Synthetic Secoisolariciresinol Diglucoside (LGM2605) Prevents Asbestos-Induced Inflammation and Genotoxic Cell Damage in Human Mesothelial Cells

Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm²). Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release (IL-1β, IL-18, IL-6, TNFα, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1β and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL ± 0.2 pg/mL and 5.4 pg/mL ± 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high (289.1 pg/mL ± 2.9 pg/mL and 511.3 pg/mL ± 10.2 pg/mL, respectively), while remaining undetectable with LGM2605 pretreatment. HMGB1 (a key inflammatory mediator and initiator of malignant transformation) release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells.     

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 β release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 β release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.     

Late Inflammation Induced by Asbestiform Fibers in Mice Is Ameliorated by a Small Molecule Synthetic Lignan

Exposure to Libby amphibole (LA) asbestos-like fibers is associated with increased risk of asbestosis, mesothelioma, pulmonary disease, and systemic autoimmune disease. LGM2605 is a small molecule antioxidant and free radical scavenger, with anti-inflammatory effects in various disease models. The current study aimed to determine whether the protective effects of LGM2605 persist during the late inflammatory phase post-LA exposure. Male and female C57BL/6 mice were administered daily LGM2605 (100 mg/kg) via gel cups for 3 days before and 14 days after a 200 µg LA given via intraperitoneal (i.p.) injection. Control mice were given unsupplemented gel cups and an equivalent dose of i.p. saline. On day 14 post-LA treatment, peritoneal lavage was assessed for immune cell influx, cytokine concentrations, oxidative stress biomarkers, and immunoglobulins. During the late inflammatory phase post-LA exposure, we noted an alteration in trafficking of both innate and adaptive immune cells, increased pro-inflammatory cytokine concentrations, induction of immunoglobulin isotype switching, and increased oxidized guanine species. LGM2605 countered these changes similarly among male and female mice, ameliorating late inflammation and altering immune responses in late post-LA exposure. These data support possible efficacy of LGM2605 in the prolonged treatment of LA-associated disease and other inflammatory conditions.     

Copper Oxide Nanoparticle-Induced Acute Inflammatory Response and Injury in Murine Lung Is Ameliorated by Synthetic Secoisolariciresinol Diglucoside (LGM2605)

Metal-oxide nanoparticles (MO-NPs), such as the highly bioreactive copper-based nanoparticles (CuO-NPs), are widely used in manufacturing of hundreds of commercial products. Epidemiological studies correlated levels of nanoparticles in ambient air with a significant increase in lung disease. CuO-NPs, specifically, were among the most potent in a set of metal-oxides and carbons studied in parallel regarding DNA damage and cytotoxicity. Despite advances in nanotoxicology research and the characterization of their toxicity, the exact mechanism(s) of toxicity are yet to be defined. We identified chlorination toxicity as a damaging consequence of inflammation and myeloperoxidase (MPO) activation, resulting in macromolecular damage and cell damage/death. We hypothesized that the inhalation of CuO-NPs elicits an inflammatory response resulting in chlorination damage in cells and lung tissues. We further tested the protective action of LGM2605, a synthetic small molecule with known scavenging properties for reactive oxygen species (ROS), but most importantly, for active chlorine species (ACS) and an inhibitor of MPO. CuO-NPs (15 µg/bolus) were instilled intranasally in mice and the kinetics of the inflammatory response in lungs was evaluated 1, 3, and 7 days later. Evaluation of the protective action of LGM2605 was performed at 24 h post-challenge, which was selected as the peak acute inflammatory response to CuO-NP. LGM2605 was given daily via gavage to mice starting 2 days prior to the time of the insult (100 mg/kg). CuO-NPs induced a significant inflammatory influx, inflammasome-relevant cytokine release, and chlorination damage in mouse lungs, which was mitigated by the action of LGM2605. Preventive action of LGM2605 ameliorated the adverse effects of CuO-NP in lung.     

The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure.     

