AQP4 regulates ferroptosis and oxidative stress of Muller cells in diabetic retinopathy by regulating TRPV4.

Diabetic retinopathy (DR) is a common microvascular complication that causes visual impairment or loss. Aquaporin 4 (AQP4) is a regulatory protein involved in water transport and metabolism. In previous studies, we found that AQP4 is related to hypoxia injury in Muller cells. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a non-selective cation channel protein involved in the regulation of a variety of ophthalmic diseases. However, the effects of AQP4 and TRPV4 on ferroptosis and oxidative stress in high glucose (HG)-treated Muller cells are unclear. In this study, we investigated the functions of AQP4 and TRPV4 in DR. HG was used to treat mouse Muller cells. Reverse transcription quantitative polymerase chain reaction was used to measure AQP4 mRNA expression. Western blotting was used to detect the protein levels of AQP4, PTGS2, GPX4, and TRPV4. Cell count kit-8, flow cytometry, 5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide staining, and glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) kits were used to evaluate the function of the Muller cells. Streptozotocin was used to induce DR in rats. Haematoxylin and eosin staining was performed to stain the retina of rats. GSH, SOD, and MDA detection kits, immunofluorescence, and flow cytometry assays were performed to study the function of AQP4 and TRPV4 in DR rats. Results found that AQP4 and TRPV4 were overexpressed in HG-induced Muller cells and streptozotocin-induced DR rats. AQP4 inhibition promoted proliferation and cell cycle progression, repressed cell apoptosis, ferroptosis, and oxidative stress, and alleviated retinal injury in DR rats. Mechanistically, AQP4 positively regulated TRPV4 expression. Overexpression of TRPV4 enhanced ferroptosis and oxidative stress in HG-treated Muller cells, and inhibition of TRPV4 had a protective effect on DR-induced retinal injury in rats. In conclusion, inhibition of AQP4 inhibits the ferroptosis and oxidative stress in Muller cells by downregulating TRPV4, which may be a potential target for DR therapy.

Transient receptor potential cation channel subfamily V member 1 (TRPV1) targeted PET imaging.

Transient receptor potential cation channel subfamily V member 1 (TRPV1) was identified using capsaicin, a pungent compound that is present in red pepper. The activation of TRPV1 induces an influx of calcium ions into cells and causes excitation of sensory neurons, associating with thermal sensing, sweating and pain. TRPV1 is also identified in various types of cancer cells. The expression of TRPV1 in cancer cells depends on the type of cancer and the stage of the disease. Therefore, TRPV1 has been considered a potential target of medicinal chemistry for drug development, and blocking its activation may lead to cancer therapy and pain relief. However, the details of the pathophysiological function of TRPV1 in vivo are still unclear. To explore practical use of TRPV1, we focused on positron emission tomography imaging and developed a 11C-radiolabeled tracer to visualize TRPV1.

Endocannabinoid signaling in microglia.

Microglia, the innate immune cells of the central nervous system (CNS), execute their sentinel, housekeeping and defense functions through a panoply of genes, receptors and released cytokines, chemokines and neurotrophic factors. Moreover, microglia functions are closely linked to the constant communication with other cell types, among them neurons. Depending on the signaling pathway and type of stimuli involved, the outcome of microglia operation can be neuroprotective or neurodegenerative. Accordingly, microglia are increasingly becoming considered cellular targets for therapeutic intervention. Among signals controlling microglia activity, the endocannabinoid (EC) system has been shown to exert a neuroprotective role in many neurological diseases. Like neurons, microglia express functional EC receptors and can produce and degrade ECs. Interestingly, boosting EC signaling leads to an anti-inflammatory and neuroprotective microglia phenotype. Nonetheless, little evidence is available on the microglia-mediated therapeutic effects of EC compounds. This review focuses on the EC signals acting on the CNS microglia in physiological and pathological conditions, namely on the CB1R, CB2R and TRPV1-mediated regulation of microglia properties. It also provides new evidence, which strengthens the understanding of mechanisms underlying the control of microglia functions by ECs. Given the broad expression of the EC system in glial and neuronal cells, the resulting picture is the need for in vivo studies in transgenic mouse models to dissect the contribution of EC microglia signaling in the neuroprotective effects of EC-derived compounds.

