Inhibiting Neutrophil Extracellular Traps Protects against Ultraviolet B-Induced Skin Damage: Effects of Hochu-ekki-to and DNase I.

UV-B radiation induces sunburn, and neutrophils are pivotal in this inflammation. In this study, we examined the potential involvement of neutrophil extracellular traps (NETs) in ultraviolet B (UVB)-induced skin inflammation, correlating the skin inflammation-mitigating effects of Hochu-ekki-to on UV-B irradiation and NETs. To elucidate NET distribution in the dorsal skin, male ICR mice, exposed to UVB irradiation, were immunohistologically analyzed to detect citrullinated histone H3 (citH3) and peptidylarginine deiminase 4 (PAD4). Reactive oxygen species (ROS) production in the bloodstream was analyzed. To establish the involvement of NET-released DNA in this inflammatory response, mice were UV-B irradiated following the intraperitoneal administration of DNase I. In vitro experiments were performed to scrutinize the impact of Hochu-ekki-to on A23187-induced NETs in neutrophil-like HL-60 cells. UV-B irradiation induced dorsal skin inflammation, coinciding with a significant increase in citH3 and PAD4 expression. Administration of DNase I attenuated UV-B-induced skin inflammation, whereas Hochu-ekki-to administration considerably suppressed the inflammation, correlating with diminished levels of citH3 and PAD4 in the dorsal skin. UV-B irradiation conspicuously augmented ROS and hydrogen peroxide (H2O2) production in the blood. Hochu-ekki-to significantly inhibited ROS and H2O2 generation. In vitro experiments demonstrated that Hochu-ekki-to notably inhibited A23187-induced NETs in differentiated neutrophil-like cells. Hence, NETs have been implicated in UV-B-induced skin inflammation, and their inhibition reduces cutaneous inflammation. Additionally, Hochu-ekki-to mitigated skin inflammation by impeding neutrophil infiltration and NETs in the dorsal skin of mice.

High-Dose Vitamin C Administration Inhibits the Invasion and Proliferation of Melanoma Cells in Mice Ovary.

Several studies have been conducted to explore the anticancer effects of vitamin C (VC). However, the effect of high-dose VC administration on melanoma is still unknown. Therefore, in this study, we investigated the effects of high-dose VC (4 g/kg) on the invasion and proliferation of melanoma cells in various organs of mice. B16 melanoma cells (1 × 106 cells/100 µL) were intravenously injected into the tails of female mice, and VC solution (4 g/kg) was orally administered once a day for 14 d. On the 15th day, samples from the liver, lungs, jejunum, and ovaries were collected and analyzed for invasion and proliferation of melanoma cells. Oral VC administration decreased the number of dihydroxyphenylalanine (DOPA)-positive cells and gp100-positive melanoma cells in the ovaries and suppressed the invasion and proliferation of melanoma. Compared to melanoma-administered mice, macrophage inflammatory protein-2 levels and number of neutrophils were increased in the VC + melanoma-administered mice. Furthermore, the concentrations of VC, iron, and hydrogen peroxide, and the number of terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells were significantly increased in the ovaries of VC + melanoma-administered mice compared to those of melanoma-administered mice. These results suggest that VC can reduce the invasion and proliferation of melanoma cells in the ovaries, and neutrophils in the ovaries play an important role in achieving this melanoma-suppressive effect.

The Preventive Effect of Coffee Compounds on Dermatitis and Epidermal Pigmentation after Ultraviolet Irradiation in Mice.

