Sulforaphane protects against acetaminophen-induced hepatotoxicity

Oxidative stress is closely associated with acetaminophen (APAP)-induced toxicity. Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, has been shown to protect against oxidant-induced tissue injury. This study investigated whether sulforaphane (SFN), as a HO-1 inducer, plays a protective role against APAP hepatotoxicity in vitro and in vivo. Pretreatment of primary hepatocyte with SFN induced nuclear factor E2-factor related factor (Nrf2) target gene expression, especially HO-1 mRNA and protein expression, and suppressed APAP-induced glutathione (GSH) depletion and lipid peroxidation, which eventually leads to hepatocyte cell death. A comparable effect was observed in mice treated with APAP. Mice were treated with 300 mg/kg APAP 30 min after SFN (5 mg/kg) administration and were then sacrificed after 6 h. APAP alone caused severe liver injuries as characterized by increased plasma AST and ALT levels, GSH depletion, apoptosis, and 4-hydroxynonenal (4-HNE) formations. This APAP-induced liver damage was significantly attenuated by pretreatment with SFN. Furthermore, while hepatic reactive oxygen species (ROS) levels were increased by APAP exposure, pretreatment with SFN completely blocked ROS formation. These results suggest that SFN plays a protective role against APAP-mediated hepatotoxicity through antioxidant effects mediated by HO-1 induction. SFN has preventive action in oxidative stress-mediated liver injury.     

ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function

This review focuses on the emerging evidence that reactive oxygen species (ROS) derived from glucose metabolism, such as H₂O₂, act as metabolic signaling molecules for glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Particular emphasis is placed on the potential inhibitory role of endogenous antioxidants, which rise in response to oxidative stress, in glucose-triggered ROS and GSIS. We propose that cellular adaptive response to oxidative stress challenge, such as nuclear factor E2-related factor 2 (Nrf2)-mediated antioxidant induction, plays paradoxical roles in pancreatic beta-cell function. On the one hand, induction of antioxidant enzymes protects beta-cells from oxidative damage and possible cell death, thus minimizing oxidative damage-related impairment of insulin secretion. On the other hand, the induction of antioxidant enzymes by Nrf2 activation blunts glucose-triggered ROS signaling, thus resulting in reduced GSIS. These two premises are potentially relevant to impairment of beta-cells occurring in the late and early stage of Type 2 diabetes, respectively. In addition, we summarized our recent findings that persistent oxidative stress due to absence of uncoupling protein 2 activates cellular adaptive response which is associated with impaired pancreatic beta-cell function.     

Eriodictyol-7-O-glucoside activates Nrf2 and protects against cerebral ischemic injury

Stroke is a complex disease that may involve oxidative stress-related pathways in its pathogenesis. The nuclear factor erythroid-2-related factor 2/antioxidant response element (Nrf2/ARE) pathway plays an important role in inducing phase II detoxifying enzymes and antioxidant proteins and thus has been considered a potential target for neuroprotection in stroke. The aim of the present study was to determine whether eriodictyol-7-O-glucoside (E7G), a novel Nrf2 activator, can protect against cerebral ischemic injury and to understand the role of the Nrf2/ARE pathway in neuroprotection. In primary cultured astrocytes, E7G increased the nuclear localization of Nrf2 and induced the expression of the Nrf2/ARE-dependent genes. Exposure of astrocytes to E7G provided protection against oxygen and glucose deprivation (OGD)-induced oxidative insult. The protective effect of E7G was abolished by RNA interference-mediated knockdown of Nrf2 expression. In vivo administration of E7G in a rat model of focal cerebral ischemia significantly reduced the amount of brain damage and ameliorated neurological deficits. These data demonstrate that activation of Nrf2/ARE signaling by E7G is directly associated with its neuroprotection against oxidative stress-induced ischemic injury and suggest that targeting the Nrf2/ARE pathway may be a promising approach for therapeutic intervention in stroke.     