Embryonic-Derived Myb− Macrophages Enhance Bacterial Clearance and Improve Survival in Rat Sepsis

Peritoneal resident macrophages play a key role in combating sepsis in the peritoneal cavity. We sought to determine if peritoneal transplantation of embryonic Myb⁻ “peritoneal-like” macrophages attenuate abdominal fecal sepsis. Directed differentiation of rodent pluripotent stem cells (PSCs) was used in factor-defined media to produce embryonic-derived large “peritoneal-like” macrophages (Ed-LPM) that expressed peritoneal macrophage markers and demonstrated phagocytic capacity. Preclinical in vivo studies determined Ed-LPM efficacy in rodent abdominal fecal sepsis with or without Meropenem. Ex vivo studies explored the mechanism and effects of Ed-LPM on host immune cell number and function, including phagocytosis, reactive oxygen species (ROS) production, efferocytosis and apoptosis. Ed-LPM reduced sepsis severity by decreasing bacterial load in the liver, spleen and lungs. Ed-LPM therapy significantly improved animal survival by ~30% and reduced systemic bacterial burden to levels comparable to Meropenem therapy. Ed-LPM therapy decreased peritoneal TNFα while increasing IL-10 concentrations. Ed-LPMs enhanced peritoneal macrophage phagocytosis of bacteria, increased macrophage production of ROS and restored homeostasis via apoptosis and efferocytosis-induced clearance of neutrophils. In conclusion, Ed-LPM reduced systemic sepsis severity, improved survival and reduced bacterial load by enhancing peritoneal macrophage bacterial phagocytosis and killing and clearance of intra-peritoneal neutrophils. Macrophage therapy may be a potential strategy to address sepsis.     

SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis,mt-DNA Damage,and Lung Fibrogenic Monocyte Recruitment

Alveolar epithelial cell (AEC) mitochondrial (mt) DNA damage and fibrotic monocyte-derived alveolar macrophages (Mo-AMs) are implicated in the pathobiology of pulmonary fibrosis. We showed that sirtuin 3 (SIRT3), a mitochondrial protein regulating cell fate and aging, is deficient in the AECs of idiopathic pulmonary fibrosis (IPF) patients and that asbestos- and bleomycin-induced lung fibrosis is augmented in Sirt3 knockout (Sirt3^−/−) mice associated with AEC mtDNA damage and intrinsic apoptosis. We determined whether whole body transgenic SIRT3 overexpression (Sirt3^Tg) protects mice from asbestos-induced pulmonary fibrosis by mitigating lung mtDNA damage and Mo-AM recruitment. Crocidolite asbestos (100 µg/50 µL) or control was instilled intratracheally in C57Bl6 (Wild-Type) mice or Sirt3^Tg mice, and at 21 d lung fibrosis (histology, fibrosis score, Sircol assay) and lung Mo-AMs (flow cytometry) were assessed. Compared to controls, Sirt3^Tg mice were protected from asbestos-induced pulmonary fibrosis and had diminished lung mtDNA damage and Mo-AM recruitment. Further, pharmacologic SIRT3 inducers (i.e., resveratrol, viniferin, and honokiol) each diminish oxidant-induced AEC mtDNA damage in vitro and, in the case of honokiol, protection occurs in a SIRT3-dependent manner. We reason that SIRT3 preservation of AEC mtDNA is a novel therapeutic focus for managing patients with IPF and other types of pulmonary fibrosis.     

New In Vitro Model of Oxidative Stress: Human Prostate Cells Injured with 2,2-diphenyl-1-picrylhydrazyl (DPPH) for the Screening of Antioxidants

The antioxidant activity of natural compounds consists in their ability to modulate gene and protein expression, thus inducing an integrated cell protective response and repair processes against oxidative stress. New screening tools and methodologies are crucial for the actual requirement of new products with antioxidant activity to boost endogenous oxidative stress responsive pathways, Reactive Oxygen Species (ROS) metabolism and immune system activity, preserving human health and wellness. In this study, we performed and tested an integrated oxidative stress analysis, using DPPH assay and PNT2 cells injured with DPPH. We firstly investigated the mechanism of action of the oxidising agent (DPPH) on PNT2 cells, studying the variation in cell viability, oxidative stress genes, inflammatory mediator and ROS levels. The results reveal that DPPH activated ROS production and release of Prostaglandin E₂ in PNT2 at low and intermediate doses, while cells switched from survival to cell death signals at high doses of the oxidising agent. This new in vitro oxidative stress model was validated by using Trolox, β-carotene and total extract of the green microalga Testraselmis suecica. Only the T. suecica extract can completely counteract DPPH-induced injury, since its chemical complexity demonstrated a multilevel protecting and neutralising effect against oxidative stress in PNT2.     

