TRPV1 participates in the protective effect of propolis on colonic tissue of ulcerative colitis / 中南大学学报(医学版)

OBJECTIVES@#Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) mainly characterized by inflammation, ulceration and erosion of colonic mucosa and submucosa. Transient receptor potential vanilloid 1 (TRPV1) is an important mediator of visceral pain and inflammatory bowel disease. This study aims to investigate the protective effect of water soluble propolis (WSP) on UC colon inflammatory tissue and the role of TRPV1.@*METHODS@#Male SD rats were randomly divided into 6 groups (n=8): a normal control (NC) group, an ulcerative colitis model (UC) group, a low-WSP (L-WSP) group, a medium-WSP (M-WSP) group, a high-WSP (H-WSP) group, and a salazosulfapyridine (SASP) group. The rats in the NC group drank water freely, and the other groups drank 4% dextran sulfate sodium (DSS) solution freely for 7 d to replicate the ulcerative colitis model. Based on the successful replication of the UC, the L-WSP, M-WSP, and H-WSP groups were given 50, 100, and 200 mg/kg of water-soluble propolis by gavage for 7 d, and the SASP group was given 100 mg/kg of sulfasalazine by gavage for 7 d. The body weight of rats in each group was measured at the same time every day, the fecal traits and occult blood were observed to record the disease activity index (DAI). After intragastric administration, the animals were sacrificed after fasted 24 h. Serum and colonic tissue were collected, and the changes of MDA, IL-6 and TNF-α were detected. The pathological changes of colon tissues were observed by HE staining, and the expression of TRPV1 in colon tissues was observed by Western blotting, immunohistochemistry, and immunofluorescence.@*RESULTS@#The animals in each group that drank DSS freely showed symptoms such as weight loss, decreased appetite, depressed state, and hematochezia, indicating that the model was successfully established. Compared with the NC group, DAI scores of other groups were increased (all P<0.05). MDA, IL-6, TNF-α in serum and colon tissues of the UC group were increased compared with the NC group (all P<0.01), and they were decreased after WSP and SASP treatment (all P<0.01). The results of showed that the colon tissue structure was obviously broken and inflammatory infiltration in the UC group, while the H-WSP group and the SASP group significantly improved the colon tissue and alleviated inflammatory infiltration. The expression of TRPV1 in colon tissues in the UC group was increased compared with the NC group (all P<0.01), and it was decreased after WSP and SASP treatment.@*CONCLUSIONS@#WSP can alleviate the inflammatory state of ulcerative colitis induced by DSS, which might be related to the inhibition of inflammatory factors release, and down-regulation or desensitization of TRPV1.

TRPV4-induced Neurofilament Injury Contributes to Memory Impairment after High Intensity and Low Frequency Noise Exposures / 生物医学与环境科学（英文）

OBJECTIVE@#Exposure to high intensity, low frequency noise (HI-LFN) causes vibroacoustic disease (VAD), with memory deficit as a primary non-auditory symptomatic effect of VAD. However, the underlying mechanism of the memory deficit is unknown. This study aimed to characterize potential mechanisms involving morphological changes of neurons and nerve fibers in the hippocampus, after exposure to HI-LFN.@*METHODS@#Adult wild-type and transient receptor potential vanilloid subtype 4 knockout (TRPV4-/-) mice were used for construction of the HI-LFN injury model. The new object recognition task and the Morris water maze test were used to measure the memory of these animals. Hemoxylin and eosin and immunofluorescence staining were used to examine morphological changes of the hippocampus after exposure to HI-LFN.@*RESULTS@#The expression of TRPV4 was significantly upregulated in the hippocampus after HI-LFN exposure. Furthermore, memory deficits correlated with lower densities of neurons and neurofilament-positive nerve fibers in the cornu ammonis 1 (CA1) and dentate gyrus (DG) hippocampal areas in wild-type mice. However, TRPV4-/- mice showed better performance in memory tests and more integrated neurofilament-positive nerve fibers in the CA1 and DG areas after HI-LFN exposure.@*CONCLUSION@#TRPV4 up-regulation induced neurofilament positive nerve fiber injury in the hippocampus, which was a possible mechanism for memory impairment and cognitive decline resulting from HI-LFN exposure. Together, these results identified a promising therapeutic target for treating cognitive dysfunction in VAD patients.

Preliminary study of TRPV4 affects chondrocyte degeneration / 中国骨伤

OBJECTIVE@#To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration.@*METHODS@#Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected.@*RESULTS@#Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05).@*CONCLUSION@#Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.

