Active Components of Leontopodium alpinum Callus Culture Extract for Blue Light Damage in Human Foreskin Fibroblasts.

Leontopodium alpinum is a source of raw material for food additives and skin health. The purpose of this study was to investigate the application of Leontopodium alpinum callus culture extract (LACCE) to prevent blue light damage to the skin. We screened and identified the blue light-damage-protecting activities and mechanisms of ten components of LACCE, including chlorogenic acid (A), isoquercitrin (B), isochlorogenic acid A (C), cynaroside (D), syringin (E), isochlorogenic acid (F), cynarin (G), rutin (H), leontopodic acid A (I), and leontopodic acid B (J), using a novel blue light-induced human foreskin fibroblast (HFF-1) cell injury model. The study examined the cytotoxicity of ten ingredients using the cell counting kit-8 (CCK-8) assay, and selecting concentrations of 5, 10, and 20 µM for experiments with a cell viability above 65%. We explored the effects and mechanisms of action of these LACCE components in response to blue light injury using Western blotting and an enzyme-linked immunosorbent assay. We also measured ROS secretion and Ca2+ influx. Our study revealed that leontopodic acid A effectively boosted COI-1 expression, hindered MMP-1 expression, curbed ROS and Ca2+ endocytosis, and reduced OPN3 expression. These results provide theoretical support for the development of new raw materials for the pharmaceutical and skincare industries.

Effects and Mechanism of the Leontopodium alpinum Callus Culture Extract on Blue Light Damage in Human Foreskin Fibroblasts.

Leontopodium alpinum is an important source of raw material for food, medicine, and modern cosmetics. The purpose of this study was to develop a new application for protection against blue light damage. To investigate the effects and mechanism of action of Leontopodium alpinum callus culture extract (LACCE) on blue light damage, a blue-light-induced human foreskin fibroblast damage model was established. The contents of collagen (COL-I), matrix metalloproteinase 1 (MMP-1), and opsin 3 (OPN3) were detected using enzyme-linked immunosorbent assays and Western blotting. The calcium influx and reactive oxygen species (ROS) levels were measured via flow cytometry and the results showed that the LACCE (10-15 mg/mL) promoted the production of COL-I, inhibited the secretion of MMP-1, OPN3, ROS and calcium influx, and may play a role in inhibiting the activation of blue light on the OPN3-calcium pathway. Thereafter, high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry were used to quantitatively analyze the contents of nine active ingredients in the LACCE. The results indicated that LACCE has an anti-blue-light-damage effect and provides theoretical support for the development of new raw materials in the natural food, medicine, and skin care industries.

Estrogen antagonizes ASIC1a-induced chondrocyte mitochondrial stress in rheumatoid arthritis.

BACKGROUND: Destruction of articular cartilage and bone is the main cause of joint dysfunction in rheumatoid arthritis (RA). Acid-sensing ion channel 1a (ASIC1a) is a key molecule that mediates the destruction of RA articular cartilage. Estrogen has been proven to have a protective effect against articular cartilage damage, however, the underlying mechanisms remain unclear. METHODS: We treated rat articular chondrocytes with an acidic environment, analyzed the expression levels of mitochondrial stress protein HSP10, ClpP, LONP1 by q-PCR and immunofluorescence staining. Transmission electron microscopy was used to analyze the mitochondrial morphological changes. Laser confocal microscopy was used to analyze the Ca2+, mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) level. Moreover, ASIC1a specific inhibitor Psalmotoxin 1 (Pctx-1) and Ethylene Glycol Tetraacetic Acid (EGTA) were used to observe whether acid stimulation damage mitochondrial function through Ca2+ influx mediated by ASIC1a and whether pretreatment with estrogen could counteract these phenomena. Furthermore, the ovariectomized (OVX) adjuvant arthritis (AA) rat model was treated with estrogen to explore the effect of estrogen on disease progression. RESULTS: Our results indicated that HSP10, ClpP, LONP1 protein and mRNA expression and mitochondrial ROS level were elevated in acid-stimulated chondrocytes. Moreover, acid stimulation decreased mitochondrial membrane potential and damaged mitochondrial structure of chondrocytes. Furthermore, ASIC1a specific inhibitor PcTx-1 and EGTA inhibited acid-induced mitochondrial abnormalities. In addition, estrogen could protect acid-stimulated induced mitochondrial stress by regulating the activity of ASIC1a in rat chondrocytes and protects cartilage damage in OVX AA rat. CONCLUSIONS: Extracellular acidification induces mitochondrial stress by activating ASIC1a, leading to the damage of rat articular chondrocytes. Estrogen antagonizes acidosis-induced joint damage by inhibiting ASIC1a activity. Our study provides new insights into the protective effect and mechanism of action of estrogen in RA.

