Studies have shown that angiotensin-converting enzyme 2 (ACE2) plays modulating roles in lung pathophysiology, including pulmonary fibrosis (PF) and acute lung injury. Pulmonary fibrosis is a common complication in these interstitial lung diseases, and PF always has a poor prognosis and short survival. To date, there are few promising methods for treating PF, and they are invariably accompanied by severe side effects. Recent studies have showed that the traditional Chinese herbal extract, osthole, had beneficial effects on lipopolysaccharide (LPS) induced acute lung injury (ALI) via an ACE2 pathway. Here we further investigated the protective effects of osthole on bleomycin induced pulmonary fibrosis and attempted to determine the underlying mechanism. PF mode rats were induced by bleomycin (BLM) and then subsequently administered osthole. Histopathological analyses were employed to identify PF changes. The results showed that BLM resulted in severe PF and diffuse lung inflammation, together with significant elevation of inflammatory factors and a marked increase in expression of angiotensin II (ANG II) and transforming growth factor-beta 1 (TGF-β1). ACE2 and angiotensin-(1–7) [ANG-(1–7)] were both greatly reduced after BLM administration. Meanwhile, osthole treatment attenuated BLM induced PF and inflammation, decreased the expression of these inflammatory mediators, ANG II, and TGF-β1, and reversed ACE2 and ANG-(1–7) production in rat lungs. We conclude that osthole may exert beneficial effects on BLM induced PF in rats, perhaps via modulating the ACE2/ANG-(1–7) axis and inhibiting lung inflammation pathways.

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The induction of lymphangiogenesis is an important process to promote cancer growth and cancer metastasis via the lymphatic system. Identifying the compounds that can prevent lymphangiogenesis for cancer therapy is urgently required. Chrysin, 5,7-dihydroxyflavone, a natural flavone extracted from Thai propolis, was used to investigate the effect on the lymphangiogenesis process of TR-LE, rat lymphatic endothelial cells. In this study, maximal nontoxic doses of chrysin on TR-LE cells were selected by performing a proliferation assay. The process of lymphangiogenesis in vitro was determined by cord formation assay, adhesion assay and migration assay. Chrysin at a nontoxic dose (25 μM) significantly inhibited cord formation, cell adhesion and migration of TR-LE cells when compared with the control group. We also found that chrysin significantly induced vascular endothelial growth factor C (VEGF-C) mRNA expression and nitric oxide (NO) production in TR-LE cells which was involved in decreasing the cord formation of TR-LE cells. In conclusion, we report for the first time that chrysin inhibited the process of lymphangiogenesis in an in vitro model. This finding may prove to be a natural compound for anti-lymphangiogenesis that could be developed for use in cancer therapy.

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Three novel series of pyridine derivatives, namely Schiff’s bases, 4-thiazolidinones and azetidin-2-ones bearing pyrazolo[3,4-b]pyridine moiety, have been synthesized. The chemical structures of the synthesized compounds were characterized. The compounds were tested for their antimicrobial activity using the agar well diffusion and broth macrodilution methods. The compounds were also evaluated for their antiproliferative activity using the sulforhodamine B (SRB) assay. The majority of the tested compounds exhibited slight to high antimicrobial activity against the test microorganisms with minimum inhibitory concentrations (MICs) of 0.12–62.5 µg/mL when compared to that of 3 standard antimicrobial agents (Ampicillin, 0.007–0.03 µg/mL; Gentamicin; 0.015–0.24 µg/mL; and Amphotericin B, 0.03–0.98 µg/mL). Compound (7b) was found to be nearly as active as the standard antimicrobial drug Amphotericin B against Fusarium oxysporum fungal strain with MIC of 0.98 µg/mL. Some of the test compounds showed remarkable cytotoxic activities against Hep G2 (hepatocellular carcinoma) cells (IC₅₀=0.0158–71.3 µM) in comparison to the standard anticancer drug doxorubicin (IC₅₀=0.008 µM). Among the compounds tested, (5), (6a), (6b), (7b), and (10) exhibited antiproliferative potency (IC₅₀=0.0001–0.0211 µM) that was found to be better than that of doxorubicin (IC₅₀=0.099 µM) against MCF7 (breast adenocarcinoma) cells. In particular, (7b) displayed the highest significant antiproliferative efficacy against both Hep G2 and MCF7 cell lines showing IC₅₀ values of 0.0158 µM and 0.0001 µM, respectively. Our findings suggest that the synthesized compounds may be promising candidates as novel antimicrobial and antiproliferative agents.

