Oral administration of a dual ETA/ETB receptor antagonist promotes neuroprotection in a rodent model of glaucoma.

Purpose: Glaucoma is a neurodegenerative disease associated with elevated intraocular pressure and characterized by optic nerve axonal degeneration, cupping of the optic disc, and loss of retinal ganglion cells (RGCs). The endothelin (ET) system of vasoactive peptides (ET-1, ET-2, ET-3) and their G-protein coupled receptors (ETA and ETB receptors) have been shown to contribute to the pathophysiology of glaucoma. The purpose of this study was to determine whether administration of the endothelin receptor antagonist macitentan was neuroprotective to RGCs and optic nerve axons when administered after the onset of intraocular pressure (IOP) elevation in ocular hypertensive rats. Methods: Male and female Brown Norway rats were subjected to the Morrison model of ocular hypertension by injection of hypertonic saline through the episcleral veins. Following IOP elevation, macitentan (5 mg/kg body wt) was administered orally 3 days per week, and rats with IOP elevation were maintained for 4 weeks. RGC function was determined by pattern electroretinography (PERG) at 2 and 4 weeks post-IOP elevation. Rats were euthanized by approved humane methods, and retinal flat mounts were generated and immunostained for the RGC-selective marker Brn3a. PPD-stained optic nerve sections were imaged by confocal microscopy. RGC and axon counts were conducted in a masked manner and compared between the treatment groups. Results: Significant protection against loss of RGCs and optic nerve axons was found following oral administration of macitentan in rats with elevated IOP. In addition, a protective trend for RGC function, as measured by pattern ERG analysis, was evident following macitentan treatment. Conclusions: Macitentan treatment had a neuroprotective effect on RGCs and their axons, independent of its IOP-lowering effect, suggesting that macitentan may complement existing treatments to prevent neurodegeneration during ocular hypertension. The findings presented have implications for the use of macitentan as an oral formulation to promote neuroprotection in glaucoma patients.

Extract of Moutan radicis cortex and Cinnamomi ramulus ameliorates laser-induced choroidal neovascularization in Brown-Norway rats.

BACKGROUND: Moutan radicis cortex (MRC) and Cinnamomi ramulus (CR) are commonly used in eastern Asian traditional medicine to treat various diseases including cerebrovascular and cardiovascular, and have wide spectrum of pharmacological activities. However, the effect against laser-induced choroidal neovascularization (CNV) of extract of MRC and CR (1:1) (MRCCR) has not yet been studied. PURPOSE: Our aim was to investigate the inhibitory effect of MRCCR on pathological CNV in laser-treated Brown-Norway (BN) rats. METHODS: MRCCR (60, 90 mg/kg) was orally administered twice per day for 15 days from the day of CNV formation in laser-treated BN rats. Effects of MRCCR or its constituents on cell migration, tube formation, hyperpermeability and phosphorylation of FAK/p38 MAPK were confirmed in humane retinal microvascular endothelial cells or human retinal pigment epithelial cells. RESULTS: MRCCR significantly reduced the CNV lesions areas and the extent of fluorescein leakage. MRCCR and its constituents such as ellagic acid, paeonol or gallic acid decreased cell migration, tube formation or hyperpermeability. MRCCR inhibited the phosphorylation of FAK and p38 MAPK. CONCLUSION: Combining the oral MRCCR and intravitreal injection of anti-VEGF medicine may result in a more potent therapeutic effect and consequently bring the reduction in eye injection numbers for patients with wet AMD.

Modulation of Glucose Production by Central Insulin Requires IGF-1 Receptors in AgRP Neurons.