Impact of the TRPV2 Inhibitor on Advanced Heart Failure in Patients with Muscular Dystrophy: Exploratory Study of Biomarkers Related to the Efficacy of Tranilast.

Cardiomyopathy is the leading cause of death in patients with muscular dystrophy (MD). Tranilast, a widely used anti-allergic drug, has displayed inhibitory activity against the transient receptor potential cation channel subfamily V member 2 and improved cardiac function in MD patients. To identify urinary biomarkers that assess improved cardiac function after tranilast administration, we performed a urinary metabolomic study focused on oxidative fatty acids. Accompanying the clinical trial of tranilast, urine specimens were collected over 24 weeks from MD patients with advanced heart failure. Urinary levels of tetranor-PGDM (tetranor-prostaglandin D metabolite), a metabolite of prostaglandin D2, significantly decreased 12 weeks after tranilast administration and were correlated with BNP. These results suggest that prostaglandin-mediated inflammation, which increases with the pathological progression of heart failure in MD patients, was attenuated. Urinary prostaglandin E3 (PGE3) levels significantly increased 4 weeks after tranilast administration. There were positive correlations between the urinary levels of PGE3 and 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker. High PGE3 levels may have a protective effect against cardiomyopathy in MD patients with high oxidative stress. Although further validation studies are necessary, urinary tetranor-PGDM and PGE3 levels may help the current understanding of the extent of advanced heart failure in patients with MD after tranilast administration.

Resilience to capsaicin-induced mitochondrial damage in trigeminal ganglion neurons.

Capsaicin is an agonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). Strong TRPV1 stimulation with capsaicin causes mitochondrial damage in primary sensory neurons. However, the effect of repetitive and moderate exposure to capsaicin on the integrity of neuronal mitochondria remains largely unknown. Our electron microscopic analysis revealed that repetitive stimulation of the facial skin of mice with 10 mM capsaicin induced short-term damage to the mitochondria in small-sized trigeminal ganglion neurons. Further, capsaicin-treated mice exhibited decreased sensitivity to noxious heat stimulation, indicating TRPV1 dysfunction, in parallel with the mitochondrial damage in the trigeminal ganglion neurons. To analyze the capsaicin-induced mitochondrial damage and its relevant cellular events in detail, we performed cell-based assays using TRPV1-expressing PC12 cells. Dose-dependent capsaicin-mediated mitochondrial toxicity was observed. High doses of capsaicin caused rapid destruction of mitochondrial internal structure, while low doses induced mitochondrial swelling. Further, capsaicin induced a dose-dependent loss of mitochondria and autophagy-mediated degradation of mitochondria (mitophagy). Concomitantly, transcriptional upregulation of mitochondrial proteins, cytochrome c oxidase subunit IV, Mic60/Mitofilin, and voltage-dependent anion channel 1 was observed, which implied induction of mitochondrial biogenesis to compensate for the loss of mitochondria. Collectively, although trigeminal ganglion neurons transiently exhibit mitochondrial damage and TRPV1 dysfunction following moderate capsaicin exposure, they appear to be resilient to such a challenge. Our in vitro data show a dose-response relationship in capsaicin-mediated mitochondrial toxicity. We postulate that induction of mitophagy and mitochondrial biogenesis in response to capsaicin stimulation play important roles in repairing the damaged mitochondrial system.

Post-mortem histology in transient receptor potential cation channel subfamily V member 6 (TRPV6) under-mineralising skeletal dysplasia suggests postnatal skeletal recovery: a case report.

