European Journal of Pharmacology (v.696, #1-3)

ATP and other extracellular nucleotides have diverse and potent effects in different organs. Evidence indicates that extracellular nucleotides and nucleosides deliver crucial signals by acting upon a wide variety of purinergic receptors, which include 19 members separated in three families. Purinergic receptors encompass adenosine-sensitive receptors (P1) as well as the ATP and ADP-responsive receptors (P2). On the other side, P2 receptors are divided into ionotropic P2X receptors and G protein-coupled receptors P2Y. This system of purinergic signaling is made further complex by the fact that ectonucleotidases, membrane bound enzymes, participate in the metabolism of extracellular nucleotides, which are released by cells. Hence, ectonucleotidases are important modulators of purinergic receptor function. It should be pointed out that the ectonucleotidases includes enzymes with different substrate preferences and by their action generate different nucleotides and nucleosides as well as phosphate and pyrophosphate. A growing body of evidence points toward the fact that the expression pattern of different ectonucleotidases and purinergic receptors is implicated in several cardiovascular disorders. In this perspective, a short account is given on the role of ectonucleotidases into the pathobiology of some cardiovascular disorders and the need to develop a novel pharmacology based on those recent findings.
Keywords: Ectonucleotidase; Pharmacology; Calcific aortic valve disease; Diabetes; Thrombosis;

Cardiovascular risk in autoimmune disorders: Role of asymmetric dimethylarginine by Xu-Meng Chen; Chang-Ping Hu; Yuan-Jian Li; Jun-Lin Jiang (5-11).
Mounting evidence indicates that cardiovascular events are a main cause of excessive mortality of autoimmune disorders like type I diabetes mellitus and rheumatic diseases. Inflammation and endothelial dysfunction, independent predictors to cardiovascular disease, are hallmarks of autoimmunity. Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, can cause or contribute to the inflammatory syndrome and endothelial dysfunction. Recently, elevated ADMA levels have been demonstrated in many autoimmune diseases, suggesting that ADMA might play an important role for the associated manifestations of cardiovascular disease. In the review, we discuss the role of ADMA in the excessive cardiovascular morbidity and mortality associated with autoimmune diseases.
Keywords: Asymmetric dimethylarginine; Autoimmune disorder; Cardiovascular disease;

Pemetrexed alters folate phenotype and inflammatory profile in EA.hy 926 cells grown under low-folate conditions by Andrea L. Hammons; Carolyn M. Summers; Jeanine Jochems; Jasbir S. Arora; Suhong Zhang; Ian A. Blair; Alexander S. Whitehead (12-17).
Elevated homocysteine is a risk marker for several major human pathologies. Emerging evidence suggests that perturbations of folate/homocysteine metabolism can directly modify production of inflammatory mediators. Pemetrexed acts by inhibiting thymidylate synthetase (TYMS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT). EA.hy 926 cells grown under low (“Lo”) and high (“Hi”) folate conditions were treated with pemetrexed. The concentrations of several intracellular folate derivatives were measured using LC-MRM/MS. Lo cells had lower total folate concentrations and a different distribution of the intracellular folate derivatives than Hi cells. Treatment with pemetrexed caused a decrease in individual folate analytes. Microarray analysis showed that several genes were significantly up or down-regulated in pemetrexed treated Lo cells. Several of the significantly up-regulated transcripts were inflammatory. Changes in transcript levels of selected targets, including C3, IL-8, and DHFR, were confirmed by quantitative RT-PCR. C3 and IL-8 transcript levels were increased in pemetrexed-treated Lo cells relative to Lo controls; DHFR transcript levels were decreased. In Lo cells, IL-8 and C3 protein concentrations were increased following pemetrexed treatment. Pemetrexed drug treatment was shown in this study to have effects that lead to an increase in pro-inflammatory mediators in Lo cells. No such changes were observed in Hi cells, suggesting that pemetrexed could not modify the inflammatory profile in the context of cellular folate sufficiency.
Keywords: Pemetrexed; Folate; Inflammation; EA.hy 926 cells;

Interaction of HM30181 with P-glycoprotein at the murine blood–brain barrier assessed with positron emission tomography by Florian Bauer; Thomas Wanek; Severin Mairinger; Johann Stanek; Michael Sauberer; Claudia Kuntner; Zahida Parveen; Peter Chiba; Markus Müller; Oliver Langer; Thomas Erker (18-27).
HM30181, a potent and selective inhibitor of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp), was shown to enhance oral bioavailability and improve antitumour efficacy of paclitaxel in mouse tumour models. In search for a positron emission tomography (PET) radiotracer to visualise Pgp expression levels at the blood–brain barrier (BBB), we examined the ability of HM30181 to inhibit Pgp at the murine BBB. HM30181 was shown to be approximately equipotent with the reference Pgp inhibitor tariquidar in inhibiting rhodamine 123 efflux from CCRF-CEM T cells (IC50, tariquidar: 8.2±2.0 nM, HM30181: 13.1±2.3 nM). PET scans with the Pgp substrate (R)-[11C]verapamil in FVB wild-type mice pretreated i.v. with HM30181 (10 or 21 mg/kg) failed to show significant increases in (R)-[11C]verapamil brain uptake compared with vehicle treated animals. PET scans with [11C]HM30181 showed low and not significantly different brain uptake of [11C]HM30181 in wild-type, Mdr1a/b (−/−) and Bcrp1 (−/−) mice and significantly, i.e. 4.7-fold (P<0.01), higher brain uptake, relative to wild-type animals, in Mdr1a/b (−/−) Bcrp1 (−/−) mice. This was consistent with HM30181 being at microdoses a dual substrate of Pgp and breast cancer resistance protein (Bcrp). In vitro autoradiography on low (EMT6) and high (EMT6Ar1.0) Pgp expressing murine breast tumour sections showed 1.9 times higher binding of [11C]HM30181 in EMT6Ar1.0 tumours (P<0.001) which was displaceable with unlabelled tariquidar, elacridar or HM30181 (1 μM). Our data suggest that HM30181 is not able to inhibit Pgp at the murine BBB at clinically feasible doses and that [11C]HM30181 is not suitable as a PET tracer to visualise cerebral Pgp expression levels.
Keywords: P-glycoprotein; Blood–brain barrier; HM30181; Tariquidar; Positron emission tomography; Microdosing;

