European Journal of Pharmacology (v.788, #C)

A-306989, an inhibitor of adenosine kinase, is renoprotective in rodent models of podocyte, basement membrane, and obstructive injury by Chang Z. Zhu; Sujatha Gopalakrishnan; Kelly Doyle; Arthur L. Nikkel; Lauren Olson; Vivek C. Abraham; Laura Leys; Deborah Widomski; Katherine Salte; Brent Putman; Steve Pratt; Ji Ma; Zhi Su; Murali Gopalakrishnan; Chih-Hung Lee; Steve P. McGaraughty (1-11).
Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15 mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30 mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50 mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50 mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on “quality of life” measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.
Keywords: Adenosine kinase inhibitor; Renoprotective; Animal model; A-306989;

Long-term topical application of preservative-free prostaglandin analogues evokes macrophage infiltration in the ocular adnexa by Anna Trzeciecka; J. Jussi Paterno; Elisa Toropainen; Ali Koskela; Lucia Podracka; Eveliina Korhonen; Anu Kauppinen; Kai Kaarniranta; Adrian Smedowski (12-20).
Success of the long-term glaucoma therapy and preservation of the visual function strongly depend on patients' compliance which may be affected by the inconvenience of treatment and its side effects. Recently, introduction of preservative-free anti-glaucoma agents has become an important step towards improved glaucoma care by eliminating the negative effects of preservatives on the eye surface. Although, newly developed eye drop formulations do not contain standard preservatives, they still can be harmful to ocular surface due to other excipients. In this study, we compared tolerability of commercial preservative-free (pf) prostaglandin analogues (pf tafluprost, pf latanoprost and pf bimatoprost) in long-term topical application in rabbits in vivo. We found that after eight weeks treatment, pf latanoprost was the worst tolerated among the tested drops. It expressed increased conjunctival redness and blinking frequency. Furthermore, it caused increased LDH release in the aqueous humour, infiltration of macrophages in the eyelids and visible defects in conjunctival goblet cells. However, we did not detect increased levels of inflammatory markers in the tear fluid or in the aqueous humour. Based on our study, we suspect that these negative effects are related to excipients included in pf latanoprost formulation.
Keywords: Preservative-free prostaglandin analogues; Topical application; Tolerability; Macrophages;

The effect of R-(-)-deprenyl administration on antioxidant enzymes in rat testis by Františka Horváthová; Viera Danielisová; Iveta Domoráková; Peter Solár; Silvia Rybárová; Ingrid Hodorová; Jozef Mihalik (21-28).
The aim of the study was to investigate the effect of R-(-)-deprenyl administration on the activity and localization of superoxide dismutases (SODs) and catalase (CAT) in rat testis. After 30 days of intraperitoneal administration of either saline (control) or R-(-)-deprenyl dissolved in saline at concentrations of 0.0025 mg/kg (low dose of deprenyl, LDD) or 0.25 mg/kg (high dose of deprenyl, HDD), males were killed by thiopental, and their testes were collected. We found that deprenyl administration significantly increased the activity of antioxidant enzymes, and this effect varied by dosage. LDD caused significant elevation of all monitored enzymes, but HDD did not increase the activity of SOD2. Employing immunohistochemistry, we detected enzymes predominantly in Leydig cells (SOD1, SOD2, CAT), in late spermatids and residual bodies (SOD1, SOD2), and in primary spermatocytes (SOD2). Histopathological examination did not reveal testicular damage in experimental groups compared to control. Deprenyl proved to be a potent stimulator of antioxidant enzymes in rat testes; therefore, it could be used in the therapy of male infertility. On the other hand, it is crucial to choose a proper dose, since lower dose was more competent compared to a dosage that was one hundred times higher.
Keywords: Selegiline; SOD; CAT; Immunohistochemistry;

Chronic treatment with resveratrol improves overactive bladder in obese mice via antioxidant activity by Eduardo C. Alexandre; Fabiano B. Calmasini; Mariana G. de Oliveira; Fábio H. Silva; Carmem P.V. da Silva; Diana M. André; Flávia C. Leonardo; Maria A. Delbin; Edson Antunes (29-36).
The objective of the present work was to evaluate whether oral intake with resveratrol ameliorates overactive bladder in high-fat fed mice. Male C57BL6 mice fed with standard chow or high-fat diet to induce obesity received a two-week therapy with resveratrol (100 mg/kg, given as a daily gavage). Weight and metabolic profile, together with cystometry and in vitro bladder contractions were evaluated. Measurements of gp91phox and SOD1 mRNA expressions and reactive-oxygen species (ROS) in bladder tissues, and serum TBARS were performed. Obese mice exhibited increases in body weight and epididymal fat mass, which were significantly reduced by oral treatment with resveratrol. Cystometric study in obese mice showed increases in non-voiding contractions, post-voiding pressure and voiding frequency that were reversed by resveratrol treatment. Likewise, the in vitro bladder overactivity in response to electrical-field stimulation (80 V, 1–32 Hz) or carbachol (1 nM to 10 mM) were normalized by resveratrol. The gp91phox and SOD1 mRNA expressions in bladder tissues were markedly higher in obese mice compared with lean group. In addition, ROS levels in bladder tissues and serum lipid peroxidation (TBARS assay) were markedly higher in obese compared with lean mice, all of which were reduced by resveratrol treatment. In lean group, resveratrol had no effect in any parameter evaluated. Our results show that two-week therapy of obese mice with resveratrol reduces the systemic and bladder oxidative stress, and greatly ameliorated the cystometry alterations and in vitro bladder overactivity. Resveratrol treatment could be an option to prevent obesity-associated overactive bladder.
Keywords: Cystometry; gp91phox; NADPH oxidase; Obesity; Reactive-oxygen species; Superoxide dismutase;

We recently reported that propofol depressed facial stimulation-evoked gamma-aminobutyric acid (GABA) transmission at cerebellar molecular layer interneuron–Purkinje cell (PC) synapses in mice in vivo, but facilitated excitatory parallel fiber inputs onto PCs. Here, we examine the effects of propofol on cerebellar granule cell layer (GCL) responses to facial stimulation in urethane-anesthetized mice, using electrophysiological and pharmacological methods. Cerebellar surface perfusion of propofol (50–1000 μM) facilitated field potentials evoked in the cerebellar GCL by air-puff stimulation of the ipsilateral whisker pad, shown by increases in the half-width and area under the curve (AUC) of the stimulus onset response (Ron). Propofol also significantly increased the amplitude of the stimulus offset response (Roff) and Roff/Ron ratio. The propofol-induced increase in Ron AUC was dose-dependent, with a 50% effective concentration (EC50) of 242.4 µM. Application of the GABAA receptor antagonist gabazine (20 μM) significantly increased the amplitude, half-width, rise tau and AUC of Ron, but these parameters were further increased by additional application of propofol (300 µM). Notably, application of the N-methyl-d-aspartate (NMDA) receptor blocker D-APV (250 µM) significantly attenuated the half-width and AUC of Ron and the amplitude of Roff, without significantly changing the amplitude of Ron. These results indicate that propofol enhanced facial stimulation-evoked responses in the cerebellar GCL via NMDA receptor activation, which resulted in the facilitation of excitatory parallel fiber inputs onto cerebellar PCs in mice in vivo.
Keywords: Propofol; Cerebellar granule cell layer (GCL); Sensory stimulation; Field potential recording; GABAA receptor; NMDA receptor;

Human biliverdin reductase promotes EMT through the ERK1/2 signal pathway in breast cancer by Min Zhang; Shasha Song; Zhi Yi; Xijuan Zhao; Li Fu; Lin Wang; Cui Ma; Min Mao; Yan Xing; Daling Zhu (45-53).
Epithelial-to-mesenchymal transition (EMT) plays an important role in the development of the invasive and metastatic potentials of breast cancer cells during progression. Human biliverdin reductase (hBVR), an enzyme in the heme metabolism pathway, is involved in hypoxia-induced renal tubular EMT. However, whether hBVR contributes to the EMT of breast cancer remains unclear. Here, we used breast cancer cell lines (MCF-7, T-47D) and normal breast epithelial cells (MCF-10A) to explore the potential role of hBVR in the EMT of breast cancer. Western blot, RT-PCR and immunofluorescence were employed to test the expression and location of hBVR in the cell lines. Small interfering RNA of hBVR (si-hBVR) was used to knockdown the expression of hBVR, and U0126 was applied to inhibit the ERK1/2 signaling in MCF-7, T-47D cells. We found that hBVR highly expressed in MCF-7 and T-47D cells compared with MCF-10A cells, and had different cellular locations between them. Our results revealed that EMT occurred in tissues from breast cancer patients and breast cancer cell lines. However, the EMT in MCF-7 and T-47D cells was suppressed by si-hBVR and U0126. Furthermore, the expression of phosphorylated ERK1/2 was down-regulated by si-hBVR. In addition, hBVR regulated EMT through the ERK1/2 signaling, but bilirubin, which is a product of hBVR in the heme metabolism pathway in breast cancer, did not. Taken together, these findings provide new evidence that hBVR plays an important role in promoting EMT in human breast cancer through the ERK1/2 signaling pathway, and hBVR may be a therapeutic target for this disease.
Keywords: EMT; hBVR; Bilirubin; ERK1/2 signal pathway; Breast cancer;

