Inhibition Of Reactive Oxygen Species Generation Research Articles

IRGM/Irgm1 facilitates macrophage apoptosis through ROS generation and MAPK signal transduction: Irgm1+/- mice display increases atherosclerotic plaque stability.

Rationale: Atherosclerosis plaque rupture (PR) is the pathological basis and chief culprit of most acute cardiovascular events and death. Given the complex and important role of macrophage apoptosis and autophagy in affecting plaque stability, an important unanswered question include is whether, and how, immunity-related GTPase family M protein (IRGM) and its mouse orthologue IRGM1 affect macrophage survival and atherosclerotic plaque stability.Methods: To investigate whether serum IRGM of ST-segment elevation myocardial infarction (STEMI) patients is related to plaque morphology, we divided 85 STEMI patients into those with and without plaque rupture (PR and non-PR, respectively) based on OCT image analysis, and quantified the patients' serum IRGM levels. Next, we engineered Irgm1 deficient mice (Irgm1+/-) and chimera mice with Irgm1 deficiency in the bone marrow on an ApoE-/- background, which were then fed a high-fat diet for 16 weeks. Pathological staining was used to detect necrotic plaque cores, ratios of neutral lipids and cholesterol crystal, as well as collagen fiber contents in these mice to characterize plaque stability. In addition, immunofluorescence, immunohistochemical staining and western blot were used to detect the apoptosis of macrophages in the plaques. In vitro, THP-1 and RAW264.7 cells were stimulated with ox-LDL to mimic the in vivo environment, and IRGM/IRGM1 expression were modified by specific siRNA (knockdown) or IRGM plasmid (knocked-in). The effect of IRGM/Irgm1 on autophagy and apoptosis of macrophages induced by ox-LDL was then evaluated. In addition, we introduced inhibitors of the JNK/p38/ERK signaling pathway to verify the specific mechanism by which Irgm1 regulates RAW264.7 cell apoptosis.Results: The serum IRGM levels of PR patients is significantly higher than that of non-PR patients and healthy volunteers, which may be an effective predictor of PR. On a high-fat diet, Irgm1-deficient mice exhibit reduced necrotic plaque cores, as well as neutral lipid and cholesterol crystal ratios, with increased collagen fiber content. Additionally, macrophage apoptosis is inhibited in the plaques of Irgm1-deficient mice. In vitro, IRGM/Irgm1 deficiency rapidly inhibits ox-LDL-induced macrophage autophagy while inhibiting ox-LDL-induced macrophage apoptosis in late stages. Additionally, IRGM/Irgm1 deficiency suppresses reactive oxygen species (ROS) production in macrophages, while removal of ROS effectively inhibits macrophage apoptosis induced by IRGM overexpression. We further show that Irgm1 can affect macrophage apoptosis by regulating JNK/p38/ERK phosphorylation in the MAPK signaling pathway.Conclusions: Serum IRGM may be related to the process of PR in STEMI patients, and IRGM/Irgm1 deficiency increases plaque stability. In addition, IRGM/Irgm1 deficiency suppresses macrophage apoptosis by inhibiting ROS generation and MAPK signaling transduction. Cumulatively, these results suggest that targeting IRGM may represent a new treatment strategy for the prevention and treatment of acute cardiovascular deaths caused by PR.

Anti-proliferative, pro-apoptotic and anti-invasive effect of the copper coordination compound Cas III-La through the induction of reactive oxygen species and regulation of Wnt/β-catenin pathway in glioma.

Gliomas are the most aggressive neoplasms that affect the central nervous system, being glioblastoma multiforme (GBM) the most malignant. The resistance of GBM to therapies is attributed to its high rate of cell proliferation, angiogenesis, invasion, and resistance to apoptosis; thus, finding alternative therapeutic approaches is vital. In this work, the anti-proliferative, pro-apoptotic, and anti-invasive effect of the copper coordination compound Casiopeina III-La (Cas III-La) on human U373 MG cells was determined in vitro and in vivo. Our results indicate that Cas III-La exerts an anti-proliferative effect, promoting apoptotic cell death and inactivating the invasive process by generating reactive oxygen species (ROS), inactivating GSK3β, activating JNK and ERK, and promoting the nuclear accumulation of β-catenin. The inhibition of ROS generation by N-acetyl-l-cysteine not only recovered cell migration and viability, but also reduced β-catenin accumulation and JNK and ERK activation. Additionally, Cas III-La significantly reduced tumor volume, cell proliferation and mitotic indices, and increased the apoptotic index in mice xenotransplanted with U373 glioma cells. Thus, Cas III-La is a promising agent to treat GBM.