Methylmercury Induces Mitochondria- and Endoplasmic Reticulum Stress-Dependent Pancreatic β-Cell Apoptosis via an Oxidative Stress-Mediated JNK Signaling Pathway

Methylmercury (MeHg), a long-lasting organic pollutant, is known to induce cytotoxic effects in mammalian cells. Epidemiological studies have suggested that environmental exposure to MeHg is linked to the development of diabetes mellitus (DM). The exact molecular mechanism of MeHg-induced pancreatic β-cell cytotoxicity is still unclear. Here, we found that MeHg (1-4 μM) significantly decreased insulin secretion and cell viability in pancreatic β-cell-derived RIN-m5F cells. A concomitant elevation of mitochondrial-dependent apoptotic events was observed, including decreased mitochondrial membrane potential and increased proapoptotic (Bax, Bak, p53)/antiapoptotic (Bcl-2) mRNA ratio, cytochrome c release, annexin V-Cy3 binding, caspase-3 activity, and caspase-3/-7/-9 activation. Exposure of RIN-m5F cells to MeHg (2 μM) also induced protein expression of endoplasmic reticulum (ER) stress-related signaling molecules, including C/EBP homologous protein (CHOP), X-box binding protein (XBP-1), and caspase-12. Pretreatment with 4-phenylbutyric acid (4-PBA; an ER stress inhibitor) and specific siRNAs for CHOP and XBP-1 significantly inhibited their expression and caspase-3/-12 activation in MeHg-exposed RIN-mF cells. MeHg could also evoke c-Jun N-terminal kinase (JNK) activation and reactive oxygen species (ROS) generation. Antioxidant N-acetylcysteine (NAC; 1mM) or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox; 100 μM) markedly prevented MeH-induced ROS generation and decreased cell viability in RIN-m5F cells. Furthermore, pretreatment of cells with SP600125 (JNK inhibitor; 10 μM) or NAC (1 mM) or transfection with JNK-specific siRNA obviously attenuated the MeHg-induced JNK phosphorylation, CHOP and XBP-1 protein expression, apoptotic events, and insulin secretion dysfunction. NAC significantly inhibited MeHg-activated JNK signaling, but SP600125 could not effectively reduce MeHg-induced ROS generation. Collectively, these findings demonstrate that the induction of ROS-activated JNK signaling is a crucial mechanism underlying MeHg-induced mitochondria- and ER stress-dependent apoptosis, ultimately leading to β-cell death.     

T-2 Toxin Induces Oxidative Stress at Low Doses via Atf3ΔZip2a/2b-Mediated Ubiquitination and Degradation of Nrf2

T-2 toxin is mainly produced by Fusarium species, which is an extremely toxic mycotoxin to humans and animals. It is well known that T-2 toxin induces oxidative stress, but the molecular mechanism is still unknown. In this study, we found that T-2 toxin significantly promoted reactive oxygen species (ROS) accumulation in MCF-7 cells at low doses which maintains cell viability at least 80%. Further analysis showed that T-2 toxin downregulated the expression of the master regulator of antioxidant defense gene, nuclear factor erythroid 2-related factor (Nrf2), and its targeted antioxidant genes. Overexpression of Nrf2 or its target gene heme oxygenase 1 (HO1) significantly blocked the ROS accumulation in MCF-7 cells under T-2 toxin treatment. Moreover, we found that T-2 toxin downregulated the antioxidant genes via inducing the expression of ATF3ΔZip2a/2b. Importantly, overexpression of ATF3ΔZip2a/2b promoted the ubiquitination and degradation of Nrf2. Altogether, our results demonstrated that T-2 toxin-induced ROS accumulation via ATF3ΔZip2a/2b mediated ubiquitination and degradation of Nrf2, which provided a new insight into the mechanism of T-2 toxin-induced oxidative stress.     

