Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 44 , Issue 1
Showing 1-25 articles out of 25 articles from the selected issue
Review
  • Hiroyuki Koide
    2021 Volume 44 Issue 1 Pages 1-6
    Published: January 01, 2021
    Released: January 01, 2021
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    Protein affinity reagents are widely used for basic research, diagnostics, and disease therapy. Antibodies and their fragments are known as the most common protein affinity reagents. They specifically and strongly bind to target molecules and inhibit their functions. Thus, antibody drugs have increased in the recent two decades for disease therapy, such as cancer. These strong protein–protein interactions are composed of a nexus of multiple weak interactions. Synthetic polymers that bind to target molecules have been developed by the imitation of protein–protein interactions. These polymers show nanomolar affinity for the target and neutralize their functions; thus, they are of significant interest as a cost-effective protein affinity reagent. We have been developing synthetic polymer nanoparticles (NPs) that bind to target peptides and proteins by the inclusion of several functional monomers, such as charged and hydrophobic monomers. In this review, the focus is on the design of synthetic polymer NPs that bind to target molecules for disease therapy. We succeeded in neutralization of toxic peptides and signaling proteins both in vitro and in vivo. Additionally, linear polymers were modified on a lipid nanoparticle surface to improve polymer biodistribution. Our recent findings should provide useful information for the development of abiotic protein affinity reagents.

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    Editor’s picks

    Synthetic polymers have the potential to work as protein affinity reagents by mimicking protein–protein interactions. The authors report the recent research in the design of synthetic polymer nanoparticles (NPs) that capture and neutralize target molecules for intractable disease therapy. The authors found that lightly crosslinked (2%) N-isopropylacrylamide (pNIPAm)-based NPs bind to target small molecules, peptides, and proteins in vivo by the inclusion of several functional monomers, such as charged and hydrophobic monomers. In addition, modification of linear polymer onto lipid nanoparticles improved polymer circulation time in vivo and binding affinity for the target. These results will provide information for the in vivo application of synthetic polymers.

Regular Articles
  • Meiyu Zhang, Taro Miura, Shunsuke Suzuki, Masako Chiyotanda, Sachiko T ...
    2021 Volume 44 Issue 1 Pages 7-17
    Published: January 01, 2021
    Released: January 01, 2021
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    Vitamin K2 is suggested to have a suppressive effect on the peripheral blood mononuclear cells (PBMCs) of pediatric atopic dermatitis patients. We examined the molecular targets of vitamin K2 to suppress proliferation and cytokine production in T-cell mitogen-activated PBMCs of atopic dermatitis patients from the viewpoint of mitogen-activated protein kinase signaling molecules. The study population included 16 pediatric vitamin K2 patients and 21 healthy subjects. The effect of vitamin K2 on concanavalin A-activated PBMC proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and cell counting assays. T-helper (Th)1/Th2/Th17 cytokine profiles in plasma and PBMC-culture supernatants were analyzed by a cytometric beads array assay. Mitogen-activated protein kinase signaling molecules in concanavalin A-activated PBMCs were examined by enzyme-linked immunosorbent assay (ELISA) assays. At 10–100 µM, vitamin K2 significantly suppressed the proliferation of mitogen-activated PBMCs derived from atopic dermatitis patients and healthy subjects (p < 0.05). The interleukin (IL)-10 concentrations in plasma and the PBMC culture supernatants of atopic dermatitis patients were significantly higher than those of healthy subjects (p < 0.05). The IL-2 concentrations in the culture supernatants of atopic dermatitis PBMCs were significantly lower than those of healthy PBMCs (p < 0.05). Vitamin K2 significantly inhibited the IL-17A, IL-10, and tumor necrosis factor α (TNF-α) production (p < 0.05), and increased the IL-2 production (p < 0.01) in the culture supernatant of atopic dermatitis PBMCs. At 10–100 µM, vitamin K2 markedly decreased the of Mek1, extracellular signal-regulated kinases (ERK)1/2 mitogen-activated protein kinase, and SAPK/c-Jun N-terminal kinase (JNK) expression in atopic dermatitis PBMCs (p < 0.05). Vitamin K2 is suggested to attenuate activated T-cell immunity in atopic dermatitis patients through the inhibition of mitogen-activated protein kinase-Mek1-ERK1/2 and SAPK/JNK signaling pathways.

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  • Hea-Ryeoun Won, Pyeongjae Lee, Sol-ra Oh, Yong-Min Kim
    2021 Volume 44 Issue 1 Pages 18-24
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Deeper wrinkles and loss of elasticity are one of the skin-aging symptoms. Collagen breakdown by matrix metalloproteinase-1 (MMP-1), which is induced by reactive oxygen species (ROS) and pro-inflammatory cytokines, has been known to be responsible for these skin-aging symptoms. Therefore, much attention has been paid to chemicals to suppress the MMP-1 activity. Epigallocatechin-3-gallate (EGCG), catechin rich in green tea, has been reported to show antioxidant and protect skin from various stimuli such as UV and chemicals. In this study, we evaluated the inhibitory effect of EGCG on MMP-1 gene expression and secretion in tumor necrosis factor-α (TNF-α)-treated human dermal fibroblast cells (Hs68 cells). Pre-treatment with EGCG (10 and 20 µM) suppressed TNF-α-induced MMP-1 expression and secretion. EGCG also reduced the phosphorylation of extracellular signal regulated kinase (ERK) significantly but not that of p38 activation and c-Jun N-terminal kinase (JNK). Besides, EGCG (10 and 20 µM) showed the inhibitory effect on mitogen-activated protein extracellular kinase (MEK) and Src phosphorylation which is reported to be upstream signal proteins of ERK signal pathway. Based on these results, EGCG might have potential activity to slow down the skin-aging through inhibition of collagen breakdown, which remains to be elucidated.

