Heme Oxygenase-1 Induction by Blood-Feeding Arthropods Controls Skin Inflammation and Promotes Disease Tolerance.

Hematophagous vectors lacerate host skin and capillaries to acquire a blood meal, resulting in leakage of red blood cells (RBCs) and inflammation. Here, we show that heme oxygenase-1 (HO-1), a pleiotropic cytoprotective isoenzyme that mitigates heme-mediated tissue damage, is induced after bites of sand flies, mosquitoes, and ticks. Further, we demonstrate that erythrophagocytosis by macrophages, including a skin-residing CD163+CD91+ professional iron-recycling subpopulation, produces HO-1 after bites. Importantly, we establish that global deletion or transient inhibition of HO-1 in mice increases inflammation and pathology following Leishmania-infected sand fly bites without affecting parasite number, whereas CO, an end product of the HO-1 enzymatic reaction, suppresses skin inflammation. This indicates that HO-1 induction by blood-feeding sand flies promotes tolerance to Leishmania infection. Collectively, our data demonstrate that HO-1 induction through erythrophagocytosis is a universal mechanism that regulates skin inflammation following blood feeding by arthropods, thus promoting early-stage disease tolerance to vector-borne pathogens.

RIP1 inhibition blocks inflammatory diseases but not tumor growth or metastases.

The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.

Inhibitory effects of superoxide dismutase 3 on Propionibacterium acnes-induced skin inflammation.

Propionibacterium acnes is a well-known commensal bacterium that plays an important role in the pathogenesis of acne and chronic inflammatory skin disease. In this study, we investigated the effect of superoxide dismutase 3 (SOD3) on P. acnes- or peptidoglycan (PGN)-induced inflammation in vitro and in vivo. Our data demonstrated that SOD3 suppressed toll-like receptor-2 (TLR-2) expression in P. acnes- or PGN-treated keratinocytes and sebocytes. Moreover, we found that SOD3 suppressed the expressions of phosphorylated nuclear factor-κB (NF-κB) and p38 in P. acnes- or PGN-treated cells. SOD3 also exhibited an anti-inflammatory role by reducing the expression of inflammasome-related proteins (NLRP3, ASC, caspase-1) and inhibiting the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1ß, interleukin-6, and interleukin-8. In addition, SOD3 reduced lipid accumulation and expression of lipogenic regulators in P. acnes-treated sebocytes. Recombinant SOD3-treated wild-type mice and SOD3 transgenic mice, which were subcutaneously infected with P. acnes, showed tolerance to inflammation through reducing inflammatory cell infiltration in skin, ear thickness, and expression of inflammatory mediators. Our result showed that SOD3 could suppress the inflammation through inhibition of TLR2/p38/NF-κB axis and NLRP3 inflammasome activation. Therefore, SOD3 could be a promising candidate for treatment of P. acnes-mediated skin inflammation.

Allelic variants of the aryl hydrocarbon receptor differentially influence UVB-mediated skin inflammatory responses in SKH1 mice.

The mouse strain SKH1 is widely used in skin research due to its hairless phenotype and intact immune system. Due to the complex nature of aryl hydrocarbon receptor (AHR) function in the skin, the development of additional in vivo models is necessary to study its role in cutaneous homeostasis and pathology. Variants of the Ah allele, exist among different mouse strains. The Ahb-1 and Ahd alleles express high and low affinity ligand binding forms of the AHR, respectively. The outbred SKH1 mice express the Ahb-2 and/or Ahd alleles. SKH1 mice were crossed with C57BL/6J mice, which harbor the Ahb-1 allele, to create useful models for studying endogenous AHR function. SKH1 mice were bred to be homozygous for either the Ahb-1 or Ahd allele to establish strains for use in comparative studies of the effects of differential ligand-mediated activation through gene expression changes upon UVB exposure. Ahb-1 or Ahd allelic status was confirmed by DNA sequence analysis. We tested the hypothesis that SKH1-Ahb-1 mice would display enhanced inflammatory signaling upon UVB exposure compared to SKH1-Ahd mice. Differential basal AHR activation between the strains was determined by assessing Cyp1a1 expression levels in the small intestine, liver, and skin of the SKH1-Ahb-1 mice compared to SKH1-Ahd mice. To determine whether SKH1-Ahb-1 mice are more prone to a pro-inflammatory phenotype in response to UVB, gene expression of inflammatory mediators was analyzed. SKH1-Ahb-1 mice expressed enhanced gene expression of the chemotactic factors Cxcl5, Cxcl1, and Ccl20, as well as the inflammatory signaling factors S100a9 and Ptgs2, compared to SKH1-Ahd mice in skin. These data supports a role for AHR activation and enhanced inflammatory signaling in skin.

