Electro-acupuncture modulated miR-214 expression to prevent chondrocyte apoptosis and reduce pain by targeting BAX and TRPV4 in osteoarthritis rats.

Osteoarthritis (OA) is a highly prevalent joint disorder characterized by progressive degeneration of articular cartilage, subchondral bone remodeling, osteophyte formation, synovial inflammation, and meniscal damage. Although the etiology of OA is multifactorial, pro-inflammatory processes appear to play a key role in disease pathogenesis. Previous studies indicate that electroacupuncture (EA) exerts chondroprotective, anti-inflammatory, and analgesic effects in preclinical models of OA, but the mechanisms underlying these potential therapeutic benefits remain incompletely defined. This study aimed to investigate the effects of EA on OA development in a rat model, as well as to explore associated molecular mechanisms modulated by EA treatment. Forty rats were divided into OA, EA, antagomiR-214, and control groups. Following intra-articular injection of monosodium iodoacetate to induce OA, EA and antagomiR-214 groups received daily EA stimulation at acupoints around the knee joint for 21 days. Functional pain behaviors and chondrocyte apoptosis were assessed as outcome measures. The expression of microRNA-214 (miR-214) and its downstream targets involved in apoptosis and nociception, BAX and TRPV4, were examined. Results demonstrated that EA treatment upregulated miR-214 expression in OA knee cartilage. By suppressing pro-apoptotic BAX and pro-nociceptive TRPV4, this EA-induced miR-214 upregulation ameliorated articular pain and prevented chondrocyte apoptosis. These findings suggested that miR-214 plays a key role mediating EA's therapeutic effects in OA pathophysiology, and represents a promising OA treatment target for modulation by acupuncture.

TRPV3 activation by different agonists accompanied by lipid dissociation from the vanilloid site.

TRPV3 represents both temperature- and ligand-activated transient receptor potential (TRP) channel. Physiologically relevant opening of TRPV3 channels by heat has been captured structurally, while opening by agonists has only been observed in structures of mutant channels. Here, we present cryo-EM structures that illuminate opening and inactivation of wild-type human TRPV3 in response to binding of two types of agonists: either the natural cannabinoid tetrahydrocannabivarin (THCV) or synthetic agonist 2-aminoethoxydiphenylborane (2-APB). We found that THCV binds to the vanilloid site, while 2-APB binds to the S1-S4 base and ARD-TMD linker sites. Despite binding to distally located sites, both agonists induce similar pore opening and cause dissociation of a lipid that occupies the vanilloid site in their absence. Our results uncover different but converging allosteric pathways through which small-molecule agonists activate TRPV3 and provide a framework for drug design and understanding the role of lipids in ion channel function.

Exploration and structure-activity relationship research of benzenesulfonamide derivatives as potent TRPV4 inhibitors for treating acute lung injury.

RN-9893, a TRPV4 antagonist identified by Renovis Inc., showcased notable inhibition of TRPV4 channels. This research involved synthesizing and evaluating three series of RN-9893 analogues for their TRPV4 inhibitory efficacy. Notably, compounds 1b and 1f displayed a 2.9 to 4.5-fold increase in inhibitory potency against TRPV4 (IC50 = 0.71 ± 0.21 µM and 0.46 ± 0.08 µM, respectively) in vitro, in comparison to RN-9893 (IC50 = 2.07 ± 0.90 µM). Both compounds also significantly outperformed RN-9893 in TRPV4 current inhibition rates (87.6 % and 83.2 % at 10 µM, against RN-9893's 49.4 %). For the first time, these RN-9893 analogues were profiled in an in vivo mouse model, where intraperitoneal injections of 1b or 1f at 10 mg/kg notably mitigated symptoms of acute lung injury induced by lipopolysaccharide (LPS). These outcomes indicate that compounds 1b and 1f are promising candidates for acute lung injury treatment.

FGF13 enhances the function of TRPV1 by stabilizing microtubules and regulates acute and chronic itch.

