Pathophysiology and inflammatory biomarkers of sulfur mustard-induced corneal injury in rabbits

Sulfur mustard (SM) is a cytotoxic, vesicating, chemical warfare agent, first used in 1917; corneas are particularly vulnerable to SM exposure. They may develop inflammation, ulceration, neovascularization (NV), impaired vision, and partial/complete blindness depending upon the concentration of SM, exposure duration, and bio-physiological conditions of the eyes. Comprehensive in vivo studies have established ocular structural alterations, opacity, NV, and inflammation upon short durations (<4 min) of SM exposure. In this study, detailed analyses of histopathological alterations in corneal structure, keratocytes, inflammatory cells, blood vessels, and expressions of cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), and cytokines were performed in New Zealand white rabbits, in a time-dependent manner till 28 days, post longer durations (5 and 7 min) of ocular SM exposure to establish quantifiable endpoints of injury and healing. Results indicated that SM exposure led to duration-dependent increases in corneal thickness, opacity, ulceration, epithelial-stromal separation, and epithelial degradation. Significant increases in NV, keratocyte death, blood vessels, and inflammatory markers (COX-2, MMP-9, VEGF, and interleukin-8) were also observed for both exposure durations compared to the controls. Collectively, these findings would benefit in temporal delineation of mechanisms underlying SM-induced corneal toxicity and provide models for testing therapeutic interventions.

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Alkylated epidermal creatine kinase as a biomarker for sulfur mustard exposure: comparison to adducts of albumin and DNA in an in vivo rat study

Archives of Toxicology ◽

10.1007/s00204-021-03005-3 ◽

2021 ◽

Author(s):

Dirk Steinritz ◽

Robin Lüling ◽

Markus Siegert ◽

Julia Herbert ◽

Harald Mückter ◽

...

Keyword(s):

Creatine Kinase ◽

Dna Adducts ◽

Sulfur Mustard ◽

Ex Vivo ◽

Immunomagnetic Separation ◽

Chemical Warfare Agent ◽

Cysteine Residue ◽

Islamic State ◽

Rat Skin

AbstractSulfur mustard (SM) is a chemical warfare agent which use is banned under international law and that has been used recently in Northern Iraq and Syria by the so-called Islamic State. SM induces the alkylation of endogenous proteins like albumin and hemoglobin thus forming covalent adducts that are targeted by bioanalytical methods for the verification of systemic poisoning. We herein report a novel biomarker, namely creatine kinase (CK) B-type, suitable as a local biomarker for SM exposure on the skin. Human and rat skin were proven to contain CK B-type by Western blot analysis. Following exposure to SM ex vivo, the CK-adduct was extracted from homogenates by immunomagnetic separation and proteolyzed afterwards. The cysteine residue Cys282 was found to be alkylated by the SM-specific hydroxyethylthioethyl (HETE)-moiety detected as the biomarker tetrapeptide TC(-HETE)PS. A selective and sensitive micro liquid chromatography-electrospray ionization high-resolution tandem-mass spectrometry (µLC-ESI MS/HRMS) method was developed to monitor local CK-adducts in an in vivo study with rats percutaneously exposed to SM. CK-adduct formation was compared to already established DNA- and systemic albumin biomarkers. CK- and DNA-adducts were successfully detected in biopsies of exposed rat skin as well as albumin-adducts in plasma. Relative biomarker concentrations make the CK-adduct highly appropriate as a local dermal biomarker. In summary, CK or rather Cys282 in CK B-type was identified as a new, additional dermal target of local SM exposures. To our knowledge, it is also the first time that HETE-albumin adducts, and HETE-DNA adducts were monitored simultaneously in an in vivo animal study.

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2-Substituted Benzoxazoles as Potent Anti-Inflammatory Agents: Synthesis, Molecular Docking and In Vivo Anti-Ulcerogenic Studies

Medicinal Chemistry ◽

10.2174/1573406418666211220125344 ◽

2021 ◽

Vol 18 ◽

Author(s):

Iqra Hamid ◽

Humaira Nadeem ◽

Sameen Fatima Ansari ◽

Sonia Khiljee ◽

Inzamam Abbasi ◽

...