Research progress of chondrocyte mechanotransduction mediated by TRPV4 and PIEZOs / 生物医学工程学杂志

Mechanical signal transduction are crucial for chondrocyte in response to mechanical cues during the growth, development and osteoarthritis (OA) of articular cartilage. Extracellular matrix (ECM) turnover regulates the matrix mechanical microenvironment of chondrocytes. Thus, understanding the mechanotransduction mechanisms during chondrocyte sensing the matrix mechanical microenvironment can develop effective targeted therapy for OA. In recent decades, growing evidences are rapidly advancing our understanding of the mechanical force-dependent cartilage remodeling and injury responses mediated by TRPV4 and PIEZOs. In this review, we highlighted the mechanosensing mechanism mediated by TRPV4 and PIEZOs during chondrocytes sensing mechanical microenvironment of the ECM. Additionally, the latest progress in the regulation of OA by inflammatory signals mediated by TRPV4 and PIEZOs was also introduced. These recent insights provide the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA. This review will shed light on the pathogenesis of articular cartilage, searching clinical targeted therapies, and designing cell-induced biomaterials.

Improvement effect of Shegan Mahuang Decoction on rats with cold-induced asthma based on TRPV1/NRF-1/mtTFA pathway / 中国中药杂志

This study investigated the therapeutic effect of Shegan Mahuang Decoction(SGMHD) on cold-induced asthma in rats and explored its underlying mechanism. Seventy-two healthy male SD rats of specific pathogen free(SPF) grade were randomly divided into a blank group, a model group, a positive control group(dexamethasone, 0.4 mg·kg~(-1)), and low-, medium-, and high-dose SGMHD groups(3.2, 6.4, and 12.8 g·kg~(-1)). The blank group received saline, while the other groups were sensitized by intraperitoneal injection of ovalbumin(OVA) solution. Subsequently, the rats were placed in a cold chamber adjustable to 0-2 ℃, and OVA solution was ultrasonically nebulized to induce cold-induced asthma in rats. After three weeks of treatment, the general behaviors of rats were observed. Hematoxylin-eosin(HE) staining was used to evaluate pathological changes in lung tissues, periodic acid-Schiff(PAS) staining assessed mucin changes, and Masson staining was performed to examine collagen deposition. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of the inflammatory factors interleukin-4(IL-4) and vascular endothelial growth factor(VEGF) in serum and bronchoalveolar lavage fluid(BALF). Real-time quantitative polymerase chain reaction(RT-PCR) was employed to assess the mRNA expression levels of transient receptor potential vanilloid subfamily member 1(TRPV1), nuclear respiratory factor 1(NRF-1), and mitochondrial transcription factor A(mtTFA) in lung tissues. Western blot was used to measure the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues. Compared with the blank group, the model group exhibited signs of rapid respiration, increased frequency of defecation with looser stools, and disheveled and dull fur. Pathological results showed significant infiltration of inflammatory cells in lung tissues, narrowing of bronchial lumens, increased mucin secretion, and enhanced collagen deposition in the model group. Additionally, the levels of IL-4 and VEGF in serum and BALF were significantly elevated, and the mRNA and protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were significantly increased. Compared with the model group, SGMHD improved the behaviors of rats, alleviated pathological changes in lung tissues, mucin production, and collagen deposition, significantly decreased the levels of IL-4 and VEGF in serum and BALF, and reduced the mRNA expression levels of TRPV1, NRF-1, and mtTFA in lung tissues, with the medium-dose SGMHD group showing the most significant effect. Moreover, the protein expression levels of TRPV1, NRF-1, and mtTFA in lung tissues were also reduced, with the medium-dose SGMHD group exhibiting the most significant effect. In conclusion, this study demonstrates that SGMHD can alleviate airway inflammation and inhibit airway remodeling in cold-induced asthma rats. These effects may be associated with the modulation of the TRPV1/NRF-1/mtTFA signaling pathway.

