Nrf2 activation by hydroxytyrosol and dimethyl fumarate ameliorates skin tissue repair in high-fat diet-fed mice by promoting M2 macrophage polarization and normalizing inflammatory response and oxidative damage.

Hydroxytyrosol (HT) or dimethyl fumarate (DMF), activators of nuclear factor erythroid 2-related factor 2 (Nrf2), may reduce obesity in high-fat diet (HFD)-fed animals; nevertheless, the role of these activators on skin tissue repair of HFD-fed animals was not reported. This study investigated whether HT or DMF could improve skin wound healing of HFD-fed obese animals. Mice were fed with an HFD, treated with HT or DMF, and full-thickness skin wounds were created. Macrophages isolated from control and obese animals were treated in vitro with HT. DMF, but not HT, reduced the body weight of HFD-fed mice. Collagen deposition and wound closure were improved by HT or DMF in HFD-fed animals. HT or DMF increased anti-inflammatory macrophage phenotype and protein Nrf2 levels in wounds of HFD-fed mice. Lipid peroxidation and protein tumor necrosis factor-α levels were reduced by HT or DMF in wounds of HFD-fed animals. In in vitro, HT stimulated Nrf2 activation in mouse macrophages isolated from obese animals. In conclusion, HT or DMF improves skin wound healing of HFD-fed mice by reducing oxidative damage and inflammatory response. HT or DMF may be used as a therapeutic strategy to improve the skin healing process in individuals with obesity.

Repeated stress-induced crosstalk between the sympathetic nervous system and mast cells contributes to delayed cutaneous wound healing in mice.

The study identifies a link between the neuroimmune interaction and the impairment of wound healing induced by repeated stress. Stress increased mast cell mobilization and degranulation, levels of IL-10, and sympathetic reinnervation in mouse wounds. In contrast to mast cells, macrophage infiltration into wounds was significantly delayed in stressed mice. Chemical sympathectomy and the blockade of mast cell degranulation reversed the effect of stress on skin wound healing in vivo. In vitro, high epinephrine levels stimulated mast cell degranulation and IL-10 release. In conclusion, catecholamines released by the sympathetic nervous system stimulate mast cells to secrete anti-inflammatory cytokines that impair inflammatory cell mobilization, leading to a delay in the resolution of wound healing under stress conditions.

Microfibril-associated protein 5 and the regulation of skin scar formation.

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5's role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring.

Dual effects of extra virgin olive oil in acute wounds.

Extra virgin olive oil (EVOO) has proved beneficial effects in skin wound healing of chronic lesions; however, the effects of EVOO in acute wounds are not completely understood. This study investigated the effects of short-term and long-term administration of a diet rich in EVOO on acute wound healing. To check this, mice were fed with a diet rich in EVOO for 1 week (short term), 1 month, or 3 months (long term). The control group received a standard diet. Mouse macrophages were treated in vitro with EVOO or hydroxytyrosol (HT), which is the main EVOO polyphenol. Short-term administration of an EVOO rich diet in vivo increased lipid peroxidation and mRNA levels of pro-inflammatory cytokine levels and impaired acute wound closure. In contrast, long-term administration of an EVOO rich diet resulted in increased mRNA levels of anti-inflammatory cytokines and enhanced acute wound closure. In both in vivo and in vitro assays, the administration of EVOO or HT resulted in a predominantly anti-inflammatory macrophage phenotype. In conclusion, a diet rich in EVOO has a positive effect on acute wound healing that is dependent on the duration of EVOO administration. Short-term EVOO diet supplementation increases oxidative damage and pro-inflammatory responses, which impaired acute wound closure. On the other hand, long-term EVOO supplementation reduces oxidative damage and enhances anti-inflammatory responses, which improved acute wound closure. The effects of EVOO on oxidation and inflammation in acute wounds are linked to the EVOO polyphenol HT.

Olive oil promotes the survival and migration of dermal fibroblasts through Nrf2 pathway activation.