基于Nrf2信号通路探讨异甘草酸镁对雷公藤甲素损伤L-02细胞中相关蛋白表达的影响

目的基于Nrf2-ARE信号通路探讨异甘草酸镁对雷公藤甲素损伤L-02细胞的保护作用及相关机制。方法体外培养正常人肝L-02细胞,通过免疫荧光技术检测异甘草酸镁(60μmol·L^-1)对雷公藤甲素(80nmol·L^-1)损伤L-02细胞中Nrf2核转移的影响;采用Western blotting测定细胞核中Nrf2及细胞中细胞色素P450 3A4(CYP3A4)、多药耐药相关蛋白2(multidrug resistance associated protein 2,MRP2)和胆盐输出泵(bile salt export pump,BSEP)的蛋白表达。结果雷公藤甲素对L-02细胞核中Nrf2具有诱导作用(P<0.01),并促进Nrf2核内转移;异甘草酸镁预处理组较雷公藤甲素组降低L-02细胞核中Nrf2的表达(P<0.01);异甘草酸镁预处理后对雷公藤甲素损伤L-02细胞中CYP3A4、MRP2和BSEP蛋白表达具有诱导作用(P<0.01)。结论异甘草酸镁可减轻雷公藤甲素对L-02细胞造成的损伤作用,其机制可能与诱导细胞核中Nrf2以及细胞中CYP3A4、MRP2和BSEP受体有关。     

Isorhamnetin protects against oxidative stress by activating Nrf2 and inducing the expression of its target genes

Isorhamentin is a 3′-O-methylated metabolite of quercetin, and has been reported to have anti-inflammatory and anti-proliferative effects. However, the effects of isorhamnetin on Nrf2 activation and on the expressions of its downstream genes in hepatocytes have not been elucidated. Here, we investigated whether isorhamnetin has the ability to activate Nrf2 and induce phase II antioxidant enzyme expression, and to determine the protective role of isorhamnetin on oxidative injury in hepatocytes. In HepG2 cells, isorhamnetin increased the nuclear translocation of Nrf2 in a dose- and time-dependent manner, and consistently, increased antioxidant response element (ARE) reporter gene activity and the protein levels of hemeoxygenase (HO-1) and of glutamate cysteine ligase (GCL), which resulted in intracellular GSH level increases. The specific role of Nrf2 in isorhamnetin-induced Nrf2 target gene expression was verified using an ARE-deletion mutant plasmid and Nrf2-knockout MEF cells. Deletion of the ARE in the promoter region of the sestrin2 gene, which is recently identified as the Nrf2 target gene by us, abolished the ability of isorhamnetin to increase luciferase activity. In addition, Nrf2 deficiency completely blocked the ability of isorhamnetin to induce HO-1 and GCL. Furthermore, isorhamnetin pretreatment blocked t-BHP-induced ROS production and reversed GSH depletion by t-BHP and consequently, due to reduced ROS levels, decreased t-BHP-induced cell death. In addition isorhamnetin increased ERK1/2, PKCδ and AMPK phosphorylation. Finally, we showed that Nrf2 deficiency blocked the ability of isorhamnetin to protect cells from injury induced by t-BHP. Taken together, our results demonstrate that isorhamnetin is efficacious in protecting hepatocytes against oxidative stress by Nrf2 activation and in inducing the expressions of its downstream genes.     

Investigation of the toxicity of a glyphosate-based herbicide in a human liver cell line: Assessing the involvement of Nrf2 pathway and protective effects of vitamin E and α-lipoic acid