Baicalin treats cerebral ischemia reperfusion-induced brain edema in rats by inhibiting TRPV4 and AQP4 of astrocytes / 中国中药杂志

This study aims to explore the pharmacodynamic effect of baicalin on rat brain edema induced by cerebral ischemia reperfusion injury and discuss the mechanism from the perspective of inhibiting astrocyte swelling, which is expected to serve as a refe-rence for the treatment of cerebral ischemia with Chinese medicine. To be specific, middle cerebral artery occlusion(suture method) was used to induce cerebral ischemia in rats. Rats were randomized into normal group, model group, high-dose baicalin(20 mg·kg~(-1)) group, and low-dose baicalin(10 mg·kg~(-1)) group. The neurobehavior, brain index, brain water content, and cerebral infarction area of rats were measured 6 h and 24 h after cerebral ischemia. Brain slices were stained with hematoxylin and eosin(HE) for the observation of pathological morphology of cerebral cortex after baicalin treatment. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the content of total L-glutathione(GSH) and glutamic acid(Glu) in brain tissue, Western blot to measure the content of glial fibrillary acidic protein(GFAP), aquaporin-4(AQP4), and transient receptor potential vanilloid type 4(TRPV4), and immunohistochemical staining to observe the expression of GFAP. The low-dose baicalin was used for exploring the mechanism. The experimental results showed that the neurobehavioral scores(6 h and 24 h of cerebral ischemia), brain water content, and cerebral infarction area of the model group were increased, and both high-dose and low-dose baicalin can lower the above three indexes. The content of GSH dropped but the content of Glu raised in brain tissue of rats in the model group. Low-dose baicalin can elevate the content of GSH and lower the content of Glu. According to the immunohistochemical staining result, the model group demonstrated the increase in GFAP expression, and swelling and proliferation of astrocytes, and the low-dose baicalin can significantly improve this situation. The results of Western blot showed that the expression of GFAP, TRPV4, and AQP4 in the cerebral cortex of the model group increased, and the low-dose baicalin reduce their expression. The cerebral cortex of rats in the model group was severely damaged, and the low-dose baicalin can significantly alleviate the damage. The above results indicate that baicalin can effectively relieve the brain edema caused by cerebral ischemia reperfusion injury in rats, possibly by suppressing astrocyte swelling and TRPV4 and AQP4.

Effects of transient receptor potential vanilloid type 4-specific activator on human vascular endothelial cell functions and blood supply of rat perforator flap and its mechanism / 中华烧伤杂志