Itching is an aversive somatosensation that triggers the desire to scratch. Transient receptor potential (TRP) channel proteins are key players in acute and chronic itch. However, whether the modulatory effect of fibroblast growth factor 13 (FGF13) on acute and chronic itch is associated with TRP channel proteins is unclear. Here, we demonstrated that conditional knockout of Fgf13 in dorsal root ganglion neurons induced significant impairment in scratching behaviors in response to acute histamine-dependent and chronic dry skin itch models. Furthermore, FGF13 selectively regulated the function of the TRPV1, but not the TRPA1 channel on Ca2+ imaging and electrophysiological recordings, as demonstrated by a significant reduction in neuronal excitability and current density induced by TRPV1 channel activation, whereas TRPA1 channel activation had no effect. Changes in channel currents were also verified in HEK cell lines. Subsequently, we observed that selective modulation of TRPV1 by FGF13 required its microtubule-stabilizing effect. Furthermore, in FGF13 knockout mice, only the overexpression of FGF13 with a tubulin-binding domain could rescue TRP channel function and the impaired itch behavior. Our findings reveal a novel mechanism by which FGF13 is involved in TRPV1-dependent itch transduction and provide valuable clues for alleviating pathological itch syndrome.

Low-Intensity Extracorporeal Shock Wave Therapy Ameliorates Detrusor Hyperactivity with Impaired Contractility via Transient Potential Vanilloid Channels: A Rat Model for Ovarian Hormone Deficiency.

This study explores low-intensity extracorporeal shock wave therapy (LiESWT)'s efficacy in alleviating detrusor hyperactivity with impaired contractility (DHIC) induced by ovarian hormone deficiency (OHD) in ovariectomized rats. The rats were categorized into the following four groups: sham group; OVX group, subjected to bilateral ovariectomy (OVX) for 12 months to induce OHD; OVX + SW4 group, underwent OHD for 12 months followed by 4 weeks of weekly LiESWT; and OVX + SW8 group, underwent OHD for 12 months followed by 8 weeks of weekly LiESWT. Cystometrogram studies and voiding behavior tracing were used to identify the symptoms of DHIC. Muscle strip contractility was evaluated through electrical-field, carbachol, ATP, and KCl stimulations. Western blot and immunofluorescence analyses were performed to assess the expressions of various markers related to bladder dysfunction. The OVX rats exhibited significant bladder deterioration and overactivity, alleviated by LiESWT. LiESWT modified transient receptor potential vanilloid (TRPV) channel expression, regulating calcium concentration and enhancing bladder capacity. It also elevated endoplasmic reticulum (ER) stress proteins, influencing ER-related Ca2+ channels and receptors to modulate detrusor muscle contractility. OHD after 12 months led to neuronal degeneration and reduced TRPV1 and TRPV4 channel activation. LiESWT demonstrated potential in enhancing angiogenic remodeling, neurogenesis, and receptor response, ameliorating DHIC via TRPV channels and cellular signaling in the OHD-induced DHIC rat model.

The ion channel TRPV5 regulates B-cell signaling and activation.

Introduction: B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. Methods: We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. Results: We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Discussion: Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Transient receptor potential vanilloid 1 involved in the analgesic effects of total flavonoids extracted from Longxuejie ().

OBJECTIVE: To evaluate the analgesic effects of total flavonoids of Longxuejie (Resina Dracaenae Cochinchinensis) (TFDB) and explore the possible analgesic mechanism associated with transient receptor potential vanilloid 1 (TRPV1). METHODS: Whole-cell patch clamp technique was used to observe the effects of TFDB on capsaicin-induced TRPV1 currents. Rat experiments in vivo were used to observe the analgesic effects of TFDB. Western blot and immunofluorescence experiments were used to test the change of TRPV1 expression in DRG neurons induced by TFDB. RESULTS: Results showed that TFDB inhibited capsaicin-induced TRPV1 receptor currents in acutely isolated dorsal root ganglion (DRG) neurons of rats and the half inhibitory concentration was (16.7 ± 1.6) mg/L. TFDB (2-20 mg/kg) showed analgesic activity in the phase Ⅱ of formalin test and (0.02-2 mg per paw) reduced capsaicin-induced licking times of rats. TFDB (20 mg/kg) was fully efficacious on complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia and capsaicin could weaken the analgesic effects. The level of TRPV1 expressions of DRG neurons was also decreased in TFDB-treated CFA-inflammatory pain rats. CONCLUSION: All these results indicated that the analgesic effect of TFDB may contribute to their modulations on both function and expression of TRPV1 channels in DRG neurons.

Elucidation of the Role of TRPV1, VEGF-A, TXA2, Redox Homeostasis, and Inflammatory Cascades in Protection against Cold Injuries by Herbosomal-Loaded PEG-Poloxamer Topical Formulation.