Consumption of Artificial Sweetener Acesulfame Potassium Increases Preterm Risk and Uterine Contraction with Calcium Influx Increased via Myosin Light Chain Kinase-Myosin Light Chain 20 Related Signaling Pathway.

SCOPE: The consumption of artificial sweeteners has been rapidly increasing, with potentially hazardous effects on human reproduction. This study aims to explore the effect of Acesulfame Potassium (Ace K) and its potential mechanism to induce uterine contraction through in vitro, ex vivo, in vivo, and clinical observation studies. METHODS AND RESULTS: Used ex vivo and in vitro studies to analyze its effect on uterine contraction and involved signaling pathway. Used the long-term, high-dose exposure to examine Ace K's affection for contractive-related protein expression. By involving a cohort of 613 participants, to assess the dose-responsiveness of Ace K consumption and calculate the odd ratio of Ace K consumption and the relationship with preterm risk. Animal studies show increasing uterine contraction, cytokine secretion, and altered contraction-related protein expression. Human data show that higher consumption of Ace K may be related to early delivery. CONCLUSION: Long-term high-dose exposure to Ace K can induce uterine hypercontraction, increase cytokine secretion, and alters contraction-related protein expression. These findings suggest that women who suffer from uterine hypercontraction causes painfulness should pay more attention to the zero- or low-calorie soft drinks or food products containing Ace K.

Neutrophil Immunomodulatory Activity of (-)-Borneol, a Major Component of Essential Oils Extracted from Grindelia squarrosa.

Grindelia squarrosa (Pursh) Dunal is used in traditional medicine for treating various diseases; however, little is known about the immunomodulatory activity of essential oils from this plant. Thus, we isolated essential oils from the flowers (GEOFl) and leaves (GEOLv) of G. squarrosa and evaluated the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of these essential oils revealed that the main components were α-pinene (24.7 and 23.2% in GEOFl and GEOLv, respectively), limonene (10.0 and 14.7%), borneol (23.4 and 16.6%), p-cymen-8-ol (6.1 and 5.8%), ß-pinene (4.0 and 3.8%), bornyl acetate (3.0 and 5.1%), trans-pinocarveol (4.2 and 3.7%), spathulenol (3.0 and 2.0%), myrtenol (2.5 and 1.7%), and terpinolene (1.7 and 2.0%). Enantiomer analysis showed that α-pinene, ß-pinene, and borneol were present primarily as (-)-enantiomers (100% enantiomeric excess (ee) for (-)-α-pinene and (-)-borneol in both GEOFl and GEOLv; 82 and 78% ee for (-)-ß-pinene in GEOFl and GEOLv), while limonene was present primarily as the (+)-enantiomer (94 and 96 ee in GEOFl and GEOLv). Grindelia essential oils activated human neutrophils, resulting in increased [Ca2+]i (EC50 = 22.3 µg/mL for GEOFl and 19.4 µg/mL for GEOLv). In addition, one of the major enantiomeric components, (-)-borneol, activated human neutrophil [Ca2+]i (EC50 = 28.7 ± 2.6), whereas (+)-borneol was inactive. Since these treatments activated neutrophils, we also evaluated if they were able to down-regulate neutrophil responses to subsequent agonist activation and found that treatment with Grindelia essential oils inhibited activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, (-)-borneol inhibited FPR-agonist-induced Ca2+ influx in neutrophils. Grindelia leaf and flower essential oils, as well as (-)-borneol, also inhibited fMLF-induced chemotaxis of human neutrophils (IC50 = 4.1 ± 0.8 µg/mL, 5.0 ± 1.6 µg/mL, and 5.8 ± 1.4 µM, respectively). Thus, we identified (-)-borneol as a novel modulator of human neutrophil function.