Glyphosate-based herbicides (GBHs) are the most widely used herbicides all over the world and has gained more attention in recent years because of health safety concerns. In this study, Roundup, one of the most popular glyphosate formulations, was used to evaluate cytotoxic, oxidative stress and apoptosis inducing effects of GBHs in a human hepatocellular cell line (HepG2). Roundup was shown to significantly increase cellular reactive oxygen species (ROS) levels, which lead to activation of the nuclear factor-erythroid-2-related factor 2 (Nrf2) antioxidant defense pathway including reduced levels of heme oxygenase 1 (HO-1). Furthermore, Roundup was found to induce apoptosis and further analysis confirmed involvement of a mitochondrial-dependent pathway verified by increased Bax/Bcl-2 ratios. Investigation of the protective effects of antioxidants vitamin E (Vit E) and α-lipoic acid (LA) against Roundup toxicity showed that both antioxidants significantly reduced the cytotoxicity, ROS formation, HO-1 downregulation, and apoptosis and that Vit E did so more efficiently than LA. In conclusion, our findings highlight the ROS producing and apoptosis inducing effects associated with GBHs, the activation of Nrf2 pathway as a defense mechanism and the protective effects of Vit E and LA against GBH toxicity.     

Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead

A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.     

Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant

Oxidative stress has been implicated as an important factor in many neurological diseases. Oxidative toxicity in a number of these conditions is induced by excessive glutamate release and subsequent glutamatergic neuronal stimulation. This, in turn, causes increased generation of reactive oxygen species (ROS), oxidative stress, excitotoxicity, and neuronal damage. Recent studies indicate that the glutamatergic neurotransmitter system is involved in lead-induced neurotoxicity. Therefore, this study aimed to (1) investigate the potential effects of glutamate on lead-induced PC12 cell death and (2) elucidate whether the novel thiol antioxidant N-acetylcysteine amide (NACA) had any protective abilities against such cytotoxicity. Our results suggest that glutamate (1 mM) potentiates lead-induced cytotoxicity by increased generation of ROS, decreased proliferation (MTS), decreased glutathione (GSH) levels, and depletion of cellular adenosine-triphosphate (ATP). Consistent with its ability to decrease ATP levels and induce cell death, lead also increased caspase-3 activity, an effect potentiated by glutamate. Exposure to glutamate and lead elevated the cellular malondialdehyde (MDA) levels and phospholipase-A(2) (PLA(2)) activity and diminished the glutamine synthetase (GS) activity. NACA protected PC12 cells from the cytotoxic effects of glutamate plus lead, as evaluated by MTS assay. NACA reduced the decrease in the cellular ATP levels and restored the intracellular GSH levels. The increased levels of ROS and MDA in glutamate-lead treated cells were significantly decreased by NACA. In conclusion, our data showed that glutamate potentiated the effects of lead-induced PC12 cell death by a mechanism involving mitochondrial dysfunction (ATP depletion) and oxidative stress. NACA had a protective role against the combined toxic effects of glutamate and lead by inhibiting lipid peroxidation and scavenging ROS, thus preserving intracellular GSH.     

Depletion of intracellular glutathione mediates butenolide-induced cytotoxicity in HepG2 cells

Butenolide, 4-acetamido-4-hydroxy-2-butenoic acid gamma-lactone is one of the mycotoxins produced by Fusarium species which are often found on cereal grains and animal feeds throughout the world. It has been implicated as the etiology of some diseases both in animals and in humans. Though butenolide represents a potential threat to animal and human heath, there are few studies on its toxicity so far, especially on the toxic mechanisms. In this study, we investigated the cytotoxicity of butenolide on HepG2 cells and its possible mechanism from the viewpoint of oxidative stress. Butenolide reduced cell viability in a concentration- and time-dependent manner. A rapid depletion of intracellular glutathione (GSH) was observed after exposure cells to butenolide, concomitantly an increase in intracellular reactive oxygen species (ROS) production prior to cell death, indicating that oxidative stress was involved in butenolide cytotoxicity. To elucidate the role of GSH in the cytotoxicity of butenolide, intracellular GSH content was modulated before exposure to butenolide. l-buthionine-[S,R]-sulfoximine (BSO), a well-known inhibitor of GSH synthesis, aggravated butenolide-induced GSH depletion, ROS production and the loss in cell viability; in contrast, GSH depletion and ROS production was strongly inhibited, and the loss in cell viability was completely abrogated by thiol-containing compounds GSH, N-acetylcysteine (NAC) and dithiothreitol (DTT). Furthermore, a ROS scavenger catalase obviously abated ROS production and cytotoxicity induced by butenolide. Together, these results clearly demonstrate that oxidative stress plays an important role in butenolide cytotoxicity, and intracellular GSH depletion may be an original trigger of the onset of butenolide cytotoxicity.     

