Combined use of hydrogen-rich water and enzyme-digested edible bird's nest improves PMA/LPS-impaired wound healing in human inflammatory gingival tissue equivalents.

Gingival wound healing plays a critical role in maintaining oral health. However, this process can be delayed by oxidative stress and excessive inflammatory responses. In this study, we established a human inflammatory gingival tissue equivalent (iGTE) to investigate the inhibitory effects of hydrogen-rich water (HW), enzyme-digested edible bird's nest (EBND) and sialic acid (SA) on PMA (an inducer of oxidative free radicals)- and LPS (an inflammatory stimulus)-impaired wound healing. The iGTE was constructed by human gingival fibroblasts (hGFs), keratinocytes and macrophages under three-dimensional conditions. Wounds in the iGTE and hGF/keratinocyte monolayers were created by mechanical injury. Tissues and cells were pretreated with HW, EBND, and SA, and then exposed to the inflammatory and oxidative environment induced by PMA (10 ng/mL) and LPS (250 ng/mL). The inflammatory cytokines IL-6 and IL-8 were quantitatively analyzed by ELISA. Histopathological image analysis was performed by HE and immunofluorescence staining. In the iGTE, PMA/LPS significantly reduced the epithelial thickness while causing a decrease in K8/18, E-cadherin, laminin and elastin expression and an increase in COX-2 expression along with ulcer-like lesions. In mechanically scratched hGFs and keratinocyte monolayers, PMA/LPS significantly impaired wound healing, and promoted the secretion of IL-6 and IL-8. Pretreatment of HW, EBND, and SA significantly suppressed PMA/LPS-induced wound healing delay and inflammatory responses in cell monolayers, as well as in the iGTE. Remarkably, the combined use of HW and EBND exhibited particularly robust results. Combined use of HW and EBND may be applied for the prevention and treatment of wound healing delay.

Hydrogen-rich bath with nano-sized bubbles improves antioxidant capacity based on oxygen radical absorbing and inflammation levels in human serum.

This study compared the effects of hydrogen-water (HW) bath on the oxygen radical absorption-based antioxidant capacity and the inflammatory indicator, C-reactive protein (CRP), in serum between healthy volunteers and inflammatory/collagen disease-patients. The HW bath apparatus supplied nano-bubbles with a diameter of 110 ± 10 nm and 338-682 µg/L of dissolved hydrogen after 120 minutes electrolysis, and nano-bubbles increased to 9.91 × 107/mL along with the increase of correlative dissolved hydrogen. Ten-minute HW bath increased the oxygen radical absorption-based antioxidant capacity to 110.9 ± 9.2% at post-bathing 120 minutes, although unaltered with 10-minute normal water bath at 40°C in healthy subjects. The CRP level was repressed to 70.2 ± 12.1% at 120 minutes after HW bath, although rather increased for normal water bath. In the patients with connective tissue diseases, the CRP level was repressed to 3-24% upon 9 days to 4 months of HW bathing. In another six patients with diverse autoimmune-related diseases, upon daily HW bathing as long as 2-25 months, the pre-bathing CRP level of 5.31 mg/dL decreased to 0.24 mg/dL being within the standard-range, with relief of visible inflammatory symptoms for some cases. Thus, the HW bath with high-density nano-bubbles has beneficial effects on serum antioxidant capacity, inflammation, and the skin appearance. The study was approved by the Committee of Ethics, Japanese Center of Anti-Aging Medical Sciences (Authorization No. H-15-03-2, on January 15, 2019), which was a non-profitable organization officially authenticated by the Hiroshima Prefecture Government of Japan.

Hydrogen Nano-Bubble Water Suppresses ROS Generation, Adipogenesis, and Interleukin-6 Secretion in Hydrogen-Peroxide- or PMA-Stimulated Adipocytes and Three-Dimensional Subcutaneous Adipose Equivalents.

