TRPA1 Expression Research Articles

A comprehensive analysis of TRP-related gene signature, and immune infiltration in patients with colorectal cancer

BackgroundTransient receptor potential (TRP) channels are involved in the development and progression of tumors. However, their role in colorectal cancer (CRC) remains unclear, and this study aims to investigate the role of TRP-related genes in CRC.MethodsData was obtained from The Cancer Genome Atlas (TCGA) database, and analyses were conducted on the GSE14333 and GSE38832 datasets to assess the prognosis and mark TRP-related genes (TRGs). Subsequently, clustering analysis and immune infiltration analysis were performed to explore the relevant TRGs. In vitro validation of key TRGs’ gene and protein expression was conducted using human colon cancer cells.ResultsCompared to normal tissues, 8 TRGs were significantly upregulated in CRC, while 11 were downregulated. TRPA1 was identified as a protective prognostic factor, whereas TRPM5 (HR = 1.349), TRPV4 (HR = 1.289), and TRPV3 (HR = 1.442) were identified as prognostic risk factors. Receiver operating characteristic (ROC) curves and Kaplan-Meier (KM) analyses yielded similar results. Additionally, lower expression of TRPA1 and higher expression of TRPV4 and TRPM5 were negatively correlated with patient prognosis, and experimental validation confirmed the underexpression of TRPA1 and overexpression of TRPV4 and TRPM5 in CRC cell lines.ConclusionThis study identifies a TRP channel-related prognosis in CRC, providing a novel approach to stratifying CRC prognosis.

Resolvin D2-induced reparative dentin and pulp stem cells after pulpotomy in a rat model

IntroductionVital pulp therapy (VPT) is performed to preserve dental pulp. However, the biocompatibility of the existing materials is of concern. Therefore, novel materials that can induce pulp healing without adverse effects need to be developed. Resolvin D2 (RvD2), one of specialized pro-resolving mediators, can resolve inflammation and promote the healing of periapical lesions. Therefore, RvD2 may be suitable for use in VPT. In the present study, we evaluated the efficacy of RvD2 against VPT using in vivo and in vitro models. MethodsFirst molars of eight-week-old male Sprague–Dawley rats were used for pulpotomy. They were then divided into three treatment groups: RvD2, phosphate-buffered saline, and calcium hydroxide groups. Treatment results were assessed using radiological, histological, and immunohistochemical (GPR18, TNF-α, Ki67, VEGF, TGF-β, CD44, CD90, and TRPA1) analyses. Dental pulp-derived cells were treated with RvD2 in vitro and analyzed using cell-proliferation and cell-migration assays, real-time PCR (Gpr18, Tnf-α, Il-1β, Tgf-β, Vegf, Nanog, and Trpa1), ELISA (VEGF and TGF-β), immunocytochemistry (TRPA1), and flow cytometry (dental pulp stem cells: DPSCs). ResultsThe formation of calcified tissue in the pulp was observed in the RvD2 and calcium hydroxide groups. RvD2 inhibited inflammation in dental pulp cells. RvD2 promoted cell proliferation and migration and the expression of TGF-β and VEGF in vitro and in vivo. RvD2 increased the number of DPSCs. In addition, RvD2 suppressed TRPA1 expression as a pain receptor. ConclusionRvD2 induced the formation of reparative dentin, anti-inflammatory effects, and decreased pain, along with the proliferation of DPSCs via the expression of VEGF and TGF-β, on the pulp surface in pulpotomy models.

Transient Receptor Potential Ankyrin 1 Channel Alters Transforming Growth Factor Beta 1/Smad Signaling in Rat Vocal Fold Fibroblasts.