Similar to insulin, central administration of IGF-1 can suppress hepatic glucose production (HGP), but it is unclear whether this effect is mediated via insulin receptors (InsRs) or IGF-1 receptors (IGF-1Rs) in the brain. To this end, we used pharmacologic and genetic approaches in combination with hyperinsulinemic-euglycemic clamps to decipher the role of these receptors in mediating central effects of IGF-1 and insulin on HGP. In rats, we observed that intracerebroventricular (ICV) administration of IGF-1 or insulin markedly increased the glucose infusion rate (GIR) by >50% and suppressed HGP (P < 0.001). However, these effects were completely prevented by preemptive ICV infusion with an IGF-1R and InsR/IGF-1R hybrid (HybridR) blocking antibody. Likewise, ICV infusion of the InsR antagonist, S961, which also can bind HybridRs, interfered with the ability of central insulin, but not IGF-1, to increase the GIR. Furthermore, hyperinsulinemic clamps in mice lacking IGF-1Rs in AgRP neurons revealed ∼30% reduction in the GIR in knockout animals, which was explained by an impaired ability of peripheral insulin to completely suppress HGP (P < 0.05). Signaling studies further revealed an impaired ability of peripheral insulin to trigger ribosomal S6 phosphorylation or phosphatidylinositol (3,4,5)-trisphosphate production in AgRP neurons lacking IGF-1Rs. In summary, these data suggest that attenuation of IGF-1R signaling in the mediobasal hypothalamus, and specifically in AgRP neurons, can phenocopy impaired regulation of HGP as previously demonstrated in mice lacking InsRs in these cells, suggesting a previously unappreciated role for IGF-1Rs and/or HybridRs in the regulation of central insulin/IGF-1 signaling in glucose metabolism.

Identification of pyrogallol from Awa-tea as an anti-allergic compound that suppresses nasal symptoms and IL-9 gene expression.

As the expression level of allergic disease sensitive genes are correlated with the severity of allergic symptoms, suppression of these gene expressions could be promising therapeutics. We demonstrated that protein kinase Cδ / heat shock protein 90-mediated H1R gene expression signaling and nuclear factor of activated T-cells (NFAT)-mediated IL-9 gene expression signaling are responsible for the pathogenesis of pollinosis. Treatment with Awa-tea combined with wild grape hot water extract suppressed these signaling and alleviated nasal symptoms in toluene-2,4-diisocyanate (TDI)-sensitized rats. However, the underlying mechanism of its anti-allergic activity is not elucidated yet. Here, we sought to identify an anti-allergic compound from Awa-tea and pyrogallol was identified as an active compound. Pyrogallol strongly suppressed ionomycin-induced up-regulation of IL-9 gene expression in RBL-2H3 cells. Treatment with pyrogallol in combination with epinastine alleviated nasal symptoms and suppressed up-regulation of IL-9 gene expression in TDI-sensitized rats. Pyrogallol itself did not inhibit calcineurin phosphatase activity. However, pyrogallol suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFAT. These data suggest pyrogallol is an anti-allergic compound in Awa-tea and it suppressed NFAT-mediated IL-9 gene expression through the inhibition of dephosphorylation of NFAT. This might be the underlying mechanism of the therapeutic effects of combined therapy of pyrogallol with antihistamine. J. Med. Invest. 67 : 289-297, August, 2020.

Preclinical Efficacy and Safety of VEGF-Grab, a Novel Anti-VEGF Drug, and Its Comparison to Aflibercept.

Purpose: VEGF-Grab is a novel anti-vascular endothelial growth factor (VEGF) candidate drug with higher affinity to both VEGF and placental growth factor (PlGF) compared to aflibercept. We investigated the preclinical efficacy of VEGF-Grab for ophthalmic therapy and compared it to that of aflibercept. Methods: The in vitro anti-VEGF efficacy of VEGF-Grab was determined using VEGF-induced cell proliferation/migration and tube formation assays. The in vivo antiangiogenic efficacy of intravitreal injection of either VEGF-Grab or aflibercept was evaluated using murine models of ocular angiogenesis: mouse oxygen-induced retinopathy (OIR) and rat laser-induced choroidal neovascularization (CNV). The in vivo retinal toxicity in the mouse eye resulting from the injection of either drug was evaluated with light and electron microscopy. Results: VEGF-Grab showed greater inhibition of VEGF-induced cell proliferation/migration than aflibercept, but it showed comparable inhibition of tube formation in vitro. In the in vivo OIR model, VEGF-Grab showed a comparable suppression of retinal neovascularization compared to aflibercept. Additionally, VEGF-Grab showed an efficacy similar to that of aflibercept in terms of CNV inhibition in the laser-induced CNV model. Histology and transmission electron microscopy showed no significant signs of toxicity in the mouse retina at 7 and 30 days following the intravitreal injection of VEGF-Grab or aflibercept. Conclusions: Compared to aflibercept, VEGF-Grab presented comparable in vivo antiangiogenic efficacy and superior in vitro anti-VEGF activity. The retinal safety profiles were comparable for the two drugs. Considering its known higher binding affinity to VEGF and PlGF compared to aflibercept, VEGF-Grab could be a potential candidate drug for neovascular retinal diseases and an alternative to aflibercept.

