Biological and Pharmaceutical Bulletin
Online ISSN : 1347-5215
Print ISSN : 0918-6158
ISSN-L : 0918-6158
Volume 43 , Issue 11
Showing 1-25 articles out of 25 articles from the selected issue
Review
  • Hidetaka Akita
    2020 Volume 43 Issue 11 Pages 1617-1625
    Published: November 01, 2020
    Released: November 01, 2020
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    Gene and nucleic acid-based medication is an ultimate strategy in the field of personalized medicine. A gene or short interference RNA (siRNA) molecule needs to be delivered to the appropriate organelle (i.e., nucleus and cytoplasm, respectively). We recently focused on improving the intrinsic activity of my original material (ssPalm) in terms of endosomal/lysosomal membrane destabilization activity by chemically modifying the tertiary amine structure. In parallel, I have been expanding the range of applications of ssPalms. The first application is a DNA or RNA vaccine. My crucial finding is that the vitamin E-scaffold ssPalm (ssPalmE) is highly immune-stimulative when combined with DNA. Thereafter, I redesigned the hydrophobic scaffold structure, and found that an oleic acid-scaffold ssPalm (ssPalmO) can confer anti-inflammatory characteristics. Based on this result, I further upgraded the ssPalmO, by inserting a newly designed linker with self-degradable properties.

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    Editor’s picks

    DNA/RNA-based medication is an ultimate strategy in the field of personalized medicine. The authors summarized the recent progress in the development of their original lipid-like material: SS-cleavable pH-Activated Lipid-like Material (ssPalm) as a platform of the DNA/RNA delivery system. The material forms a DNA/RNA-encapsulating nanoparticle with neutral charge, and it confer the endosomal escape, and subsequent release of their encapsulating cargos to the cytoplasm. Also, the trafficking and physiological functions of the particles can be controlled by the changing of the hydrophobic structure. Furthermore, the transfection activity was highly improved by the insertion of self-degradable linker. The authors summarized the concept and application of a series of ssPalm.

Regular Articles
  • Jiayao Yang, Wei Ma, Qunchao Mei, Juefei Song, Lei Shu, Shu Zhang, Che ...
    2020 Volume 43 Issue 11 Pages 1626-1633
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Fuzi Lizhong decoction (FLD) is derived from an ancient Chinese Pharmacopoeia and has been used in clinical treatment for years. The present study aimed to investigate the activities and underlying mechanisms of FLD against non-alcoholic fatty liver disease (NAFLD). Network pharmacology analysis demonstrated that FLD might affect NAFLD through regulating p53 and peroxisome proliferator activated receptor gamma (PPARG), which has been confirmed in vitro and in vivo. In vivo NAFLD was induced in rats by a high-fat diet, and in vitro studies were performed on HL-7702 cells treated with oleic acid and linoleic acid. We showed that FLD significantly improved NAFLD by regulating the immune system to induce the release of interleukin-10 (IL-10), interferon-α (IFN-α), and IFN-β through activating p53 signaling and inhibiting PPARG signaling in vivo and in vitro. P53 inhibition induced by NAFLD was recused by FLD, while PPARG overexpression induced by NAFLD was inhibited by FLD. In addition, NAFLD resulted in increased levels of total cholesterol, triglyceride, and blood glucose in the serum and free fatty acid in the liver, which were reduced by FLD treatment. Evidently, FLD exhibited potent protective effects against NAFLD via p53 and PPARG signaling. Our study could provide novel insights into the mechanisms of FLD as an anti-inflammatory candidate for the treatment of NAFLD in the future.

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  • Yuanguo Xiong, Hao Lu, Hanlin Xu
    2020 Volume 43 Issue 11 Pages 1634-1642
    Published: November 01, 2020
    Released: November 01, 2020
    [Advance publication] Released: September 04, 2020
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    Supplementary material

    Hepatic fibrosis (HF) is a common disease, with currently no available treatment. Galangin, a natural flavonoid extracted from Alpinia officinaruim Hance, has multiple effects demonstrated in previous studies. The aim of the present study was to explore the anti-fibrogenic effect of galangin in vitro, and research its potential molecular mechanisms. LX-2 cells were chosen as an in vitro HF model, and were treated with galangin in different concentrations. Cell viability was analyzed using Cell Counting Kit-8 (CCK-8) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell apoptosis was measured using flow cytometry, and the anti-fibrogenic effect of galangin was determined using RT-quantitative (q)PCR, immunofluorescence, and Western blotting. The results show that the proliferation of LX-2 cells was efficiently inhibited by galangin, and apoptosis was induced in a dose-dependent manner. Both the mRNA and protein expression of alpha-smooth muscle actin (α-SMA) and collagen I were markedly downregulated. Galangin also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt and Wnt/β-catenin signaling pathways and increased the Bax/Bcl-2 ratio. The results of this study suggest that galangin has an anti-fibrogenic effect and may represent a promising agent in the treatment of hepatic fibrosis.

