Role of transient receptor potential ankyrin 1 in idiopathic pulmonary fibrosis: modulation of M2 macrophage polarization.

Idiopathic pulmonary fibrosis (IPF) poses significant challenges due to limited treatment options despite its complex pathogenesis involving cellular and molecular mechanisms. This study investigated the role of transient receptor potential ankyrin 1 (TRPA1) channels in regulating M2 macrophage polarization in IPF progression, potentially offering novel therapeutic targets. Using a bleomycin-induced pulmonary fibrosis model in C57BL/6J mice, we assessed the therapeutic potential of the TRPA1 inhibitor HC-030031. TRPA1 upregulation was observed in fibrotic lungs, correlating with worsened lung function and reduced survival. TRPA1 inhibition mitigated fibrosis severity, evidenced by decreased collagen deposition and restored lung tissue stiffness. Furthermore, TRPA1 blockade reversed aberrant M2 macrophage polarization induced by bleomycin, associated with reduced Smad2 phosphorylation in the TGF-ß1-Smad2 pathway. In vitro studies with THP-1 cells treated with bleomycin and HC-030031 corroborated these findings, highlighting TRPA1's involvement in fibrotic modulation and macrophage polarization control. Overall, targeting TRPA1 channels presents promising therapeutic potential in managing pulmonary fibrosis by reducing pro-fibrotic marker expression, inhibiting M2 macrophage polarization, and diminishing collagen deposition. This study sheds light on a novel avenue for therapeutic intervention in IPF, addressing a critical need in the management of this challenging disease.

NCS-1 protein regulates TRPA1 channel through the PI3K pathway in breast cancer and neuronal cells.

The physical and functional interaction between transient receptor potential channel ankyrin 1 (TRPA1) and neuronal calcium sensor 1 (NCS-1) was assessed. NCS-1 is a calcium (Ca2+) sensor found in many tissues, primarily neurons, and TRPA1 is a Ca2+ channel involved not only in thermal and pain sensation but also in conditions such as cancer and chemotherapy-induced peripheral neuropathy, in which NCS-1 is also a regulatory component.We explored the interactions between these two proteins by employing western blot, qRT-PCR, co-immunoprecipitation, Ca2+ transient monitoring with Fura-2 spectrophotometry, and electrophysiology assays in breast cancer cells (MDA-MB-231) with different levels of NCS-1 expression and neuroblastoma cells (SH-SY5Y).Our findings showed that the expression of TRPA1 was directly correlated with NCS-1 levels at both the protein and mRNA levels. Additionally, we found a physical and functional association between these two proteins. Physically, the NCS-1 and TRPA1 co-immunoprecipitate. Functionally, NCS-1 enhanced TRPA1-dependent Ca2+ influx, current density, open probability, and conductance, where the functional effects depended on PI3K. Conclusion: NCS-1 appears to act not only as a Ca2+ sensor but also modulates TRPA1 protein expression and channel function in a direct fashion through the PI3K pathway. These results contribute to understanding how Ca2+ homeostasis is regulated and provides a mechanism underlying conditions where Ca2+ dynamics are compromised, including breast cancer. With a cellular pathway identified, targeted treatments can be developed for breast cancer and neuropathy, among other related diseases.

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

BACKGROUND: TRP protein is sensitive to external temperature changes, but its pathogenic mechanism in the upper airway mucosa is still unclear. OBJECTIVE: To investigate the mechanism of TRPV1and TRPA1 in regulating the secretion of inflammatory factors in nasal epithelial cells. METHODS: The 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. RESULTS: TRPV1 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. CONCLUSION: Activation 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.

TRPA1-PI3K/Akt-OPA1-ferroptosis axis in ozone-induced bronchial epithelial cell and lung injury.