Stabilization of Kv1.5 channel protein by bepridil through its action as a chemical chaperone by Sayuri Suzuki; Yasutaka Kurata; Peili Li; Tomomi Notsu; Akira Hasegawa; Nobuhito Ikeda; Masaru Kato; Junichiro Miake; Shinji Sakata; Goshi Shiota; Akio Yoshida; Haruaki Ninomiya; Katsumi Higaki; Kazuhiro Yamamoto; Yasuaki Shirayoshi; Ichiro Hisatome (28-34).
While bepridil has been reported to alter the stability of ion channel proteins, the precise mechanism of action remains unclear. We examined the effect of bepridil on the stability of Kv1.5 channel proteins expressed in COS7 cells. Bepridil at 0.3–30 μM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that bepridil delayed the degradation process of Kv1.5 channel proteins in the same manner as a proteasomal inhibitor, MG132, did. Bepridil increased the immunofluorescent signal of Kv1.5 channel proteins in the endoplasmic reticulum (ER) and Golgi apparatus and on the cell surface. The cell fraction experiment also showed bepridil-induced increases in Kv1.5 in the ER, Golgi apparatus, and the cell membrane. Bepridil at a lower concentration of 1 μM had no effect on the proteasome activity in vitro. A blocker of the ultrarapid delayed-rectifier K+ channel current, 4-aminopyridine (4AP), abolished bepridil-induced increases in Kv1.5. Kv1.5-medicated membrane currents measured as 4AP-sensitive currents were increased by bepridil. Taken together, we conclude that bepridil stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, thereby increasing the density of Kv1.5 channels in the cell membrane.
Keywords: Bepridil; Kv1.5 channel; Protein stability; Chemical chaperone;

Cleistanthoside A tetraacetate-induced DNA damage leading to cell cycle arrest and apoptosis with the involvement of p53 in lung cancer cells by Podchanart Wanitchakool; Surawat Jariyawat; Kanoknetr Suksen; Darunee Soorukram; Patoomratana Tuchinda; Pawinee Piyachaturawat (35-42).
Lung cancer is the leading cause of cancer-related death worldwide and resistance to chemotherapeutic drugs is the major obstacle for effective treatment. The present study investigated the anticancer potential of cleistanthoside A tetraacetate (CAT), a derivative of cleistanthoside A from Phyllanthus taxodiifolius Beille on human lung cancer cells, LU-1. Multiple molecular approaches were used in this study and include measuring the anti-proliferative effect of CAT in LU-1 cells using flow cytometry; evaluating the induction of apoptosis by monitoring DNA fragmentation, phosphatidylserine externalization and activation of caspase-3 activity; and assaying the expression of regulatory proteins involved in cell cycle arrest and apoptosis using immunoblots. CAT potently inhibited LU-1 proliferation through an early G1 arrest with down-regulation of cdk4/6 and cyclin D1 proteins. CAT also inhibited DNA topoisomerase IIα activity resulting in DNA damage and increased the expression of the p53 protein with the subsequent induction of apoptosis. A decrease in the Bcl-2/Bax ratio, activation of caspase-3 activity and cleavage of PARP accompanied apoptosis. CAT is highly toxic to lung cancer and its primary targets are the inhibition of topoisomerase IIα activity and inducing apoptosis through a G1 arrest. These properties indicate that CAT is a promising anticancer agent for treatment of lung cancer.Display Omitted
Keywords: Apoptosis; Cleistanthoside A tetraacetate; G1 arrest; Lung cancer cell; p53;

Reversal of P-gp and MRP1-mediated multidrug resistance by H6, a gypenoside aglycon from Gynostemma pentaphyllum, in vincristine-resistant human oral cancer (KB/VCR) cells by Hengrui Zhu; Zulong Liu; Lisha Tang; Junhua Liu; Mei Zhou; Fang Xie; Zheng Wang; Yuqi Wang; Sida Shen; Lihong Hu; Long Yu (43-53).
Multidrug resistance (MDR) to anticancer drugs is a major obstacle to successful chemotherapy in the treatment of cancers. Identification of natural compounds capable of circumventing MDR with minimal adverse side effects is an attractive goal. Here, we found that H6, a gypenoside aglycon from Gynostemma pentaphyllum, displayed potent anti-MDR activity. Average resistant fold (RF) of H6 is 1.03 and 1.04 in KB/VCR and MCF-7/ADR cells compared to their parental cells. H6 alone ranging from 2 μmol/l to 40 μmol/l (μM) did not display a significant anti-proliferative effect on KB/VCR cells and other cells, while the compound at these concentrations enhanced the cytotoxicity of vincristine (VCR) to KB/VCR cells. H6 showed a significant synergistic effect in combination with VCR. By quantification of sub-G1 fraction cells, H6 also enhanced the VCR-induced apoptosis in a dose-dependent manner. The short time treatment with H6 increased the intracellular accumulation of rhodamine 123 (Rho123) and 5(6)-carboxyfluorescein diacetate (CFDA) in KB/VCR cells. Further studies showed that H6 treatment resulted in the decrease of the RNA transcript level of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). H6 inhibited the function of P-gp by stimulating P-gp ATPase activity and decreased MRP1 expression with a blockade of STAT3 phosphorylation. These findings suggest that H6, a multi-targets reversal agent with no significant toxic effect, may be a potential candidate to circumvent the P-gp and MRP1-mediated MDR.Display Omitted
Keywords: Gypenoside aglycon; Gynostemma pentaphyllum; Multidrug resistance (MDR); KB/VCR cells; P-glycoprotein (P-gp); Multidrug resistance associated protein 1 (MRP1);

