美洲大蠊油脂对过氧化氢诱导SH-SY5Y细胞氧化损伤的保护作用

采用人神经母细胞瘤(SH-SY5Y)细胞,建立过氧化氢(H2O2)诱导的氧化损伤模型,加入H2O2前用美洲大蠊油脂(100、200、400、800、1000μg/m L)预处理,通过MTS法检测细胞存活率,比色法检测细胞乳酸脱氢酶(LDH)漏出率,相关试剂盒检测细胞内超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)系列抗氧化酶的活性,以及脂质过氧化物丙二醛(MDA)含量的变化,从细胞水平研究美洲大蠊油脂对氧化损伤细胞的保护作用,并初步探讨其作用机制。实验结果显示,与对照组相比,模型组细胞存活率明显降低,LDH漏出率明显增加,胞内SOD、GSH-Px活性显著降低,MDA含量增多。与模型组相比,美洲大蠊油脂(800、1000μg/m L)能显著增强细胞SOD(P0.01)、GSH-Px(P0.05)活性,降低MDA含量(P0.05),使得细胞存活率显著提高(P0.01),LDH漏出率降低(P0.01)。其中油脂的浓度与细胞存活率、SOD活性之间为正相关,与MDA含量为负相关,均呈现浓度依赖性。结果提示美洲大蠊油脂对H2O2所致SH-SY5Y细胞氧化损伤具有明显的保护作用,其作用机制可能与提高细胞SOD、GSH-Px活性,降低MDA含量从而增强细胞自身抗氧化能力有关。     

过表达Grx1抑制HEK293T细胞中H2O2诱导的p38MAPK信号通路

谷氧还蛋白1(glutaredoxin1, Grx1)是细胞内一种重要的巯基-二硫键氧化还原酶,在细胞内氧化还原状态的调控及抵抗氧化应激损伤过程中发挥重要作用.为进一步探讨Grx1的抗氧化机制,本实验将重组质粒pcDNA3.1(+)-hGrx1瞬时转染HEK293T细胞,经RT-PCR和Western印迹验证,细胞转染后实现了Grx1的过表达;以不同浓度H2O2为损伤因素,建立细胞氧化应激模型,检测过表达Grx1后细胞存活率,丙二醛(MDA)含量,超氧化物歧化酶(SOD)活力和乳酸脱氢酶(LDH)漏出率的变化,观察过表达Grx1后细胞的抗氧化能力;用终浓度100μmol/L H2O2作用于细胞,利用Western印迹检测120min内HEK293T细胞中p38MAPK磷酸化水平.实验结果表明,HEK293T细胞过表达Grx1后,缓解了细胞的氧化应激损伤;转染空载体组细胞p38MAPK磷酸化水平在H2O2刺激后5min开始升高,15min达到最高值,并可维持至120min左右;而过表达Grx1组细胞p38MAPK磷酸化水平在H2O2刺激后各时间段没有明显改变,提示Grx1通过抑制H2O2诱导的p38MAPK信号通路激活发挥其抗氧化作用.     

五味子对H_2O_2诱导的HaCaT细胞氧化应激损伤的保护作用

检测五味子不同组分对H2O2诱导的HaCaT细胞氧化应激损伤的保护作用,筛选得到其活性组分。利用大孔吸附树脂法,对五味子果实提取物进行分离纯化,得到不同的组分段。以DPPH自由基清除能力和总抗氧化能力(ABTS法)为考察指标,对五味子各组分的抗氧化活性进行评价;采用MTT法筛选五味子各组分对H2O2诱导的HaCaT细胞氧化应激损伤的保护作用,获得活性组分。结果显示,50%乙醇洗脱物抗氧化活性最强,能提高H2O2处理后的HaCaT细胞存活率,减少MDA的生成,上调SOD酶和GSH酶活性。研究结果证实,50%乙醇洗脱物是五味子保护皮肤细胞免受H2O2诱导氧化应激的活性组分。     

