TRPM7 Expression Research Articles

Transient Receptor Potential Melastatin 8 Contributes to Cystitis-Induced Neuronal Sprouting and Pain Hypersensitivity Through AKT/mTOR Signaling Pathway in Interstitial Cystitis/Bladder Pain Syndrome.

The aim of this study was to investigate the mechanism of TRPM8 in neuroproliferation and pain, as well as the relevance of the Akt/mTOR signaling pathway in mice with IC/BPS. The model of IC/BPS was established in wild and TRPM8-/- mice. The mechanical sensitivity was measured. The number of neurite segments, length of neurites, and density of neurites were all counted. IL-6 and norepinephrine levels were detected by ELISA, Western blot was used to detect protein levels of TRPM8, Akt, p-Akt, mTOR, p-mTOR. Immunofluorescence was used to detect TRPM8 expression and distribution in neurites, neurons, and sensory nerves in mouse bladder tissue. Pain threshold in the IC/BPS group was decreased, and neurite segments, length, and density were all significantly enhanced when compared to the control group. The parameters in the IC/BPS model + Menthol group were more statistically significant. Neurite number and density were lower in TRPM8 knockout-model mice than in IC/BPS model mice. The expression of TRPM8 and the ratios of p-Akt/Akt and p-mTOR/mTOR rose in the IC/BPS model group. In TRPM8 knockout-model mice, the ratios of p-Akt/Akt and p-mTOR/mTOR were not substantially different from those in the control group. TRPM8 knockout-model mice had considerably lower levels of serum IL-6 and urine norepinephrine than IC/BPS model mice. TRPM8 can induce pain hypersensitivity and sensory nerve proliferation by activating Akt/mTOR pathway and raising the expression of IL-6 and norepinephrine in IC/BPS models. These findings offer new perspectives on IC/BPS treatment.

Novel apocynin regulates TRPV1 activity in the trigeminal system and controls pain in a temporomandibular joint neurogenic model

ObjectiveHerein, we investigate the potential analgesic effect of a newly synthesized chalcone-derived apocynin in a neurogenic pain model. MethodsMolecular docking was used to foretell the apocynin binding features and dynamics with the TRPV1 channel, and the activity was tested in vitro, using transfected HEK 293T cells with the rat TRPV1 receptor. The analgesic effect of apocynin was investigated using a capsaicin-induced pain model. The expression of TRPV1, TRPA1, TRPM8, and MAPKs was assessed by electrophoresis, and immunosorbent assays were performed to quantify the neurotransmitters Substance P, Glutamate, and CGRP. A survival assay using Galleria mellonella was carried out to determine the toxicity. ResultsWe observed that apocynin exhibits greater thermodynamic stability. Upon apocynin ligand binding, it changes the electrostatic potential for a predominantly electronegative state in the interior and neutral in its external vanilloid pocket. Treatment of apocynin induces antinociceptive effects against the noxious challenge of capsaicin. Histologically, apocynin decreased the number of TRPV1+ immunopositive cells. Electrophoresis showed reduced phosphorylation of p44/42 (ERK1/2) and decreased protein levels of substance P, and CGRP. In the survival assay, apocynin showed low toxicity. ConclusionsIn conclusion, we provide proof-of-principles that the newly synthesized apocynin compound effectively prevented nociception in a neurogenic model of orofacial pain.

TNF inhibitors target a mevalonate metabolite/TRPM2/calcium signaling axis in neutrophils to dampen vasculitis in Behçet’s disease

TNF inhibitors have been used to treat autoimmune and autoinflammatory diseases. Here we report an unexpected mechanism underlying the therapeutic effects of TNF inhibitors in Behçet’s disease (BD), an autoimmune inflammatory disorder. Using serum metabolomics and peripheral immunocyte transcriptomics, we find that polymorphonuclear neutrophil (PMN) from patients with BD (BD-PMN) has dysregulated mevalonate pathway and subsequently increased farnesyl pyrophosphate (FPP) levels. Mechanistically, FPP induces TRPM2-calcium signaling for neutrophil extracellular trap (NET) and proinflammatory cytokine productions, leading to vascular endothelial inflammation and damage. TNF, but not IL-1β, IL-6, IL-18, or IFN-γ, upregulates TRPM2 expression on BD-PMN, while TNF inhibitors have opposite effects. Results from mice with PMN-specific FPP synthetase or TRPM2 deficiency show reduced experimental vasculitis. Meanwhile, analyses of public datasets correlate increased TRPM2 expressions with the clinical benefits of TNF inhibitors. Our results thus implicate FPP-TRPM2-TNF/NETs feedback loops for inflammation aggravation, and novel insights for TNF inhibitor therapies on BD.