The Basophil-specific Protease mMCP-8 Provokes an Inflammatory Response in the Skin with Microvascular Hyperpermeability and Leukocyte Infiltration.

Basophils have often been erroneously considered to be minor relatives or blood-circulating precursors of tissue-resident mast cells because of some phenotypic similarity between them, including basophilic secretory granules in the cytoplasm. However, recent studies revealed that the repertoire of serine proteases stored in secretory granules is distinct in them. Particularly, mouse mast cell protease 8 (mMCP-8) is specifically expressed by basophils but not mast cells despite its name. Therefore, mMCP-8 is commonly used as a basophil-specific marker, but its functional property remains uncertain. Here we prepared recombinant mMCP-8 and examined its activity in vitro and in vivo Purified recombinant mMCP-8 showed heat-sensitive proteolytic activity when α-tubulin was used as a substrate. One intradermal shot of mMCP-8, not heat-inactivated, induced cutaneous swelling with increased microvascular permeability in a cyclooxygenase-dependent manner. Moreover, repeated intradermal injection of mMCP-8 promoted skin infiltration of leukocytes, predominantly neutrophils and, to a lesser extent, monocytes and eosinophils, in conjunction with up-regulation of chemokine expression in the skin lesion. These results suggest that mMCP-8 is an important effector molecule in basophil-elicited inflammation, providing novel insights into how basophils exert a crucial and non-redundant role, distinct from that played by mast cells, in immune responses.

Brefeldin A reduces tumor necrosis factor-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR, and NF-κB pathways in human keratinocytes.

Keratinocytes may play an important role in the pathogenesis of inflammatory skin diseases. Brefeldin A has been shown to attenuate the production and secretion of chemical mediators involved in inflammation and immune responses. However, the effect of brefeldin A on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. We investigated the effect of brefeldin A on the TNF-α-stimulated production of inflammatory mediators using HaCaT cells and primary keratinocytes in relation to the Akt, mTOR, and NF-κB pathways, which regulates the transcription genes involved in immune and inflammatory responses. Brefeldin A, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), and rapamycin (mTOR inhibitor) inhibited the TNF-α-stimulated productions of inflammatory mediators, and activations of Akt, mTOR, and NF-κB in keratinocytes. The results show that brefeldin A appears to attenuate TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR, and NF-κB pathways.

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis.

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis.

An Essential Role for SHARPIN in the Regulation of Caspase 1 Activity in Sepsis.

Sepsis is burdened by high mortality due to uncontrolled inflammatory response to pathogens. Increased caspase 1 activation causing maturation of IL1ß/18 remains a therapeutic challenge in sepsis. SHARPIN (shank-associated regulator of G-protein signaling homology domain-interacting protein), a component of the LUBAC (linear ubiquitin chain-assembly complex), regulates inflammation, with unknown effects on caspase 1 activation. Mice lacking Casp1, Casp11, or both in a Sharpin-deficient background were generated, exposed to lipopolysaccharide-induced endotoxemia, and injected with caspase 1 inhibitor. We monitored survival, Il1ß/18, and caspase 1/11 levels in plasma and organs and deciphered mechanisms of SHARPIN-dependent caspase 1 inhibition. A correlation between LUBAC and active caspase 1 was found in blood mononuclear cells from septic patients. SHARPIN bound caspase 1 and disrupted p20/p10 dimer formation, the last step of caspase 1 processing, thereby inhibiting enzyme activation and maturation of IL1ß/18 in a LUBAC-independent manner. In septic patients, LUBAC-independent decline in SHARPIN correlated with enhancement of active caspase 1 in circulating mononuclear cells. Septic Sharpin-deficient mice displayed enrichment in mature Il1ß/18 and active caspase 1, and shortened survival. Inhibition of caspase 1 reduced levels of Il1ß/18 and splenic cell death, and prolonged survival in septic Sharpin-deficient mice. Our findings identify SHARPIN as a potent in vivo caspase 1 inhibitor and propose the caspase 1-SHARPIN interaction as a target in sepsis.