The incidence of type 2 diabetes mellitus is rising at alarming proportions. Central nervous system plays an important part in orchestrating glucose metabolism, with accumulating evidence linking dysregulated central nervous system circuits to the failure of normal glucoregulatory mechanisms. Pyridoxine is a water soluble vitamin and it has important role in brain function. This study aims to evaluate the role of pyridoxine in striatal glucose regulation through dopaminergic receptor expressions in streptozotocin induced diabetic rats. Radio receptor binding assays for dopamine D1, D2 receptors were done using [3H] 7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol and [3H] 5-chloro-2-methoxy-4-methylamino-N-[-2-methyl-1-(phenylmethyl)pyrrolidin-3-yl]benzamide. Gene expressions were done using fluorescently labeled Taqman probes of dopamine D1, D2 receptor, Insulin receptor, Insulin like growth factor-1(IGF-1) and Glucose transporter-3 (GLUT-3). Bmax of dopamine D1 receptor is decreased and B max of dopamine D2 was increased in diabetic rats compared to control. Gene expression of dopamine D1 receptor was down regulated and dopamine D2 receptor was up regulated in diabetic rats. Our results showed decreased gene expression of Insulin receptor, IGF-1 and increased gene expression of GLUT-3 in diabetic rats compared to control. Pyridoxine treatment restored diabetes induced alterations in dopamine D1, D2 receptors, Insulin receptor, IGF-1, GLUT-3 gene expressions in striatum compared to diabetic rats. Insulin treatment reversed dopamine D1, D2 receptor, GLUT-3 mRNA expression, D2 receptor binding parameters in the striatum compared to diabetic group. Our results suggest the potential role of pyridoxine supplementation in ameliorating diabetes mediated dysfunctions in striatal dopaminergic receptor expressions and insulin signaling. Thus pyridoxine has therapeutic significance in diabetes management.
Keywords: Dopamine receptor; Insulin receptor; IGF-1; GLUT-3; Pyridoxine;

Effect of synthetic eel calcitonin, elcatonin, on cold and mechanical allodynia induced by oxaliplatin and paclitaxel in rats by Manahito Aoki; Asami Mori; Tsutomu Nakahara; Kenji Sakamoto; Kunio Ishii (62-69).
Oxaliplatin and paclitaxel are commonly used anti-cancer drugs, but they frequently cause peripheral neuropathic pain. In this study, we investigated the effect of elcatonin, a synthetic eel calcitonin, on oxaliplatin- and paclitaxel-induced neuropathy in rats. The rats were treated with a single dose of oxaliplatin (6 mg/kg, i.p.) or repeated doses of paclitaxel (2 mg/kg, i.p.) on 4 alternate days. Both treatments resulted in cold and mechanical allodynia. We assessed the anti-allodynic effects of subcutaneously administered elcatonin (20 U/kg/day) by using a newly developed method to provide cold stimulation (8 °C) directly to the hind paw of the rats and by using the von Frey test. Elcatonin almost completely reversed the effects of both cold and mechanical allodynia. To determine the mechanism of this anti-allodynic effect, we examined the effect of elcatonin on neuropathy induced by intraplantar injection of two organic compounds: allyl isothiocyanate (1 nmol/paw), which activates transient receptor potential ankyrin-1 channels, and menthol (1.28 μmol/paw), which activates transient receptor potential ankyrin-1 and melastatin-8. Pre-administration of elcatonin almost completely prevented cold and mechanical allodynia from being induced by both compounds. These results suggest that elcatonin attenuates oxaliplatin- and paclitaxel-induced neuropathic pain by inhibiting the cellular signaling related to transient receptor potential ankyrin-1 and melastatin-8. Thus, we conclude that administration of elcatonin may improve the quality of life of cancer patients receiving chemotherapy.
Keywords: Cold allodynia; Mechanical allodynia; Oxaliplatin; Paclitaxel; Elcatonin; Transient receptor potential ankyrin-1;

Tyrosine depletion lowers in vivo DOPA synthesis in ventral hippocampus by Rodolfo Bongiovanni; Abby N. Kyser; George E. Jaskiw (70-76).
In vivo dopamine synthesis in the medial prefrontal cortex of the rat is sensitive to the availability of tyrosine. Whether other limbic cortical dopamine terminal regions are similarly tyrosine-dependent is not known. In this study we examined the effects of tyrosine depletion on dopamine synthesis and catecholamine levels in the ventral hippocampus. A tyrosine- and phenylalanine-free neutral amino acid mixture was used to lower brain tyrosine levels in rats undergoing in vivo microdialysis. In one group, NSD-1015 was included in perfusate to permit measurement of DOPA levels. In a second group, NSD-1015 was not included in perfusate so that catecholamine levels could be assayed. Tyrosine depletion significantly lowered DOPA levels in the NSD-1015 treated group and lowered DOPAC but not dopamine or noradrenaline levels in the group not exposed to NSD-1015. We conclude that while catecholamine synthesis in the ventral hippocampus declines when tyrosine availability is lowered, under basal conditions, compensatory mechanisms are able to maintain stable extracellular catecholamine levels.
Keywords: Tyrosine; Dopamine; Noradrenaline; Hippocampus; DOPA; Schizophrenia;

Morphine is a gold standard analgesic commonly used to alleviate pain. However, its use is associated with unavoidable side effects including the risk for addiction. Peripherally administered loperamide lacks effect on the central nervous system as it is a substrate for the permeability glycoprotein (P-gp) efflux pump which blocks its entry into brain. However, when administered intrathecally, loperamide has been reported to produce analgesia. The present study investigates the mechanism of the central analgesic effect of loperamide. Adult male Sprague-Dawley rats were subjected to surgery for catheter placement. Following baseline testing, different groups of rats were administered fixed intrathecal doses (1 μg, 3 μg, 10 μg and 30 μg) of loperamide and morphine. Analgesia was compared employing Hargreaves paw withdrawal apparatus at 15 min, 30 min, 60 min, 90 min and 120 min. Additionally, CTOP, a specific mu-opioid receptor antagonist was co-administered with loperamide to examine the mu-opioid receptor mediated loperamide analgesia. Furthermore, nefiracetam, a calcium channel opener, was co-administered with loperamide or morphine to evaluate the involvement of Ca2+ channels in Loperamide showed an analgesic effect which was comparable to morphine. However, loperamide produced longer analgesia and the analgesic effect was significantly better at 42 h and 49 h compared to morphine. CTOP completely reversed loperamide analgesia. Though nefiracetam significantly reversed loperamide analgesia, it did not have any effect on morphine induced analgesia. Our findings suggest that loperamide administered intrathecally produces analgesia which is mediated through mu-opioid receptor and subsequent blockade of downstream calcium channels.
Keywords: Nociception; Intrathecal catheterization; Hargreaves test; Loperamide; CTOP; Nefiracetam;