Objective: To analyze the effects of transient receptor potential vanilloid type 4 (TRPV4) activation on the function and endothelial-to-mesenchymal transition (EndMT) of human umbilical vein endothelial cells (HUVECs), as well as to explore the effects of TRPV4 activation on blood perfusion and survival of rat perforator flap and the mechanism. Methods: The experimental research methods were used. The 3rd to 6th passages of HUVECs were used for experiments and divided into 0.5 μmol/L 4α-phorbol 12, 13-didecanoate (4αPDD) group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, 10.0 μmol/L 4αPDD group, and phosphate buffer solution (PBS) group, which were cultivated in corresponding final molarity of 4αPDD and PBS, respectively. The cell proliferation activity at 6 and 12 h of culture was detected using cell counting kit-8 (CCK-8). Another batch of cells was acquired and divided into PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group, which were treated similarly as described before and then detected for cell proliferation activity at 6, 12, 24, and 48 h of culture. The residual scratch area of cells at post scratch hour (PSH) 12, 24, and 48 was detected by scratch test, and the percentage of the residual scratch area was calculated. The number of migrated cells at 24 and 48 h of culture was detected by Transwell experiment. The tube-formation assay was used to measure the number of tubular structures at 4 and 8 h of culture. The protein expressions of E-cadherin, N-cadherin, Slug, and Snail at 24 h of culture were detected by Western blotting. All the sample numbers in each group at each time point in vitro experiments were 3. A total of 36 male Sprague-Dawley rats aged 8 to 10 weeks were divided into delayed flap group, 4αPDD group, and normal saline group according to the random number table, with 12 rats in each group, and iliolumbar artery perforator flap models on the back were constructed. The flap surgical delay procedure was only performed in the rats in delayed flap group one week before the flap transfer surgery. Neither rats in 4αPDD group nor normal saline group had flap surgical delay; instead, they were intraperitoneally injected with 4αPDD and an equivalent mass of normal saline, respectively, at 10 min before, 24 h after, and 48 h after the surgery. The general state of flap was observed on post surgery day (PSD) 0 (immediately), 1, 4, and 7. The flap survival rates were assessed on PSD 7. The flap blood perfusion was detected by laser speckle contrast imaging technique on PSD 1, 4, and 7. The microvascular density in the flap's choke vessel zone was detected by immunohistochemical staining. All the sample numbers in each group at each time point in vivo experiments were 12. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results: At 6 and 12 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 0.5 μmol/L 4αPDD group, 1.0 μmol/L 4αPDD group, 3.0 μmol/L 4αPDD group, and 10.0 μmol/L 4αPDD group (P>0.05). At 6, 12, 24, and 48 h of culture, there were no statistically significant differences in cell proliferation activity in the overall comparison among PBS group, 1 μmol/L 4αPDD group, and 3 μmol/L 4αPDD group (P>0.05). At PSH 12, the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At PSH 24 and 48, compared with those in PBS group, the percentages of the residual scratch area of cells in 3 μmol/L 4αPDD group were significantly decreased (with t values of 2.83 and 2.79, respectively, P<0.05), while the percentages of the residual scratch area of cells in 1 μmol/L 4αPDD group showed no significant differences (P>0.05). At 24 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were close to that in PBS group (P>0.05). At 48 h of culture, the number of migrated cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD groups were significantly greater than that in PBS group (with t values of 6.20 and 9.59, respectively, P<0.01). At 4 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly greater than that in PBS group (with t values of 4.68 and 4.95, respectively, P<0.05 or <0.01). At 8 h of culture, the numbers of tubular structures of cells in 1 μmol/L 4αPDD and 3 μmol/L 4αPDD groups were similar to that in PBS group (P>0.05). At 24 h of culture, compared with those in PBS group, the protein expression level of E-cadherin of cells in 3 μmol/L 4αPDD group was significantly decreased (t=5.13, P<0.01), whereas there was no statistically significant difference in the protein expression level of E-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression level of N-cadherin of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.93, P<0.01), whereas there was no statistically significant difference in the protein expression level of N-cadherin of cells in 1 μmol/L 4αPDD group (P>0.05); the protein expression levels of Slug of cells in 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group were significantly increased (with t values of 3.85 and 6.52, respectively, P<0.05 or P<0.01); and the protein expression level of Snail of cells in 3 μmol/L 4αPDD group was significantly increased (t=4.08, P<0.05), whereas there was no statistically significant difference in the protein expression level of Snail of cells in 1 μmol/L 4αPDD group (P>0.05). There were no statistically significant differences in the protein expression levels of E-cadherin, N-cadherin, Slug, or Snail of cells between 1 μmol/L 4αPDD group and 3 μmol/L 4αPDD group (P>0.05). The general condition of flaps of rats in the three groups was good on PSD 0. On PSD 1, the flaps of rats in the three groups were basically similar, with bruising and swelling at the distal end. On PSD 4, the swelling of flaps of rats in the three groups subsided, and the distal end turned dark brown and necrosis occurred, with the area of necrosis in flaps of rats in normal saline group being larger than the areas in 4αPDD group and delayed flap group. On PSD 7, the necrotic areas of flaps of rats in the 3 groups were fairly stable, with the area of necrosis at the distal end of flap of rats in delayed flap group being the smallest. On PSD 7, the flap survival rates of rats in 4αPDD group ((80±13)%) and delayed flap group ((87±9)%) were similar (P>0.05), and both were significantly higher than (70±11)% in normal saline group (with t values of 2.24 and 3.65, respectively, P<0.05 or P<0.01). On PSD 1, the overall blood perfusion signals of rats in the 3 groups were basically the same, and the blood perfusion signals in the choke vessel zone were relatively strong, with a certain degree of underperfusion at the distal end. On PSD 4, the boundary between the surviving and necrotic areas of flaps of rats in the 3 groups became evident, and the blood perfusion signals in the choke vessel zone were improved, with the normal saline group's distal hypoperfused area of flap being larger than the areas in delayed flap group and 4αPDD group. On PSD 7, the blood perfusion signals of overall flap of rats had generally stabilized in the 3 groups, with the intensity of blood perfusion signal in the choke vessel zone and overall flap of rats in delayed flap group and 4αPDD group being significantly greater than that in normal saline group. On PSD 7, the microvascular density in the choke vessel zone of flap of rats in 4αPDD group and delayed flap group were similar (P>0.05), and both were significantly higher than that in normal saline group (with t values of 4.11 and 5.38, respectively, P<0.01). Conclusions: After activation, TRPV4 may promote the migration and tubular formation of human vascular endothelial cells via the EndMT pathway, leading to the enhanced blood perfusion of perforator flap and microvascular density in the choke vessel zone, and therefore increase the flap survival rate.