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  • Yang Chengyu, Zhao Long, Zhou Bin, Luan Hong, Shen Xuefei, Luo Congjua ...
    2021 Volume 44 Issue 1 Pages 25-31
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Ischemia/reperfusion injury (IRI), a participant in acute kidney injury (AKI), can occur as a series of pathological processes such as inflammation. Linarin (LIN) has been widely used for different diseases. To confirm the anti-inflammatory value and relevant mechanism of LIN during IRI, in vivo and vitro models were established. LIN or dissolvent was given, and histologic analysis, quantitative (q)RT-PCR, serum creatinine and blood urea nitrogen testing were used to evaluate kidney injury. Microarray analysis, protein–protein interaction (PPI) analysis and molecular docking were used to identify the target protein of LIN, and small interfering RNA (siRNA) transfection was applied to explore the crucial role of identified protein. First, we found that LIN inhibited kidney injury in an in vivo IRI model and decreased the expression of interleukin-12 (IL-12) p40 in vivo and in vitro IRI models. To explore the mechanism of LIN, we collected raw data from a public microarray database and identified E26 oncogene homolog 2 (ETS2) as a crucial protein of LIN according to microarray analysis and PPI. Meanwhile, qRT-PCR indicated that IL-12 p40 showed no significant difference between ETS2 knock down group and LIN treated ETS2 knock down group after hypoxia reoxygenation treatment. In addition, according to molecular docking the contact area is highly conserved and located on a PPI domain of ETS2 which indicates that LIN may alter the interaction with synergistic proteins in the regulation of IL-12 p40 expression. Our study demonstrated the anti-inflammatory effect of LIN during IRI-AKI, broadening the medicinal value of LIN and the therapeutic options for IRI-AKI.

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  • Yasuyo Morita, Tomoaki Ishida, Shumpei Morisawa, Kohei Jobu, Yangran O ...
    2021 Volume 44 Issue 1 Pages 32-38
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Sarcopenia is a disease whose symptoms include decreased muscle mass and weakened muscle strength with age. In sarcopenia, decreased production of insulin-like growth factor-1 (IGF-1) increases ubiquitin ligases, such as Atrogin1 and Muscle RING-Finger Protein-1 (MuRF1), by activating forkhead box O (FOXO), and inflammatory cytokines and oxidative stress increase the expression of ubiquitin ligases by activating the transcription factor nuclear factor-kappa B (NF-κB). In addition, increased levels of ubiquitin ligases cause skeletal muscle atrophy. Conversely, sirtuin 1 (Sirt1) is known to regulate the expression of ubiquitin ligases by suppressing the activities of NF-κB and FOXO. In this study, we evaluated the effect that juzentaihoto hot water extract (JTT) has on skeletal muscle atrophy and motor function by administering it to senescence-accelerated mouse prone-8 (SAMP8). The group treated with JTT displayed larger gastrocnemius muscle (GA) and extensor digitorum longus (EDL) weights, larger GA muscle fiber cross-sectional areas, and motor function decline during rota-rod tests. JTT also increased IGF-1 serum levels, as well as mRNA Sirt1 levels in GA. Serum levels of tumor necrosis factor-α, interleukin-6, and mRNA levels of Atrogin1 and MuRF1 in GA were reduced by JTT. The muscle fiber cross-sectional area of GA was correlated with the mRNA levels of Sirt1 in GA. The results of this study suggested that JTT administration suppresses skeletal muscle atrophy and motor function decline in SAMP8 mice. This effect may be associated with the increased expression levels of Sirt1 and IGF-1 by JTT.

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  • Hideaki Isago, Akihisa Mitani, Shiho Kohno, Saki Nagoshi, Taro Ishimor ...
    2021 Volume 44 Issue 1 Pages 39-45
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Chronic obstructive pulmonary disease (COPD) is a systemic inflammatory disorder. It often causes weight loss, which is considered a poor prognostic factor. A Japanese herbal Kampo medicine, Hochuekkito (TJ-41), has been reported to prevent systemic inflammation and weight loss in COPD patients, but the underlying biological mechanisms remain unknown. In the present study, we investigated the role of TJ-41 in vivo using a mouse model of lung emphysema. We used lung epithelium-specific Taz conditional knockout mice (Taz CKO mice) as the lung emphysema model mimicking the chronic pulmonary inflammation in COPD. Acute inflammation was induced by intratracheal lipopolysaccharide administration, simulating COPD exacerbation. Mice were fed a diet containing 2% TJ-41 or a control diet. Taz CKO mice showed increased numbers of inflammatory cells in the bronchoalveolar lavage fluid compared to control mice. This effect was reduced by TJ-41 treatment. In the acute exacerbation model, TJ-41 mitigated the increased numbers of inflammatory cells in the bronchoalveolar lavage fluid and attenuated lung inflammation in histopathological studies. Additional in vitro experiments using the human macrophage cell line U-937 demonstrated that lipopolysaccharide-induced tumor necrosis factor-alpha expression was significantly downregulated by TJ-41. These results suggest that TJ-41 has anti-inflammatory effects in lung emphysema both in the chronic phase and during an acute exacerbation. In conclusion, our study sheds light on the anti-inflammatory effects of TJ-41 in lung emphysema. This establishes its potential as a new anti-inflammatory therapy and a preventive medicine for exacerbations during the long-time maintenance of COPD patients.