Cardioprotective effects of alantolactone on isoproterenol-induced cardiac injury and cobalt chloride-induced cardiomyocyte injury.

OBJECTIVES: Alantolactone (AL) is a compound extracted from the roots of Inula Racemosa that has shown beneficial effects in cardiovascular disease. However, the cardioprotective mechanism of AL against hypoxic/ischemic (H/I) injury is still unclear. This research aimed to determine AL's ability to protect the heart against isoproterenol (ISO)-induced MI injury in vivo and cobalt chloride (CoCl2) induced H/I injury in vitro. METHODS: Electrocardiography (ECG), lactate dehydrogenase (LDH), creatine kinase (CK), and cardiac troponin I (cTnI) assays in addition to histological analysis of the myocardium were used to investigate the effects of AL in vivo. Influences of AL on L-type Ca2+ current (ICa-L) in isolated rat myocytes were observed by the patch-clamp technique. Furthermore, cell viability, apoptosis, oxidative stress injury, mitochondrial membrane potential, and intracellular Ca2+ concentration were examined in vitro. RESULTS: The results indicated that AL treatment ameliorated the morphological and ECG changes associated with MI, and decreased levels of LDH, CK, and cTnI. Furthermore, pretreatment with AL elevated antioxidant enzyme activity and suppressed ROS production. AL prevented H/I-induced apoptosis, mitochondria damage, and calcium overload while reducing ICa-L in a concentration and time dependent fashion. The 50% inhibiting concentration (IC50) and maximal inhibitory effect (Emax) of AL were 17.29 µmol/L and 57.73 ± 1.05%, respectively. CONCLUSION: AL attenuated MI-related injury by reducing oxidative stress, apoptosis, calcium overload, and mitochondria damage. These cardioprotective effects may be related to the direct inhibition of ICa-L.

Chemical constituents from the fruits of Piper longum L. and their vascular relaxation effect on rat mesenteric arteries.

Eight compounds were obtained from the dry fruits of Piper longum L., and their potential vascular relaxant activities were explored. The present study first revealed the access of Rosin (7) and Piperchabaoside (8) in the medicinal plant Piper longum L. The vessel tension studies showed that Piperine (2), (2E,4E,14Z)-N-isobutyleicosa-2,4,14-trienamide (3), and Piperlonguminine (6) exerted significant inhibitory effects on PE-induced mesenteric artery vasoconstriction. Furthermore, Calcium Imaging studies were applied to observe the effect of Piperine on the intracellular calcium in mesenteric artery smooth muscle cells (MASMCs). Piperine (2) was observed to promote the influx of extracellular calcium in MASMCs, and via an endothelium-independent mechanism involving Ca2+ entry. Piper longum L. might have a great potential to be further studied as a vascular relaxant, even to be a drug candidate of anti-hypertension.

Pharmacological effects of Pugionium cornutum (L.) Gaertn. extracts on gastrointestinal motility are partially mediated by quercetin.