一氧化氮供体对过氧化氢引起的心肌细胞损伤的保护作用

关于一氧化氮(NO)对心肌细胞是否具有保护作用目前尚存在争议,为探讨NO对过氧化氢(H2O2)引起的心肌细胞损伤是否具有保护作用及其可能的机制,实验将体外培养的新生大鼠心肌细胞分为3组(1)阴性对照组(Normal组);(2)H2O2组H2O2(0.1mmol/L)与心肌细胞共育4h;(3)S-亚硝基-N-乙酰青霉胺(SNAP)+H2O2组NO供体SNAP(0.5mmol/L)处理心肌细胞10min后,加入H2O2与心肌细胞共育4 h.用流式细胞术检测心肌细胞凋亡率,心肌细胞损伤程度以心肌细胞存活率和乳酸脱氢酶(lactate dehydrogenase,LDH)活性来表示,同时检测心肌细胞超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(MDA)含量.通过激光共聚焦显微术检测在不同处理条件下心肌细胞胞内钙的变化.结果表明,正常心肌细胞LDH活性和细胞存活率分别为631.4±75.6 U/L和93.1±6.2%,细胞凋亡率为0;H2O2处理细胞后可使细胞LDH活性显著增高(1580.5±186.7 U/L,P＜0.01),细胞存活率明显下降(58.3±7.6%,P＜0.01),流式细胞仪检测到大量心肌细胞凋亡,凋亡率为26.4±5.7%;SOD活性较正常细胞19.67±0.85 NU/ml显著下降,为14.73±1.68 NU/m(P＜0.01),MDA含量较正常细胞6.95±0.83μmol/L显著增高,为15.35±3.49μmol/L(P＜0.01).SNAP预处理细胞可显著提高心肌细胞存活率(79.7±9.3%,P＜0.01),降低LDH活性和细胞凋亡率(分别为957.8±110.9 U/L和9.1±3.3%,P＜0.01);并提高细胞抗氧化能力,表现为较H2O2处理组的SOD活性增高(21.36±3.11 NU/ml,P＜0.01),MDA含量下降(9.12±1.47 μmol/L,P＜0.01).激光共聚焦显微镜检测结果表明,H2O2可升高细胞内钙,而SNAP则可降低细胞内钙,SNAP预处理细胞后可取消H2O2升高细胞内钙的作用.上述结果提示,NO供体SNAP可对抗H2O2对心肌细胞的损伤,其机制与提高心肌细胞抗氧化损伤能力和对抗H2O2引起的细胞内钙超载有关.     

低温和氧化应激产生活酵母细胞衍生物的研究

对低温和H2O2应激条件下产生活性酵母细胞衍生物（Live Yeast Cell Derivative,简称LYCD）进行了研究。结果表明：低温预处理能够增加细胞内谷胱甘肽（GSH）含量,提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性,降低MDA含量。低温预处理可以诱导对致死浓度H2O2的抗性。通过0—15℃低温和0．2mmol／L H2O2处理酵母细胞后,提取LYCD并添加到酵母细胞培养液中,发现细胞在致死浓度H2O2作用下的存活率明显提高,说明0—15℃低温和H2O2刺激酵母细胞形成的LYCD对细胞具有抗氧化作用。     

ERK1/2信号通路对黄芪苷Ⅳ抗H_2O_2诱导H9c2细胞氧化损伤的作用

目的:研究黄芪苷Ⅳ(AST)是否通过细胞外信号调节激酶1/2(ERK1/2)通路发挥对H2O2诱导的H9c2细胞氧化损伤的保护作用。方法:用200μmol/L的H2O2处理细胞6 h,采用MTT法检测细胞存活率,建立H2O2诱导的H9c2细胞氧化损伤模型;比色法测定细胞培养液中乳酸脱氢酶(LDH)活性、总超氧化物歧化酶(T-SOD)和锰超氧化物歧化酶(Mn-SOD)活力以及丙二醛(MDA)含量;Western blot检测H9c2细胞ERK1/2蛋白的磷酸化水平。结果:在H2O2浓度为200μmol/L作用6 h条件下,细胞存活率降低程度适中,实验结果重复性好,确定后续实验采用200μmol/L H2O2作用6 h建立模型。与H2O2组比较,10 mg/L及20 mg/L AST均显著提高细胞存活率(P<0.01),使细胞培养液中LDH活性显著降低(P<0.01),T-SOD及Mn-SOD活力显著提高(P<0.01),MDA含量显著降低(P<0.01)。10 mg/L及20 mg/L AST均显著增加H2O2损伤的H9c2细胞p-ERK1/2蛋白的表达(P<0.01),当用PD98059(ERK1/2的抑制剂...     