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  • Wei Qi, Yongfu Gu, Zhen Wang, Weimin Fan
    2020 Volume 43 Issue 11 Pages 1643-1652
    Published: November 01, 2020
    Released: November 01, 2020
    [Advance publication] Released: September 02, 2020
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    Supplementary material

    Matrix metalloproteinases (MMPs) are required for collagen degradation which play a key pathological role in arthritis progression. Herein, the effect of sinomenine (SN) on Interleukin 1 beta (IL-1β)-induced MMPs production and its underlying mechanism were explored in SW1353 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that 200 and 400 µM SN significantly inhibited SW1353 cell proliferation, thus the lower dose of SN (25–100 µM) were used in the subsequent experiments. Notably, the increased mRNA and protein levels of suppressor of cytokine signaling (SOCS) 3 were dose-dependently induced by SN. SN significantly suppressed mRNA and protein levels of MMPs in IL-1β-induced SW1353 cells. Through Western blot analysis, SN showed inhibitory effect on IL-1β-induced TAK1 and p65 phosphorylation. Moreover, SN blocked the interaction of TRAF6 and TAK1 resulting in inactivation of IL-1β pathway. Mechanistically, the inhibitory effect of SN on MMPs levels alongside TRAF6 and TAK1 interactions was abrogated by silencing SOCS3. Moreover, SN did not inhibit TAK1 kinase activity. In TAK1 silencing cells, the levels of MMPs and p65 phosphorylation of SN-treatedcells were lower than dimethyl sulfoxide (DMSO)-treated cells, indicating that blocking interaction was not a unique way for SN to inhibit MMPs levels. Finally, SN significantly inhibited IL-6-induced Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in SW1353 cells. The levels of JAK2 phosphorylation and MMPs did not show a significant difference between IL-6 + SOCS3-small interfering RNA (siRNA) + SN group and IL-6 + SOCS3-siRNA + DMSO group. These findings demonstrated that SOCS3 expression was increased by SN blocked IL-1β-induced interaction between TRAF6 and TAK1 as well as IL-6 pathway activation, thereby culminating in the inhibition of MMPs levels.

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    Editor’s picks

    Sinomenine (SN), an alkaloid isolated from Sinomenium acutum, has been used therapeutically for arthritis in China over many years. Herein, it was found that SN dose-dependently increased SOCS3 expression levels and inhibited matrix metalloproteinases (MMPs) levels in human chondrocyte cells. SOCS3 expression was increased by SN blocked IL-1β-induced interaction between TRAF6 and TAK1, thereby culminating in the inhibition of MMPs levels. In addition, SN also inhibited IL-6-induced JAK2 and STAT3 phosphorylation. These findings provided a molecular basis for the potential application of SN and suggested that SN exhibited a cartilage protective property for the treatment of arthritis.

  • Yanfen Niu, Pingfen Yang, Hongjian Li, Qiang Li, Hua Lin, Lihui Gao, L ...
    2020 Volume 43 Issue 11 Pages 1653-1659
    Published: November 01, 2020
    Released: November 01, 2020
    [Advance publication] Released: August 28, 2020
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    Hyperuricemia is mainly the result of relative underexcretion of urate. Urate is mainly eliminated by kidney and several important transporters expressed on the membrane of renal tubular cells involved in urate excretion. Olsalazine sodium was screened from 3167 authorized small compounds/drugs, targeting xanthine oxidoreductase. In previous study, we reported that olsalazine sodium significantly reduced the serum urate levels, and the anti-hyperuricemic activity linked with inhibiting urate formation by reducing the activity of xanthine oxidoreductase. The current research aimed to assess olsalazine sodium renal urate excretion and likely molecular mechanism. The results showed that administration of olsalazine sodium 5.0 mg/kg decreased the levels of serum urate in hyperuricemic rats, and noticeably improved the fractional excretion of urate and urate clearance, exhibiting an uricosuric action. Moreover, olsalazine sodium (2.5, 5.0, 10.0 mg/kg) reduced the level of blood urea nitrogen in rats. Further study showed that olsalazine sodium reduced the mRNA expression of urate reabsorptive transporter glucose transporter 9 (GLUT9), increased the mRNA expression of urate secretory transporters, organic anion transporter 1 (OAT1), OAT3 and type 1 sodium-dependent phosphate transporter (NPT1) as well as the protein expression of OAT3 in the kidney in hyperuricemic mice. In conclusion, olsalazine sodium exhibited a promotion of urate excretion in kidney by increasing the expression of OAT3.

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  • Motoki Imai, Fumitaka Kawakami, Makoto Kubo, Makoto Kanzaki, Hiroko Ma ...
    2020 Volume 43 Issue 11 Pages 1660-1668
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson’s disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5′-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.

