論文 - 菅井　和久

Angiotensin-converting gene and hypoxic exercise tolerance: A randomized crossover trial

Muramoto Y., Momoi M., Nakashima D., Omae K., Sugai K., Daigo K., Iwasawa Y., Ichihara G., Okawara H., Sawada T., Kinoda A., Yamada Y., Kimura T., Sato K., Katsumata Y.

International Journal of Sports Medicine 46 （ 4 ） 246 - 254 2025年

ISSN  01724622

　概要を見る

Hypoxic training enhances endurance sports tolerance. However, individual responses vary due to physiological differences. This study investigated the relationship between genetic factors and exercise tolerance in hypoxic conditions. This randomized crossover study included 22 male university students (age 20.8±1.3 years, peak oxygen uptake 54.5±6.5 mL/min/kg). Incremental load tests were conducted to assess the symptomatic limit on separate days under normoxic and hypoxic conditions (oxygen concentration 15.4±0.8%) using an ergometer. The initial test environment was randomized. The peak oxygen uptake and blood lactate were monitored every minute, and Δ peak oxygen uptake (peak oxygen uptake under hypoxia - peak oxygen uptake under normoxia) was calculated. Sixteen genotypes linked to exercise tolerance (such as angiotensin-converting enzyme [ACE]) were examined. Peak oxygen uptake significantly decreased under hypoxia (p<0.01). Δ peak oxygen uptake varied among individuals (minimum: 0.7 and maximum: - 18.9). Among analyzed genetic polymorphisms, ACE-II genotypes showed significantly greater Δ peak oxygen uptake than ACE-ID/ACE-DD genotypes (p=0.02). ACE-II genotypes exhibited lower blood lactate elevation at peak exercise in normoxic (p=0.01) and hypoxic (p=0.03) conditions. Participants with the ACE-II genotype had lower lactate concentrations and greater reductions in peak oxygen uptake under hypoxic conditions. Optimizing hypoxic training requires individualized programs incorporating genetic analysis.

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Impact of daily administration of blackberry extract on gerbil model of transient cerebral ischemia

Oda A., Sugai K., Fujisawa M., Hakamata Y., Kobayashi S., Kobayashi E.

Acta Cirurgica Brasileira 39 2024年

ISSN  01028650

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Purpose: Blackberries are rich in polyphenols and are a human health food continuously consumed to improve health and reduce diseases caused by aging. Herein, we evaluated the effects of daily blackberry administration before and after transient cerebral ischemia in gerbils. Methods: Blackberry extract (BBE) was orally administered twice a day for two weeks to protect against ischemic events during continuous administration. On the seventh day after administration, the bilateral common carotid arteries were transiently occluded for 5 min. To verify its therapeutic effect, BBE was administered after ischemia using a similar protocol without pre-administration. In both experiments, the number of viable neurons in the CA1 region of the hippocampus was assessed seven days after ischemic treatment. Results: The number of neurons in the group treated with BBE before ischemia was higher than that in the group treated with distilled water (p = 0.0601), and similar to that in the control group. In the BBE administration experiments after ischemia, the number of neurons was significantly reduced compared to that in the control group (p < 0.0001). Conclusion: Continuous BBE intake is expected to prevent or ameliorate ischemic events such as transient cerebral ischemia.

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Establishment and application of a new 4/6 infarct nephrectomy rat model for moderate chronic kidney disease

Sugai K., Hirano M., Oda A., Fujisawa M., Shono S., Ishioka K., Tamura T., Katsumata Y., Sano M., Kobayashi E., Hakamata Y.

Acta Cirurgica Brasileira 39 2024年

ISSN  01028650

　概要を見る

Purpose: To develop a new 4/6 infarct nephrectomy (INx) model rat mimicking moderate chronic kidney disease (CKD) and to evaluate its application. Methods: We modified the conventional 5/6 INx rat model to create the 4/6 INx model by ligating the renal artery branch to induce infarction of one-third of the left kidney after right kidney removal and compared biochemically and histologically both models. To demonstrate the application of the 4/6 INx model, the effects of a supplementary compound containing calcium carbonate, chitosan, palm shell activated charcoal etc., that is effective for both CKD and its complications, were compared between both models. Results: Impairment of renal function in the 4/6 INx group was significantly more moderate than in the 5/6 INx group (P < 0.05). The 4/6 INx group showed less histological damage in kidney than in the 5/6 INx group. The supplementary compound did not improve CKD in the 5/6 INx group, but ameliorated elevation of blood urea nitrogen in the 4/6 INx group. Conclusion: We developed the 4/6 INx model, which is more moderate than the conventional 5/6 INx model. This model could potentially demonstrate the effectiveness of drugs and supplements intended to prevent CKD and its progression.

