Enhanced Nrf2 up-regulation by extracellular basic pH in a human skin equivalent system.

Extracellular basic pH regulates cellular processes in wounds, and consequently influenced wound healing. Oxidative defence system modulation in the skin helps heal wounds, inhibits skin ageing and improves the skin condition. Moreover, the role of keratinocyte growth factor (KGF) and nuclear factor erythroid 2-related factor 2 (Nrf2) in antioxidant systems has been reported in various skin models. However, the effects of extracellular basic pH on wound- or skin ageing-related skin damage have not been examined. Thus, we investigated the antioxidant systems affected by extracellular basic pH in a 3D human skin equivalent system (3HSE). Extracellular basic pH decreased KGF expression and enhanced the oxidative defence system, and thus activated Nrf2 in the 3HSE. Additionally, extracellular basic pH and KGF treatment up-regulated Nrf2 activation and its regulation of the oxidative defence system in the 3HSE. This indicates that Nrf2 up-regulation is enhanced by reactive oxygen species production, rather than KGF, and by extracellular basic pH of the skin. The inhibition of skin damage through pH imbalance and KGF regulation suggests that the development of pH-regulating or pH-maintaining materials may provide effective therapeutic strategies for maintaining a healthy skin.

Acceleration of Apoptosis by Extracellular Basic pH in a 3D Human Skin Equivalent System.

Previously, we have shown that extracellular basic pH plays a significant role in both the direct and indirect regulation of cellular processes in a wound; this in turn affects the wound-healing process. Several studies have demonstrated the importance of apoptosis modulation in the wound-healing process, especially in removing inflammatory cells and in inhibiting scar formation. However, the effects of extracellular basic pH on wound healing-related skin damage are yet to be examined. Therefore, we investigated the induction of accelerated apoptosis by extracellular basic pH in skin. Apoptosis-related protein levels were measured using an array kit, target protein expression levels were detected by immunostaining, lactate dehydrogenase was analyzed spectrophotometrically, and Annexin V levels were measured by fluorescence staining. Basic pH (8.40) strongly upregulated extrinsic apoptosis proteins (Fas, high temperature requirement A, and p21) and slightly upregulated intrinsic apoptosis proteins (cytochrome c, B-cell lymphoma 2 [Bcl-2], Bcl-2-associated death promoter, and Bcl-2-like protein 4) in a 3D human skin equivalent system. Moreover, basic pH (8.40) induced heat shock protein (HSP) 60 and 70. In addition, basic pH-exposed Fas- and HSP60-knockdown cells showed significantly decreased levels of apoptosis. Taken together, these results indicate that extracellular basic pH increases early-stage apoptosis through Fas/FasL via modulation of HSP60 and HSP70.

Acceleration of Collagen Breakdown by Extracellular Basic pH in Human Dermal Fibroblasts.

BACKGROUND/AIMS: Wound healing is a complex regeneration process involving the degradation and reassembly of connective tissues and skin layers. Previous studies have shown that pH plays a significant role in both the direct and indirect regulation of cellular processes in the wound, which, in turn, affect the wound healing process. However, the effects of pH on the collagen breakdown component of wound healing have yet to be investigated. Therefore, we investigated the induction of accelerated collagen breakdown by pH imbalance in the skin. METHODS: Na+/H+ exchanger and metalloproteinase (MMP)-1 were analyzed spectrophotometrically, and the expression of collagen type-I-alpha-1 (COL1A1) and mitogen-activated protein kinase (MAPK) was measured by Western blotting. RESULTS: Accelerated collagen breakdown induced by extracellular basic pH via the overproduction of reactive oxygen species (ROS) and MAPK signaling was examined in skin fibroblasts and in a three-dimensional human skin equivalent system. Basic pH (>7.50) upregulated MMP-1 and downregulated COL1A1 levels via ROS generation and MAPK signaling pathways. Acidic pH (<6.04) slightly upregulated MMP-1 and slightly downregulated COL1A1 levels via ROS generation and the p38 signaling pathway. CONCLUSION: Our results indicate that skin pH is an important effector of collagen formation in wound healing. This finding will aid in the development of new pH-targeted therapeutic strategies.

Isoliquiritigenin isolated from Glycyrrhiza uralensis protects neuronal cells against glutamate-induced mitochondrial dysfunction.

