This research aimed to assess the potential of irigenin to attenuate blue light (BL)-induced apoptosis in human adult retinal pigment epithelial (hARPE-19) cells loaded with N-retinylidene-N-retinylethanolamine (A2E, DA50062). Furthermore, the study investigated the associated molecular mechanisms. Cell viability was assessed using the MTT assay, and flow cytometry was employed to evaluate reactive oxygen species (ROS) production, alterations in mitochondrial membrane potential, and cytochrome c release. Lipid peroxidation levels, as well as the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and caspase enzymes, were quantified using commercially available assay kits. Bcl-2, Bax, heme oxygenase-1 (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2) expression was quantified through western blotting. Moreover, animal experiments were performed to assess BL-induced retinal damage. The results revealed that irigenin protected against BL-induced cytotoxicity and apoptosis in DA50062-laden hARPE-19 cells. Furthermore, irigenin upregulated Bcl-2 expression and downregulated Bax expression in BL-exposed DA50062-laden hARPE-19 cells. Hence, irigenin prevented cytochrome c release and inhibited BL-induced caspase-3 and caspase-9 activation in DA50062-laden hARPE-19 cells. Irigenin also effectively inhibited lipid peroxidation and ROS production in BL-exposed DA50062-laden hARPE-19 cells. Notably, irigenin upregulated Nrf2 expression, which, in turn, upregulated the expression of several antioxidative defense system, such as SOD, CAT, and GSH-Px, and HO-1 in BL-exposed DA50062-laden hARPE-19 cells. Animal studies showed that irigenin effectively protected against BL-induced retinal damage, as indicated by the increased thickness of the outer and inner nuclear layers in irigenin-treated groups compared to untreated controls. Taken together, the results suggest that irigenin inhibits the BL-induced intrinsic apoptotic pathway by activating the Nrf2-mediated antioxidative defense system.

Oxidative damage contributes to age-related macular degeneration. Irigenin possesses diverse pharmacologic properties, including antioxidative and antiapoptotic effects. Our in vivo experiments indicated that irigenin mitigates UVB-induced histopathologic changes and oxidative DNA damage. Histologic analyses and TUNEL staining revealed that this compound dose-dependently ameliorated UVB-induced retinal damage and apoptosis. Furthermore, irigenin substantially reduced the level of 8-hydroxyguanosine, a biomarker of UVB-induced oxidative DNA damage. We further explored the molecular mechanisms that mediate the protective effects of irigenin. Our findings suggested that UVB-induced generation of ROS disrupts the stability of the mitochondrial membrane, activating intrinsic apoptotic pathways; the underlying mechanisms include the release of cytochrome c, activation of caspase-9 and caspase-3, and subsequent degradation of PARP-1. Notably, irigenin reversed mitochondrial disruption and apoptosis. It also modulated the Bax and Bcl-2 expression but influenced the mitochondrial apoptotic pathways. Our study highlights the role of the Nrf2 pathway in mitigating the effects of oxidative stress. We found that UVB exposure downregulated, but irigenin treatment upregulated the expression of Nrf2 and antioxidant enzymes. Therefore, irigenin activates the Nrf2 pathway to address oxidative stress. In conclusion, irigenin exhibits protective effects against UVB-induced ocular damage, evidenced by the diminution of histological alterations. It mitigates oxidative DNA damage and apoptosis in the retinal tissues by modulating the intrinsic apoptotic pathways and the AIF mechanisms. Furthermore, irigenin effectively reduces lipid peroxidation, enhancing the activity of antioxidant enzymes by stimulating the Nrf2 pathway. This protective mechanism underscores the potential benefit of irigenin in combating UVB-mediated ocular damage.