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Geniposide is one of the main compounds in Gardenia jasminoides ELLIS and has many pharmacological activities, but its anti-hyperglycemic activity has not yet been fully explored. This study was designed to determine, for the first time, how geniposide from G. jasminoides regulates hepatic glucose production, and the underlying mechanisms. During in vitro study, we found the inhibitory effect of geniposide on the hepatic glucose production is partly through AMP-activated protein kinase (AMPK) activation in HepG2 cells. Geniposide significantly inhibited hepatic glucose production in a dose-dependent manner. AMPK, acetyl coenzyme A synthetase (ACC) and forkhead box class O1 (FoxO1) phosphorylation were stimulated by different concentrations of geniposide. In addition, the enzyme activities of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) were all significantly suppressed. What is important is that these effects were partly reversed by (1) inhibition of AMPK activity by compound C, a selective AMPK inhibitor, and by (2) suppression of AMPKα expression by small interfering RNA (siRNA). In summary, geniposide potentially ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis by modulation of the AMPK–FoxO1 signaling pathway. Geniposide or geniposide-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes on gluconeogenesis.

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Terminalia, a large genus of Combretaceae, is distributed in Tropical Asia, Africa, and America. Some Terminalia plants are used in folk medicine because they possess powerful medicinal properties. Dried fruits of Terminalia bellirica and Terminalia chebula are used as the main ingredient in Triphala, a famous polyherbal formulation in Ayurvedic medicine and Thai folk medicine, because of their laxative, detoxifying, and rejuvenating effects. To clarify the phylogenetic relationships of medicinal Terminalia species (T. bellirica, T. chebula, and T. catappa) and authenticate their crude drugs, “Samo” and Triphala, nucleotide sequencing alignments in the internal transcribed spacer one–two (ITS 1–2) regions of Terminalia plants collected in Thailand were performed. The amplified fragments of Terminalia species were approximately 800 bp in length. To compare these sequences and DDBJ registered data, a molecular phylogenetic tree was constructed. Phylogenetic analysis clearly separated the sequences into two groups: Asian Terminalia and African Terminalia with some exceptions. In the analyzed sequences, the length of the ITS1-5.8S-ITS2 region was 674 bp in T. chebula, and 677 bp in T. bellirica and T. catappa. Eighty-one single nucleotide polymorphisms (SNPs) and nine insertion–deletions (indels) were observed, and the nucleotide sequences of this region showed species-specific sequences. Based on these differences, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) were applied to identify medicinal Terminalia species. Moreover, the ARMS method was chosen for fingerprinting analysis of Samo crude drugs and Triphala formulations because it was a fast, cost-effective, and reproducible approach.

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In view of the emergence and frequency of multidrug-resistant and extensively drug-resistant tuberculosis and consequences of acquired resistance to clinically used drugs, we undertook the design and synthesis of novel prototypes that possess the advantage of the two pharmacophores of thiourea and 1,3,4-thiadiazole in a single molecular backbone. Three compounds from our series were distinguished from the others by their promising activity profiles against Mycobacterium tuberculosis strain H₃₇Rv. Compounds 11 and 19 were the most active representatives with minimum inhibitory concentration (MIC) values of 10.96 and 11.48 µM, respectively. Compound 15 was shown to inhibit M. tuberculosis strain H₃₇Rv with an MIC value of 17.81 µM. Cytotoxicity results in the Vero cell line showed that these three derivatives had selectivity indices between 1.8 and 8.7. In order to rationalize the biological results of our compounds, molecular docking studies with the enoyl acyl carrier protein reductase (InhA) of M. tuberculosis were performed and compounds 11, 15, and 19 were found to have good docking scores in the range of −7.12 to −7.83 kcal/mol.