Vocal fold scar remains a therapeutic challenge. Vocal fold fibroblasts (VFFs) secrete extracellular matrix (ECM), and transforming growth factor-beta 1 (TGF-β1)-mediated fibroblast to myofibroblast differentiation is central to the development of fibrosis. The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels, and activation of TRP ankyrin 1 (TRPA1) channel has been shown to have antifibrotic effects through TGF-β1/Smad signaling in various organs. This study aimed to elucidate expression of TRPA1 and the impact of TRPA1 activation on TGF-β1/Smad signaling in VFFs. Vocal folds were dissected from 10-week-old, male Sprague-Dawley rats and primary VFFs were established. TRPA1 was examined in VFFs and lamina propria via immunostaining. VFFs were treated with allyl isothiocyanate (AITC, TRP channel agonist, 10-5 M) ± TGF-β1 (10 ng/ml) ± A-967079 (selective TRPA1 channel antagonist, 5.0 × 10-7 M) for 4 or 24 h. Trpa1, Smad3, Smad7, Col1a1, Acta2, and Has1 mRNA expression were quantified via qPCR. TRPA1 was expressed in cultured VFFs and the lamina propria. TGF-β1 administration significantly increased Trpa1 compared to control. AITC alone did not alter Smad3, Smad7, Acta2, or ECM related genes. However, the combination of AITC and TGF-β1 significantly increased Smad3 and decreased Smad7 and Acta2 compared to TGF-β1 alone; A-967079 significantly reduced this response. VFFs expressed TRPA1, and the activation of TRPA1 regulated TGF-β1/Smad signaling in VFFs. These findings provide preliminary insights into potential anti-fibrotic mechanisms of TRPA1 activation through TGF-β1/Smad signaling in VFFs. NA Laryngoscope, 134:4593-4598, 2024.

Hypothalamic TRPM8 and TRPA1 ion channel genes in the regulation of temperature homeostasis at water balance changes

The role of the hypothalamic cold-sensitive ion channels - transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) in homeostatic systems of thermoregulation and water-salt balance – is not clear. The interaction of homeostatic systems of thermoregulation and water-salt balance without additional temperature load did not receive due attention, too. On the models of water-balance disturbance, we tried to elucidate some aspect of these problems.Body temperature (Tbody), O2 consumption, CO2 excretion, electrical muscle activity (EMA), temperature of tail skin (Ttail), plasma osmolality, as well as gene expression of hypothalamic TRPM8 and TRPA1 have been registered in rats of 3 groups: control; water-deprived (3 days under dry–eating); and hyperhydrated (6 days without dry food, drinking liquid 4 % sucrose). No relationship was observed between plasma osmolality and gene expression of Trpm8 and Trpa1. In water-deprived rats, the constriction of skin vessels, increased fat metabolism by 10 % and increased EMA by 48 % allowed the animals to maintain Tbody unchanged. The hyperhydrated rats did not develop sufficient mechanisms, and their Tbody decreased by 0.8 °C. The development of reactions was correlated with the expression of genes of thermosensitive ion channels in the anterior hypothalamus. Ttail had a direct correlation with the expression of the Trpm8 gene, whereas EMA directly correlated with the expression of the Trpa1 gene in water-deprived group. The obtained data attract attention from the point of view of management and correction of physiological functions by modulating the ion channel gene expression.

Investigation of Trpa1 and Trpc1 Immunreactivities in Colon Adenocarcinomas.

As the normal colon epithelium differentiates into adenoma, invasive cancer and metastatic cancer, the cell acquires new characteristics such as apoptosis, proliferation, differentiation, invasion and metastasis. Many mechanisms are effective in acquiring these qualities. One of these is the regulation of the functioning of ion channels. This study aimed to examine TRPA1 and TRPC1 expression in colorectal adenocarcinomas showing different degrees of differentiation. We examined the biopsy specimens of 60 patients diagnosed with colorectal adenocarcinomas, including those of patients with well-differentiated (n = 20), moderately differentiated (n = 20) and poorly differentiated (n = 20) carcinomas. Moreover, 20 biopsy specimens of individuals with normal colonic mucosa were examined. Histoscores were calculated for TRPA1 and TRPC1 based on the extent of diffusion and intensity of immunoreactivity, and these scores were compared statistically. A statistically significant increase in both TRPA1 and TRPC1 immunoreactivity was observed in low-grade and high-grade colon adenocarcinomas compared to the control group (p<0.001). A statistically significant decrease in both TRPA1 and TRPC1 immunoreactivity was observed in high-grade colon adenocarcinomas compared to low-grade colon adenocarcinomas (p<0.001). TRPA1 and TRPC1 immunoreactivites are increased in colorectal adenocarcinoma tissue compared with the healthy tissue. Furthermore, the immunoreactivity decreases as the grade of cancer increases.