α‐Lipoic acid protects against microcystin‐LR induced hepatotoxicity through regeneration of glutathione via activation of Nrf2

Microcystins (MCs), as the most dominant bloom‐forming strains in eutrophic surface water, can induce hepatotoxicity by oxidative stress. Alpha‐lipoic acid (α‐LA) is a super antioxidant that can induce the synthesis of antioxidants, such as glutathione (GSH), by nuclear factor erythroid 2‐related factor 2 (Nrf2). However, the potential molecular mechanism of α‐LA regeneration of GSH remains unclear. The present study aimed to investigate whether α‐LA could reduce the toxicity of MCs induced in human hepatoma (HepG2), Bel7420 cells, and BALB/c mice by activating Nrf2 to regenerate GSH. Results showed that exposure to 10 μM microcystin‐leucine arginine (MC‐LR) reduced viability of HepG2 and Bel7402 cells and promoted the formation of reactive oxygen species (ROS) compared with untreated cells. Moreover, the protection of α‐LA included reducing the level of ROS, increasing superoxide dismutase activity, and decreasing malondialdehyde. Levels of reduced glutathione (rGSH) and rGSH/oxidized glutathione were significantly increased in cells cotreated with α‐LA and MC‐LR compared to those treated with MC‐LR alone, indicating an ability of α‐LA to attenuate oxidative stress and MC‐LR‐induced cytotoxicity by increasing the amount of rGSH. α‐LA can mediate GSH regeneration through the Nrf2 pathway under the action of glutathione reductase in MC‐LR cell lines. Furthermore, the data also showed that α‐LA‐induced cytoprotection against MC‐LR is associated with Nrf2 mediate pathway in vivo. These findings demonstrated the potential of α‐LA to resist MC‐LR‐induced oxidative damage of liver.     

Licorice root extract and magnesium isoglycyrrhizinate protect against triptolide-induced hepatotoxicity via up-regulation of the Nrf2 pathway

Triptolide, the predominant biologically active component of the Chinese herb Tripterygium wilfordii Hook f., possesses numerous pharmacological activities, including anti-inflammatory, anti-fertility, anti-neoplastic, and immunosuppressive effects. However, toxicity and severe adverse effects, particularly hepatotoxicity, limit the clinical application of triptolide. Licorice root extract contains various bioactive compounds and is potent hepatoprotective. Magnesium isoglycyrrhizinate, a magnesium salt of the 18α-glycyrrhizic acid stereoisomer of glycyrrhizic acid, is used clinically in China to treat chronic viral hepatitis and acute drug-induced liver injury. The aim of this study was to investigate the role of the factor erythroid 2-related factor 2 pathway in the protective effects of LE and MIG against triptolide-induced hepatotoxicity. Hepatotoxicity models were established in L-02 cells and rats using triptolide, and the protective effects of LE and MIG were investigated in vitro and in vivo, respectively. LE and MIG significantly protected against triptolide-induced cytotoxicity. Additionally, triptolide decreased the mRNA and protein levels of Nrf2 and down-regulated Nrf2 target genes, including UGT1A, BSEP, and MRP2, while pretreatment with LE and MIG reversed these effects. Finally, Nrf2-involved antioxidant responses were activated in the presence of LE and MIG.     

