Protective effect of diphenyleneiodonium,a NADPH oxidase inhibitor,on hyperpermeability of endothelial cells exposed to AOPP-HSA in vitro

AIM: To investigate the effect of advanced oxidation protein product-human serum albumin (AOPP-HSA) at different concentrations on the permeability of human umbilical vein endothelial cell (HUVEC) monolayer and the protective effect of NADPH oxidase inhibitor diphenyleneiodonium (DPI) against AOPP-HSA exposure. METHODS: Cultured HUVECs were exposed to 200 mg/L HSA (control) or AOPP-HSA (50, 100 and 200 mg/L). The permeability of the endothelial monolayer was assessed by measuring CMFDA-labeled THP-1 cells across the endothelial cells. The cultured HUVECs were treated with HSA (200 mg/L), AOPP-HSA (200 mg/L), or AOPP-HSA (200 mg/L) + DPI (100 μmol/L), and the activation of NADPH oxidase, endothelial monolayer permeability and cytoskeleton rearrangement were evaluated. RESULTS: AOPP-HSA increased the permeability of the endothelial cell monolayer, and AOPP-HSA at 200 mg/L significantly increased the phosphorylation level of NADPH oxidase in the cells. Treatment with 100 μmol/L DPI obviously attenuated AOPP-HSA-induced NADPH oxidase activation, the increase in the permeability of the cell monolayer and the cytoskeleton rearrangement. CONCLUSION: AOPP-HSA increases the hyperpermeability of HUVEC monolayer via the phosphorylation of NADPH oxidase, and the NADPH oxidase inhibitor DPI reverses such effects.     

茶树NADPH氧化酶基因的克隆、亚细胞定位与表达分析

以茶树(Camellia sinensis)‘迎霜’为试验材料,采用同源克隆的方法,利用RACE和RT-PCR技术获得茶树NADPH氧化酶基因Cs RBOHA的c DNA全长(Gen Bank登录号:KJ782632)。该基因全长3 157 bp,开放阅读框2 769 bp,编码922个氨基酸。生物信息学分析显示,该基因编码的蛋白分子量为103.33 k D,理论等电点为9.28;C端序列较保守,N端序列保守性较低,与烟草和蓖麻的相似性达79%,进化关系最近;蛋白结构具有典型的家族特征。该蛋白分布于细胞质膜上,与预测结果一致。实时荧光定量PCR结果显示,该基因的表达存在组织特异性,并且在低温(4℃)、高盐(200 mmol·L-1 Na Cl)、ABA(200 mg·L-1)和干旱(10%PEG 6000)条件下出现不同程度的上调。     

Effects of oxidative stress on high-fat,high-salt diet and sucrose solution-induced metabolic syndrome in nephropathy rats

AIM: To establish a suitable animal model of nephropathy associated with metabolic syndrome (MS) induced by abnormal diet, and to investigate the effects of oxidative stress on renal damage in MS rats. METHODS: Normal 7-week-old male SD rats were randomly divided into 2 groups.The animals were fed with normal chow (control group, n=10) or high-fat and high-salt diet plus 20% sucrose solution (MS model group, n=10) for 20 weeks. Systolic blood pressure (SBP) was measured monthly. The levels of blood glucose, serum and urinary creatinine (Cr), total cholesterol (TC), triglycerides (TG), fasting insulin (FIns), urinary protein, urinary albumin and urinary sodium were determined. Insulin resistance (HOMA-IR), creatinine clearance (Ccr), urinary protein excretion (UPE), urinary albumin excretion (UAE) and urinary sodium excretion (USE) were calculated. Renal total-antioxidant capacity (T-AOC), inhibiting superoxide anion capacity (ISAC), malondialdehyde (MDA) content, and antioxidant enzyme activity were measured. Renal protein expression of Cu/Zn-SOD, NADPH oxidase subunit p47^phox and p22^phox was detected by Western blotting. In addition, pathological changes of the kidney were observed with PAS and Masson staining,and degree of glomerulosclerosis (GS) and tubulointerstitial injury was evaluated. RESULTS: Compared with control rats, SBP, TC, TG, FIns, USE and UAE were increased in MS rats. Furthermore, the MS rats showed a significant elevation of renal MDA content, p47^phox protein expression and GS score, and reduction of T-AOC, ISAC, SOD activity, and Cu/Zn-SOD protein expression in the kidney. CONCLUSION: SD rats fed with abnormal diet produce a suitable animal model of MS nephropathy that mimics the major features of human MS. Oxidative stress caused by up-regulation of NADPH oxidase expression and down-regulation of SOD expression may be one of the mechanisms leading to MS renal damage.     