Endogenous reactive oxygen species and nitric oxide have opposite roles in regulating HIF-1alpha expression in hypoxic astrocytes.

Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.

Azelaic Acid Exerts Antileukemia Effects against Acute Myeloid Leukemia by Regulating the Prdxs/ROS Signaling Pathway.

Acute myeloid leukemia (AML) is a hematological malignancy with a poor prognosis attributed to elevated reactive oxygen species (ROS) levels. Thus, agents that inhibit ROS generation in AML should be exploited. Azelaic acid (AZA), a small molecular compound, can scavenge ROS and other free radicals, exerting antitumor effects on various tumor cells. Herein, this study evaluated the antileukemic activity of AZA against AML via regulation of the ROS signaling pathway. We found that AZA reduced intracellular ROS levels and increased total antioxidant capacity in AML cell lines and AML patient cells. AZA suppressed the proliferation of AML cell lines and AML patient cells, expending minimal cytotoxicity on healthy cells. Laser confocal microscopy showed that AZA-treated AML cells surged and ruptured gradually on microfluidic chips. Additionally, AZA promoted AML cell apoptosis and arrested the cell cycle at the G1 phase. Further analysis demonstrated that peroxiredoxin (Prdx) 2 and Prdx3 were upregulated in AZA-treated AML cells. In vivo, AZA prolonged survival and attenuated AML by decreasing CD33+ immunophenotyping in the bone marrow of a patient-derived xenograft AML model. Furthermore, mice in the AZA-treated group had an increased antioxidant capacity and Prdx2/Prdx3 upregulation. The findings indicate that AZA may be a potential agent against AML by regulating the Prdxs/ROS signaling pathway.

Naringenin alleviates nonalcoholic steatohepatitis in middle-aged Apoe−/−mice: role of SIRT1

BackgroundNaringenin is naturally isolated from citrus fruits possessing many pharmacological activities. However, little is known about the effect of naringenin on nonalcoholic steatohepatitis (NASH) in the model of metabolic syndrome. PurposeThe present study is aimed to investigate the effect of naringenin on NASH in 12-mo-old male ApoE−/− mice and its possible underlying mechanism. MethodsIn vivo, 12-mo-old male ApoE−/− mice were administrated with naringenin by intragastric gavage for 12 weeks. At the end of experiment, the blood samples and liver tissues were collected. Metabolic parameters in serum, levels of triglyceride, cholesterol and hydroxyproline, activities of antioxidant enzymes, and content of inflammatory cytokines (TNF-α and IL-6) in liver were examined by corresponding assay kits. Pathological changes in liver were observed by hematoxylin-eosin, oil red O, masson's trichrome, picro-sirius red and senescence β-galactosidase staining. Dihydroethidium was used for detection of reactive oxygen species (ROS). In vitro, AML-12 cells were treated with oleic acid in the presence or absence of naringenin for 24 h. Transfection of SIRT1 siRNA was also conducted in vitro. Lipid accumulation, cellular ROS generation, malondialdehyde content, antioxidant enzyme activities and secretion levels of TNF-α and IL-6 were examined. Both in vivo and in vitro, gene expressions were detected by real-time PCR or western blot. ResultsNaringenin administration improved metabolic parameters, suppressed hepatic steatosis, regulated expression of genes involved in lipid metabolism (FASN, SCD1, PPARα and CPT1α), reduced hepatic fibrosis and cell senescence, inhibited hepatic inflammation as evidenced by the decreased macrophage recruitment and content of TNF-α and IL-6, and reduced hepatic oxidative stress by suppressing ROS generation and normalizing activities of antioxidant enzymes. Notably, naringenin administration increased hepatic SIRT1 protein expression and activity along with the increased deacetylation of liver kinase B1 (LKB1), PGC1α and NF-κB. In vitro study, the benefits of naringenin on lipid accumulation, oxidative stress and inflammation were diminished by SIRT1 siRNA transfection. ConclusionsThese results indicate that naringenin administration may be a potential curative therapy for NASH treatment and the activation of hepatic SIRT1-mediated signaling cascades is involved in its beneficial effects.