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  • Dai Majima, Ryosuke Mitsuhashi, Misaki Yamasaki, Kazuaki Kajimoto, Tat ...
    2021 Volume 44 Issue 1 Pages 46-50
    Published: January 01, 2021
    Released: January 01, 2021
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    Obesity is a pathological state related to various lifestyle-related diseases, such as diabetes and dyslipidemia, that may be prevented through the development of anti-obesity treatments. Lipid accumulation in cells could be affected by vitamin E ester α-tocopheryl succinate (TS), which has various biological activities, such as anti-cancer effect, via activation of cell signaling pathways, although the antioxidative activity of TS is lost due to esterification of the phenolic OH group. In this study, we found for the first time that TS significantly suppressed lipid accumulation in mouse 3T3-L1 adipocytes. TS treatment reduced the amount of triglycerides in the culture medium, and inhibited activity of glycerol-3-phosphate dehydrogenase, a marker of lipid synthesis. Furthermore, TS accelerated lipolysis. Treatment of adipocytes with TS for 24 h induced no significant cytotoxicity. In TS-treated cells, phosphorylation of Akt, which is involved in fatty acid synthesis via sterol regulatory element-binding proteins (SREBP), was prevented, while levels of phosphorylated protein kinase A (PKA) did not change. Taken together, these results suggest that vitamin E ester TS can suppress lipid accumulation in adipocytes by regulating lipid metabolic cell signaling.

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  • Li-Xia Guo, Bin Sun
    2021 Volume 44 Issue 1 Pages 51-56
    Published: January 01, 2021
    Released: January 01, 2021
    [Advance publication] Released: November 06, 2020
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    Supplementary material

    The bis-Schiff base of N,N′-1,10-bis(naringin) triethylenetetraamine (1) was prepared, as a copper(II) ion chelator, compound 1 was used for Alzheimer’s disease therapy in vitro. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of compound 1 showed that this Schiff base could promote PC12 cells proliferation, and also, compound 1 could inhibit Cu2+-amyloid-β (Aβ)1–42 mediated cytotoxicity on PC12 cells. The thioflavine T (ThT) assay showed that 1 can effectively attenuate Cu2+-induced Aβ1–42 aggregation. In addition, compound 1 is determined to be potent antioxidants on the basis of in vitro antioxidant assay, it can effectively decease the level of reactive oxygen species (ROS) in Cu2+-Aβ1–42-treated PC12 cells and elevate the superoxide dismutase (SOD) activity in Cu2+-Aβ1–42-treated PC12 cells. The results show that N,N′-1,10-bis(naringin) triethylenetetraamine is a potential agent for therapy of Alzheimer’s disease.

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  • Hiromi Kataoka, Kaori Tanaka, Keiko Tazuya-Murayama, Taku Yamashita, J ...
    2021 Volume 44 Issue 1 Pages 57-62
    Published: January 01, 2021
    Released: January 01, 2021
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    Fine particulate matter (PM2.5) pollution causes serious health disorders, because PM2.5 becomes deposited in the tracheobronchial and alveoli regions. In the extrathoracic region, there are more deposits of coarse particulate matter than fine particulates. As adverse health issues caused by coarse particulates have not been well investigated, this study examined the cytotoxicity of water-soluble extracts of both fine (0.05–3 µm, PM0.05–3) and coarse (> 3 µm, PM>3) particulates collected from April 2016 to March 2019 in Fukuoka, Japan. Also evaluated were concentrations of NH4+ and SO42−, multi-components of well-known secondary generation substances. The findings revealed that PM>3 showed stronger cytotoxic effects on mast cell lines than PM0.05–3. Cytotoxic effects were observed at concentrations of over 15 mM of (NH4)2SO4 and over 30 mM of NH4Cl. In contrast, Na2SO4 caused few cytotoxic effects up to a concentration of 50 mM. The causative substances for this cytotoxicity may not have been NH4+ and SO42− because their PM>3 concentrations indicating the largest cytotoxic effects were 1 and 0.4 mM, respectively. The cytotoxicities of PM>3 and PM0.05–3 were the highest in the first half of FY2016. These cytotoxicities seem to be due to cross-border pollution, although this pollution has been declining in recent years. An increasing trend of cytotoxicity was observed in the second half of FY2018. This study showed that cytotoxicity and particulate concentrations are not always correlated. Thus, we should focus not only on the quantity of atmospheric particulate matter, but also on its quality.

