Inhibition Of Transient Receptor Potential Ankyrin 1 Research Articles

Linalyl acetate exerts analgesic effects by inhibiting nociceptive TRPA1 in mice.

Clary sage essential oil (CSEO) is utilized in perfumery, aromatherapy, and skincare. Linalyl acetate (LA), a primary component of CSEO, possesses sedative, anxiolytic, and analgesic properties. However, the mechanism of its analgesic action is not clearly understood. Transient receptor potential ankyrin 1 (TRPA1) channel, a non-selective cation channel, is mainly expressed in sensory neurons and serves as a sensor of various irritants. In this study, we investigated the effects of LA on TRPA1 channel using heterologous expression system and isolated sensory neurons. To detect channel activity, we employed Ca2+ imaging and the whole-cell patch-clamp technique. The analgesic action of LA was measured in a pain-related behavioral mouse model. In cells that heterologously expressed TRPA1, LA diminished [Ca2+]i and current responses to allylisothiocyanate (AITC) and carvacrol: exogenous TRPA1 agonists, and the inhibitory effects were more pronounced for the former than for the latter. Moreover, LA suppressed [Ca2+] i and current responses to PGJ2: an endogenous TRPA1 agonist. Similar inhibitory actions were observed in native TRPA1 channels expressed in mouse sensory neurons. Furthermore, LA diminished PGJ2-induced nociceptive behaviors in mice. These findings suggest that analgesic effects of LA exert through inhibition of nociceptive TRPA1, making it a potential candidate for novel analgesic development.

Transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential melastatin 8 (TRPM8) in human odontoblast-like cells participate in lipopolysaccharide-induced immune response

ObjectivesTo investigate whether transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential melastatin 8 (TRPM8) have a function in responding to environmental stimuli in human odontoblast-like cells (hOLCs). Additionally, to explore whether activation of TRPA1 and TRPM8 in hOLCs participates in the regulation of the inflammatory process. DesignChanges in gene and protein expression levels of TRPA1 and TRPM8 in cultured hOLCs following lipopolysaccharide (LPS) stimulation, which mimics inflammation, were examined using quantitative reverse transcription-polymerase chain reaction and western blot analysis. Furthermore, we compared the expression profiles of 80 cytokines between LPS- and vehicle-treated hOLCs and investigated how the production of highly increased cytokines in LPS-treated hOLCs was affected by the pharmacological inhibition of TRPA1 and TRPM8. ResultsThe expression of TRPA1 and TRPM8 in hOLCs was observed and their mRNAs and proteins were upregulated in hOLCs after LPS treatment. Moreover, cytokine antibody assays revealed that monocyte chemoattractant protein-1 (MCP-1, CCL2), growth-regulated protein α (GROα, CXCL1), interleukin-6 (IL-6), and IL-8 (CXCL8) were significantly upregulated by LPS. The pharmacological inhibition of TRPA1 (HC-030031) during LPS treatment attenuated the expression of CCL2, CXCL1, and IL-8, whereas the pharmacological inhibition of TRPM8 (PF05105679) suppressed the expression of CCL2, CXCL1, and IL-8 as well as IL-6. ConclusionsThese results indicate that hOLCs express TRPA1 and TRPM8, which are upregulated during inflammation. In addition to being sensors of potentially harmful stimuli, TRPA1 and TRPM8 in hOLCs play important roles in regulating inflammatory responses.

Inhibition of TRPA1, Endoplasmic Reticulum Stress, Human Airway Epithelial Cell Damage, and Ectopic MUC5AC Expression by Vasaka (Adhatoda vasica; Malabar Nut) Tea.