Suppressing ROS generation by apocynin inhibited cyclic stretch-induced inflammatory reaction in HPDLCs via a caspase-1 dependent pathway

It has been reported that cyclic stretch could induce inflammatory reaction in human periodontal ligament cells (HPDLCs). Though reactive oxygen species (ROS) has been reported to be involved in pathogen-induced periodontal inflammatory reaction, its role in the force-related periodontal diseases has not been well clarified. This study inspected the role of ROS in the cyclic stretch-induced inflammatory reaction in HPDLCs and studied the inhibitory effect of antioxidant apocynin on this inflammatory reaction. Results confirmed that cyclic stretch induced inflammatory reaction and production of ROS in HPDLCs. This inflammatory reaction was inhibited by apocynin through blocking the production of ROS. The cyclic stretch also induced the expression of caspase-1 and NLRP3 inflammasome, which could also be inhibited by apocynin. Moreover, the cyclic stretch-induced inflammatory reaction was inhibited by caspase-1 inhibitor. Collectively, it is the first time that increased intracellular ROS was proved to play as an intermediate signal in the cyclic stretch-induced inflammatory reaction in HPDLCs, via a caspase-1-dependent pathway. The inhibitory effect of apocynin on the cyclic stretch-induced inflammatory reaction in HPDLCs shows the potential of antioxidants in the treatment of force-related periodontal inflammatory diseases.

Pharmacological Effects of Salvianolic Acid B Against Oxidative Damage.

Salvianolic acid B (Sal B) is one of the main active ingredients of Salvia miltiorrhiza, with strong antioxidant effects. Recent findings have shown that Sal B has anti-inflammatory, anti-apoptotic, anti-fibrotic effects and can promote stem cell proliferation and differentiation, and has a beneficial effect on cardiovascular and cerebrovascular diseases, aging, and liver fibrosis. Reactive oxygen species (ROS) include oxygen free radicals and oxygen-containing non-free radicals. ROS can regulate cell proliferation, survival, death and differentiation to regulate inflammation, and immunity, while Sal B can scavenge oxygen free radicals by providing hydrogen atoms and reduce the production of oxygen free radicals and oxygen-containing non-radicals by regulating the expression of antioxidant enzymes. The many pharmacological effects of Sal B may be closely related to its elimination and inhibition of ROS generation, and Nuclear factor E2-related factor 2/Kelch-like ECH-related protein 1 may be the core link in its regulation of the expression of antioxidant enzyme to exert its antioxidant effect. What is confusing and interesting is that Sal B exhibits the opposite mechanisms in tumors. To clarify the specific target of Sal B and the correlation between its regulation of oxidative stress and energy metabolism homeostasis will help to further understand its role in different pathological conditions, and provide a scientific basis for its further clinical application and new drug development. Although Sal B has broad prospects in clinical application due to its extensive pharmacological effects, the low bioavailability is a serious obstacle to further improving its efficacy in vivo and promoting clinical application. Therefore, how to improve the availability of Sal B in vivo requires the joint efforts of many interdisciplinary subjects.

Dan Hong Injection Protects Against Cardiomyocytes Apoptosis by Maintaining Mitochondrial Integrity Through Keap1/Nuclear Factor Erythroid 2-Related Factor 2/JNK Pathway.

Danhong injection (DHI) is used widely against cardiovascular disease in China. Recent studies have demonstrated its mitochondria-protection effect as being pivotal in treatment of myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism of action is incompletely understood. We aimed to identify the effect and mechanism of action of DHI on mitochondrial integrity and cardiomyocyte apoptosis after I/R. An I/R rat model was induced to detect the effect of DHI on myocardial repair by infarct size, apoptosis and oxidative stress. In vitro, H9C2 cells or H9C2 cells with nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown were injured under hypoxia-reoxygenation (H/R). The effects of DHI on apoptosis, antioxidant capacity and mitochondrial integrity were evaluated by mitochondrial morphology, apoptosis rate, reactive oxygen species (ROS) generation, ATP levels, mitochondrial membrane potential, and oxygen consumption in H9C2 cells treated with H/R. The underlying mechanism of action of DHI in maintenance of mitochondrial integrity and anti-apoptosis was detected in H9C2 cells with or without Nrf2 knockdown. DHI treatment significantly decreased the infarct size, inhibited apoptosis and suppressed oxidative stress in the hearts of I/R rats. Also, DHI promoted cell survival by: an anti-apoptosis action; inhibiting ROS generation; maintaining mitochondrial morphology with increased mitochondrial length; alleviating mitochondrial dysfunction with a decreased mitochondrial membrane potential; increasing ATP levels and the oxygen-consumption rate. Moreover, the Keap1/Nrf2/JNK pathway was found to be involved in DHI reducing oxidative stress and maintaining mitochondrial integrity. We revealed a novel mechanism by which DHI protected H9C2 cells against H/R injury via the Keap1/Nrf2/JNK pathway and provided a mitochondrial protectant for the treatment of myocardial I/R injury.