Imipramine protects mouse hippocampus against tunicamycin-induced cell death by Yoko Ono; Masamitsu Shimazawa; Mitsue Ishisaka; Atsushi Oyagi; Kazuhiro Tsuruma; Hideaki Hara (83-88).
Endoplasmic reticulum (ER) stress is implicated in various diseases. Recently, some reports have suggested that the sigma-1 receptor may play a role in ER stress, and many antidepressants have a high affinity for the sigma-1 receptor. In the present study, we focused on imipramine, a widely used antidepressant, and investigated whether it might protect against the neuronal cell death induced by tunicamycin, an ER stress inducer. In mouse cultured hippocampal HT22 cells, imipramine inhibited cell death and caspase-3 activation induced by tunicamycin, although it did not alter the elevated expressions of 78 kDa glucose-regulated protein (GRP78) and C/EBP-homologous protein (CHOP). Interestingly, in such cells application of imipramine normalized the expression of the sigma-1 receptor, which was decreased by treatment with tunicamycin alone. Additionally, NE-100, a selective sigma-1 receptor antagonist, abolished the protective effect of imipramine against such tunicamycin-induced cell death. Imipramine inhibited the reduction of mitochondrial membrane potential induced by tunicamycin, and NE-100 blocked this modulating effect of imipramine. Furthermore, in anesthetized mice intracerebroventricular administration of tunicamycin decreased the number of neuronal cells in the hippocampus, particularly in the CA1 and dentate gyrus (DG) areas, and 7 days' imipramine treatment (10 mg/kg/day; i.p.) significantly suppressed these reductions in CA1 and DG. These findings suggest that imipramine protects against ER stress-induced hippocampal neuronal cell death both in vitro and in vivo. Such protection may be partly due to the sigma-1 receptor.
Keywords: Endoplasmic reticulum stress; Hippocampus; Imipramine; Sigma-1 receptor; Tunicamycin;

Effects of serotonergic terminal lesion in the amygdala on conditioned fear and innate fear in rats by Takeshi Izumi; Yu Ohmura; Yukino Futami; Hirokazu Matsuzaki; Yasunori Kubo; Takayuki Yoshida; Mitsuhiro Yoshioka (89-95).
The amygdala and the medial prefrontal cortex (mPFC) are crucial brain structures for anxiety, and it is speculated that the serotonergic neural system in these structures has an important role in regulating anxiety. In our previous study, we indicated that local injections of selective serotonin reuptake inhibitor into the amygdala attenuated anxiety-related behaviors in conditioned fear in rats. In the present study, we investigated the effects of serotonergic terminal lesions in the amygdala and in mPFC induced by local injection of 5,7-dihydroxytryptamine (5,7-DHT), on anxiety-related behaviors in conditioned fear and the elevated plus-maze test in rats. A 5,7-DHT lesion in the amygdala attenuated memory-dependent fear assessed by conditioned fear, but enhanced memory-independent fear assessed by the elevated plus-maze test. These results suggest that the role of the amygdalar serotonergic system in fear is different between memory-dependent and independent fear and, in particular, it is paradoxical that an amygdalar serotonergic lesion exerts a similar effect on memory-dependent fear to SSRI. Moreover, a serotonergic lesion in the amygdala enhanced the retrieval of extinction memory in conditioned fear; however, a serotonergic lesion in mPFC did not bring about any behavioral changes.
Keywords: Amygdala; Anxiety; Fear; 5,7-dihydroxytryptamine (5,7-DHT); 5-hydroxytryptamine (5-HT);

Involvement of the sigma1 receptor in the antidepressant-like effects of fluvoxamine in the forced swimming test in comparison with the effects elicited by paroxetine by Yumi Sugimoto; Noriko Tagawa; Yoshiharu Kobayashi; Kumiko Mitsui-Saito; Yoshihiro Hotta; Jun Yamada (96-100).
We studied the involvement of the sigma1 receptor in the antidepressant-like effects of the selective serotonin reuptake inhibitor (SSRI) fluvoxamine in DBA/2 mice using the forced swimming test. The effects of the selective sigma1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine (BD1047) at 1 mg/kg significantly antagonized the anti-immobility elicited by fluvoxamine (10 mg/kg). However, the anti-immobility effects elicited by another SSRI, paroxetine (5 m/kg), were not altered by BD1047. The selective sigma1 receptor agonist 2S-(2α,6α,11R )-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol ((+)SKF-10047) elicited dose-dependent anti-immobility effects in DBA/2 mice. BD1047 significantly blocked the anti-immobility effects induced by (+)SKF-10047 at 10 mg/kg. These results suggested that the sigma1 receptor was associated with fluvoxamine-induced antidepressant-like effects but not with paroxetine-induced antidepressant-like effects.
Keywords: Fluvoxamine; Forced swimming test; Paroxetine; SSRI (Selective serotonin reuptake inhibitor); Sigma1 receptor;

Experiments were designed to determine if Wy14643 ([[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]-acetic acid), a preferential agonist at peroxisome proliferator-activated receptors (PPAR) α, improves vascular function in hypertension, and if so, the mechanism(s) involved. Isometric tension was measured in isolated thoracic aorta of spontaneously hypertensive rats (SHR). Wy14643-induced relaxations in SHR aortic rings were greater than those induced by fenofibrate or rosiglitazone (PPARα or PPARγ agonists, respectively) and were larger in rings with endothelium than those without. Both MK886 [(1-[(4-chlorophenyl)methyl]-3-1,1-dimethylethyl)thio]-(α,α-dimethyl-5-1-methylethyl)-1H-indole-2-propanoic acid (PPARα antagonist) and GW9662 (2-chloro-5-nitrobenzanilide) (PPARγ antagonists) inhibited Wy14643-induced relaxations. The inhibitory effect of MK886 was more pronounced in rings with endothelium than those without. In SHR aortic rings with endothelium, L-NAME (N ω -nitro-L-arginine methyl ester, nitric oxide synthase inhibitor), ODQ (1H-1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one, soluble guanylyl cyclase inhibitor) and compound C [adenosine monophosphate-activated protein kinase (AMPK) inhibitor] reduced Wy14643-induced relaxations. Western blotting studies indicated that Wy14643 and fenofibrate, but not rosiglitazone, increased the phosphorylation of endothelial nitric oxide synthase and AMPK; these effects were abolished by compound C but not L-NAME. Endothelium-dependent contractions evoked by acetylcholine in quiescent SHR aorta in the presence of L-NAME were reduced by Wy14643 and fenofibrate but not by rosiglitazone. MK886, but not GW9662, prevented this effect. Wy14643 and fenofibrate inhibited acetylcholine-induced prostanoid release to the same extent. These findings suggest that PPARα agonists induce nitric oxide-mediated relaxation through activation of AMPK and reduce the release of endothelium-dependent contracting factors. Because also of the ability to activate smooth muscle PPARγ to induce relaxation, Wy14643 offers additional protection against vascular dysfunction of spontaneous hypertension.
Keywords: Endothelial dysfunction; Endothelium-derived contracting factor; Nitric oxide; Peroxisome proliferator-activated receptor agonist; Prostaglandin;

