Cyclophosphamide (CPA) is a widely used chemotherapeutic drug for neoplasias. It is a DNA and protein alkylating agent having a broad spectrum of activity against a variety of neoplasms including breast cancer. The therapeutic effectiveness of CPA is limited by the high-dose hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. The present study examines the potential of combining resveratrol (RES) with CPA and aims to increase the understanding of the mechanism of cell killing. Interestingly, we found that RES significantly enhances the caspase-mediated cytotoxic activity of CPA on MCF-7 cells in vitro. RES at 50 μM decreases the IC₅₀ value of CPA from 10 to 5 mM. FACS data reveals CPA or RES alone mediated G0/G1 and S phase arrest, while the combination of these drugs released both the arrests and results in an increase in the sub G0/G1 peak. Additional analyses indicated the significant up-regulation (P = 0.001) of tumor suppressor p53 and p53-regulated pro-apoptotic Bax and Fas in MCF-7 cells following CPA treatment in combination with RES, which may contribute to the enhancement of the antitumor effect of CPA. Furthermore, downregulation of anti-apoptotic Bcl-2 (P = 0.001) was observed in MCF-7 cells treated with CPA with or without RES when compared to untreated MCF-7. These results suggest the possibility of a new combination chemotherapeutic regimen leading to improvements in the treatment of breast cancer.

抄録全体を表示

The generally accepted hypothesis for the pathogenesis of alcoholic liver disease (ALD) is the two-hit model, which proposes that fat accumulation in the liver increases the sensitivity of the liver to a second hit that leads to inflammatory liver cell damage. In this study we evaluated the effects of Magnolia officinalis (MO), which contains honokiol and magnolol as the primary pharmacological components, to eradicate fatty liver in rats fed an ethanol diet. In vitro studies showed that MO was able to protect RAW 264.7 cells from ethanol-induced production of tumor necrosis factor-α, reactive oxygen species, and superoxide anion radicals; the activation of NADPH oxidase; and subsequent cell death. We also investigated the therapeutic effects of MO on alcoholic fatty liver in Lieber-DeCarli ethanol diet–fed rats. MO treatment of the rats for the last 2 weeks of ethanol feeding completely reversed all the serum, hepatic parameters, and fatty liver changes. The increased maturation of sterol regulatory element–binding protein-1c in the liver by ethanol treatment was completely inhibited by treatment with MO. Therefore, MO may be a promising candidate for development as a therapeutic agent for ALD.

抄録全体を表示

Rapamycin (RAPA), an immunosuprpressive drug used extensively to prevent graft rejection in transplant patients, has been reported to inhibit adipogenesis in vitro. In this study, we investigated the anti-obesity effects of RAPA in C57BL/6J mice on a high-fat diet (HFD). Mice treated with RAPA (2 mg/kg per week for 16 weeks) had reduced body weight and epididymal fat pads/body weight, reduced daily food efficiency, and lower serum leptin and insulin levels compared with the HFD control mice. However, RAPA-treated mice were hyperphagic, demonstrating an increase in food intake. Dissection of RAPA-treated mice revealed a marked reduction in fatty liver scores, average fat cell size, and percentage of large adipocytes of retroperitoneal and epididymal white adipose tissue (RWAT and EWAT), compared to the HFD control mice. These results suggest that RAPA prevented the effect of the high-fat diet on the rate of accretion in body weight via reducing lipid accumulation, despite greater food intake. It is likely that RAPA may serve as a potential strategy for body weight control and/or anti-obesity therapy.

抄録全体を表示

Ginsenoside Rg1 has been reported to improve cognitive function in many memory-impaired animal models. However, little is known about the bioactivity of its metabolites in the central nervous system in vivo. In the present study, we employed the step through test and electrophysiological approach to investigate the effects of ginsenoside Rg1’s primary metabolite ginsenoside Rh1 and end metabolite protopanaxatriol (Ppt) on learning and memory as well as hippocampal excitability. The behavioral study showed that both ginsenoside Rh1 and Ppt significantly ameliorated memory-impaired models induced by scopolamine in mice. Consistently, the electrophysiological work revealed that ginsenoside Rh1 and Ppt as well as their precursor ginsenoside Rg1 all increased hippocampal excitability in the dentate gyrus of anesthetized rats. These results demonstrated that both ginsenoside Rh1 and Ppt had similar but more potent actions than ginsenoside Rg1 in improving memory and hippocampal excitability, suggesting the role of ginsenoside’s sugar moieties in biological activities is not as necessary as traditionally considered.