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Edoxaban, an oral direct factor Xa inhibitor, was developed and approved for anticoagulant thromboprophylaxis after total knee arthroplasty (TKA). We retrospectively investigated the postoperative anemia by oral administration of edoxaban 30 mg compared with fondaparinux 2.5 mg in TKA patients. Two hundred twenty nine patients who underwent TKA in National Hospital Organization Okayama Medical Center from July 2010 to June 2012 were divided into two groups; pre and post approval of edoxaban: fondaparinux-group (F-group) and edoxaban-group (E-group). As the primary endpoint, the frequency of postoperative anemia was evaluated. Blood coagulation values and relations between these parameters and postoperative anemia were also investigated. The frequency of postoperative anemia was significantly higher in E-group than F-group patients (52.7% vs. 37.8%; p<0.05). Hemoglobin (Hgb) levels were decreased with the peak at postoperative day (POD) 3 in both groups, and the change of Hgb values from POD1 (ΔHgb) was significantly increased in the E-group (p=0.04). At each POD, prothrombin time (PT) and international normalized ratio of PT (PT-INR) prolonged from the preoperative day in E-group were significantly higher than F-group. Additionally, PT and PT-INR in the E-group at POD3 were significantly prolonged in patients with postoperative anemia and the sensitivity of cut-off values to predict postoperative anemia was superior to the activated partial thromboplastin time (APTT). Thus, as the frequency of postoperative anemia tended to be higher in E-group, edoxaban 30 mg might require vigilance, and prolonged PT and PT-INR could potentially predict edoxaban-associated postoperative anemia after TKA.

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The transforming growth factor (TGF)-β–Smad signaling pathway regulates collagen biosynthesis in human dermal fibroblasts. We found that β-lapachone stimulated type I collagen expression in human dermal fibroblasts. In this study, we evaluated whether the β-lapachone-induced upregulation of collagen biosynthesis in human dermal fibroblasts is associated with the TGF-β–Smad signaling pathway. In cultured human dermal fibroblasts, both Smad 2 and Smad 3 (Smad 2/3) were phosphorylated by β-lapachone treatment in a concentration-dependent manner. SB431542, a specific inhibitor of TGF-β receptor I kinase, inhibited the β-lapachone-mediated Smad 2/3 phosphorylation and type I collagen expression, suggesting that β-lapachone stimulates collagen production via the TGF-β receptor I kinase-dependent pathway. β-Lapachone did not increase TGF-β1 synthesis in human dermal fibroblasts, suggesting that the molecular mechanism of β-lapachone for the upregulation of collagen synthesis is due to the extracellular regulation of availability and activities of TGF-β. This study provides new insights into the role of β-lapachone in collagen synthesis in human dermal fibroblasts and suggests that β-lapachone can be used as a pharmacological tool to study collagen homeostasis associated with TGF-β–Smad signaling.

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We evaluated testosterone-containing lecithin reverse wormlike micelles (reverse worms) composed of a polar substance/lecithin/isopropyl myristate for transdermal application. Water, D-ribose, or tetraglycerol were used as the polar substance and were key ingredients for forming the reverse worms. Using the reverse worms, 1 wt% of testosterone could be stably solubilized. When using D-ribose as polar substance, the maximum zero-shear viscosity of the reverse worms solution was higher than that of systems using water or tetraglycerol as the polar substance. The mechanism of skin permeation of testosterone from reverse worms solution was elucidated using skin permeation experiments with hairless mouse skin. When the structure of the reverse worms transitioned to lamellar liquid crystals at the skin/formulation interface, testosterone became supersaturated in the formulations. The structural transition occurred in systems using water or D-ribose as the polar substance, increasing the flux of testosterone. The flux of testosterone from reverse worms solution thus depends on the type of polar substance used.