BACKGROUND: The majority of global population suffer from various functional gastrointestinal disorders. Pugionium cornutum (L.) Gaertn. (PCG) is used to relieve indigestive symptoms in traditional Chinese medicine. However, little is known about the effects of bioactive components from PCG extracts on gastrointestinal motility. METHODS: Crude ethanol extract of PCG (EEP) was prepared from Pugionium cornutum (L.) Gaertn. Different solvents were used to prepare fine extracts from EEP, including water extract of PCG (WEP), petroleum ether extract of PCG (PEEP), dichloromethane extract of PCG (DEP) and ethyl acetate extract of PCG (EAEP). Smooth muscle cell model and colonic smooth muscle stripe model were used to test the bioactive effects and mechanisms of different PCG extracts on contraction and relaxation. Diverse chromatographic methods were used to identify bioactive substances from PCG extracts. RESULTS: EEP was found to promote the relaxation of gastric smooth muscle cell and inhibit the contraction of colonic smooth muscle strip. Among the fractions of EEP, EAEP mainly mediated the relaxation effect by stimulating intracellular calcium influx. Further evidences revealed that EAEP was antagonistic to acetylcholine. In addition, COX and NO-GC-PKC pathways may be also involved in EAEP-mediated relaxation effect. Quercetin was identified as a bioactive compound from PCG extract for the relaxation effect. CONCLUSION: Our research supports the notion that PCG extracts promote relaxation and inhibits contraction of gastrointestinal smooth muscle at least partially through the effect from quercetin.

Ketone Body 3-Hydroxybutyrate Ameliorates Atherosclerosis via Receptor Gpr109a-Mediated Calcium Influx.

Atherosclerosis is a chronic inflammatory disease that can cause acute cardiovascular events. Activation of the NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) inflammasome enhances atherogenesis, which links lipid metabolism to sterile inflammation. This study examines the impact of an endogenous metabolite, namely ketone body 3-hydroxybutyrate (3-HB), on a mouse model of atherosclerosis. It is found that daily oral administration of 3-HB can significantly ameliorate atherosclerosis. Mechanistically, 3-HB is found to reduce the M1 macrophage proportion and promote cholesterol efflux by acting on macrophages through its receptor G-protein-coupled receptor 109a (Gpr109a). 3-HB-Gpr109a signaling promotes extracellular calcium (Ca2+) influx. The elevation of intracellular Ca2+ level reduces the release of Ca2+ from the endothelium reticulum (ER) to mitochondria, thus inhibits ER stress triggered by ER Ca2+ store depletion. As NLRP3 inflammasome can be activated by ER stress, 3-HB can inhibit the activation of NLRP3 inflammasome, which triggers the increase of M1 macrophage proportion and the inhibition of cholesterol efflux. It is concluded that daily nutritional supplementation of 3-HB attenuates atherosclerosis in mice.

Endothelium-independent vasorelaxant effect of Asphodelus tenuifolius Cav. via inhibition of myosin light chain kinase activity in the porcine coronary artery.

ETHNOPHARMACOLOGICAL RELEVANCE: Asphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. AIM OF THE STUDY: This study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. MATERIALS AND METHODS: Aqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. RESULTS: Crude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. CONCLUSION: The butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.

Voltage-gated sodium channel Nav1.5 promotes tumor progression and enhances chemosensitivity to 5-fluorouracil in colorectal cancer.