BACKGROUND: Transient receptor potential (TRP) ankyrin 1 (TRPA1) could mediate ozone-induced lung injury. Optic Atrophy 1 (OPA1) is one of the significant mitochondrial fusion proteins. Impaired mitochondrial fusion, resulting in mitochondrial dysfunction and ferroptosis, may drive the onset and progression of lung injury. In this study, we examined whether TRPA1 mediated ozone-induced bronchial epithelial cell and lung injury by activating PI3K/Akt with the involvement of OPA1, leading to ferroptosis. METHODS: Wild-type, TRPA1-knockout (KO) mice (C57BL/6 J background) and ferrostatin-1 (Fer-1)-pretreated mice were exposed to 2.5 ppm ozone for 3 h. Human bronchial epithelial (BEAS-2B) cells were treated with 1 ppm ozone for 3 h in the presence of TRPA1 inhibitor A967079 or TRPA1-knockdown (KD) as well as pharmacological modulators of PI3K/Akt-OPA1-ferroptosis. Transcriptome was used to screen and decipher the differential gene expressions and pathways. Oxidative stress, inflammation and ferroptosis were measured together with mitochondrial morphology, function and dynamics. RESULTS: Acute ozone exposure induced airway inflammation and airway hyperresponsiveness (AHR), reduced mitochondrial fusion, and enhanced ferroptosis in mice. Similarly, acute ozone exposure induced inflammatory responses, altered redox responses, abnormal mitochondrial structure and function, reduced mitochondrial fusion and enhanced ferroptosis in BEAS-2B cells. There were increased mitochondrial fusion, reduced inflammatory responses, decreased redox responses and ferroptosis in ozone-exposed TRPA1-KO mice and Fer-1-pretreated ozone-exposed mice. A967079 and TRPA1-KD enhanced OPA1 and prevented ferroptosis through the PI3K/Akt pathway in BEAS-2B cells. These in vitro results were further confirmed in pharmacological modulator experiments. CONCLUSION: Exposure to ozone induces mitochondrial dysfunction in human bronchial epithelial cells and mouse lungs by activating TRPA1, which results in ferroptosis mediated via a PI3K/Akt/OPA1 axis. This supports a potential role of TRPA1 blockade in preventing the deleterious effects of ozone.

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

ETHNOPHARMACOLOGICAL RELEVANCE: Zhisou 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 STUDY: The 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 METHODS: Lipopolysaccharide (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. RESULTS: ZSP 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-1ß. 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. CONCLUSION: ZSP 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.

Transient receptor potential ankyrin 1 channels in the bladder mediate low temperature elicited bladder overactivity in rats.

AIMS: This study aimed to investigate whether pathways involving transient receptor potential ankyrin 1 (TRPA1) channels in the urinary bladder mediate the bladder overactivity elicited by exposure to a low temperature in rats. METHODS: At postnatal week 10, female Sprague-Dawley (SD) rats were intraperitoneally injected with the TRPA1 channel antagonist, HC030031, at room temperature (RT) and subsequently exposed to low temperature (LT). Bladder specimens treated with HC030031 were evaluated for contractions through cumulative addition of the TRPA1 channel agonist trans-cinnamaldehyde. Two days before cystometric investigation, small interfering RNA (siRNA) targeting TRPA1 was transfected into urinary bladders. Then, cystometric investigations were performed on rats subjected to TRPA1 siRNA transfection at both RT and LT. Expression of TRPA1 channels in the urinary bladder was assessed through immunohistochemistry and real-time reverse transcription-polymerase chain reaction. RESULTS: At RT, micturition patterns were unaffected by HC030031 treatment. However, upon exposure to LT, rats treated with HC030031 exhibited a reduction of LT-elicited bladder overactivity, as evidenced by inhibited decreases in voiding interval, micturition volume, and bladder capacity. Additionally, HC030031 inhibited trans-cinnamaldehyde-induced contractions. Immunohistochemical analysis showed the presence of TRPA1 channels in the urinary bladder. Notably, rats with TRPA1 siRNA-transfected bladders could partially inhibit bladder overactivity during LT exposure. CONCLUSIONS: These findings indicate that pathways involving TRPA1 channels expressed in the urinary bladder could mediate the LT-elicited bladder overactivity.

[TRPA1 channel mediates oxidative stress defense in cancer].

