质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium) 细胞壁激发子诱导人参细胞产生激发反应

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性.这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导.Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproteins)的表达.当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30min后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制.由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性.在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达.这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化.人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用.因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人参细胞产生激发反应这样一个由外及内的级联反应.     

质膜NAD（P）H氧化酶参予金瓜炭疽（Colletotr8ichum lagerarium）细胞壁激发子诱导人参细胞产生激发反应

在人参（Panax ginsengC.A.Meyer)悬浮细胞质膜上测出了NAD（P）H氧化酶活性可以被金瓜炭疽细胞壁激发子（Cle)诱导,Cle处理还能诱导人参悬浮细胞的氧迸发,促进人参悬浮细胞的皂苷合成,提高苯丙氨酸解氨酶（PAL）的活力,以及诱导查尔式酮酶（CHS）的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproteins)的表达,当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二精致苯基碘（Diphenylene iodonium,DPI)与奎吖因（quinacrine)预处理人参悬浮细胞30min后,Cle诱导的H2O2释放与Cle激活的质膜NAD（P）H氧化酶活性被抑制。同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测;人参细胞质膜NAD（P）H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性,在Cle激发人参悬浮细胞产生氧迸发的过程中,NAD（P）H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达,这一过程中还涉及到Ca^2 内流,胞内Ca^2 浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD（P）H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD（P）H氧化酶被诱导,H2O2释放,到人参细胞产生激发反应这样一个由外及内的级联反应。     

质膜NADPH氧化酶的活化与H2O2的产生介导内源激发子诱导人参悬浮细胞中PAL活性的提高与人参皂甙的积累

利用纤维素酶降解人参(Panax ginseng C.A.Meyer)悬浮细胞的细胞壁制备了内源激发子(CDW).CDW体外诱导了游离人参细胞质膜NADPH氧化酶的活性,激发了活体人参悬浮细胞产生H2O2.CDW还可以诱导提高苯丙氨酸解氨酶(PAL)活性,促进人参鲨烯环氧酶基因(sqe)的转录与人参皂甙的积累.NADPH氧化酶的抑制剂不仅可以抑制CDW体外诱导的质膜NADPH活性而且还可以抑制CDW诱导人参细胞产生H2O2.进而,这些抑制剂还可以抑制CDW诱导PAL活性的提高,以及sqe的转录与人参皂甙的合成.过氧化氢酶与H2O2的粹灭剂也可以抑制CDW激发产生的这些诱导效应.上述结果表明CDW激发质膜NADPH氧化酶的活化与H2O2的产生在介导CDW诱导人参细胞抗性反应中,包括PAL活性的提高与人参皂甙的积累,起了重要的信号转导作用.     

质膜NADPH氧化酶的活化与H2O2的产生介导内源激发子诱导人参悬浮细胞中PAL活性的提高与人参皂甙的积累

利用纤维素酶降解人参(Panax ginseng C．A．Meyer)悬浮细胞的细胞壁制备了内源激发子(CDW)。CDW体外诱导了游离人参细胞质膜NADPH氧化酶的活性,激发了活体人参悬浮细胞产生H2O2。CDW还可以诱导提高苯丙氨酸解氨酶(PAL)活性,促进人参鲨烯环氧酶基因(sqe)的转录与人参皂甙的积累。NADPH氧化酶的抑制剂不仅可以抑制CDW体外诱导的质膜NADPH活性而且还可以抑制CDW诱导人参细胞产生H2O2。进而,这些抑制剂还可以抑制CDW诱导PAL活性的提高,以及sqe的转录与人参皂甙的合成。过氧化氢酶与H2O2的粹灭剂也可以抑制CDW激发产生的这些诱导效应。上述结果表明CDW激发质膜NADPH氧化酶的活化与H2O2的产生在介导CDW诱导人参细胞抗性反应中,包括PAL活性的提高与人参皂甙的积累,起了重要的信号转导作用。     