Antioxidant Supplementation in the Treatment of Neurotoxicity Induced by Platinum-Based Chemotherapeutics-A Review.

Cancer represents one of the most pernicious public health problems with a high mortality rate among patients worldwide. Chemotherapy is one of the major therapeutic approaches for the treatment of various malignancies. Platinum-based drugs (cisplatin, oxaliplatin, carboplatin, etc.) are highly effective chemotherapeutic drugs used for the treatment of several types of malignancies, but their application and dosage are limited by their toxic effects on various systems, including neurotoxicity. Simultaneously, researchers have tried to improve the survival rate and quality of life of cancer patients and decrease the toxicity of platinum-containing drugs by combining them with non-chemotherapy-based drugs, dietary supplements and/or antioxidants. Additionally, recent studies have shown that the root cause for the many side effects of platinum chemotherapeutics involves the production of reactive oxygen species (ROS) in naive cells. Therefore, suppression of ROS generation and their inactivation with antioxidants represents an appropriate approach for platinum drug-induced toxicities. The aim of this paper is to present an updated review of the protective effects of different antioxidant agents (vitamins, dietary antioxidants and supplements, medicaments, medicinal plants and their bioactive compounds) against the neurotoxicity induced by platinum-based chemotherapeutics. This review highlights the high potential of plant antioxidants as adjuvant strategies in chemotherapy with platinum drugs.

Potential Anti-Skin Aging Effect of (-)-Catechin Isolated from the Root Bark of Ulmus davidiana var. japonica in Tumor Necrosis Factor-α-Stimulated Normal Human Dermal Fibroblasts

Reactive oxygen species (ROS) are generated during skin aging, including intrinsic (chronologic aging) and extrinsic aging (photoaging). Therefore, antioxidants that inhibit ROS generation can delay skin aging. In this study, we evaluated the potential anti-skin aging effect of (-)-phenolic compounds isolated from the root bark of Ulmus davidiana var. japonica. We preferentially investigated the possible preventive effects of isolates against the degradation of skin extracellular matrix. Among the isolates, (-)-catechin suppressed the activity of collagenase MMP-1, and reversed the degradation of collagen induced by tumor necrosis factor-α (TNF-α) in normal human dermal fibroblast. This action mechanism of (-)-catechin was validated by the suppression of tumor necrosis factor-α-induced accumulation of ROS and activation of mitogen-activated protein kinases, protein kinase B (Akt), and cyclooxygenase-2 (COX-2). The proinflammatory cytokines upregulate inflammatory reactions, and ultimately promote aging-related reactions. In this milieu, we demonstrated that (-)-catechin decreased the expression and secretion of proinflammatory cytokines, including interleukin (IL)-1β and IL-6. In conclusion, (-)-catechin is a candidate to ameliorate both intrinsic and extrinsic skin aging.

Reactive Oxygen Species and Antioxidants in Carcinogenesis and Tumor Therapy.

Strictly regulated balance between the formation and utilization of reactive oxygen species (ROS) is the basis of normal functioning of organisms. ROS play an important role in the regulation of many metabolic processes; however, excessive content of ROS leads to the development of various disorders, including oncological diseases, as a result of ROS-induced mutations in DNA. In tumors, high levels of oxygen radicals promote cell proliferation and metastasis. On the other hand, high content of ROS can trigger cell death, a phenomenon used in the antitumor therapy. Water- and lipid-soluble antioxidants, as well as antioxidant enzyme systems, can inhibit ROS generation; however, they should be used with caution. Antioxidants can suppress ROS-dependent cell proliferation and metastasis, but at the same time, they may inhibit the death of tumor cells if the antitumor therapeutic agents stimulate oxidative stress. The data on the role of antioxidants in the death of tumor cells and on the effects of antioxidants taken as dietary supplements during antitumor therapy, are contradictory. This review focuses on the mechanisms by which antioxidants can affect tumor and healthy cells.