BACKGROUND: The calcium-selective channel TRPV6 (transient receptor potential cation channel subfamily V member 6) is crucial for maternal-fetal calcium transport across the placenta. TRPV6 mutations have recently been associated with an antenatally severe under-mineralising skeletal dysplasia accompanied by postnatal biochemical abnormalities. This is the first post-mortem report in a patient with TRPV6 skeletal dysplasia. CASE PRESENTATION: The female infant had severe antenatal and postnatal skeletal abnormalities by 20 weeks gestation and was ventilator-dependent from birth. These skeletal abnormalities were apparent at an earlier gestational age than in previous reported cases and a more severe clinical course ensued. Biochemical and skeletal abnormalities, including bone density, improved postnatally but cardiac arrest at 4 months of age led to withdrawal of intensive care. Compound heterozygous TRPV6 variants (c.1978G > C p.(Gly660Arg) and c.1528C > T p.(Arg510Ter)) were identified on exome sequencing. Post-mortem identified skeletal abnormalities but no specific abnormalities in other organ systems. No placental pathology was found, multi-organ histological features reflected prolonged intensive care only. Post-mortem macroscopic examination indicated reduced thoracic size and short, pale and pliable ribs. Histological examination identified reduced number of trabeculae in the diaphyses (away from the growth plates), whereas metaphyses showed adequate mineralisation and normal number of trabeculae, but with slightly enlarged reactive chondrocytes, indicating post-natal skeletal growth recovery. Post-mortem radiological findings demonstrated improved bone density, improved rib width, healed fractures, although ribs were still shorter than normal. Long bones (especially humerus and femur) had improved from initial poorly defined metaphyses and reduced bone density to sharply defined metaphyses, prominent growth restart lines in distal diaphyses and bone-in-bone appearance along diaphyses. CONCLUSIONS: This case provide bone histological confirmation that human skeletal development is compromised in the presence of TRPV6 pathogenic variants. Post-mortem findings were consistent with abnormal in utero skeletal mineralisation due to severe calcium deficit from compromised placental calcium transfer, followed by subsequent phenotypic improvement with adequate postnatal calcium availability. Significant skeletal recovery occurs in the early weeks of postnatal life in TRPV6 skeletal dysplasia.

Capsaicin: Plants of the Genus Capsicum and Positive Effect of Oriental Spice on Skin Health.

BACKGROUND: Capsaicin, the main pungent ingredient in hot chili peppers, causes excitation of small sensory neurons. It also provides the basic pungent flavor in Capsicum fruits. SUMMARY: Capsaicin plays a vital role as an agonist for the TRPV1 (transient receptor potential cation channel, subfamily V, member 1) receptor. TRPV1 is essential for the reduction of oxidative stress, pain sensations, and inflammation. Therefore, it has many pros related to health issue. Activation and positive impact of TRPV1 via capsaicin has been studied in various dermatological conditions and in other skin-related issues. Past studies documented that capsaicin plays a vital role in the prevention of atopic dermatitis as well as psoriasis. Moreover, TRPV1 is also very important for skin health because it acts as a capsaicin receptor. It is found in nociceptive nerve fibers and nonneural structures. It prompts the release of a compound that is involved in communicating pain between the spinal cord nerves and other parts of the body. Key Messages: Here, we summarize the growing evidence for the beneficial role of capsaicin and TRPV1 and how they help in the relief of skin diseases such as inflammation, permeation, dysfunction, atopic dermatitis, and psoriasis and in pain amplification syndrome.

A distinct structural mechanism underlies TRPV1 activation by piperine.

Piperine, the principle pungent compound in black peppers, is known to activate the capsaicin receptor TRPV1 ion channel. How piperine interacts with the channel protein, however, remains unclear. Here we show that piperine binds to the same ligand-binding pocket as capsaicin but in different poses. There was no detectable detrimental effect when T551 and E571, two major sites known to form hydrogen bond with capsaicin, were mutated to a hydrophobic amino acid. Computational structural modeling suggested that piperine makes interactions with multiple amino acids within the ligand binding pocket, including T671 on the pore-forming S6 segment. Mutations of this residue could substantially reduce or even eliminate piperine-induced activation, confirming that T671 is an important site. Our results suggest that the bound piperine may directly interact with the pore-forming S6 segment to induce channel opening. These findings help to explain why piperine is a weak agonist, and may guide future efforts to develop novel pharmaceutical reagents targeting TRPV1.

CGRPα within the Trpv1-Cre population contributes to visceral nociception.