脱乙酰壳多糖处理增加人参细胞皂苷的累积和皂苷合成关键酶基因的转录

脱乙酰壳多糖处理可以诱导人参细胞产生H2 O2 ,增加人参皂苷的累积 ,提高鲨烯合酶 (squalenesynthase,GSS)与鲨烯环氧酶 (squaleneepoxidase,GSE)基因的转录水平。质膜NADPH氧化酶的抑制剂DPI,H2 O2 的淬灭剂DMTU与DHC可以抑制脱乙酰壳多糖的这些效应 ,暗示脱乙酰壳多糖可以活化质膜NADPH氧化酶而产生H2 O2 ,H2 O2 进而作为第二信使诱导gss与gse基因转录以及皂苷的合成。质膜钙通道抑制剂LaCl3与内质网钙通道抑制剂RR ,以及蛋白激酶抑制剂K2 5 2a都能削弱脱乙酰壳多糖促进皂苷积累和gss、gse转录的效应 ,说明胞内Ca2 浓度的升高与蛋白质磷酸化都参与了脱乙酰壳多糖诱导的gss、gse的转录以及皂苷的合成     

水分胁迫诱导玉米叶片质外体产生H2O2机理

通过组织化学染色、电镜观察、酶活性分析对水分胁迫诱导玉米叶片质外体产生H2O2进行了研究。结果表明:水分胁迫能够诱导玉米叶片内源ABA的积累,ABA参与了水分胁迫诱导的玉米叶片H2O2的产生,质膜NADPH氧化酶、细胞壁过氧化物酶(POD)以及质外体多胺氧化酶(PAO)是水分胁迫诱导玉米细胞在质外体产生H2O2的来源,其中质膜NADPH氧化酶是主要来源;内源ABA的积累参与了水分胁迫激活的质膜NADPH氧化酶、细胞壁POD和质外体PAO活性的提高。研究认为,水分胁迫诱导玉米细胞在质外体产生H2O2可能是由于水分胁迫下内源ABA的积累通过激活质膜NADPH氧化酶、细胞壁POD以及质外体PAO的活性而实现的。     

UV-B对拟南芥叶片不同来源H2O2的活化和气孔关闭的诱导

在UV-B调控植物许多生理过程中过氧化氢(H2O2)作为第二信使发挥着重要作用,但H2O2来源途径并不清楚。该研究借助气孔开度分析和激光扫描共聚焦显微镜技术,探讨H2O2在介导不同剂量UV-B诱导拟南芥叶片气孔关闭过程中的酶学来源途径。结果发现:0.5W.m-2 UV-B能诱导野生型拟南芥叶片保卫细胞的H2O2产生和气孔关闭,且该效应能被NADPH氧化酶抑制剂二苯基碘(DPI)抑制,而不能被细胞壁过氧化物酶抑制剂水杨基氧肟酸(SHAM)抑制,同时该剂量UV-B也不能诱导NADPH氧化酶功能缺失单突变体AtrbohD和AtrbohF以及双突变体AtrbohD/F保卫细胞的H2O2产生和气孔关闭;相反,0.65 W.m-2 UV-B既能诱导野生型也能诱导NADPH氧化酶突变体保卫细胞的H2O2产生和气孔关闭,且该效应能被SHAM抑制,却不能被DPI抑制。结果表明,不同剂量UV-B通过活化不同生成途径的H2O2来诱导拟南芥叶片气孔关闭,即低剂量UV-B主要诱导NADPH氧化酶AtrbohD和AtrbohF途径来源的H2O2生成,而高剂量UV-B主要活化细胞壁过氧化酶途径来源的H2O2。     