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  • Guadalupe Luisa Jimenez-Sánchez, Mariana Segovia-Mendoza, Alejandra Fi ...
    2021 Volume 44 Issue 1 Pages 63-68
    Published: January 01, 2021
    Released: January 01, 2021
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    Background/Aim: Hormone replacement therapy during menopause increases the risk of thromboembolic diseases and cancer, so safety alternative therapeutic strategies are needed. 17β-Aminoestrogens are a synthetic estrogens group that possess mild anticoagulant activity that contrasts with the pro-coagulant effects showed by estradiol’s (E2) in rodents. Being considered an alternative to conventional hormone replacement therapy during menopause without thrombogenic risks producing. The present study aimed to determine the estrogenic profile and anxiolytic activity of 17β-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE2), a related compound unknown until now. Methods: IE2 was assessed in immature rats by uterotrophic assay administering IE2, E2, or vehicle. In ovariectomized adult Wistar rats (Ovx) to facilitating the lordotic behavior compared with E2, estradiol benzoate, or vehicle. The effect of IE2 on anxiety was estimated in Ovx animals treated with IE2, E2, or vehicle group and evaluated in the elevated plus-maze model. Results and conclusion: IE2 produced an uterotrophic effect, lordotic behavior, and anxiolytic effect in a dose-dependent manner, similar to E2. IE2 depicted estrogenicity, indicating potential clinical use as hormone replacement therapy during menopause.

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  • Masato Taguchi, Yukako Kawasaki, Arisa Katsuma, Ayane Mito, Kentaro Ta ...
    2021 Volume 44 Issue 1 Pages 69-74
    Published: January 01, 2021
    Released: January 01, 2021
    [Advance publication] Released: October 28, 2020
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    The purpose of this study was to clarify the variability of serum concentrations of caffeine (CAF) in preterm infants, and to deliberate on a better explanation for developmental changes of systemic clearance during the neonatal period. Forty-nine serum samples were obtained from 23 preterm neonates (age, 34.1 ± 18.8 d), and additive blood sampling was conducted periodically for 10 of the 23 patients after discontinuation of CAF treatment. The concentrations of CAF and its major metabolites were determined by liquid chromatography-tandem mass spectrometory. The serum concentrations of CAF were within therapeutic levels (5–25 µg/mL) in 37 samples and exceeded 25 µg/mL in the rest of the 12 samples, although no sample was in the toxic range (> 50 µg/mL). The inter- and intra-individual variability of the concentration to dose (C/D) ratio corrected for body surface area (BSA) was more negatively associated with postmenstrual age (PMA) rather than postnatal age (PNA). The serum concentrations of major metabolites were much smaller than those of CAF throughout the study, suggesting that the contribution of hepatic metabolism to drug elimination was small in the preterm infants under 241 d of PMA. The mean values for elimination half-life and oral clearance estimated in the 10 patients were 124.6 ± 44.6 h and 2.26 ± 0.73 mL/min/1.73 m2, respectively. Consequently, we confirmed that the exposure to CAF was considerably variable and provided additive insight that the C/D ratio corrected for patient’s BSA and PMA are promising for describing and understanding the developmental change of clearance in preterm infants.

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  • Kentaro Nakanishi, Keiichi Hiramoto, Eisuke F Sato, Kazuya Ooi
    2021 Volume 44 Issue 1 Pages 75-81
    Published: January 01, 2021
    Released: January 01, 2021
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    Several studies have been conducted to explore the anticancer effects of vitamin C (VC). However, the effect of high-dose VC administration on melanoma is still unknown. Therefore, in this study, we investigated the effects of high-dose VC (4 g/kg) on the invasion and proliferation of melanoma cells in various organs of mice. B16 melanoma cells (1 × 106 cells/100 µL) were intravenously injected into the tails of female mice, and VC solution (4 g/kg) was orally administered once a day for 14 d. On the 15th day, samples from the liver, lungs, jejunum, and ovaries were collected and analyzed for invasion and proliferation of melanoma cells. Oral VC administration decreased the number of dihydroxyphenylalanine (DOPA)-positive cells and gp100-positive melanoma cells in the ovaries and suppressed the invasion and proliferation of melanoma. Compared to melanoma-administered mice, macrophage inflammatory protein-2 levels and number of neutrophils were increased in the VC + melanoma-administered mice. Furthermore, the concentrations of VC, iron, and hydrogen peroxide, and the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly increased in the ovaries of VC + melanoma-administered mice compared to those of melanoma-administered mice. These results suggest that VC can reduce the invasion and proliferation of melanoma cells in the ovaries, and neutrophils in the ovaries play an important role in achieving this melanoma-suppressive effect.