Massage as a mechanotherapy promotes skeletal muscle protein and ribosomal turnover but does not mitigate muscle atrophy during disuse in adult rats.

AIM: Interventions that decrease atrophy during disuse are desperately needed to maintain muscle mass. We recently found that massage as a mechanotherapy can improve muscle regrowth following disuse atrophy. Therefore, we aimed to determine if massage has similar anabolic effects when applied during normal weight bearing conditions (WB) or during atrophy induced by hindlimb suspension (HS) in adult rats. METHODS: Adult (10 months) male Fischer344-Brown Norway rats underwent either hindlimb suspension (HS, n = 8) or normal WB (WB, n = 8) for 7 days. Massage was applied using cyclic compressive loading (CCL) in WB (WBM, n = 9) or HS rats (HSM, n = 9) and included four 30-minute bouts of CCL applied to gastrocnemius muscle every other day. RESULTS: Massage had no effect on any anabolic parameter measured under WB conditions (WBM). In contrast, massage during HS (HSM) stimulated protein turnover, but did not mitigate muscle atrophy. Atrophy from HS was caused by both lowered protein synthesis and higher degradation. HS and HSM had lowered total RNA compared with WB and this was the result of significantly higher ribosome degradation in HS that was attenuated in HSM, without differences in ribosomal biogenesis. Also, massage increased protein turnover in the non-massaged contralateral limb during HS. Finally, we determined that total RNA degradation primarily dictates loss of muscle ribosomal content during disuse atrophy. CONCLUSION: We conclude that massage is an effective mechanotherapy to impact protein turnover during muscle disuse in both the massaged and non-massaged contralateral muscle, but it does not attenuate the loss of muscle mass.

Endothelin-1 Mediated Decrease in Mitochondrial Gene Expression and Bioenergetics Contribute to Neurodegeneration of Retinal Ganglion Cells.

Endothelin-1 (ET-1) is a vasoactive peptide that is elevated in aqueous humor as well as circulation of primary open angle glaucoma (POAG) patients. ET-1 has been shown to promote degeneration of optic nerve axons and apoptosis of retinal ganglion cells (RGCs), however, the precise mechanisms are still largely unknown. In this study, RNA-seq analysis was used to assess changes in ET-1 mediated gene expression in primary RGCs, which revealed that 23 out of 156 differentially expressed genes (DEGs) had known or predicted mitochondrial function, of which oxidative phosphorylation emerged as the top-most enriched pathway. ET-1 treatment significantly decreased protein expression of key mitochondrial genes including cytochrome C oxidase copper chaperone (COX17) and ATP Synthase, H+ transporting, Mitochondrial Fo Complex (ATP5H) in primary RGCs and in vivo following intravitreal ET-1 injection in rats. A Seahorse ATP rate assay revealed a significant decrease in the rate of mitochondrial ATP production following ET-1 treatment. IOP elevation in Brown Norway rats showed a trend towards decreased expression of ATP5H. Our results demonstrate that ET-1 produced a decrease in expression of vital components of mitochondrial electron transport chain, which compromise bioenergetics and suggest a mechanism by which ET-1 promotes neurodegeneration of RGCs in glaucoma.

Measuring the middle-ear reflex: A quantitative method to assess effects of industrial solvents on central auditory pathways.