Nav1.5, encoded by SCN5A, has been associated with metastasis in colorectal cancer (CRC). Here, we investigated the mechanism by which Nav1.5 regulates tumor progression and whether Nav1.5 influences chemosensitivity to 5-fluorouracil (5-FU) in CRCs. CRC cases were evaluated for Nav1.5 expression. Elevated Nav1.5 expression was associated with poor prognosis in CRCs, whereas stage II/III patients with upregulated SCN5A expression could have better survival after receiving 5-FU-based adjuvant chemotherapy. In CRC cells, SCN5A knockdown reduced the proliferation, migration and invasion. According to RNA sequencing, SCN5A knockdown inhibited both the cell cycle and epithelial-mesenchymal transition. In addition, Nav1.5 stabilized the KRas-calmodulin complex to modulate Ras signaling, promoting Ca2+ influx through the Na+-Ca2+ exchanger and Ca2+ release-activated calcium channel. Meanwhile, SCN5A knockdown increased the 50% inhibitory concentration to 5-FU by upregulating 5-FU-stimulated apoptosis in CRCs. In conclusion, Nav1.5 could progress to proliferation and metastasis through Ca2+/calmodulin-dependent Ras signaling in CRC, and it could also enhance 5-FU-stimulated apoptosis. Clinically, patients with stage II/III CRCs with elevated SCN5A expression demonstrated poor prognosis, yet those patients could benefit more from 5-FU-based chemotherapy than patients with lower SCN5A expression.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

The vascular dilatation induced by Hydroxysafflor yellow A (HSYA) on rat mesenteric artery through TRPV4-dependent calcium influx in endothelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hydroxysafflor yellow A (HSYA) is the principal constituent of the flowers of Carthamus tinctorius L., a traditional Chinese herbal medicine, which has been used for the treatment of cerebrovascular and cardiovascular diseases due to its property of promoting blood circulation and removing blood stasis. It is dominated in the water extract of Carthamus tinctorius L., which has been used in the clinical treatment for cardiovascular diseases. HSYA exerts a variety of pharmacological efficacy upon the vascular system. However, the underlying mechanisms remain unclear. AIM OF THE STUDY: To investigate the vascular dilatation effect of HSYA on rat mesenteric artery (MA) and its potential mechanism. MATERIALS AND METHODS: Adult male Wistar rats were applied to the study. Tension studies were conducted to determine the dilatation activity of HSYA against pre-contracted mesenteric arterial (MA) rings by U 46619 and Phenylephrine (PE). The vascular activities were measured with or without incubation with some selective inhibitors, including L-N(ω)-nitro-L-arginine methyl ester (L-NAME, a nitro oxide synthase inhibitor), HC-067047 (a selective TRPV4 antagonist), BaCl2 (a Kir channel blocker), and Indomethacin (Indo, a nonselective cyclooxygenase inhibitor), respectively. Immunocytochemistry, Calcium Imaging, NO Production detection, and Western Blot were also employed to further study the underlying mechanism. RESULTS: HSYA reversed the constriction of MAs induced by U 46619 in a manner of concentration dependency, and the dilatation capability was reversed by L-NAME. This effect was significantly dependent on the intactness of MA endothelium, accompanying an increment of NO production in mesenteric arterial endothelium cells. The increment of NO production was reversed by inhibiting the PKA. Also, the expression of p-eNOS was activated by HSYA shown in Western Blot assays. The cells imaging revealed a significant increase and drop of the influx of Ca2+ before and after treatment with HC-067047. CONCLUSIONS: These findings suggest that HSYA exerts vessel dilation effect on MAs via a TRPV4-dependent influx of Ca2+ in endothelium cells, PKA-dependent eNOS phosphorylation and NO production mechanism. The present study indicates that HSYA has the potential to be a future candidate for the treatment of hypertension.

Topical transient receptor potential ankyrin 1 antagonist treatment attenuates nociception and inflammation in an ultraviolet B radiation-induced burn model in mice.