This study tested whether a medicinal plant, Vasaka, typically consumed as a tea to treat respiratory malaise, could protect airway epithelial cells (AECs) from wood smoke particle-induced damage and prevent pathological mucus production. Wood/biomass smoke is a pneumotoxic air pollutant. Mucus normally protects the airways, but excessive production can obstruct airflow and cause respiratory distress. Vasaka tea pre- and co-treatment dose-dependently inhibited mucin 5AC (MUC5AC) mRNA induction by AECs treated with wood smoke particles. This correlated with transient receptor potential ankyrin-1 (TRPA1) inhibition, an attenuation of endoplasmic reticulum (ER) stress, and AEC damage/death. Induction of mRNA for anterior gradient 2, an ER chaperone/disulfide isomerase required for MUC5AC production, and TRP vanilloid-3, a gene that suppresses ER stress and wood smoke particle-induced cell death, was also attenuated. Variable inhibition of TRPA1, ER stress, and MUC5AC mRNA induction was observed using selected chemicals identified in Vasaka tea including vasicine, vasicinone, apigenin, vitexin, isovitexin, isoorientin, 9-oxoODE, and 9,10-EpOME. Apigenin and 9,10-EpOME were the most cytoprotective and mucosuppressive. Cytochrome P450 1A1 (CYP1A1) mRNA was also induced by Vasaka tea and wood smoke particles. Inhibition of CYP1A1 enhanced ER stress and MUC5AC mRNA expression, suggesting a possible role in producing protective oxylipins in stressed cells. The results provide mechanistic insights and support for the purported benefits of Vasaka tea in treating lung inflammatory conditions, raising the possibility of further development as a preventative and/or restorative therapy.

TRPA1 protects mice from pathogenic Citrobacter rodentium infection via maintaining the colonic epithelial barrier function.

Transient receptor potential ankyrin 1 (TRPA1) is expressed in gastrointestinal tract and plays important roles in intestinal motility and visceral hypersensitivity. However, the potential role of TRPA1 in host defense, particularly against intestinal pathogens, is unknown. Here, we show that Trpa1 knockout mice exhibited increased susceptibility to Citrobacter rodentium infection, associated with the increased severity of diarrhea and intestinal permeability associated with the disrupted tight junctions (TJs) in colonic epithelia. We further demonstrated the expression of TRPA1 in murine colonic epithelial cells (CECs) and human epithelial Caco-2 cells both at protein level and transcription level. Using calcium imaging, TRPA1 agonists allyl isothiocyanates (AITC) and hydrogen peroxide were observed to induce a transient Ca2+ response in Caco-2 cells, respectively. Moreover, TRPA1 knockdown in Caco-2 cells resulted in the decreased expression of TJ proteins, ZO-1 and Occludin, and in the increased paracellular permeabilities and the reduced TEER values of Caco-2 monolayers in vitro. Furthermore, inhibition of TRPA1 by HC-030031 in the confluent Caco-2 cells caused the altered distribution and expression of TJ proteins, ZO-1, Occludin, and Claudin-3, and exacerbated the bacterial endotoxin lipopolysaccharide (LPS)-induced damage to these TJ proteins and actin cytoskeleton. By contrast, AITC pretreatment restored the distribution and expression of these TJ proteins in the confluent Caco-2 cells upon LPS challenge. Our results identify an unrecognized protective role of TRPA1 in host defense against an enteric bacterial pathogen by maintaining colonic epithelium barrier function, at least in part, via preserving the distribution and expression of TJ proteins in CECs.

Inhibition of TRPA1 Attenuates Oxidative Stress-induced Damage After Traumatic Brain Injury via the ERK/AKT Signaling Pathway

Neuron apoptosis is a feature of secondary injury after traumatic brain injury (TBI). Evidence implies that excess calcium (Ca2+) ions and reactive oxidative species (ROS) play critical roles in apoptosis. In reaction to increased ROS, the anti-oxidative master transcription factor, Transient receptor potential Ankyrin 1 (TRPA1) allows Ca2+ ions to enter cells. However, the effect of TBI on the expression of TRPA1 and the role of TRPA1 in TBI are unclear. In the present study, TBI in the mouse brain was simulated using the weight-drop model. The process of neuronal oxidative stress was simulated in HT22 neuronal cells by treatment with hydrogen peroxide. We found that TRPA1 was significantly upregulated in neurons at 24 h after TBI. Neuronal apoptosis was increased in the in vivo and in vitro models; however, this increase was reduced by the functional inhibition of TRPA1 in both models. After TBI, TRPA1 was upregulated via nuclear factor, erythroid 2 like 2 (Nrf2) in neurons. TRPA1-mediated neuronal apoptosis after TBI might be achieved in part through the CaMKII/AKT/ERK signaling pathway. To sum up, TBI-triggered TRPA1 upregulation in neurons is mediated by Nrf2 and the functional blockade of TRPA1 attenuates neuronal apoptosis and improves neuronal dysfunction, partially mediated through the activation of the calcium/calmodulin dependent protein kinase II (CaMKII) extracellular regulated kinase (ERK)/protein kinase B (AKT) signaling pathway. Our results suggest that functional blockade of TRPA1 might be a promising therapeutic intervention related to ROS and Nrf2 in TBI.