The unscheduled proliferation of cancer cells outside their natural niches subjects the cells to multiple insults, such as metabolic aberrations, detachment from the extracellular matrix (ECM), hypoxia, and immune cell attacks. Oxidative stress is a hallmark of cancer because these insults can all lead to the accumulation of reactive oxygen species (ROS). However, it remained largely elusive how cancer cells are able to adapt to harsh oxidative environments. Here, we provide evidence that cancer cells co-opt the neuronal ROS-sensing channel TRPA1 to tolerate highly oxidative environments. While TRPA1 is usually expressed at sensory neurons, we found that the channel is also overexpressed in various types of human cancer. TRPA1 does not affect canonical ROS-neutralizing programs but senses ROS and upregulates Ca2＋-dependent anti-apoptotic programs that promotes oxidative-stress tolerance. These findings offer a significant advance in our understanding of adaptation mechanisms to oxidative stress, which represents a substantial hurdle that impedes tumor initiation and progression.

Functional TRPA1 Channels Regulate CD56dimCD16+ NK Cell Cytotoxicity against Tumor Cells.

Transient receptor potential ankyrin 1 (TRPA1) channels are expressed on the surface of different cell types, including immune cells. However, TRPA1's role in the context of innate and adaptive immune responses has not been fully elucidated so far. In this study, we aimed at investigating the expression and function of TRPA1 channels on NK cells. Among NK cells, TRPA1 was highly expressed by the CD56dimCD16+ subpopulation, but not by CD56brightCD16- cells, as detected by FACS. TRPA1 activation with the potent ligand allyl isothiocyanate (AITC) induces intracellular calcium flux in CD56dimCD16+ cells, which was prevented by the TRPA1 antagonist HC-030031. AITC treatment increased the membrane around NKp44 and strongly decreased CD16 and CD8 expression, while CD158a, CD159a, NKG2d, NKp46 were substantially unaffected. Importantly, AITC increased the granzyme production and CD107 expression and increased NK cell-mediated cytotoxicity towards the K562 cell line and two different melanoma cell lines. In parallel, TRPA1 activation also plays regulatory roles by affecting the survival of NK cells to limit uncontrolled and prolonged NK cell-mediated cytotoxicity. Our results indicate that the activation of TRPA1 is an important regulatory signal for NK cells, and agonists of TRPA1 could be used to strengthen the tumor response of the immune system.

Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel Mediates Acrolein Cytotoxicity in Human Lung Cancer Cells.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective ion channel implicated in thermosensation and inflammatory pain. It has been reported that expression of the TRPA1 channel is induced by cigarette smoke extract. Acrolein found in cigarette smoke is highly toxic and known as an agonist of the TRPA1 channel. However, the role of TRPA1 in the cytotoxicity of acrolein remains unclear. Here, we investigated whether the TRPA1 channel is involved in the cytotoxicity of acrolein in human lung cancer A549 cells. The IC50 of acrolein in A549 cells was 25 µM, and acrolein toxicity increased in a concentration- and time-dependent manner. When the effect of acrolein on TRPA1 expression was examined, the expression of TRPA1 in A549 cells was increased by treatment with 50 µM acrolein for 24 h or 500 µM acrolein for 30 min. AP-1, a transcription factor, was activated in the cells treated with 50 µM acrolein for 24 h, while induction of NF-κB and HIF-1α was observed in the cells treated with 500 µM acrolein for 30 min. These results suggest that acrolein induces TRPA1 expression by activating these transcription factors. Overexpression of TRPA1 in A549 cells increased acrolein sensitivity and the level of protein-conjugated acrolein (PC-Acro), while knockdown of TRPA1 in A549 cells or treatment with a TRPA1 antagonist caused tolerance to acrolein. These findings suggest that acrolein induces the TRPA1 channel and that an increase in TRPA1 expression promotes the cytotoxicity of acrolein.

Transient receptor potential ankyrin 1 (TRPA1) modulators: Recent update and future perspective.