BACKGROUND: Ultraviolet B (UVB) radiation exposure promotes sunburn and thereby acute and chronic inflammatory processes, contributing to pain development and maintenance. New therapeutic alternatives are necessary because typical treatments can cause adverse effects. An attractive alternative would be to target the transient receptor potential ankyrin 1 (TRPA1), a calcium-permeable, non-selective cation channel, which is involved in a variety of inflammatory pain models. OBJECTIVE: Evaluate the peripheral participation of TRPA1 using a topical treatment (HC030031 gel formulation; a selective TRPA1 antagonist) in nociception and inflammation caused by a UVB radiation-induced burn model in male mice (25-30 g). METHODS: The mice were anaesthetised, and just the right hind paw was exposed to UVB radiation (0.75 J/cm2). Topical treatments were applied immediately after irradiation and once a day for 8 days. RESULTS: HC030031 gel presented suitable pH and spreadability factor, ensuring its quality and the therapeutic effect. HC030031 0.05 % reversed UVB-induced mechanical and cold allodynia, with maximum inhibition (Imax) of 69 ± 13 % and 100 % (on day 4), respectively. HC030031 0.05 % also reduced the paw edema and MPO activity, with Imax of 77 ± 6 % (on day 5) and 69 ± 28 %, respectively. Likewise, UVB radiation increased the H2O2 levels (a TRPA1 agonist) and the Ca2+ influx in mice spinal cord synaptosomes. UVB radiation-induced Ca2+ influx was reduced by HC030031. CONCLUSION: These findings confirm the activation of the TRPA1 channel by UVB radiation, suggesting that topical TRPA1 antagonists can be a new strategy for the adjuvant treatment of sunburn-associated pain and inflammation.

Electrical Stimulation Degenerated Cochlear Synapses Through Oxidative Stress in Neonatal Cochlear Explants.

Neurostimulation devices use electrical stimulation (ES) to substitute, supplement or modulate neural function. However, the impact of ES on their modulating structures is largely unknown. For example, recipients of cochlear implants using electroacoustic stimulation experienced delayed loss of residual hearing over time after ES, even though ES had no impact on the morphology of hair cells. In this study, using a novel model of cochlear explant culture with charge-balanced biphasic ES, we found that ES did not change the quantity and morphology of hair cells but decreased the number of inner hair cell (IHC) synapses and the density of spiral ganglion neuron (SGN) peripheral fibers. Inhibiting calcium influx with voltage-dependent calcium channel (VDCC) blockers attenuated the loss of SGN peripheral fibers and IHC synapses induced by ES. ES increased ROS/RNS in cochlear explants, but the inhibition of calcium influx abolished this effect. Glutathione peroxidase 1 (GPx1) and GPx2 in cochlear explants decreased under ES and ebselen abolished this effect and attenuated the loss of SGN peripheral fibers. This finding demonstrated that ES induced the degeneration of SGN peripheral fibers and IHC synapses in a current intensity- and duration-dependent manner in vitro. Calcium influx resulting in oxidative stress played an important role in this process. Additionally, ebselen might be a potential protector of ES-induced cochlear synaptic degeneration.

Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Nectandra grandiflora essential oil and its isolated sesquiterpenoids minimize anxiety-related behaviors in mice through GABAergic mechanisms.

Nectandra grandiflora Ness (Lauraceae) essential oil (EO) main constituent, the sesquiterpenoid (+)-dehydrofukinone (DHF), has sedative and anticonvulsant effects through GABAergic mechanisms. Other DHF-related sesquiterpenoids have been identified in the EO, such as, dehydrofukinone epoxide (DFX), eremophil-11-en-10-ol (ERM) and selin-11-en-4-α-ol (SEL). However, the neuronal effects of these compounds in mammals remain unknown. Therefore, the aim of this study was to evaluate the anxiolytic potential of the N. grandiflora EO and the isolated compounds in in mice. For this purpose, mice were administered orally with vehicle, 10, 30 or 100 mg/kg EO, DHF, DFX, ERM or SEL or 1 mg/kg diazepam. Locomotion and ethological parameters in the open field (OF) and elevated plus maze (EPM) were recorded. We also examined the effect of DFX, ERM and SEL on the membrane potential and calcium influx in synaptosomes, and the presence of the compounds in the cortical tissue using gas chromatography. EOs and isolated compounds reduced anxiety-related parameters in the EPM (open arms time and entries, end activity, head dipping) and OF (center time and entries, total rearing, unprotected rearing, sniffing, grooming) without alter ambulation or induce sedation. Flumazenil (2 mg/kg, i.p.) altered the anxiolytic-like effect of all treatments and vanished the DFX, ERM and SEL-induced changes in membrane potential. However, FMZ did not blocked the DFX-, ERM- and SEL-induced inhibition of calcium influx. Therefore, our results suggest that N. grandiflora EO and isolated compounds induced anxiolytic-like effect in mice due to positive modulation of GABAa receptors and/or inhibition of neuronal calcium influx.