Pharmacological profiles of gemigliptin (LC15-0444), a novel dipeptidyl peptidase-4 inhibitor, in vitro and in vivo by Sung-Ho Kim; Eunsoo Jung; Min Kyung Yoon; O. Hwan Kwon; Dal-Mi Hwang; Dong-Wook Kim; Junghyun Kim; Sun-Mee Lee; Hyeon Joo Yim (54-64).
Gemigliptin, a novel dipeptidyl peptidase (DPP)-4 inhibitor, is approved for use as a monotherapy or in combination therapy to treat hyperglycemia in patients with type 2 diabetes mellitus. In this study, we investigated the pharmacological profiles of gemigliptin in vitro and in vivo and compared them to those of the other DPP-4 inhibitors. Gemigliptin was a reversible and competitive inhibitor with a Ki value of 7.25±0.67 nM. Similar potency was shown in plasma from humans, rats, dogs, and monkeys. The kinetics of DPP-4 inhibition by gemigliptin was characterized by a fast association and a slow dissociation rate compared to sitagliptin (fast on and fast off rate) or vildagliptin (slow on and slow off rate). In addition, gemigliptin showed at least >23,000-fold selectivity for DPP-4 over various proteases and peptidases, including DPP-8, DPP-9, and fibroblast activation protein (FAP)-α. In the rat, dog, and monkey, gemigliptin showed more potent DPP-4 inhibitory activity in vivo compared with sitagliptin.In mice and dogs, gemigliptin prevented the degradation of active glucagon-like peptide-1 by DPP-4 inhibition, which improved glucose tolerance by increasing insulin secretion and reducing glucagon secretion during an oral glucose tolerance test. The long-term anti-hyperglycemic effect of gemigliptin was evaluated in diet-induced obese mice and high-fat diet/streptozotocin-induced diabetic mice. Gemigliptin dose-dependently decreased hemoglobin A1c (HbA1c) levels and ameliorated β-cell damage. In conclusion, gemigliptin is a potent, long-acting, and highly selective DPP-4 inhibitor and can be a safe and effective drug for the long-term treatment of type 2 diabetes.
Keywords: Gemigliptin (PubChem CID: 11953153); Vildagliptin (PubChem CID: 11077541); Sitagliptin (PubChem CID: 11591741); Dipeptidyl peptidase-4 inhibitor; Gemigliptin; Incretin; Type 2 diabetes mellitus;

A neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. has bone forming effects in mice model for osteoporosis by Priyanka Kushwaha; Vikram Khedgikar; Naseer Ahmad; Anirudha Karvande; Jyoti Gautam; Padam Kumar; Rakesh Maurya; Ritu Trivedi (65-74).
Dalbergia sissoo Roxb. is a well known medicinal plant of India, enriched with various flavonoids used for treating multiple diseases. Earlier, we have shown that extract of Dalbergia sissoo Roxb. leaves mitigate ovariectomy induced bone loss and pure compounds (neoflavonoids) isolated from it, promote osteoblastogenesis in primary calvarial osteoblasts cells in vitro. Here, we hypothesize that dalsissooal (DSL), a novel neoflavonoid isolated from the heartwood of Dalbergia sissoo Roxb. is an important constituent of the extract that imparts bone forming effects. Treatment with DSL enhanced trabecular bone micro-architecture parameters, biomechanical strength, increased bone formation rate and mineral apposition rate in OVx mice comparable to 17β-estradiol. It increased bone formation by enhancing osteoblast gene expression and reduced bone turnover by decreasing osteoclastic gene expressions. Interestingly, we observed that DSL has no uterine estrogenic effects. At cellular levels, DSL promoted differentiation of bone marrow cells as well as calvaria osteoblast cells towards osteoblast lineage by enhancing differentiation and mineralizing ability to form mineralizing nodules via stimulating BMP-2 and RunX-2 expressions. Overall, our data suggest that oral supplementation of a novel neoflavonoid dalsissooal isolated from heartwood of Dalbergia sissoo Roxb. exhibited bone anabolic action by improving structural property of bone, promoting new bone formation and reducing bone turnover rate in post-menopausal model for osteoporosis with no uterine hyperplasia.Display Omitted
Keywords: Neoflavonoid; Post-menopausal osteoporosis; Micro-ct; 17β-estradiol; Bone mechanical strength; Bmp-2/runx-2 pathway;

Determinants of action of hydrophobic amines on ASIC1a and ASIC2a by Elina I. Nagaeva; Natalia N. Potapieva; Maxim V. Nikolaev; Valery E. Gmiro; Lev G. Magazanik; Denis B. Tikhonov (75-83).
Acid-sensing ion channels (ASICs) are involved in numerous physiological and pathological processes in the central nervous system. Development of pharmacological tools capable to inhibit or potentiate these channels is important for our knowledge about roles of ASICs in the neuronal network and can be promising for treatment of some disorders. Recently we described four hydrophobic monoamines that potentiate and inhibit ASICs depending on subunit composition of the channel and peculiarities of the drug structure. In the present work we performed structure-activity relationship analysis using derivatives of adamantane, phenylcyclohexyl and 9-aminoacridine to reveal the main determinants of action of amine-containing compounds on recombinant ASIC1a and ASIC2a homomers expressed in CHO cells. We found that the most active compounds are monocations with protonatable aminogroup. In general, potentiators and inhibitors of ASIC1a we found, but only potentiators for ASIC2a. Flat aromatic structure of the headgroup determines inhibition of ASIC1a while “V-shape” structure of the hydrophobic moiety favors potentiation of ASIC2a. Moreover, for some series of monoamines there was a correlation between action on ASIC1a and ASIC2a, the weaker ASIC1a inhibition, the stronger ASIC2a potentiation. Decay of response was accelerated by ASIC1a inhibitors as well as by potentiators. All compounds potentiating ASIC2a slowed down desensitization. Our results suggest that hydrophobic amines cause complex action on ASICs.
Keywords: ASICs; Monoamines; Pharmacology; Structure-activity relationships; Patch-clamp;

Role of Hypothalamic-Pituitary-Adrenal axis and corticotropin-releasing factor stress system on cue-induced relapse to alcohol seeking by Fernanda L. Galesi; Lydia O. Ayanwuyi; Miriam Garcia Mijares; Andrea Cippitelli; Nazzareno Cannella; Roberto Ciccocioppo; Massimo Ubaldi (84-89).
A large body of evidence has shown that the Corticotropin Releasing Factor (CRF) system, which plays a key role in stress modulation, is deeply involved in relapse to alcohol seeking induced by exposure to stressful events such as foot shock or yohimbine injections. Exposure to environmental cues is also known to be a trigger for alcohol relapse, nevertheless, the relationship between the relapse evoked by the cue-induced model and the CRF stress systems remains unclear.The purpose of this study was to evaluate, in male Wistar rats, the involvement of the CRF system and Hypothalamic-Pituitary-Adrenal (HPA) axis in relapse induced by environmental cues. Antalarmin, a selective CRF1 receptor antagonist, Metyrapone, a corticosterone (CORT) synthesis inhibitor and CORT were evaluated for their effects on the reinstatement test in a cue-induced relapse model.Antalarmin (20 mg/kg) blocked relapse to alcohol seeking induced by environmental cues. Metyrapone (50 and 100 mg/kg) also blocked relapse in Wistar rats but only at the highest dose (100 mg/kg). Corticosterone had no effect on relapse at the doses tested.The results obtained from this study suggest that the CRF stress system and the HPA axis are involved in cue-induced alcohol relapse.
Keywords: Alcohol; Relapse; Stress; CRF; Addiction; Reinstatement;

WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway by Shun Lu; Hao Liu; Lihe Lu; Heng Wan; Zhiqi Lin; Kai Qian; Xingxing Yao; Qing Chen; Wenjun Liu; Jianyun Yan; Zhengjun Liu (90-97).
Proliferation and migration of vascular smooth muscle cells (VSMCs) play crucial roles in the development of vascular restenosis. Our previous study showed that CCN4, namely Wnt1 inducible signaling pathway protein 1 (WISP1), significantly promotes proliferation and migration of rat VSMCs, but its mechanism remains unclear. This study aims to investigate whether and how WISP1 stimulates proliferation and migration of human VSMCs. Western blot analysis showed that FBS treatment increased WISP1 protein levels in human VSMCs in a dose-dependent manner. Overexpression of WISP1 using adenovirus encoding WISP1 (AD-WISP1) significantly increased proliferation rate of human VSMCs by 2.98-fold compared with empty virus (EV)-transfected cells, shown by EdU incorporation assay. Additionally, Scratch-induced wound healing assay revealed that adenovirus-mediated overexpression of WISP1 significantly increased cell migration compared with EV-transfected cells from 6 h (4.56±1.14% vs. 11.23±2.25%, P<0.05) to 48 h (25.25±5.51% vs. 97.54±13.12%, P<0.01) after injury. Transwell Migration Assay confirmed that WISP1 overexpression significantly promoted human VSMC migration by 2.25-fold compared with EV. Furthermore, WISP1 overexpression stimulated Akt signaling activation in human VSMCs. Blockage of Akt signaling by Akt inhibitor AZD5363 or PI3K inhibitor LY294002, led to an inhibitory effect of WISP1-induced proliferation and migration in human VSMCs. Moreover, we found that WISP1 overexpression stimulated GSK3α/β phosphorylation, and increased expression of cyclin D1 and MMP9 in human VSMCs, and this effect was abolished by AZD5363. Collectively, we demonstrated that Akt signaling pathway mediates WISP1-induced migration and proliferation of human VSMCs, suggesting that WISP1 may act as a novel potential therapeutic target for vascular restenosis.
Keywords: WISP1; Vascular smooth muscle cells; Proliferation; Migration; Restenosis;