酿酒酵母NAD(H)激酶Pos5p在细胞抵抗氧化胁迫中的作用

酿酒酵母线粒体NAD(H)激酶Pos5p显示出重要功能,其缺失将导致细胞抗氧化性能出现障碍。为了了解Pos5p的抗氧化作用机制及其与调节辅酶NAD(H)和NADP(H)之间的关系,比较了在不同类型的氧化胁迫试剂作用下,野生型BY4742、POS5基因缺失体pos5-及其回补体pos5-/POS5-YEp的生长表型,同时采用高效液相色谱测定细胞内辅酶含量。结果表明,在超氧生成试剂甲萘醌(VK3)、过氧化氢(H2O2)和GSH消耗试剂马来酸二乙酯(DEM)存在时,pos5-都表现出明显的生长缺陷,而各抗氧化基因缺失体只在其相应胁迫下表现出生长缺陷。在正常生长条件下,pos5-的NADPH含量降低,pos5-/POS5-YEp则提高,表明Pos5p对胞内NADPH的供应有重要作用。在VK3、H2O2和DEM胁迫下,BY4742、pos5-及pos5-/POS5-YEp的NADP(H)含量均有不同程度的下降,其中pos5-的NADP(H)/NAD(H)比率下降最为严重,而pos5-/POS5-YEp较pos5-有明显提高,这与其氧化胁迫表型相一致。因此,在细胞面临不同类型的氧化胁迫时,Pos5p都能有效行使其NAD(H)激酶活性,补充NADP(H)的损耗,从而对细胞起到抗氧化保护作用。     

NaCl对小麦根质膜NADPH氧化酶活性的影响

以小麦‘陇春20’为实验材料,用两相法分离根质膜微囊,研究NaCl处理对质膜NADPH氧化酶活性的影响。结果显示:(1)温和胶中酶活性条带的出现依赖于NADPH和Ca2 ,DPI(NADPH氧化酶抑制剂)完全抑制酶活性条带的出现;与0.2%的浓度相比,0.5%和1%的去垢剂TritonX-100或Chapso增溶质膜微囊明显减弱酶活性条带,表明高浓度的去垢剂抑制小麦根质膜NADPH氧化酶活性;(2)与对照相比,NaCl处理明显增强NADPH氧化酶活性温和胶染色出现的酶带;进一步研究发现,未处理质膜微囊超氧阴离子(O2.-)的产生只有7.55 nmol.mg-1protein.min-1,而100 mmol/L NaCl处理的质膜微囊O2.-的产生为13.63 nmol.mg-1protein.min-1。结果表明:质膜蛋白温和胶活性染色出现的酶带可能是小麦根质膜NADPH氧化酶,NaCl处理增强小麦根质膜NADPH氧化酶的活性。     

人参皂苷Rd通过下调Nrf2表达调控H460细胞的增殖和凋亡

核转录因子红细胞系-2p45相关因子-2(nuclear factor erythroid-2p45-related factor 2,Nrf2)是细胞应对外界应激的主要调控因子,通过调控一系列细胞保护酶,维持细胞稳态。然而,在许多肿瘤细胞中Nrf2过度激活,导致肿瘤细胞获得增殖优势并产生化疗耐药,因此,靶向抑制Nrf2是肿瘤增敏治疗的一种新思路。人参皂苷Rd是人参皂苷中的重要活性成分,具有显著的抗肿瘤作用。本研究以不同浓度的人参皂苷Rd处理人非小细胞肺癌(non-small cell lung cancer,NSCLC)H460细胞,利用CCK-8法检测细胞活力;倒置显微镜观察H460细胞的形态变化;流式细胞术检测细胞周期及凋亡率;RT-qPCR和Western印迹检测的Nrf2及其下游调控基因的表达情况;此外通过转染Nrf2-siRNA下调H460细胞中Nrf2的表达,观察其对人参皂苷Rd增敏的影响。结果显示,与对照组相比,人参皂苷Rd能够抑制H460细胞增殖活力,诱导细胞G_0/G_(1 )期阻滞,促进细胞凋亡,具有剂量依赖性(P0.05);此外,人参皂苷Rd能够下调Nrf2,醌氧化还原酶1 [NAD(P)H:quinoneoxidoreductase,NQO1],谷氨酰半胱氨酸连接酶催化亚基(glutamate--cysteine ligase catalytic subunit,GCLC)和调节亚基(glutamate--cysteine ligase regulatory subunit,GCLM)的mRNA和蛋白质水平,同时增强H460细胞对化疗药的敏感性(P0.05),而转染Nrf2-siRNA后,人参皂苷Rd的增敏作用减弱。表明人参皂苷Rd可以抑制非小细胞肺癌H460细胞活性并增敏化疗,其机制可能是通过抑制Nrf2信号通路来实现。     

Fungal elicitor induces singlet oxygen generation, ethylene release and saponin synthesis in cultured cells of Panax ginseng C. A. Meyer