Endothelial-Dependent and Independent Vascular Relaxation Effect of Tetrahydropalmatine on Rat Aorta.

Tetrahydropalmatine (THP) is an active natural alkaloid isolated from Corydalis yanhusuo W.T. Wang which has been widely used for treating pain and cardiovascular disease in traditional Chinese medicine. Previous studies suggested THP have various pharmacological effects in neural and cardio tissue while the vascular reactivity of THP was not fully established. The present study found that THP relaxed rat aorta which contracted by phenylephrine (Phe), KCl, and U46619. The vascular relaxation effect of THP was partially attenuated by PI3K inhibitor wortmannin, Akt inhibitor IV, endothelial nitric oxide synthetase (eNOS) inhibitor L-NAME, guanylate cyclase inhibitors and the mechanical removal of endothelium. Also, the eNOS substrate L-arginine reversed the inhibition effect of L-NAME on THP-induced vascular relaxation. THP also induced intracellular NO production in human umbilical vein endothelial cells. However, Pre-incubation with ß-adrenergic receptor blocker propranolol, angiotensin II receptor 1 (AT1) inhibitor losartan, angiotensin II receptor 2 (AT1) inhibitor PD123319 or angiotensin converting enzyme inhibitor enalapril enhanced the vascular relaxation effect of THP. THP did not affect the angiotensin II induced vascular contraction. Cyclooxygenase-2 (COX2) inhibitor indomethacin did not affect the vascular relaxation effect of THP. Furthermore, pre-treatment THP attenuated KCl and Phe induced rat aorta contraction in standard Krebs solution. In Ca2+ free Krebs solution, THP inhibited the Ca2+ induced vascular contraction under KCl or Phe stress and reduced KCl stressed Ca2+ influx in rat vascular smooth muscle cells. THP also inhibited intracellular Ca2+ release induced vascular contraction by blocking Ryr or IP3 receptors. In addition, the voltage-dependent K+ channel (Kv) blocker 4-aminopyridine, ATP-sensitive K+ channel (KATP) blocker glibenclamide and inward rectifying K+ channel blocker BaCl2 attenuated THP induced vascular relaxation regardless of the Ca2+-activated K+ channel (KCa) blocker tetraethylammonium. Thus, we could conclude that THP relaxed rat aorta in an endothelium-dependent and independent manner. The underlying mechanism of THP relaxing rat aorta involved PI3K/Akt/eNOS/NO/cGMP signaling path-way, Ca2+ channels and K+ channels rather than COX2, ß-adrenergic receptor and renin-angiotensin system (RAS). These findings indicated that THP might be a potent treatment of diseases with vascular dysfunction like hypertension.

TRPA1 involvement in analgesia induced by Tabernaemontana catharinensis ethyl acetate fraction in mice.