Calcium effects and systemic exposure of vitamin D3 analogues after topical treatment of active vitamin D3-containing ointments in rats by Atsushi Hosomi; Maho Hirabe; Takuya Tokuda; Hiroaki Nakamura; Toru Amano; Tadao Okamoto (98-103).
Topical agents containing vitamin D3 (VD3) analogues such as calcipotriol, maxacalcitol and tacalcitol and the combination of calcipotriol/betamethasone dipropionate (betamethasone) are prescribed for patients with psoriasis. However, they are known to occasionally cause hypercalcemia, and the frequency of hypercalcemia is suggested to vary according to the VD3 analogue used. In this study, to address the reason for these differences, the calcemic effects of maxacalcitol-, calcipotriol- and calcipotriol/betamethasone-containing ointments in rats were evaluated. The serum calcium levels in rats treated with ointments containing maxacalcitol, but not calcipotriol or calcipotriol/betamethasone, were significantly elevated, which is consistent with clinical observations. The serum concentration of VD3 analogue in rats treated with ointments containing calcipotriol and calcipotriol/betamethasone was lower than that in rats treated with maxacalcitol-containing ointment. Thus, the calcemic effects appear to be associated with the systemic exposure of VD3 analogues in rats. To understand the mechanism underlying the different systemic exposures of VD3 analogues, skin permeation and metabolic stability of VD3 analogues were evaluated. The cumulative amount of calcipotriol permeated through rat skin was significantly lower than that of maxacalcitol. On the other hand, the metabolic clearance of calcipotriol in rat hepatocytes was higher than that of maxacalcitol. Similar results were obtained using human skin and human hepatocytes. The current study demonstrates that the lower calcemic effects of calcipotriol- and calcipotriol/betamethasone-containing ointments are caused by the low systemic exposure of calcipotriol according to low skin permeability and rapid hepatic elimination after topical application.
Keywords: Vitamin D3-containing ointment; Systemic exposure; Skin permeation; Hepatic metabolism; Calcemic effect;

A novel PPARα agonist propane-2-sulfonic acid octadec-9-enyl-amide inhibits inflammation in THP-1 cells by Yun Zhao; Lu Yan; Xiu-Mei Luo; Lu Peng; Han Guo; Zuo Jing; Li-Chao Yang; Rong Hu; Xuan Wang; Xue-Feng Huang; Yi-Qing Wang; Xin Jin (104-112).
Our group synthesized propane-2-sulfonic acid octadec-9-enyl-amide (N15), a novel peroxisome proliferator activated receptor alpha (PPARα) agonist. Because PPARα activation is associated with inflammation control, we hypothesize that N15 may have anti-inflammatory effects. We investigated the effect of N15 on the regulation of inflammation in THP-1 cells stimulated with lipopolysaccharide (LPS). In particular, we assessed the production of chemokines, adhesion molecules and proinflammatory cytokines, three important types of cytokines that are released from monocytes and are involved in the development of atherosclerosis. The results showed that N15 remarkably reduced the mRNA expression of chemokines, such as monocyte chemotactic protein 1 (MCP-1 or CCL2), interleukin-8 (IL-8) and interferon-inducible protein-10 (IP-10 or CXCL10), and proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). N15 also decreased the protein expression of vascular cell adhesion molecule (VCAM) and matrix metalloproteinase (MMP) 2 and 9. The reduction in the expression of cytokine mRNAs observed following N15 treatment was abrogated in THP-1 cells treated with PPARα siRNA, indicating that the anti-inflammatory effects of N15 are dependent on PPARα activation. Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) inhibition, which are dependent on PPARα activation, were also involved in the mechanism underlying the anti-inflammatory effects of N15. In conclusion, the novel PPARα agonist, N15, exerts notable anti-inflammatory effects, which are mediated via PPARα activation and TLR4/NF-κB and STAT3 inhibition, in LPS-stimulated THP-1 cells. In our study, N15 exhibits promise for the treatment of atherosclerosis.
Keywords: Propane-2-sulfonic acid octadec-9-enyl-amide; Peroxisome proliferator activated receptor alpha; Proinflammatory cytokines; Adhesion molecules; Atherosclerosis;

Kinetic on and off rate constants for many receptor ligands are difficult to determine with regular radioligand binding technique since only few of the ligands are available in labeled form.Here we developed a new and simple radioligand binding method for determining the kinetic off-rate constant for unlabeled ligands, using whole cells expressing α2A- and α2C-adrenoceptors. The new method involves pre-incubation with unlabeled ligand, centrifugation of microtiter plates in order to adhere the cells to the bottom surface, and then upside-down centrifugation of the plates for few seconds to wash away the non-bound fraction of the pre-incubated ligand. The final on-reaction assay for the radioligand is then started by quick addition of a relatively fast-associating radioligand to the cells. The curve obtained is defined by a fairly simple mathematical formula that reflects the simultaneous dissociation of pre-incubated ligand and association of the radioligand. The method proved to produce highly reproducible results in determining the koff constants for various unlabeled ligands.The results show that the α2C-selectivity of MK912 depends mainly on a very slow off-rate at the α2C-adrenoceptor subtype. Regarding the markedly α2C- over α2A-selective compound spiroxatrine, its much faster on-rate at α2C- than α2A-adrenoceptors explains much of its exceptional α2C-selectivity.Several new techniques for determining the kinetic component of ligand-receptor interactions at molecular level are currently developing. As a reference, based on standard radioligand binding techniques, the present study describes a simple and robust experimental and mathematical procedure for determining koff constants of unlabeled drugs.Display Omitted
Keywords: Spiroxatrine; MK912; Off-rate; Residence time; [3H]-RX821002; Kinetics;

G protein coupled receptors (GPCRs) trigger various cellular and physiological responses upon the ligand binding. The ligand binding induces conformational change in GPCRs which allows G protein to interact with the receptor. The interaction of G protein also affects the active conformation of GPCRs. In this study, we have investigated the effects of Gαi1, Gαo and chimeric Gαqi5 on the active conformation of the adenosine A1 receptor, as each Gα showed difference in the interaction with adenosine A1 receptor. The conformational changes in the adenosine A1 receptor were detected as the agonist-induced decreases in efficiency of Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) fused at the two intracellular domains of the adenosine A1 receptor. Amplitudes of the agonist-induced FRET decreases were subtle when the FP-tagged adenosine A1 receptor was expressed alone, whereas they were significantly enhanced when co-expressed with Gαi1Gβ1Gγ22 (Gi1) or Gαqi5Gβ1Gγ22 (Gqi5) but not with GαοGβ1Gγ22 (Go). The enhancement of the agonist-induced FRET decrease in the presence of Gqi5 was significantly larger than that of Gi1. Furthermore, the FRET recovery upon the agonist removal in the presence of Gqi5 was significantly slower than that of Gi1. From these results it was revealed that the agonist-bound active conformation of adenosine A1 receptor is unstable without the binding of G protein and that the stabilizing effects of G protein differ depending on the types of G protein.
Keywords: G protein coupled receptor; Adenosine A1 receptor; Förster resonance energy transfer (FRET); Conformational change; G protein coupling;

Previously, we showed preventive effects of prasugrel, a P2Y12 antagonist, in a non-human primate model of thrombotic middle cerebral artery occlusion (MCAO); however, it remains unclear if P2Y12 inhibition after MCAO reduces cerebral injury and dysfunction. Here we investigated the effects of R-138727, the major active metabolite of prasugrel, on ex vivo platelet aggregation at 5 min, 15 min, 60 min, and 24 h after administration to non-human primates (n=3). A single intravenous dose of R-138727 (0.03–0.3 mg/kg) resulted in significant and sustained dose-related effects on platelets for up to 24 h. R-138727 was administered 1 h after MCAO induction, and its effects on thrombosis, cerebral infarction, and neurological deficits were determined (n=8–10). R-138727 (0.3 mg/kg) significantly increased total patency rate of the MCA (P=0.0211). Although there was no effect on the patency rate before R-138727 dosing (P=0.3975), it increased 1 h after dosing (P=0.0114). R-138727 significantly reduced total ischaemic infarction volumes (P=0.0147), including those of basal ganglia (P=0.0028), white matter (P=0.0393), and haemorrhagic infarction (P=0.0235). Additionally, treatment with R-138727 reduced overall neurological deficits (P=0.0019), including the subcategories of consciousness (P=0.0042), sensory system (P=0.0045), motor system (P=0.0079) and musculoskeletal coordination (P=0.0082). These findings support the possible utility of P2Y12 inhibition during early-onset MCAO to limit the progression and degree of cerebral ischaemia and infarction and also associated neurological deficits.
Keywords: R-138727 (PubChem CID: 10405534); P2Y12 antagonist; R-138727; Platelet; Ischaemic stroke; Non-human primate; Prasugrel;