Volatile organic solvents are frequently present in industrial atmospheres. Their lipophilic properties mean they quickly reach the brain following inhalation. Acute exposure to some solvents perturbs the middle ear reflex, which could jeopardize cochlear protection against loud noises. As the physiological mechanisms involved in this protective reflex are highly complex, in vivo rodent models are required to allow rapid and reliable identification of any adverse effects of solvents on the middle ear reflex (MER). In this study, MER amplitude was measured in anesthetized Brown-Norway rats by monitoring the decrease in distortion product otoacoustic emissions (DPOAEs) caused by a contralateral stimulation. Our screening test consisted in measuring the impact of inhalation of solvent vapors at 3000 ppm for 15 min on the MER amplitude. We had previously studied a selection of aromatic solvents with this model; here, we extended the analysis to volatile compounds from other chemical families. The results obtained shed light on the mechanisms involved in the interactions between solvents and their neuronal targets. Thus, benzene and chlorobenzene had the greatest effect on MER (≥ + 1.8 dB), followed by a group composed of toluene, styrene, p-xylene, m-xylene, tetrachloroethylene and cyclohexane, which had a moderate effect on the MER (between + 0.3 and + 0.7 dB). Finally, trichloroethylene, n-hexane, methyl-ethyl-ketone, acetone, o-xylene, and ethylbenzene had no effect on the MER. Thus, the effect of solvents on the MER is not simply linked to their lipophilicity, rather it depends on specific interactions with neuronal targets. These interactions appear to be governed by the compound's chemical structure, e.g. the presence of an aromatic ring and its steric hindrance. In addition, perturbation of the MER by a solvent is independent of its toxic effects on cochlear cells. As the MER plays a protective role against exposure to high-intensity noises, these findings could have a significant impact in terms of prevention for subjects exposed to both noise and solvents.

Lanthanum carbonate, a phosphate binder, inhibits calcification of implanted aortic allografts in a rat model.

OBJECTIVES: Calcification is one of the major postoperative problems after aortic allograft implantation. We hypothesized that phosphate binders, lanthanum carbonate and calcium carbonate inhibit calcification of implanted aortic allografts and verified this hypothesis using a rat model. METHODS: Aortas were harvested from 4-week-old Brown Norway rats and implanted into the subdermal space of 4-week-old Lewis rats. Twenty-seven recipient Lewis rats were divided into Group N, Group L, and Group C (9 rats per group), which were fed a normal diet, a normal diet containing 3% lanthanum carbonate, and a normal diet containing 3% calcium carbonate, respectively. Implanted aortic allografts were explanted 2 weeks later. Calcification of aortic allografts was evaluated using von Kossa staining and calcium content assay. Calcification score was defined in von Kossa staining as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). Serum calcium and phosphorus levels at euthanasia were measured. RESULTS: Calcification scores were 2.6, 1.2, and 0.8, and calcium content was 48.9, 15.8, and 8.9 mg/dry·g, in Groups N, L, and C, respectively. Calcification was significantly reduced in Groups L and C. Serum calcium level was 11.5, 12.2, and 13.5 mg/dl, and serum phosphorus level was 15.4, 12.5, and 11.7 mg/dl, in Groups N, L, and C, respectively. Serum calcium level in Group C was significantly higher than in the other two groups. CONCLUSIONS: Lanthanum carbonate and calcium carbonate significantly reduced calcification of implanted aortic allografts in young rats. Although calcium carbonate induced hypercalcemia, lanthanum carbonate has significant potential to inhibit calcification of implanted aortic allografts.

Effect of wild grape on the signaling of histamine H1 receptor gene expression responsible for the pathogenesis of allergic rhinitis.