NLRP3 Inflammasome Negatively Regulates RANKL-Induced Osteoclastogenesis of Mouse Bone Marrow Macrophages but Positively Regulates It in the Presence of Lipopolysaccharides

In inflammatory bone diseases such as periodontitis, the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3 (NLRP3) inflammasome accelerates bone resorption by promoting proinflammatory cytokine IL-1β production. However, the role of the NLRP3 inflammasome in physiological bone remodeling remains unclear. Here, we investigated its role in osteoclastogenesis in the presence and absence of lipopolysaccharide (LPS), a Gram-negative bacterial component. When bone marrow macrophages (BMMs) were treated with receptor activator of nuclear factor-κB ligand (RANKL) in the presence of NLRP3 inflammasome inhibitors, osteoclast formation was promoted in the absence of LPS but attenuated in its presence. BMMs treated with RANKL and LPS produced IL-1β, and IL-1 receptor antagonist inhibited osteoclastogenesis, indicating IL-1β involvement. BMMs treated with RANKL alone produced no IL-1β but increased reactive oxygen species (ROS) production. A ROS inhibitor suppressed apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) speck formation and NLRP3 inflammasome inhibitors abrogated cytotoxicity in BMMs treated with RANKL, indicating that RANKL induces pyroptotic cell death in BMMs by activating the NLRP3 inflammasome via ROS. This suggests that the NLRP3 inflammasome promotes osteoclastogenesis via IL-1β production under infectious conditions, but suppresses osteoclastogenesis by inducing pyroptosis in osteoclast precursors under physiological conditions.     

Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of β-Catenin

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced β-catenin protein, but not mRNA levels. The reduction of β-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.     

Carnosic Acid Attenuates an Early Increase in ROS Levels during Adipocyte Differentiation by Suppressing Translation of Nox4 and Inducing Translation of Antioxidant Enzymes

The objective of this study was to investigate molecular mechanisms underlying the ability of carnosic acid to attenuate an early increase in reactive oxygen species (ROS) levels during MDI-induced adipocyte differentiation. The levels of superoxide anion and ROS were determined using dihydroethidium (DHE) and 2′-7′-dichlorofluorescin diacetate (DCFH-DA), respectively. Both superoxide anion and ROS levels peaked on the second day of differentiation. They were suppressed by carnosic acid. Carnosic acid attenuates the translation of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4), p47^phox, and p22^phox, and the phosphorylation of nuclear factor-kappa B (NF-κB) and NF-κB inhibitor (IkBa). The translocation of NF-κB into the nucleus was also decreased by carnosic acid. In addition, carnosic acid increased the translation of heme oxygenase-1 (HO-1), γ–glutamylcysteine synthetase (γ-GCSc), and glutathione S-transferase (GST) and both the translation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2). Taken together, these results indicate that carnosic acid could down-regulate ROS level in an early stage of MPI-induced adipocyte differentiation by attenuating ROS generation through suppression of NF-κB-mediated translation of Nox4 enzyme and increasing ROS neutralization through induction of Nrf2-mediated translation of phase II antioxidant enzymes such as HO-1, γ-GCS, and GST, leading to its anti-adipogenetic effect.     

DHA and Its Metabolites Have a Protective Role against Methylmercury-Induced Neurotoxicity in Mouse Primary Neuron and SH-SY5Y Cells

The consumption of fish now involves a risk of methylmercury (MeHg) exposure but also provides the benefit of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) such as docosahexaenoic acid (DHA). Some epidemiological studies have suggested that the intake of DHA can alleviate the neurotoxicity of MeHg, but the underlying mechanism is not known. Herein, we observed that pretreatment with 0.1–1 µM DHA suppressed MeHg-induced cytotoxicity in human neuroblastoma (SH-SY5Y) cells and mouse primary neuronal cells. These effects of DHA were canceled in the presence of the retinoid X receptor (RXR) antagonist UVI3003. An RXR agonist, bexarotene, suppressed the cytotoxicity of MeHg. DHA also suppressed the MeHg-induced production of reactive oxygen species (ROS) via an induction of antioxidant genes (catalase and SOD1). Pretreatment with DHA did not change the incorporation of MeHg. We showed previously that in the brain, the intake of DHA increased the level of 19,20-DHDP, which is the metabolite produced by cytochrome P450 and soluble epoxide hydrolase from DHA. In the present study, we observed that 19,20-DHDP also suppressed neurotoxicity from MeHg. These results indicate that DHA and its metabolites have a protective role in MeHg-induced neurotoxicity.