Cefradine blocks solar-ultraviolet induced skin inflammation through direct inhibition of T-LAK cell-originated protein kinase.

Skin inflammation, and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induced skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. Herein, the clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. Ex vivo studies showed that SUV induced the phosphorylation of p38 and JNKs in HaCat and JB6 cells in a dose and time dependent manner. Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. The result of in vitro binding assay verified cefradine can directly bind with TOPK. In vitro kinase results showed cefradine can inhibit TOPK activity. Ex vivo studies further showed cefradine inhibited SUV-induced the phosphorylation level of p38, JNKs and H2AX through inhibiting TOPK activity in a dose and time dependent manner, and cefradine inhibited the secretion of IL6 and TNF-α in HaCat and JB6 cells. In vivo studies showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX and inhibited the secretion of IL6 and TNF-α in Babl/c mice. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation.

Therapeutic evaluation of HIV transduction basic domain-conjugated superoxide dismutase solution on suppressive effects of the formation of peroxynitrite and expression of COX-2 in murine skin.

BACKGROUND: Homeostasis of reactive oxygen species (ROS) in the skin is regulated by antioxidant defenses. The inflammatory states of skin diseases which range from acute rashes to chronic conditions are related to the level of ROS. The involvement of superoxide dismutase (SOD) in restoring the antioxidant capacity can then neutralize the inflammatory response. RESULTS: We found that denatured Tat-SOD formulated in an aqueous medium could be delivered into mouse skin and the penetration signals of Tat-SOD were detected in the epidermis and dermis. According to immunohistochemical staining, Tat-SOD successfully suppressed inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), the expression of sodium nitroferricyanide (SNP)-induced cyclooxygenase-2 (COX-2), and the production of nitrotyrosine proteins. In nerve growth factor (NGF) induced differentiated PC12 pheochromocytoma cells, we demonstrated that the denatured Tat-SOD regained its antioxidant activity and effectively protected PC12 cells from DNA fragmentation induced by paraquat. Using a luciferase reporter assay, the data was shown Tat-SOD protected PC12 cells from ROS damage, through suppression of COX-2 or nuclear factor-κB (NF-κB) activity occurred at the transcriptional level. CONCLUSION: We showed that Tat-SOD inhibited SNP-induced COX-2 expression similarly to celecoxib and prevented the formation of peroxynitrite as 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The results suggest that denatured Tat-SOD solution may perform potential protein therapy for patients suffering from disorders related to ROS.

Anti-hyaluronidase Activity in Vitro and Amelioration of Mouse Experimental Dermatitis by Tomato Saponin, Esculeoside A.

The increasing incidence of atopic dermatitis during recent decades has prompted the development of safe and effective agents for prevention of atopic diseases. Esculeoside A, a glycoside of spirosolane type, is identified as a major component in ripe tomato fruits. The present study investigated the effects of esculeoside A and its aglycon esculeogenin A on hyaluronidase activity in vitro and antiallergy in experimental dermatitis mice. Esculeogenin A/esculeoside A (esculeogenin A equivalent) with an IC50 of about 2 µM/9 µM dose-dependently inhibited hyaluronidase activity measured by a modified Morgan-Elson method. Oral treatment with esculeoside A 10 mg/kg of experimental dermatitis mice for 4 weeks significantly decreased the skin clinical score to 2.5 without any detectable side effects compared with 6.75 of the control. The scratching frequency of esculeoside A 100 mg/kg application was decreased significantly as 107.5 times compared with 296.67 times of the control. Thus, the present study showed that esculeoside A/esculeogenin A significantly blocks hyaluronidase activity in vitro and that esculeoside A ameliorates mouse experimental dermatitis.