The role of calcitonin gene-related peptide (CGRP) in visceral and somatic nociception is incompletely understood. CGRPα is highly expressed in sensory neurons of dorsal root ganglia and particularly in neurons that also express the transient receptor potential cation channel subfamily V member 1 (Trpv1). Therefore, we investigated changes in visceral and somatic nociception following deletion of CGRPα from the Trpv1-Cre population using the Cre/lox system. In control mice, acetic acid injection (0.6%, ip) caused significant immobility (time stationary), an established indicator of visceral pain. In CGRPα-mCherrylx/lx;Trpv1-Cre mice, the duration of immobility was significantly less than controls, and the distance CGRPα-mCherrylx/lx;Trpv1-Cre mice traveled over 20 min following acetic acid was significantly greater than controls. However, following acetic acid injection, there was no difference between genotypes in the writhing reflex, number of abdominal licks, or forepaw wipes of the cheek. CGRPα-mCherrylx/lx;Trpv1-Cre mice developed more pronounced inflammation-induced heat hypersensitivity above baseline values compared with controls. However, analyses of noxious acute heat or cold transmission revealed no difference between genotypes. Also, odor avoidance test, odor preference test, and buried food test for olfaction revealed no differences between genotypes. Our findings suggest that CGRPα-mediated transmission within the Trpv1-Cre population plays a significant role in visceral nociceptive pathways underlying voluntary movement. Monitoring changes in movement over time is a sensitive parameter to identify differences in visceral nociception, compared with writhing reflexes, abdominal licks, or forepaw wipes of the cheek that were unaffected by deletion of CGRPα- from Trpv1-Cre population and likely utilize different mechanisms. NEW & NOTEWORTHY The neuropeptide calcitonin gene-related peptide (CGRP) is highly colocalized with transient receptor potential cation channel subfamily V member 1 (TRPV1)-expressing primary afferent neurons, but the functional role of CGRPα specifically in these neurons is unknown in pain processing from visceral and somatic afferents. We used cre-lox recombination to conditionally delete CGRPα from TRPV1-expressing neurons in mice. We show that CGRPα from within TRPV1-cre population plays an important role in visceral nociception but less so in somatic nociception.

Histamine Receptor H1-Mediated Sensitization of TRPV1 Mediates Visceral Hypersensitivity and Symptoms in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Histamine sensitizes the nociceptor transient reporter potential channel V1 (TRPV1) and has been shown to contribute to visceral hypersensitivity in animals. We investigated the role of TRPV1 in irritable bowel syndrome (IBS) and evaluated if an antagonist of histamine receptor H1 (HRH1) could reduce symptoms of patients in a randomized placebo-controlled trial. METHODS: By using live calcium imaging, we compared activation of submucosal neurons by the TRPV1 agonist capsaicin in rectal biopsy specimens collected from 9 patients with IBS (ROME 3 criteria) and 15 healthy subjects. The sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde, and supernatants from biopsy specimens was assessed by calcium imaging of mouse dorsal root ganglion neurons. We then performed a double-blind trial of patients with IBS (mean age, 31 y; range, 18-65 y; 34 female). After a 2-week run-in period, subjects were assigned randomly to groups given either the HRH1 antagonist ebastine (20 mg/day; n = 28) or placebo (n = 27) for 12 weeks. Rectal biopsy specimens were collected, barostat studies were performed, and symptoms were assessed (using the validated gastrointestinal symptom rating scale) before and after the 12-week period. Patients were followed up for an additional 2 weeks. Abdominal pain, symptom relief, and health-related quality of life were assessed on a weekly basis. The primary end point of the study was the effect of ebastine on the symptom score evoked by rectal distension. RESULTS: TRPV1 responses of submucosal neurons from patients with IBS were potentiated compared with those of healthy volunteers. Moreover, TRPV1 responses of submucosal neurons from healthy volunteers could be potentiated by their pre-incubation with histamine; this effect was blocked by the HRH1 antagonist pyrilamine. Supernatants from rectal biopsy specimens from patients with IBS, but not from the healthy volunteers, sensitized TRPV1 in mouse nociceptive dorsal root ganglion neurons via HRH1; this effect could be reproduced by histamine and imidazole acetaldehyde. Compared with subjects given placebo, those given ebastine had reduced visceral hypersensitivity, increased symptom relief (ebastine 46% vs placebo 13%; P = .024), and reduced abdominal pain scores (ebastine 39 ± 23 vs placebo 62 ± 22; P = .0004). CONCLUSIONS: In studies of rectal biopsy specimens from patients, we found that HRH1-mediated sensitization of TRPV1 is involved in IBS. Ebastine, an antagonist of HRH1, reduced visceral hypersensitivity, symptoms, and abdominal pain in patients with IBS. Inhibitors of this pathway might be developed as a new treatment approach for IBS. ClinicalTrials.gov no: NCT01144832.