Olive oil has beneficial effects on skin wound healing due to its anti-inflammatory and antioxidant properties; however, the mechanism by which olive oil promotes wound healing is unclear. We evaluated the mechanisms involved in Nrf2 pathway activation by olive oil and its role in cell survival and migration in mouse dermal fibroblasts in a short-term exposition. Our data demonstrated that olive oil and oleic acid promoted reactive oxygen species (ROS) production, while olive oil and hydroxytyrosol stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) activation. Olive oil-mediated ROS production increased nuclear factor kappa B p65 expression, while olive oil-stimulated reactive nitrogen species production augmented the levels of Nrf2. Olive oil augmented cell proliferation, cell migration, and AKT phosphorylation, but decreased apoptotic cell number and cleaved caspase-3 levels. The effect of olive oil on cell migration and protein levels of AKT, BCL-2, and Nrf2 were reversed by an Nrf2 inhibitor. In conclusion, the activation of the Nrf2 pathway by olive oil promotes the survival and migration of dermal fibroblasts that are essential for the resolution of skin wound healing.

Imiquimod-induced ex vivo model of psoriatic human skin via interleukin-17A signalling of T cells and Langerhans cells.

Several in vitro models have been developed to study the mechanisms involved in psoriasis and to screen new antipsoriatic drugs. However, most of them use single-cell or reconstructed human skin models that did not have complex anatomy of human skin. Thus, this study aimed to create a new model of psoriasis-like dermatitis using human skin under in vitro conditions. To perform this, human skin explants were topically treated with imiquimod (IMQ) or vehicle for 2, 3 or 6 consecutive days. Some explants were treated with an anti-psoriatic drug or antibody anti-interleukin-17A (IL-17A). Topical application of IMQ increased total epidermal area, epidermal proliferation and keratinocyte differentiation at 3, 4 or 7 days. The protein levels of CD3 were augmented in the IMQ-treated human skin explants at 7 days reflecting the activation of T cells. Topical IMQ promoted a higher protein and mRNA levels of IL-17A in human skin ex vivo. Immunofluorescence analysis showed CD207-positive Langerhans cells (LCs) and CD3-positive T cells expressing IL-17A in IMQ-treated human skin explants at 7 days. In addition, administration of antibody anti-IL-17A or an anti-psoriatic drug inhibited IMQ-induced increase in the epidermal thickness in ex vivo human skin at 7 days. In conclusion, topical IMQ application promotes epidermal changes in ex vivo human skin that resemble to human psoriatic skin lesions. Moreover, IMQ-induced production of IL-17 by LCs and T cells is critical to development of psoriasis-like inflammation in our model. This new model is suitable for in vitro screening of antipsoriatic drugs.

Dietary olive oil intake aggravates psoriatic skin inflammation in mice via Nrf2 activation and polyunsaturated fatty acid imbalance.

Psoriasis is a chronic inflammatory skin disease, which does not have effective treatment options. However, olive oil has been suggested as an alternative to treat psoriasis, but no study has evaluated the mechanisms involved in the effects of olive oil on psoriasis. Thus, the current study investigated whether olive oil could ameliorate psoriasiform skin inflammation. To test this, mice received topical application of imiquimod to induce inflammation and were treated orally with olive oil. Human immortalized keratinocytes were also treated with imiquimod and olive oil. Epidermal thickness and keratinocyte proliferation were increased in imiquimod-induced lesions of olive-oil-treated animals. In both in vitro and in vivo studies, protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were elevated following imiquimod and olive oil administration. Inhibition of Nrf2 abolished the increased proliferation of keratinocytes treated with imiquimod and olive oil, demonstrating the role of Nrf2 in olive oil-mediated exacerbation of psoriasiform skin inflammation. In addition, lower levels of linoleic acid and higher levels of oleic acid were observed in imiquimod- and olive-oil-treated animals, which may also contribute to the adverse effects of olive oil on psoriasis. In conclusion, dietary intake of olive oil aggravates the symptoms of psoriatic skin lesions through the overexpression of Nrf2 and an imbalance in oleic and linoleic acids levels, suggesting that a diet rich in olive oil may have significant negative effects on psoriasis.

An ex vivo model of human skin photoaging induced by UVA radiation compatible with summer exposure in Brazil.