CD4, IL-17, and COX-2 Are Associated With Subclinical Inflammation in Malar Melasma.

The pathogenesis of melasma, a common, photo-induced hyperpigmentary disorder, is not clearly understood. Significant factors linked to melasma are ultraviolet radiation exposure and genetic predisposition. Histological analysis has demonstrated that melasma is caused by a network of cellular interactions among melanocytes, keratinocytes, mast cells, fibroblasts, and dermal vasculature exhibits, features similar to chronic sun damage. Dermal inflammation caused by ultraviolet radiation might play an important role in the hyperpigmentation and reactivation of melasma lesions through the production of melanogenic cytokines and growth factors. Because the role of inflammation in this disorder is unknown, we used histochemistry, immunohistochemistry, and quantitative real-time polymerase chain reaction to evaluate melasma lesions from healthy female patients (n = 20) with malar melasma. Lesional skin without specific solar exposure or photoprotection measures within the previous 4 weeks was compared with nonlesional skin. The increased lymphocytic infiltrate in lesional skin was mainly composed of CD4 T cells, mast cells, and macrophages. Levels of the cytokine interleukin (IL)-17 and the proinflammatory mediator cyclooxygenase (COX)-2 were significantly elevated in affected skin compared with healthy skin. In addition, the Melasma Activity and Severity Index score, fraction of solar elastosis, and epidermal melanin were positively associated with COX-2 expression. There was no statistically significant difference in IL-1α, IL-1ß, R-IL1, IL-6, IL-8, vascular endothelial growth factor, and tumor necrosis factor alpha expression levels. Together, these data indicated that melasma under unchallenged conditions is characterized by chronic inflammatory cells and mediators, which may explain its recurrent nature.

Combined deletion of p38γ and p38δ reduces skin inflammation and protects from carcinogenesis.

The contribution of chronic skin inflammation to the development of squamous cell carcinoma (SCC) is poorly understood. While the mitogen-activated protein kinase p38α regulates inflammatory responses and tumour development, little is known about the role of p38γ and p38δ in these processes. Here we show that combined p38γ and p38δ (p38γ/δ) deletion blocked skin tumour development in a chemically induced carcinogenesis model. p38γ/δ deletion reduced TPA-induced epidermal hyperproliferation and inflammation; it inhibited expression of proinflammatory cytokines and chemokines in keratinocytes in vitro and in whole skin in vivo, resulting in decreased neutrophil recruitment to skin. Our data indicate that p38γ/δ in keratinocytes promote carcinogenesis by enabling formation of a proinflammatory microenvironment that fosters epidermal hyperproliferation and tumourigenesis. These findings provide genetic evidence that p38γ and p38δ have essential roles in skin tumour development, and suggest that targeting inflammation through p38γ/δ offers a therapeutic strategy for SCC treatment and prevention.

Allergic contact dermatitis from ethylhexyl salicylate

No disponible

Deoxynivalenol induced mouse skin cell proliferation and inflammation via MAPK pathway.