Oral administration of TRPV4 inhibitor improves atrial calcium handling abnormalities in sterile pericarditis rats / 生理学报

Atrial Ca2+ handling abnormalities, mainly involving the dysfunction of ryanodine receptor (RyR) and sarcoplasmic reticulum Ca2+-ATPase (SERCA), play a role in the pathogenesis of atrial fibrillation (AF). Previously, we found that the expression and function of transient receptor potential vanilloid subtype 4 (TRPV4) are upregulated in a sterile pericarditis (SP) rat model of AF, and oral administration of TRPV4 inhibitor GSK2193874 alleviates AF in this animal model. The aim of this study was to investigate whether oral administration of GSK2193874 could alleviate atrial Ca2+ handling abnormalities in SP rats. A SP rat model of AF was established by daubing sterile talcum powder on both atria of Sprague-Dawley (SD) rats after a pericardiotomy, to simulate the pathogenesis of postoperative atrial fibrillation (POAF). On the 3rd postoperative day, Ca2+ signals of atria were collected in isolated perfused hearts by optical mapping. Ca2+ transient duration (CaD), alternan, and the recovery properties of Ca2+ transient (CaT) were quantified and analyzed. GSK2193874 treatment reversed the abnormal prolongation of time to peak (determined mainly by RyR activity) and CaD (determined mainly by SERCA activity), as well as the regional heterogeneity of CaD in SP rats. Furthermore, GSK2193874 treatment relieved alternan in SP rats, and reduced its incidence of discordant alternan (DIS-ALT). More importantly, GSK2193874 treatment prevented the reduction of the S2/S1 CaT ratio (determined mainly by RyR refractoriness) in SP rats, and decreased its regional heterogeneity. Taken together, oral administration of TRPV4 inhibitor alleviates Ca2+ handling abnormalities in SP rats primarily by blocking the TRPV4-Ca2+-RyR pathway, and thus exerts therapeutic effect on POAF.

TRPV4 channel mediates the increase of pulmonary microvascular endothelial permeability in rats with chronic hypoxic pulmonary hypertension / 生理学报

The purpose of the present study was to investigate the effect of transient receptor potential vanilloid 4 (TRPV4) channel on the permeability of pulmonary microvascular endothelial cells (PMVECs) in rats with chronic hypoxia-induced pulmonary hypertension (CHPH), so as to clarify the mechanism of vascular endothelial dysfunction during the occurrence of pulmonary hypertension (PH). CHPH rat model was established by exposure to chronic hypoxia (CH) for 21 days. Primary PMVECs were cultured by adherent tissue blocks at the edge of the lung. The permeability coefficient of primary cultured PMVECs was detected by fluorescein isothiocyanate (FITC)-dextran. The structure of tight junction (TJ) was observed by transmission electron microscope. The expression of TRPV4 and TJ-related proteins, such as, Occludin, Claudin-5, ZO-1 were examined by real-time fluorescence quantitative PCR and Western blotting. The intracellular calcium concentration ([Ca

Analysis of clinical feature and genetic basis of a rare case with Olmsted syndrome / 中华医学遗传学杂志

OBJECTIVE@#To analyze the clinical and genetic characteristics of a patient featuring autosomal dominant Olmsted syndrome.@*METHODS@#Clinical features of the patient was reviewed. High-throughput sequencing was carried out to detect potential genetic variants.@*RESULTS@#The proband, a 12-year-old girl, featured excessive keratinization on hands and feet, contracture of finger joints, and abnormal position and residual contraction of the fifth toes. Skin biopsy showed significant hyperkeratosis, epidermal hyperplasia, and mild interepidermal cell edema. A de novo heterozygous missense variant c.2016G>T(p.Met672Ile) was identified in the TRPV3 gene by high-throughout sequencing. The result was verified by Sanger sequencing.@*CONCLUSION@#The destructive palmoplantar keratosis in the child may be attributed to the c.2016G>T(p.Met672Ile) variant of the TRPV3 gene. Aboving finding has provided new evidence for the correlation of genetic variants with clinical phenotypes of Olmsted syndrome.