Reactive oxygen species (ROS)-induced oxidative stress in adipose tissue is associated with inflammation and the development of obesity-related metabolic disorders. The aim of this study is to investigate the effects of hydrogen nano-bubble water (HW) on ROS generation, adipogenesis, and interleukin-6 (IL-6) secretion in hydrogen peroxide (H2O2) or phorbol 12-myristate 13-acetate (PMA)-stimulated OP9 adipocytes, and three-dimensional (3D) subcutaneous adipose equivalents. Nanoparticle tracking analysis showed that fresh HW contains 1.17 × 108/mL of nano-sized hydrogen bubbles. Even after 8 to 13 months of storage, approximately half of the bubbles still remained in the water. CellROX® staining showed that HW could diminish H2O2- or PMA-induced intracellular ROS generation in human keratinocytes HaCaT and OP9 cells. We discovered that PMA could markedly increase lipid accumulation to 180% and IL-6 secretion 2.7-fold in OP9 adipocytes. Similarly, H2O2 (5 µM) also significantly stimulated lipid accumulation in OP9 cells and the 3D adipose equivalents. HW treatment significantly repressed H2O2- or PMA-induced lipid accumulation and IL-6 secretion in OP9 adipocytes and the 3D adipose equivalents. In conclusion, HW showed a possibility of repressing oxidative stress, inflammatory response, and adipogenesis at cellular/tissue levels. It can be used for preventing the development of metabolic disorders amongst obese people.

Electrolytic hydrogen-generating bottle supplies drinking water with free/combined chlorine and ozone repressed within safety standard under hydrogen-rich conditions.

Hydrogen molecules have attracted attention as a new antioxidant, but are left to be confirmedly verified whether the oral administration is highly safe or not, concurrently with retention of abundant hydrogen. When electrolysis was performed for 10 minutes using a direct-current electrolytic hydrogen-water generating bottle with tap water, "residual free chlorine" concurrently upon the production of molecular hydrogen (444 µg/L) could be appreciably decreased from 0.18 mg/L to 0.12 mg/L as quantified by a N,N-diethyl-p-phenylenediamine-dye colorimetric method. Moreover, the total chlorine concentration (residual bound chlorine plus free chlorine) was estimated to be decreased from 0.17 mg/L to 0.11 mg/L. Although a merit of electrolytic hydrogen-generating bottles exists in electrolysis for periods as short as 10 minutes, the 30-minute electrolysis brought about the more abundant hydrogen (479 µg/L) together with an oxidation-reduction potential of -245 mV; even upon this long-term electrolysis, the gross amounts of chlorine, hypochlorous acid and chloramine were shown not to be increased (0.09-0.10 mg/L from 0.11 mg/L for tap water) as detected by orthotolidine colorimetry. Above-mentioned levels of diverse-type chlorines might fulfill the World Health Organization guideline for drinking water below 5 mg/L. In addition, the dissolved ozone upon electrolytic generation of hydrogen-water was below the detection limit (< 0.05 mg/L) or undetectable, which fulfilled the official safety standards in Japan and the USA for drinking water below 0.1 mg/L, as evaluated by three methods such as an electrode-type ozone checker, indigo dye-utilizing ozone detector capillaries and potassium iodide-based colorimetry. Importantly, even when half the amount of tap water was poured into the tank of the apparatus and electrolyzed, both the residual chlorine and ozone concentrations measured were also below the safety standard. Thus, major potently harmful substances, such as residual free/bound chlorine, or hypochlorous-acid/chloramine, respectively, and dissolved ozone, as the drinking hydrogen-water was direct-current-electrolytically generated, were estimated to be repressed within safety concentration ranges with achievements of abundant hydrogen generation.

Hydrogen-Generating Silica Material Prevents UVA-ray-Induced Cellular Oxidative Stress, Cell Death, Collagen Loss and Melanogenesis in Human Cells and 3D Skin Equivalents.

Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana-Masson's staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

Heat-retention effects of hydrogen-rich water bath assessed by thermography for humans.

Hydrogen-rich water bath devices are commercially available, but have been scarcely clarified for heat-retention effects. In this study, heat-retention effects of hydrogen-rich water bath were assessed by thermographic clinical trials, which employed twenty-four healthy subjects. The thermograms indicated that, under the same conditions (41 °C, 10-min bathing), hydrogen-rich water bath (hydrogen concentrations: 185-548 µg/L; oxidation-reduction potentials: -167 to -91 mV, versus 0.8 µg/L and +479 mV for normal bath, respectively) brought about the heat-retention being more marked than those of normal water bath for several body-parts in the order as follows: abdomen > upper legs > arms > hands > feet, for 30- and 60-min post-bathing, being in contrast to scarce heat-retention for head, armpits and lower legs. Then, as reflection to promotive effects on blood stream, we also examined the thickness of fingertip-capillary in hands. The thickness was expanded in the hydrogen-rich water bath more markedly than that in the normal water bath, suggesting that the hydrogen-rich water bath may have the hydrogen-based promotive effect, exceeding over mere heat retention-based effects, on blood circulation of the whole body. Meanwhile, the heat-retention in hydrogen-rich water bath weakly or moderately correlated with contents of the subcutaneous fat, whole body fat and body mass index, and inversely correlated with skeletal muscle rates, although their correlation degrees did not obviously exceed over normal water bath, with a poor relation with the basal metabolism rate. Thus, the hydrogen-rich water bath was suggested to exert heat-retention effects exceeding over normal water bath, in diverse body-parts such as abdomen, upper legs, arms and hands, via promotion to blood flow which was reflected by expanding the thickness of capillary. The heat-retention after bathing can be noted as effects of the hydrogen-rich water bath, which is applicable for most of people widespread regardless of their body composition index.