胶原蛋白肽经由Nrf2信号通路对H2O2诱导的肝细胞氧化损伤的保护作用

目的：氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法：实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组（10,100,200μg/ml）。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶（LDH）的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性和丙二醛（MDA）含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果：胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶（SOD）、过氧化氢酶（CAT）的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论：总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。     

胶原蛋白肽经由Nrf2信号通路对H_2O_2诱导的肝细胞氧化损伤的保护作用(英文)

目的:氧化应激在肝脏疾病中扮演着重要的角色。胶原蛋白肽是天然的抗氧化剂,其在动物实验中已经被证实有抑制氧化应激的作用。最新研究证实胶原蛋白肽将有可能被应用在肝脏疾病的预防中,但是很少有研究报道其分子作用机制。因此本研究在胶原蛋白肽是对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用的基础上,并探索其分子作用机制。方法:实验设空白对照组,H2O2模型组,胶原蛋白肽低、中、高剂量组(10,100,200μg/ml)。胶原蛋白肽各组加入相应浓度的药物预处理12 h后,与模型组一起加入300μM H2O2的H2O2共同培养12 h,空白对照组正常培养。细胞毒性是由CCK8和乳酸脱氢酶(LDH)的释放检测。抗氧化试剂盒检测细胞内活性氧的水平,超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)含量的变化。Western blot检测细胞内Nrf2蛋白的表达水平。结果:胶原蛋白肽对H2O2诱导的正常人的肝细胞系HL7702氧化损伤有保护作用。胶原蛋白肽能够及时清除细胞内的活性氧,增加Nrf2的蛋白表达水平,提高超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的活性,减轻脂质过氧化反应,从而保护正常人的肝细胞系HL7702。结论:总之,胶原蛋白肽通过增加Nrf2的蛋白表达水平,提高抗氧化活性,对H2O2诱导损伤的肝细胞发挥保护作用。本研究为胶原蛋白肽的分子作用机制提供了新的证据,将有助于预防氧化应激所致的肝损伤。     

蓝莓提取物对H2O2诱导的体外培养的大鼠海马神经元氧化损伤的保护作用

目的：探讨蓝莓提取物对过氧化氢致大鼠海马神经元氧化应激损伤的减缓作用。方法：将培养7d的海马神经细胞分为8组：①过氧化氢组（H2O2）：培养液中加入50μmol/L的H2O2,作用24h。②不同剂量蓝莓提取物预处理组（BE＋H2O2）：在加入H2O2前24h,分别加入0.01、0.1、1.0、10.0、20.0和40.0μg/mlBE。③空白对照组（Control）：处理步骤同上组,但每次处理物质均为等量的培养液。通过测定细胞存活率和上清液中乳酸脱氢酶（LDH）的活性确定减轻海马神经元损伤的蓝莓提取物的适宜浓度。并检测细胞内丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性以及细胞凋亡率的变化。结果：①蓝莓提取物组（0.1,1.0和10.0μg/mlBE）LDH活性显著低于H2O2组,细胞存活率由H2O2组的57.44%分别上升至78.42%、87.71%、72.40%;1μg/ml蓝莓提取物组对H2O2诱导的海马神经细胞氧化应激损伤的保护作用最好。②1μg/ml蓝莓提取物组海马神经细胞培养上清液中MDA含量及细胞凋亡率显著低于H2O2组,SOD活性显著高于H2O2组。结论：适宜剂量的蓝莓提取物对氧化应激损伤的海马神经元有一定的保护作用,其机制可能与抑制海马神经元凋亡、增强神经细胞的抗氧化功能有关。     