Keyword(s):

Side Effects ◽

Therapeutic Interventions ◽

Binding Potential ◽

Spectroscopic Techniques ◽

Standard Drug ◽

Cox 2 ◽

Anti Inflammatory ◽

Cox 2 Inhibitors ◽

Protein Pocket

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) are the commonly used therapeutic interventions of inflammation and pain that competitively inhibit the cyclooxygenase (COX) enzymes. Several side effects like gastrointestinal and renal toxicities are associated with the use of these drugs. The therapeutic anti-inflammatory benefits of NSAIDs are produced by the inhibition of COX-2 enzymes, while undesirable side effects arise from the inhibition of COX-1 enzymes. Objectives: In the present study, a new series of 2-substituted benzoxazole derivatives 2(a-f) and 3(a-e) were synthesized in our lab as potent anti-inflammatory agents with outstanding gastro-protective potential. The new analogs 2(a-f) and 3(a-e) were designed depending upon the literature review to serve as ligands for the development of selective COX-2 inhibitors. Methods: The synthesized analogs were characterized using different spectroscopic techniques (FTIR, 1HNMR, 13CNMR) and elemental analysis. All synthesized compounds were screened for their binding potential in the protein pocket of COX-2 and evaluated for their anti-inflammatory potential in animals using the carrageenan-induced paw edema method. Further 5 compounds were selected to assess the in vivo anti-ulcerogenic activity in an ethanol-induced anti-ulcer rat model. Results: Five compounds (2a, 2b, 3a, 3b and 3c) exhibited potent anti-inflammatory activity and significant binding potential in the COX-2 protein pocket. Similarly, these five compounds demonstrated a significant gastro-protective effect (p<0.01) in comparison to the standard drug, Omeprazole. Conclusion: Depending upon our results, we hypothesize that 2-substituted benzoxazole derivatives have excellent potential to serve as candidates for the development of selective anti-inflammatory agents (COX-2 inhibitors). However, further assessments are required to delineate their underlying mechanisms.

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Anti-Inflammatory Activity of the Active Compounds of Sanguisorbae Radix In Macrophages and in Vivo Toxicity Evaluation in Zebrafish

Cosmetics ◽

10.3390/cosmetics6040068 ◽

2019 ◽

Vol 6 (4) ◽

pp. 68

Author(s):

Young-Ah Jang ◽

Yong Hur ◽

Jin-Tae Lee

Keyword(s):

Target Genes ◽

Inflammatory Activity ◽

Dependent Manner ◽

Active Components ◽

Necrosis Factor Alpha ◽

In Vivo Toxicity ◽

Cox 2 ◽

Anti Inflammatory ◽

Sanguisorbae Radix

Sanguisorbae Radix (SR) is the root of the Sanguisorba officinalis L., a plant native to Asian countries and used in traditional medicine. We isolated the active components of SR and investigated their anti-inflammatory potential. Quercetin (QC), (+)-catechin (CC), and gallic acid (GA) were isolated from acetone extracts of SR. To elucidate the molecular mechanism by which these compounds suppress inflammation, we analyzed the transcriptional up-regulation of inflammatory mediators, such as nuclear factor-kappa B (NF-κB) and its target genes, inducible NOS (iNOS), and cyclooxygenase (COX)-2, in lipopolysaccharide (LPS)-stimulated macrophage RAW264.7 cells. Notably, QC, CC, and GA were found to inhibit the production of nitric oxide, tumor necrosis factor-alpha, and prostaglandin in a dose-dependent manner. Western blot results indicate that the compounds decreased the expression of iNOS and COX-2 proteins. Furthermore, the compounds decreased phosphorylation of IKK, IκB, ERK, p-38, and JNK proteins in LPS-induced cells. The results support the notion that QC, CC, and GA can potently inhibit the inflammatory response, with QC showing the highest anti-inflammatory activity. In in vivo toxicity studies in zebrafish (Danio rerio), QC showed no toxicity up to 25 μg/mL. Therefore, QC has non-toxic potential as a skin anti-inflammatory biomaterial.