Carcinostatic effects of alkanoyl ascorbate plus platinum nano-colloid and stabilization of the esterolytically resultant ascorbate by hydrogen.

Carcinostatic effects of combined use of ascorbic acid (Asc), 2-O-phospho- or 6-O-palmitoyl ascorbate (Asc2Phos, Asc6Palm) or diverse alkanoyl Asc, and nano-sized platinum-poly(N-vinyl-pyrrolidone) colloid (PVP-Pt; 2-nm diameter) were examined on human esophagus carcinoma-derived cells KYSE70. Cell viability was repressed by 'Asc6Palm + PVP-Pt' mixture more markedly than by Asc6Palm or PVP-Pt alone, together with cell shrinkage and fragmentation, in contrast to no additive carcinostatic effect of 'Asc + PVP-Pt' or 'Asc2Phos + PVP-Pt'. The effects might be partly due to efficiency for intracellular uptake of PVP-Pt, as previously shown by our studies that Pt atoms composed of PVP-Pt were incorporated into human tongue carcinoma cells by 9.6-fold compared to normal human tongue epitheliocytes. Asc6Palm was advantageous for intracellular uptake, in terms of the proper balance for molecular hydrophilicity-lipophilicity (BMHL), whereas 6-O-stearoyl (C18) Asc or 2,6-O-dipalmitoyl (2 × C16) was demonstrated to be less carcinostatic owing to a lower BMHL. Although esterolytically converted from Asc6Palm, Asc was necessitated to be retained for efficient carcinostasis, and demonstrated by HPLC-coulometric ECD analysis to be appreciably stabilized in electrolytically generated hydrogen (dissolved hydrogen: 0.575 mg/L)-water, but scarcely in hydrogen-gas-bubbled water (0.427 mg/L), Mg stick-derived hydrogen (0.044 mg/L) water, or tap water, suggesting that hydrogen-rich water suppresses oxidative decomposition of Asc. Thus, Asc6Palm plus PVP-Pt with hydrogen-rich water supplement might be applicable for carcinostatic therapy.

Effects of hydrogen-rich water prepared by alternating-current-electrolysis on antioxidant activity, DNA oxidative injuries, and diabetes-related markers.

Hydrogen-rich water is conventionally prepared by direct current-electrolysis, but has been not or scarcely prepared by alternating current (AC)-electrolysis. The AC preparations from tap water for 20-30 minutes exhibit a dissolved hydrogen concentration of 1.55 mg/L, which was close to the theoretical maximum value of 1.6 mg/L. These preparations also displayed an oxidation-reduction potential of -270 mV (tap water: +576 mV) and pH of 7.7-7.8, being closer to physiological values of body fluids than general types of direct current-electrolytic hydrogen-rich water. We examined whether AC-electrolytic hydrogen-water is retained for hydrogen-abundance after boiling or for antioxidant abilities, and whether the oral administration of this water is clinically effective for diabetes and prevention against systemic DNA-oxidative injuries. 5,5-Dimethyl-1-pyrroline-N-oxide spin trapping and electron spin resonance revealed that the hydrogen-rich water generated by AC-electrolysis exhibited hydroxyl-radical-scavenging activities. Laser nanoparticle tracking method revealed that nanoparticle suspensions as abundant as 5.4 × 107/mL were efficiently retained (up to 3.5 × 107/mL) even after boiling for 10 minutes, being thermodynamically contrary to Henry's law. Oral intake of hydrogen-rich water, 1500 mL per day, lasted for 8 weeks in nine people with the diabetes-related serum markers beyond the normal ranges. The subjects exhibited significant tendencies for the decreased fasting blood glucose and fructosamine, and for the increased 1,5-anhydro-D-glucitol, concomitantly with significant decreases in urinary 8-hydroxy-2-deoxyguanosine contents and its rate of generation. Hydrogen-rich water prepared by AC-electrolysis may be effective in improving diverse diabetes-related markers and systemic DNA oxidative injuries through the formation of abundant heat-resistant nanobubbles and the increased hydrogen concentrations. The study protocol was officially approved by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences (approval No. 01S02) on September 15, 2009.