Selective loss of basal but not receptor-stimulated relaxation by endothelial nitric oxide synthase after isolation of the mouse aorta by Johanna van Langen; Paul Fransen; Cor E. Van Hove; Dorien M. Schrijvers; Wim Martinet; Guido R.Y. De Meyer; Hidde Bult (111-119).
Bioavailability of nitric oxide (NO) is mostly studied in isolated blood vessels. We investigated changes in basal and receptor-stimulated endothelial NO synthase (eNOS) activity after isolation of wild-type and Marfan mouse aorta. Starting 1 h after dissection, basal NO release was assessed at hourly intervals by its ability to suppress isometric contractions in aortic segments. Relaxation induced by acetylcholine or α 2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) was used to study stimulated NOS activity. One hour after dissection, phenylephrine- or prostaglandin F 2α -induced force attained only 17±4% or 31±7% of maximum tension in the presence of N Ω -nitro-l-arginine-methylesther ( l -NAME), and contractions increased to 63±6% and 82±11%, respectively at 5 h. In contrast, acetylcholine or UK14304 relaxation curves changed minimally. l -NAME and eNOS-deficiency abolished basal NO production, unlike inhibitors of neuronal (N Ω -propyl-l-arginine) or inducible (1400 W) NOS. Acetylcholine-induced relaxation was abolished by l -NAME, strongly suppressed by eNOS-deficiency and attenuated by N Ω -propyl-l-arginine. In a bioassay based on diethylamine NONOate concentration-response curves the suppression of contractile forces was interpolated into NO equivalents. This showed exponential decay of basal NO, which occurred three times faster in aortas from mice with Marfan syndrome, while acetylcholine-induced relaxation remained unaltered. Immunoblotting showed unchanged eNOS expression, or phosphorylation at Ser1177, Ser617 or Thr495 between 1 h and 4 h, but Akt phosphorylation declined gradually. The dramatic loss of basal NO release after tissue isolation shows that timing is crucial when studying NO responses. The preservation of receptor-induced relaxation implies differential regulation of basal and stimulated eNOS activity, and phosphoinositide-3-kinase/Akt signalling seems specifically associated with basal eNOS activity.
Keywords: Basal nitric oxide; Endothelial nitric oxide synthase; Bioassay; Mouse aorta; Marfan syndrome; ENOS knockout;

Contrary effects of sphingosine-1-phosphate on expression of α-smooth muscle actin in transforming growth factor β1-stimulated lung fibroblasts by Tatsuo Kawashima; Risa Yamazaki; Yasuo Matsuzawa; Erika Yamaura; Mamoru Takabatake; Sho Otake; Yuta Ikawa; Hiroyuki Nakamura; Hiromichi Fujino; Toshihiko Murayama (120-129).
Transforming growth factor-β1 (TGFβ1) plays a pivotal role in fibrosis in various organs including the lung. Following pulmonary injury, TGFβ1 stimulates conversion of fibroblasts to myofibroblasts that are mainly characterized by up-regulation of α-smooth muscle actin (αSMA) expression, and the resulting excess production of extracellular matrix proteins causes fibrosis with loss of alveolar function. The present study was undertaken to define the role of the sphingosine-1-phosphate (S1P) pathway in TGFβ1-induced expression of αSMA in human fetal lung fibroblasts, HFL1 cells. Analysis of mRNA revealed the existence of S1P1, S1P2, and S1P3 receptor mRNAs. Treatment with TGFβ1 increased sphingosine kinase (SphK) activity and S1P3 receptor mRNA at 24 h after stimulation, and pharmacological data showed the involvement of sphingomyelinase, SphK, and S1P3 receptor in the TGFβ1-induced up-regulation of αSMA with and without serum. Treatment with pertussis toxin and S1P1 receptor antagonist W146 enhanced αSMA expression by TGFβ1/serum, and S1P decreased and increased αSMA levels with and without serum, respectively. TGFβ1 increased cyclooxygenase-2 expression in a manner dependent on serum and the sphingomyelinase/SphK pathway, and the response was decreased by pertussis toxin. Prostaglandin E 2, formed by TGFβ1/serum stimulation, decreased the TGFβ1-induced expression of αSMA via EP prostanoid receptor. These data suggest that S1P formed by TGFβ1 stimulation has diverse effects on the expression of αSMA, inhibition via the S1P1 receptor-mediated and serum-dependent expression of cyclooxygenase-2 and the resulting formation of prostaglandin E 2, and stimulation via the S1P3 receptor in a serum-independent manner.
Keywords: TGFβ1; α-Smooth muscle actin; Sphingosine-1-phosphate; Cyclooxygenase; HFL1 cells;