Complement C3 Aggravates Post-epileptic Neuronal Injury Via Activation of TRPV1 / 神经科学通报·英文版

Epilepsy is a brain condition characterized by the recurrence of unprovoked seizures. Recent studies have shown that complement component 3 (C3) aggravate the neuronal injury in epilepsy. And our previous studies revealed that TRPV1 (transient receptor potential vanilloid type 1) is involved in epilepsy. Whether complement C3 regulation of neuronal injury is related to the activation of TRPV1 during epilepsy is not fully understood. We found that in a mouse model of status epilepticus (SE), complement C3 derived from astrocytes was increased and aggravated neuronal injury, and that TRPV1-knockout rescued neurons from the injury induced by complement C3. Circular RNAs are abundant in the brain, and the reduction of circRad52 caused by complement C3 promoted the expression of TRPV1 and exacerbated neuronal injury. Mechanistically, disorders of neuron-glia interaction mediated by the C3-TRPV1 signaling pathway may be important for the induction of neuronal injury. This study provides support for the hypothesis that the C3-TRPV1 pathway is involved in the prevention and treatment of neuronal injury and cognitive disorders.

ASIC2 Synergizes with TRPV1 in the Mechano-Electrical Transduction of Arterial Baroreceptors / 神经科学通报·英文版

Mechanosensitive ion channels (MSCs) are key molecules in the mechano-electrical transduction of arterial baroreceptors. Among them, acid-sensing ion channel 2 (ASIC2) and transient receptor potential vanilloid subfamily member 1 (TRPV1) have been studied extensively and documented to play important roles. In this study, experiments using aortic arch-aortic nerve preparations isolated from rats revealed that both ASIC2 and TRPV1 are functionally necessary, as blocking either abrogated nearly all pressure-dependent neural discharge. However, whether ASIC2 and TRPV1 work in coordination remained unclear. So we carried out cell-attached patch-clamp recordings in HEK293T cells co-expressing ASIC2 and TRPV1 and found that inhibition of ASIC2 completely blocked stretch-activated currents while inhibition of TRPV1 only partially blocked these currents. Immunofluorescence staining of aortic arch-aortic adventitia from rats showed that ASIC2 and TRPV1 are co-localized in the aortic nerve endings, and co-immunoprecipitation assays confirmed that the two proteins form a compact complex in HEK293T cells and in baroreceptors. Moreover, protein modeling analysis, exogenous co-immunoprecipitation assays, and biotin pull-down assays indicated that ASIC2 and TRPV1 interact directly. In summary, our research suggests that ASIC2 and TRPV1 form a compact complex and function synergistically in the mechano-electrical transduction of arterial baroreceptors. The model of synergism between MSCs may have important biological significance beyond ASIC2 and TRPV1.

MicroRNA-369 attenuates hypoxia-induced cardiomyocyte apoptosis and inflammation via targeting TRPV3

Hypoxia-induced apoptosis and inflammation play an important role in cardiovascular diseases including myocardial infarction (MI). miR-369 has been suggested to be a key regulator of cardiac fibrosis. However, the role of miR-369 in regulating hypoxia-induced heart injury remains unknown. Our data indicated that miR-369 expression was significantly down-regulated and TRPV3 was significantly up-regulated in myocardial tissue after MI in rats and in hypoxic-treated neonatal rat cardiomyocytes (NRCMs). In addition, we observed that hypoxia significantly promoted apoptosis and the inflammatory response, accompanied by increased caspase-3 activity and the secretion of the cytokines interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α. miR-369 overexpression significantly suppressed cell apoptosis and inflammatory factor production triggered by hypoxia, whereas miR-369 inhibition had an opposite effect. Importantly, we identified TRPV3 as a direct target of miR-369-3p. TRPV3 inhibition with small interfering RNA (siRNA) significantly inhibited hypoxia-induced inflammation and apoptosis, which can reverse the injury effects of miR-369 inhibitors. Our findings indicated that miR-369 reduced hypoxia-induced apoptosis and inflammation by targeting TRPV3.

The protective role of selenium against dental amalgam-induced intracellular oxidative toxicity through the TRPV1 channel in DBTRG glioblastoma cells

Abstract Objective The exposure to mercury (Hg) from dental amalgams is a suspected causative factor in neurological diseases. This study investigated the toxic effects of two different amalgam compositions related to Hg and the protective effects of selenium against the toxic effects of Hg through the TRPV1 channel in the human DBTRG glioblastoma cell line. Methodology Six groups of the cells were organized. Analyses of cell viability, apoptosis, caspase 3 and caspase 9 activities, mitochondrial membrane depolarization, reactive oxygen species (ROS) production, and Western Blotting for protein expression levels were performed. Results Cell viability values were lower in amalgam with high copper (HCu) and low copper (LCu) groups independently of time but were increased by selenium and capsazepine (p<0.001 and p<0.05). Conversely, apoptosis rates, caspase 3 and caspase 9 expression, ROS formation, mitochondrial membrane depolarization, and protein expression levels were higher in the HCu and LCu groups but were decreased by selenium (p<0.001 and p<0.05). Conclusions Selenium combined with an amalgam of either HCu or LCu decreases the toxic effects created by Hg in human DBTRG glioblastoma cells.