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Estradiol and mTORC2 cooperate to enhance prostaglandin biosynthesis and tumorigenesis in TSC2-deficient LAM cells

Journal of Experimental Medicine ◽

10.1084/jem.20131080 ◽

2014 ◽

Vol 211 (1) ◽

pp. 15-28 ◽

Cited By ~ 44

Author(s):

Chenggang Li ◽

Po-Shun Lee ◽

Yang Sun ◽

Xiaoxiao Gu ◽

Erik Zhang ◽

...

Keyword(s):

Exhaled Breath Condensate ◽

Tumor Development ◽

Exhaled Breath ◽

Dependent Manner ◽

Lipoxin A4 ◽

Prostaglandin Biosynthesis ◽

Cox 2 ◽

Mtorc1 Activation

Lymphangioleiomyomatosis (LAM) is a progressive neoplastic disorder that leads to lung destruction and respiratory failure primarily in women. LAM is typically caused by tuberous sclerosis complex 2 (TSC2) mutations resulting in mTORC1 activation in proliferative smooth muscle–like cells in the lung. The female predominance of LAM suggests that estradiol contributes to disease development. Metabolomic profiling identified an estradiol-enhanced prostaglandin biosynthesis signature in Tsc2-deficient (TSC−) cells, both in vitro and in vivo. Estradiol increased the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in prostaglandin biosynthesis, which was also increased at baseline in TSC-deficient cells and was not affected by rapamycin treatment. However, both Torin 1 treatment and Rictor knockdown led to reduced COX-2 expression and phospho-Akt-S473. Prostaglandin production was also increased in TSC-deficient cells. In preclinical models, both Celecoxib and aspirin reduced tumor development. LAM patients had significantly higher serum prostaglandin levels than healthy women. 15-epi-lipoxin-A4 was identified in exhaled breath condensate from LAM subjects and was increased by aspirin treatment, indicative of functional COX-2 expression in the LAM airway. In vitro, 15-epi-lipoxin-A4 reduced the proliferation of LAM patient–derived cells in a dose-dependent manner. Targeting COX-2 and prostaglandin pathways may have therapeutic value in LAM and TSC-related diseases, and possibly in other conditions associated with mTOR hyperactivation.

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Anti-inflammatory Potential of Silk Sericin

Natural Product Communications ◽

10.1177/1934578x1300800424 ◽

2013 ◽

Vol 8 (4) ◽

pp. 1934578X1300800 ◽

Cited By ~ 4

Author(s):

Pornanong Aramwit ◽

Pasarapa Towiwat ◽

Teerapol Srichana

Keyword(s):

Negative Control ◽

Dependent Manner ◽

Silk Sericin ◽

Plantar Surface ◽

Cox 2 ◽

Anti Inflammatory ◽

The Right ◽

Dose Dependent ◽

Control Water

Silk sericin was found to suppress the production of pro-inflammatory cytokines, which are related to the inflammatory reaction. The objectives of this study were to investigate the anti-inflammatory effect of sericin in vivo using the carrageenan-induced rat edema model and changes in the histology of tissues. The effects of sericin on the expression of COX-2 and iNOS were also evaluated. Sericin solutions at 0.004-0.080 mg/mL were applied topically to the top of the hind paw and carrageenan (1.0 mg) was injected subcutaneously to the plantar surface of the right hind paw. Our results indicated that sericin significantly reduced the inflammation in rats’ paw compared with the negative control (water and acetone) and its effect at 0.080 mg/mL was only slightly lower than that of 1.0% w/v indomethacin. Similar numbers of polymorphonuclear and macrophage cells were found in rats’ tissue treated with indomethacin and sericin solution, while the numbers were significantly higher in their absence. The gene expression results by RT-PCR showed that the COX-2 and iNOS genes were down-regulated in samples treated with sericin in a dose dependent manner. These data indicated that the anti-inflammatory properties of sericin may be partly attributable to the suppression of the COX-2 enzyme and nitric oxide production.