Effects of prolonged cold stress on vascular function in guinea pigs with atherosclerosis.

This study explored the effects of long-term cold stress on aortic vascular function in guinea pigs. Research Methods: Hartley guinea pigs (n=32) were divided into following groups: atherosclerosis (AS), cold stress (CS), and menthol-stimulated (M) and control (C). On days 1, 15, 30, 45, and 60, guinea pigs in the AS, CS, and M groups were intraperitoneally injected with bovine serum albumin. The C group was provided with maintenance feed and room-temperature water. The AS group was provided with a high-fat diet and room-temperature water. The CS group was maintained in a refrigerator at 4 °C，while providing a high-fat diet and iced water. The M group was administered menthol solution, and provided with a high-fat diet and room-temperature water. The modeling period lasted for 120 days. On day 121, abdominal aortic sera and aortic samples were obtained after intraperitoneal injection of sodium pentobarbital. Blood rheology tests were conducted to assess blood adhesion, biochemical tests to assess lipid levels, and enzyme-linked immunosorbent assays to detect serum nuclear factor-κB, tumor necrosis factor-α, and interleukin-1β, and endothelial nitric oxide (NO) synthase, NO, and endothelin-1(ET-1) in aortic tissue. Hematoxylin and eosin and Oil Red O staining were used to examine pathologic changes in the aorta, western blotting to detect TRPM8 and PKG protein expression, qPCR was used to measure VCAM-1 mRNA expression level. Research findings: Prolonged exposure to cold stress exacerbated lipid-metabolism disorders in guinea pigs fed a high-fat diet, increased aortic vascular cell adhesion, and exacerbated vascular inflammation, leading to endothelial injury, ultimately worsening pathologic changes associated with aortic atherosclerosis.

Evaluation of TRPM2 Channel-Mediated Autophagic Signaling Pathway in Hippocampus and Cortex Tissues of Rat Offspring Following Prenatal Exposure to Elevated Alcohol Levels.

Fetal alcohol syndrome (FAS) can occur because of high amount of alcohol intake during pregnancy and is characterized by both physical and neurological problems. Children diagnosed with FAS have difficulties in learning, memory, and coordination. Hippocampus has a major role in memory and learning. We aimed to determine whether alcohol exposure during pregnancy had any effect on offspring by evaluating learning ability as well as oxidative stress and autophagy in the hippocampus and cortex tissues of litters. Attention was also paid to sex differences. To do so, TRPM2, Beclin1, p62, LC3B, IBA1, parvalbumin, GAD65, and mGluR5 expression levels were evaluated by immunohistochemistry. Lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels, as well as total oxidant (TOS) and total antioxidant (TAS) status were determined by ELISA. Learning experiments were evaluated by the Morris water maze (MWM) test. Our findings demonstrated that IBA1, LC3B, GAD65, and mGluR5 expression levels were higher in female rats of the chronic alcohol exposure (CAE) model. Our IHC results revealed that TRPM2 expression levels were significantly increased in both males and females in the CAE group. Likewise, TAS was lower, and TOS was higher in CAE animals. Moreover, MWM outcomes supported a learning deficiency in CAE litters compared to controls and indicated that female offspring outperformed males in learning experiments. Therefore, our results revealed the detrimental effects of alcohol exposure during pregnancy on autophagy signaling in the hippocampus and cortex tissue of litters, which could affect the learning ability of animals.

TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway.

