Heparinoid and phospho-pyridoxal preserve tight junction integrity in human skin under oxidative stress.

We previously discovered that squalene monohydroperoxide (SQ-OOH) was produced on human forehead skin and suggested that skin squalene (SQ) may be the principal target lipid for oxidative stress (e.g., sunlight exposure). Because of its six double bonds, SQ peroxidation can yield various positional hydroperoxide isomers. However, the structural characterization of skin SQ-OOH isomers has never been reported. Here, we prepared pure SQ-OOH isomers and developed an analytical method for SQ-OOH isomers using a quadrupole/linear ion-trap mass spectrometer (QTRAP) MS/MS system. Collision-induced dissociation produced specific fragment ions for each SQ-OOH isomer, which permitted discrimination between SQ-OOH isomers by multiple reaction monitoring (MRM). When lipid extract from human forehead skin was subjected to LC-MS/MS with MRM, individual SQ-OOH isomers could be separated and detected with a sensitivity of 0.05 ng/injection. The total concentration of SQ-OOH isomers in forehead skin was approximately 956 microg/g skin lipids, but it increased up to 2,760 microg/g skin lipids after 3 h of sunlight exposure. The LC-MS/MS method was useful for investigating the peroxidation mechanisms of SQ as well as SQ-OOH-mediated skin disorders.

The Repair Enzyme Peptide Methionine-S-Sulfoxide Reductase Is Expressed in Human Epidermis and Upregulated by UVA Radiation

Skin aging is a complex process that may be caused by factors that are intrinsic and extrinsic to the body. Ultraviolet (UV) radiation represents one of the main sources of skin damage over the years and characterizes a process known as photoaging. Among the changes that affect cutaneous tissue with age, the loss of elastic properties caused by changes in elastin production, increased degradation and/or processing produces a substantial impact on tissue esthetics and health. The occurrence of solar elastosis is one of the main markers of cutaneous photoaging and is characterized by disorganized and non-functional deposition of elastic fibers. The occurrence of UV radiation-induced alternative splicing of the elastin gene, which leads to inadequate synthesis of the proteins required for the correct assembly of elastic fibers, is a potential explanation for this phenomenon. Innovative studies have been fundamental for the elucidation of rarely explored photoaging mechanisms and have enabled the identification of effective therapeutic alternatives such as cosmetic products. This review addresses cutaneous photoaging and the changes that affect elastin in this process.

Oxidative damage prevention in human skin and sensory neurons by a salicylic acid derivative

Deletion of the WNK1 (with no lysine kinase‐1) gene in mice causes embryonic lethality due to failed vascular development. This phenotype can be rescued by endothelial‐specific expression of WNK1 or a constitutively active form of its substrate OSR1 (Oxidative Stress Responsive‐1). Additionally, depletion of WNK1/OSR1 decreased in vitro angiogenesis and cell migration in endothelial cells. This indicates an essential function of WNK1/OSR1 in angiogenesis. However, the underlying mechanism by which WNK1/OSR1 mediates this process is unclear. WNK1 is known to regulate TGF‐β/Smad signaling which induces epithelial‐mesenchymal transition (EMT), a process utilized in cell migration and angiogenic sprouting during physiological and pathophysiological vascular remodeling. OSR1 is reported to form a complex with the TGF‐βII receptor at tight junctions which mediates dissolution of tight junction upon activation by TGF‐β. Therefore, we hypothesize that WNK1/OSR1 axis is necessary for TGF‐β/Smad‐induced disruption of cell‐cell adhesion, thereby promoting cell migration and angiogenesis. We found that WNK1 pharmacological inhibition decreased the expression of TGF‐β‐induced EMT mediators such as the receptor tyrosine kinase Axl which is known to be important for maintaining tight junction integrity in HDMECs (Human Dermal Microvascular Endothelial Cells) (Normalized densitometry values; WNKi‐ Axl: 0.2 ± 0.05 vs. siControl: 1, n=3) and in primary HUVECs (Human Umbilical Vein Endothelial Cells) (normalized densitometry values; WNK1i: 0.4 ± 0.1 vs. DMSO: 1, n=3, *p<0.05). We also determined that OSR1 and Occludin, a tight junction protein are co‐localized and that inhibition of WNK1 using a small molecule WNK1 inhibitor, decreased their co‐localization and tight junction breakdown during TGF‐β signaling in primary HUVECs (Pearson’s coefficient; Control; 0.08 ± 0.2 vs. TGF‐β: 0.61 ± 0.15 vs. TGF‐β+WNK1i: 0.12 ± 0.25, n=2, 2 replicates per experiment). Finally, we observed that bands corresponding to phosphorylated form of Occludin on a Western blot were enhanced in WNK inhibited samples compared to control (normalized densitometry values; TGF‐β+WNKi: 1 vs. TGF‐β: 0.21 ± 0.07, n=2). Since Occludin is known to be dephosphorylated during tight junction breakdown, the presence of the phosphorylated form of Occludin with WNK inhibition may indicate the disruption of cellular processes leading to TGF‐β‐dependent tight junction breakdown. These data suggest WNK1‐dependent function of OSR1 in cell migration and angiogenesis through regulating tight junction integrity. Combined, these data indicate the importance of WNK1 function in endothelial cell migration and angiogenesis. Understanding how WNK1 regulates angiogenesis and cell migration is immediately relevant during development, fibrosis as well as cancer.Support or Funding InformationAmerican Heart Association postdoctoral fellowship to Ankita Jaykumar and NIH funding R01 HL147661 to Melanie H. Cobb

