μg/μL, icv) were used to test the analgesic effect of kaempferol (1.5 mg, ICV). Morphine (10 μg, ICV) was used as a positive control. The other groups were treated with a combination of kaempferol and capsaicin, kaempferol and capsazepine, and capsaicin and capsazepine. The cannula was implanted in the cerebroventricular area. The tail-flick, acetic acid, and formalin tests were used to assess analgesic activity.For evaluation of antiinflammatory effect, the formalin-induced rat pawedema was used. Results Kaempferol significantly decreased pain in the acute pain models, including the tail-flick and the first phase of the formalin test. In the late phase of the formalin test, as a valid model of nociception, capsazepine inhibited the antinociceptive effect of kaempferol. Conclusions Kaempferol has an analgesic effect in the acute pain model and can affect inflammatory pain. Also, the TRPV1 channel plays a role in the antinociceptive activity of kaempferol.

Resumo Antecedentes A dor é uma sensação desconfortável no corpo. Kaempferol é um flavonoide com efeitos antinociceptivos. Canais receptores de potencial transitório têm sido caracterizados no sistema sensorial. Objetivo Este estudo avaliou o efeito antinociceptivo central do kaempferol e os possíveis mecanismos de ação do TRPV1. Métodos Capsaicina como agonista de TRPV (5 μg/μL, intracerebroventricular [ICV]) e capsazepina como seu antagonista (10 μg/μL, icv) foram usados para testar o efeito analgésico do kaempferol (1,5 mg, ICV). A morfina (10 μg, ICV) foi usada como controle positivo. Os outros grupos foram tratados com uma combinação de kaempferol e capsaicina, kaempferol e capsazepina e capsaicina e capsazepina. A cânula foi implantada na área cerebroventricular. Os testes de movimento de cauda, ácido acético e formalina foram usados para avaliar a atividade analgésica. Para avaliação do efeito anti-inflamatório, foi utilizado o edema de pata de rato induzido por formalina. Resultados Kaempferol diminuiu significativamente a dor nos modelos de dor aguda, incluindo o movimento da cauda e a primeira fase do teste de formalina. Na fase tardia do teste da formalina, como modelo válido de nocicepção, a capsazepina inibiu o efeito antinociceptivo do kaempferol. Conclusões Kaempferol tem efeito analgésico no modelo de dor aguda e pode afetar a dor inflamatória. Além disso, o canal TRPV1 desempenha um papel na atividade antinociceptiva do kaempferol.

Muscle pain induced by static contraction is modulated by transient receptor potential vanilloid 1 and ankyrin 1 receptors

Abstract Molecular mechanisms involved in the development of muscle pain induced by static contraction are not completely elucidated. This study aimed to evaluate the involvement of the transient receptor potential vanilloid 1 (TRPV1) and the transient receptor potential ankyrin 1 (TRPA1) receptors expressed in peripheral and central terminals of primary afferents projected to gastrocnemius muscle and spinal cord in muscle pain induced by static contraction. An electrical stimulator provided the contraction of rat gastrocnemius muscle and mechanical muscle hyperalgesia was quantified through the pressure analgesimeter Randall-Selitto. AMG9810 and HC030031 were used. When administered in ipsilateral, but not contralateral gastrocnemius muscle, drugs prevented mechanical muscle hyperalgesia induced by static contraction. Similar results were obtained by intrathecal administrations. We propose that, in an inflammatory muscle pain, peripheral and central TRPV1 and TRPA1 work together to sensitize nociceptive afferent fibers, and that TRPV1 and TRPA1 receptors are potential target to control inflammatory muscle pain.

Analgesic effects of Phα1ß toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Implications of Transient Receptor Potential Cation Channels in Migraine Pathophysiology / 神经科学通报·英文版

Migraine is a common and debilitating headache disorder. Although its pathogenesis remains elusive, abnormal trigeminal and central nervous system activity is likely to play an important role. Transient receptor potential (TRP) channels, which transduce noxious stimuli into pain signals, are expressed in trigeminal ganglion neurons and brain regions closely associated with the pathophysiology of migraine. In the trigeminal ganglion, TRP channels co-localize with calcitonin gene-related peptide, a neuropeptide crucially implicated in migraine pathophysiology. Many preclinical and clinical data support the roles of TRP channels in migraine. In particular, activation of TRP cation channel V1 has been shown to regulate calcitonin gene-related peptide release from trigeminal nerves. Intriguingly, several effective anti-migraine therapies, including botulinum neurotoxin type A, affect the functions of TRP cation channels. Here, we discuss currently available data regarding the roles of major TRP cation channels in the pathophysiology of migraine and the therapeutic applicability thereof.