BACKGROUND: Ultraviolet (UV) irradiation is well known to promote inflammation and pigmentation of skin. UVB mainly affects dermatitis and pigmentation. Coffee contains a number of polyphenols, such as caffeic acid (CA) and chlorogenic acid (CGA) but their in vivo bioactivity for photobiology remains unclear. METHODS: C57BL/6j male mice were irradiated with UVB (1.0 kJ/m2/day) for 3 days. Five days after the final session of UVB irradiation, the dorsal skin, ear epidermis, and blood samples were analyzed to investigate the inflammatory factors, melanogenesis factors and related hormones. RESULTS: After the oral administration of CA (100 mg/day) or CGA (100 mg/day) for 8 days, only CA was found to inhibit dermatitis and pigmentation. The pathway by which CA inhibits dermatitis is related to the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2/cAMP response element binding protein (CREB) pathway. Otherwise, the pathway by which CA inhibits pigmentation is related to the activation of the ß-endorphin-µ-opioid receptor and suppresses the cAMP-microphthalmia-associated transcription factor (MITF) pathway. CONCLUSION: It is suggested that the oral administration of CA prevented dermatitis and pigmentation after UVB irradiation in mice.

Role of adrenocorticotropic hormone in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: A mild restraint stressor suppressed an increase in the levels of Th2-dependent cytokines and IgE, thereby reducing the symptoms of pollinosis. In the present study, to clarify the mechanism of action of adrenocorticotropic hormone (ACTH) in improving the symptoms of pollinosis, we studied the effects of ACTH on the plasma level of histamine, mast cell number in nasal-associated lymphoid tissue (NALT) and the T cell differentiation in splenocytes. METHODS: The role of ACTH in the development of pollen antigen-induced pollinosis was studied in mice. Allergic symptoms and parameters were measured on day 17 after sensitization. To investigate the effects of ACTH on T cell differentiation, we stimulated splenocytes obtained from control mice with ACTH and CD3/CD28 in vitro, and measured the cytokine production in the culture supernatant. RESULTS: The plasma levels of IL-10, IgE and histamine and mast cell number in NALT were increased in the sensitized animals in association with a concomitant increase in the incidence of sneezing and nasal rubbing. The intraperitoneal administration of ACTH decreased the IL-10, IgE and histamine levels in the plasma and mast cell number in NALT, while increasing the IFN-γ level and suppressing the incidence of nasal rubbing. Furthermore, the production of IFN-γ increased, while the IL-4 level was suppressed after 2 days in culture. CONCLUSIONS: The present findings showed that ACTH directly affects T cell differentiation and promotes Th1-type reactions. The regulation of the Th1/Th2 balance by ACTH may result in a decrease in the pathological features of pollinosis.

Role of the hypothalamo-pituitary-adrenal axis in the modulation of pollinosis induced by pollen antigens.

BACKGROUND: To clarify the mechanism of stress-induced modification of allergic diseases, we studied the effect of restraint stress on plasma levels of cytokines and the symptoms of pollinosis in mice. METHODS: The effects of restraint stress and the role of the hypothalamo-pituitary-adrenal axis (HPA-axis) in the development of pollen antigen-induced pollinosis were studied in control, hypophysectomized, adrenalectomized or ACTH-administered mice. Twenty days after sensitization, animals were subjected to mild restraint stress for 3 hours, and plasma levels of IFN-gamma, IL-10, and IgE were measured. We analyzed the incidence of sneezing and nasal rubbing in the sensitized animals. RESULTS: Plasma levels of IL-10 and IgE increased in the sensitized animals with a concomitant increase in the incidence of sneezing and nasal rubbing. The increases in plasma IgE, IL-10 and the incidence of sneezing and nasal rubbing were suppressed by restraint stress. Adrenalectomy increased IFN-gamma, inhibited the increase in plasma IL-10 and IgE, and suppressed the incidence of sneezing. In contrast, hypophysectomy increased plasma levels of IL-10, IFN-gamma, and IgE and the incidence of sneezing. Intraperitoneal administration of ACTH decreased IL-10 in plasma but increased IFN-gamma and suppressed the incidence of nasal rubbing. CONCLUSIONS: The present findings show that the HPA-axis and ACTH play important roles in the regulation of plasma cytokines and IgE thereby modulating symptoms of pollinosis. The results also suggest that a mild restraint stress suppresses the increase in Th2-dependent cytokines and IgE to reduce the symptoms of pollinosis.

Acupuncture enhances generation of nitric oxide and increases local circulation.