Glutamate-mediated excitotoxicity, which is associated with reactive oxygen species (ROS), is hypothesized to be a major contributor to pathological cell death in the mammalian central nervous system, and to be involved in many acute and chronic brain diseases. Here, we showed that isoliquiritigenin (ISL) isolated from Glycyrrhiza uralensis (Gu), one of the most frequently prescribed oriental herbal medicines, protected HT22 hippocampal neuronal cells from glutamate-induced oxidative stress. In addition, we clarified the molecular mechanisms by which it protects against glutamate-induced neuronal cell death. ISL reversed glutamate-induced ROS production and mitochondrial depolarization, as well as glutamate-induced changes in expression of the apoptotic regulators Bcl-2 and Bax. Pretreatment of HT22 cells with ISL suppresses the release of apoptosis-inducing factor from mitochondria into the cytosol. Taken together, our results suggest that ISL may protect against mitochondrial dysfunction by limiting glutamate-induced oxidative stress. In conclusion, our results demonstrated that ISL isolated from Gu has protective effects against glutamate-induced mitochondrial damage and hippocampal neuronal cell death. We expect ISL to be useful in the development of drugs to prevent or treat neurodegenerative diseases.

Early stage ultraviolet irradiation damage to skin collagen can be suppressed by HPA axis control via controlled CYP11B.

UV rays constitute an extremely important environmental factor known to operate adaptative mechanisms that maintain biological homeostasis in the skin, adrenal glands, and the brain. The skin is extremely vulnerable to UV rays. UV rays deform collagen, the main component of elastic fibers, decreasing its normal function, and ultimately reducing skin's elasticity. We confirmed that psychological stress occurring during the early stages of UVB-irradiation degraded collagen function by inhibiting production rather than the decomposition of collagen, thereby promoting skin aging. UV irradiation for 0-2 weeks increased the level of a stress factor, corticosterone (CORT). High-performance liquid chromatography and western blot analysis confirmed that the increase was caused by enhanced CYP11B1/2 levels during steroid synthesis in the adrenal gland. Precursor levels decreased significantly during the two weeks of UV irradiation. Skin collagen and collagen fibers reduced drastically during this time. Furthermore, the administration of osilodrostat, a USFDA-approved drug that selectively inhibits CYP11B1/2, preserved skin collagen. The mechanism underlying the reduction of CORT by osilodrostat confirmed that the amount of skin collagen could be preserved with treatment. In addition, upon suppression of the CORT receptor, the amount of collagen was controlled, and skin aging was suppressed by the hypothalamic-pituitary-adrenal axis. Therefore, this study confirmed an inverse relationship between adrenal CYP11B1/2 levels and collagen during the initial stages of UV irradiation of the skin. The findings of this study may be useful for developing new detection mechanisms for aging, following their further verification.

Proteomic biomarkers for diagnosis in acute myocardial infarction.

Acute myocardial infarction (AMI) is considered the leading cause of morbidity and mortality in many industrialized nations. AMI is defined currently by detection of a rise and/or fall of cardiac biomarkers at least above the 99th percentile of the upper limit. Early detection of AMI could conceivably provide important information for understanding the molecular functions of heart disease, and would enable more effective diagnosis and treatment of patients. However, diagnostic approaches currently in use for the evaluation of patients, associated with chest pain or other symptoms suggestive of AMI are acceptable, but they are time-consuming, high-cost and labour-intensive in most cases. Thus, much work is needed in the development of biomarkers for accurate and cost-effective diagnosis of AMI and for effective management of patients. In this article, we give an overview of proteomic biomarkers for rapid and reliable diagnosis of AMI, focusing on biochemical characteristics and their clinical applications for point-of-care of AMI. We also postulate the future directions in the pursuit of integrated multiplex assay systems for multifunctional diagnosis in AMI.

Microglial peroxiredoxin V acts as an inducible anti-inflammatory antioxidant through cooperation with redox signaling cascades.

Reactive oxygen species (ROS) actively participate in microglia-mediated pathogenesis as pro-inflammatory molecules. However, little is known about the involvement of specific antioxidants in maintaining the microglial oxidative balance. We demonstrate that microglial peroxiredoxin (Prx) 5 expression is up-regulated by lipopolysaccharide (LPS) through activation of the ROS-sensitive signaling pathway and is involved in attenuation of both microglial activation and nitric oxide (NO) generation. Unlike in stimulation of oxidative insults with paraquat and hydrogen peroxide, Prx V expression is highly sensitive to LPS-stimulation in microglia. Reduction of ROS level by treatment with either NADPH oxidase inhibitor or antioxidant ablates LPS-mediated Prx V up-regulation in BV-2 microglial cells and is closely associated with the activation of the c-jun N-terminal kinase (JNK) signaling pathway. This suggests the involvement of ROS/JNK signaling in LPS-mediated Prx V induction. Furthermore, NO induces Prx V up-regulation that is ablated by the addition of inducible nitric oxide synthase inhibitor or deleted mutation of inducible nitric oxide synthase in LPS-stimulated microglia. Therefore, these results suggest that Prx V is induced by cooperative action among the ROS, RNS, and JNK signaling cascades. Interestingly, knockdown of Prx V expression causes the acceleration of microglia activation, including augmented ROS generation and JNK-dependent NO production. In summary, we demonstrate that Prx V plays a key role in the microglial activation process through modulation of the balance between ROS/NO generation and the corresponding JNK cascade activation.