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  • Asami Mori, Erika Yano, Kenji Sakamoto, Kunio Ishii, Tsutomu Nakahara
    2021 Volume 44 Issue 1 Pages 82-87
    Published: January 01, 2021
    Released: January 01, 2021
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    CYP epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to endothelium-dependent hyperpolarization (EDH)-related dilation in multiple vascular beds. The present study aimed to determine the role of EETs in the acetylcholine (ACh)-induced dilation of retinal arterioles in rats in vivo. The vasodilator responses were assessed by determining the change in diameter of the retinal arterioles on images of the ocular fundus. The intravitreal injection of 17-octadecynoic acid (1.4 nmol/eye), an inhibitor of CYP epoxygenase, and 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EE-5(Z)-E; 2 nmol/eye), an antagonist of EETs, reduced the ACh (0.3–10 µg/kg/min)-induced dilation of the retinal arterioles. The EET antagonist attenuated the vasodilator response to ACh under blockade of nitric oxide (NO) synthases and cyclooxygenases with NG-nitro-L-arginine methyl ester (30 mg/kg) plus indomethacin (5 mg/kg). Intravitreal injection of 14,15-EET (0.5 nmol/eye) dilated retinal arterioles and the response was prevented by iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (BKCa) channels (20 pmol/eye). These results suggest that ACh stimulates the production of EETs, thereby dilating the retinal arterioles via activation of BKCa channels. CYP epoxygenase-derived EETs may be involved in the EDH-related component of the ACh-induced dilation of the retinal arterioles.

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    Editor’s picks

    Acetylcholine (ACh) dilates retinal blood vessels through nitric oxide (NO)-dependent and NO-independent mechanisms. In the rat retinal arteriole, NO stimulates the cyclooxygenase-1 (COX-1)/prostaglandin I2 (PGI2)/prostanoid IP receptor/cAMP signaling pathway and activates 4-aminopyridine-sensitive KV channels (KV). The article by Mori et al. provides evidence suggesting that cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) activate large-conductance Ca2+-activated K+ channels (BKCa) in rat retinal arterioles. The NO-independent component of ACh-induced retinal vasodilator response is mediated partly by an endothelium-dependent hyperpolarization mechanism, through CYP epoxygenase-derived EETs. EETs may function as an endothelium-derived hyperpolarizing factor in rat retinal arterioles.

  • Man Liu, Yao Meng, Keren He, Chonglin Luan
    2021 Volume 44 Issue 1 Pages 88-95
    Published: January 01, 2021
    Released: January 01, 2021
    [Advance publication] Released: November 05, 2020
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    Supplementary material

    Osteoporosis (OP) is increasingly becoming one of a major health concerns all over the world. However, the limitations of current therapeutic drugs for OP are including considerable side effects and low efficacy. Therefore, it is required to develop new therapeutic drugs for OP. This study aimed to investigate the role of hsa_circ_0002060 in the regulation of osteoporosis. Osteoblast cells (hFOB 1.19) were transfected with hsa_circ_0002060 small interfering RNA (siRNA), following by stimulated with dexamethasone (DEX) to mimic OP in vitro. Cell counting kit-8, apoptosis, and JC-1 mitochondrial membrane potential assays were used to evaluate the cell viability, apoptosis, and mitochondrial membrane potential, respectively. Western blot was conducted to detect the expression of proteins. In addition, the levels of reactive oxygen species, superoxide dismutase, glutathione and malondialdehyde were measured with enzyme-linked immunosorbent assay (ELISA). The putative target of hsa_circ_0002060 was verified by dual luciferase reporter assay and RNA pull down. At last, the role of hsa_circ_0002060 in the progression of OP was investigated with an ovariectomy (OVX)-induced OP mouse model. The results indicated DEX could induce cell viability decline in hFOB 1.19 cells, which was ameliorated by hsa_circ_0002060 knockdown. Consistently, DEX-induced cell apoptosis of hFOB 1.19 was ameliorated by hsa_circ_0002060 knockdown as well. As for the underlying mechanisms study, hsa_circ_0002060 was proved to regulate the viability of hFOB 1.19 cells through targeting miR-198-5p/Bax axis. Additionally, hsa_circ_0002060 knockdown alleviated ovariectomy-induced OP in a mouse model. Taken together, hsa_circ_0002060 knockdown alleviated the progression of OP by targeting miR-198-5p. Hsa_circ_0002060 might possibly be served as a therapeutic target for treating OP.

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  • Moeko Kojina, Keiichiro Suzuki, Akane Nishiwaki, Tetsuya Aiba
    2021 Volume 44 Issue 1 Pages 96-102
    Published: January 01, 2021
    Released: January 01, 2021
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    The effects of inflammation on hypoglycemic agents were evaluated in male rats with acute peripheral inflammation (API). Nateglinide (NTG) was utilized as a model compound, since it is a hepatically-metabolized compound and its metabolism is mainly mediated by CYP 2C11 enzyme. In the experiments, rats were subjected to carrageenan injection into their hind paws for API induction, and the plasma concentration profiles of NTG were then examined. In addition, pooled liver microsomes were prepared from control and API rats, and the hepatic drug-metabolizing activity toward NTG and the hepatic expression of CYP2C11 protein were evaluated. It was shown that the plasma concentration of NTG following its intravenous administration decreases at a slower rate in API rats than that in control rats. It was also indicated in the incubation study with the liver microsomes that the hepatic drug-metabolizing activity toward NTG decreases in API rats. Additionally, it was revealed in Western immunoblotting that the hepatic expression of CYP2C11 protein decreases in API rats. These findings suggest that inflammation occurring in peripheral tissues brings about a decrease in hepatic NTG metabolism by suppressing the hepatic expression of CYP2C11 protein, causing an alteration of the plasma concentration profile of NTG with its impaired elimination.

