Nrf 2抗氧化系统在慢性阻塞性肺疾病中的作用

C O PD可致肺功能进行性减退,严重影响患者的生活质量,且目前为止尚无确切有效的治疗方法。尽管确切的病因仍不清楚,但是氧化应激被认为在疾病发病过程中扮演着重要角色。核因子相关因子2（Nrf2）是体内调节抗氧化系统的关键性转录因子之一,其目的基因表达的增加能增强细胞抗氧化作用。本文通过对氧化应激和 Nrf2介导的抗氧化系统,及其在COPD中的作用进行综述。     

无机砷对角质细胞红系相关因子及其下游抗氧化酶蛋白表达的影响

目的探讨无机砷暴露对皮肤角质细胞红系相关因子2(Nrf2)及其调控的下游抗氧化酶NAD(P)H醌氧化还原酶1(NQO1)和血红素单加氧酶-1(HO-1)蛋白表达的影响。方法 25μmol/L亚砷酸钠(NaAsO2)作用于人类皮肤角质细胞系HaCaT细胞株0.5、3、6、12、24 h,采用western blot法检测细胞内Nrf2、NQO1和HO-1的蛋白表达情况。结果25μmol/L NaAsO2暴露0.5、3、6、12、24 h均能够显著诱导HaCaT细胞的Nrf2蛋白表达(P<0.01),Nrf2蛋白在NaAsO2暴露3、6、12 h表达量呈现高峰,24 h则呈下降趋势,但仍显著高于对照组;NQO1的蛋白表达仅在暴露后3 h显著高于对照组(P<0.01),暴露时间延长后则显著下降,6、12、24 h的NQO1蛋白表达水平明显低于对照组(P<0.05或P<0.01);HO-1蛋白则从暴露3 h开始呈现明显的诱导表达,且随染毒时间的延长(6、12、24 h)表达持续增强,具有明显的时间-效应关系(P<0.01)。结论无机砷暴露能够诱导人类皮肤角质细胞系HaCaT细胞的Nrf2及其调控的下游抗氧化酶NQO1和HO-1的蛋白表达。     

Nrf2信号通路及其对ARDS肺脏的保护作用

肺部或全身失控的炎症反应在急性呼吸窘迫综合征(ARDS)发病过程中起核心作用。核因子E2相关因子2(Nrf2)及其相关信号通路是细胞炎症反应及氧化应激调节中的关键通路,在细胞防御保护中发挥重要作用。研究表明Nrf2激活对ARDS炎性和氧化应激损伤具有重要保护作用。本文对Nrf2及其相关通路在ARDS发病机制中的最新研究...     

p38MAPK信号通路与内皮细胞激活

内皮细胞激活是内皮细胞损伤的始动因素,是引起各种不同程度的炎症反应和细胞凋亡的前提条件 ;丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK) 是哺乳动物细胞中重要的信号转导通路,其中 p38MAPK 通路在细胞应激、细胞生长、凋亡和炎症等多种生理和病理过程中起重要作用,越来越引起业界的广泛关注,本文就 p38MAPK 信号通路及其与内皮细胞激活的相关性做一综述,旨在进一步对内皮细胞损伤引发心血管疾病研究提供参考资料。     

无机砷对红系相关因子-2及其抑制因子mRNA表达的影响

目的观察亚砷酸钠（NaAsO2,sodium arsenite）对Chang肝细胞株核转录因子红系相关因子（nuclear factor ery-throid 2-related factor 2,Nrf2）及其胞浆抑制因子Keap1（Kelch-like ECH-associated protein 1）的mRNA表达水平的影响。方法分别以不同浓度NaAsO2（0、50、200、400μmol/L）暴露人类Chang肝细胞株12 h,采用AlamarBlue法测定细胞增殖活性,采用RT-PCR法测定Nrf2和Keap1的mRNA表达水平。结果 50μmol/L NaAsO2暴露组的细胞增殖活性与对照组相比差异无统计学意义（P〉0.05）,而200μmol/L和400μmol/L NaAsO2暴露组的细胞增殖活性均显著低于对照组（P〈0.05）;50、200、400μmol/L的NaAsO2暴露12 h,Nrf2和Keap1的mRNA表达水平与对照组比较均显著下降（P〈0.05）,且呈剂量-反应关系。结论高浓度无机砷暴露能抑制Chang肝细胞株Nrf2和Keap1的mRNA表达水平,并可能与无机砷造成机体的高氧化应激水平有关。     