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A neural cell automated analysis system based on pathological specimens in a gerbil brain ischemia model

Katsumata E., Ranjan A.K., Tashima Y., Takahata T., Sato T., Kobayashi M., Ishii M., Takahashi T., Oda A., Hirano M., Hakamata Y., Sugai K., Kobayashi E.

Acta Cirurgica Brasileira 39 2024年

ISSN  01028650

　概要を見る

Purpose: Amid rising health awareness, natural products which has milder effects than medical drugs are becoming popular. However, only few systems can quantitatively assess their impact on living organisms. Therefore, we developed a deep-learning system to automate the counting of cells in a gerbil model, aiming to assess a natural product’s effectiveness against ischemia. Methods: The image acquired from paraffin blocks containing gerbil brains was analyzed by a deep-learning model (fine-tuned Detectron2). Results: The counting system achieved a 79%-positive predictive value and 85%-sensitivity when visual judgment by an expert was used as ground truth. Conclusion: Our system evaluated hydrogen water’s potential against ischemia and found it potentially useful, which is consistent with expert assessment. Due to natural product’s milder effects, large data sets are needed for evaluation, making manual measurement labor-intensive. Hence, our system offers a promising new approach for evaluating natural products.

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Anaerobic threshold using sweat lactate sensor under hypoxia

Okawara H., Iwasawa Y., Sawada T., Sugai K., Daigo K., Seki Y., Ichihara G., Nakashima D., Sano M., Nakamura M., Sato K., Fukuda K., Katsumata Y.

Scientific Reports 13 （ 1 ）  2023年12月

　概要を見る

We aimed to investigate the reliability and validity of sweat lactate threshold (sLT) measurement based on the real-time monitoring of the transition in sweat lactate levels (sLA) under hypoxic exercise. In this cross-sectional study, 20 healthy participants who underwent exercise tests using respiratory gas analysis under hypoxia (fraction of inspired oxygen [FiO2], 15.4 ± 0.8%) in addition to normoxia (FiO2, 20.9%) were included; we simultaneously monitored sLA transition using a wearable lactate sensor. The initial significant elevation in sLA over the baseline was defined as sLT. Under hypoxia, real-time dynamic changes in sLA were successfully visualized, including a rapid, continual rise until volitionary exhaustion and a progressive reduction in the recovery phase. High intra- and inter-evaluator reliability was demonstrated for sLT’s repeat determinations (0.782 [0.607–0.898] and 0.933 [0.841–0.973]) as intraclass correlation coefficients [95% confidence interval]. sLT correlated with ventilatory threshold (VT) (r = 0.70, p < 0.01). A strong agreement was found in the Bland–Altman plot (mean difference/mean average time: − 15.5/550.8 s) under hypoxia. Our wearable device enabled continuous and real-time lactate assessment in sweat under hypoxic conditions in healthy participants with high reliability and validity, providing additional information to detect anaerobic thresholds in hypoxic conditions.

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Estimation of maximal lactate steady state using the sweat lactate sensor

Muramoto Y., Nakashima D., Amano T., Harita T., Sugai K., Daigo K., Iwasawa Y., Ichihara G., Okawara H., Sawada T., Kinoda A., Yamada Y., Kimura T., Sato K., Katsumata Y.

Scientific Reports 13 （ 1 ）  2023年12月

　概要を見る

A simple, non-invasive algorithm for maximal lactate steady state (MLSS) assessment has not been developed. We examined whether MLSS can be estimated from the sweat lactate threshold (sLT) using a novel sweat lactate sensor for healthy adults, with consideration of their exercise habits. Fifteen adults representing diverse fitness levels were recruited. Participants with/without exercise habits were defined as trained/untrained, respectively. Constant-load testing for 30 min at 110%, 115%, 120%, and 125% of sLT intensity was performed to determine MLSS. The tissue oxygenation index (TOI) of the thigh was also monitored. MLSS was not fully estimated from sLT, with 110%, 115%, 120%, and 125% of sLT in one, four, three, and seven participants, respectively. The MLSS based on sLT was higher in the trained group as compared to the untrained group. A total of 80% of trained participants had an MLSS of 120% or higher, while 75% of untrained participants had an MLSS of 115% or lower based on sLT. Furthermore, compared to untrained participants, trained participants continued constant-load exercise even if their TOI decreased below the resting baseline (P < 0.01). MLSS was successfully estimated using sLT, with 120% or more in trained participants and 115% or less in untrained participants. This suggests that trained individuals can continue exercising despite decreases in oxygen saturation in lower extremity skeletal muscles.