荭草花醇提物对H_2O_2诱导的H9c2细胞氧化损伤的保护作用机制研究

研究荭草花醇提物对H9c2心肌细胞氧化损伤的保护作用机制。采用200μmol/L H_2O_2作用H9c2细胞0.5 h,建立H9c2细胞氧化损伤模型。将细胞分为正常对照组、H9c2细胞氧化损伤模型组、不同浓度荭草花醇提物(20、40、80μg/m L)预处理组。采用MTS法检测细胞存活率;生化试剂盒检测细胞乳酸脱氢酶(LDH)释放量,细胞内丙二醛(MDA)含量及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活力;Western blot测定cleavedcaspase-3、Bcl-2、Bax、p-AKT和AKT的表达。与模型组比较,荭草花醇提物能明显增加心肌细胞存活率,降低LDH释放量和MDA含量,提高SOD及CAT活性,并呈剂量依赖性抑制H_2O_2诱导的氧化应激损伤。荭草花醇提物下调cleaved caspase-3和Bax的蛋白水平,上调Bcl-2的表达,减少心肌细胞凋亡;增加细胞中p-AKT的表达,且这种表达可被PI3Ks抑制剂LY294002抵消。表明荭草花醇提物可减轻H_2O_2诱导的H9c2心肌细胞氧化应激损伤,其机制可能与减少细胞凋亡,平衡氧化应激产物有关,且部分依赖于磷脂酰肌醇3-激酶(PI3K/Akt)通路。     

The antioxidant effects of ribonuclease inhibitor

Ribonuclease inhibitor (RI) is an acidic cytosolic glycoprotein with molecular weight of about 50 kDa, which contains 32 cysteine residues. It is possibly that RI may have antioxidant effect by thiol-disulfide exchange reaction. We studied the effects of RI over-expression on the rat glial cell line C6 injured with H2O2. The transfected C6 cells with RI cDNA (C6') had higher viability, less LDH leakage and MDA contents, but more GSH contents compare that in the control C6 cells. In transfected C6 cells, the activities of CAT and GST were higher than that in the control C6 cells. Without H202 stress, the activities of CAT and GST in the C6' cells were 1.73 and 3.62 times that in the control C6 cells, respectively; With 1.00 mmol/L H2O2 stress, the activities of CATand GSTin the C6' cells were 3.38 and 2.11 times that in the C6 cells, respectively. These results suggest that the over-expression RI has antioxidant activity and it is able to protect cells from per-oxidative injuries. Moreover, we investigated whether RI has a protective role against mouse hepatic damage in vivo. The mice pretreated with different doses of human RI were injected by CC14. The results show that the SOD activities of therapy groups were significantly higher than that of the control group (p < 0.01), while the contents of MOD and activities of ALT and AST in blood were remarkably lower than that of the control group (p < 0.01). Pathological examination shows that the degree of damage was alleviated with RI therapy. These results suggest that RI has the protective role against mouse hepatic damage induced by CC14. The anti-oxidative effects of RI may play an important role in cell protection from per-oxidative injuries.     

Ethanol-induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty-two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol-fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long-term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and gamma-glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, gamma-glutamyltranspeptidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose-6-phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long-term ethanol administration.     

Antioxidant status,lipid peroxidation,mixed function oxidase and UDP-glucuronyl transferase activities in livers from control and DOCA salt-hypertensive male Sprague Dawley rats

The effects of DOCA-salt hypertensive treatment on hepatic glutathione-dependent defense system, antioxidant enzymes, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities were investigated in male Sprague Dawley rats.Compared with controls, DOCA-salt hypertensive rats had lower body weights (linked to liver hypertrophy). Mixed function oxidase and p-nitrophenol-UGT activities were not affected by the treatment but a significant lower rate of the glucuronoconjugation rate of bilirubin (p < 0.001) was observed in DOCA-salt hypertensive rats. While cytosolic glutathione contents and glutathione reductase activity were not affected, glutathione peroxidase (p < 0.001), glutathione transferase (p < 0.001) and catalase (p < 0.01) activities were decreased and associated with higher malondialdehyde contents (p < 0.001) in treated rats. The imbalance in liver antioxidant status (increasing generation of cellular radical species), associated with increases in lipid peroxidation, suggests that oxidative stress might be directly related to arterial hypertension in DOCA-salt treated male Sprague Dawley rats.     