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  • Shin-ichi Akanuma, Kahori Hashimoto, Yukiko Yoshida, Yoshiyuki Kubo, K ...
    2020 Volume 43 Issue 11 Pages 1669-1677
    Published: November 01, 2020
    Released: November 01, 2020
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    Prostaglandin (PG) D2 is a lipid mediator, and in the brain, overproduction of PGD2 is reportedly involved in the progression and exacerbation of neuroinflammation. The objective of this study was to elucidate PGD2 efflux transport, under normal and inflammatory conditions, across the blood–brain barrier (BBB), which is formed by brain capillaries. Elimination of [3H]PGD2 across the BBB of normal and lipopolysaccharide (LPS)-induced inflammatory rats was examined by the intracerebral microinjection technique. After intracerebral injection, the percentage of [3H]PGD2 remaining in the ipsilateral cerebrum decreased with time, with a half-life of 13 min. This [3H]PGD2 elimination across the BBB was significantly inhibited by the co-administration of unlabeled PGD2, which suggests carrier-mediated PGD2 efflux transport at the BBB. In isolated rat brain capillaries, mRNA expression of organic anion transporter (Oat) 3, organic anion-transporting polypeptide (Oatp) 1a4, and multidrug resistance-associated protein (Mrp) 4 was observed. In addition, co-administration of substrates/inhibitors for Oat3, Oatp1a4, and/or Mrp4, such as benzylpenicillin and cefmetazole, reduced [3H]PGD2 elimination across the BBB. Data suggest that Oat3 and Mrp4, but not Oatp1a4 are involved in PGD2 elimination across the BBB, as Oatp1a4-expressing Xenopus (X.) oocytes did not show the significant [3H]PGD2 uptake compared with water-injected X. oocytes. In LPS-treated rats, [3H]PGD2 elimination across the BBB and mRNA expression levels of Oat3 and Mrp4 were significantly decreased. Our data suggest that Oat3- and Mrp4-mediated PGD2 elimination across the BBB is attenuated under inflammatory conditions.

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  • Riyo Morimoto-Kamata, Daiki Tsuji, Satoru Yui
    2020 Volume 43 Issue 11 Pages 1678-1686
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells. It has been suggested that tumor cell aggregates are associated with tumor embolism, thus CG-induced cell aggregation may promote tumor metastasis. We have revealed that cell aggregation is caused by elevated free insulin-like growth factor (IGF)-1 in the medium, followed by activation of IGF-1 receptor (IGF-1R). However, the molecular mechanism underlying IGF-1 elevation induced by CG remains unclear. Here, we aimed to elucidate the mechanism by examining the degradative effects of CG on IGF-1, and the IGF binding proteins (IGFBPs), which interfere with the binding of IGF-1 to its receptor. CG specifically evoked MCF-7 cell aggregation at less than 1 nM in a dose-dependent manner, however, neutrophil elastase (NE), chymotrypsin, and trypsin did not. Free IGF-1 concentration was continuously elevated in the medium of cells treated with CG, whereas treatments with other serine proteases resulted in only a transient or slight increase. IGFBP-2, the predominant IGFBP in MCF-7 cells, was gradually digested by CG. CG did not cleave IGF-1 for at least 48 h, whereas other proteases completely digested it. Moreover, CG induced continuous phosphorylation of IGF-1R and Akt, whereas NE-induced phosphorylation was transient, possibly due to insulin receptor substrate (IRS)-1 digestion. These results indicated that CG-specific IGF-1 elevation in the medium is caused by digestion of IGFBP-2, not IGF-1. Hence, this study clarifies the molecular mechanism of CG-specific cell aggregation.

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  • Seiichi Yoshida, Takamichi Ichinose, Takayuki Shibamoto
    2020 Volume 43 Issue 11 Pages 1687-1692
    Published: November 01, 2020
    Released: November 01, 2020
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    Several studies show that maternal conventional cigarette smoking during pregnancy has been associated with reduced sperm concentration in sons. The development of heat-not-burn (HnB) tobacco has gained a growing following. However, the effects of prenatal HnB tobacco smoking on male offspring are as yet unknown. Pregnant CD-1 mice were exposed to I-Quit-Ordinary-Smoking (IQOS) (HnB tobacco) aerosol from heat sticks, mainstream smoke from 3R4F (conventional cigarettes) or clean air, using a whole-body exposure system. Adult male offspring mice were divided into six groups: control (5- and 15-weeks-old offspring), IQOS (5 and 15-weeks-old) and 3R4F (5 and 15-weeks-old). Spermatogenesis, sperm characteristics, serum testosterone, and seminiferous tubule morphology were evaluated. Prenatal IQOS exposure increased abnormal seminiferous tubule morphology and decreased sperm production at 5 weeks, but 3R4F exposure did not. Prenatal exposure to IQOS aerosol delays sexual maturation of male offspring or adversely affects the male testicular function of the offspring more than smoke from a combustion cigarette.