The 11β-hydroxysteroid dehydrogenase type 1 inhibitor protects against the insulin resistance and hepatic steatosis in db/db mice by Xiaohuan Yuan; Hongzhi Li; He Bai; Xiaojin Zhao; Chunlei Zhang; Haifeng Liu; Yufei Zhang; Binghai Zhao; Yan Wu; Jieting Liu; Qi Xiang; Biao Feng; Yanhui Chu; Yadong Huang (140-151).
Glucocorticoids (GCs) metabolism is regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). When GCs are present in excess, they can impair glucose-dependent insulin sensitivity. We have previously synthesized several curcumin analogues, of which four compounds were selective inhibitors of 11β-HSD1. Here, we present data supporting that the 11β-hydroxysteroid dehydrogenase type 1 inhibitor (H8) inhibits insulin resistance and ameliorates hepatic steatosis in db/db mice. We compared glucose and lipid metabolism in db/db mice with or without administration of H8, which significantly decreased fasting blood glucose levels and protected against insulin resistance and hepatic steatosis compared to when glucose and lipid metabolism were measured following curcumin administration. The hepatic enzyme was reduced significantly in the plasma samples from db/db mice which were treated with H8. Serum corticosterone (active) levels, which are regulated by 11β-HSD1 were reduced when mice received H8. H8 administration suppressed phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-pase) expression, which are related to gluconeogenesis and enhanced glucose transporter 4 (GLUT4) protein content in liver. Treatment with H8 improved obesity and metabolic disorders, such as insulin resistance and hepatic steatosis by suppressing activity of 11β-HSD1, suggesting that H8 might be a beneficial drug for the treatment of obesity and Type-2 diabetes (T2D).Display Omitted
Keywords: Curcumin analogue; Glucose and lipid metabolic; 11β-HSD1; CortiCosterone; Type-2 diabetes;

Role of selective blocking of bradykinin receptor subtypes in attenuating allergic airway inflammation in guinea pigs by Mohamed M. El-Kady; Zarif I. Girgis; Eman A. Abd El-Rasheed; Olfat Shaker; Magdy I. Attallah; Ahmed A. Soliman (152-159).
The present study was designed to evaluate the potential role of bradykinin antagonists (R-715; bradykinin B1 receptor antagonist and icatibant; bradykinin B2 receptor antagonist) in treatment of allergic airway inflammation in comparison to dexamethasone and montelukast. R-715 as dexamethasone significantly decreased peribronchial leukocyte infiltration, bronchoalveolar lavage fluid (BALF) albumin and interleukin 1β as well as serum OVA-specific IgE level. Also, R-715 like montelukast significantly decreased BALF cell count (total and eosinophils). Icatibant showed negative results. The current findings suggest that selective bradykinin B1 receptor antagonists may have the therapeutic potential for the treatment of allergic airway inflammation.
Keywords: Bradykinin; R-715; Icatibant; Airway hyperresponsiveness; Bronchoalveolar lavage; Asthma;

The effects of metformin and simvastatin on the growth of LNCaP and RWPE-1 prostate epithelial cell lines by Pasi Pennanen; Heimo Syvälä; Merja Bläuer; Kimmo Savinainen; Timo Ylikomi; Teuvo L.J. Tammela; Teemu J. Murtola (160-167).
The anti-diabetic drug metformin and cholesterol-lowering statins inhibit prostate cancer cell growth in vitro and have been linked with lowered risk of prostate cancer in epidemiological studies. We evaluated the effects of these drugs on cancerous and non-cancerous prostate epithelial cell lines. Cancer (LNCaP) and normal (RWPE-1) prostate epithelial cell lines were treated with pharmacologic concentrations of metformin and simvastatin alone and in combinations. Relative changes in cell number were measured with crystal violet staining method. Drug effects on apoptosis and cell cycle were measured with flow cytometry. We also measured changes in the activation and expression of a set of reported target proteins of metformin and statins with Western blotting. Metformin decreased the relative cell number of LNCaP cells by inducing G1 cell cycle block, autophagy and apoptosis, and slightly increased cytosolic ATP levels, whereas RWPE-1 cells were resistant to metformin. However, RWPE-1 cells were sensitive to simvastatin, which induced G2 cell cycle block, autophagy and apoptosis, and increased cytosolic ATP levels in these cells. Combination of metformin and simvastatin synergistically decreased cytosolic ATP levels, increased autophagy and instead of apoptosis, induced necrosis in LNCaP cells. Synergistic effects were not observed in RWPE-1 cells. These results suggest, that prostate cancer cells may be more vulnerable to combined growth-inhibiting effects of metformin and simvastatin compared to normal cells. The data presented here provide evidence for the potency of combined metformin and statin, also at pharmacologic concentrations, as a chemotherapeutic option for prostate cancer.
Keywords: Prostate; Cancer; Metformin; Statin; Chemotherapy; Pharmacologic;

Ketoprofen and antinociception in hypo-oestrogenic Wistar rats fed on a high sucrose diet by Osmar Antonio Jaramillo-Morales; Josué Vidal Espinosa-Juárez; Betzabeth Anali García-Martínez; Francisco Javier López-Muñoz (168-175).
Non-steroidal anti-inflammatory drugs such as ketoprofen are the most commonly used analgesics for the treatment of pain. However, no studies have evaluated the analgesic response to ketoprofen in conditions of obesity. The aim of this study was to analyse the time course of nociceptive pain in Wistar rats with and without hypo-oestrogenism on a high sucrose diet and to compare the antinociceptive response using ketoprofen. Hypo-oestrogenic and naïve rats received a hyper caloric diet (30% sucrose) or water ad libitum for 17 weeks, the thermal nociception (“plantar test” method) and body weight were tested during this period. A biphasic response was observed: thermal latency decreased in the 4th week (hyperalgesia), while from 12th to 17th week, thermal latency increased (hypoalgesia) in hypo-oestrogenic rats fed with high sucrose diet compared with the hypo-oestrogenic control group. At 4th and 17th weeks, different doses of ketoprofen (1.8–100 mg/kg p.o.), were evaluated in all groups. The administration of ketoprofen at 4th and 17th weeks showed dose-dependent effects in the all groups; however, a greater pharmacological efficacy was observed in the 4th week in the hypo-oestrogenic animals that received sucrose. Nevertheless, in all the groups significantly diminish the antinociceptive effects in the 17th week. Our data showed that nociception was altered in the hypo-oestrogenic animals that were fed sucrose (hyperalgesia and hypoalgesia). Ketoprofen showed a dose-dependent antinociceptive effect at both time points. However, hypo-oestrogenism plus high-sucrose diet modifies the antinociceptive effect of ketoprofen.Display Omitted
Keywords: High-sucrose diet; Nociception; Ketoprofen; Efficacy;

Preventive and alleviative effects of the dual enkephalinase inhibitor (Denki) PL265 in a murine model of neuropathic pain by Elisabeth Bonnard; Hervé Poras; Marie-Claude Fournié-Zaluski; Bernard P. Roques (176-182).
Neuropathic pain remains difficult to treat due to the involvement of various pathophysiological mechanisms in its pathogeny. Among the different opioidergic systems the enkephalinergic one is primarily recruited via activation of delta opioid receptor (DOP) in chronic pain and of mu opioid receptor (MOP) in acute pain. To investigate the role of their endogenous ligands Met and Leu-enkephalin in neuropathic pain control, a dual inhibitor of their degrading enzymes, PL265, which acts restrictively at the level of peripheral nociceptors, was administered per os to assess its efficacy in pain prevention and alleviation using a partial sciatic nerve ligation model (PSNL) in mice. We demonstrated here that the pre-injury oral administration of PL265 (50 mg/kg) during the 9 days of neuropathy development reduces thermal hyperalgesia and mechanical allodynia for two weeks after the end of treatment. The repeated administration (50 mg/kg daily, during 10 days) does not induce tolerance. Therefore, protecting the enkephalins released at the peripheral level during neuropathic pain with oral PL265 seems to be a promising approach to prevent and alleviate the painful symptoms of neuropathic pain in humans without the unwanted effects of exogenous opiates such as morphine.
Keywords: Dual enkephalinase inhibitor; Endogenous enkephalins; Neuropathic pain; Prevention; Tolerance;

In vivo dopamine agonist properties of rotigotine: Role of D1 and D2 receptors by Sandro Fenu; Elena Espa; Augusta Pisanu; Gaetano Di Chiara (183-191).
Rotigotine acts in vitro as a full agonist of dopamine D1 receptors at concentrations almost superimposable to those at which it acts on D2 receptors. However in vivo evidence of the differences between the agonist activity of rotigotine at D1 receptors from that on the D2 receptors has not been provided yet. In order to test the ability of rotigotine to stimulate dopamine D1 and D2 receptors in vivo, we studied the effect of SCH39166 and eticlopride, selective dopamine D1 and D2/D3 receptor antagonists respectively, on rotigotine-induced contralateral turning behavior in 6-hydroxydopamine lesioned rats. Furthermore, the expression of the immediate-early gene c-fos in the caudate-putamen, was evaluated. As a comparison, we tested the D2/D3 agonist pramipexole. In primed rats, rotigotine (0.035, 0.1 and 0.35 mg/kg) induced dose-dependent contralateral turning. Turning induced by 0.1 mg/kg of rotigotine was reduced by pretreatment with the D1 antagonist SCH39166 and the D2 antagonist eticlopride. In drug-naive rats, rotigotine was less effective in eliciting turning but SCH39166 still reduced turning induced by rotigotine (0.35 mg/kg). Pramipexole induced contralateral turning only in primed rats. SCH39166 potentiated and eticlopride abolished pramipexole-induced turning. Rotigotine induced Fos expression in the caudate-putamen and SCH39166 completely blocked it. Pramipexole failed to induce Fos. These results indicate that rotigotine acts in vivo as an agonist of D1 and D2 receptors while pramipexole is devoid of D1 activity in vivo. Given their differing DA receptor profiles, rotigotine and pramipexole might differ in their spectrum of application to the therapy of Parkinson's disease.
Keywords: Dopamine D1 receptor; Dopamine D2 receptor; Parkinson's disease; Pramipexole; Rotigotine; Turning behavior;