抄録全体を表示

KR-31378 is a newly developed K_ATP-channel opener. To investigate the ability of KR-31378 to protect retinal ganglion cells (RGC), experiments were conducted using two retinal ischemia models. Retinal ischemia was induced by transient high intraocular pressure (IOP) for acute ischemia and by three episcleral vein occlusion for chronic retinal ischemia. KR-31378 was injected intraperitoneally and administered orally in the acute and chronic ischemia models, respectively. Under the condition of chronic ischemia, RGC density in the KR-31378–treated group was statistically higher than that in the non-treated group, and IOP was reduced. In the acute retinal ischemia model, 90% of RGC were degenerated after one week in non-treated retina, but, RGC in KR-31378–treated retina were protected from ischemic damage in a dose-dependent manner and showed inhibited glial fibrillary acidic protein (GFAP) expression. Furthermore, the KR-31378 protective effect was inhibited by glibenclamide treatment in acute ischemia. These findings indicate that systemic KR-31378 treatment may protect against ischemic injury–induced ganglion cell loss in glaucoma.

抄録全体を表示

We have previously reported that sleep patterns are significantly affected by both physical and psychological stress induced by a communication box; however, the mechanism by which stress alters sleep patterns was not established. In the present study, we investigated the role of γ-aminobutyric acid (GABA), acting through the GABA_B receptor, on stress-induced changes in sleep patterns. Our results show that physical stress increased the total wakefulness time by increasing sleep latency and inhibiting both rapid eye movement (REM) and non rapid eye movement (NREM) sleep during a 6 h sleep–recording period. The GABA_B agonist baclofen (20 pmol/2 μl) attenuated the effects of physical stress on sleep latency, total wakefulness, and NREM sleep, but not total REM sleep. In contrast, psychological stress enhanced total REM sleep and shortened REM sleep latency without altering other sleep patterns. The effect of psychological stress on total REM sleep was also reversed by baclofen. These results suggest that GABA via GABA_B receptors may play a role in the regulation of specific sleep patterns by both physical and psychological stress.

抄録全体を表示

Aloe vera has long been used as a traditional medicine for inducing wound healing. Gingival fibroblasts (GFs) play an important role in oral wound healing. In this study, we investigated the effects of acemannan, a polysaccharide extracted from Aloe vera gel, on GF proliferation; keratinocyte growth factor-1 (KGF-1), vascular endothelial growth factor (VEGF), and type I collagen production; and oral wound healing in rats. [³H]-Thymidine incorporation assay and ELISA were used. Punch biopsy wounds were created at the hard palate of male Sprague Dawley rats. All treatments (normal saline; 0.1% triamcinolone acetonide; plain 1% Carbopol^¨; and Carbopol^¨ containing 0.5%, 1%, and 2% acemannan (w/w)) were applied daily. Wounded areas and histological features were observed at day 7 after treatment. From our studies, acemannan at concentrations of 2, 4, 8, and 16 mg/ml significantly induced cell proliferation (P<0.05). Acemannan concentrations between 2 – 16 mg/ml significantly stimulated KGF-1, VEGF, and type I collagen expressions (P<0.05). Wound healing of animals receiving Carbopol^¨ containing 0.5% acemannan (w/w) was significantly better than that of the other groups (P<0.05). These findings suggest that acemannan plays a significant role in the oral wound healing process via the induction of fibroblast proliferation and stimulation of KGF-1, VEGF, and type I collagen expressions.

抄録全体を表示

This study was conducted to make a new mouse model of neuropathic pain due to injury to a branch of the sciatic nerve. One of three branches (sural, tibial, and common peroneal nerves) of the sciatic nerve was tightly ligated, and mechanical and cool stimuli were applied to the medial part (tibial and common peroneal nerve territories) of the plantar skin. The three types of nerve injuries produced behavioral mechanical hypersensitivities, and the extent of the hypersensitivities after sural and tibial nerve ligation was larger than that of common peroneal nerve ligation. Sural nerve ligation did not affect motor function of the affected hind paw, but tibial and common peroneal nerve ligation produced motor dysfunction. These results suggest that the ligation of the sural nerve is the most suitable for behavioral study. Sural nerve ligation induced behavioral hypersensitivities to mechanical and cool stimuli, which were almost completely inhibited by gabapentin (30 mg/kg). Sural nerve ligation increased spontaneous activity and responses of the wide-dynamic range neurons in the lumbar dorsal horn, which were also almost completely inhibited by gabapentin (30 mg/kg). Sural nerve ligation provides a new mouse model of neuropathic pain, which is easy to prepare and sensitive to gabapentin.