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Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia in infants. The lack of proper prophylactics and therapeutics for controlling hRSV infection has been of great concern worldwide. Xylitol is a well-known sugar substitute and its effect against bacteria in the oral cavity is well known. However, little is known of its effect on viral infections. In this study, the effect of dietary xylitol on hRSV infection was investigated in a mouse model for the first time. Mice received xylitol for 14 d prior to virus challenge and for a further 3 d post challenge. Significantly larger reductions in lung virus titers were observed in the mice receiving xylitol than in the controls receiving phosphate-buffered saline (PBS). In addition, fewer CD3⁺ and CD3⁺CD8⁺ lymphocytes, whose numbers reflect inflammatory status, were recruited in the mice receiving xylitol. These results indicate that dietary xylitol can ameliorate hRSV infections and reduce inflammation-associated immune responses to hRSV infection.

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A dual readout assay based on fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) exhibits many advantages over single assay technology in terms of screening quality and efficiency. In this study, we developed a dual readout assay combining FP and TR-FRET to identify ribosomal S6 kinase 1 (RSK1) inhibitors. This dual readout assay can monitor both FP and TR-FRET signals from a single RSK1 kinase reaction by using the immobilized metal affinity for phosphochemical (IMAP)-based assay. The Z′ value and signal to background (S/B) ratio were 0.85 and 4.0 using FP, and 0.79 and 10.6 using TR-FRET, which led to performance of a pilot library screening against the drug repositioning set consisting of 2320 compounds with a reasonable reproducibility. From this screening, we identified 16 compounds showing greater than 50% inhibition against RSK1 for both FP and TR-FRET; 6 compounds with greater than 50% inhibition only for FP; and 4 compounds with greater than 50% inhibition only for TR-FRET. In a cell-based functional assay to validate the hit compounds, 10 compounds identified only in a single assay had little effect on the RSK-mediated phosphorylation of liver kinase B1, whereas 5 compounds showing greater than 80% inhibition for both FP and TR-FRET reduced the phosphorylation of liver kinase B1. These results demonstrate that the dual readout assay can be used to identify hit compounds by subsequently monitoring both FP and TR-FRET signals from one RSK1 reaction.

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Lung allergic diseases sometimes accompany pulmonary vaso- and broncho-constriction. Rats are currently used for the experimental study of lung allergies. However, their hemodynamic mechanisms are not fully understood. Therefore the effects of allergic mediators were determined systematically in vivo in rats in terms of pulmonary vascular resistance (PVR), airway pressure (AWP) and total peripheral resistance (TPR). We directly measured pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C₄, and prostaglandin (PG) D₂ in anesthetized open-chest artificially ventilated Sprague-Dawley (SD) rats. PVR was dose-dependently increased by consecutive administration of PAF, LTC₄, and PGD₂, with the maximal responsiveness being PAF>LTC₄>PGD₂. However, neither histamine nor serotonin changed PVR. TPR was decreased by all agents except LTC₄ which actually increased it. PAF and serotonin, but not the other agents, increased AWP. In conclusion, allergic mediators exert non-uniform actions on pulmonary and systemic circulation and airways in anesthetized SD rats: PAF, LTC₄ and PGD₂, but not histamine or serotonin, caused substantial pulmonary vasoconstriction; LTC₄ yielded systemic vasoconstriction, while the others caused systemic vasodilatation; only two mediators, PAF and serotonin, induce airway constriction.

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Vesicular nucleotide transporter (VNUT) is a membrane protein that is responsible for vesicular storage and subsequent vesicular release of nucleotides, such as ATP, and plays an essential role in purinergic chemical transmission. In the present study, we investigated whether VNUT is present in the rodent retina to define the site(s) of vesicular ATP release. In the mouse retina, reverse transcription polymerase chain reaction (RT-PCR) and immunological analyses using specific anti-VNUT antibodies indicated that VNUT is expressed as a polypeptide with an apparent molecular mass of 59 kDa. VNUT is widely distributed throughout the inner and outer retinal layers, particularly in the outer segment of photoreceptors, outer plexiform layer, inner plexiform layer, and ganglion cell layer. VNUT is colocalized with vesicular glutamate transporter 1 and synaptophysin in photoreceptor cells, while it is colocalized with vesicular γ-aminobutyric acid (GABA) transporter in amacrine cells and bipolar cells. VNUT is also present in astrocytes and Müller cells. The retina from VNUT knockout (VNUT^−/−) mice showed the loss of VNUT immunoreactivity. The retinal membrane fraction took up radiolabeled ATP in diisothiocyanate stilbene disulfonic acid (DIDS)-, an inhibitor of VNUT, and bafilomycin A1-, a vacuolar adenosine triphosphatase (ATPase) inhibitor, in a sensitive manner, while membranes from VNUT^−/− mice showed the loss of DIDS-sensitive ATP uptake. Taken together, these results indicate that functional VNUT is expressed in the rodent retina and suggest that ATP is released from photoreceptor cells, bipolar cells, amacrine cells, and astrocytes as well as Müller cells to initiate purinergic chemical transmission.