Transient Receptor Potential Ankyrin-1 (TRPA1) Block Protects against Loss of White Matter Function during Ischaemia in the Mouse Optic Nerve.

Oligodendrocytes produce myelin, which provides insulation to axons and speeds up neuronal transmission. In ischaemic conditions, myelin is damaged, resulting in mental and physical disabilities. Recent evidence suggests that oligodendrocyte damage during ischaemia can be mediated by Transient Receptor Potential Ankyrin-1 (TRPA1), whose activation raises intracellular Ca2+ concentrations and damages compact myelin. Here, we show that TRPA1 is constitutively active in oligodendrocytes and the optic nerve, as the specific TRPA1 antagonist, A-967079, decreases basal oligodendrocyte Ca2+ concentrations and increases the size of the compound action potential (CAP). Conversely, TRPA1 agonists reduce the size of the optic nerve CAP in an A-967079-sensitive manner. These results indicate that glial TRPA1 regulates neuronal excitability in the white matter under physiological as well as pathological conditions. Importantly, we find that inhibition of TRPA1 prevents loss of CAPs during oxygen and glucose deprivation (OGD) and improves the recovery. TRPA1 block was effective when applied before, during, or after OGD, indicating that the TRPA1-mediated damage is occurring during both ischaemia and recovery, but importantly, that therapeutic intervention is possible after the ischaemic insult. These results indicate that TRPA1 has an important role in the brain, and that its block may be effective in treating many white matter diseases.

Inhibition of TRPA1 reduces airway inflammation and hyperresponsiveness in mice with allergic rhinitis.

This study was conducted to investigate whether a transient receptor potential ankyrin 1 (TRPA1) antagonist (HC-030031) can reduce airway inflammation and hyperresponsiveness in a murine allergic rhinitis (AR) model. BALB/c mice were sensitized and challenged by ovalbumin (OVA) to induce AR. HC-030031 or vehicle was administrated to mice via intraperitoneal injection prior to OVA challenges. Nose-scratching events, histopathologic alterations of the airways, and bronchial hyperresponsiveness (BHR) were assessed. Differential cells and proinflammatory cytokines in the nasal lavage (NAL) and bronchoalveolar lavage (BAL) fluid were measured. Expressions of TRPA1 in nasal mucosa were examined by immunohistochemistry. TRPA1-expressing vagal neurons were labeled by immunofluorescent staining. HC-030031-treated AR mice had markedly reduced type-2 inflammation in nasal mucosa and ameliorated-nose-scratching events than AR mice received vehicle. HC-030031 treatment also dramatically reduced leucocyte numbers and IL-8 level in the BAL fluid, inhibited lower airway remodeling and fibrosis, and nearly abolished BHR. HC-0300031 treatment significantly inhibited the upregulated number of TRPA1 expressing nasal epithelial cells and TRPA1 expressing sensory neurons, leading to downregulation of SP in both upper and lower airways. Targeting TRPA1 may represent a promising strategy for treating AR and AR-related asthma.

Transient receptor potential ankyrin 1 contributes to the ATP-elicited oxidative stress and inflammation in THP-1-derived macrophage.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1β (IL-1β) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.

Transient receptor potential ankyrin 1 contributes to the lysophosphatidylcholine-induced oxidative stress and cytotoxicity in OLN-93 oligodendrocyte.

