NADPH Oxidase NOX4 Mediates Stellate Cell Activation and Hepatocyte Cell Death during Liver Fibrosis Development

A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2^−/−/p19^ARF−/−, Stat3^Δhc/Mdr2^−/−) and a model of experimental induced fibrosis (CCl₄) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes.     

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4 ^−/−) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4 ^−/− mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy.     

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H₂O₂ production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H₂O₂ production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H₂O₂ production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.     

砷暴露诱导人肝细胞氧化应激过程中NADPH氧化酶作用机制

目的：探讨砷暴露诱导细胞氧化应激的分子机制。方法：采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽（reducedglutathione,GSH）预处理,检测细胞超氧阴离子（02。）和细胞整体ROS的水平。WestemBlot方法检测细胞氧化／抗氧化重要酶微粒体谷胱甘肽硫转移酶（microsomalglutathioneS-transferase-l,Mgst．1）、半胱氨酸双加氧酶l（cysteinedioxygenasel,Cd01）和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂（diphenyleneiodoniumchloride,DPI）进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果：砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷（亚砷酸钠,A矿）诱导氧化应激作用显著强于五价砷（砷酸钠,As5＋）。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论：NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。     

Gap-junctional Hemichannels Are Activated by ATP Depletion in Human Renal Proximal Tubule Cells

We present evidence suggesting that gap-junctional hemichannels (GJH) may be involved in acute ischemic injury of human renal proximal tubule cells (hPT cells). Two GJH, from neighboring cells, join to form an intercellular gap junction channel (GJC). Undocked GJH are permeable to hydrophilic molecules up to 1 kDa, and their opening can significantly alter cell homeostasis. Both GJC and GJH formed by connexin 43 (Cx43) are activated by dephosphorylation. Hence, we tested whether GJH activation during ATP depletion contributes to cell damage in renal ischemia. We found that hPT cells in primary culture express Cx43 (RT-PCR and Western-blot analysis) at the plasma membrane region (immunofluorescence). Divalent-cation removal or pharmacological ATP depletion increased cell loading with the hydrophilic dye 5/6 carboxy-fluorescein (CF, 376 Da) but not with fluorescein-labeled dextran (>1500 Da). Endocytosis and activation of P2X channels were experimentally ruled out. Several GJC blockers inhibited the loading elicited by PKC inhibition. Double labeling (CF and propidium iodide) showed that both Ca(2+) removal and ATP depletion increase the percentage of necrotic cells. Gadolinium reduced both the loading and the degree of necrosis during divalent-cation removal or ATP depletion. In conclusion, GJH activation may play an important role in the damage of human renal proximal tubule cells during ATP depletion. These studies are the first to provide evidence supporting a role of GJH in causing injury in epithelial cells in general and in renal-tubule cells in particular.     

Uric Acid Promotes Osteogenic Differentiation and Inhibits Adipogenic Differentiation of Human Bone Mesenchymal Stem Cells

To investigate the effect of uric acid on the osteogenic and adipogenic differentiation of human bone mesenchymal stem cells (hBMSCs). The hBMSCs were isolated from bone marrow of six healthy donors. Cell morphology was observed by microscopy and cell surface markers (CD44 and CD34) of hBMSCs were analyzed by immunofluorescence. Cell morphology and immunofluorescence analysis showed that hBMSCs were successfully isolated from bone marrow. The number of hBMSCs in uric acid groups was higher than that in the control group on day 3, 4, and 5. Alizarin red staining showed that number of calcium nodules in uric acid groups was more than that of the control group. Oil red‐O staining showed that the number of red fat vacuoles decreased with the increased concentration of uric acid. In summary, uric acid could promote the proliferation and osteogenic differentiation of hBMSCs while inhibit adipogenic differentiation of hBMSCs.     

The Role of the NADPH Oxidase NOX2 in Prion Pathogenesis

Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis.     