北京黑猪NADPH生成酶活性的遗传分析

本文收集１３０２头北京黑猪个体测定资料,利用最小二乘模型估计北京黑猪背膘脂肪组织ＮＡＤＰＨ生成酶活性、背膘厚、日增重、饲料转化率等性状的遗传力、遗传相关、表型相关、环境相关等参数,同时估计了１９９１～１９９５年间这些性状的变化以及采样体重、性别等因素对ＮＡＤＰＨ生成酶活性的影响。结果表明：①ＮＡＤＰＨ生成酶总活性的估计遗传力为０．２５±０．０３（Ｐ＜０．０１）;②ＮＡＤＰＨ生成酶活性与背膘厚、日增重、饲料转化率的遗传相关分别为０．５１,０．７５及０．４８;③在５０ｋｇ左右体重采样时,不同性别测定猪ＮＡＤＰＨ生成酶总活性有显著差别（Ｐ＜０．０５）。     

亚精胺对渗透胁迫的玉米幼苗叶片NADPH氧化酶和抗氧化酶活性的影响

为了探讨多胺增强植物抗旱性的机理,研究了渗透胁迫下,玉米品种农大108(抗旱性较强)和掖单13号(抗旱性较弱)幼苗叶片中亚精胺(spermidine:Spd)含量与质膜NADPH氧化酶和3种抗氧化酶——超氧物歧化酶(SOD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)活性的变化.结果表明,渗透胁迫48 h,抗旱性弱的掖单13号幼苗叶片的NADPH氧化酶活性的上升幅度明显大于抗旱性强的农大108,而农大108幼苗叶片Spd含量以及SOD,CAT和APX活性的上升幅度明显大于掖单13号.外源Spd处理明显提高了掖单13号幼苗叶片的Spd含量以及SOD,CAT和APX活性,也明显抑制了NADPH氧化酶活性的上升.Spd的生物合成专一性抑制剂——甲基乙二醛-双(鸟嘌呤腙)(MGBG)处理,则明显抑制了渗透胁迫下农大108叶片中SOD,CAT和APX活性以及Spd含量的上升,也明显提高了NADPH氧化酶活性.结果还表明,渗透胁迫下玉米幼苗叶片的Spd可能通过抑制NADPH氧化酶活性以及促进抗氧化酶活性而降低叶片内的活性氧水平,从而减轻活性氧对幼苗的伤害.     

FBW7 inhibits ox-LDL-induced granulosa cell apoptosis by promoting NOX1 degradation

AIM To investigate the effect of F-box and WD repeat domain containing protein 7 （FBW7） on the injury of granulosa cells （GCs） induced by oxidized low-density lipopretion （ox-LDL） stimulation and its potential mechanism. METHODS The GCs isolated from women of reproductive age with different obesity levels, the GCs isolated from high fatty diet （HFD）-fed rats, and the ox-LDL-treated rat GCs were collected, and the expression of FBW7 was detected by Western blot. After the rat GCs were infected with adenovirus encoding FBW7 （Ad-FBW7）, the cells were treated with ox-LDL （80 mg/L） for 48 h, and then the cell viability, apoptosis, NADPH oxidase （NOX） activity and the key subunit proteins of NOX were detected by CCK-8 assay, Annexin V/PI staining, lucigenin chemiluminescence and Western blot, respectively. The effect of FBW7 over-expression on NOX1 degradation was evaluated by cycloheximide （CHX） chase assay. RESULTS The expression of FBW7 was lower in the GCs isolated from obese women of reproductive age, the GCs isolated from HFD-fed rats, and the ox-LDL-treated rat GCs （P<0.05）. Over-expression of FBW7 inhibited ox-LDL-induced injury of GCs, NOX activity, and NOX1 expression （P<0.05）. The results of CHX chase assay showed that over-expression of FBW7 accelerated the degradation of NOX1 protein under ox-LDL condition （P<0.05）. CONCLUSION Over-expression of FBW7 reduces ox-LDL-induced injury of GCs by accelerating NOX1 degradation and then weakening NOX activity.     

Over-expression of ACE2 gene alleviates Ang Ⅱ-induced oxidative stress in neural cells

AIM:To investigate the effect of over-expression of angiotensin-converting enzyme 2 (ACE2) gene on angiotensin Ⅱ (Ang Ⅱ)-induced oxidative stress and NADPH oxidase (NOX) expression in mouse neuroblastoma Neuro-2A cells. METHODS:The recombinant lentivirus encoding ACE2 gene was constructed and transfected into the Neuro-2A cells at a multiplicity of infection (MOI) of 10 for 72 h. The transfection efficiency of ACE2 gene and protein expression of ACE2 were detected, and the Neuro-2A cells were identified by detection of a neural cell marker. The Neuro-2A cells were divided into 7 groups:control group, eGFP group, ACE2-eGFP group, Ang Ⅱ treatment group, Ang Ⅱ-eGFP group, Ang Ⅱ-ACE2-eGFP group and Ang Ⅱ-ACE2-eGFP-A779 group. The Ang(1-7) level was determined by ELISA. The level of reactive oxygen species (ROS) in the cells was measured with a method of DHE staining. The protein expression of MAS receptor and NOX subunits (NOX2, NOX4, p47^phox and p67^phox) was detected by Western blot. RESULTS:Ang Ⅱ signi-ficantly increased ROS levels (P<0.01) and up-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox (P<0.01), but down-regulated MAS protein expression (P<0.01). Over-expression of ACE2 inhibited Ang Ⅱ-induced increase in ROS, down-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox,and still increased the Ang(1-7) level (P<0.01) and MAS receptor expression (P<0.01). An antagonist of the MAS receptor, A779, blocked the down-regulating effect of ACE2 on NOX expression (P<0.05). CONCLUSION:ACE2 over-expression antagonizes Ang Ⅱ-induced oxidative stress via MAS receptor in the neural cells.     