抄録全体を表示

The present study was undertaken to clarify the characteristics of lateral geniculate nucleus (LGN) kindling in rats, especially the efficacies of antiepileptics, in comparison with those of amygdala (AMG) kindling. Daily electrical stimulation of the LGN led to the development of a generalized convulsion (kangaroo posture and falling back) in all subjects, similar to AMG kindling. The kindling response of the LGN differed from that of the AMG in a number of respects, that is, a high after-discharge (AD) threshold, a large number of stimulations for completion of kindling, and a different pattern of electroencephalogram (EEG) development. On the other hand, the oral administration of sodium valproate, carbamazepine, clobazam, or zonisamide caused dose-dependent inhibitions of both seizure stage and AD duration of LGN-kindled seizures, whereas ethosuximide had no significant effects. In addition, seizure stage was more potently inhibited than AD duration by these antiepileptics, particularly with clobazam. In conclusion, LGN kindling possesses characteristics that are different from AMG kindling. In addition, it was demonstrated that LGN kindling is a useful model, similar to other types of limbic system kindling, for the evaluation of antiepileptics.

抄録全体を表示

The objective of this study was to compare intracellular calcium concentration ([Ca²⁺]_i) and contractile responses in isolated rat middle cerebral artery (MCA) with those in basilar artery (BA) employing real-time confocal laser microscopy. KCl elicited transient [Ca²⁺]_i elevation and sustained contraction in both arteries; moreover, nearly equal responses were evident in both arteries. Application of 5-hydroxytryptamine (5-HT), vasopressin (VP), and α,β-methylene adenosine 5'-triphosphate (α,β-me ATP) also induced elevation of [Ca²⁺]_i and contraction in both arteries. The maximum response of 5-HT and VP necessary to increase [Ca²⁺]_i and to constrict the MCA was less in comparison to the BA; however, a linear relationship emerged between the maximum response of [Ca²⁺]_i and that of contraction. Additionally, the slope of the correlation regression line of MCA was nearly identical to that of BA. On the other hand, cyclopiazonic acid (CPA)-induced Ca²⁺ release from store sites following contraction of MCA was distinct from that of BA. In MCA, velocity of [Ca²⁺]_i elevation in smooth muscle cells and Ca²⁺-wave propagation along smooth muscle cells induced by 5-HT were slower than those in BA. These observations revealed that different regions of arteries along the same cerebral tissue may display distinct [Ca²⁺]_i response; moreover, this difference may be one reason for the distinct contractile response.

抄録全体を表示

We have previously reported that peroxynitrite is involved in dysfunction of nitric oxide (NO)-mediated vasorelaxation in SHR/NDmcr-cp rats (SHR-cp), which display typical symptoms of metabolic syndrome. This study investigated whether peroxynitrite is actually generated in the vascular wall with angiotensin II–induced NADPH-oxidase activation, thus contributing to the dysfunction. In isolated mesenteric arteries of male 18-week-old SHR-cp, relaxations in response to acetylcholine and sodium nitroprusside were impaired compared with that in Wistar-Kyoto rats. This impaired relaxation was not restored by treatment with apocynin, an NADPH-oxidase inhibitor. Protein expression of endothelial NO synthase increased while that of soluble guanylyl cyclase (sGC) decreased in the artery. We observed increased production of superoxide anions and peroxynitrite from the artery and their inhibition by apocynin, and also increased contents of nitrotyrosine, a biomarker of peroxynitrite, in mesenteric arteries and angiotensin II in aortas. Long-term (8 weeks) administration of telmisartan, an angiotensin II type 1–receptor antagonist, prevented the impaired vasorelaxation, decreased sGC expression and increased nitrotyrosine content in mesenteric arteries. These findings suggest that in the vascular wall of SHR-cp, peroxynitrite is continually produced by the reaction of NO with NADPH oxidase–derived superoxide via angiotensin II and gradually denatures sGC protein, leading to vasorelaxation dysfunction.