Role of TRPV1 and TRPA1 in TSLP production in nasal epithelial cells

BackgroundTRP protein is sensitive to external temperature changes, but its pathogenic mechanism in the upper airway mucosa is still unclear. ObjectiveTo investigate the mechanism of TRPV1and TRPA1 in regulating the secretion of inflammatory factors in nasal epithelial cells. MethodsThe expression of TRPV1 and TRPA1 in nasal mucosal epithelial cells was investigated using immunofluorescence assays. Epithelial cells were stimulated with TRPV1 and TRPA1 agonists and antagonists, and changes in Ca2+ release and inflammatory factor secretion in epithelial cells were detected. TSLP secretion stimulated with the calcium chelating agent EGTA was evaluated. The transcription factor NFAT was observed by immunofluorescence staining. ResultsTRPV1 and TRPA1 expression was detected in nasal epithelial cells, and Ca2+ influx was increased after stimulation with agonists. After the activation of TRPV1 and TRPA1, the gene expression of TSLP, IL-25, and IL-33 and the protein expression levels of TSLP and IL-33 were increased, and only TSLP could be inhibited by antagonists and siRNAs. After administration of EGTA, the secretion of TSLP was inhibited significantly, and the expression of the transcription factor NFAT in the nucleus was observed after activation of the TRPV1 and TRPA1 proteins in epithelial cells. ConclusionActivation of TRPV1 and TRPA1 on nasal epithelial cells stimulates the generation of TSLP through the Ca2+/NFAT pathway. It also induces upregulation of IL-25 and IL-33 gene expression levels and increased levels of IL-33 protein, leading to the development of airway inflammation.

Molecular classification reveals the sensitivity of lung adenocarcinoma to radiotherapy and immunotherapy: multi-omics clustering based on similarity network fusion

BackgroundDue to individual differences in tumors and immune systems, the response rate to immunotherapy is low in lung adenocarcinoma (LUAD) patients. Combinations with other therapeutic strategies improve the efficacy of immunotherapy in LUAD patients. Although radioimmunotherapy has been demonstrated to effectively suppress tumors, the underlying mechanisms still need to be investigated.MethodsTotal RNA from LUAD cells was sequenced before and after radiotherapy to identify differentially expressed radiation-associated genes. The similarity network fusion (SNF) algorithm was applied for molecular classification based on radiation-related genes, immune-related genes, methylation data, and somatic mutation data. The changes in gene expression, prognosis, immune cell infiltration, radiosensitivity, chemosensitivity, and sensitivity to immunotherapy were assessed for each subtype.ResultsWe used the SNF algorithm and multi-omics data to divide TCGA-LUAD patients into three subtypes. Patients with the CS3 subtype had the best prognosis, while those with the CS1 and CS2 subtypes had poorer prognoses. Among the strains tested, CS2 exhibited the most elevated immune cell infiltration and expression of immune checkpoint genes, while CS1 exhibited the least. Patients in the CS2 subgroup were more likely to respond to PD-1 immunotherapy. The CS2 patients were most sensitive to docetaxel and cisplatin, while the CS1 patients were most sensitive to paclitaxel. Experimental validation of signature genes in the CS2 subtype showed that inhibiting the expression of RHCG and TRPA1 could enhance the sensitivity of lung cancer cells to radiation.ConclusionsIn summary, this study identified a risk classifier based on multi-omics data that can guide treatment selection for LUAD patients.

Zhisou powder suppresses airway inflammation in LPS and CS-induced post-infectious cough model mice via TRPA1/TRPV1 channels