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MRP1-Dependent Extracellular Release of Glutathione Induces Cardiomyocyte Ferroptosis After Ischemia-Reperfusion

Ichihara G., Katsumata Y., Sugiura Y., Matsuoka Y., Maeda R., Endo J., Anzai A., Shirakawa K., Moriyama H., Kitakata H., Hiraide T., Goto S., Ko S., Iwasawa Y., Sugai K., Daigo K., Goto S., Sato K., Yamada K.I., Suematsu M., Ieda M., Sano M.

Circulation Research 133 （ 10 ） 861 - 876 2023年10月

ISSN  00097330

　概要を見る

BACKGROUND: The membrane components of cardiomyocytes are rich in polyunsaturated fatty acids, which are easily oxidized. Thus, an efficient glutathione-based lipid redox system is essential for maintaining cellular functions. However, the relationship between disruption of the redox system during ischemia-reperfusion (IR), oxidized lipid production, and consequent cell death (ferroptosis) remains unclear. We investigated the mechanisms underlying the disruption of the glutathione-mediated reduction system related to ferroptosis during IR and developed intervention strategies to suppress ferroptosis. METHODS: In vivo fluctuations of both intra- and extracellular metabolite levels during IR were explored via microdialysis and tissue metabolome analysis. Oxidized phosphatidylcholines were assessed using liquid chromatography high-resolution mass spectrometry. The areas at risk following IR were assessed using triphenyl-tetrazolium chloride/Evans blue stain. RESULTS: Metabolomic analysis combined with microdialysis revealed a significant release of glutathione from the ischemic region into extracellular spaces during ischemia and after reperfusion. The release of glutathione into extracellular spaces and a concomitant decrease in intracellular glutathione concentrations were also observed during anoxia-reperfusion in an in vitro cardiomyocyte model. This extracellular glutathione release was prevented by chemical inhibition or genetic suppression of glutathione transporters, mainly MRP1 (multidrug resistance protein 1). Treatment with MRP1 inhibitor reduced the intracellular reactive oxygen species levels and lipid peroxidation, thereby inhibiting cell death. Subsequent in vivo evaluation of endogenously oxidized phospholipids following IR demonstrated the involvement of ferroptosis, as levels of multiple oxidized phosphatidylcholines were significantly elevated in the ischemic region 12 hours after reperfusion. Inhibition of the MRP1 transporter also alleviated intracellular glutathione depletion in vivo and significantly reduced the generation of oxidized phosphatidylcholines. Administration of MRP1 inhibitors significantly attenuated infarct size after IR injury. CONCLUSIONS: Glutathione was released continuously during IR, primarily in an MRP1-dependent manner, and induced ferroptosis. Suppression of glutathione release attenuated ferroptosis and reduced myocardial infarct size following IR.

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Direct evidence of hydrogen absorption from the skin – a pig study.

Iwai S, Kobayashi S, Kobayashi E, Sugai K, Hakamata Y, Shirakawa K, Katsumata Y, Sano M.

Archives of Medical Science – Civilization Diseases. 2023年

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-<inf>L</inf>-Lysine Induce Antibody Production as Vaccine Adjuvants

Zhao D., Tu A.T.T., Shobo M., Le N.B.T., Yoshikawa C., Sugai K., Hakamata Y., Yamazaki T.

Biomolecules 12 （ 12 ）  2022年12月

　概要を見る

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-β. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

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Pharmacokinetics of hydrogen administered intraperitoneally as hydrogen-rich saline and its effect on ischemic neuronal cell death in the brain in gerbils

Hirano M., Sugai K., Fujisawa M., Kobayashi E., Katsumata Y., Hakamata Y., Sano M.