Analgesic effects of Phalfa1 beta toxin: a review of mechanisms of action involving pain pathways

Phα1ß is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Phα1ß to treat chronic pain reverted opioid tolerance with a safer profile than ω-conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Phα1ß (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Phα1ß antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Analgesic effects of Ph1 toxin: a review of mechanisms of action involving pain pathways

Abstract Ph1 is a neurotoxin purified from spider venom that acts as a high-voltage-activated (HVA) calcium channel blocker. This spider peptide has shown a high selectivity for N-type HVA calcium channels (NVACC) and an analgesic effect in several animal models of pain. Its activity was associated with a reduction in calcium transients, glutamate release, and reactive oxygen species production from the spinal cord tissue and dorsal ganglia root (DRG) in rats and mice. It has been reported that intrathecal (i.t.) administration of Ph1 to treat chronic pain reverted opioid tolerance with a safer profile than -conotoxin MVIIA, a highly selective NVACC blocker. Following a recent development of recombinant Ph1 (CTK 01512-2), a new molecular target, TRPA1, the structural arrangement of disulphide bridges, and an effect on glial plasticity have been identified. CTK 01512-2 reproduced the antinociceptive effects of the native toxin not only after the intrathecal but also after the intravenous administration. Herein, we review the Ph1 antinociceptive activity in the most relevant pain models and its mechanisms of action, highlighting the impact of CTK 01512-2 synthesis and its potential for multimodal analgesia.

Establishment of HEK-293T cell model stably expressing TRPA1 channel / 中国药理学通报

Aim: To establish a HEK-293T cell line model stably expressing TRPA1 channel, and to verify the successful establishment of the model. Methods: The eukaryotic expression plasmid of TRPA1 was constructed and transfected into HEK-293T cells by liposome transfection method, the stable expression strain was screened by G418, and the transcription and protein expression of TRPA1 gene in HEK-293T cells were detected by RT-PCR and immunohistochemical techniques. Results: After restriction enzyme digestion and sequencing, it proved recombinant cloning plasmid of TRPA1 gene was successfully constructed; the results of PCR and immunohistochemistry showed that this recombinant plasmid could be transferred into HEK-293T cells with TRPA1 gene stable expression. Conclusions: A HEK-293T cell line with stable expression of TRPA1 channel is successfully constructed, which lays the foundation for studying the physiological and pathological functions of TRPA1 in vitro and screening of relevant TR-PA1 channel regulators.

Vagal Transient Receptor Potential Ankyrin 1 Mediates Stress-exacerbated Visceral Mechanonociception After Antral Cold Exposure

BACKGROUND/AIMS: Abdominal pain can be evoked or exacerbated after gastrointestinal cold stimulation in some patients with diarrhea-predominant irritable bowel syndrome (IBS-D), indicating a low temperature-induced sensitization of visceral perception. We investigated the role of vagal transient receptor potential ankyrin 1 (TRPA1, a cold-sensing ion channel) in cold-aggravated visceral mechanonociception in a stress-induced IBS animal model. METHODS: TRPA1 expression was examined in antral biopsies of healthy controls and IBS-D patients. Abdominal symptoms were assessed before and after warm or cold water intake. The visceromotor response (VMR) to colorectal distention (CRD) following intra-antral infusion of cold saline was measured in animals undergoing sham or chronic water avoidance stress. TRPA1 expression, extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation, and neuronal calcium influx in vagal afferents were assessed. RESULTS: Compared to healthy controls, IBS-D patients displayed elevated antral TRPA1 expression, which was associated with symptom scores after cold (4°C) water intake. Intra-antral infusion of cold saline increased VMR to CRD in naive rats, an effect dependent on vagal afferents. In stressed rats, this effect was greatly enhanced. Functional blockade and gene deletion of TRPA1 abolished the cold effect on visceral nociception. TRPA1 expression in vagal (but not spinal) afferents increased after stress. Moreover, the cold-induced, TRPA1-dependent ERK1/2 activation and calcium influx in nodose neurons were more robust in stressed rats. CONCLUSIONS: Stress-exaggerated visceral mechanonociception after antral cold exposure may involve up-regulation of TRPA1 expression and function on vagal afferents. Our findings reveal a novel mechanism for abnormal gastrointestinal cold sensing in IBS.