Singlet oxygen is a high-energy molecular oxygen species. As one of the most active intermediates involved in chemical and biochemical reactions, singlet oxygen plays essential roles in plant responses to UV and strong light. Here, we report that Cle, an elicitor derived from fungal cell walls, induces the generation of singlet oxygen in cell cultures of ginseng, Panax ginseng. Cle treatment also triggers the activation of plasma membrane NADPH oxidase and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), subsequently leading to ethylene release and increased saponin synthesis, as shown by increased mRNA expression of squalene synthase (SQS) and squalene epoxidase (SQE), and accumulation of beta-amyrin synthase (beta-AS). Suppression of Cle-induced singlet oxygen generation or inhibition of ethylene production blocks saponin synthesis, whereas treatment of ginseng cells with ethylene or singlet oxygen induces the synthesis of saponin. Together, these results indicate that Cle-induced production of both singlet oxygen and ethylene is required for saponin synthesis, and that singlet oxygen may function upstream of ethylene during Cle-induced saponin synthesis.     

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.     

H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury

Non-phagocytic NAD(P)H oxidases have been implicated as major sources of reactive oxygen species in blood vessels. These oxidases can be activated by cytokines, thereby generating O(2), which is subsequently converted to H(2)O(2) and other oxidant species. The oxidants, in turn, act as important second messengers in cell signaling cascades. We hypothesized that reactive oxygen species, themselves, can activate the non-phagocytic NAD(P)H oxidases in vascular cells to induce oxidant production and, consequently, cellular injury. The current report demonstrates that exogenous exposure of non-phagocytic cell types of vascular origin (smooth muscle cells and fibroblasts) to H(2)O(2) activates these cell types to produce O(2) via an NAD(P)H oxidase. The ensuing endogenous production of O(2) contributes significantly to vascular cell injury following exposure to H(2)O(2). These results suggest the existence of a feed-forward mechanism, whereby reactive oxygen species such as H(2)O(2) can activate NAD(P)H oxidases in non-phagocytic cells to produce additional oxidant species, thereby amplifying the vascular injury process. Moreover, these findings implicate the non-phagocytic NAD(P)H oxidase as a novel therapeutic target for the amelioration of the biological effects of chronic oxidant stress.     

Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng

The mitogen-activated protein kinase (MAPK) cascade is a key signaling pathway responsible for the transduction of signals from the cell surface to the cell interior and the nucleus. MAPKs are involved in vari-ety of physiological process including cell growth, development, meiosis, cell death and cell differentia-tion[1—3]. Typically, the components of MAPK cas-cades include the MAPK, a mitogen-activated protein kinase kinase (MAPKK) and a mitogen-activated pro-tein kinase kinase kin…     

Homocysteine enhances cell proliferation in vascular smooth muscle cells: role of p38 MAPK and p47phox

Elevation of blood homocysteine levels (hyperhomocysteinemia) is a risk factor for cardiovascular disorders. One of the mechanisms by which homocysteine induces atherosclerosis is to promote the proliferation of vascular smooth muscle cells (VSMCs) in a reactive oxygen species (ROS)-dependent manner. It has been shown that homocysteine induces the production of ROS through the activation of NAD(P)H oxidases in VSMCs. In this study, we investigated the signal transduction pathways involved in the activation of NAD(P)H oxidases. Homocysteine promoted DNA synthesis in VSMCs. Inhibition of ROS by N-acetyl-L-cysteine (an antioxidant) and apocynin (an inhibitor of NAD(P)H oxidases) significantly blocked homocysteine-induced proliferation in VSMCs. Homocysteine induced a rapid increase in the phosphorylation of p38-mitogen-activated protein kinase (p38 MAPK). p38 MAPK in turn activated NAD(P)H oxidases by inducing the phosphorylation of p47phox, resulting in the generation of ROS. ROS induced the phosphorylation of Akt, which was probably responsible for proliferation in VSMCs. These findings demonstrate that homocysteine induces an increase in the activity of NAD(P)H oxidases in VSMCs by activating p38 MAPK and enhancing the phosphorylation of p47phox.