Citrus hystrix Extracts Protect Human Neuronal Cells against High Glucose-Induced Senescence.

Citrus hystrix (CH) is a beneficial plant utilized in traditional folk medicine to relieve various health ailments. The antisenescent mechanisms of CH extracts were investigated using human neuroblastoma cells (SH-SY5Y). Phytochemical contents and antioxidant activities of CH extracts were analyzed using a gas chromatograph–mass spectrometer (GC-MS), 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay and 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) assay. Effects of CH extracts on high glucose-induced cytotoxicity, reactive oxygen species (ROS) generation, cell cycle arrest and cell cycle-associated proteins were assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) assay, non-fluorescent 2′, 7′-dichloro-dihydrofluorescein diacetate (H2DCFDA) assay, flow cytometer and Western blot. The extracts protected neuronal senescence by inhibiting ROS generation. CH extracts induced cell cycle progression by releasing senescent cells from the G1 phase arrest. As the Western blot confirmed, the mechanism involved in cell cycle progression was associated with the downregulation of cyclin D1, phospho-cell division cycle 2 (pcdc2) and phospho-Retinoblastoma (pRb) proteins. Furthermore, the Western blot showed that extracts increased Surtuin 1 (SIRT1) expression by increasing the phosphorylation of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Collectively, CH extracts could protect high glucose-induced human neuronal senescence by inducing cell cycle progression and up-regulation of SIRT1, thus leading to the improvement of the neuronal cell functions.

Brazilian red propolis extract enhances expression of antioxidant enzyme genes in vitro and in vivo.

Brazilian red propolis reportedly has reactive oxygen species (ROS) scavenging effects in vitro, but the cellular mechanisms remain unclear. In the present study, the effects of an ethanol extract of Brazilian red propolis (EERP) on the Nrf2-ARE intracellular antioxidant pathway were examined in vitro and in vivo. EERP and its constituents transactivated the reporter gene through the ARE sequence and enhanced the expression of Nrf2-regulated genes in HEK293 cells. It also increased Nrf2 protein in the nucleus, which was partially inhibited by kinase inhibitors. Furthermore, EERP suppressed ROS generation and cytotoxicity induced by tert-butyl hydroperoxide. In vivo, orally administered EERP increased the expression of Nrf2-regulated genes in mice liver. These results suggest that EERP is a potential resource for preventing oxidative stress-related diseases as an Nrf2 inducer.

Rutin-protected BisGMA-induced cytotoxicity, genotoxicity, and apoptosis in macrophages through the reduction of the mitochondrial apoptotic pathway and induction of antioxidant enzymes.

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.

Loganin Attenuates High Glucose-Induced Schwann Cells Pyroptosis by Inhibiting ROS Generation and NLRP3 Inflammasome Activation.

Diabetic peripheral neuropathy (DPN) is caused by hyperglycemia, which induces oxidative stress and inflammatory responses that damage nerve tissue. Excessive generation of reactive oxygen species (ROS) and NOD-like receptor protein 3 (NLRP3) inflammasome activation trigger the inflammation and pyroptosis in diabetes. Schwann cell dysfunction further promotes DPN progression. Loganin has been shown to have antioxidant and anti-inflammatory neuroprotective activities. This study evaluated the neuroprotective effect of loganin on high-glucose (25 mM)-induced rat Schwann cell line RSC96 injury, a recognized in vitro cell model of DPN. RSC96 cells were pretreated with loganin (0.1, 1, 10, 25, 50 μM) before exposure to high glucose. Loganin’s effects were examined by CCK-8 assay, ROS assay, cell death assay, immunofluorescence staining, quantitative RT–PCR and western blot. High-glucose-treated RSC96 cells sustained cell viability loss, ROS generation, NF-κB nuclear translocation, P2 × 7 purinergic receptor and TXNIP (thioredoxin-interacting protein) expression, NLRP3 inflammasome (NLRP3, ASC, caspase-1) activation, IL-1β and IL-18 maturation and gasdermin D cleavage. Those effects were reduced by loganin pretreatment. In conclusion, we found that loganin’s antioxidant effects prevent RSC96 Schwann cell pyroptosis by inhibiting ROS generation and suppressing NLRP3 inflammasome activation.