Skin is the largest body organ and can be affected by several factors, such as ultraviolet (UV) radiation. UV radiation is subdivided in UVA, UVB and UVC according to the radiation wavelength. UVC radiation does not cross the ozone layer; UVB cause DNA damage and is closely related to carcinogenesis; UVA radiation penetrates deeply into the skin, reaching epidermis and dermis and is considered the main promoter of skin aging, known as photoaging. In order to understand photoaging mechanisms and propose efficient therapies, several photoaging study models have been developed, each with benefits and limitations, but most of them use very high doses of UVA radiation, which is not compatible with our daily sun exposure. The objective of this work was to develop a human ex vivo photoaging model induced by UVA exposure compatible to a summer in Brazil. For this, human skin fragments were obtained from healthy donors who underwent otoplasty surgery and skin explants were prepared and placed in plates, with the epidermis facing upwards. Skin explants were exposed to UVA at 16 J/cm2 carried out by protocols of 2 or 4 exposures. Results showed an increase of oxidative damage, inflammatory cells, collagenolytic and elastolytic MMPs expression as well as a decrease of elastin expression, suggesting that the experimental model based on skin explants is able to evaluate UVA-induced aging in human skin.

Dermal fibroblast phagocytosis of apoptotic cells: A novel pathway for wound resolution.

The effective clearance of apoptotic cells is an essential step in the resolution of healing wounds. In particular, blood vessel regression during wound resolution produces a significant number of apoptotic endothelial cells (ApoEC) that must be cleared. In considering the fate of ApoEC and the presence of fibroblasts during wound resolution, we hypothesized that fibroblasts might serve as phagocytes involved in endothelial cell removal. The current study investigated whether dermal fibroblasts engulf ApoEC, whether this uptake alters the phenotype of dermal fibroblasts, and the biological molecules involved. In both in vitro and in vivo studies, following ApoEC engulfment, fibroblasts acquired a pro-healing phenotype (increased cell migration, contractility, α-smooth muscle actin expression, and collagen deposition). In addition, fibroblast uptake of ApoEC was shown to be mediated in part by the milk fat globule-EGF factor 8 protein/integrin αv ß5 pathway. Our study demonstrates a novel function of fibroblasts in the clearance of ApoEC and suggests that this capability has significant implications for tissue repair and fibrosis.

Dimethyl Fumarate Attenuates Lung Inflammation and Oxidative Stress Induced by Chronic Exposure to Diesel Exhaust Particles in Mice.

Air pollution is mainly caused by burning of fossil fuels, such as diesel, and is associated with increased morbidity and mortality due to adverse health effects induced by inflammation and oxidative stress. Dimethyl fumarate (DMF) is a fumaric acid ester and acts as an antioxidant and anti-inflammatory agent. We investigated the potential therapeutic effects of DMF on pulmonary damage caused by chronic exposure to diesel exhaust particles (DEPs). Mice were challenged with DEPs (30 µg per mice) by intranasal instillation for 60 consecutive days. After the first 30 days, the animals were treated daily with 30 mg/kg of DMF by gavage for the remainder of the experimental period. We demonstrated a reduction in total inflammatory cell number in the bronchoalveolar lavage (BAL) of mice subjected to DEP + DMF as compared to those exposed to DEPs alone. Importantly, DMF treatment was able to reduce lung injury caused by DEP exposure. Intracellular total reactive oxygen species (ROS), peroxynitrite (OONO), and nitric oxide (NO) levels were significantly lower in the DEP + DMF than in the DEP group. In addition, DMF treatment reduced the protein expression of kelch-like ECH-associated protein 1 (Keap-1) in lung lysates from DEP-exposed mice, whereas total nuclear factor κB (NF-κB) p65 expression was decreased below baseline in the DEP + DMF group compared to both the control and DEP groups. Lastly, DMF markedly reduced DEP-induced expression of nitrotyrosine, glutathione peroxidase-1/2 (Gpx-1/2), and catalase in mouse lungs. In summary, DMF treatment effectively reduced lung injury, inflammation, and oxidative and nitrosative stress induced by chronic DEP exposure. Consequently, it may lead to new therapies to diminish lung injury caused by air pollutants.

Pigment epithelium-derived factor attenuates angiogenesis and collagen deposition in hypertrophic scars.