Low proarrhythmic potential of citalopram and escitalopram in contrast to haloperidol in an experimental whole-heart model by Gerrit Frommeyer; Benedict Brücher; Henning von der Ahe; Sven Kaese; Dirk G. Dechering; Simon Kochhäuser; Harilaos Bogossian; Peter Milberg; Lars Eckardt (192-199).
In several case reports proarrhythmic effects of citalopram and escitalopram have been reported. Systematic analyses on prorarrhythmic effects of these drugs are not yet available.The aim of the present study was to investigate if application of citalopram, escitalopram or haloperidol provokes polymorphic ventricular tachycardia in a sensitive model of proarrhythmia.In isolated rabbit hearts monophasic action potentials and ECG showed a significant QT-prolongation after application of citalopram (2 µM: +47 ms, 4 µM: +56ms, P<0.05) accompanied by an increase of action potential duration (APD) but not dispersion of repolarization. Reduced potassium concentration in bradycardic AV-blocked hearts provoked early afterdepolarizations (EAD) in 2 of 12 hearts but no polymorphic ventricular tachycardia (pVT). Application of escitalopram also increased QT-interval (2 µM: +3 ms, 4 µM: +30 ms, P<0.05) and APD without effects on dispersion. 3 of 10 hearts showed EAD and pVT in 2 of 10 hearts (32 episodes).The results were compared to 12 rabbits treated with haloperidol which led to an increase in QT-interval (1 µM:+62 ms; 2 µM:+96ms; P<0.01), APD and dispersion (1 µM:+15 ms, 2 µM:+40 ms; P<0.01) and induced EAD in all 12 and pVT in 10 of 12 hearts (152 episodes).Citalopram and escitalopram demonstrated a rather safe electrophysiologic profile despite significant QT prolongation. In contrast, haloperidol led to significant increase of dispersion of repolarization while this parameter remained stable under the influence of citalopram or escitalopram. These results imply that application of citalopram or escitalopram is not as proarrhythmic as some case reports might suggest while haloperidol is torsadogenic.
Keywords: Citalopram; Escitalopram; Haloperidol; Dispersion of repolarization; Proarrhythmia; Sudden cardiac death;

(S)YS-51, a novel isoquinoline alkaloid, attenuates obesity-associated non-alcoholic fatty liver disease in mice by suppressing lipogenesis, inflammation and coagulation by Eun Jung Park; Young Min Kim; Hye Jung Kim; Se-Yun Jang; Moo Hyun Oh; Duck-Hyung Lee; Ki Churl Chang (200-209).
Obesity-associated non-alcoholic fatty liver disease (NAFLD) increases coagulation and inflammation. We hypothesized that (S)YS-51, an agent found to be beneficial in animal models of sepsis, may reduce NAFLD in high-fat diet (HFD) mice by reducing coagulation and inflammation. C57BL/6 mice were fed either a chow diet or HFD and each was supplemented with or without (S)YS-51 (10 mg/kg, daily, i.p.) for 16 weeks. The results showed that HFD caused significant increases in lipogenesis [CD36, fatty acid synthase (FAS) and sterol response element binding protein (SREBP)-1c mRNA and protein], inflammation [monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, intercellular cell adhesion molecule-1 (ICAM-1), TGF-β, and procollagen type 1 mRNA, macrophage infiltration] and coagulation [tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) mRNA and thrombin antithrombin complex (TAT)] in the liver, adipose tissue and serum, which were significantly reduced by (S)YS-51. These results of (S)YS-51 were accompanied by significant reduction of weight gain, liver size, hepatic steatosis and fibrosis, blood cholesterol, hepatic triglyceride, and macrophage infiltration and inflammatory cytokines in adipose tissue without affecting food intake in HFD mice. Interestingly, (S)YS-51 increased SIRT1 mRNA and protein and AMPK expression in the liver of HFD mice by increasing both NAD+/NADH ratio and LKB1 phosphorylation. In HepG2 cells, (S)YS-51 activated SIRT1 followed by AMPK. Finally, (S)YS-51 improved glucose tolerance and insulin resistance in HFD mice. We concluded that (S)YS-51 attenuates NAFLD and insulin resistance in HFD mice by, at least, activation of SIRT1/AMPK signals. Thus, (S)YS-51 may be beneficial in NAFLD treatment.Display Omitted
Keywords: AMPK; SIRT1; Inflammation; Coagulation; High fat-diet;

Trans fat intake across gestation and lactation increases morphine preference in females but not in male rats: Behavioral and biochemical parameters by Karine Roversi; Camila Simonetti Pase; Katiane Roversi; Luciana Taschetto Vey; Verônica Tironi Dias; Vinícia Garzella Metz; Marilise Escobar Burger (210-217).
The abuse of morphine has risen considerably in recent years, mainly due to the increase of its prescription in clinical medicine. Also, increased consumption of processed foods, rich in trans fatty acids (TFA), has caused concerns about human health. Thus, the aim of our study was to determine whether trans fat consumption in the perinatal period may affect preference for morphine in adolescent female and male rats. Dams were orally supplemented with water (C-control) or hydrogenated vegetable fat (HVF-rich in TFA) during gestation and lactation periods. On post-natal day 43, pups were exposed to morphine (4 mg/kg i.p., for 4 days) and assessed in the conditioned place preference paradigm. Anxiety-like symptoms were assessed, and oxidative status of the brain was estimated by reactive species (RS) generation. Female rats with HVF supplementation showed increased morphine preference and less anxiety-like symptoms. Additionally, both male and female rats from HVF-supplementation showed increased RS generation in the ventral tegmental area, whose level was similar in morphine-conditioned female rats. RS generation was increased in the hippocampus of morphine-conditioned female rats, regardless of the supplementation of their dams. We may infer that gender is a predictive factor to opioid preference, since adolescent female rats showed more susceptibility to addiction than males. Furthermore, trans fat consumption across the perinatal period is able to modify parameters of opioid preference in female rats, possibly due to TFA incorporation in phospholipid membranes, modifying the endogenous opioid system and the oxidative status in brain areas related to drug addiction.
Keywords: Trans fat; Opioid addiction; Gender; Conditioned place preference; Oxidative status; Adolescent rats;

GHSR-1a is a novel pro-angiogenic and anti-remodeling target in rats after myocardial infarction by Ming-Jie Yuan; Tao Wang; Bin Kong; Xin Wang; Cong-Xin Huang; Danny Wang (218-225).
Cardiac remodeling and subsequent heart failure is an increasing public health problem after myocardial infarction (MI). The aim of our research is to investigate whether gene therapy of growth hormone secretagogue receptor 1a may regulate cardiac remodeling and function after MI. Adenoviral vector expressing GHSR-1a or empty adeno-null was injected into rat peri-infarct myocardium after left anterior descending coronary artery ligation. GHSR-1a expression was confirmed by western blotting. 14 days after operation, haemodynamic and echocardiographic analysis demonstrated that GHSR-1a treatment significantly improved survive rate, increased scar thickness, preserved LV diameter, restored cardiac function and increased angiogenesis. There was no difference in infarct size between MI+Ad-GHSR-1a group and MI + Ad-null group. Additionally, increased protein expression of Akt phosphorylation and AMP-activated protein kinase phosphorylation in the infarct border myocardium were also observed. Moreover, GHSR-1a overexpression significantly enhanced tube formation in human umbilical endothelial cells (HUVECs) under ischemia condition. Knockdown of GHSR-1a by siRNA markedly decreased vascular endothelial growth factor expression as well as mRNA levels of Akt and AMPK. In conclusion, GHSR-1a gene therapy improves cardiac remodeling and function in rats after myocardial infarction. This may be a new anti-remodeling target to MI.
Keywords: Myocardial infarction; GHSR-1a; Angiogenesis; Cardiac remodeling;

