Icaritin inhibits oxidative stress in murine astrocytes by binding to Orai1 to block store-operated calcium channel

Previous study has shown that icaritin (ICT) has meaningful protective effect on cerebral ischemic stroke, and this study aimed to investigate its mechanism from the aspect of protecting astrocytes from oxidative stress. Murine primary astrocytes were pretreated by ICT and exposed to H₂O₂ to induce oxidative stress. The results indicated that ICT inhibited H₂O₂-induced astrocytes apoptosis, decreased Bax and cleaved caspase-3, and increased Bcl-2. In addition, ICT inhibited H₂O₂-induced oxidative stress, increased mitochondrial membrane potential (ΔΨ_m), and maintained mitochondrial morphology. ICT decreased the synthesis of malondialdehyde and increased the activity of glutathione peroxidase, catalase, and superoxide dismutase. Moreover, ICT suppressed the transient and resting intracellular Ca²⁺ overload. Further investigation revealed that ICT could target the combination with Orai1 to block store-operated calcium channel induced by H₂O₂. However, ICT did not enhance the protective effect of RO2959, a selective blocker of Orai1. These results indicate that ICT can play a neuroprotective role against oxidative stress injury by binding to Orai1 to block SOCC.     

Oxyresveratrol protects human lens epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis by activation of Akt/HO-1 pathway

Oxidative stress induced by hydrogen peroxide (H₂O₂) triggers human lens epithelial cell (HLEC) apoptosis and initiates cataract formation. Oxyresveratrol (Oxy) was reported to possess antioxidant and free radical scavenging activities. Herein, we investigated the effects of Oxy on H₂O₂-induced oxidative stress and apoptosis in HLECs and the associated mechanisms. Cell viability was detected by MTT assay. The oxidative damage was assessed by measuring the activities of superoxide dismutases-1 (SOD-1), catalase (CAT), glutathione reductase (GSH), and malondialdehyde (MDA). Apoptosis was analyzed by flow cytometry analysis. The changed expressions of heme oxygenase-1 (HO-1) and protein kinase B (Akt) pathways were evaluated by qRT-PCR and western blot. We found that exposure to H₂O₂ dose-dependently reduced cell viability, and induced oxidative stress and apoptosis in HLECs, which were reversed by pretreatment with Oxy. Oxy increased p-Akt and HO-1 expressions in H₂O₂-stimulated HLECs. Akt and HO-1 expressions form a regulatory axis and Oxy activated the Akt/HO-1 pathway in H₂O₂-stimulated HLECs. Inhibition of the Akt/HO-1 pathway by LY294002 or ZnPP attenuated the effects of Oxy on oxidative stress and apoptosis in H₂O₂-stimulated HLECs. In conclusion, Oxy protected H₂O₂-induced oxidative stress and apoptosis through activating the Akt/HO-1 pathway, suggesting the protective effect of Oxy against H₂O₂-induced cataract.     

p38(MAPK) and ERK1/2 dictate cell death/survival response to different pro-oxidant stimuli via p53 and Nrf2 in neuroblastoma cells SH-SY5Y

Redox changes are often reported as causative of neoplastic transformation and chemoresistance, but are also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derived tumor histotypes are resistant to ROS-based treatments by means of the redox activation of Nrf2, but highly sensitive to disulfide stressors triggering apoptosis via the redox induction of Trx1/p38^MAPK/p53 signaling pathway.Here, we provide evidence that neuroblastoma SH-SY5Y has a complete opposite behavior, being sensitive to H₂O₂, but resistant to the glutathione (GSH)-oxidizing molecule diamide. Consistent with these observations, the apoptotic pathway activated upon H₂O₂ treatment relies upon Trx1 oxidation, and is mediated by the p38^MAPK/p53 signaling axis. Pre-treatment with different antioxidants, pharmacological inhibitor of p38^MAPK, or small interfering RNA against p53 rescue cell viability. On the contrary, cell survival to diamide relies upon redox activation of Nrf2, in a way independent on Keap1 oxidation, but responsive to ERK1/2 activation. Chemical inhibition of GSH neo-synthesis or ERK1/2 phosphorylation, as well as overexpression of the dominant-negative form of Nrf2 sensitizes cells to diamide toxicity. In the searching for the molecular determinant(s) unifying these phenomena, we found that SH-SY5Y cells show high GSH levels, but exhibit very low GPx activity. This feature allows to efficiently buffer disulfide stress, but leaves them being vulnerable to H₂O₂-mediated insult. The increase of GPx activity by means of selenium supplementation or GPx1 ectopic expression completely reverses death phenotype, indicating that the response of tumor cells to diverse oxidative stimuli deeply involves the entire GSH redox system.     