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  • Sirima Soodvilai, Paranee Meetam, Lawan Siangjong, Ratchanaporn Chokch ...
    2020 Volume 43 Issue 11 Pages 1693-1698
    Published: November 01, 2020
    Released: November 01, 2020
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    Cisplatin is a widely used chemotherapy for solid tumors; however, its benefits are limited by serious nephrotoxicity, particularly in proximal tubular cells. The present study investigated the renoprotective effect and mechanisms of germacrone, a bioactive terpenoid compound found in Curcuma species on cisplatin-induced toxicity of renal cells. Germacrone (50 and 100 µM) attenuated apoptosis of human renal proximal tubular cells, RPTEC/TERT1 following treatment with 50 µM cisplatin and for 48 h. Co-treating RPTEC/TERT1 cells with cisplatin and germacrone significantly reduced cellular platinum content compared with cisplatin treatment alone. The effect of germacrone on organic cation transporter 2 (OCT2) which is a transporter responsible for cisplatin uptake was determined. Germacrone showed an inhibitory effect on OCT2-mediated methyl-4-phenylpyridinium acetate (3H-MPP+) uptake with IC50 of 15 µM with less effect on OCT1. The germacrone’s protective effect on cisplatin-induced cytotoxicity was not observed in cancer cells; cisplatin’s anti-cancer activity was preserved. In conclusion, germacrone prevents cisplatin-induced toxicity in renal proximal tubular cells via inhibition OCT2 transport function and reducing cisplatin accumulation. Thus germacrone may be a good candidate agent used for reducing cisplatin-induced nephrotoxicity.

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    Editor’s picks

    Nephrotoxicity induced by chemotherapy is common and limits the use of anticancer drug especially cisplatin. To improve the therapeutic efficiency of cisplatin, minimizing nephrotoxicity is very important. The present study investigated whether germacrone, a bioactive compound found in Curcuma species, has a protective effect on renal proximal tubular cells toxicity induced by cisplatin. The authors found that germacrone reduces cisplatin-induced cytotoxicity in renal proximal tubular cells by inhibition of OCT2-mediated cisplatin uptake into renal proximal tubular cells.  This result suggests that germacrone may be a candidate agent for reducing nephrotoxicity during cisplatin chemotherapy.

  • Hikaru Sada, Makoto Kajizono, Souichiro Ushio, Satoru Esumi, Yoshihisa ...
    2020 Volume 43 Issue 11 Pages 1699-1706
    Published: November 01, 2020
    Released: November 01, 2020
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    Lubiprostone is an effective drug for various types of constipation in patients without cancer; however, there is no report on its efficacy and safety in patients with cancer. Our purpose was to evaluate the efficacy and safety of lubiprostone for constipation in cancer patients. We retrospectively studied 124 patients (cancer, N = 67) who were treated with lubiprostone for constipation in our hospital between June 2013 and May 2016. The number of bowel movements (BMs) increased in the both the cancer and non-cancer groups. The mean change in BM frequency did not differ between the two groups. Approximately 70% of patients in both groups had an initial BM within 24 h after administration of lubiprostone. The most common lubiprostone-related adverse events in both groups were diarrhea (38.8 vs. 14%), and nausea (22.4 vs. 8.8%). No lubiprostone-related serious adverse events occurred. Discontinuation due to the side effects of lubiprostone was more frequent in cancer patients (p = 0.023). Logistic regression analysis showed that the risk of discontinuation of lubiprostone in cancer patients was high in patients with a body-mass index (BMI) <22, and low in patients using opioids and magnesium oxide dosage ≥1000 mg/d. Our study showed that while lubiprostone was as effective in cancer patients as in non-cancer patients, in cancer patients it was associated with a high incidence of diarrhea and nausea side effects and warranted caution, especially in patients with a low BMI.

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    Editor’s picks

    Lubiprostone is an effective drug for various types of constipation in patients without cancer; however, efficacy and safety in patients with cancer have not been clarified. In the present study, Sada et al. retrospectively compared the efficacy and safety of lubiprostone on constipation in patients with and without cancer. It is shown that while lubiprostone was as effective in cancer patients as in non-cancer patients, in cancer patients it was associated with a high incidence of diarrhea and nausea side effects, especially in patients with a low body-mass index.