Enzyme-digested Colla Corii Asini (E'jiao) accelerates wound healing and prevents ultraviolet A-induced collagen synthesis decline and wrinkle formation in three-dimensional skin equivalents.

Cutaneous wound healing delay, collagen synthesis decline and wrinkle formation are the common features of skin aging. The aim of this study is to investigate repressive effects of Colla Corii Asini (CCA) (a traditional Chinese medicine which has been used for anti-aging) on hydrogen peroxide (300 µM, 2 h) and ultraviolet A (UVA) (3.2 mJ/cm2)-induced skin aging in vitro. To simulate the in vivo condition of CCA, CCA was digested by gastrointestinal enzymes and added to human gingival fibroblasts (HGF) and three dimensional (3D) skin equivalents at different concentrations. Cell viability assay showed that the enzyme-digested CCA (CCAD) exhibited significant preventive effects on hydrogen peroxide- and UVA-induced cell death. The in vitro scratch assay showed that CCAD was able to prevent hydrogen peroxide-induced wound healing delay in HGF cell sheets. Immunostaining and imaging analysis showed that CCAD could suppress UVA-reduced expression of type IV collagen and elastin in both HGF cells and the 3D skin equivalents. Using a tissue stretching system, wrinkles were formed on UVA-irradiated 3D skin equivalents. Without CCAD-treatment, the wrinkles on the skin were deep, whereas CCAD markedly reduced the depth of wrinkles. In conclusion, CCAD could protect skin cells from oxidative stress and UVA-induced harmful effects, accelerate wound healing, promote synthesis of collagen and elastin, and reduce wrinkles formation. CCAD might be developed as an anti-skin aging reagent in the cosmetic industry.

Hydrogen-bubbled platinum-colloid suppresses human esophagus- or tongue-carcinoma cells with intracellular platinum-uptake and the diminished normal-cell mortality.

Carcinostatic effects of combined use of hydrogen nano-bubbles (nano-H) and platinum-povidone (PVP--Pt) were examined. Hydrogen-dissolved medium was prepared by hydrogen-gas bubbling with a microporous gas-emittance-terminal into a medium in the absence or presence of PVP-Pt (nano-H, nano-H/PVP-Pt). Human esophagus-derived carcinoma cells KYSE70 were repressed for cell proliferation with nano-H/PVP-Pt more markedly than with nano-H, indicating the hydrogen-intensification for PVP-Pt-alone-carcinostasis. However, the intensified carcinostasis required co-administration of nano-H and PVP-Pt, and no intensified carcinostasis was shown in two-step separate administration of nano-H and PVP-Pt. Furthermore, hydrogen bubbling into PVP-Pt-containing medium achieved more appreciable carcinostasis than mere addition of PVP-Pt into nano-H-containing medium, indicating the potent interaction of hydrogen and PVP-Pt. The nano-H/PVP-Pt-administered human tongue-derived carcinoma cells HSC-4 were repressed for cell proliferation more markedly than pre-malignant human tongue-derived epitheliocytes DOK, concurrently with more abundant intracellular Pt-intake into HSC-4 cells than DOK as analyzed by ICP-MS. Thus, PVP-Pt is able to adsorb hydrogen nano-bubbles on Pt and applicable for cancer therapy by diminishing the side-effects to normal cells.

A New Method for Testing Filtration Efficiency of Mask Materials Under Sneeze-like Pressure.