Deciphering physiological role of the mechanosensitive TRPV4 channel in the distal nephron.

Long-standing experimental evidence suggests that epithelial cells in the renal tubule are able to sense osmotic and pressure gradients caused by alterations in ultrafiltrate flow by elevating intracellular Ca(2+) concentration. These responses are viewed as critical regulators of a variety of processes ranging from transport of water and solutes to cellular growth and differentiation. A loss in the ability to sense mechanical stimuli has been implicated in numerous pathologies associated with systemic imbalance of electrolytes and to the development of polycystic kidney disease. The molecular mechanisms conferring mechanosensitive properties to epithelial tubular cells involve activation of transient receptor potential (TRP) channels, such as TRPV4, allowing direct Ca(2+) influx to increase intracellular Ca(2+) concentration. In this review, we critically analyze the current evidence about signaling determinants of TRPV4 activation by luminal flow in the distal nephron and discuss how dysfunction of this mechanism contributes to the progression of polycystic kidney disease. We also review the physiological relevance of TRPV4-based mechanosensitivity in controlling flow-dependent K(+) secretion in the distal renal tubule.

Pharmacological activity of capsaicin: Mechanisms and controversies (Review).

Capsaicin, which is abundant in chili peppers, exerts antioxidative, antitumor, antiulcer and analgesic effects and it has demonstrated potential as a treatment for cardiovascular, gastrointestinal, oncological and dermatological conditions. Unique among natural irritants, capsaicin initially excites neurons but then 'calms' them into long­lasting non­responsiveness. Capsaicin can also promote weight loss, making it potentially useful for treating obesity. Several mechanisms have been proposed to explain the therapeutic effects of capsaicin, including antioxidation, analgesia and promotion of apoptosis. Some of the mechanisms are proposed to be mediated by the capsaicin receptor (transient receptor potential cation channel subfamily V member 1), but some are proposed to be independent of that receptor. The clinical usefulness of capsaicin is limited by its short half­life. The present review provided an overview of what is known about the therapeutic effects of capsaicin and the mechanisms involved and certain studies arguing against its clinical use were mentioned.

Puerarin attenuates remifentanil­induced postoperative hyperalgesia via targeting PAX6 to regulate the transcription of TRPV1.

Remifentanil­induced hyperalgesia (RIH) is characterized by the emergence of stimulation­induced pain, including phenomena such as allodynia and thermal hyperalgesia following remifentanil infusion. As a sequence­specific DNA binding transcription factor, PAX6 positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system. It was hypothesized that puerarin could relieve RIH via targeting PAX6 to regulate transcription of transient receptor potential cation channel subfamily V Member 1 (TRPV1). A total of 32 rats were randomly divided into five groups, namely control group, RI group, RI + 10 mg/kg puerarin group (RI + puerarin10), RI + 20 mg/kg puerarin group (RI + puerarin20), and RI + 40 mg/kg puerarin group (RI + puerarin40). Mechanical and thermal hyperalgesia were tested at ­24, 2, 6, 24 and 48 h after remifentanil infusion. Following the sacrifice of rats after the last behavioral test, western blot was used to detect the expression levels of TRPV1 in the tissues; Immunofluorescence staining and western blotting were used to detect the expression of PAX6 in the spinal cord. PharmMapper and JASPAR were used to predict the binding sites of puerarin/PAX6/TRPV1. Chromatin immunoprecipitation­PCR and dual luciferase reporter assay were used to verify the targeting relationship between PAX6 and TRPV1. Immunofluorescence was used to detect the expression levels of TRPV1 and p­NR2B. The results revealed that puerarin (10, 20, 40 mg/kg) dose­dependently reduced thermal and mechanical hyperalgesia from 2 to 48 h after remifentanil infusion. Remifentanil infusion remarkably stimulated the expression of phosphorylated (p­)NR2B. Nevertheless, the increased amount of p­NR2B by RIH was dose­dependently suppressed by puerarin in rats. In conclusion, puerarin was revealed to attenuate postoperative RIH via targeting PAX6 to regulate the transcription of TRPV1.