Transient receptor potential ankyrin 1 (TRPA1), the non-selective cation channel, was found that can mediate the generation of multiple sclerosis, while the mechanism is still controversial. Lysophosphatidylcholine (LPC) is a critical trigger of multiple sclerosis which results from the syndrome of neuronal inflammation and demyelination. In this work, we suggested that TRPA1 can mediate the LPC-induced oxidative stress and cytotoxicity in OLN-93 oligodendrocyte. The expression of TRPA1 in OLN-93 was detected by using quantitative real-time PCR (qRT-PCR) and immunofluorescence. The calcium overload induced by LPC via TRPA1 was detected by calcium imaging. The mechanism of LPC-induced mitochondrial reactive oxygen species (mtROS) generation, mitochondria membrane depolarization, nitric oxide (NO) increase, and development of superoxide production via TRPA1 was verified by using confocal imaging. The cell injury elicited by LPC via TRPA1 was confirmed by both CCK-8 and LDH cytotoxicity detection. These results indicate that TRPA1 plays an important role of the LPC-induced oxidative stress and cell damage in OLN-93 oligodendrocyte. Therefore, inhibition of TRPA1 may protect the LPC-induced demyelination.

The neuro-immune interaction in airway inflammation through TRPA1 expression in CD4+ T cells of asthmatic mice

Asthma is an inflammatory disorder of the airways dominated by a Th2-type pattern. Recently, an emerging interest arises whether transient receptor potential ankyrin 1 (TRPA1) plays a potential role in the adaptive immune response. In this study, the role of TRPA1 in the development and exacerbation of asthma was explored. The classic OVA-induced asthma and OVA plus PM2.5-induced exacerbated asthma model were used. The CD4+ T cells were sorted from spleen in asthmatic and exacerbated asthmatic mice. In the BALB/c mice treated with OVA, the increased phenotype of asthma was obtained, accompanied by the high expression of TRPA1 in lung tissue and levels of IL-4, IL-13, NGF, PGD2 in BAL. In contrast, genetic deletion or pharmacological inhibition of TRPA1 alleviated the phenotype of asthma. Similarly, in wild type (WT) C57BL/6 mice treated with OVA, the high expression of TRPA1 in lung tissues was obtained, and the levels of IL-4, IL-13, NGF, PGD2 in BAL remarkably increased when compared with those in the TRPA1 deleted mice. Furthermore, high expression of TRPA1 was detected in CD4+ T cells of OVA-treated WT C57BL/6 mice. Additional detection in the asthmatic mice exacerbated by OVA plus PM2.5 also showed high TRPA1 expression in lung tissue and CD4+ T cells. All evidence confirmed that TRPA1 is essential for the development and exacerbation of asthma. More importantly, the expression of TRPA1 in CD4+ T cells of different asthmatic mice suggested that it might be involved in neuro-immune interactions in airway inflammation of asthmatic mice.

Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid that stimulates an increase in mitochondrial reactive oxygen species (mtROS) and the release of cytokines under inflammasome activation. However, the potential receptors of LPC in macrophages are poorly understood. Members of the transient receptor potential (TRP) channel superfamily, which is crucially involved in transducing environmental irritant stimuli into nociceptor activity, are potential receptors of LPC. In this study, we investigated whether LPC can induce the activation of transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily. The functional expression of TRPA1 was first detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and calcium imaging in human acute monocytic leukemia cell line (THP-1)-derived macrophages. The mechanism by which LPC induces the activation of macrophages through TRPA1 was verified by cytoplasmic and mitochondrial calcium imaging, mtROS detection, a JC-1 assay, enzyme-linked immunosorbent assay, the CCK-8 assay and the lactate dehydrogenase (LDH) cytotoxic assay. LPC induced the activation of THP-1-derived macrophages via calcium influx, and this activation was suppressed by potent and selective inhibitors of TRPA1. These results indicated that TRPA1 can mediate mtROS generation, mitochondrial membrane depolarization, the secretion of IL-1β and cytotoxicity through cellular and mitochondrial Ca2+ influx in LPC-treated THP-1-derived macrophages. Therefore, the inhibition of TRPA1 may protect THP-1-derived macrophages against LPC-induced injury.

Inhibition of TRPA1 and IL-6 signal alleviates neuropathic pain following chemotherapeutic bortezomib.