Role of Nrf2 in preventing ethanol-induced oxidative stress and lipid accumulation

Oxidative stress and lipid accumulation play important roles in alcohol-induced liver injury. Previous reports showed that, in livers of nuclear factor erythroid 2-related factor 2 (Nrf2)-activated mice, genes involved in antioxidant defense are induced, whereas genes involved in lipid biosynthesis are suppressed. To investigate the role of Nrf2 in ethanol-induced hepatic alterations, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation, were treated with ethanol (5 g/kg, po). Blood and liver samples were collected 6 h thereafter. Ethanol increased alanine aminotransferase and lactate dehydrogenase activities as well as thiobarbituric acid reactive substances in serum of Nrf2-null and wild-type mice, but not in Nrf2-enhanced mice. After ethanol administration, mitochondrial glutathione concentrations decreased markedly in Nrf2-null mice but not in Nrf2-enhanced mice. H₂DCFDA staining of primary hepatocytes isolated from the four genotypes of mice indicates that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. Ethanol increased serum triglycerides and hepatic free fatty acids in Nrf2-null mice, and these increases were blunted in Nrf2-enhanced mice. In addition, the basal mRNA and nuclear protein levels of sterol regulatory element-binding protein 1(Srebp-1) were decreased with graded Nrf2 activation. Ethanol further induced Srebp-1 mRNA in Nrf2-null mice but not in Nrf2-enhanced mice. In conclusion, Nrf2 activation prevented alcohol-induced oxidative stress and accumulation of free fatty acids in liver by increasing genes involved in antioxidant defense and decreasing genes involved in lipogenesis.     

DL0410 attenuates oxidative stress and neuroinflammation via BDNF/TrkB/ERK/CREB and Nrf2/HO-1 activation

Oxidative stress and neuroinflammation have been deeply associated with Alzheimer’s disease. DL0410 is a novel acetylcholinesterase inhibitor with potential anti-oxidative effects in AD-related animal models, while the specific mechanism has not been fully clarified. In this study, DL0410 was predicted to be related to the modification of cell apoptosis, oxidation-reduction process, inflammatory response and ERK1/ERK2 cascade by in silico target fishing and GO enrichment analysis. Then the possible protective effects of DL0410 were evaluated by hydrogen peroxide (H₂O₂)-induced oxidative stress model and lipopolysaccharides (LPS)-induced neuroinflammation model H₂O₂ decreased the viability of SH-SY5Y cells, induced malondialdehyde (MDA) accumulation, mitochondrial membrane potential (Δψm) loss and cell apoptosis, which could be reversed by DL0410 dose-dependently, indicating that DL0410 protected SH-SY5Y cells against H₂O₂-mediated oxidative stress. Western blot analysis showed that DL0410 increased the H₂O₂-triggered down-regulated TrkB, ERK and CREB phosphorylation and the expression of BDNF. In addition, TrkB inhibitor ANA-12, ERK inhibitor SCH772984 and CREB inhibitor 666-15 eliminated the inhibition of DL0410 on MDA accumulation and Δψm loss. Furthermore, DL0410 attenuates inflammatory responses and ROS production in LPS-treated BV2 cells, which is responsible for Nrf2 and HO-1 up-regulation. The present study demonstrates that DL0410 is a potential activator of the BDNF/TrkB/ERK/CREB and Nrf2/HO-1 pathway and may be a potential candidate for regulating oxidative stress and neuroinflammatory response in the brain. Together, the results showed that DL0410 is a promising drug candidate for treating AD and possibly other nervous system diseases associated with oxidative stress and neuroinflammation.     