Several toxicological manifestations of deoxynivalenol (DON), a mycotoxin, are well documented; however, dermal toxicity is not yet explored. The effect of topical application of DON to mice was studied using markers of skin proliferation, inflammation and tumor promotion. Single topical application of DON (84-672nmol/mouse) significantly enhanced dermal hyperplasia and skin edema. DON (336 and 672nmol) caused significant enhancement in [(3)H]-thymidine uptake in DNA along with increased myeloperoxidase and ornithine decarboxylase activities, suggesting tissue inflammation and cell proliferation. Furthermore, DON (168nmol) caused enhanced expression of RAS, and phosphorylation of PI3K/Akt, ERK, JNK and p38 MAPKs. DON exposure also showed activation of transcription factors, c-fos, c-jun and NF-κB along with phosphorylation of IkBα. Enhanced phosphorylation of NF-κB by DON caused over expression of target proteins, COX-2, cyclin D1 and iNOS in skin. Though a single topical application of DMBA followed by twice weekly application of DON (84 and 168nmol) showed no tumorigenesis after 24weeks, however, histopathological studies suggested hyperplasia of the epidermis and hypertrophy of hair follicles. Interestingly, intestine was also found to be affected as enlarged Peyer's patches were observed, suggesting inflammatory effects which were supported by elevation of inflammatory cytokines after 24weeks of topical application of DON. These results suggest that DON induced cell proliferation in mouse skin is through the activation of MAPK signaling pathway involving transcription factors NFκB and AP-1, further leading to transcriptional activation of downstream target proteins c-fos, c-jun, cyclin D1, iNOS and COX-2 which might be responsible for its inflammatory potential.

Feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling.

BACKGROUND: Feline eosinophilic dermatoses (FEDs) are common diseases of cats with an unknown pathogenesis. They are histologically characterized by an eosinophilic infiltration and often by the presence of flame figures (FFs) and/or areas of loss of tissue architecture, here termed necrotic foci (NF). It has been postulated that an alteration in the degradation of the extracellular matrix could be responsible for these histological features. Matrix metalloproteinases (MMPs) are a group of proteases that are fundamental in extracellular matrix remodelling. HYPOTHESIS/OBJECTIVES: The aim of the study was to investigate retrospectively the expression of a subgroup of MMPs, in particular MMP-2 and MMP-9 gelatinases, in FEDs. The expression of one of their inhibitors, TIMP-2, was also investigated in order to establish the role of these molecules in the pathogenesis of FEDs. The ultrastructural characteristics of extracellular matrix in FFs and NF were subsequently assessed. METHODS: Fifty-one formalin-fixed, paraffin-embedded specimens from cutaneous and mucosal biopsies diagnosed as FEDs were investigated immunohistochemically. Two selected samples were processed for electron microscopy. RESULTS: This study revealed an increased expression of MMP-2 in NF and a decreased expression of this gelatinase in FFs. An imbalance between MMP-2 and TIMP-2 was evident using immunohistochemistry. No significative results were observed for MMP-9 expression. Electron microscopy confirmed the lack of normal collagen fibres in NF, whereas in FFs only occasional, amorphous material was observed among normal collagen fibres. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that an imbalance in the expression of matrix metalloproteinases could be responsible for different morphological findings in FEDs. Further studies are needed to assess the role of matrix metalloproteinases in the pathogenesis of FEDs.

Enhanced in vitro and in vivo skin deposition of apigenin delivered using ethosomes.

The aim of this study was to develop and evaluate a novel topical delivery system for apigenin by using ethosomes. An optimal apigenin-loaded ethosome formulation was identified by means of uniform design experiments. Skin deposition and transdermal flux of apigenin loaded in ethosomes, liposomes, and deformable liposomes were compared in vitro and in vivo. The efficiency of apigenin encapsulation increased with an increase in the amount of phospholipids in ethosome formulations. Moreover, skin deposition and transdermal flux of apigenin improved with an increase in the levels of phospholipids (Lipoid S 75) and short-chain alcohols (propylene glycol and ethanol), but decreased with an increase in the ratio of propylene glycol to ethanol. Profiles of skin deposition versus time for ethosomes varied markedly between in vivo and in vitro studies compared with those of liposomes or deformable liposomes. Optimized ethosomes showed superior skin targeting both in vitro and in vivo. Moreover, they had the strongest effect on reduction of cyclooxygenase-2 levels in mouse skin inflammation induced by ultraviolet B (UVB) light. Therefore, apigenin-loaded ethosomes represent a promising therapeutic approach for the treatment of UVB-induced skin inflammation.

The role of sterol-C4-methyl oxidase in epidermal biology.

