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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., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

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 (C 18 ) Asc or 2,6-O-dipalmitoyl (2 × C 16 ) 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.

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/cm 2 )-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.

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.

The aim of the present study is to investigate protective effects of hydrogen-rich water (HW) against reactive oxygen species (ROS)-induced cellular harmful events and cell death in human gingival fibroblasts (HGF) and three-dimensional (3D-) gingival tissue equivalents. HW was prepared with a magnesium stick in 600-mL double distilled water (DDW) overnight. Dissolved hydrogen was about 1460 ± 50 μg/L versus approximately 1600 μg/L for the saturated hydrogen. Under cell-free conditions, HW, dose-dependently, significantly scavenged peroxyl radicals (ROO·) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). Extract from HW-treated HGF cells scavenged ROO· more markedly than that from DDW-treated cells, suggesting that HW can increase the intracellular antioxidant capacity. Hydrogen peroxide dose-dependently increased the intracellular ROS generation, which was significantly repressed by HW, both in the cytoplasm and nuclei. LIVE/DEAD staining and our original cell viability dye-extraction assay showed that HW significantly protected HGF cells from hydrogen peroxide-induced cell death. Hydrogen peroxide also diminished the contents of intracellular glutathione, which were appreciably relieved by HW-pretreatment. Additionally, HW noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-gingival equivalents. The in vitro scratch assay showed that HW was able to diminish physical injury-induced ROS generation and promote wound healing in HGF cell monolayer sheets. In summary, HW was able to increase intracellular antioxidative capacity and to protect cells and tissue from oxidative damage. Thus, HW might be used for prevention/treatment of oxidative stress-related diseases.

Neuronal cells as many as 40-70% in several regions of the brain are destined to suffer from programmed cell death at the terminal stage of the ontogenesis. At the stage the mouse brain secretes some species of cell growth-inhibitory factors including a novel glycoprotein (62KD, pI9.1) which inhibits DNA synthesis and proliferation of tumor cells preferentially over those of normal cells. The factor was designated as NBCF (neonatal brain-derived carcinostatic factor) because it is not produced at stages other than neonatal and prenatal stages or from other organs. The activity is exhibited through a protenic body of NBCF as an active principle, whereas the glycan moiety contributes assumedly to secretion and/or activity stabilization. NBCF is cytocidal more markedly to neuroblastoma cells than a diversity of other tumor cells; the susceptibility to NBCF becomes marked when neuroblastoma cells are undifferentiated.