Oxidative Stress and Bio-Regulation.

Reactive oxygen species (ROS) and free radicals work to maintain homeostasis in the body, but their excessive production causes damage to the organism. The human body is composed of a variety of cells totaling over 60 trillion cells. Each cell performs different functions and has a unique lifespan. The lifespan of cells is preprogrammed in their genes, and the death of cells that have reached the end of their lifespan is called apoptosis. This is contrary to necrosis, which is the premature death of cells brought about by physical or scientific forces. Each species has its own unique lifespan, which in humans is estimated to be up to 120 years. Elucidating the mechanism of the death of a single cell will lead to a better understanding of human death, and, conversely, the death of a single cell will lead to exploring the mechanisms of life. In this sense, research on active oxygen and free radicals, which are implicated in biological disorders and homeostasis, requires an understanding of both the physicochemical as well as the biochemical aspects. Based on the discussion above, it is clear to see that active oxygen and free radicals have dual functions of both injuring and facilitating homeostasis in living organisms.

The Effect of Oxidative Stress on the Human Voice.

The vocal fold vibrates in high frequency to create voice sound. The vocal fold has a sophisticated histological "layered structure" that enables such vibration. As the vibration causes fricative damage to the mucosa, excessive voicing can cause inflammation or injury to the mucosa. Chronic inflammation or repeated injury to the vocal fold occasionally induces scar formation in the mucosa, which can result in severe dysphonia, which is difficult to treat. Oxidative stress has been proven to be an important factor in aggravating the injury, which can lead to scarring. It is important to avoid excessive oxidative stress during the wound healing period. Excessive accumulation of reactive oxygen species (ROS) has been found in the injured vocal folds of rats during the early phase of wound healing. Antioxidants proved to be useful in preventing the accumulation of ROS during the period with less scar formation in the long-term results. Oxidative stress is also revealed to contribute to aging of the vocal fold, in which the mucosa becomes thin and stiff with a reduction in vibratory capacity. The aged voice can be characterized as weak and breathy. It has been confirmed that ROS gradually increases in rat vocal fold mucosa with age, which may cause further damage to the vocal fold. Antioxidants have also proved effective in avoiding aging of the vocal fold in rat models. Recently, human trials have shown significant effects of the antioxidant Twendee X for maintaining the voice of professional opera singers. In conclusion, it is suggested that oxidative stress has a great impact on the damage or deterioration of the vocal folds, and the use of antioxidants is effective for preventing damage of the vocal fold and maintaining the voice.

Role of Oxidative Stress in Sensorineural Hearing Loss.

Hearing is essential for communication, and its loss can cause a serious disruption to one's social life. Hearing loss is also recognized as a major risk factor for dementia; therefore, addressing hearing loss is a pressing global issue. Sensorineural hearing loss, the predominant type of hearing loss, is mainly due to damage to the inner ear along with a variety of pathologies including ischemia, noise, trauma, aging, and ototoxic drugs. In addition to genetic factors, oxidative stress has been identified as a common mechanism underlying several cochlear pathologies. The cochlea, which plays a major role in auditory function, requires high-energy metabolism and is, therefore, highly susceptible to oxidative stress, particularly in the mitochondria. Based on these pathological findings, the potential of antioxidants for the treatment of hearing loss has been demonstrated in several animal studies. However, results from human studies are insufficient, and future clinical trials are required. This review discusses the relationship between sensorineural hearing loss and reactive oxidative species (ROS), with particular emphasis on age-related hearing loss, noise-induced hearing loss, and ischemia-reperfusion injury. Based on these mechanisms, the current status and future perspectives of ROS-targeted therapy for sensorineural hearing loss are described.