Fasudil evokes vasodilatation of rat mesenteric vascular bed via Ca2+ channels and Rho/ROCK pathway by Yu-cai Chen; Tian-yi Yuan; Hui-fang Zhang; Dan-shu Wang; Zi-ran Niu; Li Li; Lian-hua Fang; Guan-hua Du (226-233).
As a Rho kinase (ROCK) inhibitor, fasudil has been used in clinical trials of several cardiovascular diseases. This study was to investigate the vasorelaxant effect of fasudil on resistance arterial rings including mesenteric, renal, ventral tail and basilar artery. We also examined the potential mechanisms of its vasodilatory action using mesenteric artery rings.A DMT multiwire myograph system was used to test the tension of isolated small arteries. K+ channel blockers, NO-cGMP pathway blockers and Ca2+-free physiological salt solution (PSS) were employed to verify the underlying mechanisms.Fasudil (10−7–10−4  M) relaxed four types of small artery rings pre-contracted by 60 mmol/l KCl (pEC50: 6.01±0.09, 5.47±0.03, 5.54±0.04, and 5.72±0.10 for mesenteric, renal, ventral tail and basilar artery rings, respectively). Pre-incubation with fasudil (1, 3, or 10 μmol/l) attenuated KCl (10–60 mmol/l) and angiotensin II (Ang II; 1 μmol/l)-induced vasoconstriction in mesenteric artery rings. Fasudil at the concentration of 10−6  mol/l showed different relaxant potency in endothelium intact (pEC50:6.01±0.09) or denued (5.75±0.06) mesenteric artery. The influx and release of Ca2+ were inhibited by fasudil. In addition, fasudil could block the increased phosphorylation level of myosin light chain (MLC) and myosin-binding subunit of myosin phosphatase (MYPT1) induced by Ang II. However, pretreatment with various K+ channel blockers did not affect the relaxant effects of fasudil remarkably.The present results demonstrate that fasudil has a vasorelaxant effect on isolated rat resistance arteries, including mesenteric, renal, ventral tail and basilar artery, and may exert its action through the endothelium, Ca2+ channels, and the Rho/ROCK pathway.
Keywords: Fasudil; Rho-kinase; Mesenteric artery; Endothelium; Calcium blocker; Nitric oxide;

The protective role of neuregulin-1: A potential therapy for sepsis-induced cardiomyopathy by Qin Zhou; Xia Pan; Long Wang; Xi Wang; Dongsheng Xiong (234-240).
The extremely high mortality of sepsis in intensive care units, caused primarily by sepsis-induced cardiomyopathy, is a pressing issue. Current studies have revealed the importance of the neuregulin-1 (NRG-1)/ErbB signaling axis at the cardiovascular level and the positive effect of NRG-1 on cardiac function in patients with heart failure. To investigate the protective mechanism of NRG-1 against myocardial injury in septic rats, a cecal ligation and puncture (CLP) model was applied. Animals were administered either a vehicle or recombinant human NRG-1 (rhNRG-1, 10 μg/kg). Their survival rates were noted 24 h after CLP. The hemodynamic method was used to evaluate their cardiac function. The myocardial morphology was observed. An enzyme-linked immunosorbent assay was used to detect the level of cardiac troponin-T (cTn-T), cytokines, and angiotensin II (Ang II) in the serum and myocardium. Compared with the vehicle, rhNRG-1 improved survival of rats and prevented hemodynamic derangement, as reflected in the increased mean arterial pressure, left ventricular systolic pressure, ±dp/dt max, and decreased left ventricular end-diastolic pressure (P<0.05). Furthermore, the serum levels of cTn-T and pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6) were significantly increased in vehicle-treated rats but reduced in rhNRG-1-treated rats. The latter also showed decreased concentration of macrophage inhibitory factor and Ang II in the myocardium (P<0.05). These results suggest that NRG-1 improved cardiac function and protected cardiomyocytes of rats from CLP-induced sepsis by suppressing the immune inflammatory response and excessive activation of the renin–angiotensin–aldosterone system. Ultimately, NRG-1 increased the survival rate of rats.
Keywords: Neuregulin-1; Sepsis; Cardiac function; Inflammation; Macrophage inhibitory factor; Angiotensin II;

Pretreatment of 6-shogaol attenuates oxidative stress and inflammation in middle cerebral artery occlusion-induced mice by Ji-Young Na; Kibbeum Song; Ju-Woon Lee; Sokho Kim; Jungkee Kwon (241-247).
6-Shogaol can be extracted from ginger and has been shown to exert anti-inflammatory and antioxidant activities, which are potentially relevant to the treatment of central nervous system disorders. Oxidative stress and inflammation are closely associated with ischemic injury and can eventually result in neuronal death. The aim of this study was to evaluate if 6-shogaol exerts neuroprotective activity. To this end, we determined its effects on oxidative stress and inflammation in a mouse model of middle cerebral artery occlusion (MCAO)-induced brain damage. In this model, MCAO was induced in C57BL/6 mice (30–35 g, 9 weeks) for 1 h, followed by 24 h reperfusion. Mice were treated orally with 6-shogaol (0.1 ml, 5 or 20 mg/kg) once daily for 7 consecutive days prior to MCAO. We found that 6-shogaol significantly reduced neurological deficit scores and the mean infarct area. Moreover, 6-shogaol improved the behavioral deficits in the MCAO group. In addition, 6-shogaol pretreatment dampened MCAO-mediated production of reactive oxygen species and inflammatory cytokines. Mechanistic studies revealed that 6-shogaol inhibits the cysteinyl leukotriene 1 receptor (CysLT1R) and mitogen-activated protein kinase (MAPK) signaling proteins, thus providing a potential pharmacological mechanism for our observations. These results suggest that 6-shogaol can ameliorate the outcomes of MCAO and could thus be used as a potential preventive of stroke.Display Omitted
Keywords: 6-Shogaol; MCAO; Ischemia; Oxidative stress; Inflammation;

Interaction of atorvastatin with the human glial transporter SLC16A1 by Shotaro Sasaki; Yuya Futagi; Masaya Ideno; Masaki Kobayashi; Katsuya Narumi; Ayako Furugen; Ken Iseki (248-254).
Solute carrier (SLC) 16A1 is a pH-dependent carrier of 5-oxoproline, a derivative of the amino acid. SLC16A1 interacts with carboxylate group-containing substrates, which are also present in atorvastatin, and might be the reason for its ability to interact with atorvastatin. Does atorvastatin interact with the carrier? Does it also interact with the carrier via the substrate recognition site? This study was carried out to answer these questions. Polymerase chain reaction was used to determine the expression of SLC16A1 in normal human astrocytes. We induced SLC16A1 expression in a mammalian cell line and in Xenopus laevis oocytes. We used [3H] 5-oxoproline for direct measurement of SLC16A1-specific transport activity. SLC16A1 was clearly observed in normal human astrocytes. 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors inhibited the SLC16A1-specific transport of 5-oxoproline. Atorvastatin was the most potent inhibitor, with an inhibition constant of 40 μM. The drug was a non-competitive inhibitor of SLC16A1. In the present study, we showed non-competitive inhibition of SLC16A1-specific transport activity by atorvastatin. However, the affinity between the drug and the carrier was extremely low. Therefore, the interaction of atorvastatin with SLC16A1 is unlikely to be a problem in clinical practice.
Keywords: Atorvastatin; Solute carrier; Human monocarboxylate transporter; 5-oxoproline;

In 2013, we discovered that the entitled “calcium paradox” phenomenon, which means a paradoxical sympathetic hyperactivity produced by l-type Ca2+ channel blockers (CCBs), used in antihypertensive therapy, is due to interaction between the intracellular signalling pathways mediated by Ca2+ and cAMP (Ca2+/cAMP interaction). In 2015, we proposed that the pharmacological manipulation of this interaction could be a new therapeutic strategy for increasing neurotransmission in psychiatric disorders, and producing neuroprotection in the neurodegenerative diseases. Besides the paradoxical sympathetic hyperactivity produced by CCBs, several clinical studies have been demonstrating pleiotropic effects of CCBs, including neuroprotective effects. CCBs genuinely exhibit cognitive-enhancing abilities and reduce the risk of dementia, including Alzheimer’s, Parkinson´s disease and others. The molecular mechanisms involved in these pleiotropic effects remain under debate. Our recent discovery that the “calcium paradox” phenomenon is due to Ca2+/cAMP interaction may provide new insights for the pharmacological treatment of neurological and psychiatric disorders, including enhancement of current therapies mainly by reducing adverse effects, and improving effectiveness of modern medicines. Whether Ca2+/cAMP interaction is involved in CCBs pleiotropic effects also deserves special attention. Then, the pharmacological manipulation of the Ca2+/cAMP interaction could be a more efficient therapeutic strategy for increasing neurotransmission in psychiatric disorders, and producing neuroprotection in the neurodegenerative diseases. Thus, in this review we summarize the current knowledge of this field, making new directions and future perspectives.
Keywords: Neurological and psychiatric disorders; Ca2+/cAMP signalling interaction;

Pattern recognition receptors in chronic pain: Mechanisms and therapeutic implications by Jungo Kato; Nilesh M. Agalave; Camilla I. Svensson (261-273).
For the individual, it is vital to promptly detect and recognize a danger that threatens the integrity of the body. Pattern recognition receptors (PRRs) are several classes of protein families originally classified as receptors detecting exogenous pathogens. PRRs are also capable of recognizing molecules released from damaged tissues (damage-associated molecular pattern molecules; DAMPs) and thereby contribute to danger recognition. Importantly, it is now evident that PRRs, such as toll-like receptors (TLRs) and receptors for advanced glycation end products (RAGE), are not only expressed in peripheral immune cells but also present in neurons and glial cells in the nervous system. These PRR-expressing cells work in concert, enabling highly sensitive danger recognition. However, this sensitiveness can act as a double-edged sword. Accumulated evidence has led to the hypothesis that aberrant activation of PRRs may play a crucial role in the pathogenesis of pathological pain. Indeed, numerous studies employing gene deletion or pharmacological inhibition of PRRs successfully reversed or prevented pathological pain in experimental animal models. Furthermore, a number of preclinical studies have shown the therapeutic potential of targeting PRRs for chronic pain. Here, we review the current knowledge regarding the role of PRRs in chronic pain and discuss the promise and challenges of targeting PRRs as a novel therapeutic approach for chronic pain.
Keywords: PRRs; DAMPs; TLR; RAGE; Chronic pain;