As expression level of allergic disease-sensitive genes are correlated with allergic symptom severity, suppression of these gene expressions could be good therapeutics. We have demonstrated that PKCδ signaling and NFAT signaling, involve in histamine H1 receptor (H1R) and IL-9 gene expressions, respectively, are responsible for the pathogenesis of allergic rhinitis. We explore anti-allergic compounds that suppress these signaling pathways and found that wild grape (WG) contains such compounds. Here, we investigated the effect of WG hot water extract (WGE) on the signaling pathways for PKCδ-mediated H1R and NFAT-mediated IL-9 gene expressions. WGE suppressed histamine/PMA-induced H1R gene up-regulation in HeLa cells. Toluene-2,4-diisocyanate (TDI)-induced H1R mRNA elevation in TDI-sensitized rats was also suppressed by WGE treatment. Treatment with WGE in combination with Awa-tea, suppresses NFAT signaling-mediated IL-9 gene, markedly alleviated nasal symptoms. Furthermore, WGE suppressed PMA-induced IL-33 gene up-regulation in Swiss 3T3 cells. Data suggest that combination of WGE, suppresses PKCδ signaling with Awa-tea, suppresses NFAT signaling would have distinct clinical and therapeutic advantages as a substitute for anti-allergic drugs. In addition, as the expression level of IL-33 mRNA was correlated with the blood eosinophils number in patients with pollinosis, WG could alleviate eosinophilic inflammation through the suppression of IL-33 gene expression. J. Med. Invest. 65:242-250, August, 2018.

Preventing acute asthmatic symptoms by targeting a neuronal mechanism involving carotid body lysophosphatidic acid receptors.

Asthma accounts for 380,000 deaths a year. Carotid body denervation has been shown to have a profound effect on airway hyper-responsiveness in animal models but a mechanistic explanation is lacking. Here we demonstrate, using a rat model of asthma (OVA-sensitized), that carotid body activation during airborne allergic provocation is caused by systemic release of lysophosphatidic acid (LPA). Carotid body activation by LPA involves TRPV1 and LPA-specific receptors, and induces parasympathetic (vagal) activity. We demonstrate that this activation is sufficient to cause acute bronchoconstriction. Moreover, we show that prophylactic administration of TRPV1 (AMG9810) and LPA (BrP-LPA) receptor antagonists prevents bradykinin-induced asthmatic bronchoconstriction and, if administered following allergen exposure, reduces the associated respiratory distress. Our discovery provides mechanistic insight into the critical roles of carotid body LPA receptors in allergen-induced respiratory distress and suggests alternate treatment options for asthma.

Stereological Method in Optical Coherence Tomography for In Vivo Evaluation of Laser-Induced Choroidal Neovascularization.

BACKGROUND AND OBJECTIVE: To evaluate a stereological method in optical coherence tomography (OCT) as an in vivo volume measurement of laser-induced choroidal neovascularization (L-CNV) lesion size. PATIENTS AND METHODS: Laser photocoagulation was applied in rats to rupture Bruch's membrane and induce L-CNV. In vivo OCT images of neovascular lesions were acquired with a spectral-domain OCT system at days 0, 3, 7, 10, and 14 after laser surgery. A stereological image-processing method was used to calculate lesion volumes from the OCT images. Rats were euthanized at day 14, and confocal microscopy was used to obtain accurate volume measurements of the lesions ex vivo. Lesion sizes calculated from OCT and confocal were compared. RESULTS: In vivo assessment by OCT allowed three distinct stages of L-CNV to be visualized: the initial early reaction, neovascular proliferation, and regression. At day 14, correlations between OCT and confocal lesion volumes showed a positive association (Pearson's r = 0.50, P < .01). Except for the largest lesions, volumes measured by OCT were statistically similar to those measured by the confocal gold standard (P = .90). CONCLUSION: The stereological approach used to measure neovascular lesion volume from OCT images offers an accurate means to track L-CNV lesion size in vivo. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:e65-e74.].

DenTimol as A Dendrimeric Timolol Analogue for Glaucoma Therapy: Synthesis and Preliminary Efficacy and Safety Assessment.