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The mechanism of serotonin 5-HT₂ receptor subtype-stimulated DNA synthesis and proliferation was investigated in primary cultures of adult rat hepatocytes to elucidate the intracellular signal transduction pathways. DNA synthesis and proliferation were detected in hepatocyte parenchymal cells grown in serum-free, defined medium containing 5-HT (10⁻⁶ M) or the selective 5-HT_2B receptor agonist BW723C86 (10⁻⁶ M). In addition, exogenous transforming growth factor (TGF)-α (1.0 ng/mL) significantly increased hepatocyte DNA synthesis and proliferation, which reached plateau after 4 h of culture. Use of blocking monoclonal antibodies demonstrated that TGF-α, but not insulin-like growth factor-I, was involved in hepatocyte proliferation mediated by 5-HT or BW723C86. TGF-α levels in the culture medium increased significantly versus baseline within 5 min in response to 5-HT (10⁻⁶ M) or BW723C86 (10⁻⁶ M), and the maximum TGF-α level (30 pg/mL) was reached 10 min after 5-HT or BW723C86 stimulation. Secretion of TGF-α into the culture medium was inhibited by addition of the selective phospholipase C (PLC) inhibitor, U-73122 (10⁻⁶ M), or somatostatin (10⁻⁷ M). These results indicate that the proliferative mechanism of action of 5-HT is mediated mainly through a 5-HT_2B receptor/Gq/PLC-stimulated increase in autocrine secretion of TGF-α from primary cultured hepatocytes.

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As one of the adverse effects of oxaliplatin, a key agent in colon cancer chemotherapy, a taste disorder is a severe issue in a clinical situation because it decreases the quality of life of patients. However, there is little information on the mechanism underlying the oxaliplatin-induced taste disorder. Here, we examined the molecular and behavioral characteristics of the oxaliplatin-induced taste disorder in rats. Oxaliplatin (4–16 mg/kg) was administered to Sprague-Dawley (SD) rats intraperitoneally for 2 d. Expression levels of mRNA and protein of taste receptors in circumvallate papillae (CP) were measured by real-time quantitative polymerase chain reaction (PCR) and immunohistochemistry, respectively. Taste sensitivity was assessed by their behavioral change using a brief-access test. Morphological change of the taste buds in CP was evaluated by hematoxyline–eosin (HE) staining, and the number of taste cells in taste buds was counted by immunohistochemical analysis. Among taste receptors, the expression levels of mRNA and protein of T1R2, a sweet taste receptor subunit, were increased transiently in CP of oxaliplatin-administered rats on day 7. In a brief-access test, the lick ratio was decreased in oxaliplatin-administered rats on day 7 and the alteration was recovered to the control level on day 14. There was no detectable alteration in the morphology of taste buds, number of taste cells or plasma zinc level in oxaliplatin-administered rats. These results suggest that decreased sensitivity to sweet taste in oxaliplatin-administered rats is due, at least in part, to increased expression of T1R2, while these alterations are reversible.