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Hepatocytes are a rich source of novel aspirin-triggered 15-epi-lipoxin A4

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.5.c870 ◽

1999 ◽

Vol 277 (5) ◽

pp. C870-C877 ◽

Cited By ~ 36

Author(s):

Esther Titos ◽

Nan Chiang ◽

Charles N. Serhan ◽

Mario Romano ◽

Joan Gaya ◽

...

Keyword(s):

Rat Hepatocytes ◽

Arachidonic Acid Metabolism ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Potent Inducer ◽

Biologically Relevant ◽

In Vivo Experiments ◽

Cox 2 ◽

Cytochrome P 450

Novel aspirin (ASA)-triggered 15-epi-lipoxins (ATL) comprise new potent bioactive eicosanoids that may contribute to the therapeutic effect of this drug. ATL biosynthesis is initiated by ASA acetylation of cyclooxygenase (COX)-2 and was originally identified during the interaction of leukocytes with either endothelial or epithelial cells. Here, we examined ATL biosynthesis in rat hepatocytes either alone or in coincubation with nonparenchymal liver cells (NPC) and in liver homogenates from ASA-treated rats. Rat hepatocytes and CC-1 cells, a rat hepatocyte cell line, displayed COX-1 but not COX-2 mRNA expression and predominantly produced thromboxane A2(TXA2) and 15-hydroxyeicosatetraenoic acid (15-HETE). In these cells, ASA shifted the arachidonic acid metabolism from TXA2 to 15-HETE in a concentration-dependent manner. In contrast, neither indomethacin, ibuprofen, valeryl salicylate, nor nimesulide was able to trigger 15-HETE biosynthesis. SKF-525A, a cytochrome P-450 inhibitor, significantly reduced the effect of ASA on 15-HETE biosynthesis. Furthermore, phenobarbital, a potent inducer of cytochrome P-450 activity, further increased ASA-induced 15-HETE production. ASA treatment of hepatocyte-NPC coincubations resulted in the generation of significant amounts of ATL. In addition, in vivo experiments demonstrated augmented hepatic levels of 15-epi-lipoxin A4 in ASA-treated rats. Taken together and considering that ASA is hydrolyzed on its first pass through the portal circulation, these data indicate that, during ASA's consumption, liver tissue generates biologically relevant amounts of ATL by COX-2-independent mechanisms.

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The E3 ubiquitin ligase RNF216/TRIAD3 is a central regulator of the hypothalamic-pituitary-gonadal axis

10.1101/2021.03.21.436306 ◽

2021 ◽

Author(s):

Arlene J. George ◽

Yarely C. Hoffiz ◽

Christopher Ware ◽

Bin Dong ◽

Ning Fang ◽

...

Keyword(s):

Hypogonadotropic Hypogonadism ◽

Cell Activity ◽

Calcium Transient ◽

Therapeutic Interventions ◽

List Type ◽

Dependent Manner ◽

Hpg Axis ◽

Knock Out ◽

Soma Size

SummaryRNF216/TRIAD3 is an E3 ligase that ubiquitinates substrates in the nervous system. Recessive mutations in RNF216/TRIAD3 cause Gordon Holmes syndrome (GHS), where hypogonadotropic hypogonadism is a core phenotype. However, the functions of RNF216/TRIAD3 within the neuroendocrine system are not well-understood. Here, we used the CRISPR-Cas9 system to knock out Rnf216/Triad3 in GT1-7 cells, a GnRH immortalized cell line derived from mouse hypothalamus. Rnf216/Triad3 knockout cells had decreased steady state Gnrh and reduced calcium transient frequency. To address functions of RNF216/TRIAD3 in vivo, we generated a Rnf216/Triad3 constitutive knockout (KO) mouse. KO mice of both sexes showed reductions in GnRH and soma size. Furthermore, KO mice exhibited sex-specific phenotypes with males showing gonadal impairment and derangements in gonadotropin release compared to KO females, which only had irregular estrous cyclicity. Our work shows that dysfunction of RNF216/TRIAD3 affects the HPG axis in a sex-dependent manner, implicating sex-specific therapeutic interventions for GHS.HighlightsRnf216/Triad3 controls Gnrh and intrinsic hypothalamic cell activityRnf216/Triad3 knockout male mice have greater reproductive impairments than femalesRnf216/Triad3 controls the HPG axis at multiple levels