Scar forming wounds are often characterized by higher levels of vascularity than non-scarring wounds and normal skin, and inhibition of angiogenesis has been shown to inhibit scar formation in some model systems. The rabbit ear hypertrophic scar (HS) model has been widely used to study the mechanisms that underlie the development of HS as well as the effectiveness of various treatments. Although the rabbit ear HS model is well characterized in terms of scar formation, the rate and level of angiogenesis has not been investigated in this model, and the cause-effect relationship between angiogenesis and rabbit HSs has not been examined. In the current study, full-thickness excisional wounds were created on the ventral side of New Zealand White rabbit ears to induce HS formation, and the dynamic pattern of angiogenesis and the expression of angiogenic regulatory factors were examined over time. Blood vessel density was found to peak at 2.7% on day 14 post-wounding, decreasing to 1.7% by day 28. mRNA levels of the proangiogenic factor VEGF-A peaked at day 14, while the expression of the antiangiogenic factors pigment epithelium-derived factor (PEDF) and thrombospondin 1 (TSP1) peaked at day 28 post-wounding. To examine whether inhibition of angiogenesis influences HS formation in this model, wounds were treated with exogenous soluble antiangiogenic agents including recombinant PEDF (rPEDF) and a functional PEDF peptide (PEDF-335). rPEDF and PEDF-335 were administered intradermally from day 4 post-wounding every 3 days until day 19. Intradermal injection of rPEDF or PEDF-335 both led to decreased angiogenesis and decreased collagen deposition at the wound site. The results support the utility of antiangiogenic therapies, including rPEDF/PEDF-335, as a potential new strategy for the prevention or treatment of HSs.

Topical application of a commercially available formulation of vitamin C stabilized by vitamin E and ferulic acid reduces tissue viability and protein synthesis in ex vivo human normal skin.

BACKGROUND: Aqueous formulations of vitamin C stabilized by vitamin E and ferulic acid at low pH effectively protect skin against reactive oxygen species-induced damage. However, the effects of these formulations on human skin have not clearly been described. The aim of this study was to investigate whether topical application of two commercially available formulations of vitamin C alter human skin using an ex vivo model. METHODS: Human skin explants were topically treated on alternate days with commercially available formulation 1 (15% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline or formulation 2 (20% vitamin C) at 100% (without dilution), 50%, or 10% diluted in saline. Only saline was applied to control skin explants. RESULTS: Topical formulation 1 at 100%, 50%, or 10%, but not formulation 2 at 100%, 50%, or 10%, reduced the viability of ex vivo human skin compared to the control after 7, 10, and 13 days. In addition, compared to the control, ex vivo human skin treated with formulation 1 at 50%, but not formulation 2 at 50%, also decreased mRNA levels of actin and ribosomal protein L10 and gene expression of extracellular matrix components after 10 days. Furthermore, after 10 days, topical application of formulation 1 at 50%, but not formulation 2 at 50%, decreased the protein expression of proliferating cellular nuclear antigen, lysyl oxidase, ß-actin, and glyceraldehyde-3-phosphate dehydrogenase compared to the control. CONCLUSIONS: Topical formulation 1, but not formulation 2, may reduce the viability of and protein synthesis in ex vivo human skin. Those effects might be due to action of vehicle of formulation 1 on ex vivo human skin.

Oleic acid and hydroxytyrosol present in olive oil promote ROS and inflammatory response in normal cultures of murine dermal fibroblasts through the NF-κB and NRF2 pathways.