NK4拮抗HGF促进肿瘤细胞凋亡的生物学效应

观察NK4通过拮抗肝细胞生长因子（HGF）诱导不同肿瘤细胞凋亡,研究其生物学作用及分子机制.以足叶乙甙（VP-16）诱导凋亡,分别或经HGF蛋白、NK4蛋白处理5种肿瘤细胞（B细胞淋巴瘤细胞系Raji、人急性粒细胞白血病细胞系HL-60、宫颈癌细胞系HeLa、前列腺癌细胞系PC-3、非小细胞肺癌细胞系A549）,采用流式细胞术（FCM）、吖啶橙 (AO) 染色法、苏木素 伊红（HE）染色法定量观察5种肿瘤细胞的凋亡情况,并进行相关分析. FCM发现,经VP-16处理5种肿瘤细胞凋亡率均显著高于对照组(P<0.001),而HGF+VP-16组凋亡率明显下降(P<0.01),HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.05). AO染色和HE染色结果也证实,5种肿瘤细胞经VP-16处理后凋亡率均显著增高 (P<0.001,P<0.001),而HGF+VP-16组细胞凋亡率均明显低于VP-16组(P<0.001,P<0.01), HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.001,P<0.05).此外,发现NK4+VP-16组、HGF+ NK4+VP-16组、VP-16组等3组间凋亡率无统计学差异(P>0.05). 以上结果提示,HGF蛋白可抵抗凋亡诱导剂VP-16的作用, 明显降低细胞凋亡;NK4通过竞争性抑制HGF从而促进肿瘤细胞的凋亡,具有潜在的肿瘤治疗价值.     

葡萄糖氧化酶诱导肝细胞氧化应激模型的建立

目的:研究不同浓度葡萄糖氧化酶(GO)对人肝细胞L02氧化应激水平的影响,以确定建立肝细胞氧化应激模型的合适浓度。方法:用不同浓度GO干预L02肝细胞2h,MTT法检测细胞的存活率,流式细胞术检测细胞内活性氧簇(ROS),荧光强度(FI)来表示ROS水平。分光光度法检测检测细胞MDA、GSH,速率法检测细胞培养液LDH、AST和ALT的水平。结果:①随GO浓度增加,肝细胞的存活率逐渐降低,其中75U/L、100U/L和125U/L组存活率显著低于对照组(P<0.05)。②随GO浓度增加,MDA含量逐渐增高,其中50U/L、75U/L、100U/L、125U/L组MDA水平较对照组显著增高(P<0.05)。GSH水平随GO浓度增高而逐渐减低,各干预组较对照组均显著降低(P<0.05)。GO各干预组FI均较对照组显著降低(P<0.05)。③各干预组LDH活性均显著高于对照组(P<0.05),50U/L、75U/L、100U/L、125U/L干预组AST与ALT水平均较对照组显著增高(P<0.05)。结论:GO能引起的肝细胞氧化应激损伤有剂量依赖性,100U/L是建立肝细胞氧化应激的合适浓度。     

葡萄糖氧化酶诱导肝细胞氧化应激模型的建立

目的：研究不同浓度葡萄糖氧化酶（GO）对人肝细胞L02氧化应激水平的影响,以确定建立肝细胞氧化应激模型的合适浓度。方法：用不同浓度GO干预L02肝细胞2h,MTT法检测细胞的存活率,流式细胞术检测细胞内活性氧簇（ROS）,荧光强度（FI）来表示ROS水平。分光光度法检测检测细胞MDA、GSH,速率法检测细胞培养液LDH、AST和ALT的水平。结果：①随GO浓度增加,肝细胞的存活率逐渐降低,其中75U/L、100U/L和125U/L组存活率显著低于对照组（P〈0.05）。②随GO浓度增加,MDA含量逐渐增高,其中50U/L、75U/L、100U/L、125U/L组MDA水平较对照组显著增高（P〈0.05）。GSH水平随GO浓度增高而逐渐减低,各干预组较对照组均显著降低（P〈0.05）。GO各干预组FI均较对照组显著降低（P〈0.05）。③各干预组LDH活性均显著高于对照组（P〈0.05）,50U/L、75U/L、100U/L、125U/L干预组AST与ALT水平均较对照组显著增高（P〈0.05）。结论：GO能引起的肝细胞氧化应激损伤有剂量依赖性,100U/L是建立肝细胞氧化应激的合适浓度。     

Homocysteine-Induced Apoptosis in Endothelial Cells Coincides With Nuclear NOX2 and Peri-nuclear NOX4 Activity