MUC1­ND interacts with TRPV1 to promote corneal epithelial cell proliferation in diabetic dry eye mice by partly activating the AKT signaling pathway.

Although both mucin1 (MUC1) and transient receptor potential cation channel subfamily V member 1 (TRPV1) have been reported to be associated with dry eye (DE) disease, whether they interact and their regulatory roles in diabetic DE disease are unknown. Diabetic DE model mice were generated by streptozotocin induction and assessed by corneal fluorescein staining, tear ferning (TF) tests, phenol red thread tests, hematoxylin and eosin staining of corneal sections and periodic acid Schiff staining of conjunctival sections. Cell proliferation was measured by CCK8 assay. Western blotting was performed to measure protein expression. Primary mouse corneal epithelial cells (MCECs) were cultured after enzymatic digestion. Immunofluorescence staining of MCECs and frozen corneal sections was conducted to assess protein expression and colocalization. Coimmunoprecipitation was performed to detect protein­protein interactions. It was found that, compared with control mice, diabetic DE mice exhibited increased corneal epithelial defects, reduced tear production, poorer TF pattern grades and impaired corneal and conjunctival tissues. In vivo and in vitro experiments showed that hyperglycemia impaired cell proliferation, accompanied by decreased levels of the MUC1 extracellular domain (MUC1­ND) and TRPV1. Additionally, it was found that capsazepine (a TRPV1 antagonist) inhibited the proliferation of MCECs. Notably, MUC1­ND was shown to interact with the TRPV1 protein in the control group but not in the diabetic DE group. It was also found that the AKT signaling pathway was attenuated in the diabetic DE mice and downstream of TRPV1. MUC1­ND interacted with TRPV1, partly activating the AKT signaling pathway to promote MCEC proliferation. The present study found that the interaction of MUC1­ND with TRPV1 promotes MCEC proliferation by partly activating the AKT signaling pathway, providing new insight into the pathogenesis of corneal epithelial dysfunction in diabetic DE disease.

Temporal changes in tissue 1α,25-dihydroxyvitamin D3, vitamin D receptor target genes, and calcium and PTH levels after 1,25(OH)2D3 treatment in mice.

The vitamin D receptor (VDR) maintains a balance of plasma calcium and 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], its natural active ligand, by directly regulating the calcium ion channel (TRPV6) and degradation enzyme (CYP24A1), and indirectly regulating the parathyroid hormone (PTH) for feedback regulation of the synthetic enzyme CYP27B1. Studies that examined the intricate relationships between plasma and tissue 1,25(OH)2D3 levels and changes in VDR target genes and plasma calcium and PTH are virtually nonexistent. In this study, we investigated temporal correlations between tissue 1,25(OH)2D3 concentrations and VDR target genes in ileum and kidney and plasma calcium and PTH concentrations in response to 1,25(OH)2D3 treatment in mice (2.5 µg/kg ip, singly or q2d × 4). After a single ip dose, plasma 1,25(OH)2D3 peaked at ∼0.5 h and then decayed biexponentially, falling below basal levels after 24 h and then returning to baseline after 8 days. Upon repetitive ip dosing, plasma, ileal, renal, and bone 1,25(OH)2D3 concentrations rose and decayed in unison. Temporal profiles showed increased expressions of ileal Cyp24a1 and renal Cyp24a1, Mdr1/P-gp, and VDR but decreased renal Cyp27b1 mRNA after a time delay in VDR activation. Increased plasma calcium and attenuated PTH levels and increased ileal and renal Trpv6 expression paralleled the changes in tissue 1,25(OH)2D3 concentrations. Gene changes in the kidney were more sustained than those in intestine, but the magnitudes of change for Cyp24a1 and Trpv6 were lower than those in intestine. The data revealed that 1,25(OH)2D3 equilibrates with tissues rapidly, and VDR target genes respond quickly to exogenously administered 1,25(OH)2D3.