Few studies have evaluated the effects of olive oil on normal tissues like skin and its components. Hence, we investigated whether olive oil could increase the production of ROS and oxidative damage in murine dermal fibroblast cultures in a short-term exposition. In addition, we evaluated the role of oleic acid and hydroxytyrosol, which are the two most important components of olive oil, in the associated mechanisms of action, and the metabolism of long-chain fatty acids from olive oil. To study this, neonatal murine dermal fibroblasts (NMDF) were incubated with olive oil, oleic acid, or hydroxytyrosol for 24 or 72 h. The NMDF incubated with olive oil or oleic acid showed an increase in the production of ROS after 24 h, lipid peroxidation, and protein carbonylation after 72 h, as well as increased expression of nuclear factor-kappa B (NF-κB) p65 and cyclooxygenase-2 (COX-2) after 72 h. However, NMDF treated with olive oil or hydroxytyrosol demonstrated an increase in the expression of nuclear factor-erythroid2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) after 72 h. In addition, NMDF treated with olive oil also showed an increase in the protein expression of diacylglycerol acyltransferase1 (DGAT1), which promotes triacylglycerol synthesis, and in the levels of triacylglycerols. The microscopic analysis showed Nile red-positive lipid droplets inside olive oil-treated NMDF after 72 h. Moreover, gas chromatography-mass spectrometry demonstrated high levels of oleic acid in the olive oil-treated NMDF after 72 h. In conclusion, oleic acid present in the olive oil promotes the production of ROS and oxidative damage in murine dermal fibroblasts, which leads to NF-κB p65 and COX-2 expression, while hydroxytyrosol promotes NRF2 and HO-1 expression. In addition, NMDF area capable of absorbing long-chain fatty acids derived from olive oil, which promotes the synthesis and the accumulation of triacylglycerols into cytoplasm of NMDF through DGAT1 activation.

Short-Term Administration of a High-Fat Diet Impairs Wound Repair in Mice.

High intake of dietary fat plays an important role in obesity development in animals and humans, and prolonged intake of high-fat diet might lead to low-grade chronic inflammation. Previous study showed that diet-induced overweight delays cutaneous wound healing in both obesity-prone and obesity-resistant animals, highlighting the importance of diet composition in the wound healing process. This study evaluated the hypothesis that a short-term administration of high-fat diet could affect cutaneous wound healing. Male mice (C57/bl6) were randomly divided into standard (10% energy from fat) or high-fat (60% energy from fat) chow groups. After 10 days of diet administration, an excisional lesion was performed and the animals were sacrificed 6 or 10 days later. There was no difference in the fasting blood glucose between groups. Ten days after wounding, high-fat chow group presented increased inflammatory infiltrate, levels of inducible nitric oxide synthase and cyclo-oxygenase-2 proteins, and lipid peroxidation. The high-fat chow group presented delayed wound closure, increased amount of myofibroblasts and vessels, and decreased deposition of type I collagen. These findings support the hypothesis that short-term administration of high-fat diet exerts negative effects on mice cutaneous wound healing, due to the interference in the inflammatory phase.

Eucalyptol promotes lung repair in mice following cigarette smoke-induced emphysema.

BACKGROUND: Eucalyptol is a monoterpenoid oil present in many plants, principally the Eucalyptus species, and has been reported to have anti-inflammatory and antioxidative effects. HYPOTHESIS/PURPOSE: Since the potential effect of eucalyptol on mouse lung repair has not yet been studied, and considering that chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide, the aim of this study was to investigate eucalyptol treatment in emphysematous mice. STUDY DESIGN: Male mice (C57BL/6) were divided into the following groups: control (sham-exposed), cigarette smoke (CS) (mice exposed to 12 cigarettes a day for 60 days), CS + 1 mg/ml (CS mice treated with 1 mg/ml eucalyptol for 60 days), and CS + 10 mg/ml (CS mice treated with 10 mg/ml eucalyptol for 60 days). Mice in the CS and control groups received vehicle for 60 days. Eucalyptol (or the vehicle) was administered via inhalation (15 min/daily). Mice were sacrificed 24 h after the completion of the 120-day experimental procedure. METHODS: Histology and additional lung morphometric analyses, including analysis of mean linear intercept (Lm) and volume density of alveolar septa (Vv[alveolar septa]) were performed. Biochemical analyses were also performed using colorimetric assays for myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) activity, in addition to using ELISA kits for the determination of inflammatory marker levels (tumor necrosis factor alpha [TNF-α], interleukin-1 beta [IL-1ß], interleukin 6 [IL-6], keratinocyte chemoattractant [KC], and tumor growth factor beta 1 [TGF-ß1]). Finally, we investigated protein levels by western blotting (nuclear factor (erythroid-derived 2)-like 2 [Nrf2], nuclear factor kappa B [NF-κB], matrix metalloproteinase 12 [MMP-12], tissue inhibitor of matrix metalloproteinase 1 [TIMP-1], neutrophil elastase [NE], and elastin). RESULTS: Eucalyptol promoted lung repair at the higher dose (10 mg/ml), with de novo formation of alveoli, when compared to the CS group. This result was confirmed with Lm and Vv[alveolar septa] morphometric analyses. Moreover, collagen deposit around the peribronchiolar area was reduced with eucalyptol treatment when compared to the CS group. Eucalyptol also reduced all inflammatory (MPO, TNF-α, IL-1ß, IL-6, KC, and TGF-ß1) and redox marker levels (MDA) when compared to the CS group (at least p < 0.05). In general, 10 mg/ml eucalyptol was more effective than 1 mg/ml and, at both doses, we observed an upregulation of SOD activity when compared to the CS group (p < 0.001). Eucalyptol upregulated elastin and TIMP-1 levels, and reduced neutrophil elastase (NE) levels, when compared to the CS group. CONCLUSION: In summary, eucalyptol promoted lung repair in emphysematous mice and represents a potential therapeutic phytomedicine in the treatment of COPD.