Bortezomib (BTZ) is used as a chemotherapeutic agent for the treatment of multiple myeloma. Nevertheless, one of the significant limiting complications of BTZ is painful peripheral neuropathy during BTZ therapy. Thus, in this study we examined signaling pathways of interleukin-6 (IL-6) and transient receptor potential ankyrin 1 (TRPA1) in the sensory nerves responsible for neuropathic pain induced by BTZ and further determined if influencing the pathways can improve neuropathic pain. ELISA and western blot analysis were used to examine the levels of IL-6, and IL-6 receptor (IL-6R), TRPA1 and p38-MAPK and JNK signal in the lumbar dorsal root ganglion. Behavioral test was performed to determine mechanical and cold sensitivity in a rat model. Our results showed that systemic injection of BTZ increased mechanical pain and cold sensitivity as compared with control animals. Data also showed that protein expression of TRPA1 and IL-6R was upregulated in the dorsal root ganglion of BTZ rats and blocking TRPA1 attenuated mechanical and cold sensitivity in control rats and BTZ rats. Notably, the inhibitory effect of blocking TRPA1 was smaller in BTZ rats than that in control rats. In addition, a blockade of IL-6 signal attenuated intracellular p38-MAPK and JNK in the sensory neuron. This also decreased TRPA1 expression and alleviated mechanical hyperalgesia and cold hypersensitivity in BTZ rats. In conclusion, we revealed specific signaling pathways leading to neuropathic pain induced by chemotherapeutic BTZ, including IL-6-TRPA1, suggesting that blocking these signals is beneficial to alleviate neuropathic pain during BTZ intervention.

TRPA1-expressing lamina propria mesenchymal cells regulate colonic motility.

The physiological process of defecation is directly controlled by colorectal motility. The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in small intestine enterochromaffin cells and is involved in gastrointestinal motility via serotonin release. In the colorectum, however, enterochromaffin cell localization is largely distinct from that in the small intestine. Here, we investigated the role of lower gastrointestinal tract TRPA1 in modulating colorectal motility. We found that in colonic tissue, TRPA1 is predominantly expressed in mesenchymal cells of the lamina propria, which are clearly distinct from those in the small intestine. These cells coexpressed COX1 and microsomal prostaglandin E synthase-1. Intracolonic administration of TRPA1 agonists induced colonic contraction, which was suppressed by a prostaglandin E2 (PGE2) receptor 1 antagonist. TRPA1 activation induced calcium influx and PGE2 release from cultured human fibroblastic cells. In dextran sulfate sodium-treated animals, both TRPA1 and its endogenous agonist were dramatically increased in the colonic lamina propria, accompanied by abnormal colorectal contractions. Abnormal colorectal contractions were significantly prevented by pharmacological and genetic inhibition of TRPA1. In conclusion, in the lower gastrointestinal tract, mesenchymal TRPA1 activation results in PGE2 release and consequently promotes colorectal contraction, representing what we believe is a novel physiological and inflammatory bowel disease-associated mechanism of gastrointestinal motility.

Transient receptor potential ankyrin 1 (TRPA1) as a factor and drug target in osteoarthritis: TRPA1 mediates fibroblast growth factor 2 expression in chondrocytes