The transient receptor potential ankyrin 1 (TRPA1) channel is a non-selective cation channel that senses irritant chemicals. Its activation is closely associated with pain, inflammation, and pruritus. TRPA1 antagonists are promising treatments for these diseases, and there has been a recent upsurge in their application to new areas such as cancer, asthma, and Alzheimer's disease. However, due to the generally disappointing performance of TRPA1 antagonists in clinical studies, scientists must pursue the development of antagonists with higher selectivity, metabolic stability, and solubility. Moreover, TRPA1 agonists provide a deeper understanding of activation mechanisms and aid in antagonist screening. Therefore, we summarize the TRPA1 antagonists and agonists developed in recent years, with a particular focus on structure-activity relationships (SARs) and pharmacological activity. In this perspective, we endeavor to keep abreast of cutting-edge ideas and provide inspiration for the development of more effective TRPA1-modulating drugs.

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

BACKGROUND: Transient 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 RESULTS: TRPA1 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-1ß; 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-1ß. 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. CONCLUSIONS: TRPA1 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.

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-1ß (IL-1ß) 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-1ß 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-1ß-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.

Transient Receptor Potential Ankyrin 1 (TRPA1) Channel as a Sensor of Oxidative Stress in Cancer Cells.

Moderate levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), fuel tumor metastasis and invasion in a variety of cancer types. Conversely, excessive ROS levels can impair tumor growth and metastasis by triggering cancer cell death. In order to cope with the oxidative stress imposed by the tumor microenvironment, malignant cells exploit a sophisticated network of antioxidant defense mechanisms. Targeting the antioxidant capacity of cancer cells or enhancing their sensitivity to ROS-dependent cell death represent a promising strategy for alternative anticancer treatments. Transient Receptor Potential Ankyrin 1 (TRPA1) is a redox-sensitive non-selective cation channel that mediates extracellular Ca2+ entry upon an increase in intracellular ROS levels. The ensuing increase in intracellular Ca2+ concentration can in turn engage a non-canonical antioxidant defense program or induce mitochondrial Ca2+ dysfunction and apoptotic cell death depending on the cancer type. Herein, we sought to describe the opposing effects of ROS-dependent TRPA1 activation on cancer cell fate and propose the pharmacological manipulation of TRPA1 as an alternative therapeutic strategy to enhance cancer cell sensitivity to oxidative stress.

Aconitine - A promising candidate for treating cold and mechanical allodynia in cancer induced bone pain.

BACKGROUND AND AIMS: Patients suffering from cancer induced bone pain (CIBP) have a poor quality of life that is exacerbated by the lack of effective therapeutic drugs. Monkshood is a flowering plant that has been used in traditional Chinese medicine where it has been used to relieve cold pain. Aconitine is the active component of monkshood, but the molecular mechanism for how this compound reduces pain is unclear. METHODS AND RESULTS: In this study, we employed molecular and behavioral experiments to explore the analgesic effect of aconitine. We observed aconitine alleviated cold hyperalgesia and AITC (allyl-isothiocyanate, TRPA1 agonist) induced pain. Interestingly, we found aconitine directly inhibits TRPA1 activity in calcium imaging studies. More importantly, we found aconitine alleviated cold and mechanical allodynia in CIBP mice. Both the activity and expression of TRPA1 in L4 and L5 DRG (Dorsal Root Ganglion) neurons were reduced with the treatment of aconitine in the CIBP model. Moreover, we observed aconiti radix (AR) and aconiti kusnezoffii radix (AKR), both components of monkshood that contain aconitine, alleviated cold hyperalgesia and AITC induced pain. Furthermore, both AR and AKR alleviated CIBP induced cold allodynia and mechanical allodynia. CONCLUSIONS: Taken together, aconitine alleviates both cold and mechanical allodynia in cancer induced bone pain via the regulation of TRPA1. This research on the analgesic effect of aconitine in cancer induced bone pain highlights a component of a traditional Chinese medicine may have clinical applications for pain.

Curcumin suppresses cell proliferation and reduces cholesterol absorption in Caco-2 cells by activating the TRPA1 channel.