Apoptosis of endothelial cells related to homocysteine (Hcy) has been reported in several studies. In this study, we evaluated whether reactive oxygen species (ROS)-producing signaling pathways contribute to Hcy-induced apoptosis induction, with specific emphasis on NADPH oxidases. Human umbilical vein endothelial cells were incubated with 0.01–2.5 mM Hcy. We determined the effect of Hcy on caspase-3 activity, annexin V positivity, intracellular NOX1, NOX2, NOX4, and p47^phox expression and localization, nuclear nitrotyrosine accumulation, and mitochondrial membrane potential (ΔΨ _m). Hcy induced caspase-3 activity and apoptosis; this effect was concentration dependent and maximal after 6-h exposure to 2.5 mM Hcy. It was accompanied by a significant increase in ΔΨ _m. Cysteine was inactive on these parameters excluding a reactive thiol group effect. Hcy induced an increase in cellular NOX2, p47^phox, and NOX4, but not that of NOX1. 3D digital imaging microscopy followed by image deconvolution analysis showed nuclear accumulation of NOX2 and p47^phox in endothelial cells exposed to Hcy, but not in control cells, which coincided with accumulation of nuclear nitrotyrosine residues. Furthermore, Hcy enhanced peri-nuclear localization of NOX4 coinciding with accumulation of peri-nuclear nitrotyrosine residues, a reflection of local ROS production. p47^phox was also increased in the peri-nuclear region. The Hcy-induced increase in caspase-3 activity was prevented by DPI and apocynin, suggesting involvement of NOX activity. The data presented in this article reveal accumulation of nuclear NOX2 and peri-nuclear NOX4 accumulation as potential source of ROS production in Hcy-induced apoptosis in endothelial cells.     

Dopamine Promotes the Survival of Embryonic Striatal Cells: Involvement of Superoxide and Endogenous NADPH Oxidase

The dopaminergic system appears early in mammalian brain development, and a neurodevelopmental role for dopamine (DA) has been suggested. In the present study, we found that DA markedly promoted the survival of embryonic striatal cells in cultures. The failure of DA receptor antagonists to block this survival-promoting effect and the capability of S-apomorphine, which is devoid of DA receptor agonist activity but possesses antioxidative activity as R-apomorphine and DA, to completely mimic this effect suggested that DA receptor activation was not required in the survival-promoting effect elicited by DA, and its antioxidative activity might be involved. Moreover, it was found that mRNA of NADPH oxidase was expressed in the embryonic striatum. Furthermore, DPI or apocynin, NADPH oxidase inhibitors, promoted the survival of embryonic striatal cells. Addition of either DA or DPI into striatal cell cultures decreased the superoxide level. These results indicate that the mechanisms underlying the neuroprotective effects of DA were likely associated with its antioxidative activity. NADPH oxidase might contribute, at least in part, to ROS generation.     

敲减NADPH氧化酶4能降低STAT3活性抑制人黑色素瘤A375细胞的增殖

NADPH氧化酶参与细胞活性氧族（ROS）的生成过程,而ROS与肿瘤细胞增殖密切相关. 为了阐明NADPH氧化酶影响黑色素瘤A375细胞增殖的分子机制,本文首先应用荧光定量PCR和Western 印迹证实NOX4为人黑色素瘤A375细胞的NADPH氧化酶功能核心亚基;随后根据NOX4基因设计3条干扰序列和对照序列并连接到pSuper-retro-puro载体,经鉴定后转化E.coli DH5α感受态细胞、筛选有效干扰序列并用于逆转录病毒包装,病毒液感染A375细胞并经嘌呤霉素筛选10 d,构建了NOX4缺陷的A375稳转细胞珠（A375 NOX4Δ）,其NOX4的mRNA和蛋白表达分别下降了66.02%和77.35%,伴随NADPH氧化酶活性和ROS水平分别下降了79.17%和64.16%;MTT、EdU法检测显示,A375-NOX4Δ细胞的增殖能力比A375-WT细胞明显降低、倍增时间延长,增殖细胞数量下降了68.27%（P＜0.01）,呈现G1→S期阻滞;Western blot检测表明A375 NOX4Δ细胞的 cyclin D1、CDK4分别下降了55.7%（P＜0.01）和64.8%（P＜0.01）,而P53、P21分别增加了6.89 倍（P＜0.01）和3.27 倍（P＜0.01）,STAT3、P-STAT3分别下降了51.80%（P＜0.05）和82.58%（P＜0.01）;电泳迁移率变动分析（EMSA）表明,A375 NOX4Δ细胞的STAT3-DNA结合活性明显降低. 上述结果提示,敲减A375细胞的NOX4表达可能通过减少ROS生成使得STAT3磷酸化水平及其结合DNA的活性下降,最终导致A375-NOX4Δ细胞增殖减少、呈现G1→S期阻滞,这为黑色素瘤发病机制研究提供了新思路及可能的药物作用靶点.     