Effect of San'ao Decoction on ovalbum induced asthmatic mice and expression of TRPV2 in lung / 中国中药杂志

To observe the efficacy of San'ao Decoction(SAD) in diffusing the lung and relieving asthma, and its intervention effect on the expression of transient receptor potential V2(TRPV2) during alleviating asthma, this study replicated an ovalbumin(OVA)-induced asthmatic mice model, and investigated the intervention effect of SAD on the airway inflammation and airway hyperresponsiveness. The regulatory mechanisms of SAD on the mRNA and protein expressions of TRPV2 in lung tissues and the levels of interleukin-4(IL-4),-10(IL-10), nerve growth factor(NGF), prostaglandin D_2(PGD_2) in bronchoalveolar lavage fluid(BALF) were discussed. Compared with the control group, the model group showed typical asthmatic phenotype, the level of eosinophils(EOS) in peripheral blood and BALF as well as the airway hyperresponsiveness were increased(P<0.01), and pathological damage in lung tissue was serious. The mRNA and protein expressions of TRPV2 in lung tissue were increased significantly, while the levels of IL-4, IL-10, NGF and PGD_2 in BALF were elevated(P<0.05,P<0.01). SAD could relieve bronchial asthma manifested as repaired lung patholo-gical changes(P<0.05), reduce the level of EOS in blood and BALF(P<0.05, P<0.01), and improve pulmonary resistance and lung compliance(P<0.05, P<0.01). SAD could also regulate the inflammatory cytokine levels of IL-4, IL-10, NGF, PGD_2 in BALF, and reduce the gene and protein expression of TRPV2 in the lung tissue(P<0.05, P<0.01). It is verified that SAD could reduce the lung inflammation, and improve lung function in asthmatic mice. The regulatory mechanism of SAD on asthma induced by OVA might be related to the regulation of TRPV2 expression and the induced decrease of Th2-related cytokines and neuropeptides, which provides the evidences for the treatment of asthma with SAD.

Cannabis and the exocannabinoid and endocannabinoid systems. Their use and controversies

Cannabis (marijuana) is one of the most consumed psychoactive substances in the world. The term marijuana is of Mexican origin. The primary cannabinoids that have been studied to date include cannabidiol and delta-9-tetrahydrocannabinol, which is responsible for most cannabis physical and psychotropic effects. Recently, the endocannabinoid system was discovered, which is made up of receptors, ligands and enzymes that are widely expressed in the brain and its periphery, where they act to maintain balance in several homeostatic processes. Exogenous cannabinoids or naturally-occurring phytocannabinoids interact with the endocannabinoid system. Marijuana must be processed in a laboratory to extract tetrahydrocannabinol and leave cannabidiol, which is the product that can be marketed. Some studies suggest cannabidiol has great potential for therapeutic use as an agent with antiepileptic, analgesic, anxiolytic, antipsychotic, anti-inflammatory and neuroprotective properties; however, the findings on cannabinoids efficacy and cannabis-based medications tolerability-safety for some conditions are inconsistent. More scientific evidence is required in order to generate recommendations on the use of medicinal cannabis.

Transient receptor potential Vanilloid 1-based gene therapy alleviates orthodontic pain in rats / 国际口腔科学杂志·英文版

Orthodontic pain that is induced by tooth movement is an important sequela of orthodontic treatment and has a significant effect on patient quality of life. Studies have shown that the high expression of transient receptor potential vanilloid 1 (TRPV1) in trigeminal ganglions plays a vital role in the transmission and modulation of orofacial pain. However, little is known about the role of TRPV1 in orthodontic pain. In this study, male Sprague-Dawley rats were randomly assigned to six groups to study the role of TRPV1 in the modulation of tooth-movement pain. The expression levels of TRPV1 mRNA and protein were determined by real-time PCR and western blot, respectively. Moreover, pain levels were assessed using the rat grimace scale (RGS). The role of TRPV1 in modulating tooth-movement pain was examined by injecting a TRPV1 antagonist into the trigeminal ganglia of rats. A lentivirus containing a TRPV1 shRNA sequence was constructed and transduced into the rats' trigeminal ganglia. The results showed that the expression levels of TRPV1 protein and mRNA were elevated following tooth-movement pain. Pain levels increased rapidly on the 1 day, peaked on the 3 day and returned to baseline on the 14 day. The TRPV1 antagonist significantly reduced tooth-movement pain. The lentivirus containing a TRPV1 shRNA sequence was able to inhibit the expression of TRPV1 and relieved tooth-movement pain. In conclusion, TRPV1-based gene therapy may be a treatment strategy for the relief of orthodontic pain.