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The steroid hormones synthesized by the male gonads play diverse roles in biological processes. Androgens, the primary hormones produced by the male gonads, are key regulators of fat homeostasis, hence androgen-deprivation therapies often induce obesity. However, the molecular mechanism by which male gonadal dysfunction leads to obesity remains unclear, because results from animal studies regarding fat accumulation in the context of gonadal defects do not reflect clinical findings. Here, we investigated the mechanism underlying the development of obesity in animals with male gonadal dysfunction by analyzing the long-term physiological changes in adult male mice with surgical castration. Nine weeks after surgery, white adipose tissue (WAT) mass was higher in the castrated (Cas) mice than in sham-operated (Sham) mice. In addition, castration induced hyperlipidemia and hyperglycemia. However, genes involved in lipid metabolism, including hormone-sensitive lipase, were unchanged in the adipose tissue of the Cas mice, despite the increase in WAT. In contrast, a hepatic gluconeogenesis gene, glucose-6-phosphatase, was significantly upregulated in the Cas mice than in Sham mice. Our findings suggest that long-term hypogonadism in mice mimics the effects in humans, and a potential molecular basis for the induction of obesity in this model is impairment of hepatic gluconeogenesis.

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The aim of this study was to investigate the effect of deer bone oil extract (DBOE) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264.7 cells. DBOE was fractionated by liquid–liquid extraction to obtain two fractions: methanol fraction (DBO-M) and hexane fraction (DBO-H). TLC showed that DBO-M had relatively more hydrophilic lipid complexes, including unsaturated fatty acids, than DBOE and DBO-H. The relative compositions of tetradecenoyl carnitine, α-linoleic acid, and palmitoleic acid increased in the DBO-M fraction by 61, 38, and 32%, respectively, compared with DBOE. The concentration of sugar moieties was 3-fold higher in the DBO-M fraction than DBOE and DBO-H. DBO-M significantly decreased LPS-induced nitric oxide (NO) production in RAW264.7 cells in a dose-dependent manner. This DBO-M-mediated decrease in NO production was due to downregulation of mRNA and protein levels of inducible nitric oxide synthase (iNOS). In addition, mRNA expression of pro-inflammatory mediators, such as cyclooxygenase (COX-2), interleukin (IL)-1β, and IL-12β, was suppressed by DBO-M. Our data showed that DBO-M, which has relatively higher sugar content than DBOE and DBO-H, could play an important role in suppressing inflammatory responses by controlling pro-inflammatory cytokines and mediators.

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The aim of this study was to develop novel microemulsions (MEs) for the transdermal delivery of capsaicin. Microemulsion-based nonionic surfactants consisting of isopropyl myristate as the oil phase, various nonionic surfactants as the surfactant (S), various glycols or alcohol as the co-surfactant (CoS), and reverse osmosis water as the aqueous phase were formulated. Based on the optimal ME obtained from Design Expert^®, MEs containing a fixed concentration of oil, water or surfactant were prepared while varying the amounts of the other two fractions. The results indicated that the skin permeation flux of low dose capsaicin (0.15% (w/w)) was significantly higher for the selected ME than the commercial product and capsaicin in ethanol (control) by approximately two- and four-fold, respectively. We successfully demonstrated the feasibility of the transdermal delivery of capsaicin-loaded ME using a low concentration of nonionic surfactant and ethanol. Moreover, the optimization using computer program helped to simplify the development of a pharmaceutical product.

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Antiemetic prophylaxis with aprepitant, a 5-hydroxytryptamine₃ (5-HT₃) receptor antagonist and dexamethasone is recommended for patients receiving intravenous cisplatin chemotherapy. Whether the same antiemetic regime is superior for hepatic transcatheter arterial infusion chemotherapy with cisplatin (CDDP-TAI) is unknown. We conducted a retrospective study of antiemetic prophylaxis protection against chemotherapy-induced nausea and vomiting (CINV) in CDDP-TAI at Nagasaki University Hospital. The rate of complete response (CR) to antiemetics in the acute (<24 h) and delayed phases (24–120 h) was measured. Twenty-four patients were treated with a 5-HT₃ receptor antagonist (granisetron or azasetron) and dexamethasone on the day of chemotherapy (day 1 only). There was a significant difference between the CR rates in the acute and delayed phases, 91.6, and 69.7%, respectively. Combination of a 5-HT₃ antagonist and dexamethasone on day 1 is effective against acute CINV, but not delayed CINV during CDDP-TAI. These results may help guide the management of nausea and vomiting during CDDP-TAI to achieve better tolerance and compliance for fewer interventions and increased favorable therapeutic outcomes.