The Potential of Twendee X® as a Safe Antioxidant Treatment for Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease characterized by systemic skin hardening, which combines Raynaud's phenomenon and other vascular disorders, skin and internal organ fibrosis, immune disorders, and a variety of other abnormalities. Symptoms vary widely among individuals, and personalized treatment is sought for each patient. Since there is no fundamental cure for SSc, it is designated as an intractable disease with patients receiving government subsidies for medical expenses in Japan. Oxidative stress (OS) has been reported to play an important role in the cause and symptoms of SSc. HOCl-induced SSc mouse models are known to exhibit skin and visceral fibrosis, vascular damage, and autoimmune-like symptoms observed in human SSc. The antioxidant combination Twendee X® (TwX) is a dietary supplement consisting of vitamins, amino acids, and CoQ10. TwX has been proven to prevent dementia in humans with mild cognitive impairment and significantly improve cognitive impairment in an Alzheimer's disease mouse model by regulating OS through a strong antioxidant capacity that cannot be achieved with a single antioxidant ingredient. We evaluated the effectiveness of TwX on various symptoms of HOCl-induced SSc mice. TwX-treated HOCl-induced SSc mice showed significantly reduced lung and skin fibrosis compared to untreated HOCl-induced SSc mice. TwX also significantly reduced highly oxidized protein products (AOPP) in serum and suppressed Col-1 gene expression and activation of B cells involved in autoimmunity. These findings suggest that TwX has the potential to be a new antioxidant treatment for SSc without side effects.

A Blended Vitamin Supplement Improves Spatial Cognitive and Short-Term Memory in Aged Mice.

Although many types of antioxidant supplements are available, the effect is greater if multiple types are taken simultaneously rather than one type. However, it is difficult to know which type and how much to take, as it is possible to take too many of some vitamins. As it is difficult for general consumers to make this choice, it is important to provide information based on scientific evidence. This study investigated the various effects of continuous administration of a blended supplement to aging mice. In 18-month-old C57BL/6 mice given a blended supplement ad libitum for 1 month, spatial cognition and short-term memory in the Morris water maze and Y-maze improved compared with the normal aged mice (spontaneous alternative ratio, normal aged mice, 49.5%, supplement-treated mice, 68.67%, p < 0.01). No significant differences in brain levels of secreted neurotrophic factors, such as nerve growth factor and brain-derived neurotrophic factor, were observed between these two groups. In treadmill durability tests before and after administration, the rate of increase in running distance after administration was significantly higher than that of the untreated group (increase rate, normal aged mice, 91.17%, supplement-treated aged mice, 111.4%, p < 0.04). However, training had no reinforcing effect, and post-mortem serum tests showed a significant decrease in aspartate aminotransferase, alanine aminotransferase, and total cholesterol values. These results suggest continuous intake of a blended supplement may improve cognitive function and suppress age-related muscle decline.

A mixed antioxidant supplement improves cognitive function, and coordination in aged mice.

Accumulation of oxidative damage increases the risk of several disorders. To prevent these diseases, people consume supplements. However, there is little evidence of the impact of supplement intake on cognitive function. Recently, frailty and sarcopenia have become serious issues, and these phenomena include a risk of mild cognitive impairment. In this study, aged mice were fed the combination supplement and cognitive and motor functions were measured. Following 1 month of treatment with the supplement, significant improvements in cognitive function and neuromuscular coordination were observed. Following 2 weeks of treadmill training, treatment with the supplement dramatically increased running distance compared to that in untreated normal aged mice. Serum indices such as triglyceride and total cholesterol were significantly decreased in the supplement-treated aged mice compared to untreated aged mice. These results indicate that the combination supplement may play a role in maintaining cognitive function, coordination ability and improving lipid metabolism.

Controlling Gut Microbiota by Twendee X® May Contribute to Dementia Prevention.