Impact of TRPV3 on the development of allergic dermatitis as a dendritic cell modulator.

The transient receptor potential channel vanilloid subfamily V member 3 (TRPV3), which functions as a thermosensor in keratinocytes, plays an important role in the development of allergic and itchy dermatitis in rodents. Although real-time PCR analysis using lesional and non-lesional skin samples from patients with atopic dermatitis showed that TRPV3 was expressed in lesional skin, the role that TRPV3 plays in patients with dermatitis is still relatively obscure. Here, we determined whether TRPV3 was a dendritic cell (DC) modulator using DS-Nh mice with a gain-of-function mutation in TRPV3 (TRPV3Gly573Ser), because increasing skin temperature is associated with the modulation of dermal dendritic cells (DCs). Interestingly, increased responses to haptens by skin and DCs were observed in DS-Nh mice compared with those from DS mice with wild-type TRPV3. Increased thymic stromal lymphopoietin (TSLP) responses were also observed in keratinocytes from DS-Nh mice compared with those from DS mice. Taken together, we propose that the DS-Nh mouse is a good model to use in order to better understand the role of this orphan channel and that TRPV3 may represent a new therapeutic target in certain types of dermatitis through the control of DCs.

Shexiang Baoxin Pill treats acute myocardial infarction by promoting angiogenesis via GDF15-TRPV4 signaling.

Angiogenesis has been considered a pivotal strategy for treating ischemic heart disease. One possible approach, the Shexiang Baoxin Pill (MUSKARDIA), has been noted to promote angiogenesis, but its underlying mechanism is still largely unknown. We aimed to determine the effects of MUSKARDIA on acute myocardial infarction (AMI), as well as the underlying mechanistic bases. AMI was induced in rats, using left anterior descending coronary arterial occlusion, and either 6 (low) or 12 (high-dose) mg/kg/day of MUSKARDIA was administered for 56 days. We found that MUSKARDIA improved cardiac function and counteracted against adverse remodeling among AMI rats, which most likely is due to it promoting angiogenesis. Transcriptome analysis by RNA-sequencing found that MUSKARDIA up-regulated cardiac pro-angiogenic genes, particularly growth differentiation factor 15 (GDF15), which was confirmed by RT-qPCR. This up-regulation was also correlated with elevated serum GDF15 levels. In vitro analyses with human umbilical vein endothelial cells found that increased GDF15, stimulated by MUSKARDIA, resulted in enhanced cell migration, proliferation, and tubular formation, all of which were reversed after GDF15 knockdown using a lentiviral vector. Gene Ontology, as well as Kyoto Genes and Genomes enrichment analyses identified calcium signaling pathway as a major contributor to these outcomes, which was verified by Western blot and Cal-590 AM loading showing that transient receptor potential cation channel subfamily V member 4 protein (TRPV4) and intracellular Ca2+ levels increased in accordance with MUSKARDIA-induced GDF15 up-regulation, and decreased with GDF15 knock-down. Therefore, MUSKARDIA may exert its cardioprotective effects via stimulating the GDF15/TRPV4/calcium signaling/angiogenesis axis.

Hydrogen Sulphide Alleviates Senescence of Human Periodontal Ligament Stem Cells by TRPV4 Channel Mediated Calcium Flux.