Although it is widely used, the mechanisms and effects of acupuncture on pain are not completely understood. Recently, increased nitric oxide (NO) synthase activity has been found in meridians and acupoints. Because NO is a key regulator of local circulation, and because change in circulation can affect the development and persistence of pain, we propose that acupuncture might regulate NO levels. We studied the effects of acupuncture on local NO levels and circulation in a randomized, double-blind, crossover study with 20 volunteers, each of whom underwent one session each of real and noninvasive sham acupuncture in a single hand and forearm with a 1-wk interval between treatments. NO concentration in the plasma from the acupunctured arm was significantly increased by 2.8 +/- 1.5 micromol/L at 5 min and 2.5 +/- 1.4 micromol/L at 60 min after acupuncture. Blood flow in palmar subcutaneous tissue of the acupunctured arm also increased, and this correlated with the NO increase. These changes were not observed in noninvasive sham-acupunctured hands and forearms. In conclusion, acupuncture increases the NO level in treated regions and thereby increases local circulation. These regulatory effects might contribute to pain relief provided by acupuncture.

Stimulation of membrane permeability transition by alpha-lipoic acid and its biochemical characteristics.

Mitochondria play an important role in apoptosis by generating reactive oxygen species (ROS) and inducing membrane permeability transition (MPT). Recent studies on alpha-lipoic acid (LA) and its reduced form, dihydrolipoic acid, suggest that these agents (LAs) inhibit apoptosis of cells by means of their antioxidant activity. On the other hand, LAs also stimulate Ca2+-dependent mitochondrial MPT and induce apoptosis of certain cells. Thus, the role of LAs in apoptotic cell death remains obscure. We investigated the mechanism of LA-induced MPT of mitochondria. Biochemical analysis revealed, in the presence of Ca2+, inorganic phosphate and succinate, LA induced uncoupling of oxidative phosphorylation, stimulated oxidation of pyridine nucleotides and enhanced Ca2+-induced MPT, as characterized by decrease in Ca2+ loading, ROS generation, oxidation of thiol groups of adenine nucleotide translocator, membrane depolarization, swelling, and cytochrome c release in an incubation time and concentration dependent manner. LA also stimulated hydroxyl radical-induced MPT in a alpha-tocopherol-inhibitable manner. Cyclosporine A, a potent inhibitor of mitochondrial MPT, inhibited all these events induced by LA. These results indicate that, under certain conditions, LA stimulates Ca2+-induced MPT through the decrease in loading capacity of Ca2+ and that MPT is involved in LA-induced apoptotic cell death. Since fairly high doses of LA have been used as a dietary supplement, the possible occurrence of such side effects, including mitochondrial dysfunction and induction of apoptosis in normal tissues, should be studied.

Effect of endocrine disruptor para-nonylphenol on the cell growth and oxygen radical generation in Escherichia coli mutant cells deficient in catalase and superoxide dismutase.

para-Nonylphenol (NP) had previously been found to have strong suppressive effects of growth of bacterial and yeast cells, and these effects were associated with NP-induced generation of radical oxygen species (ROS). In the present study, we determined that wild-type strains of Escherichia coli (CSH 7, SY-11, and IFO-3545) were resistant to NP compared with other sensitive microorganisms reported previously. To elucidate the relationship between NP-induced ROS generation and cell growth inhibition in more detail, we analyzed the effect of NP on cell growth and survival of wild-type and mutant E. coli strains deficient in ROS-scavenging enzymes such as catalase and superoxide dismutase (SOD). The SOD-deficient strain QC 774 (sod A- and sod B-) was much more sensitive to NP than wild-type (CSH 7) and catalase-deficient (UM 1 kat E- and kat G-) strains. As a comparative experiment, when hydrogen peroxide was applied to the same growth and survival assays, UM 1 cells were more sensitive to hydrogen peroxide than QC 774 and CSH 7. A chemiluminescence (CHL) experiment using MCLA (2-methyl-6-Lf-methylphenyl]-3,7-dihydroimidazc [1,2-alpha] pyrazin-3-one) reflecting predominantly superoxide generation showed that NP caused marked CHL generation in QC 774 cells, but not in CSH 7 and UM 1 cells. However, the CHL experiment using L-012 reflecting predominantly hydroxyl radical and hypochlorite did not exhibit significant CHL generation in QC 774 cells at the same concentrations of NP. Furthermore, supplementation with SOD prevented NP-induced ROS generation and cell survival inhibition of QC 774 cells, but the catalase and metal-chelating agent deferoxamine did not have significant effects. These results suggest that one of the primary actions of NP in cells is the generation of superoxide which may be responsible for NP-induced cell growth inhibition.