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Physiologically relevant aspirin concentrations trigger immunostimulatory cytokine production by human leukocytes

PLoS ONE ◽

10.1371/journal.pone.0254606 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0254606

Author(s):

Regine Brox ◽

Holger Hackstein

Keyword(s):

Acetylsalicylic Acid ◽

Inflammatory Cytokines ◽

Whole Blood ◽

Plasma Concentrations ◽

Immunomodulatory Activity ◽

Prostaglandin E ◽

Dependent Manner ◽

Cox 2 ◽

Anti Inflammatory

Acetylsalicylic acid is a globally used non-steroidal anti-inflammatory drug (NSAID) with diverse pharmacological properties, although its mechanism of immune regulation during inflammation (especially at in vivo relevant doses) remains largely speculative. Given the increase in clinical perspective of Acetylsalicylic acid in various diseases and cancer prevention, this study aimed to investigate the immunomodulatory role of physiological Acetylsalicylic acid concentrations (0.005, 0.02 and 0.2 mg/ml) in a human whole blood of infection-induced inflammation. We describe a simple, highly reliable whole blood assay using an array of toll-like receptor (TLR) ligands 1–9 in order to systematically explore the immunomodulatory activity of Acetylsalicylic acid plasma concentrations in physiologically relevant conditions. Release of inflammatory cytokines and production of prostaglandin E2 (PGE2) were determined directly in plasma supernatant. Experiments demonstrate for the first time that plasma concentrations of Acetylsalicylic acid significantly increased TLR ligand-triggered IL-1β, IL-10, and IL-6 production in a dose-dependent manner. In contrast, indomethacin did not exhibit this capacity, whereas cyclooxygenase (COX)-2 selective NSAID, celecoxib, induced a similar pattern like Acetylsalicylic acid, suggesting a possible relevance of COX-2. Accordingly, we found that exogenous addition of COX downstream product, PGE2, attenuates the TLR ligand-mediated cytokine secretion by augmenting production of anti-inflammatory cytokines and inhibiting release of pro-inflammatory cytokines. Low PGE2 levels were at least involved in the enhanced IL-1β production by Acetylsalicylic acid.

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Cross-talk between SIM2s and NFκB regulates cyclooxygenase 2 expression in breast cancer

Breast Cancer Research ◽

10.1186/s13058-019-1224-y ◽

2019 ◽

Vol 21 (1) ◽

Cited By ~ 2

Author(s):

Garhett L. Wyatt ◽

Lyndsey S. Crump ◽

Chloe M. Young ◽

Veronica M. Wessells ◽

Cole M. McQueen ◽

...

Keyword(s):