Protective mechanisms of Aralia continentalis extract against tert-butyl hydroperoxide-induced hepatotoxicity: in vivo and in vitro studies

In the present work, we investigated the protective effects of the ethanol extract of Aralia continentalis roots (AC) on tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in a cultured Hepa1c1c7 cell line and in mouse liver. Pretreatment with AC prior to the administration of t-BHP significantly prevented the increase in serum levels of hepatic enzyme markers (ALT, AST) and lipid peroxidation and reduced oxidative stress, as measured by glutathione content, in the liver. Histopathological evaluation of the livers also revealed that AC reduced the incidence of liver lesions. The in vitro study showed that AC significantly reduced t-BHP-induced oxidative injury in Hepa1c1c7 cells, as determined by cell cytotoxicity, intracellular glutathione content, lipid peroxidation, reactive oxygen species (ROS) levels, and caspase-3 activation. Also, AC up-regulated phase II genes including heme oxygenase-1 (HO-1), NAD(P)H:quinone reductase, and glutathione S-transferase. Moreover, AC induced Nrf2 nuclear translocation and ERK1/2 and p38 activation, pathways that are involved in inducing Nrf2 nuclear translocation. Taken together, these results suggest that the protective effects of AC against t-BHP-induced hepatotoxicity may, at least in part, be due to its ability to scavenge ROS and to regulate the antioxidant enzyme HO-1 via the ERK1/2 and p38/Nrf2 signaling pathways.     

Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells

Due to the interesting magnetic and electrical properties with good chemical and thermal stabilities, nickel ferrite nanoparticles are being utilized in many applications including magnetic resonance imaging, drug delivery and hyperthermia. Recent studies have shown that nickel ferrite nanoparticles produce cytotoxicity in mammalian cells. However, there is very limited information concerning the toxicity of nickel ferrite nanoparticles at the cellular and molecular level. The aim of this study was to investigate the cytotoxicity, oxidative stress and apoptosis induction by well-characterized nickel ferrite nanoparticles (size 26 nm) in human lung epithelial (A549) cells. Nickel ferrite nanoparticles induced dose-dependent cytotoxicity in A549 cells demonstrated by MTT, NRU and LDH assays. Nickel ferrite nanoparticles were also found to induce oxidative stress evidenced by generation of reactive oxygen species (ROS) and depletion of antioxidant glutathione (GSH). Further, co-treatment with the antioxidant L-ascorbic acid mitigated the ROS generation and GSH depletion due to nickel ferrite nanoparticles suggesting the potential mechanism of oxidative stress. Quantitative real-time PCR analysis demonstrated that following the exposure of A549 cells to nickel ferrite nanoparticles, the level of mRNA expressions of cell cycle checkpoint protein p53 and apoptotic proteins (bax, caspase-3 and caspase-9) were significantly up-regulated, whereas the expression of anti-apoptotic proteins (survivin and bcl-2) were down-regulated. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in nickel ferrite nanoparticles exposed cells. To the best of our knowledge this is the first report showing that nickel ferrite nanoparticles induced apoptosis in A549 cells through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways.     

Necroptosis contributes to the cyclosporin A-induced cytotoxicity in NRK-52E cells

Cyclosporin A (CsA) induces renal tubular epithelial cells apoptosis and necrosis following in vitro exposure. The mechanisms of CsA-induced apoptosis have been studied intensively, whereas the mechanisms of necrosis remain to be elucidated. Necroptosis has been described as programmed necrosis. This study investigated the ability of CsA to induce necroptosis in the rat tubular cell line NRK-52E. The NRK-52E cells were incubated with CsA for 24 hours with or without necrostatin-1 (Nec-1). The majority of the NRK-52E cells died of necrosis as indicated by LDH leakage, Hoechst 33342/PI staining, and flow cytometry analysis. Cell death was significantly reduced by Nec-1 pretreated before CsA exposure. CsA-induced apoptosis and necrosis were also compared in NRK-52E cells with or without knockdown of receptor interaction protein 3 (RIP3) expression using small interfering RNA. Moreover, the role of reactive oxygen species (ROS) in CsA-induced cell death was also attempted. The result suggests that necroptosis contributes to the CsA-induced cytotoxicity in NRK-52E cells. Meanwhile, RIP3 and ROS are involved in CsA-induced necroptosis. To our knowledge, this is the first report on necroptosis in CsA-induced renal tubular cell death pathways, which might offer a novel protective target for CsA nephrotoxicity.