  • Yung Hyun Choi
    2020 Volume 43 Issue 11 Pages 1707-1714
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Oxidative stress, which is characterized by overproduction of reactive oxygen species (ROS), is considered a major risk factor associated with fibroblast death in severe lung diseases such as idiopathic pulmonary fibrosis. trans-Cinnamaldehyde (tCA), the major phytochemical constituent in cinnamon, is known to possess strong anti-oxidant activity. However, whether tCA can defend lung fibroblasts against oxidative injury remains to be elucidated. Therefore, this study was conducted to investigate the protective effects of tCA on oxidative stress in V79-4 Chinese hamster lung fibroblasts. The current results showed that tCA inhibited hydrogen peroxide (H2O2)-induced cytotoxicity by blocking abnormal accumulation of ROS in V79-4 Chinese hamster lung fibroblasts. tCA attenuated apoptosis by suppressing of mitochondrial dysfunction and cytosolic release of cytochrome c, increasing the rate of Bcl-2/Bax expression and reducing the activity of caspase-9 and caspase-3 in H2O2-stimulated V79-4 cells, suggesting that tCA protected V79-4 cells from the induction of mitochondria-mediated apoptosis by H2O2. Additionally, the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) was markedly promoted by tCA in the presence of H2O2, which was associated with the enhanced expression of heme oxygenase-1 (HO-1). However, inhibiting the activity of HO-1 by zinc protoporphyrin IX, a potent inhibitor of HO-1, eliminated the ROS scavenging and protective effects of tCA, indicating that tCA was able to protect V79-4 lung fibroblasts from H2O2-induced oxidative stress by activating the Nrf2 signaling pathway. Therefore, it is suggested that tCA may be useful as a candidate for the treatment of oxidative stress-mediated lung injuries in the future.

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  • Misa Tokorodani, Hirona Ichikawa, Katsutoshi Yuasa, Tetsuyuki Takahash ...
    2020 Volume 43 Issue 11 Pages 1715-1728
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    SV40-encoded microRNA (miRNA), miR-S1, downregulates the large and small T antigens (LTag and STag), which promote viral replication and cellular transformation, thereby presumably impairing LTag and STag functions essential for the viral life cycle. To explore the functional significance of miR-S1-mediated downregulation of LTag and STag as well as the functional roles of miR-S1, we evaluated viral DNA replication and proinflammatory cytokine induction in cells transfected with simian virus 40 (SV40) genome plasmid and its mutated form lacking miR-S1 expression. The SV40 genome encodes two mature miR-S1s, miR-S1-3p and miR-S1-5p, of which miR-S1-3p is the predominantly expressed form. MiR-S1-3p exerted strong repressive effects on a reporter containing full-length sequence complementarity, but only marginal effect on one harboring a sequence complementary to its seed sequence. Consistently, miR-S1-3p downregulated LTag and STag transcripts with complete sequence complementarity through miR-S1-3p-Ago2-mediated mRNA decay. Transfection of SV40 plasmid induced higher DNA replication and lower LTag and STag transcripts in most of the examined cells compared to that miR-S1-deficient SV40 plasmid. However, miR-S1 itself did not affect DNA replication without the downregulation of LTag transcripts. Both LTag and STag induced the expression of tumor necrosis factor α (TNFα) and interleukin (IL)-17F, which was slightly reduced by miR-S1 due to miR-S1-mediated downregulation of LTag and STag. Forced miR-S1 expression did not affect TNFα expression, but increased IL-17F expression. Overall, our findings suggest that miR-S1-3p is a latent modifier of LTag and STag functions, ensuring efficient viral replication and attenuating cytokine expression detrimental to the viral life cycle.

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  • Tatsuya Fukuta, Natsu Nakatani, Shintaro Yoneda, Kentaro Kogure
    2020 Volume 43 Issue 11 Pages 1729-1734
    Published: November 01, 2020
    Released: November 01, 2020
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    Technologies that overcome the barrier presented by vascular endothelial cells are needed to facilitate targeted delivery of drugs into tissue parenchyma by intravenous administration. We previously reported that weak electric current treatment (ET: 0.3–0.5 mA/cm2) applied onto skin tissue in a transdermal drug delivery technique termed iontophoresis induces cleavage of intercellular junctions that results in permeation of macromolecules such as small interfering RNA and cytosine-phosphate-guanine (CpG) oligonucleotide through the intercellular space. Based on these findings, we hypothesized that application of ET to blood vessels could promote cleavage of intercellular junctions that artificially induces increase in vascular permeability to enhance extravasation of drugs from the vessels into target tissue parenchyma. Here we investigated the effect of ET (0.34 mA/cm2) on vascular permeability using embryonated chicken eggs, which have blood vessels in the chorioallantoic membrane (CAM), as an animal model. ET onto the CAM of the eggs significantly increased extravasation of intravenously injected calcein (M.W. 622.6), a low molecular weight compound model, and the macromolecule fluorescein isothiocyanate (FITC)-dextran (M.W. 10000). ET-mediated promotion of penetration of FITC-dextran through vascular endothelial cells was also observed in transwell permeability assay using monolayer of human umbilical vein endothelial cells without induction of obvious cellular damage. Confocal microscopy detected remarkable fluorescence derived from injected FITC-dextran in blood vessel walls. These results in embryonated chicken eggs suggest that ET onto blood vessels could artificially enhance vascular permeability to facilitate extravasation of macromolecules from blood vessels.