The cytoprotective effect of <Emphasis Type="Italic">Rumex Aquaticus Herba</Emphasis> extract against hydrogen peroxide-induced oxidative stress in AGS cells

The Rumex Aquaticus Herba extract containing quercetin-3-β-D-glucuronopyranoside (ECQ) has been reported to exhibit various pharmacological activities, including anti-inflammatory and anti-oxidative effects. This plant has been traditionally used for the treatment of diarrhea, disinfestation, edema and jaundice, and as an antipyretic drug. The aim of the present study was to investigate the ability of ECQ to protect against oxidative damage and to determine its signaling mechanism in AGS cells. The protein expressions of heme oxygenase-1 (HO-1) and nuclear factor-erythroid 2 related factor 2 (Nrf2) were measured by Western blots. Cell viability was measured by MTT assay. Intracellular reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorofluorescein diacetate. Glutathione peroxidase levels were measured using kits. The protein expressions of HO-1 and its upstream mediator, Nrf2, increased after ECQ treatment. The HO-1 inhibitor, ZnPP, repressed the protective effect of ECQ on H₂O₂-induced cell damage. We found that LY294002, a specific PI3 K/Akt inhibitor, suppressed ECQ-induced HO-1 expression. ECQ significantly attenuated H₂O₂-induced cytotoxicity and ROS generation. Also, ECQ enhanced the antioxidant enzyme activities of glutathione peroxidase. These results suggest that ECQ exerts a cytoprotective effect against H₂O₂-induced oxidative stress by upregulation of Nrf2/HO-1 via the PI3 K/Akt pathway.     

Apoptotic effects of cigarette smoke extracts on mouse embryonic stem cells via oxidative stress

Previous studies have reported that cigarette smoke and cigarette smoke extract (CSE) have negative effects on embryonic development. However, no studies have investigated the mechanism through which CSE affects the cellular signaling pathway leading to apoptosis and oxidative stress in embryonic cells, or how the two pathways are cross‐linked. Thus, we studied the effects of CSE on apoptosis and oxidative stress in mouse embryonic stem cells (mESCs). Specifically, we measured changes in cell viability in response to CSEs (3R4F and two domestic cigarettes CSE 1 and 2) using a water soluble tetrazolium (WST) assay and a neutral red uptake (NRU) assay, which revealed that cell viability decreased in a concentration‐dependent manner. Western blot analysis revealed that the expression of cyclin D1 and cyclin E1 was decreased and that of p21 and p27 was increased by CSE. Additionally, the number of terminal deoxynucleotidyl transferase (TUNEL)‐stained cells was increased by CSE, while the levels of Bax and Caspase‐3 increased and Bcl‐2 decreased. Moreover, a 2′,7′‐dichlorofluorescin diacetate (DCF‐DA) assay and reactive oxygen species (ROS)‐Glo H₂O₂ assay confirmed that ROS were generated in response to CSE and that they were associated with up‐regulated Keaf‐1 and CHOP. Overall, the results revealed that cigarette smoke extract (CSE) inhibited cell proliferation by regulating cell cycle‐related protein expression and increased oxidative stress by regulating the expression of Kelch‐like ECH‐associated protein 1 (Keap‐1) and CCAAT/enhancer‐binding protein homologous protein (CHOP), resulting in apoptosis in mESCs.     

Kurarinone attenuates hydrogen peroxide-induced oxidative stress and apoptosis through activating the PI3K/Akt signaling by upregulating IGF1 expression in human ovarian granulosa cells