The role of endogenous hydrogen sulfide in pathogenesis of chronotropic dysfunction in rats with cirrhosis by Mohammad Babaei-Karamshahlou; Bita Hooshmand; Sohrab Hajizadeh; Ali R. Mani (130-135).
Endogenous hydrogen sulfide is produced by cystathionine-γ-lyase and cystathionine-β-synthase in a variety of tissues and has recently been implicated in the regulation of cardiac functions. Acceleration of the heart rate in response to catecholamines is impaired in patients with cirrhosis. The present study was aimed to examine the role of endogenous hydrogen sulfide in the pathogenesis of chronotropic dysfunction in rats with cirrhosis. Cirrhosis was induced by surgical ligation of bile duct in rats. There was no significant difference in atrial cystathionine-γ-lyase and cystathionine-β-synthase mRNA levels in control and cirrhotic rats as assessed by quantitative RT-PCR. Four weeks after bile duct ligation or sham surgery the atria were isolated and chronotropic responsiveness to adrenergic stimulation was assessed using standard organ bath. Incubation of the atria with propargylglycine (PAG, a cystathionine-γ-lyase inhibitor) and amino-oxyacetic acid (AOAA, a cystathionine-β-synthase inhibitor) was associated with a significant desensitization of chronotropic response to adrenergic stimulation in controls rats. This indicates that endogenous hydrogen sulfide might be involved in modulation of adrenergic signaling in the atrium. Bile duct ligation was associated with impaired chronotropic responsiveness to adrenergic stimulation in comparison with sham-operated rats. In contrast to control group, incubation of the atria with PAG and AOAA was able to partially improve the chronotropic responsiveness to adrenergic stimulation in cirrhotic rats. Our data shows that local inhibition of endogenous hydrogen sulfide in atria has opposite effect in cirrhotic versus control rats and may play a role in physiological modulation of adrenergic signaling in the atrium.
Keywords: Adrenergic; Cardiomyopathy; Cirrhosis; Hydrogen sulfide; (Rat);

Multidrug resistance-associated protein 1 and lung function decline with or without long-term corticosteroids treatment in COPD by Simona E. Budulac; Dirkje S. Postma; Pieter S. Hiemstra; Thérèse S. Lapperre; Lisette I.Z. Kunz; Judith M. Vonk; H. Marike Boezen; Wim Timens; the GLUCOLD study group (136-142).
Multidrug resistance-associated protein-1 (MRP1) reduces the oxidative stress generated by smoking, a risk factor for Chronic Obstructive Pulmonary Disease (COPD). We previously showed that MRP1 variants are associated with the level and decline of annual forced expiratory volume in one second (FEV1) in the general population. Moreover, we showed that MRP1 variants are also associated with FEV1 level and inflammatory markers in COPD patients.We investigate in the current study the association of MRP1 protein expression in bronchial biopsies with FEV1 decline in COPD patients using placebo, or inhaled corticosteroids (ICS) with or without long-acting β2-agonists. Additionally we investigate the association of MRP1 variants with FEV1 decline. MRP1 variants (rs212093, rs4148382, rs504348, rs4781699, rs35621) were genotyped in 110 COPD patients. Associations of MRP1 variants and MRP1 protein expression in bronchial biopsies (obtained at baseline, 6 and 30 months) with FEV1 decline were analyzed using linear mixed-effect models. During 30-month ICS treatment, subjects with a moderate staining for MRP1 had less FEV1 decline than those with a weak staining. In subjects stopping ICS after 6 months followed by 24-month placebo, moderate staining for MRP1 was associated with faster FEV1 decline than in those with a weak staining. None of the variants was associated with FEV1 decline. Our unique study suggests a role of MRP1 protein expression in bronchial biopsies in FEV1 decline occurring selectively in COPD patients with long-term (30-month) ICS therapy.
Keywords: Chronic Obstructive Pulmonary Disease (COPD); Lung function decline; Multidrug resistance-associated protein-1; Protein expression; Single nucleotide polymorphisms (SNPs); Treatment;

Involvement of autophagy in the pharmacological effects of the mTOR inhibitor everolimus in acute kidney injury by Shunsaku Nakagawa; Kumiko Nishihara; Ken-ichi Inui; Satohiro Masuda (143-154).
Inhibitors of mammalian target of rapamycin (mTOR) have immunosuppressive and anti-cancer effects, but their effects on the progression of kidney disease are not fully understood. Using cells from normal kidney epithelial cell lines, we found that the antiproliferative effects of mTOR inhibitor everolimus accompanied the accumulation of a marker for cellular autophagic activity, the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 (LC3-II) in cells. We also showed that the primary autophagy factor UNC-51-like kinase 1 was involved in the antiproliferative effects of everolimus. Levels of LC3-II decreased in the kidneys of rats treated with ischemia-reperfusion or cisplatin; however, renal LC3-II levels increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment. Simultaneously, increased signals for kidney injury molecule-1 and single-stranded DNA and decreased signals for Ki-67 in the proximal tubules were observed after treatment with everolimus, indicating that everolimus diminished renal function after acute tubular injury. We also found leakage of LC3 protein into rat urine after treatment with everolimus, and urinary LC3 protein was successfully measured between 0.1 and 500 ng/mL by using an enzyme-linked immunosorbent assay. Urinary LC3 levels were increased after administration of everolimus to rats subjected to ischemia-reperfusion or cisplatin treatment, suggesting that renal LC3-II and urinary LC3 protein are new biomarkers for autophagy in acute kidney injury. Taken together, our results demonstrated that the induction of autophagy by everolimus aggravates tubular dysfunction during recovery from kidney injury.
Keywords: Mammalian target of rapamycin; Autophagy; Proximal tubule; Acute kidney injury; Biomarker;

d-serine modulates non-adrenergic non-cholinergic contraction of lower esophageal sphincter in rats by Maryam Ghasemi-Kasman; Ahmad R. Dehpour; Ali R. Mani (155-160).
Endogenous d-serine is known to modulate glutamatergic transmission via interaction with the glycine site of N-methyl-d-aspartate (NMDA) receptors. d-serine is synthesized by racemization of l-serine using an enzymatic reaction catalyzed by serine racemase. Although much attention has been focused on the role of d-serine within the central nervous system, the physiological role of d-serine in enteric nervous system has not been investigated. Lower esophageal sphincter (LES) function is known to be modulated by NMDA-dependent mechanisms. The present study was aimed to study the expression of enzymes involved in d-serine metabolism and the function of d-serine in lower esophageal sphincter in rats. Reverse transcription polymerase chain reaction (RT-PCR) and western blotting showed the expression of serine racemase in isolated rat LES. Electrical field stimulation was used to induce non-adrenergic non-cholinergic (NANC) contraction/relaxation of isolated rat LES in an organ bath using an isometric force transducer. The organ bath studies on isolated rat LES showed that incubation with d-serine (100 μM) is associated with a significant increase in the NANC contraction of isolated LES. This effect of exogenous d-serine was inhibited by NMDA receptor antagonists (MK-801), suggesting that NMDA receptors are involved in the effects of d-serine on NANC contraction of LES. Incubation with d-serine did not show a significant effect on NANC relaxation within our experimental setting. The results of this study suggest that serine racemase is expressed in LES and d-serine modulates contraction of the lower esophageal sphincter in rats.
Keywords: d-Serine;