BACKGROUND: Sneezes produce many pathogen-containing micro-droplets with high velocities of 4.5-50.0 m/s. Face masks are believed to protect people from infection by blocking those droplets. However, current filtration efficiency tests can't evaluate masks under sneeze-like pressure. The goal of this study was to establish a method to evaluate the filtration efficiency of mask materials under extreme conditions. MATERIALS AND METHODS: Efficiency of surgical masks, gauze masks, gauze, cotton, silk, linen and tissue paper on blocking micro-droplet sized starch particles (average 8.2 µm) and latex microspheres (0.75 µm) with a velocity of 44.4 m/s created by centrifugation was qualitatively analyzed by using imaging-based analysis. RESULTS: The 4 layers of silk could block 93.8% of microspheres and 88.9% of starch particles, followed by the gauze mask (78.5% of microspheres and 90.4% of starch particles) and the 2 layers of cotton (74.6% of microspheres and 87.5-89.0% of particles). Other materials also blocked 53.2-66.5% of microspheres and 76.4%-87.9% of particles except the 8 layers of gauze which only blocked 36.7% of particles. The filtration efficiency was improved by the increased layers of materials. CONCLUSION: Centrifugation-based filtration efficiency test not only compensates shortcomings of current tests for masks, but also offers a simple way to explore new mask materials during pandemics. Common mask materials can potentially provide protection against respiratory droplet transmission.

Dependencies of hydrogen-water on mineral-based hardness, temperatures and the container materials, and effects of the oral washing and drinking.

Widely distributed electrolytic-generators for hydrogen-water are not fully considered for the dependencies of post-electrolytic values of the dissolved hydrogen concentration (DH) and the oxidation-reduction potential (ORP) on the properties of the pre-electrolytic water. We investigated the dependencies of DH and ORP on mineral-based hardness, temperatures and the container materials, and effects on the oral cavity by oral washing or drinking. Along with an increase in mineral-based water-hardness, DH decreased from 960 to 870 µg/L and the ORP unexpectedly increased from -460 to -320 mV. Purified water of almost zero hardness, however, caused a post-electrolytic DH as low as 80 µg/L and an ORP as high as +20 mV. Post-electrolytic DHs were not significantly changed (780-900 µg/L) upon electrolysis at 1.5-30°C and decreased at 40-50°C. The diffusion of hydrogen from the inside to the outside of the container was extremely small even after 12 hours for an aluminum- or stainless steel-made container, but not for containers made of diverse plastics. The ORP of the intact saliva was +136 mV, and decreased to +90 mV at 20 minutes after 1-minute oral-cramming of hydrogen-water, but returned to +135 mV after 60-minute leaving, showing a transient ORP-decrease in the saliva. Drinking-pause for 4 weeks after drinking hydrogen-water, however, saliva ORP, gradually but not instantly, increased to +60 to +80 mV, but upon drinking-resumption and 2 weeks thereafter, decreased again to -100 to -110 mV, suggesting that several-week hydrogen-water drinking caused a certain decrease in the saliva ORP. Thus, the present study provided the appropriate conditions such as hardness and temperatures for hydrogen-water production by the electrolytic generator, and the container materials suitable for hydrogen-water preservation. Furthermore, we clarified ORP changes of human saliva, being an indicator for human oxidative stress. The study was approved by the Medical Ethics Committee of the NPO (Non-Profitable Organization)-Corporate Japanese Center for Anti-Aging Medical Sciences (approval No. 09S02) on May 2, 2012.

Enzyme-digested Colla Corii Asini (E'jiao) prevents hydrogen peroxide-induced cell death and accelerates amyloid beta clearance in neuronal-like PC12 cells.

As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aß), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aß removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H2O2)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H2O2-treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H2O2-induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aß1-42-treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aß clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease.

ACE2: The key Molecule for Understanding the Pathophysiology of Severe and Critical Conditions of COVID-19: Demon or Angel?

Recently, the SARS-CoV-2 induced disease COVID-19 has spread all over the world. Nearly 20% of the patients have severe or critical conditions. SARS-CoV-2 exploits ACE2 for host cell entry. ACE2 plays an essential role in the renin-angiotensin-aldosterone system (RAAS), which regulates blood pressure and fluid balance. ACE2 also protects organs from inflammatory injuries and regulates intestinal functions. ACE2 can be shed by two proteases, ADAM17 and TMPRSS2. TMPRSS2-cleaved ACE2 allows SARS-CoV-2 cell entry, whereas ADAM17-cleaved ACE2 offers protection to organs. SARS-CoV-2 infection-caused ACE2 dysfunction worsens COVID-19 and could initiate multi-organ failure. Here, we will explain the role of ACE2 in the pathogenesis of severe and critical conditions of COVID-19 and discuss auspicious strategies for controlling the disease.