Ethnopharmacological relevanceZhisou Powder (ZSP), a traditional Chinese medicine (TCM) prescription, has been widely used in the clinic for the treatment of post-infectious cough (PIC). However, the exact mechanism is not clear. Aim of the studyThe aim of this study was to investigate the ameliorative effect of ZSP on PIC in mice. The possible mechanisms of action were screened based on network pharmacology, and the potential mechanisms were explored through molecular docking and in vivo experimental validation. Materials and methodsLipopolysaccharide (LPS) (80μg/50 μL) was used to induce PIC in mice, followed by daily exposure to cigarette smoke (CS) for 30 min for 30 d to establish PIC model. The effects of ZSP on PIC mice were observed by detecting the number of coughs and cough latency, peripheral blood and bronchoalveolar lavage fluid (BALF) inflammatory cell counts, enzyme-linked immunosorbent assay (ELISA), and histological analysis. The core targets and key pathways of ZSP on PIC were analyzed using network pharmacology, and TRPA1 and TRPV1 were validated using RT-qPCR and western blotting assays. ResultsZSP effectively reduced the number of coughs and prolonged the cough latency in PIC mice. Airway inflammation was alleviated by reducing the expression levels of the inflammatory mediators TNF-α and IL-1β. ZSP modulated the expression of Substance P, Calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF) in BALF. Based on the results of network pharmacology, the mechanism of action of ZSP may exert anti-neurogenic airway-derived inflammation by regulating the expression of TRPA1 and TRPV1 through the natural active ingredients α-spinastero, shionone and didehydrotuberostemonine. ConclusionZSP exerts anti-airway inflammatory effects through inhibition of TRPA1/TRPV1 channels regulating neuropeptides to alleviate cough hypersensitivity and has a favorable therapeutic effect on PIC model mice. It provides theoretical evidence for the clinical application of ZSP.

TRP-CHANNELS IN TENDONS: FIRST INSIGHTS, NOVEL QUESTIONS

Tendinopathy is a disease associated with pain and tendon degeneration, leading to a decreased range of motion and an increased risk of tendon rupture. The etiology of this frequent disease is still unknown. In other musculoskeletal tissues like cartilage and intervertebral discs, transient receptor potential channels (TRP- channels) were shown to play a major role in the progression of degeneration. Due to their responsiveness to a wide range of stimuli like temperature, pH, osmolarity and mechanical load, they are potentially relevant factors in tendon degeneration as well. We therefore hypothesize that TRP- channels are expressed in tendon cells and respond to degeneration inducing stimuli.By immunohistochemistry, qRT-PCR and western blot analyses, we found three TRP channel members, belonging to the vanilloid (TRPV), and ankyrin (TRPA) subfamily, respectively, to be expressed in healthy human tendon tissue as well as in rodent tendon, with expression being located to cells within the dense tendon proper, as well as to endotenon resident cells. In vitro-inflammatory and ex vivo-mechanical stimulation led to a significant upregulation of TRPA1 expression in tendon cells, which correlates well with the fact that TRPA1 is considered as mechanosensitive channel being sensitized by inflammatory mediators.This is the first description of TRP- channels in human and rodent tendon. As these channels are pharmacologically targetable by both agonists and antagonists, they may represent a promising target for novel treatments of tendinopathy.

TRPA1 channel mediates methylglyoxal-induced mouse bladder dysfunction.

Introduction: The transient receptor potential ankyrin 1 channel (TRPA1) is expressed in urothelial cells and bladder nerve endings. Hyperglycemia in diabetic individuals induces accumulation of the highly reactive dicarbonyl compound methylglyoxal (MGO), which modulates TRPA1 activity. Long-term oral intake of MGO causes mouse bladder dysfunction. We hypothesized that TRPA1 takes part in the machinery that leads to MGO-induced bladder dysfunction. Therefore, we evaluated TRPA1 expression in the bladder and the effects of 1 h-intravesical infusion of the selective TRPA1 blocker HC-030031 (1nmol/min) on MGO-induced cystometric alterations. Methods: Five-week-old female C57BL/6 mice received 0.5% MGO in their drinking water for 12weeks, whereas control mice received tap water alone. Results: Compared to the control group, the protein levels and immunostaining for the MGO-derived hydroimidazolone isomer MG-H1 was increased in bladders of the MGO group, as observed in urothelium and detrusor smooth muscle. TRPA1 protein expression was significantly higher in bladder tissues of MGO compared to control group with TRPA1 immunostaining both lamina propria and urothelium, but not the detrusor smooth muscle. Void spot assays in conscious mice revealed an overactive bladder phenotype in MGO-treated mice characterized by increased number of voids and reduced volume per void. Filling cystometry in anaesthetized animals revealed an increased voiding frequency, reduced bladder capacity, and reduced voided volume in MGO compared to vehicle group, which were all reversed by HC-030031 infusion. Conclusion: TRPA1 activation is implicated in MGO-induced mouse overactive bladder. TRPA1 blockers may be useful to treat diabetic bladder dysfunction in individuals with high MGO levels.