Anti-inflammatory effects of troxerutin are mediated through elastase inhibition

Context Obesity is a chronic low-grade inflammatory state associated with immune cell infiltration into the adipose tissue (AT). We hypothesize that the anti-obesity and anti-inflammatory effects of troxerutin (TX) are mediated through inhibition of elastase. Objective To determine the inhibitory effect of TX on elastase in vitro and in tumor necrosis factor alpha (TNFα) induced 3T3-L1 adipocytes and the molecular interaction of TX with human neutrophil elastase (HNE). Materials and Methods Differentiated 3T3-L1 adipocytes were pretreated with TX, elastatinal (ELAS) or sodium salicylate (SAL) before exposure to TNFα. Lipid accumulation, reactive oxygen species (ROS) generation and oxidant-antioxidant balance were examined. The mRNA and protein expression of TNFα, interleukin-6, monocyte chemoattractant protein-1, adiponectin, leptin, resistin, chemerin, and elastase were analyzed. Elastase inhibition by TX and ELAS in a cell free system and docking studies for HNE with TX and ELAS were performed. Results TX, ELAS or SAL pretreatment had lowered lipid droplets formation and TG content. TX suppressed ROS generation, oxidative stress and improved antioxidant status. The expression of inflammatory cytokines and elastase was downregulated while that of adiponectin was upregulated by TX. The concentration required to produce 50% inhibition in vitro (IC50) was 11.5 μM for TX and 16.9 μM for ELAS. TX showed hydrogen bonding and hydrophobic interactions with elastase. Discussion TNFα induces inflammation of 3T3-L1 cells through elastase activation. TX inhibits elastase activity, downregulates expression and binds with elastase. Conclusion The antioxidant and anti-inflammatory activities of TX in AT could be of relevance in the management of obesity.

Brassinosteroids mediate the effect of high temperature during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile rice lines

Brassinosteroids (BRs) play critical roles in a wide range of plant developmental processes. However, it is unknown whether and how BRs mediate the effect of high temperature (HT) stress during anthesis on the pistil activity of photo-thermosensitive genetic male-sterile (PTSGMS) rice (Oryza sativa L.) lines. This study investigated the question. Three pot-grown PTSGMS rice lines were subjected to HT stress during anthesis. The contents of 24-epibrassinolide (24-EBL) and 28-homobrassinolide (28-HBL), the major forms of BR in rice plants, and levels of reactive oxygen species (ROS) or antioxidants (AOS), hydrogen peroxide (H2O2), 1-aminocylopropane-1-carboxylic acid (ACC), ascorbic acid (AsA), and catalase activity in pistils, were determined. HT stress significantly reduced the contents of both 24-EBL and 28-EBL relative to those under normal temperatures, but the reduction varied by PTSGMS line. A line with higher BR contents under HT stress showed lower contents of ACC and H2O2, higher catalase activity and AsA content in pistils, and higher fertilization rate, seed-setting rate, and seed yield when the line was crossed with a restorer line, indicating that higher levels of BRs increase HT stress resistance. Applying 24-EBL, 28-HBL or an inhibitor of BR biosynthesis confirmed the roles of BRs in response to HT stress. The results suggest that BRs mediate the effect of HT stress on pistil activity during anthesis and alleviate the harm of HT stress by increasing AOS and suppressing ROS generation.

Rosa multiflora Thunb Flower Extract Attenuates Ultraviolet-Induced Photoaging in Skin Cells and Hairless Mice.