Macrophages play an important role in defending the body against invading pathogens. In the face of pathogens, macrophages become activated and release toxic materials that disrupt the pathogens. Macrophage overactivation can lead to severe illness and inflammation. Wogonin has several therapeutic effects, including anti-inflammatory, anticancer, antioxidant, and neuroprotective effects. No studies have investigated the cytotoxic effects of wogonin at concentrations of more than 0.1 mM in RAW264.7 cells. In this study, RAW 264.7 cells were treated with wogonin, which, at concentrations of more than 0.1 mM, had cytotoxic and genotoxic effects in the RAW264.7 cells, leading to apoptosis and necrosis. Further, wogonin at concentrations of more than 0.1 mM induced caspase-3, caspase-8, and caspase-9 activation and mitochondrial dysfunction and death receptor expression. These results suggest that wogonin induces apoptosis through upstream intrinsic and extrinsic pathways by exhibiting cytotoxic and genotoxic effects.

Rutin, also called quercetin-3-rhamnosyl glucoside, is a natural flavonol glycoside present in many plants. Rutin is used to treat various diseases, such as inflammation, diabetes, and cancer. For polymeric biomaterials, triethylene glycol dimethacrylate (TEGDMA) is the most commonly used monomer and serves as a restorative resin, a dentin bonding agent and sealant, and a bone cement component. Overall, TEGDMA induces various toxic effects in macrophages, including cytotoxicity, apoptosis, and genotoxicity. The aim of this study was to investigate the protective mechanism of rutin in alleviating TEGDMA-induced toxicity in RAW264.7 macrophages. After treatment with rutin, we assessed the cell viability and apoptosis of TEGDMA-induced RAW264.7 macrophages using an methylthiazol tetrazolium (MTT) assay and Annexin V-FITC/propidium iodide assay, respectively. Subsequently, we assessed the level of genotoxicity using comet and micronucleus assays, assessed the cysteinyla aspartate specific proteinases (caspases) and antioxidant enzyme (AOE) activity using commercial kits, and evaluated the generation of reactive oxygen species (ROS) using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay. We evaluated the expression of heme oxygenase (HO)-1, the expression of nuclear factor erythroid 2 related factor (Nrf-2), and phosphorylation of AMP activated protein kinase (AMPK) using the Western blot assay. The results indicated that rutin substantially reduced the level of cytotoxicity, apoptosis, and genotoxicity of TEGDMA-induced RAW264.7 macrophages. Rutin also blocked the activity of caspase-3, caspase-8, and caspase-9 in TEGDMA-stimulated RAW264.7 macrophages. In addition, it decreased TEGDMA-induced ROS generation and AOE deactivation in macrophages. Finally, we found that TEGDMA-inhibited slightly the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation would be revered by rutin. In addition, the HO-1 expression, Nrf-2 expression, and AMPK phosphorylation was enhanced by rutin. These findings indicate that rutin suppresses TEGDMA-induced caspase-mediated toxic effects through ROS generation and antioxidative system deactivation through the Nrf-2/AMPK pathway. Therefore, rutin has the potential to serve as a novel antitoxicity agent for TEGDMA in RAW264.7 macrophages.

Patients with NTG or POAG with more than one outpatient or discharge diagnosis from the ophthalmology department were included in the study. These data were merged with the PM2.5 data from the Air Quality Monitoring Network for analysis. This was a case–control study, with 1006 participants in the NTG group and 2533 in the POAG group. To investigate fine particulate matter (PM2.5) exposure levels in patients with normal-tension glaucoma (NTG) and primary open-angle glaucoma (POAG), patient data were obtained from Taiwan’s Longitudinal Health Insurance Database 2000 for the 2008 to 2013 period. We used a multivariate logic regression model to assess the risk for each participant. The PM2.5 exposure levels were divided into four groups: <25th percentile (Q1), <617 μg/mm3; 25th to 50th percentile (Q2), 617 to 1297 μg/mm3; 50th to 75th percentile (Q3), 1297 to 2113 μg/mm3; and >75th percentile (Q4), >2113 μg/mm3. The results are expressed in terms of odds ratio (OR) and 95% CI. A multiple logistic regression was used to compare the results of the NTG group with those of the POAG group. Compared with the PM2.5 Q1 level, the OR of the PM2.5 Q2 level was 1.009 (95% CI 0.812–1.254), the PM2.5 Q3 level was 1.241 (95% CI 1.241–1.537, p < 0.05), and the PM2.5 Q4 level was 1.246 (95% CI 1.008–1.539, p < 0.05). Our research reveals that compared with POAG, the risk of developing NTG is more closely related with PM2.5 exposure, and PM2.5 has a concentration–dose effect. It is hoped that in the future, in the clinical judgment of NTG and POAG, the level of PM2.5 in the environment can be taken as a risk factor.