Aqueous extract of Terminalia arjuna attenuates tert‐butyl hydroperoxide–induced oxidative stress in HepG2 cell model

Arjuna (Terminalia arjuna) is a medicinal plant used in many polyherbal hepatoprotective formulations. Although widely claimed to be antioxidant, data supporting such actions of Arjuna are limited. In the present study, we have investigated the efficacy of the aqueous extract of T. arjuna (AETA) using a standard pro‐oxidant [tertiary butyl hydroperoxide (TBHP)] in HepG2 cells. Cells were incubated with AETA (5–100 µg/ml) for a range of time points (4–24 h) with or without TBHP (500 μM), and biochemical markers of oxidative stress (OS) were determined. Cells incubated with TBHP showed the significant induction of OS response in cytosol manifested as lipid hydroperoxide (76%–198%) and the generation of reactive oxygen species (60%–127%). Diminished levels of reduced glutathione (35%–60%) and total antioxidant capacity (20%–61%) suggested an altered redox state. Significant perturbations in the activities of antioxidant enzymes such as catalase (30%–56%), superoxide dismutase (25%–68%), glutathione S‐transferase (29%–67%), glutathione peroxidase (24%–68%) and glutathione reductase (38%–49%) were discernible suggesting the ongoing OS in the cells. However, cells treated with AETA (100 µg/ml) along with TBHP offered significant protection by reducing levels of lipid hydroperoxide (33%–62%) and ROS (69%) and by increasing antioxidant capacity (54%–81%) and levels of reduced glutathione (49%–82%). Further, it also enhanced the activities of endogenous antioxidant enzymes (superoxide dismutase, 60%; catalase, 35%–82%; glutathione peroxidase, 42–65 %; glutathione reductase, 48%–62%; and glutathione S‐transferase, 22%–100%). Taken together, these data suggest that Arjuna can protect against the oxidative damage induced by TBHP and may be effectively used as a hepatoprotective adjuvant to abrogate OS in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

轮状病毒VP7基因在大肠杆菌中的表达及其免疫原性

轮状病毒是世界范围内引起婴幼儿病毒性腹泻的主要病原体。VP7是轮状病毒的主要外壳蛋白和中和抗原,是发展基因工程疫苗的首选。把包含全部3个主要抗原性区域的轮状病毒SA11 VP7基因片段以谷胱甘肽S转移酶融合蛋白的形式在大肠杆菌中进行表达,表达产物占菌体总蛋白的30%左右。经一步Glutathione Sepharose4B亲和纯化,重组蛋白纯度超过90%。Western blot实验表明,重组蛋白可被抗SA11的多抗特异地识别。动物实验表明,重组抗原可在小鼠和家兔体内诱导VP7特异的抗体和一定水平的SA11中和抗体。     

Ethanol‐induced alterations of the antioxidant defense system in rat kidney

We report here the effects of chronic ethanol consumption on the antioxidant defense system in rat kidney. Thirty‐two male Wistar rats were randomly divided in two identical groups and were treated as follows: control group (water for fluid) and the ethanol‐fed group (2 g/kg body weight/24 h). The animals were sacrificed after 10 weeks, and respectively 30 weeks of ethanol consumption, and the renal tissue was isolated and analyzed. Results revealed that kidney alcohol dehydrogenase activities increased significantly after ethanol administration, but the electrophoretic pattern of alcohol dehydrogenase isoforms was unmodified. The SDS polyacrylamidegel electrophoretic study of kidney proteins has revealed the appearance of two new protein bands after long‐term ethanol consumption. The kidney reduced glutathione/oxidized glutathione ratio decreased, indicating an oxidative stress response due to ethanol ingestion. The malondialdehyde contents and xanthine oxidase activities were unchanged. The antioxidant enzymatic defense system showed a different response during the two periods of ethanol administration. After 10 weeks, catalase, glutathione peroxidase, glutathione reductase, and glucose‐6‐phosphate dehydrogenase were activated, while superoxide dismutase, glutathione transferase, and γ‐glutamyltranspeptidase levels were stationary. After 30 weeks, superoxide dismutase and glutathione peroxidase activities were unmodified, but catalase, glutathione transferase, γ‐glutamyltranspeptidase, glutathione reductase, and glucose‐6‐phosphate dehydrogenase activities were significantly increased. Remarkable changes have been registered after 30 weeks of ethanol administration for glutathione reductase and glucose‐6‐phosphate dehydrogenase activities, including an increase by 106 and 216' of control values, respectively. These results showed specific changes in rat kidney antioxidant system and glutathione status as a consequence of long‐term ethanol administration. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:386‐395, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20101     