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  • Masakazu Ozaki, Kyoji Kouda, Takashi Kitahara
    2020 Volume 43 Issue 11 Pages 1735-1741
    Published: November 01, 2020
    Released: November 01, 2020
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    Cisplatin is classified as a drug with high emetic risk; thus, the use of aprepitant or fosaprepitant in addition to a 5-hydroxytryptamine-3 (5-HT3) receptor antagonist and dexamethasone is recommended for antiemetic therapy. Further, hydration is required to prevent renal dysfunction, and the use of magnesium has been proposed as a part of the hydration procedure. When fosaprepitant is chosen for antiemetic therapy because the patient has dysphagia, and magnesium is added to the hydration procedure, there may be an incompatibility between the two drugs that reduces the antiemetic effect. In our hospital, in a former regimen, these two drugs were administered concurrently as premedication for regimens containing cisplatin. We varied the conditions so that in a revised regimen the two drugs did not come into contact due to pharmaceutical support, and we conducted a retrospective study to determine the difference in the antiemetic effect. The observation period was 2 years (from October 2015 to September 2017) for the former regimen group (n = 89) and 2 years (from October 2017 to September 2019) for the revised regimen group (n = 177). Comparison of the former and revised regimen groups revealed sex (p = 0.012); anticancer drug dosage (p = 0.006); and variation of premedication condition (p = 0.043) as factors affected by the revised regimen. Optimization of the premedication regimen was a form of necessary pharmaceutical support to maintain the patient’s QOL.

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  • Toshinori Hirai, Ryosuke Yamaga, Motoki Kei, Keiko Hosohata, Toshimasa ...
    2020 Volume 43 Issue 11 Pages 1742-1748
    Published: November 01, 2020
    Released: November 01, 2020
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    Although hypokalemia is an adverse effect of Yokukansan preparation, especially in geriatric patients, its association with age is unclear. We investigated whether age is a risk factor for hypokalemia. This single-center retrospective cohort study, conducted at Tokyo Women’s Medical University, Medical Center East between June 2015 and May 2019, included patients who received the Yokukansan preparation. The primary outcome was hypokalemia (serum potassium level: < 3.0 mEq/L). A multivariate Cox proportional hazard model was used to determine risk factors, hazard ratio (HR) and 95% confidence interval (95% CI). The cut-off age was also examined. Of 665 patients (median age: 78 years; interquartile range: 68–84 years), 55 (8.3%) developed hypokalemia associated with Yokukansan preparation. Risk factors for hypokalemia were age (HR: 1.013, 95% CI: 1.006–1.021, p < 0.001), dementia (HR: 0.500, 95% CI: 0.357–0.682, p < 0.001), serum albumin level (HR: 0.754, 95% CI: 0.669–0.850, p < 0.001), and daily Yokukansan preparation dose ≥ 7.5 g (HR: 1.446, 95% CI: 1.144–1.850, p = 0.002). The cut-off ages were >75 and >80 years but not 65 years and >70 years. Clinicians should assess risk factors and monitor serum potassium levels to avoid hypokalemia associated with the Yokukansan preparation.

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  • Yangyang Wang, Jie Meng, Lu Men, Boran An, Xiaoxu Jin, Wenjuan He, Suc ...
    2020 Volume 43 Issue 11 Pages 1749-1759
    Published: November 01, 2020
    Released: November 01, 2020
    [Advance publication] Released: September 04, 2020
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    Rosmarinic acid (RA) is extensively utilized in herbal medicine in China. The AMP-activated protein kinase (AMPK) signaling can be activated by RA and inhibited by the synthetic, reversible AMP-competitive inhibitor, Compound C (CC). The objective of this study was to investigate the role of AMPK signaling involving the protective effects of RA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) in mice. BALB/c mice were treated with RA, with or without CC, followed by the pretreatment with Con A. Analysis of serum aminotransferases and cytokines were conducted and liver tissue histology was performed to evaluate hepatic injury. Cytokine levels in serum and hepatic tissue were respectively measured by enzyme-linked immunoassay (ELISA) and used quantitative (q)PCR. Levels of phosphorylated acetyl CoA carboxylase in the liver, representing AMPK activation, were detected by Western blotting. Compared with the Con A group, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in RA group (100 and 150 mg/kg/d) were significantly reduced. RA also reduced hepatocyte swelling, cell death, and infiltration of leukocytes in the liver of Con A-treated mice. Serum levels of cytokines, such as interferon-γ (IFN-γ), interleukin-2 (IL-2) and interleukin-1β (IL-1β), were reduced by RA pretreatment, while the levels of serum interleukin-10 (IL-10), an anti-inflammatory cytokine, was elevated. These protective effects were reversed by treatment with CC. RA treatment reduced the hepatic damage via the activation of AMPK in the mice of Con A-induced. So RA acts as a potential part in the therapy of autoimmune hepatitis.