Phospholipases Involved in Activation of the Neutrophil NADPH Oxidase

Stimulation of neutrophils with a variety of stimuli can result in the activation of phospholipases A₂, C, or D with the resultant hydrolysis of plasma membrane phospholipids and the formation of important second messenger molecules. In the neutrophil, the activities of these phospholipases have been implicated in the processes of both stimulating and maintaining oxidase activation. In this review, some of the methods currently used to measure the products of phospholipase activation in the neutrophil are described, along with the possible role of their products in reactive oxidant production by the neutrophil NADPH oxidase.     

SiO2NPs通过诱导氧化应激激活Fas/FasL通路促进TM4细胞凋亡

目的 探讨纳米二氧化硅（silicon dioxide nanoparticles,SiO₂NPs）对小鼠睾丸支持细胞（TM4）的毒性作用及其分子机制。方法 将TM4细胞暴露于不同浓度的SiO₂NPs（0、1、10、100 mg/L）培养液24 h,处理结束后,采用光学显微镜和CCK-8法检测小鼠睾丸支持细胞的形态和活性。利用荧光探针DCFH-DA检测细胞内活性氧（ROS）水平,MDA和SOD试剂盒检测细胞内的丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。利用Annexin V-FITC/PI凋亡试剂盒分析TM4细胞的凋亡水平,免疫印迹法检测Fas、FasL、Caspase-8、Caspase-3、Bax和Bcl-2等细胞凋亡信号分子的蛋白质表达水平。结果 研究发现,SiO₂NPs呈剂量依赖性地抑制TM4细胞增殖,降低细胞存活率和数量,并影响细胞的形态结构。此外,SiO₂NPs诱导TM4细胞产生过量ROS,引起脂质过氧化产物MDA含量以及抗氧化酶SOD活性增加导致氧化应激。进一步研究显示,SiO₂NPs显著增加TM4细胞凋亡水平,并激活Fas/FasL死亡受体介导的细胞凋亡信号通路。有意思的是,抗氧化剂NAC可以通过降低氧化应激水平有效缓解SiO₂NPs引起的TM4细胞损伤和细胞凋亡。结论 综上,SiO₂NPs通过诱导氧化应激激活Fas/FasL信号通路促进TM4细胞凋亡。     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Arsenic Sulfide Promotes Apoptosis in Retinoid Acid Resistant Human Acute Promyelocytic Leukemic NB4-R1 Cells through Downregulation of SET Protein

Tetra-arsenic tetra-sulfide (As₄S₄) is an arsenic compound with anti-tumor activity, especially in acute promyelocytic leukemia (APL) that are resistant to retinoic acid (RA). Although recent studies revealed that the therapeutic action of As₄S₄ is closely associated with the induction of cellular apoptosis, the exact molecular mechanism of action of As₄S₄ in RA-resistant APL remains to be clarified. In this study, we found that As₄S₄-induced apoptosis was accompanied by reduced mRNA and protein expression of SET gene in RA-resistant NB4-R1 cells. Moreover, RNAi knockdown of SET gene further promoted As₄S₄-induced apoptosis, while SET over-expression inhibited it, suggesting that As₄S₄ induces apoptosis through the reduction of SET protein in NB4-R1 cells. We also demonstrated that the knockdown of SET gene resulted in the upregulation of protein phosphatase 2 (PP2A) expression and the downregulation of promyelocytic leukemia and retinoic acid receptor α fusion gene (PML-RARα) expression, which were enhanced by As₄S₄ treatments. By contrast, over-expression of SET gene resulted in PP2A downregulation and PML-RARα upregulation, which were abolished by As₄S₄ pretreatment. Since PP2A is a pro-apoptotic factor and PMLRARα is an anti-apoptotic factor, our results suggest that As₄S₄-induced apoptosis in NB4-R1 cells is through the downregulation of SET protein expression, which in turn increases PP2A and reduces PML-RARα expressions to lead to cell apoptosis.