Hepatocellular carcinoma (HCC) is a malignant tumour associated with high morbidity and mortality rates worldwide. Recently, TRPM8 was reported to play an important role in tumour progression. However, the precise role of TRPM8 in HCC remains unclear. In this study, we explored the expression levels, molecular functions and underlying mechanisms of TRPM8 in HCC. Tissue samples were used to analyse the expression of TRPM8 to assess its diagnostic value for prognosis. Cell Counting Kit-8, EdU and colony formation assays were performed to evaluate the effects of TRPM8 on cell proliferation, whereas the Transwell assay was used to assess cell migration and invasion. The role of TRPM8 invivo was evaluated using a mouse subcutaneous xenograft tumour model. We performed PPI network analyses to understand the possible mechanisms of TRPM8 action. TRPM8 expression was decreased in HCC tissues and was correlated with histological grade and poor patient prognosis. Functionally, TRPM8 repressed the proliferation and metastasis of HCC cells both invitro and invivo by modulating the RTP3/STAT3 signalling pathway. Our findings underscore the critical role of the TRPM8-RTP3-STAT3 axis in maintaining the malignant progression of HCC. Moreover, our study demonstrates that AD80 is involved in anti-tumour processes by upregulating the expression of TRPM8.

The neurotrophic factor artemin and its receptor GFRα3 mediate migraine-like pain via the ion channel TRPM8.

Migraine has a strong genetic foundation, including both monogenic and polygenic types. The former are rare, with most migraine considered polygenic, supported by genome-wide association studies (GWAS) identifying numerous genetic variants associated with migraine risk. Surprisingly, some of the most common mutations are associated with TRPM8, a non-selective cation channel that is the primary sensor of cold temperatures in primary afferent neurons of the somatosensory system. However, it is unlikely that the temperature sensitivity of TRPM8 underlies its role in migraine pathogenesis. To define the basis of the channel's involvement, we reasoned that cellular processes that increase cold sensitivity in the skin, such as the neurotrophic factor artemin, via its receptor GFRα3, also mediate TRPM8-associated migraine-like pain in the meninges. To investigate the role of artemin and GFRα3 in preclinical rodent migraine models, we infused nitroglycerin acutely and chronically, and measured changes in periorbital and hind paw mechanical sensitivity in male and female mice lacking GFRα3, after neutralization of free artemin with specific monoclonal antibodies, or by systemic treatment with a TRPM8-specific antagonist. Further, in wildtypes and mice lacking either GFRα3 or TRPM8, we tested the effects of supradural infusions of a mix of inflammatory mediators, artemin, and a TRPM8-specific agonist on migraine-related pain in mice. We find that mechanical allodynia induced by systemic nitroglycerin, or supradural infusion of inflammatory mediators, involves GFRα3. In addition, neutralization of circulating artemin reduces the nitroglycerin phenotype, demonstrating the importance of this neurotrophic pathway. Further, we show TRPM8 expression in the meninges and that direct supradural infusion of either a TRPM8-specific agonist or artemin itself produces mechanical allodynia, the latter dependent on TRPM8 and ameliorated by concurrent treatment with sumatriptan. These results indicate that neuroinflammatory events in the meninges can produce migraine-like pain in mice via artemin and GFRα3, likely acting upstream of TRPM8, providing a novel pathway that may contribute to migraine pathogenesis.

Dynamic Transcriptome Profile Analysis of Mechanisms Related to Melanin Deposition in Chicken Muscle Development.