The human gut microbiota (GM) is a complex and dynamic ecosystem that hosts trillions of commensal and potentially pathogenic microorganisms. It is crucial in protecting humans from pathogens and in maintaining immune and metabolic homeostasis. Numerous studies have demonstrated that GM has a significant impact on health and disease, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disorder characterized by impaired short-term memory and cognitive deficits. Patients with AD have been reported to exhibit abnormalities in GM density and species composition. Oxidative stress (OS) has been implicated in the onset and progression of AD; however, the relationship between OS and gut microbiota in AD onset and progression is not clear. Twendee X® (TwX), an oral supplement consisting of eight active ingredients, has been shown to prevent dementia in mild cognitive impairment (MCI) in humans and substantially improve cognitive impairment in mouse models of AD. This positive effect is achieved through the potency of the combined antioxidants that regulate OS; therefore, similar results cannot be achieved by a single antioxidant ingredient. To examine the impact of long-term OS elevation, as seen in AD on the body and GM, we examined GM alterations during the initial OS elevation using a two-week OS loading rat model, and examined the effects of TwX on OS and GM. Furthermore, using a questionnaire survey and fecal samples, we analyzed the impact of TwX on healthy individuals' gut bacteria and the associated effect on their quality of life (QOL). TwX was found to increase the number of bacteria species and their diversity in GM, as well as butyrate-producing bacteria, which tend to be reduced in AD patients. Additionally, TwX improved defecation condition and QOL. The gut bacteria function as part of the homeostatic function during OS elevation, and the prophylactic administration of TwX strengthened this function. The results suggest that the preventative effect of TwX on dementia may involve the GM, in addition to the other previously demonstrated effects.

Why Does the Antioxidant Complex Twendee X® Prevent Dementia?

Alzheimer's disease (AD) is a complex neurodegenerative disease characterized by cognitive and short-term memory impairments. The disease involves multiple pathological factors such as amyloid plaque formation, mitochondrial dysfunction, and telomere shortening; however, oxidative stress and diabetes mellitus are significant risk factors. The onset of AD begins approximately 20 years before clinical symptoms manifest; therefore, treating AD after symptoms become evident is possibly too late to have a significant effect. As such, preventing AD or using an effective treatment at an early stage is important. Twendee X® (TwX) is an antioxidant formulation consisting of eight ingredients. TwX has been proven to prevent the progression to dementia in patients with mild cognitive impairment (MCI) in a multicenter, randomized, double-blind, placebo-controlled, prospective intervention trial. As well, positive data has already been obtained in several studies using AD model mice. Since both diabetes and aging are risk factors for AD, we examined the mechanisms behind the effects of TwX on AD using the spontaneous hyperglycemia model and the senescence model of aged C57BL/6 mice in this study. TwX was administered daily, and its effects on diabetes, autophagy in the brain, neurogenesis, and telomere length were examined. We observed that TwX protected the mitochondria from oxidative stress better than a single antioxidant. TwX not only lowered blood glucose levels but also suppressed brain neurogenesis and autophagy. Telomeres in TWX-treated mice were significantly longer than those in non-treated mice. There are many factors that can be implicated in the development and progression of dementia; however, multiple studies on TwX suggest that it may offer protection against dementia, not only through the effects of its antioxidants but also by targeting multiple mechanisms involved in its development and progression, such as diabetes, brain neurogenesis, telomere deficiency, and energy production.

Twendee X, a mixed antioxidant supplement, improves cognitive function, coordination, and neurotrophic factor expression in long-term vitamin E-deficient mice.

Oxidation products gradually accumulate during senescence, enhancing the risk of onset of many severe diseases. One such disease is dementia, and the number of cases of dementia, including Alzheimer's disease, has been increasing world-wide. These diseases can be prevented via attenuation of age-related physiological dysfunction; one preventive approach is the ingestion of antioxidants such as vitamin C and vitamin E. Many antioxidants are readily available commercially. Ingestion of mixed antioxidants is expected to provide further beneficial effects for human health. In this study, we used vitamin E-deficient mice as an animal model of increased oxidative stress and assessed the effects of dosing with mixed antioxidants. Administration of a commercial mixed antioxidant formula, Twendee X significantly improved cognitive function and coordination compared to untreated vitamin E-deficient animals. Furthermore, the levels of brain-derived neurotrophic factor and nerve growth factor were significantly increased in the cerebral cortex of Twendee X-dosed vitamin E-deficient mice compared to untreated animals. These results indicate that intake of a mixed antioxidant supplement may be beneficial to human health, even after oxidative stress has begun. In the next stage, it will be necessary to compare with other antioxidants and consider whether it is effective in the aged model.

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy.

BACKGROUND AIMS: Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. METHODS: ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. RESULTS: ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. CONCLUSIONS: Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.

The stent-eluting drugs sirolimus and paclitaxel suppress healing of the endothelium by induction of autophagy.