Purpose: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, which is known to be involved in nociception and neurogenic inflammation. TRPA1 is widely expressed in neurons, and according to more recent findings, also in non-neuronal cells, including synoviocytes and keratinocytes. TRPA1 acts as a chemosensor of pungent environmental compounds, but it is also activated by agents formed endogenously in inflammatory and hypoxic conditions such as those found in osteoarthritic joints. We have recently shown in monosodium iodoacetate-induced experimental osteoarthritis that TRPA1 activation mediates inflammation, cartilage degradation and pain (Moilanen et al. Osteoarthritis Cartilage 2015). We have also shown that TRPA1 is functionally expressed in human chondrocytes, where it mediates inflammatory effects (Nummenmaa et al. Arthritis Res Ther 2016). Further, we found that anti-inflammatory drugs dexamethasone and aurothiomalate downregulate the expression of TRPA1 in human chondrocytes (Nummenmaa et al. RMD Open 2017), revealing TRPA1 as a potential mediator and drug target in osteoarthritis. Fibroblast growth factor 2 (FGF-2) belongs to the fibroblast growth factor family, which is a large group of molecules involved in the regulation of connective tissue development and metabolism. FGF-2 is found in the synovial fluid and cartilage of osteoarthritis patients, and has previously been associated with the pathogenesis of osteoarthritis. In the present study, we investigated the functional consequences of TRPA1 activation in chondrocytes focusing in particularly on the expression of FGF-2. Methods: Chondrocytes were isolated from cartilage tissue from wild type (WT) and TRPA1 deficient (knock-out, KO) mice and cultured in the absence or presence of IL-1β. Total RNA was isolated and genome-wide expression analysis was carried out with next generation RNA sequencing (NGS) method with Illumina HiSeq2500 at the Institute for Molecular Medicine Finland. Results from the RNA sequencing analysis were verified by quantitative RT-PCR. Primary human OA chondrocytes were isolated from cartilage samples obtained from patients undergoing knee replacement surgery. Chondrocytes were cultured in the presence of the TRPA1-inducing cytokine IL-1β alone and together with the selective TRPA1 antagonist HC-030031. For experiments investigating the effects of FGF-2 in chondrocytes, the cells were cultured in the presence of FGF-2. Expression of FGF-2 subsequent to TRPA1 activation was measured by quantitative RT-PCR, and aggrecan, collagen II and MMP-enzymes by quantitative RT-PCR/immunoassay. Results: The expression of FGF-2 was significantly downregulated in chondrocytes from TRPA KO mice compared to WT mice. The downregulation of FGF-2 was verified by performing quantitative RT-PCR analysis on the sequenced samples, and on a larger set of corresponding samples. The results were confirmed using primary human OA chondrocytes. Pharmacological inhibition of TRPA1 with the selective antagonist HC-030031 downregulated FGF-2 expression in these cells. We further investigated the effects of FGF-2 in human OA chondrocytes, and found that FGF-2 upregulated the production of cartilage matrix degrading enzymes MMP-1 and MMP-13, and downregulated the expression of cartilage matrix components aggrecan and collagen II. Conclusions: The results revealed a hitherto unknown role for TRPA1 in the regulation of FGF-2 expression in chondrocytes. Further, FGF-2 was confirmed to have catabolic and anti-anabolic effects in human OA chondrocytes. Inhibition of TRPA1 and subsequent downregulation of FGF-2 could therefore have beneficial effects on the balance of catabolic and anabolic mediators within OA cartilage. These results together with our recent data discussed above support the concept of TRPA1 as a potential mediator and drug target in osteoarthritis.

Roles of tumor necrosis factor-α and interleukin-6 in regulating bone cancer pain via TRPA1 signal pathway and beneficial effects of inhibition of neuro-inflammation and TRPA1.

BackgroundPain is one of the most common and distressing symptoms suffered by patients with progression of bone cancer; however, the mechanisms responsible for hyperalgesia are not well understood. The purpose of our current study was to determine contributions of the sensory signaling pathways of inflammatory tumor necrosis factor-α and interleukin-6 and downstream transient receptor potential ankyrin 1 (TRPA1) to neuropathic pain induced by bone cancer. We further determined whether influencing these pathways can improve bone cancer pain.MethodsBreast sarcocarcinoma Walker 256 cells were implanted into the tibia bone cavity of rats to induce mechanical and thermal hyperalgesia. ELISA and western blot analysis were used to examine (1) the levels of tumor necrosis factor-α and interleukin-6 in dorsal root ganglion and (2) protein expression of tumor necrosis factor-α and interleukin-6 receptors (TNFR1 and IL-6R) and TRPA1 as well as intracellular signals (p38-MAPK and JNK).ResultsTumor necrosis factor-α and interleukin-6 were elevated in the dorsal root ganglion of bone cancer rats, and expression of TNFR1, IL-6R, and TRPA1 was upregulated. In addition, inhibition of TNFR1 and IL-6R alleviated mechanical and thermal hyperalgesia in bone cancer rats, accompanied with downregulated TRPA1 and p38-MAPK and JNK.ConclusionsWe revealed specific signaling pathways leading to neuropathic pain during the development of bone cancer, including tumor necrosis factor-α-TRPA1 and interleukin-6-TRPA1 signal pathways. Overall, our data suggest that blocking these signals is beneficial to alleviate bone cancer pain.