Ultraviolet B irradiation of the eye activates a nitric oxide-dependent hypothalamopituitary proopiomelanocortin pathway and modulates functions of alpha-melanocyte-stimulating hormone-responsive cells.

Ultraviolet B radiation increases DOPA-positive melanocytes in the skin specifically at the site of exposure. We found unexpectedly that ultraviolet B irradiation of the eye increased the concentration of alpha-melanocyte-stimulating hormone in plasma and systemically stimulated epidermal melanocytes in mice. To test the possible involvement of hypothalamopituitary proopiomelanocortin system in the systemic activation of skin melanocytes, ultraviolet B was also irradiated to the eye after hypophysectomy. Hypophysectomy strongly inhibited the ultraviolet B-induced stimulation of melanocytes. To elucidate the pathway by which ultraviolet B irradiation of the eye activated the hypothalamopituitary system, we examined the effect of bilateral ciliary ganglionectomy and denervation of the optic nerves on the ultraviolet B-induced melanocyte stimulation. Ciliary ganglionectomy, but not optic nerve denervation, strongly inhibited melanocyte stimulation by localized irradiation of the eye. Furthermore, melanocyte stimulation by localized ultraviolet B irradiation of the eye was not observed in mice that lack the inducible type of nitric oxide synthase. These results clearly indicate that a signal evoked by ultraviolet B irradiation of the eye is transmitted in a nitric oxide-dependent manner through the ciliary ganglia involving the first branch of the trigeminal nerve to the hypothalamopituitary proopiomelanocortin system, resulting in upregulation of alpha-melanocyte-stimulating hormone secretion and consequent stimulation of melanocytes in the skin. The novel network involving the trigeminal nerve and nitric oxide-dependent signaling pathway might play important parts in the activation of proopiomelanocortin-dependent biologic reactions, such as alpha-melanocyte-stimulating hormone-induced stimulation of melanocytes in the skin, in ultraviolet B-enriched environments.

Antioxidant protection of propofol and its recycling in erythrocyte membranes.

alpha-Tocopherol is a potent antioxidant that effectively protects biological membranes against oxidative injury through coordination with ascorbic acid. Because propofol has a phenolic structure similar to that of alpha-tocopherol, this intravenous anesthetic may also have similar antioxidant activity. To test this hypothesis, the effect of propofol on oxidative injury of human erythrocytes was examined. Propofol inhibited oxidative hemolysis and cis-parinaric acid oxidation in erythrocyte membranes (ED(50) = 6 microM). Although ascorbic acid alone has no appreciable effect, the protective effect of propofol was enhanced by ascorbic acid. An electron spin resonance (ESR) study showed that propofol-derived radicals (g = 2.005) were continuously generated during the oxidation of erythrocyte membranes by an ascorbic acid-inhibitable mechanism. These and other results suggest that propofol interacts with ascorbic acid, thereby exhibiting potent antioxidant activity in and around membranes as does alpha-tocopherol. Kinetic analysis revealed that propofol increased the membrane fluidity of erythrocytes, thereby increasing their resistance to physical and hemodynamic stress. Further, a greater preservation of red blood cell counts was seen after surgery with propofol compared with conventional sevoflurane anesthesia. Thus, propofol may protect erythrocytes against both oxidative and physical stress, indicating its potential as an efficient and safe antioxidant.