Deficiency of sterol C4 methyl oxidase, encoded by the SC4MOL gene, has recently been described in four patients from three different families. All of the patients presented with microcephaly, congenital cataracts, and growth delay in infancy. The first patient has suffered since the age of six years from severe, diffuse, psoriasiform dermatitis, sparing only her palms. She is now 20 years old. The second patient is a 5 year old girl who has just started to develop dry skin and hair changes. The third and fourth patients are a pair of affected siblings with a severe skin condition since infancy. Quantitative sterol analysis of plasma and skin scales from all four patients showed marked elevation of 4α-methyl- and 4, 4'-dimethylsterols, consistent with a deficiency in the first step of sterol C4 demethylation in cholesterol biosynthesis. Mutations in the SC4MOL have been identified in all of the patients. SC4MOL deficiency is the first autosomal recessive disorder identified in the sterol demethylation complex. Cellular studies with patient-derived fibroblasts have shown a higher mitotic rate than control cells in cholesterol-depleted medium, with increased de novo cholesterol biosynthesis and accumulation of methylsterols. Immunologic analyses of granulocytes and B cells from patients and obligate carriers in the patients' families indicated dysregulation of immune-related receptors. Inhibition of sterol C4 methyl oxidase in human transformed lymphoblasts induced activation of the cell cycle. Additional studies also demonstrated diminished EGFR signaling and disrupted vesicular trafficking in cells from the affected patients. These findings suggest that methylsterols play an important role in epidermal biology by their influence on cell proliferation, intracellular signaling, vesicular trafficking and immune response. SC4MOL is situated within the psoriasis susceptibility locus PSORS9, and may be a genetic risk factor for common skin conditions. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

[Effects of zhuhong ointment on MMPs activities and production by HSF].

HuaFu Shengji is the primary traditional Chinese medicine (TCM) therapy for treating chronic skin ulcer. The high activities of the protein enzyme in the wound fluids is one of the main cause of healing delay. In order to investigate the effect of TCM Zhuhong ointment for promoting wound healing. This research focused on its influence on matrix metalloproteinase (MMP) activities in wound fluids with TCM Yang syndromes, directly on the activated MMP-1,2 activities in vitro and on MMP-1,-2,-9 production by HSF. 8 wound fluid samples were collected, which were diagnosed Yang Syndromes in TCM. Wound fluid activities of MMP-2 and MMP-9 were measured by gelatin zymogram assay. MMP-1 and MMP-2 activities in vitro were measured by substrate cleavage. CCK-8 was used to observe the toxicity of Zhuhong ointment on HSF. MMP-1,-2,-9 production by HSF were detected by confocal microscope. Zhuhong ointment from 1 to 25 g x L(-1) obviously inhibited MMP-2 activity in wound fluid. When Zhuhong ointment was over 5 g x L(-1), it showed significantly inhibitory effect on wound fluid MMP-9 activity. In vitro study, when the mercury concentration was 320 mg x L(-1), Zhuhong ointment solution directly inhibited both MMP-1 activity and MMP-2. But mercury concentration from 0.51-2.56 mg x L(-1), it could activate MMP-1 activity, and from 0.51-64 mg x L(-1), activate MMP-2 activity instead. The mercury concentration when Zhuhong ointment saturated in DMEM was 39.6 mg x L(-1). When the mercury concentration was over 1.23 mg x L(-1), Zhuhong ointment showed toxicity to HSF. At 1.23, 0.62, 0.31 mg x L(-1) of mercury concentration, it increased MMP-1 expression by HSF, and at 1.23, 0.62 mg x L(-1), decreased MMP-2 expression. However, at 1.23, 0.62, 0.31 mg x L(-1), it decreased MMP-9 expression. At higher concentration, Zhuhong ointment can inhibit MMP-2, MMP-9 activities in wound fluid with dose-dependent way and show a direct inhibitory effect on activated MMP-1 and MMP-2 in vitro. But at a lower concentration, it showed two-way adjustment, with increased MMP-1, MMP-2 activities and its expression by HSF and decreased MMP-9 activity.

PXS-4681A, a potent and selective mechanism-based inhibitor of SSAO/VAP-1 with anti-inflammatory effects in vivo.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.