Expression of transient receptor potential channels on peripheral blood leukocytes of patients with chronic obstructive pulmonary disease

It is known that transient receptor potential (TRP) channels are receptors for reactive oxygen species and components of cigarette smoke, and therefore may be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD).Aim: To investigate the expression of TRPV1, TRPV4, TRPM8 and TRPA1 channels on peripheral blood leukocytes of COPD patients and healthy individuals and to establish its possible relationships with inflammatory markers and lung function parameters.Methodology and Research Methods. 23 patients with COPD of varying severity and 17 individuals without bronchial obstruction were examined. The expression of TRP channels on monocytes, lymphocytes and granulocytes was determined by indirect flow cytometry. Plasma concentrations of cytokines were measured in a multiplex assay. Lung function parameters were assessed by spirometry.Results. Monocytes from COPD patients were characterized by increased expression of TRPV1 (98.8 (97.6; 99.6)% vs. 97.8 (96.3; 98.6)%, p = 0.03) and TRPA1 (99.2 (98.7; 99.8)% vs. 95.1 (94.2; 98.4)%, p = 0.003). In addition, COPD was associated with an increased ratio of TRPV4/TRPM8 expression on monocytes (0.66 (0.51, 0.90) vs. 0.27 (0.12, 0.70), p = 0.02) and granulocytes (0.97 (0.86; 0.92) vs. 0.29 (0.06; 0.89), p = 0.003). TRPV4 expression on monocytes showed correlation with CXCL10 level (ρ = 0.55, p = 0.01), and for TRPM8 inverse relationships were found with the concentrations of IL-6 (ρ = –0.56, p = 0.02) and IL-10 (ρ = –0.55, p = 0.02). We did not find any associations between the expression of TRP channels and lung function parameters.Conclusion. Increased expression of TRPV1 and TRPA1, as well as an increased TRPV4/TRPM8 ratio on peripheral blood monocytes of COPD patients may indicate the contribution of these receptors to the proinflammatory activation of the cells and indicate possible prospects of TRP channels pharmacological modulation for the purpose of the disease therapy.

Identification and functional characterization of a novel TRPA1 gene from sea cucumber Apostichopus japonicus and interaction with miR-2013 in response to salt stress.

Salinity is important abiotic factor influencing sea cucumber aquaculture. This study aimed to identify and functional study of a novel transient receptor potential cation channel subfamily A member 1 (TRPA1) involved in salinity stress through interaction with miR-2013 in the sea cucumber. The full-length cDNA sequence was 1369bp in length and encoded 138 amino acids. The TRPA1 homolog protein was a hydrophilic protein without a signal peptide and was predicted to a spatial structure of seven helices and eight random coils and two major ANK functional domains. Bioinformatic analysis and luciferase reporter assays confirmed TRPA1 as a target gene of miR-2013. Quantitative PCR revealed that miR-2013 was induced upregulation after salinity stress, while TRPA1 showed upregulated expression with maximum expression at 24h. The expression of miR-2013 and TRPA1 was negatively regulated. Transfection experiments were conducted to validate the role of miR-2013 and TRPA1 in salinity response. The results showed that miR-2013 was upregulated and TRPA1 was downregulated after transfection with miR-2013 mimics, while miR-2013 was downregulated and TRPA1 was upregulated after transfection with miR-2013 inhibitor. Transfection with si-TRPA1 homolog resulted in upregulation of miR-2013 and downregulation of TRPA1 homolog. These findings suggest that miR-2013 can regulate the expression of TRPA1 under salt stress, and highlight the importance of miR-2013 and TRPA1 in salt stress response. miR-2013 mimics improved the survival rate, while miR-2013 inhibitor and si-TRPA1 reduced it. These findings suggest that miR-2013 and TRPA1 play important roles in sea cucumbers adaptation to salinity changes.