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The purpose of this study was to evaluate the association between therapy-induced hemoglobin (Hb) decreasing rapidity and severity with erythrocyte inosine triphosphatase (ITPase) activity and ATP concentration in chronic hepatitis C patients receiving chronic hepatitis C (HCV) treatment. Forty-three Japanese patients were included in the study. Erythrocyte ITPase activity before therapy was determined by HPLC-UV. Erythrocyte ATP concentrations before and during therapy were determined by luciferase assay. Genotyping for ITPA 94C>A (rs1127354) and IVS2+21 A>C (rs7270101) was conducted using TaqMan probes. The median ITPase activity (µmol/h/g hemoglobin) of ITPA 94 CC, CA, and AA genotypes was 136.8 (range, 80.4–289.6), 41.1 (24.3–93.1), and 11.8, respectively. ITPase activity and Hb decreasing showed a significantly inverse relationship at therapeutic weeks 2, 4, and 6 (p<0.01). Erythrocyte ATP concentration was decreased by therapy, and Hb decreasing was significantly and inversely correlated with erythrocyte ATP concentration at week 4 and after week 8 (p<0.001 and 0.05, respectively). ATP concentration for patients with ITPA 94CA was significantly lower than ITPA 94CC at week 4 (p=0.045). We concluded that ITPase activity plays an important function and that ATP concentration changes due to therapy are related to the Hb decreasing mechanism in the early period of therapy with HCV treatment.

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Pain is sensed, transmitted, and modified by a variety of mediators and receptors. Histamine is a well-known mediator of pain. In addition to their anti-histaminic effects, the classical, or 1st generation, anti-histamines (1st AHs) possess, to various degrees, anti-muscarinic, anti-serotonergic, anti-adrenergic, and other pharmacologic effects. Although there have been attempts to use 1st AHs as analgesics and/or analgesic adjuvants, the advent of non-steroidal anti-inflammatory drugs (NSAIDs) discouraged such trials. We previously reported that in patients with temporomandibular disorders, osteoporosis, and/or osteoarthritis, the analgesic effects of certain 1st AHs (chlorpheniramine and diphenhydramine) are superior to those of the NSAIDs flurbiprofen and indomethacin. Here, we compared analgesic effects among 1st AHs and NSAIDs against responses shown by mice to intraperitoneally injected 0.7% acetic acid. Since 1st AHs are water soluble, we selected water-soluble NSAIDs. For direct comparison, drugs were intravenously injected 30 min before the above tests. Histamine-H1-receptor-deficient (H1R-KO) mice were used for evaluating H1-receptor-independent effects. The tested 1st AHs (especially cyproheptadine) displayed or tended to display analgesic effects comparable to those of NSAIDs in normal and H1R-KO mice. Our data suggest that the anti-serotonergic and/or anti-adrenergic effects of 1st AHs make important contributions to their analgesic effects. Moreover, combination of a 1st AH with an NSAID (cyclooxygenase-1 inhibitor) produced remarkably potent analgesic effects. We propose that a 1st AH, by itself or in combination with a cyclooxygenase-1 inhibitor, should undergo testing to evaluate its usefulness in analgesia.

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Parathyroid hormone (PTH) is a potential medicine for osteoporosis, and subcutaneous (s.c.) PTH treatment enhances bone mass; however, continuous infusion of PTH elicits bone resorption and induces bone loss. To clarify this contradictory phenomenon, we examined bone markers and bone mass in rats to assess the optimal duration of PTH(1–34) infusion. Continuous infusion of PTH at 1 µg/kg/h (C_ss, steady-state concentration ca. 300 pg/mL) for 1–4 h clearly stimulated the expression both of bone formation-related genes (c-fos, Wnt4, EphrinB2) and of bone resorption-related genes (tnfsf11, tnfsf11b, encoding receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG)), but s.c. treatment stimulated these genes only 1-h after the injection. Rats were treated with 1-, 2-, or 4-h infusions of PTH daily using a totally implanted catheter system, and the femoral bone mineral density (BMD) was measured at 4 weeks. The 1-h infusion of PTH significantly stimulated serum bone formation markers (procollagen I N-terminal propeptide (PINP) and osteocalcin) on day 14 and femoral BMD at 2 and 4 weeks, but the 4-h infusion of PTH did not enhance BMD. Since the 4-h infusion increased the levels of both the bone formation markers and a bone resorption marker (urinary C-terminal telopeptide of type 1 collagen (CTx)), the increased bone resorption may predominate over bone formation. The intermittent elevation of plasma PTH to 300 pg/mL for 1-h each day is optimal for increasing bone mass in rats. In osteoporosis therapy in human, using the optimal duration for the clinical dose of PTH may selectively stimulate bone formation.