Clinical studies have indicated that the stent-eluting drugs sirolimus and paclitaxel impact restenosis; however, it is still elusive how these drugs affect the vascular endothelium at the molecular and cellular levels. The purpose of this study was to determine whether sirolimus and paclitaxel induce molecular and cellular alterations in the vascular endothelium. Endothelial regrowth was assessed in human aortic endothelial cells and rat aortic endothelium. Molecular and cellular alterations were analyzed in human aortic endothelial cells by Western blot analysis, transmission electron microscopy, and immunofluorescence staining. Green fluorescent protein-LC3 mice were used to analyze autophagic endothelium. Here, we show that sirolimus and paclitaxel differentially induce self-digesting autophagy in vascular endothelial cells with changes in expression of LC3B, p53, and Bcl-2, considerably suppressing re-endothelialization and revascularization. These results suggest that phenotypic alteration in the endothelium by sirolimus or paclitaxel might affect the rates of late stent thrombosis, myocardial infarction, and mortality.

Overexpression of immediate early gene X-1 (IEX-1) enhances gamma-radiation-induced apoptosis of human glioma cell line, U87-MG.

Malignant gliomas are usually incurable even if adjuvant therapy is delivered after neurosurgical treatment. Therefore, to enhance their radiation-induced apoptosis, it is important to detect the mechanism(s) leading to the death of malignant glioma cells. We report that apoptosis was induced in a time-dependent manner after gamma-radiation and that irradiated U87-MG cells (human glioblastoma cell line) expressed immediate early gene X-1 (IEX-1) with p53. We also document that their apoptotic sensitivity to gamma-radiation was enhanced by the overexpression of IEX-1. Our findings suggest that IEX-1 may represent a new factor for the enhancement of radiation-induced apoptosis of human glioma cells.

Clinical Benefits of Antioxidative Supplement Twendee X for Mild Cognitive Impairment: A Multicenter, Randomized, Double-Blind, and Placebo-Controlled Prospective Interventional Study.

Oxidative stress is part of the entire pathological process that underlies the development of Alzheimer's disease (AD), including the mild cognitive impairment (MCI) stage. Twendee X (TwX) is a supplement containing a strong antioxidative mix of eight antioxidants, which has been shown to have a clinical and therapeutic benefit in AD model mice. Here, we conducted a multicenter, randomized, double-blind, and placebo-controlled prospective interventional study to evaluate the efficacy of TwX in mitigating MCI. The primary outcomes were differences in Mini-Mental State Examination (MMSE) and Hasegawa Dementia Scale-revised (HDS-R) scores between baseline and six months for placebo and TwX groups. Seventy-eight subjects with MCI were randomized into placebo (n = 37) and TwX (n = 41) groups. MMSE scores at six months differed significantly between the TwX and placebo groups (p = 0.018), and HDS-R scores for the TwX group exhibited a significant improvement at six months relative to baseline (p = 0.025). The TwX group did not show any change in affective or activities of daily living scores at six months. The present study indicates that strong antioxidative supplement TwX is clinical beneficial for cognitive function in subjects with MCI.

AKT plays a pivotal role in the acquisition of resistance to 5-fluorouracil in human squamous carcinoma cells.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic agent that inhibits the growth and initiates the apoptosis of epithelial tumors, including squamous cell carcinoma of the head and neck region. However, resistance to this drug is often observed in a clinical setting. The primary mode of action of 5-FU is believed to be the inhibition of thymidylate synthase. Overexpression of the enzymes involved in thymidine synthesis has been shown in some cases to be associated with resistance. However, the detailed mechanisms of resistance of squamous cell carcinoma are not fully understood. In the present study, we examined the involvement of survival signaling pathways in the resistance of squamous carcinoma cells to 5-FU. 5-FU induced the activation of the ERK and Akt kinases in UM-SCC-23 human squamous carcinoma cells, indicating that this anticancer drug activates survival signaling pathways as well as apoptotic signals. In 5-FU-resistant UM-SCC-23 cells established by our group, ERK and Akt signals were constitutively activated. U0126 is an inhibitor of MEK, which is an upstream activator for ERK. U0126 failed to sensitize resistant UM-SCC-23 cells to 5-FU-induced apoptotic cell death. This is in sharp contrast to LY294002, which is an inhibitor of phosphatidylinositol 3-kinase, an upstream activator for Akt. LY294002 drastically enhanced 5-FU-induced apoptotic cell death in resistant UM-SCC-23 cells. These results indicate that the Akt survival signal plays an important role in the resistance of squamous carcinoma cells to 5-FU treatment, and suggest that the modification of Akt activity might provide a new strategy for human 5-FU-resistant squamous carcinoma therapy.