抄録全体を表示

The expression levels of the Na⁺/Ca²⁺ exchanger type 1 (NCX1) change under various cardiac pathophysiological conditions, but the mechanism is unknown. We previously demonstrated that lysophosphatidylcholine (LPC) increased NCX1 expression by activating RhoB in H9c2 cardiomyoblasts. Conversely, fluvastatin (Flv), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, decreased NCX1 mRNA and protein expression by inhibiting RhoB. RhoB can be isoprenylated by either geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP). Here we investigated which of GGPP or FPP is involved in the NCX1-increasing effect of LPC. When LPC was added with GGPP to the Flv-treated H9c2 cells, NCX1 mRNA was increased to a level significantly higher than that in the control cells. Only GGPP, but not FPP, allowed LPC to increase NCX1 mRNA in the presence of Flv. Furthermore, geranylgeranyltransferase 1 inhibitor (GGTI), but not farnesyltransferase inhibitor (FTI), inhibited the LPC-induced NCX1 mRNA increase. We conclude that geranylgeranylation, but not farnesylation, of RhoB mediates LPC-induced NCX1 mRNA increase in H9c2 cells.

抄録全体を表示

Our previous study showed that organobismuth compounds induce apoptosis in human promyelocytic leukemia cells, although solid tumor cell lines were relatively resistant. Herein, we investigated the primary cellular target of these compounds in HeLa cells. One organobismuth compound, bi-chlorodibenzo[c,f][1,5]thiabismocine (compound 3), arrested the cell cycle at G₂/M as assessed by flow cytometry and by upregulating the expression of cyclin B1. At a low concentration (0.5 μM), compound 3 caused cell cycle arrest at the mitotic phase and induced apoptosis. At a higher concentration (>1.0 μM), it induced an arrest in the G₂/M phase, leading to apoptosis. In many cells blocked at the M phase, the organization of microtubules was affected, indicating depolymerization of the microtubule network. Western blotting demonstrated that compound 3 depolymerized microtubules similar to colchicine and nocodazole. Experiments in vitro also showed that compound 3 inhibited the assembly of purified tubulin in a concentration-dependent manner by interacting with the colchicine-binding site of tubulin through its SH groups. Heterocyclic organobismuth compounds are novel tubulin ligands.

抄録全体を表示

Manassantin A (MSA) inhibits nitric oxide production by macrophages through the inhibition of NF-κB activation, but the effect of MSA on dendritic cells has not been elucidated yet. Here we investigated the inhibitory effects of MSA on immune functions of dendritic cells (DCs). MSA inhibited lipopolysaccharide (LPS)-induced phenotypic maturation of DCs, which was proved by the decreased expression of CD40, CD80, CD86, MHC-I, and MHC-II. MSA also inhibited functional maturation of DCs, that is, decreased the gene expression of IL-12, IL-1β, TNF-α, and IFN-α/β; enhanced antigen capture capacity of DCs; and impaired induction of allogenic T cell activation. As a mechanism of action, MSA inhibited LPS-induced activation of NF-κB, ERK, p38, and JNK, which played pivotal roles in toll-like receptor 4–mediated DC maturation. Collectively, these results suggested that MSA might be used for the treatment of DC-related immune diseases.

抄録全体を表示

We studied the effects of tandospirone, a 5-HT_1A agonistic anxiolytic agent, on haloperidol-induced catalepsy and forebrain Fos expression in mice. Haloperidol (0.5 mg/kg, i.p.) markedly increased the catalepsy time and enhanced Fos expression in the shell (AcS) and core (AcC) regions of the nucleus accumbens, the dorsolateral striatum (dlST), and the lateral septal nucleus (LSN). Tandospirone (0.1 – 1 mg/kg, s.c.) significantly alleviated haloperidol-induced catalepsy in a dose-dependent manner, which was antagonized by WAY-100135 (a selective 5-HT_1A antagonist). The anticataleptic dose of tandospirone (1 mg/kg, s.c.) significantly reduced haloperidol-induced Fos expression in the dlST. This inhibition by tandospirone was regionally specific, and it failed to affect haloperidol-induced Fos expression either in the AcS, AcC, or LSN. In addition, the reversal of haloperidol-induced striatal Fos expression by tandospirone was antagonized by WAY-100135. These results support the notion that stimulation of 5-HT_1A receptors region-specifically counteracts the D₂-blocking actions of haloperidol in the striatum, which may account for the ameliorative effects of 5-HT_1A agonists on antipsychotic-associated extrapyramidal disorders.