Ethanol extract (RET) of Rosa multiflora Thunb flowers and its subfractions in ethylacetate (REA) or n-butanol subfractions (RBT) were reported to have potent antioxidative and anti-inflammatory activities. In this study, we investigated if those Rosa multiflora flower (RMF) extracts prevent ultraviolet (UV)-induced biochemical damages leading to photoaging. In keratinocyte or dermal fibroblasts, RET, REA, and RBT treatments with UV irradiation significantly decreased reactive oxygen species (ROS), interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-1 levels through suppression of nuclear factor kappa B and mitogen-activated protein kinases. In the animal experiment, mice were orally supplemented with RET (RET group) or REA and RBT mixture (RM group) for 10 weeks, concomitantly with UV exposure. Tumor necrosis factor alpha production and MMP-13 expression were reduced in the mouse skin of RET and RM groups compared with those in the UV control (UVC) group. UV-induced IL-6 production and epidermal thickening were reduced in RM group compared with those in UVC group. Eight phenolic compounds, including quercitrin (quercetin-3-O-rhamnoside), were identified in RMF extracts. Quercitrin treatment to dermal fibroblasts significantly attenuated an increase of MMP-1 expression and a decrease of type I procollagen expression caused by UV. Collectively, RMF extracts showed protective effects from UV-induced photoaging in the skin through suppression of ROS generation, proinflammatory cytokine production, and MMP expression. Quercitrin is suggested to be one of the effective compounds.

Induction of oxidative stress and DNA damage in human renal proximal tubular cells by aristolochic acid

Aristolochic acid 1 (AAI) is found primarily in the plant Aristolochia. Consumption of products containing AAI has been linked with permanent kidney damage and urothelial carcinoma. This study applied human proximal tubule epithelial cell line (HK-2) to examine the relationship among AAI-induced intracellular oxidative stress, DNA damage and MAP kinase activation. High concentrations of AAI caused a decrease in cell viability and an increase in the activity of caspase 3. AAI treatment also led to a dose-dependent increase of reactive oxygen species (ROS) in HK-2 cells, and the presence of antioxidant glutathione (GSH) effectively inhibited ROS generation. Stimulating HK-2 cultures with AAI also led to GSH depletion. Results from single cell gel electrophoresis (SCGE) assays demonstrated that AAI showed the ability to increase the levels of DNA strand breaks in HK-2 cells. Up-regulation of luciferase activity driven by the Nrf2 binding element was also observed after 200 μM AAI treatment. Exposure of HK-2 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only the MEK1/2 inhibitor, U0126, significantly decreased the levels of AAI-mediated ROS. In addition, both U0126 and SB202190 effectively reversed the levels of DNA damage triggered by AAI. This suggests that AAI treatment of HK-2 results in ROS formation and DNA damage. Furthermore, ROS generation occurs via the MEK/ERK1/2 signaling pathway, whereas DNA damage occurs via both the ERK1/2 and p38 pathways.

Indole 6 Carboxaldehyde Prevents Oxidative Stress-Induced Mitochondrial Dysfunction, DNA Damage and Apoptosis in C2C12 Skeletal Myoblasts

Abstract Objectives Indole-6-carboxaldehyde (I6CA) is an indole derivative derived from the brown algae Sargassum thunbergii, and some pharmacological activity is known, but the antioxidant effect of I6CA has not been reported. In the present study, the protective capacity of I6CA against oxidative stress-induced cytotoxicity in C2C12 skeletal myoblasts was investigated. Methods I6CA's effect on hydrogen peroxide (H2O2)-disrupted cell viability, mitochondrial function, and intracellular ATP level was measured. Furthermore, I6CA's potency to reduce H2O2-induced DNA damage, reactive oxygen species (ROS) generation, AMP-activated protein kinase (AMPK) activation and apoptosis was monitored. Results The results indicate that preincubation of cells with I6CA prior to H2O2 stimulation resulted in significant inhibition of cytotoxicity and DNA damage associated with the inhibition of ROS generation. In addition, H2O2-induced loss of mitochondrial membrane potential and cytosolic release of cytochrome c were significantly protected in the presence of I6CA. I6CA also attenuated the decrease in ATP content by H2O2 and restored H2O2-induced activation of AMPK, indicating that I6CA maintains energy metabolism through the preservation of mitochondrial function while eliminating the ROS generated by H2O2. Furthermore, I6CA rescued C2C12 cells from H2O2-triggered apoptosis, as evidenced by reduced caspase-3 activation, PARP degradation and DNA fragmentation. Conclusions Collectively, our findings conclude that I6CA effectively protected C2C12 myoblasts against oxidative damage, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS. Funding Sources The research was supported by the Basic Science Research Program through the National Research Foundation of Korea grant funded by the Korea government (2018R1A2B2005705).