The pectoral muscle is an important component of skeletal muscle. The blackness of pectoral muscles can directly affect the economic value of black-boned chickens. Although the genes associated with melanogenesis in mammals and birds have been thoroughly investigated, only little is known about the key genes involved in muscle hyperpigmentation during embryonic development. Here, we analyzed melanin deposition patterns in the pectoral muscle of Yugan black-boned chickens and compared differentially expressed genes (DEGs) between the muscles of Wenchang (non-black-boned chickens) and Yugan black-boned chickens on embryonic days 9, 13, 17, and 21. Melanin pigments were found to gradually accumulate in the muscle fibers over time. Using RNA-seq, there were 40, 97, 169, and 94 genes were identified as DEGs, respectively, between Yugan black-boned chicken muscles and Wenchang chickens at embryonic day 9, 13, 17, and 21 stages (fold change ≥2.0, false discovery rate (FDR) < 0.05). Thirteen DEGs, such as MSTRG.720, EDNRB2, TYRP1, and DCT, were commonly identified among the time points observed. These DEGs were mainly involved in pigmentation, melanin biosynthetic and metabolic processes, and secondary metabolite biosynthetic processes. Pathway analysis of the DEGs revealed that they were mainly associated with melanogenesis and tyrosine metabolism. Moreover, weighted gene co-expression network analysis (WGCNA) was used to detect core modules and central genes related to melanogenesis in the muscles of black-boned chickens. A total of 24 modules were identified. Correlation analysis indicated that one of them (the orange module) was positively correlated with muscle pigmentation traits (r > 0.8 and p < 0.001). Correlations between gene expression and L* values of the breast muscle were investigated in Yugan and Taihe black-boned chickens after hatching. The results confirmed that EDNRB2, GPNMB, TRPM1, TYR, and DCT expression levels were significantly associated with L* values (p < 0.01) in black-boned chickens (p < 0.05). Our results suggest that EDNRB2, GPNMB, TRPM1, TYR, and DCT are the essential genes regulating melanin deposition in the breast muscle of black-boned chickens. MSTRG.720 is a potential candidate gene involved in melanin deposition in the breast muscles of Yugan black-boned chickens.

TRPM2-AS promotes ovarian cancer cell proliferation and inhibits cell apoptosis by upregulating the nearby gene TRPM2 via miR-6764-5p

Ovarian cancer (OC) becomes a fatal gynecologic malignant cancer in females worldwide. Target therapy is a promising therapeutical choice for patients with OC, and identifying biomarkers and exploring molecular mechanisms are necessary. In this study, the functions and mechanism of long noncoding RNA transient receptor potential cation channel subfamily M member 2 antisense RNA (TRPM2-AS) in OC were explored. TRPM2-AS expression in OC cells was analyzed utilizing reverse transcription quantitative polymerase chain reaction (RT-qPCR). Cell counting kit-8 (CCK-8) and colony forming assays were carried out to explore the influence of TRPM2-AS on OC cell viability and proliferation. Cell apoptosis was detected using TdT-mediated dUTP Nick-End labeling (TUNEL) and flow cytometry analysis. Protein expression of apoptotic markers was subjected to western blotting. RNA pulldown or luciferase reporter assays were applied to explore the interaction between TRPM2-AS and miR-6764-5p or the binding of miR-6764-5p and TRPM2. The results showed that TRPM2-AS is highly expressed in OC cells and was mainly localized in cytoplasm. TRPM2-AS depletion suppressed OC cell viability and proliferation while increasing cell apoptotic rate. TRPM2 displayed a high level in OC cells and was positively regulated by TRPM2-AS. TRPM2-AS interacted with miR-6764-5p and thereby upregulated TRPM2 expression. In addition, TRPM2 overexpression reversed the repressive impact of TRPM2-AS depletion on malignant OC cellular process. In conclusion, TRPM2-AS promotes OC cell viability and proliferation while enhancing cell apoptosis through interaction with miR-6764-5p to regulate TRPM2 level.

Fentanyl induces analgesic effect through miR-381-3p/TRPM7 when combined with bupivacaine in subarachnoid injection

Fentanyl combined with bupivacaine in subarachnoid anesthesia exerts a strong synergistic analgesic effect, extending the duration of analgesia. However, the mechanism of enhanced analgesic effect of fentanyl remains elusive. The present study investigated the potential mechanism of the analgesic effect of fentanyl when combined with bupivacaine. The subarachnoid injection (SI) rat model was employed, and SI of fentanyl or/and bupivacaine was used to investigate their analgesic effect. Dorsal root ganglion (DRG)’ RNA sequencing (RNA-Seq) and bioinformatics analysis were performed to evaluate the downstream mechanisms of MicroRNAs (miRNAs). Further validation tests included RT-PCR, Western blot, and immunofluorescence. A single SI of fentanyl or bupivacaine decreased the positive responses to stimulation when used alone or in combination. RNA-seq results revealed that miR-381-3p played a role in the fentanyl-driven promotion of analgesia. Bioinformatics analysis and dual-luciferase reporter identified TRPM7 as a direct downstream target gene of miR-381-3p. In vitro, overexpression of miR-381-3p could further block fentanyl-induced expression of TRPM7, p-ERK1/2, CGRP, and SP. In addition, antagomir-381-3p reversed the inhibitory effect of fentanyl on the expression of TRPM7, p-ERK1/2, CGRP, and SP, in vivo; however, TRPM7 siRNA rescued the effect of antagomir-381-3p. In conclusion, fentanyl inhibits p-ERK by targeting TRPM7 via miR-381-3p, lowering the production of CGRP and SP, and ultimately inducing analgesic effects.