Transient receptor potential ankyrin 1 (trpa1) mediates il-1\u03b2-induced apoptosis in rat chondrocytes via calcium overload and mitochondrial dysfunction

BackgroundChondrocyte apoptosis is a central feature in the progression of osteoarthritis (OA), and would be triggered by sustained elevation of intracellular calcium ion (Ca2+), also known as a cellular second messenger. Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, and the activation of which causes an influx of cation ions, in particularly Ca2+, into the activated cells. Therefore, we investigate the potential role of TRPA1 in mediating Ca2+ influx to promote chondrocyte apoptosis in OA.MethodsThe expression of TRPA1 in interleukin (IL)-1β-treated rat chondrocytes was assessed by Polymerase chain reaction (PCR) and Western blot (WB), and the functionality of TRPA1 channel by Ca2+ influx measurements. Meanwhile, the chondrocyte apoptosis in IL-1β-treated cells was measured by TUNEL assay and flow cytometry. The measurement of mitochondrial membrane potential and apoptosis-associated proteins after inhibition of TRPA1 were also performed in IL-1β-treated rat chondrocytes.ResultsAfter being induced by IL-1β, the gene and protein expression of TRPA1 was increased in the dose-dependent manner. Meanwhile, Ca2+ influx mediated by TRPA1 in rat chondrocytes was also enhanced. Pharmacological inhibition of TRPA1 downregulated the apoptotic rate in IL-1β-treated rat chondrocytes. In addition, the membrane potential depolarization was improved and significantly increased expression of apoptosis-associated proteins also reduced by the TRPA1 antagonist.ConclusionsWe found the IL-1β caused the increased functional expression of TRPA1, the activation of which involved IL-1β-induced apoptosis in rat chondrocytes. The potential mechanism may be linked to the intracellular calcium overload mediated by TRPA1 and attendant mitochondrial dysfunction.

TRPA1 inhibition ameliorates pressure overload-induced cardiac hypertrophy and fibrosis in mice.

BackgroundRecent evidence has indicated that the transient receptor potential ankyrin 1 (TRPA1) is expressed in the cardiovascular system and implicated in the development and progression of several cardiovascular diseases. However, the effects of TRPA1 on cardiac hypertrophy development remain unclear. The aim of this study was to determine the role of TRPA1 in cardiac hypertrophy and fibrosis development.MethodsC57BL/6J mice were subjected to transverse aortic constriction (TAC) and were orally treated with the TRPA1 selective inhibitors HC-030031 (HC) and TCS-5861528 (TCS). Morphological assessments, echocardiographic parameters, histological analyses and flow cytometry were used to evaluate cardiac hypertrophy and fibrosis.ResultsHuman and mouse hypertrophic hearts presented with noticeably increased TRPA1 protein levels. Inhibition of TRPA1 by HC and TCS attenuated cardiac hypertrophy and preserved cardiac function after chronic pressure overload, as evidenced by increased heart weight/body weight ratio, cardiomyocyte cross-sectional area and mRNA expression of hypertrophic markers, including ANP, BNP and β-MHC. Dramatic interstitial fibrosis was observed in the mice subjected to TAC surgery, and this was markedly attenuated in the HC and TCS treated mice. Mechanistically, the results revealed that TRPA1 inhibition ameliorated pressure overload-induced cardiac hypertrophy by negatively regulating Ca2+/calmodulin-dependent protein kinase II (CaMKII) and calcineurin signaling pathways. We also demonstrated that blocking TRPA1 decreased the proportion of M2 macrophages and reduced profibrotic cytokine levels, thereby improving cardiac fibrosis.ConclusionsTRPA1 inhibition protected against cardiac hypertrophy and suppressed cardiac dysfunction via Ca2+-dependent signal pathways and inhibition of the M2 macrophages transition. These results suggest that TRPA1 may represent a potential therapeutic drug target for cardiac hypertrophy and fibrosis.