In this work, we report the synthesis and characterization of DenTimol, a dendrimer-based polymeric timolol analog, as a glaucoma medication. A timolol precursor ( S)-4-[4-(oxiranylmethoxy)-1,2,5-thiadiazol-3-yl]morpholine (OTM) was reacted with the heterobifunctional amine polyethylene glycol acetic acid (amine-PEG-acetic acid, Mn = 2000 g/mol) via a ring opening reaction of an epoxide by an amine to form the OTM-PEG conjugate. OTM-PEG was then coupled to an ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimer G3 to generate DenTimol using the N-(3-(dimethylamino)propyl)- N'-ethylcarbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) coupling reaction. MALDI mass spectrometry, 1H NMR spectroscopy, and HPLC were applied to characterize the intermediate and final products. Ex vivo corneal permeation of DenTimol was assessed using the Franz diffusion cell system mounted with freshly extracted rabbit cornea. The cytotoxicity of DenTimol was assessed using the WST-1 assay. Our results show that DenTimol is nontoxic up to an OTM equivalent concentration of 100 µM. DenTimol is efficient at crossing the cornea. About 8% of the dendrimeric drug permeated through the cornea in 4 h. Its IOP-lowering effect was observed in normotensive adult Brown Norway male rats. Compared to the undosed eye, an IOP reduction by an average of 7.3 mmHg (∼30% reduction from baseline) was observed in the eye topically treated with DenTimol (2 × 5 µL, 0.5% w/v timolol equivalent) in less than 30 min. Daily dosing of DenTimol for a week did not cause any irritation or toxicity as confirmed by the histological examination of ocular tissues, including the cornea, ciliary body, and retina.

Enhanced skeletal muscle regrowth and remodelling in massaged and contralateral non-massaged hindlimb.

KEY POINTS: Muscle fibre cross sectional area is enhanced with massage in the form of cyclic compressive loading during regrowth after atrophy. Massage enhances protein synthesis of the myofibrillar and cytosolic, but not the mitochondrial fraction, in muscle during regrowth. Focal adhesion kinase activation and satellite cell number are elevated in muscles undergoing massage during regrowth. Muscle fibre cross sectional area and protein synthesis of the myofibrillar fraction, but not DNA synthesis, are elevated in muscle of the contralateral non-massaged limb. Massage in the form of cyclic compressive loading is a potential anabolic intervention during muscle regrowth after atrophy. ABSTRACT: Massage, in the form of cyclic compressive loading (CCL), is associated with multiple health benefits, but its potential anabolic effect on atrophied muscle has not been investigated. We hypothesized that the mechanical activity associated with CCL induces an anabolic effect in skeletal muscle undergoing regrowth after a period of atrophy. Fischer-Brown Norway rats at 10 months of age were hindlimb unloaded for a period of 2 weeks. The rats were then allowed reambulation with CCL applied at a 4.5 N load at 0.5 Hz frequency for 30 min every other day for four bouts during a regrowth period of 8 days. Muscle fibre cross sectional area was enhanced by 18% with massage during regrowth compared to reloading alone, and this was accompanied by elevated myofibrillar and cytosolic protein as well as DNA synthesis. Focal adhesion kinase phosphorylation indicated that CCL increased mechanical stimulation, while a higher number of Pax7+ cells likely explains the elevated DNA synthesis. Surprisingly, the contralateral non-massaged limb exhibited a comparable 17% higher muscle fibre size compared to reloading alone, and myofibrillar protein synthesis, but not DNA synthesis, was also elevated. We conclude that massage in the form of CCL induces an anabolic response in muscles regrowing after an atrophy-inducing event. We suggest that massage can be used as an intervention to aid in the regrowth of muscle lost during immobilization.

Effects of long-term exposures to low iron and branched-chain amino acid containing diets on aging skeletal muscle of Fisher 344 × Brown Norway rats.