PLoS ONE 17 （ 12 December ）  2022年12月

　概要を見る

Intraperitoneal administration of hydrogen (H2)-containing saline inhibited neuronal cell death in ischemic stroke in a number of animal models, but it is unknown whether H2 is absorbed from the abdominal cavity into the blood and reaches the brain. In this study, we investigated whether intraperitoneal administration of saline containing H2 inhibits neuronal cell death caused by cerebral ischemia and measured the concentration of H2 in the carotid artery and inferior vena cava (IVC). Gerbils were subjected to transient unilateral cerebral ischemia twice, and saline or H2-rich saline was administered intraperitoneally three or seven times every 12 hours. We evaluated the number of apoptotic cells in the hippocampus and cerebral cortex on day 3 and the number of viable neurons in the hippocampus and cerebral cortex on day 7. In addition, a single dose of saline or H2-rich saline was administered intraperitoneally, and blood H2 levels in the carotid artery and IVC were measured. On day 3 of ischemia/reperfusion, the number of neurons undergoing apoptosis in the cortex was significantly lower in the H2-rich saline group than in the saline group, and on day 7, the number of viable neurons in the hippocampus and cerebral cortex was significantly higher in the H2-rich saline group. Intraperitoneal administration of H2-rich saline resulted in large increases in H2 concentration in the IVC ranging from 0.00183 mg/L (0.114%) to 0.00725 mg/L (0.453%). In contrast, carotid H2 concentrations remained in the range of 0.00008 mg/ L (0.0049%) to 0.00023 (0.0146%). On average, H2 concentrations in carotid artery were 0.04 times lower than in IVC. These results indicate that intraperitoneal administration of H2rich saline significantly suppresses neuronal cell death after cerebral ischemia, even though H2 hardly reaches the brain.

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H2 Inhibits the Formation of Neutrophil Extracellular Traps

Shirakawa K., Kobayashi E., Ichihara G., Kitakata H., Katsumata Y., Sugai K., Hakamata Y., Sano M.

JACC: Basic to Translational Science 7 （ 2 ） 146 - 161 2022年02月

ISSN  2452302X

　概要を見る

Neutrophil extracellular traps (NETs) contribute to inflammatory pathogenesis in numerous conditions, including infectious and cardiovascular diseases, and have attracted attention as potential therapeutic targets. H2 acts as an antioxidant and has been clinically and experimentally proven to ameliorate inflammation. This study was performed to investigate whether H2 could inhibit NET formation and excessive neutrophil activation. Neutrophils isolated from the blood of healthy volunteers were stimulated with phorbol-12-myristate-13-acetate (PMA) or the calcium ionophore A23187 in H2-exposed or control media. Compared with control neutrophils, PMA- or A23187-stimulated human neutrophils exposed to H2 exhibited reduced neutrophil aggregation, citrullination of histones, membrane disruption by chromatin complexes, and release of NET components. CXCR4high neutrophils are highly prone to NETs, and H2 suppressed Ser-139 phosphorylation in H2AX, a marker of DNA damage, thereby suppressing the induction of CXCR4 expression. H2 suppressed both myeloperoxidase chlorination activity and production of reactive oxygen species to the same degree as N-acetylcysteine and ascorbic acid, while showing a more potent ability to inhibit NET formation than these antioxidants do in PMA-stimulated neutrophils. Although A23187 formed NETs in a reactive oxygen species–independent manner, H2 inhibited A23187-induced NET formation, probably via direct inhibition of peptidyl arginine deiminase 4-mediated histone citrullination. Inhalation of H2 inhibited the formation and release of NET components in the blood and bronchoalveolar lavage fluid in animal models of lipopolysaccharide-induced sepsis (mice and aged mini pigs). Thus, H2 therapy can be a novel therapeutic strategy for NETs associated with excessive neutrophil activation.

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A microsurgical technique for catheter insertion in the rat femoral artery.

Sugai K, Hakamata Y, Tamura T, Kataoka M, Fujisawa M, Sano M, Koyabashi E.

Acta Cirúrgica Brasileira. 2020年

Daily inhalation of hydrogen gas has a blood pressure-lowering effect in a rat model of hypertension.

Sugai K, Tamura T, Sano M, Uemura S, Fujisawa M, Katsumata Y, Endo J, Yoshizawa J, Homma K, Suzuki M, Kobayashi E, Sasaki J, Hakamata Y.

Scientific Reports. 2020年