抄録全体を表示

Cyclooxygenase (COX) contributes to neuropathic pain after peripheral nerve injury, yet COX inhibitors are generally ineffective against mechanical allodynia and hyperalgesia in neuropathic pain patients and animal models. In the present study, we investigated the effects of etodolac, a selective COX-2 inhibitor, on mechanical allodynia in mice after partial sciatic nerve ligation (PSNL) compared to indomethacin (a nonselective COX inhibitor) or celecoxib (a selective COX-2 inhibitor). PSNL decreased the paw-withdrawal threshold (PWT) as assessed by the von Frey hair test, and etodolac, but not indomethacin or celecoxib, administered daily for two weeks, partially or wholly reversed the decrease. The efficacy of etodolac gradually increased throughout the administration period, and the higher dosages restored preligation PWT values by day 21. The positive control pregabalin also partially or wholly reversed the decrease in PWT, but in contrast to etodolac, it showed no increase in efficacy throughout the administration period. In normal mice, etodolac did not affect the PWT, whereas pregabalin increased it. These findings suggest that the mechanisms of inhibition of mechanical allodynia by etodolac and pregabalin are different and demonstrate that in contrast to other COX inhibitors, etodolac is effective against mechanical allodynia in a mouse neuropathic pain model.

抄録全体を表示

Kujin, the dried root of Sophorae flavescensis, has been used in Chinese folklore medicine against allergy. Evaluation of its anti-allergic potential as well as its mechanism of action has rarely been established. We investigated the effect of Kujin on toluene-2,4-diisocyanate (TDI)-induced allergic behavior and related histamine signaling including mRNA levels of histamine H₁ receptor (H1R) and histidine decarboxylase (HDC), H1R and HDC activities, and histamine content in rat nasal mucosa. We also investigated the effect of Kujin on the mRNA levels of helper T cell type 2 (Th2)-cytokine genes closely related to histamine signaling. TDI provocation caused acute allergic symptoms accompanied with up-regulations of H1R and HDC mRNAs and increases in HDC activity, histamine content, and [³H]mepyramine binding activity in the nasal mucosa, all of which were significantly suppressed by pretreatment with Kujin for 3 weeks. Kujin also suppressed the TDI-induced IL-4 and IL-5 mRNA elevations. These data suggest that oral administration of Kujin showed anti-allergic activity through suppression of histamine signaling by the inhibition of TDI-induced H1R and HDC mRNA elevations followed by decrease in H1R, HDC protein level, and histamine content in the nasal mucosa of TDI-sensitized rats. Suppression of Th2-cytokine signaling by Kujin also suggests that it could affect the histamine-cytokine network.

抄録全体を表示

To elucidate the mechanism of action of prostaglandin I₂ (PGI₂) and carbaprostacyclin, we studied their effect on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes. Hepatocyte parenchymal cells, maintained in a serum-free, defined medium, synthesized DNA and proliferated in the presence of PGI₂ or carbaprostacyclin in a time- and dose-dependent manner. PGI₂ was less potent than carbaprostacyclin in stimulating hepatocyte mitogenesis. These effects of PGI₂ and carbaprostacyclin were abolished by treatment with a specific IP-receptor antagonist, CAY10441 (10⁻⁹ – 10⁻⁷ M). Hepatocyte mitogenesis induced by the IP-receptor agonists was almost completely blocked by specific inhibitors of growth-related signal transducers such as AG1478 (5 × 10⁻⁷ M), LY294002 (10⁻⁷ M), PD98059 (10⁻⁶ M), and rapamycin (10 ng/ml). In addition, PGI₂ or carbaprostacyclin significantly increased the kinase activity of a (p175 kDa) receptor tyrosine kinase and the phosphorylation of extracellular signal-regulated kinase (ERK) 2. Addition of a monoclonal antibody against transforming growth factor (TGF)-α, but not insulin-like growth factor-I, to the culture dose-dependently inhibited the PGI₂- or carbaprostacyclin-induced hepatocyte mitogenesis. Furthermore, treatment with the IP-receptor agonists significantly increased the secretion of TGF-α to the culture medium. These results indicate that the IP receptor agonist–induced hepatocyte mitogenesis is mediated by autocrine secretion of TGF-α followed by activation of a receptor tyrosine kinase / ERK pathway.

抄録全体を表示

We investigated the effect of cisplatin on glutamate release in the hypothalamus of Suncus murinus measured by brain microdialysis. Dialysis samples were collected every 20 min for 1 h before and 3 h after the cisplatin (30 mg/kg, i.p.) administration with the animals also being observed for the development of emesis. Cisplatin increased glutamate levels within 1 h and this was closely associated with the occurrence of emesis. Pretreatment with the 5-HT₃–receptor antagonist ondansetron (2 mg/kg, i.p.) inhibited both the emesis and the increased glutamate levels. These results suggest that hypothalamic glutamate is involved in cisplatin-induced emesis in Suncus murinus.

抄録全体を表示