Ginger Extract and Omega-3 Fatty Acids Supplementation: A Promising Strategy to Improve Diabetic Cardiomyopathy.

Diabetic cardiomyopathy may result from the overproduction of ROS, TRPM2 and TRPV2. Moreover, the therapeutic role of ginger, omega-3 fatty acids, and their combinations on the expression of TRPM2 and TRPV2 and their relationship with apoptosis, inflammation, and oxidative damage in heart tissue of rats with type 2 diabetes have not yet been determined. Therefore, this study aimed to investigate the therapeutic effects of ginger and omega-3 fatty acids on diabetic cardiomyopathy by evaluating the cardiac gene expression of TRPM2 and TRPV2, oxidative damage, inflammation, and apoptosis in male rats. Ninety adult male Wistar rats were equally divided into nine control, diabetes, and treated diabetes groups. Ginger extract (100 mg/kg) and omega-3 fatty acids (50, 100, and 150 mg/kg) were orally administrated in diabetic rats for 6 weeks. Type 2 diabetes was induced by feeding a high-fat diet and a single dose of STZ (40 mg/kg). Glucose, cardiac troponin I (cTnI), lipid profile, insulin in serum, and TNF-alpha IL-6, SOD, MDA, and CAT in the left ventricle of the heart were measured. The cardiac expression of TRPM2, TRPV2, NF-kappaB, Bcl2, Bax, Cas-3, and Nrf-2 genes was also measured in the left ventricle of the heart. An electrocardiogram (ECG) was continuously recorded to monitor arrhythmia at the end of the course. The serum levels of cTnI, glucose, insulin, and lipid profile, and the cardiac levels of MDA, IL-6, and TNF-alpha increased in the diabetic group compared to the control group (p<0.05). Moreover, the cardiac levels of SOD and CAT decreased in the diabetic group compared to the control group (p<0.05). The cardiac expression of TRPM2, TRPV2, NF-kappaB, Bax, and Cas-3 increased and Bcl2 and Nrf-2 expression decreased in the diabetic group compared to the control group (p<0.05). However, simultaneous and separate treatment with ginger extract and omega-3 fatty acids (50, 100, and 150 mg/kg) could significantly moderate these changes (p<0.05). The results also showed that the simultaneous treatment of ginger extract and different doses of omega-3 fatty acids have improved therapeutic effects than their individual treatments (p<0.05). It can be concluded that ginger and omega-3 fatty acids showed protective effects against diabetic cardiomyopathy by inhibiting inflammation, apoptosis and oxidative damage of the heart and reducing blood glucose and cardiac expression of TRPM2 and TRPV2. Combining ginger and omega-3 in the diet may provide a natural approach to reducing the risk or progression of diabetic cardiomyopathy while preserving heart structure and function.

Neuregulin 1 mitigated prolactin deficiency through enhancing TRPM8 signaling under the influence of melatonin in senescent pituitary lactotrophs