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells / Caracterização farmacológica das vias de influxo de cálcio envolvidas na produção de óxido nítrico por células endoteliais

ABSTRACT Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

Targeting the hTRPV6 with capsaicinoid inhibitors

Capsaicin is a substance produced by Capsicum peppers with extensive biological activity reported in the literature. Among these studies, it was suggested that the anti-tumor activity is related to modulation of the Transient Potential Receptor Vanilloid (TRPV) channels. Capsaicin is known to bind with very high affinity to TRPV1 (IC50 ≈ 7 nM), triggering the burning sensation followed by analgesia. However, recent studies have suggested that the pro-apoptotic effects of capsaicin are TRPV6-mediated. Herein we report the development of a novel inhibitor of the TRPV6 using two different strategies for compounds design. We generated a series of direct and chimeric capsaicinoids based on the literature compounds, capsaicin, and cis-22a. These analogs were probed against HEK-hTRPV6 and the hits were further optimized. Based on the previous SAR and chemical optimization, we found 56h, named MRC-130, a derivative that remarkably inhibited TRPV6 in the nanomolar range (IC50 = 83 ± 4 nM), possess high selectivity and stability in vitro, and lesser hERG inhibition compared to the reference compound, cis-22a. It is expected that these new molecules would contribute significantly to the study on the TRPV6 function and its role in tumor pathophysiology

Capsaicina é uma substância produzida por pimentas do gênero Capsicum com extensa atividade biológica relatada na literatura. Entre esses estudos, sugeriu-se que a atividade antitumoral esteja relacionada à modulação dos canais TRPV (do inglês, Transient Potential Receptor Vanilloid). Sabe-se que a capsaicina se liga com altíssima afinidade ao TRPV1 (IC50 ≈ 7 nM), desencadeando a sensação de queimação seguida de analgesia. No entanto, estudos recentes sugeriram que os efeitos pró-apoptóticos da capsaicina são mediados pelo TRPV6. Visando o exposto, este trabalho relata o desenvolvimento de um novo inibidor do TRPV6 usando duas estratégias diferentes para o planejamento dos compostos. Geramos séries de capsaicinoides diretos e quiméricos com base nos compostos da literatura, capsaicina e cis-22a. Esses análogos foram analisados contra HEK-hTRPV6 e os análogos mais promissores foram otimizados. Com base na REA e em otimizações químicas anteriores, encontramos 56h, chamado MRC-130, um derivado que inibiu notavelmente o TRPV6 na faixa nanomolar (IC50 = 83 ± 4 nM), possui alta seletividade e estabilidade in vitro e menor inibição de hERG em comparação com o composto de referência, cis-22a. Espera-se que essas novas moléculas contribuam significativamente para o estudo da função do TRPV6 e seu papel na fisiopatologia tumoral

Olmsted Syndrome Caused by a Heterozygous p.Gly568Val Missense Mutation in TRPV3 Gene

Olmsted syndrome (OS) is a rare congenital skin disorder characterized by severe palmoplantar and periorificial keratoderma, alopecia, onychodystrophy, and severe pruritus. Recently, pathogenic ‘gain-of-function‘ mutations of the transient receptor potential vanilloid 3 gene (TRPV3), which encodes a cation channel involved in keratinocyte differentiation and proliferation, hair growth, inflammation, pain and pruritus, have been identified to cause OS. Due to the rarity, the pattern of inheritance of OS is still unclear. We report a case of OS in a 3-year-old Korean girl and its underlying gene mutation. The patient presented with a disabling, bilateral palmoplantar keratoderma with onychodystrophy. She also exhibited pruritic eczematous skin lesions around her eyes, ears and gluteal fold. Genetic analysis identified a heterozygous p.Gly568Val missense mutation in the exon 13 of TRPV3. To our knowledge, this is the first case of OS in the Korean population showing a missense mutation p.Gly573Ser.