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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Radiation mitigating properties of the lignan component in flaxseed

BACKGROUND: Wholegrain flaxseed (FS), and its lignan component (FLC) consisting mainly of secoisolariciresinol diglucoside (SDG), have potent lung radioprotective properties while not abrogating the efficacy of radiotherapy. However, while the whole grain was recently shown to also have potent mitigating properties in a thoracic radiation pneumonopathy model, the bioactive component in the grain responsible for the mitigation of lung damage was never identified. Lungs may be exposed to radiation therapeutically for thoracic malignancies or incidentally following detonation of a radiological dispersion device. This could potentially lead to pulmonary inflammation, oxidative tissue injury, and fibrosis. This study aimed to evaluate the radiation mitigating effects of FLC in a mouse model of radiation pneumonopathy. METHODS: We evaluated FLC-supplemented diets containing SDG lignan levels comparable to those in 10% and 20% whole grain diets. 10% or 20% FLC diets as compared to an isocaloric control diet (0% FLC) were given to mice (C57/BL6) (n=15-30 mice/group) at 24, 48, or 72-hours after single-dose (13.5 Gy) thoracic x-ray treatment (XRT). Mice were evaluated 4 months post-XRT for blood oxygenation, lung inflammation, fibrosis, cytokine and oxidative damage levels, and survival. RESULTS: FLC significantly mitigated radiation-related animal death. Specifically, mice fed 0% FLC demonstrated 36.7% survival 4 months post-XRT compared to 60–73.3% survival in mice fed 10%-20% FLC initiated 24–72 hours post-XRT. FLC also mitigated radiation-induced lung fibrosis whereby 10% FLC initiated 24-hours post-XRT significantly decreased fibrosis as compared to mice fed control diet while the corresponding TGF-beta1 levels detected immunohistochemically were also decreased. Additionally, 10-20% FLC initiated at any time point post radiation exposure, mitigated radiation-induced lung injury evidenced by decreased bronchoalveolar lavage (BAL) protein and inflammatory cytokine/chemokine release at 16 weeks post-XRT. Importantly, neutrophilic and overall inflammatory cell infiltrate in airways and levels of nitrotyrosine and malondialdehyde (protein and lipid oxidation, respectively) were also mitigated by the lignan diet. CONCLUSIONS: Dietary FLC given early post-XRT mitigated radiation effects by decreasing inflammation, lung injury and eventual fibrosis while improving survival. FLC may be a useful agent, mitigating adverse effects of radiation in individuals exposed to incidental radiation, inhaled radioisotopes or even after the initiation of radiation therapy to treat malignancy

The Synthetic Lignan Secoisolariciresinol Diglucoside Prevents Asbestos-Induced NLRP3 Inflammasome Activation in Murine Macrophages

Background. The interaction of asbestos with macrophages drives two key processes that are linked to malignancy: (1) the generation of reactive oxygen species (ROS)/reactive nitrogen species (RNS) and (2) the activation of an inflammation cascade that drives acute and chronic inflammation, with the NLRP3 inflammasome playing a key role. Synthetic secoisolariciresinol diglucoside (SDG), LGM2605, is a nontoxic lignan with anti-inflammatory and antioxidant properties and was evaluated for protection from asbestos in murine peritoneal macrophages (MF). Methods. MFs were exposed to crocidolite asbestos ± LGM2605 given 4 hours prior to exposure and evaluated at various times for NLRP3 expression, secretion of inflammasome-activated cytokines (IL-1β and IL-18), proinflammatory cytokines (IL-6, TNFα, and HMGB1), NF-κB activation, and levels of total nitrates/nitrites. Results. Asbestos induces a significant (p<0.0001) increase in the NLRP3 subunit, release of proinflammatory cytokines, NLRP3-activated cytokines, NF-κB, and levels of nitrates/nitrites. LGM2605 significantly reduced NLRP3 ranging from 40 to 81%, IL-1β by 89–96%, and TNFα by 67–78%, as well as activated NF-κB by 48-49% while decreasing levels of nitrates/nitrites by 85–93%. Conclusions. LGM2605 reduced asbestos-induced NLRP3 expression, proinflammatory cytokine release, NF-κB activation, and nitrosative stress in MFs supporting its possible use in preventing the asbestos-induced inflammatory cascade leading to malignancy

Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605)

The interaction of asbestos fibers with macrophages generates harmful reactive oxygen species (ROS) and subsequent oxidative cell damage that are key processes linked to malignancy. Secoisolariciresinol diglucoside (SDG) is a non-toxic, flaxseed-derived pluripotent compound that has antioxidant properties and may thus function as a chemopreventive agent for asbestos-induced mesothelioma. We thus evaluated synthetic SDG (LGM2605) in asbestos-exposed, elicited murine peritoneal macrophages as an in vitro model of tissue phagocytic response to the presence of asbestos in the pleural space. Murine peritoneal macrophages (MFs) were exposed to crocidolite asbestos fibers (20 µg/cm2) and evaluated at various times post exposure for cytotoxicity, ROS generation, malondialdehyde (MDA), and levels of 8-iso Prostaglandin F2α (8-isoP). We then evaluated the ability of LGM2605 to mitigate asbestos-induced oxidative stress by administering LGM2605 (50 µM) 4-h prior to asbestos exposure. We observed a significant (p &lt; 0.0001), time-dependent increase in asbestos-induced cytotoxicity, ROS generation, and the release of MDA and 8-iso Prostaglandin F2α, markers of lipid peroxidation, which increased linearly over time. LGM2605 treatment significantly (p &lt; 0.0001) reduced asbestos-induced cytotoxicity and ROS generation, while decreasing levels of MDA and 8-isoP by 71%–88% and 41%–73%, respectively. Importantly, exposure to asbestos fibers induced cell protective defenses, such as cellular Nrf2 activation and the expression of phase II antioxidant enzymes, HO-1 and Nqo1 that were further enhanced by LGM2605 treatment. LGM2605 boosted antioxidant defenses, as well as reduced asbestos-induced ROS generation and markers of oxidative stress in murine peritoneal macrophages, supporting its possible use as a chemoprevention agent in the development of asbestos-induced malignant mesothelioma

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

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

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2−/−) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm2) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1β (IL-1β), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2−/− macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation

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 &micro;M) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 &micro;g/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-1&beta;, IL-18, IL-6, TNF&alpha;, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2&alpha;). Asbestos induced a time-dependent ROS increase that was significantly (p &lt; 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% &plusmn; 1.0%. Although levels of inflammasome-activated cytokines, IL-1&beta; and IL-18, reached 29.2 pg/mL &plusmn; 0.7 pg/mL and 43.9 pg/mL &plusmn; 0.8 pg/mL, respectively, LGM2605 treatment significantly (p &lt; 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL &plusmn; 0.2 pg/mL and 5.4 pg/mL &plusmn; 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNF&alpha; in asbestos-exposed mesothelial cells were high (289.1 pg/mL &plusmn; 2.9 pg/mL and 511.3 pg/mL &plusmn; 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% &plusmn; 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2&alpha;, markers of oxidative cell injury, were significantly (p &lt; 0.001) reduced by 80.5% &plusmn; 0.1% and 76.6% &plusmn; 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