The age-related alterations in pituitary function, including changes in prolactin (PRL) production contributes to the systemic susceptibility to age-related diseases. Our previous research has shown the involvement of Nrg1 in regulating the expression and secretion of PRL. However, the precise role of Nrg1 in mitigating the senescence of pituitary lactotrophs and the underlying mechanisms are yet to be comprehended. Here, data from the GEPIA database was used to evaluate the association between transient receptor potential cation channel subfamily M member 8 (TRPM8) and PRL in normal human pituitary tissues, followed by immunofluorescence verification using a human pituitary tissue microarray. TRPM8 levels showed a significant positive association with PRL expression in normal human pituitary tissues, and both TRPM8 and PRL levels declined during aging, suggesting that TRPM8 may regulate pituitary aging by affecting PRL production. It was also found that treatment with exogenous neuregulin 1 (Nrg1) markedly delayed the senescence of GH3 cells (rat lactotroph cell line) generated by D-galactose (D-gal). In addition, melatonin reduced the levels of senescence-related markers in senescent pituitary cells by promoting Nrg1 / ErbB4 signaling, stimulating PRL expression and secretion. Further investigation showed that Nrg1 attenuated senescence in pituitary cells by increasing TRPM8 expression. Downregulation of TRPM8 activation eliminated Nrg1-mediated amelioration of pituitary cell senescence. These findings demonstrate the critical function of Nrg1 / ErbB signaling in delaying pituitary lactotroph cell senescence and enhancing PRL production via promoting TRPM8 expression under the modulation of melatonin.

TRPM4 mRNA stabilization by METTL3-mediated m6A modification promotes calcific aortic valve inflammation

BackgroundTransient receptor potential melastatin 4 (TRPM4) affects immune responses by regulating calcium homeostasis, but its role in calcific aortic valve inflammation remains unclear. This study aimed to assess the expression and function of TRPM4 in patients with or without calcific aortic valve disease (CAVD). MethodsThe mRNA and protein expression levels of TRPM4 and related factors in calcified and noncalcified tissues were measured using qRT-PCR and Western blot. The proteins interacting with TRPM4 were confirmed by RNA pull-down and RNA immunoprecipitation assays. Dual-Luciferase Reporter Assay was performed to confirm the m6A site of TRPM4. ResultsThe mRNA expression levels of TRPM4, TLR4, IL-6, MCP-1, TNF-α, and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues, and TRPM4 was significantly positively correlated with inflammation-related factors. The protein expression level of TRPM4, TLR4 and NF-κB p65 were significantly higher in calcified aortic valve tissues than in noncalcified tissues. N6-methyladenosine (m6A) modification of TRPM4 mRNA by METTL3-YTHDF1 up-regulated its expression in CAVD. And TRPM4 promoted the level of inflammation via activation of the JNK-MAPK signaling pathway, after knockdown TRPM4, the production of proinflammatory cytokines was significantly suppressed. ConclusionThe results indicate the pivotal role of TRPM4 in CAVD and highlight METTL3-mediated m6A modification of TRPM4 in promoting inflammation through JNK-MAPK signaling pathway. This work provides potential therapeutic strategy to impede inflammation in CAVD.

TRPA1 up-regulation mediates oxidative stress in a pulpitis model in vitro.

Pulpitis is associated with tooth hypersensitivity and results in pulpal damage. Thermosensitive transient receptor potential (TRP) ion channels expressed in the dental pulp may be key transducers of inflammation and nociception. We aimed at investigating the expression and role of thermo-TRPs in primary human dental pulp cells (hDPCs) in normal and inflammatory conditions. Inflammatory conditions were induced in hDPC cultures by applying polyinosinic:polycytidylic acid (poly(I:C)). Gene expression and pro-inflammatory cytokine release were measured by RT-qPCR and ELISA. Functions of TRPA1 channels were investigated by monitoring changes in intracellular Ca2+ concentration. Mitochondrial superoxide production was measured using a fluorescent substrate. Cellular viability was assessed by measuring the activity of mitochondrial dehydrogenases and cytoplasmic esterases. TRPA1 activity was modified by agonists, antagonists, and gene silencing. Transcripts of TRPV1, TRPV2, TRPV4, TRPC5, and TRPA1 were highly expressed in control hDPCs, whereas TRPV3, TRPM2, and TRPM3 expressions were much lower, and TRPM8 was not detected. Poly(I:C) markedly up-regulated TRPA1 but not other thermo-TRPs. TRPA1 agonist-induced Ca2+ signals were highly potentiated in inflammatory conditions. Poly(I:C)-treated cells displayed increased Ca2+ responses to H2O2, which was abolished by TRPA1 antagonists. Inflammatory conditions induced oxidative stress, stimulated mitochondrial superoxide production, resulted in mitochondrial damage, and decreased cellular viability of hDPCs. This inflammatory cellular damage was partly prevented by the co-application of TRPA1 antagonist or TRPA1 silencing. Pharmacological blockade of TRPA1 channels may be a promising therapeutic approach to alleviate pulpitis and inflammation-associated pulpal damage.