BACKGROUND: Ionic channels such as the transient receptor potential ankyrin 1 (TRPA1) are essential for the detection and transmission of painful stimuli. In this sense, new TRPA1 antagonists have been searched as analgesics. PURPOSE: Preclinical studies support the antinociceptive activity of Tabernaemontana catharinensis ethyl acetate fraction (Eta), which has constituents previously identified as TRPA1 antagonists (gallic acid). It was verified for the first time the involvement of the TRPA1 on Eta's antinociceptive and anti-inflammatory effects in mice pain models. STUDY DESIGN: It was evaluated the Eta's effect (0.01-100 mg/kg, oral route) on nociceptive (spontaneous nociception, mechanical and cold allodynia) and inflammatory (paw edema) parameters in pain models involved with TRPA1 activation. METHODS: Firstly, it was investigated the ability of Eta to act on TRPA1 or TRPV1 channels (Ca2+influx and binding assays in mice spinal cords). Next, it was evaluated the Eta's antinociceptive and anti-inflammatory effects after intraplantar injection of TRPA1 agonists (hydrogen peroxide, cinnamaldehyde or allyl isothiocyanate) in male Swiss mice (30-35 g). Moreover, the Eta's antinociceptive effects were evaluated on complete Freund's adjuvant (CFA)-induced chronic inflammatory pain (CIP), postoperative pain and on paclitaxel-induced peripheral neuropathy (PIPN). Oxidative parameters were evaluated in mice paw utilized for CFA induced-CIP model. RESULTS: Eta inhibited the TRPA1 agonist-induced Ca2+ influx [Imax = 72.4 ±â€¯1.5%; IC50 = 0.023(0.004-0.125)µg/ml], but not TRPV1 agonist-induced, nor was able to displace [3H]-resiniferatoxin (TRPV1 agonist) binding. Eta (0.1-100 mg/kg) inhibited the spontaneous nociception [ID50 = 0.043(0.002-0.723)mg/kg], mechanical [ID50 = 7.417(1.426-38.570)mg/kg] and cold allodynia, and edema development caused by TRPA1 agonists. Moreover, Eta (100 mg/kg) prevented and reversed the CFA-induced CIP (Imax = 55.8 ±â€¯13.7%, Imax = 80.4 ±â€¯5.1%, respectively) and postoperative pain (Imax = 88.0 ±â€¯11.6%, Imax = 51.3 ±â€¯14.9%, respectively), been also effective in reversing the acute (Imax = 94.4 ±â€¯12.4%) and chronic (Imax = 86.8 ±â€¯8.6%) PIPN. These effects seem to occur by TRPA1 channels pathway, and independently of TRPV1 or oxidative mechanisms. CONCLUSION: Our results demonstrate that Eta-induced antinociception and anti-inflammatory effects occur by TRPA1 inhibition making possible the use of this preparation as a potential therapeutic agent to treat pathological pains.

Susceptibility of epithelial tumour cell lines to hyperthermia.

BACKGROUND: Human skin or mucosa exposes cells to both an internal and exogeneous thermal environment and the cells survive within a certain range of temperature. Exogeneous hyperthermia has been applied for the treatment of various types of cancers, fungal disease, and warts. OBJECTIVES: To determine whether different cellular components in the skin adapt to hyperthermic conditions differentially and further elucidate the mechanisms involved. MATERIALS & METHODS: Cell lines derived from normal and tumour epithelial cells were treated with hyperthermic conditions and tested for viability (using an MTS assay), apoptosis (using a FITC-conjugated annexin V apoptosis detection kit), and changes in intracellular calcium (using a calcium-sensitive fluorescent single-wavelength dye, Fluo-4 AM). RESULTS: Thermo-resistance of different cell types was different when cells were subjected to heat at 45̊C for 30 minutes. Stronger effects of hyperthermia were noted on cell viability and apoptosis in epidermal cells relative to their malignant counterparts, except for cell lines harbouring human papillomavirus (HPV). Hyperthermia had a much greater effect on cell viability and apoptosis in a HPV-negative cell line compared to HPV-positive cell lines. We further found that hyperthermia treatment resulted in a strong calcium influx which led to apoptotic cells. However, no obvious increase in apoptosis was observed in cells treated with the CRAC channel selective inhibitor, BTP2, before application of hyperthermia in all cell types, except three cervical cell lines harbouring HPV. CONCLUSION: We propose that hyperthermia results in a CRAC-related strong calcium influx which induces apoptosis, with the exception of HPV-positive cells.