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  • Guangya Xu, Xueling Yan, Jie Chen, Xiaoheng Guo, Xiaolan Guo, Yong Tan ...
    2020 Volume 43 Issue 11 Pages 1760-1766
    Published: November 01, 2020
    Released: November 01, 2020
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    Ulcerative colitis (UC) is chronic, idiopathic disease that affects the colon and the rectum and the underlying pathogenesis of UC remains to be known. The clinical drugs are mainly work based on anti-inflammation and immune system. However, most of them are expensive and have severe side effects. Therefore, identification of novel targets and exploring new drugs are urgently needed. In this study, several bioinformatics approaches were used to discover key genes and further in order to explore the pathogenesis of UC. Two microarray datasets, GSE38713 and GSE9452 were selected from NCBI-Gene Expression Omnibus database. Differentially expression genes (DEGs) were identified by using LIMMA Package of R. Then, we filtered clustered candidate genes into Gene Ontology (GO) and pathway enrichment analysis with the Database for Annotation, Visualization and Integrated Discovery (DAVID), KEGG pathway based on functions and signaling pathways with significant enrichment analysis. The protein–protein interaction (PPI) network was constructed by the Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis, and visualized by Cytoscape and further analyzed by Molecular Complex Detection. Lastly, 353 up-regulated and 145 down-regulated genes were than recognized. After consulting a number of references and network degree analysis, four hub genes, namely FCGR2A, C3, INPP5A, and ACAA1 were identified, and these genes were mainly enriched in complement and coagulation cascades, mineral absorption, and Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathways. In conclusion, this study would provide new clues for the pathogenesis and identification of drug targets of UC in the near future.

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  • Hitomi Kawakami, Hiroyuki Fuchino, Nobuo Kawahara
    2020 Volume 43 Issue 11 Pages 1767-1775
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

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  • Kazuki Kurihara, Hajime Moteki, Hideshi Natsume, Masahiko Ogihara, Mit ...
    2020 Volume 43 Issue 11 Pages 1776-1784
    Published: November 01, 2020
    Released: November 01, 2020
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    Two-thirds partial hepatectomy (PHx) was performed in rats, and the differences in effects between S-allylcysteine (SAC) and other sulfur-containing compounds on regeneration of the remaining liver and restoration of the injury were examined. Three days after two-thirds PHx, rats treated with 300 mg/kg/d, per os (p.o.) SAC showed a 1.2-fold increase in liver weight per 100 g body weight compared with saline-treated controls. In contrast, S-methylcysteine (SMC) (300 mg/kg/d, p.o.) or cysteine (Cys) (300 mg/kg/d, p.o.) did not have a regeneration-promoting effect. In the comparison with control rats, the regenerating liver of SAC-treated rats showed a significantly higher 5-bromo-2′-deoxyuridine labeling index on day 1. In contrast, serum alanine aminotransferase activity, which increases following PHx, was significantly inhibited by SAC and SMC (but not Cys) on day 1 after two-thirds PHx. In addition, SAC induced increases in insulin-like growth factor (IGF)-1 and its receptor mRNA expressions at 1 h after two-thirds PHx, and it increased phosphorylation of extracellular signal-regulated kinase (ERK)2 and Akt at 3 h after two-thirds PHx without affecting serum growth hormone levels. These results demonstrate that SAC is a mitogenic effector of normal remnant liver and promotes recuperation of liver function after two-thirds PHx. Moreover, SAC-induced proliferative effects are mediated via increased mRNA expressions of IGF-1 and its receptor and subsequent phosphorylation of ERK2 and Akt.

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  • Yusuke Kono, Akihiro Miyamoto, Serina Hiraoka, Ryosuke Negoro, Takuya ...
    2020 Volume 43 Issue 11 Pages 1785-1791
    Published: November 01, 2020
    Released: November 01, 2020
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    Supplementary material

    Mesenchymal stem cells (MSCs) are capable of repairing skeletal muscle via paracrine mechanisms. This regenerative effect of MSCs on skeletal muscle is based on promoting the proliferation and differentiation of myogenic cells and inhibiting the inflammatory response of immune cells. However, it is unclear whether MSCs affect the inflammatory response of skeletal muscle cells. In this study, we evaluated the paracrine effect of mouse MSCs on the inflammatory response of lipopolysaccharide (LPS)-stimulated C2C12 mouse myoblasts. Interleukin (IL)-6 production from LPS-stimulated C2C12 cells was significantly increased by coculture with MSCs or culture in conditioned medium of MSCs. This increased IL-6 production from C2C12 cells was not significantly suppressed by inhibiting mitogen-activated protein kinase pathways, but it was significantly suppressed by pretreatment with nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) inhibitors. In addition, IL-6 and inducible nitric oxide synthase (iNOS) mRNA expression was increased significantly in C2C12 cells cocultured with MSCs, while tumor necrosis factor (TNF)-α and IL-1β mRNA expression was decreased. Furthermore, conditioned medium of C2C12 cells cocultured with MSCs exerted remarkable anti-inflammatory effects on LPS-stimulated mouse macrophages.