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Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4′-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment.

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Vitamin A constituents include retinal, which plays a role in vision, and retinoic acid (RA), which has been used in the therapy of human acute promyelocytic leukemia. However, the effects on cancer of retinol (Rol) and its ester, retinyl palmitate (RP) are not known well. In the current study, we examined the effects of these agents on proliferation and adhesion of various cancer cells. Rol exhibited dose-dependent inhibition of the proliferation of human refractory and prostate cancer cells, while RA and RP showed little or no effect. In contrast, RA inhibited the growth of human breast cancer cells to a greater extent than Rol at low concentrations, but not at high concentrations. Rol suppressed adhesion of refractory and prostate cancer cells to a greater extent than RA, while it suppressed adhesion of breast cancer cells as well as RA and of JHP-1 cells less effectively than RA. These results indicate that Rol is a potent suppressor of cancer cell growth and adhesion, which are both linked to metastasis and tumor progression. Rol might be useful for the clinical treatment of cancer.

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Hypoxia-inducible factor (HIF)-1α is a transcription factor belonging to the HIF family that is activated in mammalian cells during conditions of low oxygen tension or hypoxia to induce an adaptive response and promote cell survival. Some of the genes targeted by HIF-1α are important for angiogenesis and proliferation. Here, we found that the E26 transformation-specific (ETS) transcription factor myeloid elf-1-like factor (MEF)/Elf4 is activated by HIF-1α. MEF induces genes such as human beta-defensin 2 (HβD2) and perforin (PRF1), and is known to affect the cell cycle. Treatment with hypoxia mimetic CoCl₂ or low O₂ incubation up-regulated MEF mRNA and protein levels in various cell lines. HIF-1α overexpression in HEK293 cells also increased MEF mRNA and protein levels. In contrast, HIF-1α knockdown by small interfering RNA (siRNA) suppressed the induction of MEF in response to hypoxia. HIF-1α binds to the hypoxia response element in the MEF promoter region (−200 bp) and activates MEF promoter under hypoxia condition. The induction of MEF by hypoxia/HIF-1α correlated with the increase of MEF target genes HβD2 and PRF1. Intriguingly, the hypoxia-induced expression of HIF-1α target gene vascular endothelial growth factor (VEGF) was enhanced by the exogenous addition of MEF. Overall, these data indicate that hypoxia or HIF-1α positively regulates MEF expression and function.

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The present study examined the dissolution of magnesium oxide (MgO) from MgO tablets placed in a food thickening agent (food thickener) and its effects on laxative activity. We prepared mixtures of MgO tablets suspended in an aqueous suspension and food thickeners in order to evaluate the dissolution of MgO. The results of the dissolution tests revealed that agar-based food thickeners did not affect the MgO dissolution. In contrast, some xanthan gum-based food-thickener products show dissolution rates with certain mixtures containing disintegrated MgO tablets suspended in a food thickener that decrease over time. However, other xanthan gum-based food-thickener products show dissolution rates that decrease immediately after mixing, regardless of the time they were allowed to stand. In order to investigate the laxative activity of MgO, we orally administered a mixture of MgO suspension and food thickener to mice and observed their bowel movements. The animal experiments showed that when agar-based food thickeners were used, the laxative activity of MgO was not affected, but it decreased when xanthan gum-based food thickeners were used.

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