Dysregulated follicular development may lead to follicular atresia, and this is associated with oxidative stress in granulosa cells. Kurarinone is a natural compound possessing multiple activities, including antioxidative ability. However, the role of kurarinone in granulosa cell damage during follicular atresia remains unknown. Human ovarian granulosa KGN cells were treated with hydrogen peroxide (H₂O₂) to induce cellular damage. Cytotoxicity was investigated by lactate dehydrogenase (LDH) release assay. Oxidative stress was evaluated by detection of reactive oxygen species (ROS) generation and oxidative biomarker levels. Cell apoptosis was evaluated by flow cytometry, a Cell Death Detection ELISA Kit, and a Caspase-3 Assay Kit. The downstream target and related signaling pathway were analyzed by western blotting. Kurarinone attenuated H₂O₂-induced LDH release in KGN cells. Kurarinone relieved H₂O₂-induced increase in ROS generation and malondialdehyde level as well as decrease in superoxide dismutase-1 activity and heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 mRNA levels. Kurarinone inhibited H₂O₂-induced apoptosis in KGN cells. Kurarinone targeted insulin-like growth factor 1 (IGF1) and upregulated IGF1 expression to activate the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling. IGF1 silencing attenuated the suppressive effects of kurarinone on H₂O₂-induced oxidative stress and apoptosis in KGN cells. In conclusion, kurarinone attenuates H₂O₂-induced oxidative stress and apoptosis in KGN cells through activating the PI3K/Akt signaling by upregulating IGF1 expression, indicating the therapeutic potential of kurarinone in follicular atresia.     

大鼠心脏骤停后行心肺复苏对海马脑组织的影响及机制

目的探讨大鼠心脏骤停后,实施心肺复苏术对大鼠海马脑组织的保护作用和机制。方法SD大鼠40只,随机均分为对照组、缺血组、心肺复苏组、依达拉奉治疗组,采取窒息法使大鼠心脏骤停造模,缺血组大鼠心脏停跳10 min;复苏组实施心脏骤停/心肺复苏(CA/CPR),即使大鼠心脏停跳3 min后,再行心肺复苏7 min。10 min后处死各组大鼠,留取静脉血检测氧化应激指标,HE染色和电镜观察海马脑组织结构改变,实时荧光定量PCR和Western blot检测大鼠海马中Nrf2、Keap1基因和蛋白表达。结果与对照组大鼠比较,缺血组大鼠血清氧化应激水平升高,海马内神经细胞尼氏体减少,线粒体嵴破坏,Nrf2、Keap1基因和蛋白表达增加;与缺血组比较,心肺复苏组大鼠血清氧化应激水平降低,海马内神经细胞尼氏体增多,线粒体嵴破坏减轻,Nrf2、Keap1基因和蛋白表达降低。结论心脏骤停后心肺复苏术对大鼠海马脑组织具有保护作用,其机制与改善氧化应激损伤Nrf2/Keap1通路有关。     

HJ22, a Novel derivative of piperine,Attenuates ibotenic acid-induced cognitive impairment,oxidativestress, apoptosis and inflammation via inhibiting the protein-protein interaction of Keap1-Nrf2

Kelch-like ECH-associated protein (Keap1)-nuclear factor erythroid-2-related factor 2 (Nrf2) protein-protein interaction has become an important drug target for the treatment of Alzheimer’s disease. In this study, we found a novel piperine derivative (HJ22) synthesized by our group with great ability to bind to Keap-1 and activate Keap1-Nrf2-ARE signaling pathway in vitro, driving us to investigate the beneficial effects of HJ22 on ibotenic acid (IBO)-induced neurological disorders in rats and underlying mechanisms. Interestingly, HJ22 significantly ameliorated IBO-induced cognitive impairment in Morris water maze, Y-maze and passive avoidance tests. Moreover, HJ22 significantly attenuated cholinergic dysfunction and neuronal morphological changes via inhibiting apoptotic cell death induced by IBO. Notably, HJ22 inhibited the interaction between Keap1 and Nrf2, and subsequently up-regulated nuclear Nrf2 expression, thereby inhibiting oxidative stress and Thioredoxin-interacting protein (TXNIP)-mediated Nod-like receptor protein 3 (NLRP3) inflammasome activation. These findings demonstrated that HJ22 exhibited potent therapeutic effects against IBO-induced cognitive impairment by alleviating cholinergic damage, oxidative stress, apoptosis and neuroinflammation, which might be partly attributed to its inhibitory activity on Keap1-Nrf2 protein-protein interaction.     