Resistance arteries constrict in response to mechanical stress. This response is myogenic in nature, and reliant on membrane depolarization, activation of L-type Ca2+ channels, Ca2+-induced Ca2+-release and large conductance Ca2+-dependent K+ channels (BKCa). Airway smooth muscle is also affected by mechanical stress: a deep inspiration produces a bronchodilation in healthy individuals, but bronchoconstriction in moderate to severe asthmatics. In this study, our objective was to investigate the regulation of this airway stretch-activated contractile response (R stretch), and explore its similarities to the vascular myogenic response. Using a pharmacological approach in intact bovine bronchial segments cannulated horizontally in an organ bath, we showed the ability of carbachol (2-carbamoyloxyethyl-trimethyl-azanium), KCl, neurokinin-A, and U46619 (9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z, 13E-dien-1-oic acid) to generate R stretch in a concentration-dependent manner. R stretch was significantly reduced by nifedipine, ryanodine, and iberiotoxin, suggesting that it possesses characteristics similar to those of the vascular smooth muscle myogenic response, such as a role for membrane depolarization, L-type Ca2+ channel, ryanodine receptors and BKCa channel activation.This study demonstrates a novel role for the L-type Ca2+ channel in airway smooth muscle and provides new insights into possible mechanisms regulating the deep inspiration-induced bronchoconstriction seen in asthmatics.
Keywords: Airway hyperresponsiveness; Deep inspiration; Bronchoconstriction;

Zinc sulphate attenuates chloride secretion in Human colonic mucosae in vitro by Mekki Medani; Victoria A Bzik; Ailin Rogers; Danielle Collins; Rory Kennelly; Des C Winter; David J Brayden; Alan W Baird (166-171).
Zinc's usefulness in the treatment of diarrhoea is well established as an addition to oral rehydration. Mechanisms of action of zinc have been explored in intestinal epithelia from rodents and in cell lines. The aim was to examine how zinc alters ion transport and signal transduction in human colon in vitro. Voltage clamped colonic sheets obtained at the time of surgical resection were used to quantify ion transport responses to established secretagogues. Nystatin permeabilisation was used to study basolaterally-sited ion channels. Direct actions of zinc were determined using preparations of colonic crypts isolated from human mucosal sheets. Electrophysiological measurements revealed zinc to be an inhibitor of electrogenic ion transport stimulated by forskolin, PGE2, histamine and carbachol in isolated human colonic epithelium. Basolateral addition of zinc sulphate had no direct effect on the epithelium. To further outline the mechanism of action, levels of secondary intracellular messengers (3’, 5’-cyclic adenosine monophosphate; cAMP) were determined in isolated colonic crypts, and were found to be reduced by zinc sulphate. Finally, indirect evidence from nystatin-permeabilised mucosae further suggested that zinc inhibits basolateral K+ channels, which are critical for transepithelial Cl secretion linked to water flux. Anti-secretory, and therefore anti-diarrhoeal, actions of exogenous zinc are due, at least in part, to direct basolateral epithelial K+ channel inhibition.
Keywords: Zinc sulphate; Colon; Potassium channel; Secretion; Anti-diarrhoeal;

Toxicity of cytotoxic agents to granulocyte–macrophage progenitors is increased in obese Zucker and non-obese but insulin resistant Goto-Kakizaki rats by Krisztina Géresi; Klára Benkő; Boglárka Szabó; Attila Megyeri; Barna Peitl; Zoltán Szilvássy; Ilona Benkő (172-178).
Increased risk of anticancer chemotherapy in seriously obese patients is known. Obesity may be among factors that predict treatment-related toxicity during chemotherapy. We investigated whether functional changes in granulopoiesis may also contribute to increased myelotoxicity in addition to the known alterations of pharmacokinetic parameters in obesity. Hemopoiesis – as measured by cellularity, frequency of granulocyte–macrophage progenitors (CFU-GM) and total CFU-GM content of the femoral bone marrow – did not differ in obese, insulin resistant Zucker rats compared with Wistar rats. Nevertheless increased sensitivity of their CFU-GM progenitor cells to cytotoxic drugs was found by culturing them in vitro in the presence of carboplatin, doxorubicin and 5-fluorouracil. All drugs were more toxic on CFU-GM progenitor cells of insulin resistant Zucker rats than on CFU-GM cells of the control strain. This might be based on metabolic disorders, at least in part, because we could demonstrate a similar increase in toxicity of the studied anticancer drugs to the CFU-GM progenitors originated from the non-obese but insulin resistant Goto-Kakizaki rats in the same dose ranges. After in vivo administration of rosiglitazone, an insulin sensitizer, the anticancer drug sensitivity of CFU-GM progenitors of Goto-Kakizaki rats was decreased concurrently with improvement of insulin resistance. Although the increased treatment-related myelotoxicity and mortality are well-known among obese patients with malignant diseases, only the altered half lives, volumes of distribution and clearances of cytotoxic drugs are thought to be the underlying reasons. According to our knowledge the results presented here, are the first observations about an impaired granulopoiesis in obese animals.
Keywords: Granulocyte–macrophage progenitor cells (CFU-GM); Obesity; Myelotoxicity; Carboplatin; Doxorubicin; 5-fluorouracil;