N-Acetylserotonin is an oxidation-responsive activator of Nrf2 ameliorating colitis in rats

N-Acetylserotonin (NAS) is an intermediate in the melatonin biosynthetic pathway. We investigated the anti-inflammatory activity of NAS by focusing on its chemical feature oxidizable to an electrophile. NAS was readily oxidized by reaction with HOCl, an oxidant produced in the inflammatory state. HOCl-reacted NAS (Oxi-NAS), but not NAS, activated the anti-inflammatory nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase (HO)-1 pathway in cells. Chromatographic and mass analyses demonstrated that Oxi-NAS was the iminoquinone form of NAS and could react with N-acetylcysteine possessing a nucleophilic thiol to form a covalent adduct. Oxi-NAS bound to Kelch-like ECH-associated protein 1, resulting in Nrf2 dissociation. Moreover, rectally administered NAS increased the levels of nuclear Nrf2 and HO-1 proteins in the inflamed colon of rats. Simultaneously, NAS was converted to Oxi-NAS in the inflamed colon. Rectal NAS mitigated colonic damage and inflammation. The anticolitic effects were significantly compromised by the coadministration of an HO-1 inhibitor.     

还原型谷胱甘肽对大鼠肝星状细胞HSC-T6增殖及核转录因子NF-E2相关因子2/血红素加氧酶-1信号通路的影响

目的 探讨还原型谷胱甘肽(GSH)对大鼠肝星状细胞HSC-T6增殖及核转录因子NF-E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)信号通路的影响.方法 分别用浓度为0、1、2、5、10 mmol/L的GSH作用于经0.1 μg/ml脂多糖活化的大鼠HSC-T6细胞24h后,四甲基偶氮唑盐比色法检测HSC-T6增殖情况,放射免疫法检测细胞上清液中透明质酸(HA)及Ⅳ型胶原的含量,实时荧光定量PCR检测Nrf2和HO-1的mRNA表达水平,免疫细胞化学染色法检测HSC-T6中Nrf2和HO-1的蛋白质表达情况,分光光度计检测HO-1的活性变化.两两比较采用t检验,两变量间的关系采用曲线拟合方法. 结果 GSH能抑制HSC-T6增殖,1、2、5、10 mmol/L组的吸光度值分别为0.79±0.02、0.74±0.03、0.70±0.02、0.62±0.01,均低于0mmol/L组的0.88±0.03(t值分别为3.16、6.09、7.17、11.94,P值均＜0.05).GSH 1、2、5、10mmol/L组的HA表达量分别为(372.98±11.01)μg/L、(320.76±16.37) μg/L、(284.46±13.17)μg/L、(239.08±16.95)μg/L,明显低于0 mmol/L组的(415.74±14.52)μg/L(t值分别为4.07、7.52、11.59、13.71,P值均＜0.05);Ⅳ型胶原表达量分别为(191.27±17.49)μg/L、(163.85±16.26) μg/L、(133.03±13.14)μg/L、(103.31±12.52) μg/L,也低于0mmol/L组的(251.47±14.06) μg/L(t值分别为4.65、7.58、10.66、13.63,P值均＜0.05).0 mmol/L组HSC-T6中Nrf2 mRNA相对表达量(1.21±0.11)低于1、2、5、10 mmol/L组(分别为1.51±0.06、1.92±0.08、2.69±0.07、3.43±0.07),Nrf2蛋白表达的累积吸光度值(17.84±0.61)也低于1、2、5、10 mmol/L组(分别为23.85±0.20、27.90±0.32、33.69±0.75、38.64±0.38); HO-1 mRNA相对表达量(1.25±0.09)低于1、2、5、10 mmol/L组(分别为1.43±0.08、1.73±0.07、2.10±0.08、2.64±0.07),HO-1蛋白表达的累积吸光度值(16.77±0.31)也低于1、2、5、10 mmol/L组(20.75±0.30、24.84±0.24、28.89±0.19、33.88±0.19),差异均有统计学意义(P值均＜0.05).HO-1的活性也随GSH浓度增加而上升.结论 GSH可抑制HSC-T6增殖,减少其细胞外基质HA及Ⅳ型胶原分泌,其机制可能与GSH对HSC-T6的Nrf2/HO-1信号通路调控有关.     