HnRNPA1 SUMOylation promotes cold hypersensitivity in chronic inflammatory pain by stabilizing TRPA1 mRNA

TRPA1 is pivotal in cold hypersensitivity, but its regulatory mechanisms in inflammatory cold hyperalgesia remain poorly understood. We show here that the upregulation of SUMO1-conjugated protein levels in a complete Freund's adjuvant (CFA)-induced inflammatory pain model enhances TRPA1 mRNA stability, ultimately leading to increased expression levels. We further demonstrate that hnRNPA1 binds to TRPA1 mRNA, and its SUMOylation, upregulated in CFA-induced inflammatory pain, contributes to stabilizing TRPA1 mRNA by accumulating hnRNPA1 in the cytoplasm. Moreover, we find that wild-type hnRNPA1 viral infection in dorsal root ganglia neurons, and not infection with the SUMOylation-deficient hnRNPA1 mutant, can rescue the reduced ability of hnRNPA1-knockdown mice to develop inflammatory cold pain hypersensitivity. These results suggest that hnRNPA1 is a regulator of TRPA1 mRNA stability, the capability of which is enhanced upon SUMO1 conjugation at lysine 3 in response to peripheral inflammation, and the increased expression of TRPA1 in turn underlies the development of chronic inflammatory cold pain hypersensitivity.

Neuroinflammation in the Dorsal Root Ganglia and Dorsal Horn Contributes to Persistence of Nociceptor Sensitization in SIV-Infected Antiretroviral Therapy-Treated Macaques

Despite the development of antiretroviral therapy (ART), HIV-associated distal sensory polyneuropathy remains prevalent. Using SIV-infected rhesus macaques, this study examined molecular mechanisms of peripheral and central sensitization to infer chronic pain from HIV infection. Previous studies identified atrophy in nociceptive neurons during SIV infection, which was associated with monocyte infiltration into the dorsal root ganglia (DRG). However, the sensory signaling mechanism connecting this pathology to symptoms remains unclear, especially because pain persists after resolution of high viremia and inflammation with ART. We hypothesized that residual DRG and dorsal horn neuroinflammation contributes to nociceptive sensitization. Using three cohorts of macaques [uninfected (SIV-), SIV-infected (SIV+), and SIV infected with ART (SIV+/ART)], this study showed an increase in the cellular and cytokine inflammatory profiles in the DRG of SIV+/ART macaques compared with uninfected animals. It found significant increase in the expression of nociceptive ion channels, TRPV1, and TRPA1 among DRG neurons in SIV+/ART compared with uninfected animals. SIV-infected and SIV+/ART animals showed reduced innervation of the nonpeptidergic nociceptors into the dorsal horn compared with uninfected animals. Finally, there were a significantly higher number of CD68+ cells in the dorsal horn of SIV+/ART macaques compared with uninfected animals. In summary, these data demonstrate that neuroinflammation, characteristics of nociceptor sensitization, and central terminal atrophy persists in SIV+/ART animals.

Transient receptor potential M2 channel in the hypothalamic preoptic area and its impact on thermoregulation during menopause

BackgroundDecreased estrogen levels can cause abnormal thermosensitivity of the preoptic area (POA) in the hypothalamus during menopause, which may cause hot flashes. Thermosensitive transient receptors (ThermoTRPs) affect the thermosensitivity of neurons. It is worth exploring whether ThermoTRPs change under low estrogen state and participate in the abnormal thermoregulation of POA. MethodsAdult female Sprague–Dawley rats were randomly divided into sham operation (SHAM), ovariectomy (OVX) and estrogen treatment after ovariectomy (OVX+E) groups. Under 10 ℃, 18 ℃, 25 ℃, 37 ℃ and 45 ℃ incubations, their skin temperature was monitored and the expression of TRPA1, TRPM8, TRPM2, and TRPV1 in POA were investigated. ResultsThe skin temperature of ovariectomized rats changed faster and more dramatically under different incubation temperatures. The results at mRNA level show that only the expression of TRPM2 decreased in POA of OVX group compared with the other two groups at 25 ℃, TRPA1 expression in POA of the three groups increased at 10 ℃, TRPM8 increased at 10 ℃ and 18 ℃, TRPV1 increased at 10 ℃ and 45 ℃, while the expression of TRPM2 decreased at 10 ℃ and 18 ℃ and increased at 37 ℃ and 45 ℃. In all these cases, the magnitudes of the changes were less in the OVX group relative to the other two groups. The further immunohistochemical and Western blot results of TRPM2 and the activated TRPM2 positive cells labeled by c-Fos were consistent with the results of mRNA level. ConclusionsThe expression and thermosensitivity of TRPM2 in POA changed greatly under different incubation temperatures, but the changes in ovariectomized rats were less. This may be the key factor triggering thermoregulation dysfunction under low estrogen and may cause hot flashes.