2′-Benzoyloxycinnamaldehyde inhibits nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells via regulation of AP-1 pathway by Jung-Yeon Kwon; Su-Hyung Hong; Sun-Dong Park; Sang-Gun Ahn; Jung-Hoon Yoon; Byoung-Mog Kwon; Soo-A Kim (179-186).
Cinnamaldehyde, an active compound of cinnamon, has been reported to exert various biological functions such as anti-inflammatory and anti-tumor activities. Previously, we showed that 2′-hydroxycinnamaldehyde (HCA) has an inhibitory effect on nitric oxide (NO) production through the inhibition of NF-κB signaling. In an effort to find a more effective anti-atherosclerotic agent, here we evaluated the anti-inflammatory effect of 2′-benzoyloxycinnamaldehyde (BCA) in RAW 264.7 murine macrophage cells. We showed that BCA more effectively inhibited NO production than HCA with less cytotoxicity. We also demonstrated that BCA inhibited the lipopolysaccharide (LPS)-induced expression of iNOS in a concentration-dependent manner. Signal transduction studies showed that BCA significantly inhibited the phosphorylation of SAPK/JNK and AP-1-dependent reporter gene activity. LPS-induced expression levels of JunB, c-Jun and c-Fos were also decreased by BCA treatment. Moreover, the LPS-induced DNA binding activity of AP-1 was markedly inhibited by BCA. The direct injection of BCA into mice inhibited the LPS-induced increase in plasma nitrite levels, confirming the anti-inflammatory effect of BCA in vivo. Overall, these observations suggest that BCA has the potential for use as an anti-atherosclerotic agent.
Keywords: Cinnamaldehyde; 2′-benzoyloxycinnamaldehyde; Lipopolysaccharide; Nitric oxide; AP-1; Atherosclerosis;

A compound (DW1182v) protecting high glucose/palmitate-induced glucolipotoxicity to INS-1 beta cells preserves islet integrity and improves hyperglycemia in obese db/db mouse by Soo-Jin Lee; Sung-E Choi; Yun Cheong Hwang; Ik-Rak Jung; Sang-A Yi; Jong Gab Jung; Jin-Mo Ku; Kwiwan Jeoung; Seung Jin Han; Hae Jin Kim; Dae Jung Kim; Kwan-Woo Lee; Yup Kang (187-193).
Loss of beta cells is a pathogenic cause for the development of type 2 diabetes. High glucose/free fatty acid (HG/FFA)-induced glucolipotoxicity was thought to play a role in the beta cell loss. Thus, application of small molecules capable of preventing HG/FFA-induced glucolipotoxicty to beta cells could be an avenue for a therapeutic intervention for the development of type 2 diabetes. We screened a representative library supplied from Korean Chemical Bank for prevention of high glucose/palmitate (HG/PA)-induced viability reduction of INS-1 beta cells and were able to identify a new small molecule (DW1182v) with a function to protect HG/PA-induced glucolipotoxicity. The protective effect was specific to HG/PA-induced beta cell death since DW1182v did not protect streptozotocin- or cytokine-induced INS-1 cell death. The protective effect by DW1182v was likely due to the reduction of death-promoting endoplasmic reticulum (ER) stress responses such as phospho-C-Jun N-terminal kinase (JNK) and C/EBP homologous protein (CHOP). Treatment of obese diabetic db/db mice with DW1182v preserved islet integrity and thus increased insulin secretion and lowered blood glucose after glucose infusion. These results suggest that a small molecule protecting HG/PA-induced glucolipotoxicity to beta cells can be a new therapeutic candidate to prevent the development of type 2 diabetes.
Keywords: CHOP; ER stress; Glucolipotoxicity; INS-1 beta cell; JNK; Type 2 diabetes;

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been demonstrated to improve glycemic control, in particular postprandial hyperglycemic control, in patients with type 2 diabetes. Teneligliptin is a novel chemotype prolylthiazolidine-based DPP-4 inhibitor. The present study aimed to characterize the pharmacological profiles of teneligliptin in vitro and in vivo. Teneligliptin competitively inhibited human plasma, rat plasma, and human recombinant DPP-4 in vitro, with IC50 values of approximately 1 nmol/l. Oral administration of teneligliptin in Wistar rats resulted in the inhibition of plasma DPP-4 with an ED50 of 0.41 mg/kg. Plasma DPP-4 inhibition was sustained even at 24 h after administration of teneligliptin. An oral carbohydrate-loading test in Zucker fatty rats showed that teneligliptin at ≥0.1 mg/kg increased the maximum increase in plasma glucagon-like peptide-1 and insulin levels, and reduced glucose excursions. This effect was observed over 12 h after a dose of 1 mg/kg. An oral fat-loading test in Zucker fatty rats also showed that teneligliptin at 1 mg/kg reduced triglyceride and free fatty acid excursions. In Zucker fatty rats, repeated administration of teneligliptin for two weeks reduced glucose excursions in the oral carbohydrate-loading test and decreased the plasma levels of triglycerides and free fatty acids under non-fasting conditions. The present studies indicate that teneligliptin is a potent, competitive, and long-lasting DPP-4 inhibitor that improves postprandial hyperglycemia and dyslipidemia by both single and repeated administrations.
Keywords: Teneligliptin; Dipeptidyl peptidase-4; Glucagon-like peptide-1; Type 2 diabetes; Postprandial hyperglycemia; Dyslipidemia;

Astragaloside IV attenuates Toll-like receptor 4 expression via NF-κB pathway under high glucose condition in mesenchymal stem cells by Mincai Li; Liangzu Yu; Tonghui She; Yapin Gan; Fuxin Liu; Zhengwu Hu; Yongbin Chen; Suqin Li; Hongli Xia (203-209).
Diabetic hyperglycemia causes a variety of pathological changes. Astragaloside IV (AS-IV) was widely used for the treatment of cardiovascular diseases in China. The aim of this study was to determine the effect of AS-IV on bone marrow mesenchymal stem cells (MSCs) and the underlying mechanism in diabetes. We used reverse transcription polymerase chain reaction and western blotting to determine the expression of Toll-like receptor 4 (TLR4), matrix metalloproteinase-2 (MMP-2) and NF-κB p65 in MSCs under high glucose (HG) with or without pretreatment with AS-IV. The surface expression of TLR4 was checked by flow cytometry and the expression of TNF-α and MCP-1 were detected by ELISA in diabetes patients treated with AS-IV. AS-IV promoted the proliferation of MSCs and attenuated the increased expression of TLR4 induced by HG. In addition, AS-IV decreased the HG-induced translocation of NF-κB p65 and increased the MMP-2 expression in MSCs. AS-IV decreased the TLR4, TNF-α and MCP-1 expression in patients. Collectively,our data revealed that AS-IV attenuated TLR4 expression through the NF-κB signaling pathway in MSCs.
Keywords: Astragaloside IV; Toll-like receptor 4; NF-κB pathway; Bone mesenchymal stem cell; Diabetes;