Breast Cancer ◽

Cyclooxygenase 2 ◽

Luciferase Reporter ◽

Signaling Proteins ◽

Dependent Manner ◽

Mcf7 Cells ◽

Human Breast Carcinomas ◽

Cox 2

Abstract Background Breast cancer is a leading cause of cancer-related death for women in the USA. Thus, there is an increasing need to investigate novel prognostic markers and therapeutic methods. Inflammation raises challenges in treating and preventing the spread of breast cancer. Specifically, the nuclear factor kappa b (NFκB) pathway contributes to cancer progression by stimulating proliferation and preventing apoptosis. One target gene of this pathway is PTGS2, which encodes for cyclooxygenase 2 (COX-2) and is upregulated in 40% of human breast carcinomas. COX-2 is an enzyme involved in the production of prostaglandins, which mediate inflammation. Here, we investigate the effect of Singleminded-2s (SIM2s), a transcriptional tumor suppressor that is implicated in inhibition of tumor growth and metastasis, in regulating NFκB signaling and COX-2. Methods For in vitro experiments, reporter luciferase assays were utilized in MCF7 cells to investigate promoter activity of NFκB and SIM2. Real-time PCR, immunoblotting, immunohistochemistry, and chromatin immunoprecipitation assays were performed in SUM159 and MCF7 cells. For in vivo experiments, MCF10DCIS.COM cells stably expressing SIM2s-FLAG or shPTGS2 were injected into SCID mice and subsequent tumors harvested for immunostaining and analysis. Results Our results reveal that SIM2 attenuates the activation of NFκB as measured using NFκB-luciferase reporter assay. Furthermore, immunostaining of lysates from breast cancer cells overexpressing SIM2s showed reduction in various NFκB signaling proteins, as well as pAkt, whereas knockdown of SIM2 revealed increases in NFκB signaling proteins and pAkt. Additionally, we show that NFκB signaling can act in a reciprocal manner to decrease expression of SIM2s. Likewise, suppressing NFκB translocation in DCIS.COM cells increased SIM2s expression. We also found that NFκB/p65 represses SIM2 in a dose-dependent manner, and when NFκB is suppressed, the effect on the SIM2 is negated. Additionally, our ChIP analysis confirms that NFκB/p65 binds directly to SIM2 promoter site and that the NFκB sites in the SIM2 promoter are required for NFκB-mediated suppression of SIM2s. Finally, overexpression of SIM2s decreases PTGS2 in vitro, and COX-2 staining in vivo while decreasing PTGS2 and/or COX-2 activity results in re-expression of SIM2. Conclusion Our findings identify a novel role for SIM2s in NFκB signaling and COX-2 expression.

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Impaired endothelial dysfunction in diabetes mellitus rats was restored by oral administration of prostaglandin I2 analogue

Journal of Endocrinology ◽

10.1677/joe.0.1750217 ◽

2002 ◽

Vol 175 (1) ◽

pp. 217-223 ◽

Cited By ~ 21

Author(s):

K Matsumoto ◽

R Morishita ◽

N Tomita ◽

A Moriguchi ◽

K Yamasaki ◽

...

Keyword(s):

Diabetes Mellitus ◽

Endothelial Dysfunction ◽

Endothelial Function ◽

Blood Vessels ◽

Northern Blotting ◽

Endothelial Injury ◽

Dependent Manner ◽

Vasodilator Response

We have previously reported that a decrease in hepatocyte growth factor (HGF), which has many protective functions against endothelial damage by high d-glucose, might be a trigger of endothelial injury. However, the regulation of vascular HGF in diabetes mellitus (DM) has not been clarified in vivo, although vascular disease is frequently observed in DM patients. In addition, our previous report revealed that a prostaglandin I(2) (PGI(2)) analogue prevented endothelial cell death through the induction of vascular HGF production in cultured human epithelial cells. Thus, in this study, we examined the effects of a PGI(2) analogue in the regulation of the local HGF system using DM rats. A PGI(2) analogue (beraprost sodium; 300 and 600 micro g/kg per day) or vehicle was administered to 16-week-old DM rats induced by administration of streptozotocin for 28 days. Endothelial function was evaluated by the vasodilator response to acetylcholine, and the expression of vascular HGF mRNA was measured by Northern blotting. Of importance, expression of HGF mRNA was significantly decreased in the blood vessels of DM rats as compared with non-DM (P<0.01). In addition, the in vitro vasodilator response of the abdominal aorta to acetylcholine was markedly impaired in DM rats. Importantly, the vasodilator response was restored by PGI(2) treatment in a dose-dependent manner (P<0.01), whereas N(omega)-nitro-l-arginine methyl ester inhibited the restoration of endothelial function. Of particular interest, vascular HGF mRNA and protein were significantly increased in the blood vessels of DM rats treated with PGI(2) as compared with vehicle. Similarly, an increase in HGF protein was also confirmed by immunohistochemical analysis. In addition, the specific HGF receptor, c-met, was also increased by PGI(2) treatment. Overall, this study demonstrated that treatment with a PGI(2) analogue restored endothelial dysfunction in DM rats, accompanied by the induction of vascular HGF and c-met expression. Increased local vascular HGF production by a PGI(2) analogue may prevent endothelial injury, potentially resulting in the improvement of endothelial dysfunction.

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