BACKGROUND: Curcumin (Cur) is a bioactive dietary polyphenol of turmeric with various biological activities against several cancers. Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Intestinal cholesterol homeostasis is associated with CRC. Chemotherapy for CRC is related to varied adverse effects. Therefore, natural products with anti-cancer properties represent a potential strategy for primary prevention of CRC. METHODS: The present study used Cur as a therapeutic approach against CRC using the Caco-2 cell line. The cells were treated with different concentrations of Cur for different duration of time and then the proliferation ability of cells was assessed using Cell Counting Kit-8 and 5-Ethynyl-2'-deoxyuridine assays. Oil red O staining and cholesterol assay kit were used to evaluate cellular lipid content and cholesterol outward transportation. Finally, the protein expressions of cholesterol transport-related protein and signal transduction molecules were assessed using Western blot assay. RESULTS: Cur inhibited cell proliferation in Caco-2 cells in a dose- and time-dependent manner by activating the transient receptor potential cation channel subfamily A member 1 (TRPA1) channel. Activation of the TRPA1 channel led to increased intracellular calcium, peroxisome proliferator-activated receptor gamma (PPARγ) upregulation, and the subsequent downregulation of the specificity protein-1 (SP-1)/sterol regulatory element-binding protein-2 (SREBP-2)/Niemann-Pick C1-like 1 (NPC1L1) signaling pathway-related proteins, and finally reduced cholesterol absorption in Caco-2 cells. CONCLUSIONS: Cur inhibits cell proliferation and reduces cholesterol absorption in Caco-2 cells through the Ca2+/PPARγ/SP-1/SREBP-2/NPC1L1 signaling by activating the TRPA1 channel, suggesting that Cur can be used as a dietary supplement for the primary prevention of CRC. In Caco-2 cells, Cur first stimulates calcium influx by activating the TRPA1 channel, further upregulates PPARγ and downregulates SP-1/SREBP-2/NPC1L1 signaling pathway, and finally inhibits the absorption of cholesterol. TRPA1, transient receptor potential cation channel subfamily A member 1; NPC1L1, Niemann-Pick C1-like 1; PPARγ, peroxisome proliferator-activated receptor gamma; SP-1, specificity protein-1; SREBP-2, sterol regulatory element-binding protein-2; Cur, curcumin.

Anti-Inflammatory Effects of Cannabigerol in Rheumatoid Arthritis Synovial Fibroblasts and Peripheral Blood Mononuclear Cell Cultures Are Partly Mediated by TRPA1.

Since its medical legalization, cannabis preparations containing the major phytocannabinoids (cannabidiol (CBD) and δ9-tetrahydrocannabinol (THC)) have been used by patients with rheumatoid arthritis (RA) to alleviate pain and inflammation. However, minor cannabinoids such as cannabigerol (CBG) also demonstrated anti-inflammatory properties, but due to the lack of studies, they are not widely used. CBG binds several cellular target proteins such as cannabinoid and α2-adrenergic receptors, but it also ligates several members of the transient potential receptor (TRP) family with TRPA1 being the main target. TRPA1 is not only involved in nnociception, but it also protects cells from apoptosis under oxidative stress conditions. Therefore, modulation of TRPA1 signaling by CBG might be used to modulate disease activity in RA as this autoimmune disease is accompanied by oxidative stress and subsequent activation of pro-inflammatory pathways. Rheumatoid synovial fibroblasts (RASF) were stimulated or not with tumor necrosis factor (TNF) for 72 h to induce TRPA1 protein. CBG increased intracellular calcium levels in TNF-stimulated RASF but not unstimulated RASF in a TRPA1-dependent manner. In addition, PoPo3 uptake, a surrogate marker for drug uptake, was enhanced by CBG. RASF cell viability, IL-6 and IL-8 production were decreased by CBG. In peripheral blood mononuclear cell cultures (PBMC) alone or together with RASF, CBG-modulated interleukin (IL)-6, IL-10, TNF and immunoglobulin M and G production which was dependent on activation stimulus (T cell-dependent or independent). However, effects on PBMCs were only partially mediated by TRPA1 as the antagonist A967079 did inhibit some but not all effects of CBG on cytokine production. In contrast, TRPA1 antagonism even enhanced the inhibitory effects of CBG on immunoglobulin production. CBG showed broad anti-inflammatory effects in isolated RASF, PBMC and PBMC/RASF co-cultures. As CBG is non-psychotropic, it might be used as add-on therapy in RA to reduce IL-6 and autoantibody levels.