Aging skeletal muscle displays an altered iron status that may promote oxidative stress and sarcopenia. A diet containing low iron (LI) could reduce muscle iron status and attenuate age-related muscle atrophy. Supplemental branched-chain amino acids (BCAA) may also alleviate sarcopenia by promoting muscle protein synthesis and iron status improvement. This study examined individual and combined effects of LI and BCAA diets on anabolic signaling and iron status in skeletal muscle of aging rats. Twenty-nine-month-old male Fisher 344 × Brown Norway rats consumed the following control-base diets: control + regular iron (35 mg iron/kg) (CR; n = 11); control + LI (∼6 mg iron/kg) (CL; n = 11); 2×BCAA + regular iron (BR; n = 10); and 2×BCAA + LI (BL; n = 12) for 12 weeks. Although LI and/or 2×BCAA did not affect plantaris muscle mass, 2×BCAA groups showed lower muscle iron content than did CR and CL groups (P < 0.05). p70 ribosomal protein S6 kinase phosphorylation was greater in 2×BCAA and LI animals compared with CR animals (P < 0.05). Interactions between IRON and BCAA were observed for proteins indicative of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1 alpha) and oxidative capacity (cytochrome c oxidase subunit 2 and citrate synthase) (P < 0.05) wherein the combined diet (BL) negated potential benefits of individual diets. Antioxidant capacity, superoxide dismutase activity, and oxidative injury (3-nitrotyrosine, protein carbonyls, and 4-hydroxynonenal) were similar between groups. In conclusion, 12 weeks of LI and 2×BCAA diets showed significant impacts on increasing anabolic signaling as well as ameliorating iron status; however, these interventions did not affect muscle mass.

Discovery and Optimization of Thiazolidinyl and Pyrrolidinyl Derivatives as Inhaled PDE4 Inhibitors for Respiratory Diseases.

Phosphodiesterase 4 (PDE4) is a key cAMP-metabolizing enzyme involved in the pathogenesis of inflammatory disease, and its pharmacological inhibition has been shown to exert therapeutic efficacy in chronic obstructive pulmonary disease (COPD). Herein, we describe a drug discovery program aiming at the identification of novel classes of potent PDE4 inhibitors suitable for pulmonary administration. Starting from a previous series of benzoic acid esters, we explored the chemical space in the solvent-exposed region of the enzyme catalytic binding pocket. Extensive structural modifications led to the discovery of a number of heterocycloalkyl esters as potent in vitro PDE4 inhibitors. (S*,S**)-18e and (S*,S**)-22e, in particular, exhibited optimal in vitro ADME and pharmacokinetics properties and dose-dependently counteracted acute lung eosinophilia in an experimental animal model. The optimal biological profile as well as the excellent solid-state properties suggest that both compounds have the potential to be effective topical agents for treating respiratory inflammatory diseases.

Effect of High-Salt Diet on Age-Related High Blood Pressure and Hypothalamic Redox Signaling.

AIM: The aim of this study was to investigate the effect of a high salt (HS) diet on age-related changes in blood pressure (BP) and the possible role played by regulatory central mechanisms. METHODS: Young (5 months) and old (27 months) male Fischer 344 × Brown Norway (F344/BN) rats were fed standard chow or 8% HS diet for 12 days. BP and heart rate (HR) were measured by telemetry. RESULTS: Mean arterial BP (MAP) was significantly elevated in old rats during the day and night when compared with young animals. The HS diet further elevated MAP in both age groups, and the increase was more pronounced in the old animals, while HR was not altered by age or HS diet. In addition, cardiovascular responses to restraint stress were diminished in the old when compared with the young and were unchanged with HS diet in either age group. Both age and the HS diet elevated the adrenomedullary mRNA levels of tyrosine hydroxylase, an indicator for sympathoexcitation. HS diet enhanced intracerebroventricular angiotensin II (AngII)-induced BP and HR elevations in both age groups. AngII type 1 receptor mRNA increased significantly in the hypothalamus with age and HS diet. Furthermore, hypothalamic p22phox mRNA and gp91phox protein, subunits of NADPH oxidase, as well as NADPH oxidase activity increased with the HS diet in the old animals, whereas antioxidant enzymes that decreased with age yet remained unaltered with the HS diet. CONCLUSION: Our findings indicate that sensitivity of BP to HS diet increases with age, and that central AngII-induced pressor responses are diminished in old rats compared with the young both under control conditions and during HS diet treatment. These changes are paralleled by increases in the expression and NADPH oxidase activity in the hypothalamus, possibly leading to central oxidative stress-mediated sympathoexcitation and high BP.