Interactions between calcium channels and SK channels in midbrain dopamine neurons and their impact on pacemaker regularity: Contrasting roles of N- and L-type channels by Veronne de Vrind; Jacqueline Scuvée-Moreau; Guillaume Drion; Cyrine Hmaied; Fabian Philippart; Dominique Engel; Vincent Seutin (274-279).
Although small-conductance Ca2+-activated K+ (SK) channels and various types of voltage-gated Ca2+ (Cav) channels have been described in midbrain dopaminergic neurons, the nature of their interactions is unclear. More particularly, the role of various Cav channel types in either promoting irregularity of firing (by generating an inward current during SK channel blockade) or promoting regularity of firing (by providing the source of Ca2+ for the activation of SK channels) has not been systematically explored. We addressed this question using intracellular and extracellular recordings from substantia nigra, pars compacta (SNc), dopaminergic neurons in rat midbrain slices. Neurons were pharmacologically isolated from their differences. When examining the ability of various Cav channel blockers to inhibit the SK-mediated afterhyperpolarization (AHP), we found that only the N-type Cav channel blocker ω-conotoxin-GVIA was able to reduce the apamin-sensitive AHP, but only partially (~40%). Specific blockers of L, P/Q, T or R channels had no effect on this AHP. Combining ω-conotoxin-GVIA and other specific blockers did not yield greater block and even the broad Cav blocker Cd2+ induced a submaximal (~75%) effect. Extracellular recordings examining firing regularity yielded congruent results: none of the specific blockers was able to increase firing irregularity to the extent that the specific SK blocker apamin did. The irregularity of firing observed with apamin could only be reversed by blocking L-type Ca2+ channels. Thus various sources of Ca2+ appear to be required for SK channel activation in SNc neurons (some of them still unidentified), ensuring robustness of pacemaking regularity.
Keywords: SK channel; Cav channels; Dopaminergic neurons;

U0126 attenuates ischemia/reperfusion-induced apoptosis and autophagy in myocardium through MEK/ERK/EGR-1 pathway by Anxing Wang; Huijun Zhang; Zeming Liang; Kai Xu; Weifeng Qiu; Yongbo Tian; Hong Guo; Junzheng Jia; Erke Xing; Rufei Chen; Zongxing Xiang; Jia Liu (280-285).
Myocardial ischemia is one of the main causes of sudden cardiac death worldwide. Depending on the cell type and stimulus, ERK activity mediates different anti-proliferative events, such as apoptosis, autophagy, and senescence. The aim of this study was to determine the protective effect of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126), an ERK kinase inhibitor, on myocardial ischemia/reperfusion (I/R) injury and the mechanisms involved. An I/R model was established in vivo in C57BL/6 mice and in vitro using mouse cardiomyocytes, respectively. To evaluate the protective effects of U0126 on I/R injury, we measured the myocardial infarct area, apoptosis, and autophagy. Our data indicated that pretreatment with U0126 significantly reduced the infarct area caused by I/R. Moreover, U0126 reduced the caspase-3 activity and the number of TUNEL-positive cardiomyocytes, which together indicate decreased apoptosis. Additionally, U0126 remarkable reduced the level of Beclin-1 and LC3 and increased p62 expression, which indicates that U0126 suppressed H/R-induced autophagy. Furthermore, the relationship between U0126 and MEK/ERK pathway activation in H/R-induced cardiomyocytes was also investigated. U0126 ameliorated H/R injury through inhibition of the MEK/ERK pathway and by suppressing in the downstream EGR-1 expression. Together, our research suggests that U0126 may protect against H/R injury by preventing H/R-induced myocardium apoptosis and autophagy via the MEK/ERK/EGR-1 pathway, and may be a potential therapeutic approach for attenuating myocardial I/R injury.
Keywords: U0126; Ischemia/reperfusion (I/R); MEK/ERK; EGR-1; Myocardium;

Block of Na+/Ca2+ exchanger by SEA0400 in human right atrial preparations from patients in sinus rhythm and in atrial fibrillation by Torsten Christ; Peter P. Kovács; Karoly Acsai; Michael Knaut; Thomas Eschenhagen; Norbert Jost; András Varró; Erich Wettwer; Ursula Ravens (286-293).
The Na+/Ca2+ exchanger (NCX) plays a major role in myocardial Ca2+ homoeostasis, but is also considered to contribute to the electrical instability and contractile dysfunction in chronic atrial fibrillation (AF). Here we have investigated the effects of the selective NCX blocker SEA0400 in human right atrial cardiomyocytes from patients in sinus rhythm (SR) and AF in order to obtain electrophysiological evidence for putative antiarrhythmic activity of this new class of drugs. Action potentials were measured in right atrial trabeculae using conventional microelectrodes. Human myocytes were enzymatically isolated. Rat atrial and ventricular cardiomyocytes were used for comparison. Using perforated-patch, NCX was measured as Ni2+-sensitive current during ramp pulses. In ruptured-patch experiments, NCX current was activated by changing the extracellular Ca2+ concentration from 0 to 1 mM in Na+-free bath solution (100 mM Na+ intracellular, “Hilgemann protocol”). Although SEA0400 was effective in rat cardiomyocytes, 10 µM did not influence action potentials and contractility, neither in SR nor AF. SEA0400 (10 μM) also failed to affect human atrial NCX current measured with perforated patch. With the “Hilgemann protocol” SEA0400 concentration-dependently suppressed human atrial NCX current, and its amplitude was larger in AF than in SR cardiomyocytes. Our results confirm higher NCX activity in AF than SR. SEA0400 fails to block Ni2+-sensitive current in human atrial cells unless unphysiological conditions are used. We speculate that block of NCX with SEA0400 depends on intracellular Na+ concentration.
Keywords: Sodium-calcium exchanger; NCX current; SEA0400; Human right atrial cardiomyocytes; Atrial fibrillation;

Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD by Jürgen Knobloch; Yakup Yakin; Sandra Körber; Barbara Grensemann; Zeynep Bendella; Niyazi Boyaci; Willem-Jakob Gallert; Sarah Derya Yanik; David Jungck; Andrea Koch (294-305).
T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity.Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma.T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.
Keywords: COPD; Asthma; Simvastatin; Anti-inflammatory activity; T-cells;

Effects of the new thiazolidine derivative LPSF/GQ-02 on hepatic lipid metabolism pathways in non-alcoholic fatty liver disease (NAFLD) by Shyrlene Araújo; Amanda Soares e Silva; Fabiana Gomes; Edlene Ribeiro; Wilma Oliveira; Amanda Oliveira; Ingrid Lima; Maria do Carmo Lima; Ivan Pitta; Christina Peixoto (306-314).
Non-alcoholic fatty liver disease (NAFLD) is considered the most common manifestation of metabolic syndrome. One of its most important features is the accumulation of triglycerides in the hepatocyte cells. Thiazolidinediones (TZDs) act as insulin sensitizers and are used to treat patients with type 2 diabetes and other conditions that are resistant to insulin, such as hepatic steatosis. Controversially, TZDs are also associated with the development of cardiovascular events and liver problems. For this reason, new therapeutic strategies are necessary to improve liver function in patients with chronic liver diseases. The aim of the present study was to evaluate the effects of LPSF/GQ-02 on the liver lipid metabolism in a murine model of NAFLD. Eighty male LDLR-/- mice were divided into 3 groups: 1–fed with a high-fat diet (HFD); 2–HFD+Pioglitazone (20 mg/kg/day); 3–HFD+LPSF/GQ-02 (30 mg/kg/day). The experiments lasted 12 weeks and drugs were administered daily by gavage in the final four weeks. The liver was processed for optical microscopy, Oil Red O, immunohistochemistry, immunofluorescence and western blot analysis. LPSF/GQ-02 effectively decreased fat accumulation, increased the hepatic levels of p-AMPK, FoxO1, ATGL, p-ACC and PPARα, and reduced the expression of LXRα, SREBP-1c and ACC. These results suggest that LPSF/GQ-02 acts directly on the hepatic lipid metabolism through the activation of the PPAR-α/AMPK/FoxO1/ATGL lipolytic pathway, and the inhibition of the AMPK/LXR/SREBP-1c/ACC/FAS lipogenic pathway.
Keywords: NAFLD; Thiazolidinediones; LPSF/GQ-02; PPARα; AMPK and SREBP-1c;