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  • Hiroshi Arakawa, Natsumi Amezawa, Yu Kawakatsu, Ikumi Tamai
    2020 Volume 43 Issue 11 Pages 1792-1798
    Published: November 01, 2020
    Released: November 01, 2020
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    Xanthine and hypoxanthine are intermediate metabolites of uric acid and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR), suggesting that facilitating their elimination is beneficial. Since they are reabsorbed in renal proximal tubules, we investigated their reabsorption mechanism by focusing on the renal uric acid transporters URAT1 and GLUT9, and examined the effect of clinically used URAT1 inhibitor on their renal clearance when their plasma concentration is increased by XOR inhibitor. Uptake study for [3H]xanthine and [3H]hypoxanthine was performed using URAT1- and GLUT9-expressing Xenopus oocytes. Transcellular transport study for [3H]xanthine was carried out using Madin–Darby canine kidney (MDCK)II cells co-expressing URAT1 and GLUT9. In in vivo pharmacokinetic study, renal clearance of xanthine was estimated based on plasma concentration and urinary recovery. Uptake by URAT1- and GLUT9-expressing oocytes demonstrated that xanthine is a substrate of URAT1 and GLUT9, while hypoxanthine is not. Transcellular transport of xanthine in MDCKII cells co-expressing URAT1 and GLUT9 was significantly higher than those in mock cells and cells expressing URAT1 or GLUT9 alone. Furthermore, dotinurad, a URAT1 inhibitor, increased renal clearance of xanthine in rats treated with topiroxostat to inhibit XOR. It was suggested that xanthine is reabsorbed in the same manner as uric acid through URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with XOR and URAT1 inhibitors will effectively decrease purine pools in the body and prevent cell injury due to ROS generated during XOR-mediated reactions.

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    Editor’s picks

    Xanthine and hypoxanthine are intermediate metabolites of uric acid formation and a source of reactive oxidative species (ROS) by xanthine oxidoreductase (XOR). They are reabsorbed in renal proximal tubules, but the mechanism has not been clarified yet. This study showed that xanthine is reabsorbed in the same manner as uric acid through uric acid transporters URAT1 and GLUT9, while hypoxanthine is not. Accordingly, it is expected that treatment with URAT1 inhibitor along with XOR inhibitor will effectively decrease purine pools in the body by suppressing reabsorption of xanthine increased by XOR inhibitor, thereby preventing cell injury due to ROS generated during XOR-mediated reactions.

Notes
  • Emiko Akashi, Sayaka Nagata, Motoo Yamasaki, Kazuo Kitamura
    2020 Volume 43 Issue 11 Pages 1799-1803
    Published: November 01, 2020
    Released: November 01, 2020
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    Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8–37) or AM (22–52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.

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  • Takahiro Horinouchi, Yuichi Mazaki, Koji Terada, Soichi Miwa
    2020 Volume 43 Issue 11 Pages 1804-1809
    Published: November 01, 2020
    Released: November 01, 2020
    [Advance publication] Released: September 01, 2020
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    Acrolein (ACR), a highly reactive α,β-unsaturated aldehyde, is a major cytotoxic factor in nicotine- and tar-free cigarette smoke extract (CSE). There are conflicting results regarding endothelial functions despite the fact that both CSE and ACR cause cellular damage. Several lines of evidence indicate that CSE impairs endothelium-derived nitric oxide (NO)-dependent vasodilation by reducing the activity and protein expression of endothelial NO synthase (eNOS), whereas ACR elicits endothelium-dependent vasorelaxation by increasing the production of NO and expression of eNOS. To clarify whether CSE and its cytotoxic factor ACR cause endothelial dysfunction, this study examined the effects of CSE and ACR on human vascular endothelial EA.hy926 cells. CSE and ACR reduced the phosphorylation of eNOS at serine (Ser)1177 and total expression of eNOS. The CSE- and ACR-induced decrease in the phosphorylation and expression of eNOS was counteracted by glutathione (reduced form), an antioxidant. Basal NO production was inhibited by CSE, ACR, NG-nitro-L-arginine methyl ester (a competitive eNOS inhibitor), and nominally Ca2+-free solution supplemented with BAPTA-AM (a membrane permeable Ca2+ chelator). These results indicate that CSE and ACR increase oxidative stress, and reduce NO production by reducing the activity and total protein level of eNOS.

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  • Noriyuki Akahoshi, Waka Kamichatani, Isao Ishii
    2020 Volume 43 Issue 11 Pages 1810-1813
    Published: November 01, 2020
    Released: November 01, 2020
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    An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth−/−) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth−/− dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth−/− male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth−/− female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth−/− L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth−/− mice. Therefore, we hypothesize that the blood–brain barrier protects the CNS from high levels of circulatory homocysteine in Cth−/− dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

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