Investigation of Some Ion Channel Expressions in Cochlear Nucleus of Tinnitus Induced Rats

Aim: The aim of this study is to gain a better understanding of how certain ion channels play a role in the molecular mechanisms of salicylate- and noise-induced tinnitus. Method: The present study was conducted on thirty-two, 4-month-old, male Wistar Albino rats. Rats were equally divided into four groups; two experimental groups and two control groups. The assessment of tinnitus was based on a behavioral test which was modified from the conditional suppression method. Tinnitus was induced by sodium salicylate administration and noise exposure in rats in which the suppression ratios were zero (0). All animals in both experimental and control groups were decapitated in deep anaesthesia for 2 h after salicylate or saline administration and noise exposure, consecutively. Tissues from the left and right cochlear nucleus were dissected immediately in ice-cold RNA later (Invitrogen). Before reverse transcription, the RNA pools were arranged. Quantitative changes in HCN1, HCN2, HCN4, SCN1A, SCN2A1, SCN3A, TRPM2, TRPM7 and GAPDH mRNA expressions in the cochlear nucleus in both experimental and control groups were examined by quantitative real-time PCR method. Statistical data were analysed using the SPSS 21 program (Version 21.0, SPSS Inc., Chicago, IL, USA) with the Kruskal-Wallis and Mann-Whitney U tests. Results: Fold changes in the expression levels of SCNA1, SCN2A1, SCN3A, TRPM2, TRPM7, CACNA1B, HCN1, HCN2 and HCN4 genes in both salicylate-induces tinnitus (SAT) and noise-induced tinnitus (NT) groups compared with the control group. According to these data, it is seen that the mRNA levels of all genes are lower in the cochlear nucleus area of the rats in both SAT and NT groups than in the control group. Considering each of these genes in NT group: SCNA1, SCN3A, TRPM7 genes slightly decreased; SCN2A1, TRPM2, HCN1 and HCN4 genes slightly increased compared with the SAT group. For HCN2 gene fold changes were nearly the same in the NT and SAT groups. Conclusion: The findings of this study suggest that tinnitus generation may be closely related to alterations in several key ion channel families activity including voltage-gated calcium channels, hyperpolarization-activated cyclic nucleotide–gated (HCN) channels, transient receptor potential (TRP) channels, voltage-gated sodium channels within the CN, specifically in response to salicylate-induced and noise-induced tinnitus models.

Thermosensitive TRP Channels Are Functionally Expressed and Influence the Lipogenesis in Human Meibomian Gland Cells.

While the involvement of thermosensitive transient receptor potential channels (TRPs) in dry eye disease (DED) has been known for years, their expression in the meibomian gland (MG) has never been investigated. This study aims to show their expression and involvement in the lipogenesis of the MG, providing a possible new drug target in the treatment of DED. Our RT-PCR, Western blot and immunofluorescence analysis showed the expression of TRPV1, TRPV3, TRPV4 and TRPM8 in the MG at the gene and the protein level. RT-PCR also showed gene expression of TRPV2 but not TRPA1. Calcium imaging and planar patch-clamping performed on an immortalized human meibomian gland epithelial cell line (hMGECs) demonstrated increasing whole-cell currents after the application of capsaicin (TRPV1) or icilin (TRPM8). Decreasing whole-cell currents could be registered after the application of AMG9810 (TRPV1) or AMTB (TRPM8). Oil red O staining on hMGECs showed an increase in lipid expression after TRPV1 activation and a decrease after TRPM8 activation. We conclude that thermo-TRPs are expressed at the gene and the protein level in MGs. Moreover, TRPV1 and TRPM8's functional expression and their contribution to their lipid expression could be demonstrated. Therefore, TRPs are potential drug targets and their clinical relevance in the therapy of meibomian gland dysfunction requires further investigation.