Topical retinol attenuates stress-induced ageing signs in human skin ex vivo, throughEGFR activation viaEGF, but notERK andAP-1 activation.

Stress-induced oxidative damage and the inflammatory response lead to degradation of collagen and elastic fibres and wrinkle formation. Topical retinol (or vitamin A) can be a strategy to attenuate the effects of stress in skin as it promotes collagen and elastic fibre production and reduces protease synthesis. This study investigated the effect of topical retinol in stressed human skin using in vitro and ex vivo models. Human skin explants were treated with high levels of epinephrine (as observed in stressed patients) and topically with retinol for 13 days. Human dermal fibroblasts were treated with conditioned medium of ex vivo retinol-treated and non-stressed (without epinephrine) human skin for 24 hours. In ex vivo human skin, retinol reversed the epinephrine-induced reduction in epidermal proliferation and differentiation, normalizing epidermal thickness. Retinol also inhibited the epinephrine-induced reduction in elastic fibre deposition and organization, restoring dermal thickness. In addition, retinol reversed the epinephrine-induced increase in c-JUN protein expression, but it did not alter extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in ex vivo human skin. Conditioned medium of ex vivo retinol-treated and non-stressed human skin presented an increased protein expression of epidermal growth factor (EGF). In human dermal fibroblasts, conditioned medium of ex vivo retinol-treated and non-stressed human skin increased protein and gene expression of fibrillin-1 and protein expression of EGF receptor (EGFR). In conclusion, topical retinol attenuates stress-induced skin ageing signs in human skin ex vivo, probably through EGFR activation via EGF, but not by the stress-activated ERK 1/2 and c-JUN pathways.

Acute Exposure to Diesel-Biodiesel Particulate Matter Promotes Murine Lung Oxidative Stress by Nrf2/HO-1 and Inflammation Through the NF-kB/TNF-α Pathways.

Air pollution caused by fuel burning contributes to respiratory impairments that may lead to death. We aimed to investigate the effects of biodiesel (DB) burning in mouse lungs. DB particulate matter was collected from the exhaust pipes of a bus engine. Mice were treated with 250 µg or 1000 µg of DB particulate matter by intranasal instillation over 5 consecutive days. We demonstrated that DB particulate matter penetrated the lung in the 250-µg and 1000-µg groups. In addition, the DB particulate matter number in pulmonary parenchyma was 175-fold higher in the 250-µg group and 300-fold higher in the 1000-µg group compared to control mice. The instillation of DB particulate matter increased the macrophage number and protein levels of TNF-alpha in murine lungs. DB particulate matter enhanced ROS production in both exposed groups and the malondialdehyde levels compared to the control group. The protein expression levels of Nrf2, p-NF-kB, and HO-1 were higher in the 250-µg group and lower in the 1000-µg group than in control mice and the 250-µg group. In conclusion, DB particulate matter instillation promotes oxidative stress by activating the Nrf2/HO-1 and inflammation by p-NF-kB/TNF-alpha pathways.

Caffeic acid phenethyl ester promotes wound healing of mice pressure ulcers affecting NF-κB, NOS2 and NRF2 expression.