High-altitude regions, cold deserts, permafrost regions, and the polar region have some of the severest cold conditions on earth and pose immense perils of cold injuries to exposed individuals. Accidental and unintended exposures to severe cold, either unintentionally or due to occupational risks, can greatly increase the risk of serious conditions including hypothermia, trench foot, and cold injuries like frostbite. Cold-induced vasoconstriction and intracellular/intravascular ice crystal formation lead to hypoxic conditions at the cellular level. The condition is exacerbated in individuals having inadequate and proper covering and layering, particularly when large area of the body are exposed to extremely cold environments. There is a paucity of preventive and therapeutic pharmacological modalities that have been explored for managing and treating cold injuries. Given this, an efficient modality that can potentiate the healing of frostbite was investigated by studying various complex pathophysiological changes that occur during severe cold injuries. In the current research, we report the effectiveness and healing properties of a standardized formulation, i.e., a herbosomal-loaded PEG-poloxamer topical formulation (n-HPTF), on frostbite. The intricate mechanistic pathways modulated by the novel formulation have been elucidated by studying the pathophysiological sequelae that occur following severe cold exposures leading to frostbite. The results indicate that n-HPTF ameliorates the outcome of frostbite, as it activates positive sensory nerves widely distributed in the epidermis transient receptor potential vanilloid 1 (TRPV1), significantly (p < 0.05) upregulates cytokeratin-14, promotes angiogenesis (VEGF-A), prominently represses the expression of thromboxane formation (TXA2), and significantly (p < 0.05) restores levels of enzymatic (glutathione reductase, superoxide dismutase, and catalase) and nonenzymatic antioxidants (glutathione). Additionally, n-HPTF attenuates oxidative stress and the expression of inflammatory proteins PGF-2α, NFκB-p65, TNF-α, IL-6, IL-1ß, malondialdehyde (MDA), advanced oxidative protein products (AOPP), and protein carbonylation (PCO). Masson's Trichrome staining showed that n-HPTF stimulates cellular proliferation, and increases collagen fiber deposition, which significantly (p < 0.05) promotes the healing of frostbitten tissue, as compared to control. We conclude that protection against severe cold injuries by n-HPTF is mediated via modulation of pathways involving TRPV1, VEGF-A, TXA2, redox homeostasis, and inflammatory cascades. The study is likely to have widespread implications for the prophylaxis and management of moderate-to-severe frostbite conditions.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

OBJECTIVE: This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. METHODS: The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 µM) or TRPV1 inhibitor capsazepine (1 µM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. RESULTS: After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. CONCLUSION: ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Calcitonin gene­related peptide alleviates hyperoxia­induced human alveolar cell injury via the CGRPR/TRPV1/Ca2+ axis.

Although exogenous calcitonin gene­related peptide (CGRP) protects against hyperoxia­induced lung injury (HILI), the underlying mechanisms remain unclear. The present study attempted to elucidate the molecular mechanism by which CGRP protects against hyperoxia­induced alveolar cell injury. Human alveolar A549 cells were treated with 95% hyperoxia to establish a hyperoxic cell injury model. ELISA was performed to detect the CGRP secretion. Immunofluorescence, quantitative (q)PCR, and western blotting were used to detect the expression and localization of CGRP receptor (CGRPR) and transient receptor potential vanilloid 1 (TRPV1). Cell counting kit­8 and flow cytometry were used to examine the proliferation and apoptosis of treated cells. Digital calcium imaging and patch clamp were used to analyze the changes in intracellular Ca2+ signaling and membrane currents induced by CGRP in A549 cells. The mRNA and protein expression levels of Cyclin D1, proliferating cell nuclear antigen (PCNA), Bcl­2 and Bax were detected by qPCR and western blotting. The expression levels of CGRPR and TRPV1 in A549 cells were significantly downregulated by hyperoxic treatment, but there was no significant difference in CGRP release between cells cultured under normal air and hyperoxic conditions. CGRP promoted cell proliferation and inhibited apoptosis in hyperoxia, but selective inhibitors of CGRPR and TRPV1 channels could effectively attenuate these effects; TRPV1 knockdown also attenuated this effect. CGRP induced Ca2+ entry via the TRPV1 channels and enhanced the membrane non­selective currents through TRPV1 channels. The CGRP­induced increase in intracellular Ca2+ was reduced by inhibiting the phospholipase C (PLC)/protein kinase C (PKC) pathway. Moreover, PLC and PKC inhibitors attenuated the effects of CGRP in promoting cell proliferation and inhibiting apoptosis. In conclusion, exogenous CGRP acted by inversely regulating the function of TRPV1 channels in alveolar cells. Importantly, CGRP protected alveolar cells from hyperoxia­induced injury via the CGRPR/TRPV1/Ca2+ axis, which may be a potential target for the prevention and treatment of the HILI.