Sulforaphane protects against cytokine- and streptozotocin-induced β-cell damage by suppressing the NF-κB pathway

Sulforaphane (SFN) is an indirect antioxidant that protects animal tissues from chemical or biological insults by stimulating the expression of several NF-E2-related factor-2 (Nrf2)-regulated phase 2 enzymes. Treatment of RINm5F insulinoma cells with SFN increases Nrf2 nuclear translocation and expression of phase 2 enzymes. In this study, we investigated whether the activation of Nrf2 by SFN treatment or ectopic overexpression of Nrf2 inhibited cytokine-induced β-cell damage. Treatment of RIN cells with IL-1β and IFN-γ induced β-cell damage through a NF-κB-dependent signaling pathway. Activation of Nrf2 by treatment with SFN and induction of Nrf2 overexpression by transfection with Nrf2 prevented cytokine toxicity. The mechanism by which Nrf2 activation inhibited NF-κB-dependent cell death signals appeared to involve the reduction of oxidative stress, as demonstrated by the inhibition of cytokine-induced H₂O₂ production. The protective effect of SFN was further demonstrated by the restoration of normal insulin secreting responses to glucose in cytokine-treated rat pancreatic islets. Furthermore, pretreatment with SFN blocked the development of type 1 diabetes in streptozotocin-treated mice.     

淫羊藿苷对H2O2诱导的软骨细胞氧化损伤的保护作用及其机制

目的 探究淫羊藿苷（icariin，ICA）对H₂O₂诱导的软骨细胞氧化损伤的保护作用及相关机制。方法 分离SD新生大鼠软骨细胞，随机分为对照组、H₂O₂模型组、ICA低剂量组、ICA中剂量组、ICA高剂量组；采用CCK8法检测各组细胞增殖能力的变化；采用ELISA试剂盒检测各组细胞中活性氧（reactive oxygen，ROS）、超氧化物歧化酶（superoxide dismutase，SOD）、丙二醛（malondialdehyde，MDA）、过氧化氢酶（catalase，CAT）以及谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）的表达情况；流式细胞术检测各组细胞周期情况，并计算增殖指数（proliferation index，PI）；Hoechst染色观察各组细胞核凋亡情况；分别采用荧光定量PCR （qRT-PCR）和Western blotting检测凋亡相关因子及Nrf2/HO-1通路的表达情况。结果 与对照组相比，H₂O₂模型组细胞增殖能力降低，ROS、MDA含量升高，SOD、CAT及GSH-Px含量下降，细胞凋亡情况加重；经ICA干预后，软骨细胞的增殖能力上升，ROS、MDA含量下降，SOD、CAT及GSH-Px含量增加，并且ICA能够有效抑制软骨细胞凋亡，上调Nrf2和HO-1蛋白的表达。结论 ICA对H₂O₂诱导的软骨细胞氧化损伤具有保护作用，能够抑制软骨细胞凋亡，其机制跟Nrf2/HO-1信号通路有关。     

Epigallocatechin‐3‐gallate partially restored redox homeostasis in arsenite‐stressed keratinocytes

Arsenite (AsIII) is known for inducing severe oxidative stress and skin carcinogenesis. Contrastingly, phytochemical, epigallocatechin‐3‐gallate (EGCG) combats toxic insults. Our study focused on the effect of EGCG on redox status of AsIII‐stressed normal human keratinocytes, HaCaT cells. EGCG (50 μm ) increased the cell viability by 29% in AsIII (50 μm ) insulted HaCaT cells but exhibited pro‐oxidant activity by elevated expression of the oxidative stress markers. EGCG was effective not only in reducing AsIII‐induced nuclear expression of Nrf2 and Nrf2Ser40 but also in increasing nuclear expression of Keap1 both at protein and mRNA level. EGCG did not have similar effects on all Nrf2 downstream targets. EGCG elevated expression of HO‐1 and γ‐GCL,showed no change in MRP1 but decreased superoxide dismutase, NAD(P)H dehydrogenase quinone 1 and glutathione S transferase activity in AsIII‐treated HaCaT cells. EGCG along with AsIII caused decreased phosphorylation of Nrf2 at ser40 residue, which might have facilitated Keap1‐mediated nuclear export and degradation of Nrf2 and paved the pro‐survival signal for AsIII‐insulted HaCaT cells. In conclusion, it might be indicated that EGCG in spite of inducing the pro‐oxidant effect was effective in increasing the viability of AsIII‐treated HaCaT cells by partially restoring the Nrf2/Keap1‐mediated signaling axis.