磷脂酰肌醇3激酶/蛋白激酶B/核转录因子E2相关因子2信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的作用及机制

目的研究核转录因子E2相关因子2(Nrf2)及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路对高糖状态下人甲状腺乳头状癌K1细胞增殖的影响,并探讨糖代谢相关酶在Nrf2及PI3K/Akt影响K1细胞增殖中的作用。方法根据不同处理因素将K1细胞分为5组:正糖组(正糖5.5 mmol/L培养48 h)、高糖组(正糖5.5 mmol/L培养24 h后,换25 mmol/L高糖继续培养24 h)、Nrf2siRNA组(正糖条件下siRNA转染细胞24 h后,换高糖继续培养24 h)、NcsiRNA组(正糖条件下阴性转染细胞24 h后,换高糖继续培养24 h)、LY294002组(正糖条件下培养24 h后,加50μmol/L LY294002预孵育0.5 h再换用高糖培养24 h)。K1细胞按照上述分组处理后分别用以下方法检测相关指标:MTT法检测细胞增殖,细胞免疫荧光法检测Nrf2蛋白的分布,Western blot检测PI3K、p-PI3K、Akt、p-Akt、核Nrf2、浆Nrf2、葡萄糖-6-磷酸脱氢酶(G6PD)、丙酮酸激酶M2(PKM2)蛋白的表达水平。两组数据比较采用独立样本t检验,多组数据比较采用单因素方差分析。结果与正糖组比较,高糖组K1细胞增殖率、PI3K、p-Akt/Akt、Nrf2、G6PD、PKM2表达显著升高[分别为(87.31±3.67)%比(126.64±5.41)%、0.272±0.039比0.425±0.019、0.168±0.035比0.446±0.021、0.308±0.026比0.597±0.014、0.421±0.024比0.626±0.026、0.198±0.023比0.314±0.023,t=6.109~16.951,均P<0.05],Nrf2在细胞核分布的比例显著升高[(21.6±4.5)%比(91.2±3.5)%,χ2=98.497,P<0.01];与高糖组比较,Nrf2siRNA组K1细胞增殖率明显下降,G6PD、PKM2的蛋白表达明显下调,差异有统计学意义(t=8.936、7.056、8.843,均P<0.01),LY294002组也呈现相似的趋势(t=7.228、6.351、7.910;均P<0.01);与高糖组比较,LY294002组K1细胞PI3K、Akt蛋白水平无明显改变,而p-PI3K、p-Akt、核Nrf2、浆Nrf2蛋白水平及Nrf2核浆蛋白比值均被显著抑制(t=5.748~23.572,均P<0.01)。结论高糖可激活PI3K/Akt信号通路,上调Nrf2表达与核转位,上调磷酸戊糖和糖酵解途径相关酶PKM2、G6PD的表达,促进K1细胞增殖。     