Acute lung injury (ALI) is an acute and life-threatening inflammatory disease of the lung parenchyma that is associated with high mortality worldwide. No therapeutic strategies have been developed for the mitigation of the proinflammatory response that characterizes ALI. Kirenol has anti-inflammatory, antiarthritic, and immunoregulatory effects. In the present study, we investigated the protective effects of kirenol against lipopolysaccharides (LPS)-induced ALI in mice. Kirenol reduced the LPS-induced histopathology changes involving edema and thickening of the interstitial or alveolar walls, infiltration of leukocytes, formation of hyaline membrane. Pretreatment with kirenol reduced leukocytes infiltration in bronchoalveolar lavage fluid (BALF), the alveolar-capillary barrier disruption and lipid peroxidation in lung tissues induced by LPS. Kirenol significantly inhibited the secretion of cytokines, IL-1β, IL6, and TNFα, into the BALF of the mice with LPS-induced ALI through NFκB activation. Moreover, kirenol attenuated the downregulation of the antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and catalase that was induced by LPS. HO-1 expression and the phosphorylation of Nrf2 and AMPK2 were also induced by kirenol. The results indicate that kirenol can be developed as a treatment strategy for ALI, and its effects are induced through the inhibition of the NF-κB proinflammatory pathway and promotion of AMPK2/Nrf2-mediated HO-1 and antioxidant enzymes (AOE) activation.

Genotoxic stress from environmental pollutants plays a critical role in cytotoxicity. The most abundant nitro-polycyclic aromatic hydrocarbon in environmental pollutants, 1-nitropyrene (1-NP), is generated during fossil fuel, diesel, and biomass combustion under sunlight. Macrophages, the key regulators of the innate immune system, provide the first line of defense against pathogens. The toxic effects of 1-NP on macrophages remain unclear. Through a lactate dehydrogenase assay, we measured the cytotoxicity induced by 1-NP. Our results revealed that 1-NP induced genotoxicity also named DNA damage, including micronucleus formation and DNA strand breaks, in a concentration-dependent manner. Furthermore, 1-NP induced p53 phosphorylation and nuclear accumulation; mitochondrial cytochrome c release; caspase-3 and -9 activation and cleavage; and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in a concentration-dependent manner. Pretreatment with the PARP inhibitor, 3-aminobenzamide, significantly reduced cytotoxicity, genotoxicity, and PARP-1 cleavage induced by 1-NP. Pretreatment with the caspase-3 inhibitor, z-DEVD-fmk, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, and caspase 3 activation induced by 1-NP. Pretreatment with the p53 inhibitor, pifithrin-α, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, caspase 3 activation, and p53 phosphorylation induced by 1-NP. We propose that cytotoxicity and genotoxicity induced by 1-NP by PARP-1 cleavage via caspase-3 and -9 activation through cytochrome c release from mitochondria and its upstream p53-dependent pathway in macrophages.