家蚕氟中毒后中肠NADPH-细胞色素P450还原酶和NADPH-细胞色素C还原酶活性的变化

为了探究家蚕对NaF的代谢机制,以家蚕耐氟品种T6和氟化物敏感品种734为材料,从5龄起蚕开始分别添食50、100、200、400 mg/kg NaF溶液处理后的桑叶,检测蚕体中肠微粒体酶液中的黄素蛋白NADPH-细胞色素P450还原酶(CPR)和NADPH-细胞色素C还原酶(CR)的活性变化。氟物化敏感品种734的4个NaF处理组第3天的中肠CPR活性低于对照组,其余时间几乎都高于对照组,400 mg/kg NaF处理组在第4天的CPR活性最高,且各NaF处理组的CPR活性差异显著(P<0.05);耐氟品种T6 NaF处理组和对照组的中肠CPR活性整体上呈先升后降的趋势,几乎都在第2天达到最高值,各组之间的差异不显著(P>0.05)。氟化物敏感品种734的4个NaF处理组的中肠CR活性呈现先升后降的趋势,而对照组呈下降趋势;耐氟品种T6的50、100、400 mg/kg NaF处理组在第1～2天的中肠CR活性呈明显下降趋势,之后的变化相对较小,对照组的CR活性仅在第3～4天略高于NaF处理组,而其余时间NaF处理组的CR活性较高。2个家蚕品种添食不同浓度NaF后的中肠CR活性差异均不显著(P>0.05)。试验结果显示,耐氟品种T6在NaF作用下中肠的CPR和CR活性变化范围(分别为对照组的0.4～2.0倍和0.3～2.9倍)远小于氟化物敏感品种734这2种酶的活性变化范围(分别为对照组的0.6～9.3倍和0.4～4.6倍)。初步推测CPR和CR与家蚕对氟化物代谢具有一定的关联。     

Roles of peroxidases and NADPH oxidases in the oxidative response of wheat (Triticum aestivum) to brown rust (Puccinia triticina) infection

The goal of this work was to establish which enzymes – peroxidases or NADPH oxidases – play the most important role in the resistance‐related oxidative burst response of wheat to infection by brown rust (Puccinia triticina). The expression of four peroxidases and two NADPH oxidases was analysed in the susceptible wheat cv. Thatcher and isogenic lines with different Lr resistance genes after pathogen inoculation. Of the peroxidases, TaPrx118 and TaPrx112 were induced several times more strongly than TaPrx103 and TaPrx107. The induction of peroxidases was more pronounced than that of NADPH oxidases. The patterns of peroxidase expression clearly differentiated moderately resistant from highly resistant lines and corresponded to oxidative response profiles. The possible involvement of peroxidases or NADPH oxidases was verified with enzyme‐specific inhibitors. The oxidative burst in the susceptible cv. Thatcher and in the lines TcLr24, TcLr25, TcLr9 was peroxidase‐dependent, while the response in line TcLr26 was NADPH‐oxidase‐dependent. It is postulated that class III peroxidases play a leading role in the formation of reactive oxygen species molecules during the response of wheat to pathogen infection. The results suggest a high level of redundancy of some peroxidase genes induced in biotic stress. The role of both enzyme systems in wheat response/resistance to brown rust is discussed in relation to the oxidative response, the efficiency of resistance, and the presence and origin of particular Lr resistance genes.     

NbRbohB基因在本氏烟与疫霉菌互作中的功能分析

活性氧(active oxygen species, AOS)在植物抗病中发挥着重要作用,主要由NADPH氧化酶(nicotinamide adenine dinucleotide phosphate oxidase)系统产生.为明确NADPH氧化酶NbRbohB基因在本氏烟与疫霉菌亲和与非亲和性互作中的功能,采用荧光定量PCR技术以及病毒诱导的基因沉默方法探究了NbRbohB基因在本氏烟中对2种疫霉菌抗性中的作用,并利用NADPH氧化酶抑制剂对辣椒疫霉的抗性进行了检测.结果发现:2种疫霉菌均能诱导本氏烟发生氧迸发,且NbRbohB基因可能参与了疫霉菌诱导本氏烟发生的氧迸发过程.该基因沉默后降低了本氏烟对亲和互作辣椒疫霉菌的抗性,但对非亲和互作疫霉菌的抗性没有肉眼可见的影响;NADPH氧化酶抑制剂处理本氏烟后也能降低其对辣椒疫霉的抗性.表明该基因通过介导AOS产生,参与植物对亲和性与非亲和性互作疫霉的抗病反应,在亲和互作中尤为重要.