AIMS: In pressure ulcers, the synthesis of reactive oxygen species induced by ischemia and reperfusion leads to chronic inflammation and tissue damage, which impair the closure of these lesions. Caffeic acid phenethyl ester (CAPE), found in propolis, promotes cutaneous wound healing of acute lesions and severe burns. However, the effects of CAPE on wound healing of pressure ulcers have not been investigated. This study investigated the effects of CAPE administration in a murine model of pressure ulcers. MAIN METHODS: To induce pressure ulcers, two cycles of ischemia and reperfusion by external application of two magnetic plates were performed in the skin dorsum of mice. After the last cycle, animals were treated daily with CAPE or vehicle until they were euthanized. KEY FINDINGS: The nitric oxide synthesis, lipid peroxidation, macrophage migration, protein nuclear factor kappa B and nitric-oxide synthase-2 expression were increased 3 days after ulceration but decreased 7 days later, in pressure ulcers of the CAPE group compared to that of the control group. CAPE reduced the protein expression of nuclear factor-erythroid2-related factor 2 in pressure ulcers 3 days after ulceration, but increased 7 days later. Myofibroblast density was increased in the CAPE group 7 days after ulceration, but reduced 12 days later when compared to control group. In addition, CAPE promoted collagen deposition, re-epithelialization and wound closure of mice pressure ulcers 12 days after ulceration. SIGNIFICANCE: CAPE brings forward inflammatory response and oxidative damage involved in injury by ischemia and reperfusion, promoting dermal reconstruction and closure of pressure ulcers.

Propolis reversed cigarette smoke-induced emphysema through macrophage alternative activation independent of Nrf2.

Chronic obstructive pulmonary disease (COPD) is an incurable and progressive disease. Emphysema is the principal manifestation of COPD, and the main cause of this condition is cigarette smoke (CS). Natural products have shown antioxidant and anti-inflammatory properties that can prevent acute lung inflammation and emphysema, but there are few reports in the literature regarding therapeutic approaches to emphysema. We hypothesized that supplementation with natural extracts would repair lung damage in emphysema caused by CS exposure. Mice were exposed to 60days of CS and then treated or not with three different natural extracts (mate tea, grape and propolis) orally for additional 60days. Histological analysis revealed significant improvements in lung histoarchitecture, with recovery of alveolar spaces in all groups treated with natural extracts. Propolis was also able to recovery alveolar septa and elastic fibers. Propolis also increased MMP-2 and decreased MMP-12 expression, favoring the process of tissue repair. Additionally, propolis recruited leukocytes, including macrophages, without ROS release. These findings led us to investigate the profile of these macrophages, and we showed that propolis could promote macrophage alternative activation, thus increasing the number of arginase-positive cells and IL-10 levels and favoring an anti-inflammatory microenvironment. We further investigated the participation of Nrf2 in lung repair, but no Nrf2 translocation to the nucleus was observed in lung cells. Proteins and enzymes related to Nrf2 were not altered, other than NQO1, which seemed to be activated by propolis in a Nrf2-independent manner. Finally, propolis downregulated IGF1 expression. In conclusion, propolis promoted lung repair in a mouse emphysema model via macrophage polarization from M1 to M2 in parallel to the downregulation of IGF1 expression in a Nrf2-independent manner.

Exercise prior to, but not concomitant with, stress reverses stress-induced delayed skin wound healing.

Stress-induced prolonged inflammation impairs cutaneous wound healing. Exercise may inhibit this effect via an anti-inflammatory mechanism. Our aim was to investigate the effect of moderate exercise on skin wound healing in chronically stressed mice. Mice were trained five times per week on a treadmill or received no training. Mice underwent daily rotational stress from the 6th week until euthanasia. During the 8th week, two wounds were created in the dorsum and collected 10 days later. A control group only received wounds. Exercise was performed prior to and simultaneous with stress for 2 weeks or only prior to stress. Stress increased normetanephrine levels 10 days after wounding, resulting in an increased amount of inflammatory cells and reduced expression of inflammatory cytokines as well as angiogenesis, myofibroblast differentiation and matrix deposition. Concomitant exercise and stress potentiated these effects, intensifying the delayed wound contraction. When exercise was performed only prior to stress, however, the mice showed reduced inflammatory cells in granulation tissue 10 days after wounding and improved wound healing compared with animals with exercise and concomitant stress. Moderate exercise in association with stress potentiates the stress effect; however, when exercise was performed prior to stress, wound healing was improved.