Correlation of TRPA1 RNAscope and Agonist Responses.

The TRPA1 ion channel is a sensitive detector of reactive chemicals, found primarily on sensory neurons. The phenotype exhibited by mice lacking TRPA1 suggests its potential as a target for pharmacological intervention. Antibody-based detection for distribution analysis is a standard technique. In the case of TRPA1, however, there is no antibody with a plausible validation in knockout animals or functional studies, but many that have failed in this regard. To this end we employed the single molecule in situ hybridization technique RNAscope on sensory neurons immediately after detection of calcium responses to the TRPA1 agonist allyl isothiocyanate. There is a clearly positive correlation between TRPA1 calcium imaging and RNAscope detection (R = 0.43), although less than what might have been expected. Thus, the technique of choice should be carefully considered to suit the research question. The marginal correlation between TRPV1 RNAscope and the specific agonist capsaicin indicates that such validation is advisable for every RNAscope target. Given the recent description of a long-awaited TRPA1 reporter mouse, TRPA1 RNAscope detection might still have its use cases, for detection of RNA at particular sites, for example, defined structurally or by other molecular markers.

TRPV1-Dependent Antiproliferative Activity of Dioecious Maclura pomifera Extracts in Estrogen Receptor-Positive Breast Cancer Cell Lines Involves Multiple Apoptotic Pathways.

Globally, breast cancer is a significant cause of mortality. Recent research focused on identifying compounds regulating the transient receptor potential vanilloid 1 (TRPV1) ion channel activity for the possibility of developing cancer therapeutics. In this study, the antiproliferative properties and mechanisms of action through TRPV1 of Maclura pomifera, a dioecious tree native to the south-central USA, have been investigated. Male and female extracts of spring branch tissues and leaves (500 µg/mL) significantly reduced the viability of MCF-7 and T47D cells by 75-80%. M. pomifera extracts induced apoptosis by triggering intracellular calcium overload via TRPV1. Blocking TRPV1 with the capsazepine antagonist and pretreating cells with the BAPTA-AM chelator boosted cell viability, revealing that M. pomifera phytochemicals activate TRPV1. Both male and female M. pomifera extracts initiated apoptosis through multiple pathways, the mitochondrial, ERK-induced, and endoplasmic reticulum-stress-mediated apoptotic pathways, demonstrated by the expression of activated caspase 3, caspase 9, caspase 8, FADD, FAS, ATF4, and CHOP, the overexpression of phosphorylated PERK and ERK proteins, and the reduction of BCL-2 levels. In addition, AKT and pAKT protein expressions were reduced in female M. pomifera-treated cells, revealing that female plant extract also inhibits PI3K/Akt signaling pathways. These results suggest that phytochemicals in M. pomifera extracts could be promising for developing breast cancer therapeutics.

Gain-of-function mutations of TRPV4 acting in endothelial cells drive blood-CNS barrier breakdown and motor neuron degeneration in mice.

Blood-CNS barrier disruption is a hallmark of numerous neurological disorders, yet whether barrier breakdown is sufficient to trigger neurodegenerative disease remains unresolved. Therapeutic strategies to mitigate barrier hyperpermeability are also limited. Dominant missense mutations of the cation channel transient receptor potential vanilloid 4 (TRPV4) cause forms of hereditary motor neuron disease. To gain insights into the cellular basis of these disorders, we generated knock-in mouse models of TRPV4 channelopathy by introducing two disease-causing mutations (R269C and R232C) into the endogenous mouse Trpv4 gene. TRPV4 mutant mice exhibited weakness, early lethality, and regional motor neuron loss. Genetic deletion of the mutant Trpv4 allele from endothelial cells (but not neurons, glia, or muscle) rescued these phenotypes. Symptomatic mutant mice exhibited focal disruptions of blood-spinal cord barrier (BSCB) integrity, associated with a gain of function of mutant TRPV4 channel activity in neural vascular endothelial cells (NVECs) and alterations of NVEC tight junction structure. Systemic administration of a TRPV4-specific antagonist abrogated channel-mediated BSCB impairments and provided a marked phenotypic rescue of symptomatic mutant mice. Together, our findings show that mutant TRPV4 channels can drive motor neuron degeneration in a non-cell autonomous manner by precipitating focal breakdown of the BSCB. Further, these data highlight the reversibility of TRPV4-mediated BSCB impairments and identify a potential therapeutic strategy for patients with TRPV4 mutations.