Effects of hydrogen-rich water bath on visceral fat and skin blotch, with boiling-resistant hydrogen bubbles.

Hydrogen-dissolved water has been shown to improve diverse oxidation stress-related diseases, which drove us to examine effects of hydrogen-rich water on oxidation stress-related skin troubles and lipid-metabolism markers. The purpose of this study is whether the dissolved hydrogen in hydrogen-rich water was kept even after boiling, and whether hydrogen-bath utilization improves cosmetic effects such as skin-blotch repression and the visceral-fat-based slimming effects. The subjects were two men and two women, aged 48, 43, 42, and 41 years (n = 4). They took warm (41°C) water bath of dissolved hydrogen 300-310 µg/L (< 10 µg/L for normal water) for 10-minute once daily for 1-6 months, followed by examination of skin blotch, visceral fat, and cholesterol and glucose metabolisms. The dissolved hydrogen concentration was measured after 15-minute boiling and the subsequent cooling naturally. The wide-ranging, dense, and irregularly shaped skin blotches became markedly smaller and thinner, assumedly through reductive bleaching of melanin and lipofuscin and promotion of dermal cell renewal by the hydrogen-rich warm water. Ultrasonic resonance-based analysis on the abdominal cross-section revealed that the visceral fat area decreased from 47 to 36 cm[2], and the abdominal circumference decreased from 91 to 82 cm, in the two female subjects bathing in hydrogen-water. After 6-month hydrogen-water bathing, the low-density lipoprotein cholesterol level was decreased by 16.2% and the fasting blood glucose level increased by 13.6% in the blood of a female subject. Before boiling, the dissolved hydrogen and an oxidation-reduced potential were 300 µg/L and -115 mV, respectively. Dissolved hydrogen was retained at 300-175 µg/L and 200 µg/L, even 1-6 hours and 24 hours, respectively, after boiling. Therefore, a hydrogen-rich water-bath apparatus can electrolytically generate abundant boiling-resistant hydrogen bubbles, improving visceral fat and blotches on the skin. The study was approved by the Medical Ethics Committee of the Japanese Center for Anti-Aging Medical Sciences and that was officially authenticated by the Hiroshima Prefecture Government of Japan (approval number 15C1) in 2016.

Effects of hydrogen-occluding-silica microparticles on wound repair and cell migratory behavior of normal human esophageal epitheliocytes.

Many conventional studies on molecular hydrogen have not examined cell migration ability and the relationship between apoptosis and the cytoskeleton. Here we investigated the influence of hydrogen-occluding silica microparticles (H2-silica) on cell migration motility and changes of the cytoskeleton (F-actin) in normal human esophageal epithelial cells (HEEpiCs). As the results, cell migration was promoted, and formation of microvilli was activated in the 100 ppm (low concentration) scratched group. After performing a wound healing assay, cells exhibited migration after 48 hours and 72 hours for both 10 ppm and 100 ppm groups, suggesting that the wound-repairing effects could be attributed to the antioxidant ability of H2-silica. In scratched groups, high levels of activated caspase-3 were relatively expressed and presented a tendency to increase the observed Bax/Bcl-2 ratio at more than 300 ppm groups. The above-mentioned results show that H2-silica induced apoptosis in HEEpiCs, especially in the scratched cells. Toxicity may cause an exaggerated apoptosis. Furthermore, since the ratio of fascin/tubulin in the 100, 300, and 600 ppm groups tended to increase in both the scratched and the non-scratched control groups, H2-silica was thought to be able to promote fascin action on normal cells and may be have a proliferative effect.

Electrolytically generated hydrogen warm water cleanses the keratin-plug-clogged hair-pores and promotes the capillary blood-streams, more markedly than normal warm water does.