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  • Kentaro Yano, Itsuki Todokoro, Hiroki Kamioka, Takumi Tomono, Takuo Og ...
    2021 Volume 44 Issue 1 Pages 103-111
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Our previous report indicated that Snail-induced epithelial–mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human non-small cell lung cancer (NSCLC) cell line, HCC827. Our objective is to evaluate the changes in the mRNA and protein expression levels and the functions of multidrug resistance-associated protein (MRP) 2, MRP5 and breast cancer resistance protein (BCRP). Snail-expressing HCC827 cells showed increased mRNA levels of Snail and a mesenchymal marker vimentin, and decreased mRNA levels of an epithelial marker E-cadherin after transduction, indicating that Snail had induced EMT consistent with our previous reports. The mRNA level of MRP2 was significantly decreased, while that of MRP5 remained unchanged, in Snail-expressing cells. The expression levels of MRP2 and MRP5 proteins in whole-cell homogenate were unchanged in Snail-expressing cells, but MRP5 protein showed significantly increased membrane localization. Snail-transduction increased the efflux transport of 5-(and-6)-carboxy-2′,7′-dichlorofluorescein (CDCF), a substrate of MRP2, 3 and 5. This increase was blocked by MK571, which inhibits MRP1, 2, and 5. Toxicity of cisplatin, a substrate of MRP2 and 5, was significantly decreased in Snail-expressing cells. BCRP mRNA and protein levels were both decreased in Snail-expressing cells, which showed an increase in the intracellular accumulation of 7-ethyl-10-hydroxycamptothecin (SN-38), a BCRP substrate, resulting in reduced viability. These results suggested that MRP5 function appears to be increased via an increase in membrane localization, whereas the BCRP function is decreased via a decrease in the expression level in HCC827 cells with Snail-induced EMT.

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    Editor’s picks

    Snail-induced epithelial-mesenchymal transition (EMT) enhanced P-glycoprotein (P-gp) function and drug resistance to P-gp substrate anticancer drug in a human NSCLC cell line, HCC827. This study indicates that multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP) are regulated differently in HCC827 cells with Snail-induced EMT. Specifically, the function of MRP5 appears to be enhanced via an increase in membrane localization, whereas the function of BCRP is reduced via a decrease in the expression level. Therefore, it was suggested that MRPs and BCRP are regulated differently in HCC827 cells with Snail-induced EMT.

  • Jun-ichi Takino, Takuma Sato, Kentaro Nagamine, Akiko Sakasai-Sakai, M ...
    2021 Volume 44 Issue 1 Pages 112-117
    Published: January 01, 2021
    Released: January 01, 2021
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    Advanced glycation end-products (AGEs) are produced by the non-enzymatic reaction of sugars with proteins. It has been revealed that glyceraldehyde-derived toxic AGEs (TAGE) are elevated in the serum of non-alcoholic steatohepatitis (NASH) patients. NASH causes liver fibrosis and progresses to cirrhosis and hepatocellular carcinoma. However, the impact of TAGE in liver fibrosis caused by extracellular matrix accumulation remains poorly understood. In this study, we examined the effect of TAGE on the activation of hepatic stellate cells that are involved in liver fibrosis. LX-2 cells treated with transforming growth factor-β1 (TGF-β1) significantly reduced cell viability by apoptosis. However, the decrease in cell viability with TGF-β1 treatment was significantly suppressed by TAGE co-treatment. The levels of α-smooth muscle actin (α-SMA) and platelet-derived growth factor (PDGF)-Rβ and its ligand PDGF-B were increased in LX-2 cells following TGF-β1 treatment, suggesting that these cells were activated; however, these increases were unaffected by TAGE co-treatment. Moreover, collagen I level was increased with TGF-β1 treatment, and this increase was further increased by TAGE co-treatment. These results suggested that the suppression of apoptosis in activated LX-2 cells by TGF-β1 and TAGE co-treatment is related to an increase in the production of the extracellular matrix such as collagen I. Therefore, it was suggested that TAGE might aggravate the liver fibrosis of chronic hepatitis, such as NASH.

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    Editor’s picks

    Advanced glycation end-products (AGEs) are produced by the non-enzymatic reaction of sugars with proteins. It has been revealed that glyceraldehyde-derived toxic AGEs (TAGE) are elevated with the non-alcoholic steatohepatitis (NASH) that causes such as liver fibrosis. Liver fibrosis is caused by activation of hepatic stellate cells (HSCs). Herein, it was found that the suppression of apoptosis in activated LX-2 cells which are HSCs by TGF-β1 and TAGE co-treatment is related to an increase in the production of the extracellular matrix such as collagen Ⅰ. This result suggests that TAGE might aggravate the liver fibrosis of chronic hepatitis, such as NASH.