Naringenin Alleviates Radiation-Induced Intestinal Injury by Inhibiting TRPV6 in Mice.

SCOPE: Naringenin (NAR) possesses unique anti-inflammatory, antiapoptosis effects and various bioactivities; however, its role against radiation-induced intestinal injury (RIII) remains unclear. This study aims to investigate whether NAR has protective effects against radiation-induced intestinal injury and the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are exposed to a single dose of 13 Gy X-ray total abdominal irradiation (TAI), then gavaged with NAR for 7 days. NAR treatment prolongs the survival rate, protects crypts and villi from damage, alleviates the level of radiation-induced inflammation, and mitigates intestinal barrier damage in the irradiated mice. Additionally, NAR reduces immune cell infiltration and intestinal epithelial cell apoptosis. NAR also shows radioprotective effects in human colon cancer cells (HCT116) and human intestinal epithelial cells (NCM460). It reduces cell damage by reducing intracellular calcium ion levels and reactive oxygen species (ROS) levels. NAR-mediated radioprotection is associated with the downregulation of transient receptor potential vanilloid 6 (TRPV6), and inhibition of apoptosis pathway. Notably, treatment with NAR fails to further increase the protective effects of the TRPV6 inhibitor 2-APB, indicating that TRPV6 inhibition is essential for NAR activity. CONCLUSION: NAR inhibits the apoptosis pathway by downregulating TRPV6 and reducing calcium ion level, thereby alleviating RIII. Therefore, NAR is a promising therapeutic drug for RIII.

Discovery of N-(1,4-Benzoxazin-3-one) urea analogs as Mode-Selective TRPV1 antagonists.

A series of 1,4-benzoxazin-3-one analogs were investigated to discover mode-selective TRPV1 antagonists, since such antagonists are predicted to minimize target-based adverse effects. Using the high-affinity antagonist 2 as the lead structure, the structure activity relationship was studied by modifying the A-region through incorporation of a polar side chain on the benzoxazine and then by changing the C-region with a variety of substituted pyridine, pyrazole and thiazole moieties. The t-butyl pyrazole and thiazole C-region analogs provided high potency as well as mode-selectivity. Among them, antagonist 36 displayed potent and capsaicin-selective antagonism with IC50 = 2.31 nM for blocking capsaicin activation and only 47.5 % inhibition at 3 µM concentration toward proton activation, indicating that more than a 1000-fold higher concentration of 36 was required to inhibit proton activation than was required to inhibit capsaicin activation. The molecular modeling study of 36 with our homology model indicated that two π-π interactions with the Tyr511 and Phe591 residues by the A- and C-region and hydrogen bonding with the Thr550 residue by the B-region were critical for maintaining balanced and stable binding. Systemic optimization of antagonist 2, which has high-affinity but full antagonism for activators of all modes, led to the mode-selective antagonist 36 which represents a promising step in the development of clinical TRPV1 antagonists minimizing side effects such as hyperthermia and impaired heat sensation.

NETosis Drives Blood Pressure Elevation and Vascular Dysfunction in Hypertension.

BACKGROUND: Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. METHODS: NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. RESULTS: Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. CONCLUSIONS: These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.

Ammonium chloride-induced hypothermia is attenuated by transient receptor potential channel vanilloid-1, but augmented by ankyrin-1 in rodents.

AIMS: Systemic administration of ammonium chloride (NH4Cl), an acidifying agent used in human patients and experimental conditions, causes hypothermia in mice, however, the mechanisms of the thermoregulatory response to NH4Cl and whether it develops in other species remained unknown. MAIN METHODS: We studied body temperature (Tb) changes in rats and mice induced by intraperitoneal administration of NH4Cl after blockade of transient receptor potential vanilloid-1 (TRPV1) or ankyrin-1 (TRPA1) channels. KEY FINDINGS: In rats, NH4Cl decreased Tb by 0.4-0.8°C (p < 0.05). The NH4Cl-induced hypothermia also developed in Trpv1 knockout (Trpv1-/-) and wild-type (Trpv1+/+) mice, however, the Tb drop was exaggerated in Trpv1-/- mice compared to Trpv1+/+ controls with maximal decreases of 4.0 vs. 2.1°C, respectively (p < 0.05). Pharmacological blockade of TRPV1 channels with AMG 517 augmented the hypothermic response to NH4Cl in genetically unmodified mice and rats (p < 0.05 for both). In contrast, when NH4Cl was infused to mice genetically lacking the TRPA1 channel, the hypothermic response was significantly attenuated compared to wild-type controls with maximal mean Tb difference of 1.0°C between the genotypes (p = 0.008). Pretreatment of rats with a TRPA1 antagonist (A967079) also attenuated the NH4Cl-induced Tb drop with a maximal difference of 0.7°C between the pretreatment groups (p = 0.003). SIGNIFICANCE: TRPV1 channels limit, whereas TRPA1 channels exaggerate the development of NH4Cl-induced hypothermia in rats and mice, but other mechanisms are also involved. Our results warrant for regular Tb control and careful consideration of NH4Cl treatment in patients with TRPA1 and TRPV1 channel dysfunctions.