A pharmacological profile of the high-affinity GluK5 kainate receptor by Stine Møllerud; Jette Sandholm Kastrup; Darryl S. Pickering (315-320).
Mouse GluK5 was expressed in Sf9 insect cells and radiolabelled with [3H]-kainate in receptor binding assays (Kd=6.9 nM). Western immunoblotting indicated an Sf9 GluK5 band doublet corresponding to the glycosylated (128 kDa) and deglycosylated (111 kDa) protein, which was identical to the band pattern of native rat brain GluK5. A pharmacological profile of the high-affinity kainate receptor GluK5 is described which is distinct from the profiles of other kainate receptors (GluK1-3). The 27 tested ligands generally show a preferential affinity to GluK1 over GluK5, the exceptions being: dihydrokainate, (S)-5-fluorowillardiine, (S)-glutamate and quisqualate, where the affinity is similar at GluK1 and GluK5. In contrast, quisqualate shows 40-fold higher affinity at GluK5 over GluK3 whereas (S)-1-(2′-amino-2′-caboxyethyl)thienol[3,4-d]pyrimidin-2,4-dione (NF1231), (RS)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionate (ATPA), dihydrokainate and (2S,4R)-4-methyl-glutamate (SYM2081) have higher affinity at GluK3 compared to GluK5. Since some studies have indicated that GluK5 is associated with various diseases in the central nervous system (e.g. schizophrenia, temporal lobe epilepsy, bipolar disorder), selective GluK5 ligands could have therapeutic potential. The distinct pharmacological profile of GluK5 suggests that it would be possible to design ligands with selectivity towards GluK5.
Keywords: Ionotropic glutamate receptor; Radioligand binding; Kinetics; Affinity; Glycosylation; Sf9;

Heme oxygenase-1 (HO-1) significantly contributes to survival of cancer cells and is being considered as one of therapeutic targets for cancer treatment. Propyl gallate (PG) is a synthetic phenolic compound that possess a potent anti-oxidant and anti-inflammatory activities. In the present study, we investigated whether PG exhibit an anti-cancer effect through modulating HO-1 activation. In human non-small cell lung cancer (NSCLC) cells, treatment with PG dose-dependently diminished HO-1 protein levels without changing its mRNA levels and consequently decreased HO-1 activity. PG also significantly enhanced the sensitivity of NSCLC cells to cisplatin-induced apoptosis, and this effect was attenuated by overexpression of HO-1. Mechanistically, PG exerted its chemosensitization effect by down-regulating HO-1 protein expression through a TRC8 (translocation in renal carcinoma, chromosome 8)-mediated ubiquitin-proteasome pathway. Collectively, our data provide the potential application of PG in combination chemotherapy to enhance drug sensitivity in lung cancer by targeting HO-1.
Keywords: Propyl gallate; Drug sensitivity; Lung cancer; HO-1 degradation; TRC8 E3 ligase;

Nitric oxide and Kir6.1 potassium channel mediate isoquercitrin-induced endothelium-dependent and independent vasodilation in the mesenteric arterial bed of rats by Arquimedes Gasparotto Junior; Renê dos Reis Piornedo; Jamil Assreuy; José Eduardo Da Silva-Santos (328-334).
The vascular effect of flavonoid isoquercitrin was investigated in the perfused mesenteric vascular bed of rats. In preparations with functional endothelium isoquercitrin (100, 300 and 1000 nmol) dose-dependently reduced the perfusion pressure by 13±2.2, 33±3.9, and 58±3.7 mm Hg, respectively. Endothelium removal or inhibition of the nitric oxide synthase enzymes by l-NAME did not change the effects of 100 and 300 nmol isoquercitrin, but reduced by 30–40% the vasodilation induced by 1000 nmol isoquercitrin. Perfusion with nutritive solution containing 40 mM KCl abolished the vasodilatory effect of all isoquercitrin doses. Treatment with glibenclamide, a Kir6.1 (ATP-sensitive) potassium channel blocker, inhibited vasodilation induced by 100 and 300 nmol isoquercitrin, but only partially reduced the effect of 1000 nmol isoquercitrin. The non-selective KCa (calcium-activated) potassium channel blocker tetraethylammonium, but not the selective KCa1.1 channel blocker iberiotoxin, reduced by around 60% vasodilation induced by all isoquercitrin doses. In addition, association of tetraethylammonium and glibenclamide, or l-NAME and glibenclamide, fully inhibited isoquercitrin-induced vasodilation. Our study shows that isoquercitrin induces vasodilation in resistance arteries, an effect mediated by K+ channel opening and endothelial nitric oxide production.
Keywords: Isoquercitrin; Mesenteric arterial bed; Potassium channel; Vasodilation;

Neuroprotective effects of Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside on cerebral ischemia-reperfusion injury in rats by Shuaijun Wang; Huali Xu; Ying Xin; Maowei Li; Wenwen Fu; Yuchen Wang; Zeyuan Lu; Xiaofeng Yu; Dayun Sui (335-342).
In the present study, we aim to evaluate the potential neuroprotective effect and the underlying mechanism of Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside (A-F-B) against cerebral I/R injury. Adult male rats were pretreated with A-F-B by intragastric administration once a day for 3 days. One hour after the third day administration, animals were subjected to 2 h of transient middle cerebral artery occlusion (MCAO) followed by 24 h of reperfusion. Neurological deficit, infarct volume, histopathological changes, oxidative stress-related biochemical parameters, neuronal apoptosis, apoptosis-related proteins and the expression of pro-inflammator cytokines genes were measured. A-F-B significantly decreased neurological and histological deficits, reduced the infarct volume, and decreased neuroapoptosis. Meanwhile, A-F-B inhibited the expression of Bax, cleaved caspase-3, cleaved caspase-9, and promoted Bcl-2 expression. In addition, the expression of pro-inflammator cytokines, including phospho-NF-kBp65, interleukin-1β, interleukin-6, tumor necrosis factor-α, intercellular adhesion molecule-1, cyclooxygenase-2 and inducible nitric oxide synthase, were also suppressed by A-F-B pretreatment. Furthermore, pretreatment with A-F-B could significantly increase the activities of superoxide dismutase, glutathione peroxidase, but decrease the content of malondiadehyde in blood serum. These results suggest that A-F-B has the neuroprotective effect in ischemic stroke by suppressing neuroinflammation, reactive oxygen species and neuroapoptosis.
Keywords: Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside; Cerebral ischemia; Reperfusion; Oxidative stress; Apoptosis;

Recent studies have implicated that Hypoxia-inducible factor 1α (HIF-1α) plays an integral role in the pathogenesis of allergic rhinitis and asthma. In the present study, we showed that HIF-1α antagonist YC-1, 3-(5-hydroxymethyl-2-furyl)-1-benzylindazole, elicited a potent allergy-ameliorating effect in a rat model of ovalbumin (OVA)-sensitized combined allergic rhinitis and asthma syndrome (CARAS). We revealed that YC-1 administration markedly impaired the total number and percentage of eosinophil in bronchoalveolar lavage fluid (BAL Fluid) of the rats, suggesting that YC-1 might attenuate lung and nasal mucosal inflammation in OVA-sensitized rats. Moreover, histological examination found that OVA-induced pathological alterations were evidently attenuated following YC-1 administration. In addition, immunohistochemistrial analysis indicated that YC-1 treatment decreased the expression of HIF-1α in rat lungs and nasal mucosa. Notably, Nuclear factor kappa B (NF-κB) p65 and Peroxisome proliferator-activated receptor α (PPARα), two important regulators of inflammatory responses, were also significantly down-regulated following YC-1 administration. Real-time PCR analysis confirmed that YC-1 impaired the expression of HIF-1α, NF-κB and PPARα in CARAS model. These findings together indicated that YC-1 exerted remarkable anti-allergic effects through the modulation of inflammatory pathways, implying that YC-1 may potentially serve as a novel anti-CARAS medicine in clinical patients.
Keywords: HIF-1α; Combined allergic rhinitis and asthma syndrome; YC-1; Inflammation; Rat;

Osteoporosis is caused by a functional imbalance between osteoblasts and osteoclasts. The increased activation of osteoclasts that is a hallmark of osteoporosis results in the progressive loss of bone mass and therefore in an increased susceptibility to bone fractures. Diarylheptanoids are a group of phytoestrogens that have been isolated from a number of plant species, including the rhizomes of Curcuma comosa Roxb. In this study, the effect of one of diarylheptanoids, (3S)-1-(3,4-dihydroxyphenyl)-3-hydroxy-7-phenyl-(6E)-6-heptene (DHPH), was investigated for anti-inflammatory and anti-osteoclastogenic activity. DHPH significantly inhibited nitric oxide production in RAW264.7 cell line following their activation by lipopolysaccharide and interferon-γ, with no cytotoxicity. In primary mouse bone-marrow-derived macrophage precursors, DHPH suppressed osteoclastogenesis induced by receptor activator of nuclear factor-κB (RANK) ligand at an inhibitory concentration 50 of 325±1.37 nM. DHPH treatment delayed and reduced the expression of master regulators of osteoclast differentiation, NFATc1 and c-Fos. Consistent with this result, the mRNA level of cathepsin K, associated with osteoclast differentiation, was decreased whereas the reduction in the mRNA of irf8, a negative regulator of osteoclast differentiation, was similar to that measured in the vehicle-treated control cells. DHPH reduced the phosphorylation of p38 MAPK, ERK (p44/42). Furthermore, DHPH suppressed the bone absorption activity of osteoclasts and enhanced osteoblast differentiation. Taken together, DHPH interrupts the immediate downstream signaling cascade of RANK and interferes with osteoclast differentiation and its function while enhances osteoblast differentiation. These results demonstrate the potential of this diarylheptanoid as a new therapeutic agent in osteoporosis.Display Omitted
Keywords: Osteoclast; Receptor activator of nuclear factor-κB ligand; Diarylheptanoid; Nuclear factor of activated T cells c1;