  • Toshinori Hirai, Ryosuke Yamaga, Motoki Kei, Keiko Hosohata, Toshimasa ...
    2021 Volume 44 Issue 1 Pages 118-124
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    The time course of acute kidney injury and hypokalemia remains unelucidated. We investigated whether altered renal function impacts hypokalemia and clinical predictors for acute kidney injury in patients who used Yokukansan preparation. We performed a secondary analysis of retrospective observational cohort data from adult patients who started Yokukansan preparation. The study was conducted from June 2015 to May 2019 at Tokyo Women’s Medical University, Medical Center East. The effect of acute kidney injury (>1.5-fold increase from baseline serum creatinine level) or renal function recovery on hypokalemia (serum potassium level <3.0 mEq/L) was investigated. The clinical predictors for acute kidney injury were determined using a multivariate Cox proportional hazard analysis. Out of 258 patients, 12 patients had both outcomes, and all but one patient experienced in the order of acute kidney injury and hypokalemia. Excluding one patient, hypokalemia occurred in 11/34 (32%) patients after acute kidney injury and 27/223 (12%) patients without acute kidney injury (p = 0.005). Hypokalemia occurred in 9/25 (36%) of acute kidney injury with recovery, 2/9 (22%) of acute kidney injury without recovery, and 27/223 (12%) of no acute kidney injury (p = 0.014). Patients with acute kidney injury showed a late onset of hypokalemia compared with those without acute kidney injury (p = 0.001). In 258 patients, multivariate Cox proportional hazard analysis showed that high systolic blood pressure and mean arterial pressure increased the risk of acute kidney injury. Clinicians should remember that hypokalemia developed after acute kidney injury while Yokukansan preparation treatment.

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    Editor’s picks

    This retrospective observational cohort study evaluated the chronological order of acute kidney injury (AKI) and hypokalemia with Yokukansan preparation in 258 patients. Among the patients with AKI and hypokalemia, 92% developed hypokalemia associated with Yokukansan preparation after AKI. Excluding one patient with hypokalemia before AKI, the incidence of hypokalemia was higher in patients with AKI than in those without AKI (32% vs. 12%; p = 0.005). Furthermore, the prevalence of hypokalemia differed according to AKI recovery (AKI with recovery, 36%; AKI without recovery, 22%; and no AKI, 12%; p = 0.014).

  • Yoshihiro Kobashigawa, Mana Namikawa, Mitsuhiro Sekiguchi, Yuki Inada, ...
    2021 Volume 44 Issue 1 Pages 125-130
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug–drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

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Notes
  • Koji Mizuno, Michika Komiya, Katsuki Okuyama, Keisuke Imada, Takashi S ...
    2021 Volume 44 Issue 1 Pages 131-135
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Rheumatoid arthritis (RA) is an inflammatory disease with joint dysfunction following cartilage degradation. The level of lysophosphatidic acid (LPA) has been reported to be augmented in human synovial fluid from patients with RA. However, it remains to be elucidated whether LPA participates in cartilage destruction. In the present study, we have demonstrated that the production of promatrix metalloproteinases (proMMPs)-1 and -3 was augmented along with an increase of extracellular signal-regulated kinase (ERK)1/2 phosphorylation through LPA receptor 1 (LPAR1) in human synovial fibroblasts. These results suggest that LPA transcriptionally increases MMP production by the activation of an LPAR1/ERK1/2 signal pathway in human synovial fibroblasts. Thus, LPA is likely to be a pathological candidate for cartilage degradation in RA.

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  • Toshio Niwa, Shin-ichiro Yokoyama, Mika Mochizuki, Toshihiko Osawa
    2021 Volume 44 Issue 1 Pages 136-139
    Published: January 01, 2021
    Released: January 01, 2021
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    A hexahydrocurcumin-producing bacterium named 2a1-2b was isolated from human feces. It was observed that the bacterium had more than 99% similarity with Enterococcus avium ATCC14025T according to 16S ribosomal DNA (rDNA) sequence. The strain 2a1-2b produced optically active 5R-hexahydrocurcumin (enantiomeric excess (e.e.) > 95%) from tetrahydrocurcumin but not from curcumin. Our results showed that intestine is an important place for producing hexahydrocurcumin.

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  • Chihiro Naito, Tomoko Yamaguchi, Hidemasa Katsumi, Suyo Kimura, Sachi ...
    2021 Volume 44 Issue 1 Pages 140-143
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    To establish a system for assessing drug permeation and irritation of the skin, the permeation of benzoic acid and isosorbide dinitrate, which are listed in the Pharmacopoeia, and the chemical irritation were evaluated using skin generated from human induced pluripotent stem cells (iPSCs). Multilayer structures and cellular markers (keratin 14 and 10, which are in basal and suprabasal epidermal layers) were clearly detected in our iPSC-based skin. Transepidermal water loss (TEWL) decreased after iPSC-derived keratinocytes were cultured on collagen gels from human primary fibroblasts. These results indicate that the barrier function was partly increased by formation of the living epidermis. The cumulative amount of benzoic acid and isosorbide dinitrate across human iPSC-based skin gradually increased after an initial lag time. Moreover, the irritancy of various chemicals (non-irritants: ultrapure water, allyl phenoxy-acetate, isopropanol, and hexyl salicylate and irritants: 5% sodium dodecyl sulfate (SDS), heptanal, potassium hydroxide (5% aq.) and cyclamen aldehyde) to iPSC-based skin was almost met the irritation criteria of the Organisation for Economic Co-operation and Development (OECD) guideline. The results of our iPSC-based skin evaluation provide useful basic information for developing an assessment system to predict the permeation and safety of new transdermal drugs in human skin.