Lactobacillus helveticus-Derived Whey-Calcium Chelate Promotes Calcium Absorption and Bone Health of Rats Fed a Low-Calcium Diet.

This study investigated the characteristics of Lactobacillus helveticus-derived whey-calcium chelate (LHWCC) and its effect on the calcium absorption and bone health of rats. Fourier-transform infrared spectroscopy showed that carboxyl oxygen atoms, amino nitrogen atoms, and phosphate ions were the major binding sites with calcium in LHWCC, which has a sustained release effect in simulated in vitro digestion. LHWCC had beneficial effects on serum biochemical parameters, bone biomechanics, and the morphological indexes of the bones of calcium-deficient rats when fed at a dose of 40 mg Ca/kg BW for 7 weeks. In contrast to the inorganic calcium supplement, LHWCC significantly upregulated the gene expression of transient receptor potential cation V5 (TRPV5), TRPV6, PepT1, calcium-binding protein-D9k (Calbindin-D9k), and a calcium pump (plasma membrane Ca-ATPase, PMCA1b), leading to promotion of the calcium absorption rate, whereas Ca3(PO4)2 only upregulated the TRPV6 channel in vivo. These findings illustrate the potential of LHWCC as an organic calcium supplement.

Transient receptor potential vanilloid-1 (TRPV1) channels act as suppressors of the growth of glioma.

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 1 (TRPV1) in glioma. We found that the expression of TRPV1 mRNA and protein were upregulated in glioma compared with normal brain by qPCR and western blot analysis. In order to investigate the function of TRPV1 in glioma, short hairpin RNA (shRNA) and the inhibitor of TRPV1 were used. In vitro, the activation of TRPV1 induced cell apoptosis with decreased migration capability and inhibited proliferation, which was abolished upon TRPV1 pharmacological inhibition and silencing. Mechanistically, TRPV1 modulated glioma proliferation through the protein kinase B (Akt) signaling pathway. More importantly, in immunodeficient (NOD-SCID) mouse xenograft models, tumor size was significantly increased when TRPV1 expression was disrupted by a shRNA knockdown approach in vivo. Altogether, our findings indicate that TRPV1 negatively controls glioma cell proliferation in an Akt-dependent manner, which suggests that targeting TRPV1 may be a potential therapeutic strategy for glioma.

Ca2+ Overload Decreased Cellular Viability in Magnetic Hyperthermia without a Macroscopic Temperature Rise.

Magnetic hyperthermia is a crucial medical engineering technique for treating diseases, which usually uses alternating magnetic fields (AMF) to interplay with magnetic substances to generate heat. Recently, it has been found that in some cases, there is no detectable temperature increment after applying an AMF, which caused corresponding effects surprisingly. The mechanisms involved in this phenomenon are not yet fully understood. In this study, we aimed to explore the role of Ca2+ overload in the magnetic hyperthermia effect without a perceptible temperature rise. A cellular system expressing the fusion proteins TRPV1 and ferritin was prepared. The application of an AMF (518 kHz, 16 kA/m) could induce the fusion protein to release a large amount of iron ions, which then participates in the production of massive reactive oxygen radicals (ROS). Both ROS and its induced lipid oxidation enticed the opening of ion channels, causing intracellular Ca2+ overload, which further led to decreased cellular viability. Taken together, Ca2+ overload triggered by elevated ROS and the induced oxidation of lipids contributes to the magnetic hyperthermia effect without a perceptible temperature rise. These findings would be beneficial for expanding the application of temperature-free magnetic hyperthermia, such as in cellular and neural regulation, design of new cancer treatment methods.