BCL-2 is involved in preventing oxidant-induced cell death and in decreasing oxygen radical production

It has been hypothesized that programmed cell death is mediated, in part, through the formation of free radicals via oxidative pathways. Furthermore, it has been proposed that BCL-2 acts to inhibit cell death by interfering with the production of oxygen-derived free radicals induced by a wide variety of stimuli. In order to examine the antioxidant function of BCL-2, we transfected mouse epidermal cells JB6 clone 41 with the expression vector pD5-Neo-BCL-2 and studied the effect of BCL-2 overexpression on oxidant-induced cell death and on the production of reactive oxygen species. Compared to Neo control cells, BCL-2-expressing cells are more resistant to the killing and growth retardation induced by hydrogen peroxide, superoxide, or by the oxygen radical-generating quinone-containing compounds menadione, diaziquone and adriamycin. The latter compounds generate reactive oxygen species during bioreductive metabolism. In addition, the exposed cells die by necrosis rather than apoptosis. Hydroxyl radical levels generated by the quinone-containing agents were low in BCL-2-expressing JB6 cells compared to control Neo cells. BCL-2, however, does not change the activities of the major cellular antioxidant enzymes superoxide dismutase, catalase or glutathione peroxidase. On the other hand, the glutathione concentrations increased in BCL-2 overexpressing cells after oxidative challenge, while the opposite was true for control cells. Thus, our results suggest that BCL-2 inhibition of oxidant-induced cell death is mediated, at least in part, through an antioxidant pathway, and that this pathway involves glutathione.     

硒性白内障大鼠模型晶状体中GR和GSH-Px的表达

 为探讨硒性白内障大鼠晶状体中谷胱甘肽过氧化物酶 (GSH Px)和谷胱甘肽还原酶 (GR)的活性调节在硒性白内障形成中的作用及调节方式 ,采用半定量RT PCR方法 ,比较正常晶状体、核中心混浊晶状体 (核白 )和完全混浊晶状体 (全白 )中GSH Px和GR的mRNA水平及酶活性的变化 .研究发现 ,核白晶状体中 2种酶的活性和mRNA水平均升高 ,其中酶活性的升高幅度小于mRNA水平 .随着白内障的发展 ,2种酶的活性和mRNA水平均逐渐下降 .至晶状体全白时 ,2种酶的活性均显著低于正常 ;全白时GR的mRNA水平降至正常 ,GSH Px的mRNA水平则仍高于正常 .结果表明 ,硒性白内障形成与细胞内GSH Px和GR的活性调节密切相关 ,GSH Px和GR的活性调节可能主要发生在转录水平     

Shift of the Cellular Oxidation-Reduction Potential in Neural Cells Expressing Bcl-2

Abstract: Expression of the protooncogene bcl-2 inhibits both apoptotic and in some cases necrotic cell death in many cell types, including neural cells, and in response to a wide variety of inducers. The mechanism by which the Bcl-2 protein acts to prevent cell death remains elusive. One mechanism by which Bcl-2 has been proposed to act is by decreasing the net cellular generation of reactive oxygen species. To evaluate this proposal, we measured activities of antioxidant enzymes as well as levels of glutathione and pyridine nucleotides in control and bcl-2 transfectants in two different neural cell lines—rat pheochromocytoma PC12 and the hypothalamic GnRH cell line GT1-7. Both neural cell lines overexpressing bcl-2 had elevated total glutathione levels when compared with control transfectants. The ratios of oxidized glutathione to total glutathione in PC12 and GT1-7 cells overexpressing bcl-2 were significantly reduced. In addition, the NAD⁺/NADH ratio of bcl-2 -expressing PC12 and GT1-7 cells was two- to threefold less than that of control cell lines. GT1-7 cells overexpressing bcl-2 had the same level of glutathione peroxidase, catalase, superoxide dismutase, and glutathione reductase activities as control cells. PC12 cells overexpressing bcl-2 had a twofold increase in superoxide dismutase and catalase activity when compared with matched control transfected cells. The levels of glutathione peroxidase and glutathione reductase in PC12 cells overexpressing bcl-2 were similar to those of control cells. These results indicate that the overexpression of bcl-2 shifts the cellular redox potential to a more reduced state, without consistently affecting the major cellular antioxidant enzymes.