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  • Takeshi Terada, Jayesh A Kulkarni, Ariel Huynh, Yuen Yi C. Tam, Pieter ...
    2021 Volume 44 Issue 1 Pages 144-149
    Published: January 01, 2021
    Released: January 01, 2021
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    Supplementary material

    Liposomes containing ionizable cationic lipids have been widely used for the delivery of nucleic acids such as small-interfering RNA and mRNA. The utility of cationic lipids with a permanent positive charge, however, is limited to in vitro transfection of cultured cells due to its dose-limiting toxic side effects observed in animals. Several reports have suggested that the permanently charged cationic lipids induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. We therefore hypothesized that the concomitant use of ROS inhibitor could reduce toxicity and improve drug efficacy. In this study, suppression of the cationic toxicity was evaluated using an ROS scavenger, edaravone, which is a low-molecular-weight antioxidant drug clinically approved for acute-phase cerebral infarction and amyotrophic lateral sclerosis. Cell viability assay in the mouse macrophage-like cell line RAW264 indicated that the concomitant use of edaravone were not able to suppress the cytotoxicity induced by cationic liposomes comprised of monovalent cationic lipid N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA) over a short period of time. Cationic lipids-induced necrosis was assumed to be involved in the cytotoxicity upon short-term exposure to cationic liposomes. On the other hand, the significant improvement of cell viability was observed when the short treatment with cationic liposomes was followed by exposure to edaravone for 24 h. It was also confirmed that apoptosis inhibition by ROS elimination might have contributed to this effect. These results suggest the utility of continuous administration with edaravone as concomitant drug for suppression of adverse reactions in therapeutic treatment using cationic liposomes.

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  • Hiroki Yoshioka, Sarah Tominaga, Mai Nishikawa, Yasuro Shinohara, Mako ...
    2021 Volume 44 Issue 1 Pages 150-153
    Published: January 01, 2021
    Released: January 01, 2021
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    Bromobenzene (BB) is known to pose a serious threat to human health. We previously demonstrated that BB showed chronotoxicity, that is, daily fluctuations in the severity of hepatotoxicity induced in mice. Although BB showed mild nephrotoxicity, a daily fluctuation was not observed in this toxicity. This might be attributed to the fact that BB-induced chronotoxicity is observed only in the liver and not in the kidneys and that the damage caused by BB is prominent in the liver, masking the daily fluctuation in nephrotoxicity. To confirm these two possibilities, we examined the daily fluctuations in nephrotoxicity due to BB intermediate metabolites that target the kidneys: 3-bromophenol, bromohydroquinone, and 4-bromocatechol. Mice were injected with 3-bromophenol, bromohydroquinone, or 4-bromocatechol intraperitoneally at six different time points in a day (zeitgeber time (ZT): ZT2, ZT6, ZT10, ZT14, ZT18, or ZT22). Mortality was monitored for 7 d post-injection. Mice were more sensitive to the acute toxicity of these metabolites around at ZT14 (dark-phase) exposure than around at ZT2 (light-phase) exposure. Furthermore, mice administered with a non-lethal dose of 4-bromocatechol showed significant increases in the levels of plasma blood urea nitrogen and renal malondialdehyde at ZT14 exposure. Moreover, glutathione peroxidase-4, a ferroptosis indicator, was attenuated at ZT14 exposure. These results indicate the toxicity of BB metabolites was higher during the dark-phase exposure, and demonstrate the reason why the diurnal variation of nephrotoxicity by BB was not observed in our previous report is that renal damage was masked due to severe hepatic damage.

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  • Iyuki Namekata, Ryosuke Odaka, Shunsuke Hamazaki, Hina Nisaka, Shogo H ...
    2021 Volume 44 Issue 1 Pages 154-157
    Published: January 01, 2021
    Released: January 01, 2021
    [Advance publication] Released: October 30, 2020
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    We developed a method to evaluate the activity of the Na+–Ca2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) with fluorescence microscopy in mouse ventricular cardiomyocytes. In non-beating ventricular cardiomyocytes, α-adrenoceptor stimulation by phenylephrine caused a decrease in the cytoplasmic Ca2+ concentration, which was inhibited by SEA0400, an NCX inhibitor, but not cyclopiazonic acid, a SERCA inhibitor. β-Adrenoceptor stimulation by isoprenaline caused a decrease in the cytoplasmic Ca2+ concentration, which was inhibited by cyclopiazonic acid but not SEA0400. Ellagic acid, a phenolic phytochemical, also decreased the basal Ca2+ concentration, which was inhibited by cyclopiazonic acid, but not SEA0400. Thus, this method using fluorescent microscopy and specific inhibitors would be useful for the evaluation of pharmacological agents acting on NCX and SERCA.

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