Roles of TRPV1 and TRPA1 in Spontaneous Pain from Inflamed Masseter Muscle

Craniofacial muscle pain, such as spontaneous pain and bite-evoked pain, are major symptoms in patients with temporomandibular disorders and infection. However, the underlying mechanisms of muscle pain, especially mechanisms of highly prevalent spontaneous pain, are poorly understood. Recently, we reported that transient receptor potential vanilloid 1 (TRPV1) contributes to spontaneous pain but only marginally contributes to bite-evoked pain during masseter inflammation. Here, we investigated the role of transient receptor potential ankyrin 1 (TRPA1) in spontaneous and bite-evoked pain during masseter inflammation, and dissected the relative contributions of TRPA1 and TRPV1. Masseter inflammation increased mouse grimace scale (MGS) scores and face wiping behaviors. Pharmacological or genetic inhibition of TRPA1 significantly attenuated MGS but not face wiping behaviors. MGS scores were also attenuated by scavenging putative endogenous ligands for TRPV1 or TRPA1. Simultaneous inhibition of TRPA1 by AP18 and TRPV1 by AMG9810 in masseter muscle resulted in robust inhibition of both MGS and face wiping behaviors. Administration of AP18 or AMG9810 to masseter muscle induced conditioned place preference (CPP). The extent of CPP following simultaneous administration of AP18 and AMG9810 was greater than that induced by the individual antagonists. In contrast, inflammation-induced reduction of bite force was not affected by the inhibition of TRPA1 alone or in combination with TRPV1. These results suggest that simultaneous inhibition of TRPV1 and TRPA1 produces additive relief of spontaneous pain, but does not ameliorate bite-evoked pain during masseter inflammation. Our results provide further evidence that distinct mechanisms underlie spontaneous and bite-evoked pain from inflamed masseter muscle.

Transient Receptor Potential Ankyrin 1 (TRPA1) Mediates Lipopolysaccharide (LPS)-Induced Inflammatory Responses in Primary Human Osteoarthritic Fibroblast-Like Synoviocytes.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal and non-neuronal cells. Recently, emerging evidences suggested the crucial role of TRPA1 in the disease progression of osteoarthritis (OA). Therefore, we aimed to investigate whether TRPA1 mediate lipopolysaccharide (LPS)-induced inflammatory responses in primary human OA fibroblast-like synoviocytes (OA-FLS). The expression of TRPA1 in LPS-treated OA-FLS was assessed by polymerase chain reaction (PCR) and western blot (WB), and the functionality of TRPA1 channel by Ca2+ influx measurements. Meanwhile, production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, matrix metalloproteinase (MMP)-1, and MMP-3 in LPS-treated cells was measured by immunoassay. Histological observation after inhibition of TRPA1 was also performed in rats with LPS-induced inflammatory arthritis. After being induced by LPS, the gene and protein expression of TRPA1 was increased in the time-dependent or dose-dependent manner. Meanwhile, Ca2+ influx mediated by TRPA1 in human OA-FLS was also enhanced. In addition, pharmacological inhibition and gene silencing of TRPA1 downregulated the production of IL-1β, TNF-α, IL-6, MMP-1, and MMP-3 in LPS-treated FLS. Finally, synovial inflammation and cartilage degeneration were also reduced by the TRPA1 antagonist. We found the LPS caused the increased functional expression of TRPA1, the activation of which involved in LPS-reduced inflammatory responses in primary human OA-FLS, and the inhibition of TRPA1 produces protective effect in LPS-induced arthritis.

Inhibition of TRPA1 Attenuates Doxorubicin-Induced Acute Cardiotoxicity by Suppressing Oxidative Stress, the Inflammatory Response, and Endoplasmic Reticulum Stress.

The transient receptor potential ankyrin 1 (TRPA1) channel is expressed in cardiomyocytes and involved in many cardiovascular diseases. However, the expression and function of TRPA1 in doxorubicin- (Dox-) induced acute cardiotoxicity have not been elucidated. This study aimed at investigating whether blocking the TRPA1 channel with the specific inhibitor HC-030031 (HC) attenuates Dox-induced cardiac injury. The animals were randomly divided into four groups: control, HC, Dox, and Dox + HC. Echocardiography was used to evaluate cardiac function, and the heart was removed for molecular experiments. The results showed that the expression of TRPA1 was increased in the heart after Dox treatment. Cardiac dysfunction and increased serum CK-MB and LDH levels were induced by Dox, but these effects were attenuated by HC treatment. In addition, HC mitigated Dox-induced oxidative stress, as evidenced by the decreased MDA level and increased GSH level and SOD activity in the Dox + HC group. Meanwhile, HC treatment lowered the levels of the proinflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α induced by Dox. Furthermore, HC treatment mitigated endoplasmic reticulum (ER) stress and cardiomyocyte apoptosis induced by Dox. These results indicated that inhibition of TRPA1 could prevent Dox-induced cardiomyocyte apoptosis in mice by inhibiting oxidative stress, inflammation, and ER stress.