Prostaglandin E2 downregulates TNF-alpha-induced production of matrix metalloproteinase-1 in HCS-2/8 chondrocytes by inhibiting Raf-1/MEK/ERK cascade through EP4 prostanoid receptor activation.

Matrix metalloproteinase-1 (MMP-1, collagenase-1) plays a pivotal role in the process of joint destruction in degenerative joint diseases. We have examined the regulation of MMP-1 production in human chondrocytic HCS-2/8 cells stimulated by tumor necrosis factor-alpha (TNF-alpha). In response to TNF-alpha, MMP-1 is induced and actively released from HCS-2/8 cells. The induction of MMP-1 expression correlates with activation of ERK1/2, MEK, and Raf-1, and is potently prevented by U0126, a selective inhibitor of MEK1/2 activation. In contrast, SB203580, a selective p38 mitogen-activated protein kinases (MAPK) inhibitor, had no effects on TNF-alpha-induced MMP-1 release. A serine/threonine kinase, Akt was not activated in TNF-alpha-stimulated HCS-2/8 cells. TNF-alpha stimulated the production of PGE(2) in addition to MMP-1 in HCS-2/8 cells. Exogenously added PGE(2) potently inhibited TNF-alpha-induced both MMP-1 production and activation of ERK1/2. The effects of PGE(2) were mimicked by ONO-AE1-329, a selective EP4 receptor agonist but not by butaprost, a selective EP2 agonist. In contrast, blockade of endogenously produced PGE(2) signaling by ONO-AE3-208, a selective EP4 receptor antagonist, enhanced TNF-alpha-induced MMP-1 production. Furthermore, the suppression of MMP-1 production by exogenously added PGE(2) was reversed by ONO-AE3-208. Activation of EP4 receptor resulted in cAMP-mediated phosphorylation of Raf-1 on Ser259, a negative regulatory site, and blocked activation of Raf-1/MEK/ERK cascade. Taken together, these findings indicate that Raf-1/MEK/ERK signaling pathway plays a crucial role in the production of MMP-1 in HCS-2/8 cells in response to TNF-alpha, and that the produced PGE(2) downregulates the expression of MMP-1 by blockage of TNF-alpha-induced Raf-1 activation through EP4-PGE(2) receptor activation.

Immediate early gene IEX-1 induces astrocytic differentiation of U87-MG human glioma cells.

The immediate early response gene IEX-1 is involved in the regulation of apoptosis and cell growth. In order to increase the apoptotic sensitivity to chemotherapeutic drugs and gamma-ray, we attempted to establish U87-MG human glioma cell line expressing IEX-1. Unexpectedly, however, transfection of IEX-1 into U87-MG glioma cells resulted in morphological changes to astrocytic phenotype and increase in glial differentiation marker proteins, S-100 and glial fibrillary acidic protein (GFAP). Glial cell differentiation was used to examine in rat C6 glioma cell line, since this cell line express astrocytic phenotypes by increase in intracellular cAMP concentration. Stimulation of human U87-MG glioma cells by membrane-permeable dibutyryl cAMP (dbcAMP) not only elicited their morphological changes but also induced expression of IEX-1 as well as S-100 and GFAP. H89, an inhibitor of protein kinase A (PKA), blocked dbcAMP-induced morphological changes of U87-MG cells and expression of IEX-1. In contrast, morphological changes and expression of S-100 and GFAP induced by IEX-1 were not affected by H89. Morphological changes induced by dbcAMP were totally abolished by functional disruption of IEX-1 expression by anti-sense RNA. These results indicate that IEX-1 plays an important role in astrocytic differentiation of human glioma cells and that IEX-1 functions at downstream of PKA.