Concentration dependent dual effect of thrombin in endothelial cells via Par-1 and Pi3 Kinase.

Disruption of endothelial barrier is a critical pathophysiological factor in inflammation. Thrombin exerts a variety of cellular effects including inflammation and apoptosis through activation of the protease activated receptors (PARs). The activation of PAR-1 by thrombin is known to have a bimodal effect in endothelial cell permeability with a low concentration (pM levels) eliciting a barrier protective and a high concentration (nM levels) eliciting a barrier disruptive response. It is not known whether this PAR-1-dependent activity of thrombin is a unique phenomenon specific for the in vitro assay or it is part of a general anti-inflammatory effect of low concentrations of thrombin that may have a physiological relevance. Here, we report that low concentrations of thrombin or of PAR-1 agonist peptide induced significant anti-inflammatory activities. However, relatively high concentration of thrombin or of PAR-1 agonist peptide showed pro-inflammatory activities. By using function-blocking anti-PAR-1 antibodies and PI3 kinase inhibitor, we show that the direct anti-inflammatory effects of low concentrations of thrombin are dependent on the activation of PAR-1 and PI3 kinase. These results suggest a role for cross communication between PAR-1 activation and PI3 kinase pathway in mediating the cytoprotective effects of low concentrations of thrombin in the cytokine-stimulated endothelial cells. J. Cell. Physiol. 219: 744-751, 2009. (c) 2009 Wiley-Liss, Inc.

Efficacy on anaplastic thyroid carcinoma of valproic acid alone or in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative, or lactacystin.

The present study investigated the mechanism underlying the antitumor activity of the histone deacetylases inhibitor valproic acid (VPA), alone and in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative (HS-1200), or the proteasome inhibitor lactacystin on cultured anaplastic thyroid carcinoma KAT-18 cells. Cell viability was evaluated by trypan-blue exclusion. Western blotting determined caspase and histone deacetylase activities and expression of poly(ADP)-ribose polymerase. Induction of apoptosis was identified by Hoechst staining, DNA electrophoresis, DNA hypoploidy and cell cycle phase analysis, and measurement of mitochondrial membrane potential. Subcellular translocation of apoptosis inducing factor and caspase-activated DNase after treatment was determined by confocal microscopy following immunofluorescent staining. VPA treatment increased apoptotic death of KAT-18 cells. VPA treatment was also associated with degradation of procaspase-3, procaspase-7, and poly(ADP)-ribose polymerase; induction of histone hyperacetylation; condensation of peripheral chromatin; decreased mitochondrial membrane potential and DNA content; and decreased translocation of apoptosis inducing factor and caspase-activated DNase. VPA in combination with doxorubicin, HS-1200, or lactacystin, applied at the highest concentrations that did not induce KAT-18 cell death, efficiently induced apoptosis in KAT-18 cells. The results suggest VPA combination therapy may represent an alternative therapeutic strategy for anaplastic thyroid carcinoma.

Melatonin inhibits attention-deficit/hyperactivity disorder caused by atopic dermatitis-induced psychological stress in an NC/Nga atopic-like mouse model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with the hallmark characteristics of pruritus, psychological stress, and sleep disturbance, all possibly associated with an increased risk of attention-deficit/hyperactivity disorder (ADHD). However, the etiology of the possible association between AD and ADHD is still not well understood. 2,4-dinitrochlorobenzene or corticosterone was used to evaluate the atopic symptom and its psychologic stress in the atopic mice model. Melatonin, corticotropin-releasing hormone, corticotropin-releasing hormone receptor, urocortin, proopiomelanocortin, adrenocorticotropic hormone, corticosterone, cAMP, cAMP response element-binding protein, dopamine and noradrenaline were analyzed spectrophotometrically, and the expression of dopamine beta-hydroxylase and tyrosine hydroxylase were measured by Western blotting or immunohistochemistry. AD-related psychological stress caused an increase in the levels of dopamine beta-hydroxylase and tyrosine hydroxylase, degradation of melatonin, hyper-activity of the hypothalamic-pituitary-adrenal axis, and dysregulation of dopamine and noradrenaline levels (ADHD phenomena) in the locus coeruleus, prefrontal cortex, and striatum of the AD mouse brain. Notably, melatonin administration inhibited the development of ADHD phenomena and their-related response in the mouse model. This study demonstrated that AD-related psychological stress increased catecholamine dysfunction and accelerated the development of psychiatric comorbidities, such as ADHD.