Role and mechanism of TRP ion channels in pruritus / 中国药理学通报

Pruritus is an unpleasant noxious sensation induced by a variety of causes. Although pruritus does not cause death directly, long-term chronic pruritus severely reduces the quality of life of patients. TRP (transient receptor potential ion channels) participate in the physiological process from the sensation to skin balance. A large number of studies have confirmed that many itch media are involved in signal transduction related to TRP ion channels. We review the difference between itch and pain, and the role and mechanism of TRP in itch, so as to provide new research targets for treatment of pruritus.

A curcumin derivative J147 ameliorates diabetic peripheral neuropathy in streptozotocin (STZ)-induced DPN rat models through negative regulation AMPK on TRPA1

Abstract Purpose: To investigate the specific molecular mechanisms and effects of curcumin derivative J147 on diabetic peripheral neuropathy (DPN). Methods: We constructed streptozotocin (STZ)-induced DPN rat models to detected mechanical withdrawal threshold (MWT) in vivo using Von Frey filaments. In vitro, we measured cell viability and apoptosis, adenosine 5'-monophosphate-activated protein kinase (AMPK) and transient receptor potential A1 (TRPA1) expression using MTT, flow cytometry, qRT-PCR and western blot. Then, TRPA1 expression level and calcium reaction level were assessed in agonist AICAR treated RSC96cells. Results: The results showed that J147reduced MWT in vivo, increased the mRNA and protein level of AMPK, reduced TRPA1 expression and calcium reaction level in AITCR treated RSC96 cells, and had no obvious effect on cell viability and apoptosis. Besides, AMPK negative regulated TRPA1 expression in RSC96 cells. Conclusions: J147 could ameliorate DPN via negative regulation AMPK on TRPA1 in vivo and in vitro. A curcumin derivative J147might be a new therapeutic potential for the treatment of DPN.

Gingerol activates noxious cold ion channel TRPA1 in gastrointestinal tract / 中国天然药物

TRPA1 channels are non-selective cation channels that could be activated by plant-derived pungent products, including gingerol, a main active constituent of ginger. Ginger could improve the digestive function; however whether ginger improves the digestive function through activating TRPA1 receptor in gastrointestinal tract has not been investigated. In the present study, gingerol was used to stimulate cell lines (RIN14B or STC-1) while depletion of extracellular calcium. TRPA1 inhibitor (rethenium red) and TRPA1 gene silencing via TRPA1-specific siRNA were also used for mechanistic studies. The intracellular calcium and secretion of serotonin or cholecystokinin were measured by fura-2/AM and ELISA. Stimulation of those cells with gingerol increased intracellular calcium levels and the serotonin or cholecystokinin secretion. The gingerol-induced intracellular calcium increase and secretion (serotonin or cholecystokinin) release were completely blocked by ruthenium red, EGTA, and TRPA1-specific siRNA. In summary, our results suggested that gingerol derived from ginger might improve the digestive function through secretion releasing from endocrine cells of the gut by inducing TRPA1-mediated calcium influx.

Involvement of TRPA1 in the cinnamaldehyde-induced pulpal blood flow change in the feline dental pulp

OBJECTIVES: The purpose of this study was to investigate the involvement of TRPA1 in the cinnamaldehyde-induced pulpal blood flow (PBF) change in the feline dental pulp. MATERIALS AND METHODS: Mandibles of eight cats were immobilized and PBF was monitored with a laser Doppler flowmetry at the mandibular canine tooth. To evaluate the effect of cinnamaldehyde on PBF, cinnamaldehyde was injected into the pulp through the lingual artery at a constant rate for 60 seconds. As a control, a mixture of 70% ethanol and 30% dimethyl sulfoxide (DMSO, vehicle) was used. To evaluate the involvement of transient receptor potential ankyrin 1 (TRPA1) in PBF change, AP18, a specific TRPA1 antagonist, was applied into the pulp through the Class V dentinal cavity followed by cinnamaldehyde-administration 3 minutes later. The paired variables of experimental data were statistically analyzed using paired t-test. A p value of less than 0.05 was considered as statistically significant. RESULTS: Administration of cinnamaldehyde (0.5 mg/kg, intra-arterial [i.a.]) induced significant increases in PBF (p 0.05), administration of cinnamaldehyde (0.5 mg/kg, i.a.) following the application of AP18 (2.5 - 3.0 mM, i.c.) resulted in an attenuation of PBF increase from the control level (p < 0.05). As a result, a TRPA1 antagonist, AP18 effectively inhibited the vasodilative effect of cinnamaldehyde (p < 0.05). CONCLUSIONS: The result of the present study provided a functional evidence that TRPA1 is involved in the mechanism of cinnamaldehyde-induced vasodilation in the feline dental pulp.