Biomedical properties of hydrogen water have been extensively investigated, but the effect of hydrogen on good healthy subjects remains unclear. This study was designed to explore the hygiene improvement by electrolytically generated hydrogen warm water (40°C) on capillary blood streams, skin moisture, and keratin plugs in skin pores in normal good healthy subjects with their informed consents. Fingertip-capillary blood stream was estimated after hand-immersing in hydrogen warm water by videography using a CCD-based microscope, and the blood flow levels increased to about 120% versus normal warm water, after 60 minutes of the hand-immersing termination. Skin moisture of subjects was assessed using an electro-conductivity-based skin moisture meter. Immediately after taking a bath filled with hydrogen warm water, the skin moisture increased by 5-10% as compared to before bathing, which was kept on for the 7-day test, but indistinct, because of lower solubility of hydrogen in "warm" water than in room-temperature water. Cleansing of keratin plugs in skin-pores was assessed by stereoscopic microscopy and scanning electron microscopy. After hydrogen warm water bathing, the numbers of cleansed keratin plugs also increased on cheek of subjects 2.30- to 4.47-fold as many as the control for normal warm water. And areas of cleansed keratin plugs in the cheeks increased about 1.3-fold as much as the control. More marked improvements were observed on cheeks than on nostrils. Hydrogen warm water may thoroughly cleanse even keratin-plugs of residual amounts that could not be cleansed by normal warm water, through its permeability into wide-ranged portions of hair-pores, and promote the fingertip blood streams more markedly than merely through warmness due to normal warm water.

Radiosensitization by Liposome-Encapsulated Fullerenes to Mitochondria/DNA-Damages on Human Melanoma Cells.

The polyhydroxy small-gap fullerenes [C120O30(OH)30 · 30H2O · 25Na+: SGFs] were encapsulated in multilamellar liposomes (Lpsm) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), which are designated as LpsmSGFs (DOPC/DOPS/SGFs = 35 mM:15 mM:246-445 µM, diameter = 141.2 nm, ζ-potential = -35.65 mV). Radiosensitization by LpsmSGFs under X-ray irradiation was evaluated on human melanoma HMV-II cells. On 7th day after X-ray irradiation, cell proliferation degree assessed by WST-8 decreased more markedly on cells pretreated with LpsmSGFs than Lpsm or free-SGFs. Fluorescent imaging of cells with Rhodamine123, dihydroethidium or anti-8-hydroxydeoxyguanosine antibody was monitored as an indicator for mitochondrial membrane potentials, intracellular superoxide anion radicals (O˙-2) or oxidative DNA-damages, respectively. After X-ray irradiation, LpsmSGFs obviously exhibited more augmented mitochondrial membrane potentials on perinuclear region of cells than Lpsm or free-SGFs. Without X-ray irradiation, superoxide anion radicals were found principally in the cytoplasm, but, when exposed to X-ray, they were found in cell nuclei associated with oxidative DNA-damages on cells pretreated with LpsmSGFs. Meanwhile, the oxidation-reduction potentials of SGFs aqueous solution increased by X-ray irradiation. These results suggest that LpsmSGFs-mediated generation of reactive oxygen species results in damages to cellular components such as mitochondria and DNA on cells, and thereby cell proliferation decreased. The LpsmSGFs has a potential as a pro-oxidative type radiosensitizer.

Influence of hydrogen-occluding-silica on migration and apoptosis in human esophageal cells in vitro.

In the last decade, many studies have shown that hydrogen gas or hydrogen water can reduce the levels of reactive oxygen species in the living body. Molecular hydrogen has antioxidant and antiapoptotic effects and a preventive effect on oxidative stress-induced cell death. In the present study, we investigated solidified hydrogen-occluding-silica (H2-silica) that can release molecular hydrogen into cell culture medium because the use of hydrogen gas has strict handling limitations in hospital and medical facilities and laboratories, owing to its physicochemical characteristics. Human esophageal squamous cell carcinoma (KYSE-70) cells and normal human esophageal epithelial cells (HEEpiCs) were used to investigate the effects of H2-silica on cell viability and proliferation. Cell migration was examined with wound healing and culture-insert migration assays. The intracellular levels of reactive oxygen species were evaluated with a nitroblue tetrazolium assay. To assess the apoptotic status of the cells, the Bax/Bcl-2 ratio and cleaved caspase-3 were analyzed by western blot. The results showed that KYSE-70 cells and HEEpiCs were generally inhibited by H2-silica administration, and there was a significant proliferation-inhibitory effect in an H2-silica concentration-dependent manner compared with the control group (P < 0.05) in KYSE-70. Apoptosis-inducing effect on KYSE-70 cells was observed in 10, 300, 600, and 1,200 ppm H2-silica, and only 1,200 ppm H2-silica caused a 2.4-fold increase in apoptosis in HEEpiCs compared with the control group as the index of Bax/Bcl-2. H2 silica inhibited cell migration in KYSE-70 cells, and high concentrations had a cytotoxic effect on normal cells. These findings should provide insights into the mechanism of inhibition of H2-silica on human cancer cells in vitro.