The regulation of TRPA1 expression and function by Th1 and Th2-type inflammation in human A549 lung epithelial cells

BackgroundTransient Receptor Potential Ankyrin 1 (TRPA1) is a cation channel that mediates pain, itch, cough, and neurogenic inflammation in response to pungent compounds such as acrolein in cigarette smoke. TRPA1 is also activated by endogenous factors and promotes inflammation in asthma models. We have recently shown that TRPA1 is upregulated by inflammatory cytokines in A549 human lung epithelial cells. Here, we explored the effects of Th1 and Th2-type inflammation on TRPA1.Methods and resultsTRPA1 expression and function was studied in A549 human lung epithelial cells. To induce inflammation, the cells were exposed to a combination of cytokines TNF-α and IL-1β; and to model Th1 or Th2-type responses, IFN-γ or IL-4/IL-13 was added, respectively. TRPA1 expression (measured by RT-PCR and Western blot) and function (assessed by Fluo-3AM intracellular calcium measurement) was enhanced under the influence of TNF-α + IL-1β. IFN-γ further enhanced TRPA1 expression and function, whereas IL-4 and IL-13 suppressed them. The effects of IFN-γ and IL-4 on TRPA1 expression were reversed by the Janus kinase (JAK) inhibitors baricitinib and tofacitinib, and those of IL-4 also by the STAT6 inhibitor AS1517499. The glucocorticoid dexamethasone downregulated TRPA1 expression, whereas the PDE4 inhibitor rolipram had no effect. Under all conditions, TRPA1 blockade was found to reduce the production of LCN2 and CXCL6.ConclusionsTRPA1 expression and function in lung epithelial cells was upregulated under inflammatory conditions. IFN-γ further increased TRPA1 expression while IL-4 and IL-13 suppressed that in a JAK-STAT6 dependent manner which is novel. TRPA1 also modulated the expression of genes relevant to innate immunity and lung disease. We propose that the paradigm of Th1 and Th2 inflammation is a major determinant of TRPA1 expression and function, which should be considered when targeting TRPA1 for pharmacotherapy in inflammatory (lung) disease.

TRPA1 deficiency attenuates cardiac fibrosis via regulating GRK5/NFAT signaling in diabetic rats

BackgroundTransient receptor potential ankyrin 1 (TRPA1) has been linked to the development of various cardiovascular diseases, but its role in diabetic cardiomyopathy is not well understood. This study aimed to investigate the protective effects of TRPA1 deficiency on diabetic cardiomyopathy in rats with streptozotocin-induced diabetes and in neonatal rat cardiac fibroblasts (CFs) exposed to high glucose (HG). MethodsCardiac TRPA1 expression levels were measured in diabetic rats. Cardiac function, remodeling, and fibrosis were analyzed in Sprague-Dawley (SD) rats and TRPA1-deficient rats with diabetic cardiomyopathy. In vitro, fibrosis was measured in CFs exposed to HG. Additionally, 1,8-cineole, a natural inhibitor of TRPA1, was used to treat SD rats with diabetic cardiomyopathy. ResultsTRPA1 expression was increased in the heart tissue of diabetic rats and in CFs treated with HG. TRPA1 deficiency significantly improved cardiac function in diabetic rats, as evidenced by improved echocardiography and reduced cardiac hypertrophy and fibrosis. In vitro, TRPA1 deficiency suppressed the transformation of HG-induced CFs into myofibroblasts. The cardioprotective effect of TRPA1 deficiency was found to inhibit cardiac fibrosis by regulating GRK5/NFAT signaling. Furthermore, inhibition of GRK5/NFAT signaling abolished the promotion of CF transformation into myofibroblasts by TRPA1 activation. Inhibition of TRPA1 activation by 1,8-cineole reduced cardiac dysfunction and remodeling in diabetic rats by regulating GRK5/NFAT signaling. ConclusionsTRPA1 deficiency reduced cardiac fibrosis in diabetic rats and inhibited HG-induced CF activation in vitro by regulating GRK5/NFAT signaling. The TRPA1 inhibitor 1,8-cineole may serve as a novel therapeutic agent for the treatment of diabetic cardiomyopathy.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging.TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found.In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Human Osteoarthritic Chondrocytes Express Nineteen Different TRP-Genes-TRPA1 and TRPM8 as Potential Drug Targets.