Bisphenol-A-glycidyldimethacrylate (BisGMA) is a resin monomer frequently used in dentin restorative treatments. The leakage of BisGMA monomer from BisGMA-based polymeric resins can lead to cytotoxicity in macrophages. Rutin has various beneficial bioeffects, including antioxidation and antiinflammation. In this study, we found that pretreatment of RAW264.7 macrophages with rutin-inhibited cytotoxicity induced by BisGMA in a concentration-dependent manner. BisGMA-induced apoptosis, which was detected by levels of phosphatidylserine from the internal to the external membrane and formation of sub-G1, and genotoxicity, which was detected by cytokinesis-blocked micronucleus and single-cell gel electrophoresis assays, were inhibited by rutin in a concentration-dependent manner. Rutin suppressed the BisGMA-induced activation of caspase-3 and -9 rather than caspase-8. Rutin inhibited the activation of the mitochondrial apoptotic pathway, including cytochrome C release and mitochondria disruption, after macrophages were treated with BisGMA. Finally, BisGMA-induced reactive oxygen species (ROS) generation and antioxidant enzyme (AOE) deactivation could be reversed by rutin. Parallel trends were observed in the elevation of AOE activation and inhibition of ROS generation, caspase-3 activity, mitochondrial apoptotic pathway activation, and genotoxicity. These results suggested that rutin suppressed BisGMA-induced cytotoxicity through genotoxicity, the mitochondrial apoptotic pathway, and relatively upstream factors, including reduction of ROS generation and induction of AOE.

OBJECTIVE:Safrole, also called shikimol and Sassafras, is the carcinogenic and phenylpropanoid compound extracted from Sassafras tree and anise, betel, and camphor. Moreover, a high concentration of safrole can be occur in the saliva because of betel nut or areca quid chewing which a common habit observed in Southern and Southeastern Asia. Notably, macrophages are crucial phagocytic cells of the immune system. Nonetheless, to date, no evidence has been reported regarding safrole-induced proinflammatory response and the corresponding mechanism in macrophages.MATERIALS AND METHODS:In the present study, the cytokines expression, NO generation, protein phosphorylation, and expression were assessed by enzyme-linked immunosorbent assay, Griess reagent, and Western blot assay, respectively.RESULTS:In this study, we determined that safrole induces the generation of nitric oxide and proinflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and IL-6 in RAW264.7 macrophages in a concentration-dependent manner. Furthermore, inhibitor of κB (IκB) degradation was caused by safrole in a concentration-dependent manner. In addition, the phosphorylation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) family, including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase, was induced by safrole began to increase at 10 μM and attained a plateau at 100 μM.CONCLUSION:These results indicated that safrole induces the expression of proinflammatory responses in macrophages through the NF-κB/IκB pathway and its upstream factor, MAPK family phosphorylation.

Manganese (Mn) is an essential trace element maintained at an optimal level in human body. Mn plays the important activator of enzymes in metabolism for protein digestion and utilization. The major exposure source of Mn is from the occupational environment, including welders and miners are the occupational exposure population. Manganese nitrate (Mn(NO3)2), a Mn compound, is the major material in alloy and battery. There are evidence have demonstrated that Mn is a potential risk factor in neurological disorders. Up to now, the evidence for Mn(NO3)2-induced cytotoxicity and genotoxicity and its related mechanism in macrophages is not supportive. At the present study, we first found that Mn (NO3)2 induced cytotoxicity in RAW 264.7 cells in a concentration- and time-dependent manners. Genotoxicty, including microcucleus (MN) formation and DNA damage, was induced by Mn(NO3)2 in a concentration-dependent manner via alkaline single cell gel electrophoresis (COMET) and cytokinesis-block MN assays. In addition, apoptosis and related signal molecular mechanism, including caspases (-3, -8, and -9) activation and mitochondria disruption, were induced by Mn(NO3)2. ROS generation play an important role in apoptosis. Here, we also found intracellular ROS generation induced by Mn(NO3)2. More, N-acetyl-cysteine (NAC), a ROS scavenger, decreased the effects of cytotoxicity and genotoxicity induced by Mn(NO3)2.In conclusion, we had demonstrated that Mn(NO3)2-induced cytotoxicity and genotoxicity on RAW 264.7cells were mediated by caspase-3,-8,and -9 activation.