Culture pH affects exopolysaccharide production in submerged mycelial culture of Ganoderma lucidum.

In submerged culture of Ganoderma lucidum, the pH optimum for cell growth has been shown to be lower than that for exopolysaccharides (EPS) formation. Therefore, in the present study, a two-stage pH-control strategy was employed to maximize the productions of mycelial biomass and EPS. When compared, a batch culture without pH control had a maximum concentration of EPS and endopolysaccharides, which was much lower than those with pH control. Maximum mycelial growth (12.5 g/L) and EPS production (4.7 g/L) were achieved by shifting the controlled pH from 3.0 to 6.0 after day 4. The contrast between the controlled-pH process and uncontrolled pH was marked. By using various two-stage culture processes, it was also observed that culture pH has a significant affect on the yield of product, mycelial morphology, chemical composition, and molecular weight of EPS. A detailed observation of mycelial morphology revealed that the productive morphological form for EPS production was a dispersed pellet (controlled pH shifting from 3.0 to 6.0) rather than a compact pellet with a dense core area (controlled pH 4.5) or a feather-like pellet (controlled pH shifting from 6.0 to 3.0). Three different polysaccharides were obtained from each pH conditions, and their molecular weights and chemical compositions were significantly different.

Anti-inflammatory Steroid from Phragmitis rhizoma Modulates LPS-Mediated Signaling Through Inhibition of NF-κB Pathway.

Lipopolysaccharides (LPS) strongly stimulate immune cells, and unabated activation of immune system by LPS may lead to an exacerbation of sickness and depression. In this study, stigmasta-3,5-dien-7-one (ST) was isolated from Phragmitis rhizoma as a negative regulator of LPS-induced inflammation in macrophages. ST effectively reduced nitric oxide (NO), prostaglandin E2, and pro-inflammatory cytokine levels, which were markedly raised by LPS treatment. In addition, ST blocked the nuclear factor-kappa B (NF-κB) signaling pathway via down-regulation of phospho-p38 mitogen-activated protein kinase and phosphorylation and degradation of the inhibitor of NF-κB α. To our knowledge, this is the first study showing anti-inflammatory activities of ST isolated from Phragmitis rhizoma.

A new cytotoxic guaianolide from Chrysanthemum boreale.

A new cytotoxic guaianolide was isolated from Chrysanthemum boreale Makino. The structure of guaianolide was elucidated as 8-acetoxy-4,10-dihydroxy-2,11(13)-guaiadiene-12,6-olide (1). Compound 1 exhibited cytotoxic activity (IC(50)< or =4 microg/ml) against all five human cancer cell lines tested.

Sorption of the fumigant 1,3-dichloropropene on soil.

The fumigant 1,3-dichloropropene (1,3-D) is considered a major replacement to methyl bromide, which is to be phased out of use in the United States by 2005. The main purpose of this study was to evaluate soil-water partitioning of 1,3-D in two California agricultural soils (Salinas clay loam and Arlington sandy loam). The partition coefficients (Kd and Kf) were determined by directly measuring the concentration of 1,3-D in the solid phase (Cs) and aqueous phase (Cw) after batch equilibration. In the Salinas clay loam, the Kf of cis-1,3-D in adsorption and desorption isotherms was 0.47 and 0.54, respectively, with respective values of 0.39 and 0.49 for trans-1,3-D. This slight hysteric effect suggests that a different range of forces are involved in the adsorption and desorption process. Since n was near unity in the Freundlich equation, the Freundlich isotherms can also be approximated using the liner isotherm. At 25 degrees C, the Kd of the 1,3-D isomers in both soils ranged from 0.46 to 0.56, and the Koc (organic matter partition coefficient) ranged from 58 to 70. The relatively low Kd values and a Koc that falls within the range of 50-150, suggests that 1,3-D is weakly sorbed and highly mobile in these soils. Understanding the sorption behavior of 1,3-D in soil is important when developing fumigation practices to reduce the movement of 1,3-D to the air and groundwater.