Upregulation of calprotectin in mild IgE-mediated ovalbumin hypersensitivity.

Calprotectin, also known as S100A8/A9, has been linked to gut inflammation caused by IgE-mediated food hypersensitivities, but the pathophysiologic abnormalities it causes remain to be determined. We created a mild food hypersensitivity model through oral gavage of ovalbumin in Norway brown rats without using immune adjuvant. Changes in the levels of calprotectin and inflammation-associated cytokines were then observed over time. We found that fecal calprotectin as well as jejunal and liver TLR4, TNF-α, NF-κB, IL-1ß, and IL-6 were upregulated in hypersensitive rats. Additionally, the influence of calprotectin on CD4+ T and dendritic cells was observed by co-culturing CD4+ T cells with dendritic cells, which revealed a shift toward increased Th2 T cells in calprotectin-treated cultures. These results suggest that calprotectin, along with other inflammatory factors, promotes the inflammation seen in mild food allergy.

Evaluation of anaphylactoid constituents in vitro and in vivo.

Natural medicine injections have been widely used in clinics, while adverse reaction reports also have increased rapidly in recent years. To examine the anaphylactoid constituents of natural medicine injections, RBL-2H3 cell degranulation and human serum complement activation models were used to screen the anaphylactoid constituents, and the BN rat model was used to explore the anaphylactoid mechanism of these constituents. The result of an in vitro study showed that the individual compounds of natural medicine injections (chlorogenic acid, hyodeoxycholic acid, cholalic acid, ginkgolic acid, phillyrin, schisandrin B, schisandrin A, puerarin, and tanshinone IIA) and polysaccharide could not induce RBL-2H3 to release histamine and ß-hexosaminidase, while proteins Tween-80 and tannic acid were the main anaphylactoid constituents in the natural medicine injections. The in vivo study also indicated that >10kDa molecules (proteins) activated classical complement pathways through direct stimulation to cause an anaphylactoid reaction. Tween-80 activated direct stimulation and coagulation pathways through classical and alternative pathways; tannic acid induced anaphylactoid reaction through co-activation of the kallikrein-kinin system, coagulation, integrated, classical and alternative complement pathways. This is the first study to evaluate the anaphylactoid constituents systematically through in vitro and in vivo study. And tannic acid, >10kDa molecules (proteins), and injection additives such as Tween-80 are the main anaphylactoid constituents of natural medicine injections.

Intracerebroventricular tempol administration in older rats reduces oxidative stress in the hypothalamus but does not change STAT3 signalling or SIRT1/AMPK pathway.

Hypothalamic inflammation and increased oxidative stress are believed to be mechanisms that contribute to obesity. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), a free radical scavenger, has been shown to reduce inflammation and oxidative stress. We hypothesized that brain infusion of tempol would reduce oxidative stress, and thus would reduce food intake and body weight and improve body composition in rats with age-related obesity and known elevated oxidative stress. Furthermore, we predicted an associated increase in markers of leptin signalling, including the silent mating type information regulator 2 homolog 1 (SIRT1)/5'AMP-activated protein kinase (AMPK) pathway and the signal transducer and activator of transcription 3 (STAT3) pathway. For this purpose, osmotic minipumps were placed in the intracerebroventricular region of young (3 months) and aged (23 months) male Fischer 344 x Brown Norway rats for the continuous infusion of tempol or vehicle for 2 weeks. Tempol significantly decreased (p < 0.01) nicotinamide adenine dinucleotide phosphate oxidase activity in the hypothalamus but failed to reduce food intake or weight gain and did not alter body composition. SIRT1 activity and Acetyl p53 were decreased and phosphorylation of AMPK was increased with age, but they were unchanged with tempol. Basal phosphorylation of STAT3 was unchanged with age or tempol. These results indicate that tempol decreases oxidative stress but fails to alter feeding behaviour, body weight, or body composition. Moreover, tempol does not modulate the SIRT1/AMPK/p53 pathway and does not change leptin signalling. Thus, a reduction in hypothalamic oxidative stress is not sufficient to reverse age-related obesity.