OBJECTIVE: To explore whether hydrogen sulphide (H2S) could protect human periodontal ligament stem cells (PDLSCs) from senescence and the possible underlying mechanisms. METHODS: Cell cycle assay and Ki-67 assay were used to measure proliferation of PDLSCs. Real-time polymerase chain reaction (PCR) was used to measure cellular senescence-related p16 and p21. Calcium influx was detected by measurement of Ca2+ imaging. In addition, we analysed the possible mechanisms underlying H2S acting on PDLSCs by microarray. RESULTS: The cell proliferation rate of aging PDLSCs decreased significantly. The expression of cellular senescence-related p16 and p21 significantly increased in aging PDLSCs. H2S donor (GYY4137) treatment increased the proliferation rate of senescence PDLSCs. Furthermore, the donor of H2S treatment effectively prevented cell cycle arrest of PDLSCs during the aging process and inhibited the expression of cellular senescence-related markers. Mechanically, H2S donor treatment could activate the calcium influx in PDLSCs. Moreover, pretreatment with TRPV4 inhibitors significantly attenuated the calcium influx induced by H2S donor treatment in PDLSCs. It also alleviated the protective effect of H2S on the senescence of PDLSCs. CONCLUSION: H2S alleviated the senescence of human PDLSCs by TRPV4 channel mediated calcium flux. These results provide a potential strategy to deal with cell aging and may facilitate cell therapy for oral diseases.

Potential mechanism of transient receptor potential cation channel subfamily V member 1 combined with an ATP­sensitive potassium channel in severe preeclampsia.

Severe preeclampsia is one of the most serious obstetric diseases. However, the pathogenesis of the disease is not fully understood. In the present study, placental artery and blood serum was collected from patients with severe preeclampsia, as well as from normal pregnant women. The results of reverse transcription-quantitative (q)PCR, western blotting, and immunohistochemical staining revealed markedly decreased transient receptor potential cation channel subfamily V member 1 (TRPV1), ATP-sensitive potassium channel (KATP) subtype Kir6.1/SUR2B and endothelial nitric oxide synthase (eNOS) expression in severe preeclampsia tissue specimens compared with those in samples from normal pregnant women. The nitrate reduction method indicated lower NO levels in the tissue specimens and serum of patients with severe preeclampsia. Moreover, hematoxylin-eosin staining showed that the endothelial cell layer in the placental artery of patients with severe preeclampsia was notably damaged. To investigate the potential role of TRPV1-KATP channels in severe preeclampsia, HUVECs were used for in vitro experiments. The samples were divided into a control group, a TRPV1 agonist group (capsaicin) and a TRPV1 inhibitor group (capsazepine). qPCR and western blotting revealed that the relative gene and protein expression levels of TRPV1, Kir6.1, SUR2B and eNOS in the control group were significantly lower than those in the capsaicin group and considerably higher than those in the capsazepine group. Based on previous studies and the results of the present study, we hypothesized that impairment of the endothelial TRPV1-KATP channels results in decreased eNOS/NO pathway activity, which may be one of the mechanisms involved in severe preeclampsia. The increase in NO generation mediated by TRPV1-KATP may be a suitable target for the management of severe preeclampsia.

A novel frameshift mutation in TRPV6 is associated with hereditary pancreatitis.

Introduction: Hereditary pancreatitis (HP) is a rare debilitating disease with incompletely understood etio-pathophysiology. The reduced penetrance of genes such as PRSS1 associated with hereditary pancreatitis indicates a role for novel inherited factors. Methods: We performed whole-exome sequencing of three affected members of an Indian family (Father, Son, and Daughter) with chronic pancreatitis and compared variants with those seen in the unaffected mother. Results: We identified a novel frameshift mutation in exon 11 of TRPV6 (c.1474_1475delGT; p.V492Tfs*136), a calcium channel, in the patients. Functional characterization of this mutant TRPV6 following heterologous expression revealed that it was defective in calcium uptake. Induction of pancreatitis in mice induced Trpv6 expression, indicating that higher expression levels of the mutant protein and consequent dysregulation of calcium levels in patients with chronic pancreatitis could aggravate the disease. Discussion: We report a novel frameshift mutation in TRPV6 in an Indian family with HP that renders the mutant protein inactive. Our results emphasize the need to expand the list of genes used currently for evaluating patients with hereditary pancreatitis.