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.

Paclitaxel Regulates TRPA1 Function and Expression Through PKA and PKC.

Paclitaxel (PTX) is a frequently used anticancer drug that causes peripheral neuropathy. Transient receptor potential ankyrin 1 (TRPA1), a plasma membrane calcium channel, has been associated with PTX toxicity and with other chemotherapy agents such as oxaliplatin and vincristine. However, the effect of PTX on the functional expression and calcium currents of TRPA1 has not been determined. The present study shows the effect of PTX on TRPA1 activity in a neuronal cell line (SH-SY5Y). The effect of PTX on the expression of TRPA1 was assessed through quantitative PCR and Western blot analyses to determine the relative mRNA and protein expression levels. To assess the effect on calcium flux and currents, cells were exposed to PTX; simultaneously, a specific agonist and antagonist of TRPA1 were added to evaluate the differential response in exposed versus control cells. To assess the effect of PKA, PKC and PI3K on PTX-induced TRPA1 increased activity, selective inhibitors were added to these previous experiments. PTX increased the mRNA and protein expression of TRPA1 as well as the TRPA1-mediated Ca2+ currents and intracellular Ca2+ concentrations. This effect was dependent on AITC (a selective specific agonist) and was abolished with HC-030031 (a selective specific antagonist). The inhibition of PKA and PKC reduced the effect of PTX on the functional expression of TRPA1, whereas the inhibition of PI3K had no effects. PTX-induced neuropathy involves TRPA1 activity through an increase in functional expression and is regulated by PKA and PKC signaling. These findings support the role of the TRPA1 channel in the mechanisms altered by PTX, which can be involved in the process that lead to chemotherapy-induced neuropathy.

Ectopic endometriosis in the pelvic cavity evokes bladder hypersensitivity via transient receptor potential ankyrin 1 hyperexpression in rats.

INTRODUCTION AND HYPOTHESIS: In women with chronic pelvic pain (CPP), interstitial cystitis/bladder pain syndrome (IC/BPS) and endometriosis frequently coexist. The mechanism of these diseases coexisting is explained by cross-sensitization between endometriosis and IC/BPS. The overlapped symptoms may be related to cross-sensitization with transient receptor potential vanilloid 1 (TRPV1) and/or transient receptor potential ankyrin 1 (TRPA1) hyperexpression. This study was aimed at exploring whether bladder hypersensitivity is evoked in the surgically induced ectopic endometriosis rat and whether TRPV1 and/or TRPA1 play a vital role. METHODS: A total of 63 Sprague-Dawley female rats were divided into two groups, 39 for physiological examination and 24 for molecular analysis. Surgical induction of ectopic endometriosis (ENDO, n=27), surgical sham treatment (n=18), and treatment for endometriosis by GnRH analog (ENDO-G) (n=18) were performed. Bladder function was investigated by cystometry (for TRPV1 in the sham [n=6] and ENDO [n=9] groups and for TRPA1 in the sham [n=6], ENDO [n=9], and ENDO+G [n=9] groups), and TRPV1 and TRPA1 mRNA expressions were measured using real-time qPCR in the bladder and dorsal root ganglia (DRGs). RESULTS: On cystometry, the relative intercontraction interval (ICI) after/before resiniferatoxin (RTx; TRPV1 activator) infusion to the bladder showed no significant difference between the two groups, whereas relative ICI after/before allyl isothiocyanate (AITC; TRPA1 activator) infusion was significantly lower in the ENDO group than in the sham group. TRPA1 mRNA expression in the bladder and L5 DRG was considerably higher in the ENDO group than in the sham group on real-time qPCR. TRPA1 mRNA hyperexpression and bladder hypersensitivity after AITC infusion were reduced in the ENDO-G group. CONCLUSIONS: Bladder cross-sensitization in ENDO rats occurs in association with hyperexpression of TRPA1 at both the DRG and the bladder mucosa. This can be understood by the "cross-sensitization of endometriosis to bladder" theory explaining overlapping symptoms among BPS/IC and ectopic endometriosis.