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.

Anti-cancer agent piperlongumine is an inhibitor of transient receptor potential melastatin 7 channel in oral squamous cell carcinoma

ObjectivesTo elucidate the association between the anticancer activities of piperlongumine (PL) and its potential target, transient receptor potential melastatin 7 channel (TRPM7), in oral squamous cell carcinoma (OSCC). MethodsThe expression levels and electrical characteristics of TRPM7 as well as cell viability in response to various PL treatments were investigated in the OSCC cell line Cal27. ResultsPL treatment resulted in a concentration- and time-dependent reduction in TRPM7 mRNA and protein expression in Cal27 cells. Furthermore, PL treatment inhibited TRPM7-like rectifying currents in Cal27 cells; however, this inhibition was less effective than that of the TRPM7 antagonist waixenicin A. Rapid perfusion and washout experiments revealed an immediate inhibitory effect of PL on TRPM7-like currents. The antagonistic effect of PL occurred within 1 min and was not completely reversed following washout. Notably, the extracellular Ca2+ concentration still influenced PL-induced changes in the TRPM7-like current, indicating that PL can directly but gently antagonize the TRPM7 channel. Functional changes in TRPM7 correlated with the observed antiproliferative and cytotoxic effects of PL in Cal27 cells. ConclusionsThese findings suggest that PL exhibits potent inhibitory effects on TRPM7 and exerts its anti-cancer effects by downregulating TRPM7 expression and antagonizing channel currents.

Methyl Jasmonate-induced Increase in Intracellular Magnesium Promotes Apoptosis in Breast Cancer Cells.

Methyl jasmonate (MeJa) is a botanical stress hormone that serves as a defense mechanism to inhibit growth in stressed plants. It is well known that MeJa exhibits an anticancer effect by reducing intracellular ATP, activating reactive oxygen species (ROS) production, and promoting mitogen-activated protein kinase (MAPK) activity. Presently, no report has been published on MeJa-induced changes in intracellular Mg2+ concentration ([Mg2+]i), and TRPM7 as an Mg2+ transporter in cancer cells. Therefore, this study aimed to investigate the Mg2+ homeostatic changes and apoptotic effects following MeJa treatment using the MCF-7 human breast cancer cell line. The MTT assay was used to assess the cell viability and half-inhibitory concentration, microscopic two-photon excitation wavelength spectrophotometry was used to measure the [Mg2+]i, a luminescent assay determined intracellular ATP levels, western blot assay measured TRPM7 levels, antioxidant capacities, endoplasmic reticulum (ER) stress, and MAPK signaling pathways, while the fluorescence assay evaluated ROS concentrations and the cell apoptotic index. This study provides evidence that MeJa has an antiapoptotic effect on MCF-7 cells. The increase in [Mg2+]i led to decreased TRPM7 expression, which is related to elevated ROS production, in addition to elevated ER stress and MAPK signaling pathway activity and decreased ATP content. The increase in [Mg2+]i leads to decreased TRPM7 expression and may be the epicenter of MeJa-induced apoptotic cell death in MCF-7 cells.

Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer

Inflammation is a common condition of prostate tissue, whose impact on carcinogenesis is highly debated. Microbial colonization is a well-documented cause of a small percentage of prostatitis cases, but it remains unclear what underlies the majority of sterile inflammation reported. Here, androgen- independent fluctuations of PSA expression in prostate cells have lead us to identify a prominent function of the Transient Receptor Potential Cation Channel Subfamily M Member 8 (TRPM8) gene in sterile inflammation. Prostate cells secret TRPM8 RNA into extracellular vesicles (EVs), which primes TLR3/NF-kB-mediated inflammatory signaling after EV endocytosis by epithelial cancer cells. Furthermore, prostate cancer xenografts expressing a translation-defective form of TRPM8 RNA contain less collagen type I in the extracellular matrix, significantly more infiltrating NK cells, and larger necrotic areas as compared to control xenografts. These findings imply sustained, androgen-independent expression of TRPM8 constitutes as a promoter of anticancer innate immunity, which may constitute a clinically relevant condition affecting prostate cancer prognosis.