Transient receptor potential (TRP) ion channels are expressed in neuronal and some non-neuronal cells and are involved particularly in pain and thermosensation. We previously showed that TRPA1 is functionally expressed in human osteoarthritic (OA) chondrocytes and mediates inflammation, cartilage degradation, and pain in monosodium-iodoacetate-induced experimental OA. In the present study, we explored the expression of TRP-channels in primary human OA chondrocytes and investigated whether drugs used in the treatment of OA, ibuprofen and glucocorticoids, have effects on TRP-channel expression. OA cartilage was obtained from knee replacement surgery and chondrocytes were isolated with enzyme digestion. NGS analysis showed the expression of 19 TRP-genes in OA chondrocytes, with TRPM7, TRPV4, TRPC1, and TRPM8 having the highest counts in unstimulated cells. These results were verified with RT-PCR in samples from a different group of patients. Interleukin-1β (IL-1β) significantly increased TRPA1 expression, while TRPM8 and TRPC1 expression was decreased, and TRPM7 and TRPV4 expression remained unaffected. Furthermore, dexamethasone attenuated the effect of IL-1β on TRPA1 and TRPM8 expression. The TRPM8 and TRPA1 agonist menthol increased the expression of the cartilage-degrading enzymes MMP-1, MMP-3, and MMP-13 and the inflammatory factors iNOS and IL-6 in OA chondrocytes. In conclusion, human OA chondrocytes express 19 different TRP-genes, of which the significant TRPM8 expression is a novel finding. Dexamethasone attenuated IL-1β-induced TRPA1 expression. Interestingly, the TRPM8 and TRPA1 agonist menthol increased MMP expression. These results support the concept of TRPA1 and TRMP8 as potential novel drug targets in arthritis.

Intraganglionic reactive oxygen species mediate inflammatory pain and hyperalgesia through TRPA1 in the rat.

Reactive oxygen species (ROS) are generated in nociceptive pathways in response to inflammation and injury. ROS are accumulated within the sensory ganglia following peripheral inflammation, but the functional role of intraganlionic ROS in inflammatory pain is not clearly understood. The aims of this study were to investigate whether peripheral inflammation leads to prolonged ROS accumulation within the trigeminal ganglia (TG), whether intraganglionic ROS mediate pain hypersensitivity via activation of TRPA1, and whether TRPA1 expression is upregulated in TG during inflammatory conditions by ROS. We demonstrated that peripheral inflammation causes excess ROS production within TG during the period when inflammatory mechanical hyperalgesia is most prominent. Additionally, scavenging intraganglionic ROS attenuated inflammatory mechanical hyperalgesia and a pharmacological blockade of TRPA1 localized within TG also mitigated inflammatory mechanical hyperalgesia. Interestingly, exogenous administration of ROS into TG elicited mechanical hyperalgesia and spontaneous pain-like responses via TRPA1, and intraganglionic ROS induced TRPA1 upregulation in TG. These results collectively suggest that ROS accumulation in TG during peripheral inflammation contributes to pain and hyperalgesia in a TRPA1 dependent manner, and that ROS further exacerbate pathological pain responses by upregulating TRPA1 expression. Therefore, any conditions that exacerbate ROS accumulation within somatic sensory ganglia can aggravate pain responses and treatments reducing ganglionic ROS may help alleviate inflammatory pain.