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

When the skin is exposed to solar irradiation, UVA photons interact with skin tissues and induce excessive reactive oxygen species, resulting in oxidative stress. We have shown in a previous study that in vivo chemiluminescence's measurement can be used to evaluate the overall level of UVA-induced oxidative stress in human skin. However, the origin of the observed chemiluminescence signals remains unclear. UVA-induced chemiluminescence measurements were conducted: (a) in vitro on collagen solutions and solid collagen sheet preparations, (b) ex vivo on human and mouse skin biopsies, and (c) in vivo on human skin of various constitutive pigmentation levels. Fluorescence was measured on collagen in vitro as well as on skin for the in vivo experiments. We found in the in vitro experiments that UVA-induced chemiluminescence increases with the presence of collagen cross-links. When dermal sides were exposed to UVA irradiation, both mouse and human skin biopsies demonstrated significantly higher chemiluminescence levels than when epidermal sides were exposed to UVA. The amount of collagen cross-links decreases slightly following UVA exposure, as shown both by in vivo fluorescence and by UVA-induced chemiluminescence. Finally, there was less measurable UVA-induced chemiluminescence in dark skin compared with light pigmented skin in vivo. The dermis is very sensitive to UVA photons. Dermal cross-links are potential UVA sensitizers. The oxidative stress induced by UVA and measured by chemiluminescence may largely be attributed to the breakdown of dermal collagen cross-links.

The Multiple Roles of Urocanic Acid in Health and Disease

Fibroblast senescence

In order to assess the effect of the different spectral solar regions on the development of free radicals in skin, in vivo electron paramagnetic resonance investigations with human volunteers and ex vivo studies on excised human and porcine skin were carried out. For all skin probes, the ultraviolet spectral region stimulates the most intensive radical formation, followed by the visible and the near infrared regions. A comparison between the different skin models shows that for both regions, the fastest and highest production of free radicals could be detected in vivo, followed by excised porcine and human skin. Additional studies indicated that the intake of vegetable extract and the topical application of formulations rich in antioxidants could reduce the oxidative stress in the skin.

Oxidative stress is a central pathogenic mechanism in a wide range of dermatological and inflammatory disorders, driving DNA damage, impaired epidermal barrier function and defective tissue repair. Although endogenous melanin provides intrinsic redox protection in skin, its therapeutic translation has been limited by poor solubility, structural heterogeneity and insufficient mechanistic validation in human tissues. To determine whether commercially available synthetic melanin is internalised by human keratinocytes and confers intracellular protection against oxidative injury, with functional and molecular validation in ex vivo human skin. Keratinocyte-based assays were integrated with ex vivo human skin explants to evaluate the effects of synthetic melanin (1-6 µM) under chemically induced oxidative stress (100 µM tert-butyl hydroperoxide). Intracellular reactive oxygen species (ROS), phosphorylated histone H2AX (γ-H2AX) associated DNA damage, keratinocyte migration, tight-junction organisation and epithelial permeability were assessed in vitro. Label-free Raman spectroscopy was employed to map oxidative biochemical perturbations and molecular recovery in human skin tissue. Synthetic melanin was efficiently internalised by human keratinocytes in a dose-dependent and well-tolerated manner, functioning as an intracellular redox modulator. Melanin pre-treatment significantly reduced intracellular ROS accumulation and attenuated oxidative DNA damage, improved keratinocyte migration, preserved tight-junction organisation and maintained epithelial barrier integrity under oxidative stress. In human skin explants, oxidative injury caused marked attenuation of Raman spectral signatures associated with proteins, lipids, and aromatic amino acids. Melanin treatment restored these biochemical features, reduced DNA damage-associated spectral bands and preserved epidermal biochemical architecture. Synthetic melanin functions as a bioinspired intracellular redox active compound that mitigates oxidative stress induced damage at cellular and tissue levels. By combining mechanistic in vitro assays with label-free molecular validation in human skin, this study provides proof-of-concept evidence supporting further translational investigation of synthetic melanin in oxidative stress associated dermatological conditions.

Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells

Background: Piperine, an alkaloid from Piper nigrum, modulates oxidative stress, proliferation, and survival pathways in several cancer models; however, its mechanistic effects in colorectal epithelial Caco-2 cells remain insufficiently defined. Objective: This study aimed to investigate the cytotoxic, antiproliferative, oxidative, autophagic, and anti-migratory effects of piperine in Caco-2 cells. Methods: Caco-2 cells were treated with piperine (0.001-0.1 mg/mL) for up to 72 h. Cell viability, proliferation, and migration were assessed using SRB and scratch assays. Oxidative stress, apoptosis, autophagy, and tight junction integrity were evaluated through ROS quantification, Western blotting, gene expression analysis, confocal microscopy, and transmission electron microscopy (TEM). NACET was used to determine the contribution of oxidative stress to piperine-induced cytotoxicity and autophagy. Results: Piperine induced a time- and dose-dependent reduction in viability, with viability decreasing to 53.0 ± 2.88% at 0.1 mg/mL after 72 h. Proliferation decreased to 51% of control levels (p < 0.001), accompanied by p21 upregulation (p < 0.05), indicating G2/M cell cycle arrest. Piperine markedly increased intracellular ROS (p < 0.001), downregulated NRF2 (p < 0.05), and suppressed GSTA1 expression (p < 0.001), while NACET co-treatment restored viability (p < 0.001). No activation of caspase-dependent apoptosis was observed. Piperine significantly enhanced autophagic flux, as shown by the increased LC3B-II/LC3B-I ratio (p < 0.01), elevated LC3B-II/LAMP-1 co-localization (p < 0.01), and chloroquine-induced accumulation of LC3B-II and p62 (p < 0.01), with preserved lysosomal function. TEM analysis confirmed a marked increase in double-membrane autophagosomes in piperine-treated cells compared with controls. NACET reduced LC3B-II/LC3B-I levels, increased p21 expression, and significantly improved cell viability, indicating that piperine-induced autophagy is cytotoxic and driven by oxidative stress. Additionally, piperine upregulated occludin (p < 0.01) and reduced cell migration independently of proliferation (p < 0.01). Conclusions: Piperine exerts antiproliferative effects in Caco-2 cells through ROS-mediated stress, p21-dependent G2/M arrest, and activation of cytotoxic autophagy. Its ability to impair migration and enhance tight junction integrity further supports its potential as a complementary therapeutic agent in colon cancer.

Mitochondrial tolerance to single and repeat exposure to simulated sunlight in human epidermal and dermal skin cells

Ethanol is metabolized into acetaldehyde in most tissues. In this study, we investigated the synergistic effect of ethanol and acetaldehyde on the tight junction integrity in Caco-2 cell monolayers. Expression of alcohol dehydrogenase sensitized Caco-2 cells to ethanol-induced tight junction disruption and barrier dysfunction, whereas aldehyde dehydrogenase attenuated acetaldehyde-induced tight junction disruption. Ethanol up to 150 mM did not affect tight junction integrity or barrier function, but it dose-dependently increased acetaldehyde-mediated tight junction disruption and barrier dysfunction. Src kinase and MLCK inhibitors blocked this synergistic effect of ethanol and acetaldehyde on tight junction. Ethanol and acetaldehyde caused a rapid and synergistic elevation of intracellular calcium. Calcium depletion by BAPTA or Ca2+-free medium blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. Diltiazem and selective knockdown of TRPV6 or CaV1.3 channels, by shRNA blocked ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. Ethanol and acetaldehyde induced a rapid and synergistic increase in reactive oxygen species by a calcium-dependent mechanism. N-acetyl-L-cysteine and cyclosporine A, blocked ethanol and acetaldehyde-induced barrier dysfunction and tight junction disruption. These results demonstrate that ethanol and acetaldehyde synergistically disrupt tight junctions by a mechanism involving calcium, oxidative stress, Src kinase and MLCK.