Effect of cold-sensitive channel activity induced by cold air on expression of inflammatory cytokines in rat lungs / 军事医学

Objective To investigate the effect of rapid repeated cold air inhalation stimulation on cold-sensitive chan-nel transient receptor potential ankyrin 1(TRPA1) associated inflammatory cytokines in living lungs .Methods A total of 20 male Wistar rats were randomly and evenly divided into 4 groups:cold air inhalation group , warm air inhalation group , TRPA1 channel agonist inhalation group and normal group , respectively . Tracheal intubation was carried out after anesthesia in rats of cold air inhalation group , and the tubes were linked to the air temperature controlled device with the temperature controlled at ( 2 ±1 )℃.The rats of warm air inhalation group were treated in the same way as cold air inhalation group except for the temperature at (36 ±1)℃.The rats of both groups were treated for 3 times, 1 hour each time.There were 12 hour intervals between treatments .The rats of TRPA1 channel agonist inhalation group were treated with atomizing inhalation of 60 mmol/L acrolein at room temperature (24 ±2)℃for 3 times, 1 hour each time.There were 12 hour intervals between two treatments .The rats were sacrificed after the last treatment .The tracheas and left lung tissues of all rats were taken and total RNA was extracted .The mRNA expressions of TRPA1, interleukin 1 beta ( IL-1β), interleukin 5(IL-5),and neutrophil chemoattractant chemokine (CXCL-1/KC)in the rat lungs were detected by real-time quantitative PCR .Results TRPA1 was expressed in rat lung tissues .In the short term cold air inhalation group , the expressions of IL-1β,IL-5 and CXCL-1/KC mRNA in the rat lungs were higher than those of the warm air inhalation or the normal groups .Similar results were also found in the specific TRPA 1 channel positive agonist ( acrolein ) inhalation group . Conclusion In living animals, the TRPA1 channels of the lungs can be activated by short-term cold air(<17℃),which results in the increasing expression of some inflammatory cytokines in lungs .

Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats / 대한통증학회지

BACKGROUND: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats. METHODS: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated. RESULTS: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG. CONCLUSIONS: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.

Effects of Nefopam on Streptozotocin-Induced Diabetic Neuropathic Pain in Rats

BACKGROUND: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats. METHODS: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated. RESULTS: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG. CONCLUSIONS: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.

Naringenin stimulates cholecystokinin secretion in STC-1 cells

BACKGROUND/OBJECTIVES: Cholecystokinin (CCK), a hormone or neuropeptide, is secreted in response to intraluminal nutrients by enteroendocrine I-cells of the intestine and has important physiological actions related to appetite regulation and satiety. The stimulation on CCK secretion from the intestine is of potential relevance for body weight management. Naringenin (4',5,7-trihydroxyflavanone) and its glycoside naringin (naringenin 7-rhamnoglucoside) have been reported to have many biological functions. In the current study, we investigated the question of whether naringenin and naringin could stimulate CCK secretion and then examined the mechanisms involved in CCK release. MATERIALS/METHODS: STC-1 cells were used as a model of enteroendocrine cells. CCK release and changes in intracellular Ca2+ ([Ca2+]i) were measured after incubation of cells with naringenin and naringin for 1 h. RESULTS: Naringenin caused significant (P < 0.05) stimulation of CCK secretion, but naringin did not. In addition, regarding the secretory mechanisms, naringenin-induced CCK secretion involved increases in [Ca2+]i, influx of extracellular Ca2+, at least in part, and activation of TRP channels, including TRPA1. CONCLUSION: Findings of this study suggest that naringenin could have a role in appetite regulation and satiety.

Gabapentin Attenuates the Activation of Transient Receptor Potential A1 by Cinnamaldehyde

Gabapentin is used as an effective drug for relieving pain, but the main mechanism is still unclear. Recently, voltage-gated Ca2+ channel subunits are suggested for the main target for the analgesic action of gabapentin. We wonder whether gabapentin directly modulates other specific ion channels peripherally expressed in the sensory neurons. To test this, we used a heterologous expression system in which the cell lines transiently expressed thermosensitive transient receptor potential ion channels (thermoTRPs) as well as the primary cultured mouse trigeminal neurons. The application of gabapentin reduced the increases in the intracellular Ca2+ level caused by TRPA1 activation in the heterologous expression system whereas the responses via actions of other thermoTRPs were not dramatically affected by the gabapentin treatment. Gabapentin also attenuated the TRPA1-mediated intracellular Ca2+ increases in the cultured trigeminal neurons. These findings suggest TRPA1 in the peripheral sensory neurons as a novel target for the analgesic of gabapentin.