大鼠脑出血急性期核因子Nrf2信号通路表达变化及其与继发性脑损伤的关系

目的观察大鼠脑出血(ICH)急性期不同时间点核因子Nrf2信号通路相关因子的表达与继发性脑损伤的关系。方法将120只大鼠随机分为出血组和假手术组,每组60只。采用自体血立体定向注入大脑基底核区,建立大鼠ICH模型,根据ICH后不同时间点,将大鼠随机分为2、8、24 h,3、5、10 d组,各时间点设相应假手术组,每组10只。分别于造模后2、8、24 h,3、5、10 d,检测Nrf2及其下游基因的mRNA及蛋白水平表达的变化,并在相应时间点行神经行为学评价及脑含水量的测定。结果①从ICH后24 h开始,出血组大鼠的前肢抬起的比例均低于假手术组,出血组的右向转角比例均高于假手术组,差异均有统计学意义,P<0.05。②在ICH后24 h,3、5 d,出血组的脑含水量高于假手术组,差异有统计学意义,P<0.05。出血组的脑含水量在3 d达到高峰,为(86.9±5.4)%,此后逐渐降低。③与假手术组比较,Nrf2 mRNA在ICH后2 h至5 d均高于假手术组,2 h达到高峰,差异有统计学意义。Nrf2下游基因血红素加氧酶(HO)、谷胱甘肽-S-转移酶α1(GST-α1)、谷胱甘肽(GSH)及硫氧还蛋白(TRX)mRNA表达亦有不同程度的升高。其中HO-1mRNA表达上调最为显著,ICH后2 h至5 d均高于假手术组,3 d时达高峰,差异有统计学意义。④与假手术组比较,Nrf2蛋白在ICH后2 h至5 d均高于假手术组,24 h达到高峰,相对表达量为0.39±0.05,差异有统计学意义。HO-1蛋白在ICH后8 h至10 d均高于假手术组,3 d达到高峰,相对表达量为0.99±0.08,差异有统计学意义。结论核因子Nrf2信号通路在大鼠ICH急性期被激活,影响下游基因的表达,其中HO-1表达变化与神经行为学、脑含水量变化趋势相近,可能在反映ICH继发性脑损伤程度方面具有重要意义。     

PTEN在心力衰竭心肌MAPK/PKC信号通路的作用

目的 观察充血性心力衰竭病人心肌重构病理过程中肿瘤抑制因子PTEN（phosphatase and tensin homolog tumor suppressor,PTEN）及丝裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）与蛋白激酶C（protein kinase C,PKC）的作用.方法 通过手术取材,选择因瓣膜性心脏病接受二尖瓣置换术的心力衰竭病人39例,正常对照38例（其中8例来自意外伤亡的器官捐献者）.竞争蛋白结合法检测心肌组织PKC及MAPK活性,免疫沉淀法检测PTEN蛋白表达.结果 心力衰竭病人心肌组织呈典型的重构心肌的病理改变.心肌组织PTEN表达蛋白光吸收（absorbance,A）与β肌动蛋白光吸收比值（PTEN/β-actin）随心功能恶化而降低,各心力衰竭组与正常组相比,差异有统计学意义（P＜0.01）;相反,心力衰竭病人心肌组织PKC和MAPK活性明显高于对照组（P＜0.01）,并随心功能恶化其表达逐渐增加,各心力衰竭组与正常组比较,差异有统计学意义（P＜0.01）.结论 PTEN及MAPK与PKC信号通路共同参与调节CHF病人心肌重构的病理过程,PTEN在心肌重构病理过程中起负性调节作用.     

Impaction of factors associated with oxidative stress on the pathogenesis of gestational hypertension and preeclampsia: A Chinese patients based study

We aimed to investigate the effect of Kelch-like ECH-associated protein 1/NF-E2 p45-related factor 2 (Keap1/Nrf2) pathway on the biological function of trophoblast cells in oxidative stress model at the cellular level, and analyzed the expression level and clinical significance of Keap1/Nrf2 pathway and related antioxidant factors in placental tissues of Preeclampsia (PE) patients at clinical level. In present study, we found that under hypoxia/reoxygenation conditions, the activity of oxidative stress-related enzymes (CAT, GSH-Px, SOD) in HTR8/SVneo cells was significantly lower than that before treatment (P < .01). The activities of CAT, GSH-Px and SOD in HTR8/SVneo cells in SiRNA+H/R group decreased significantly (P < .01), indicating the important defense effect of Keap1/Nrf2 signaling pathway in oxidative stress. As a control group of Nrf2 SiRNA+H/R group, Si-NC+H/R group had CAT, GSH-Px and SOD activities decreasing, which was similar to that in H/R group. Moreover, the activities of oxidative stress-related active enzymes in patients with PE were further confirmed by detecting and comparing the activities of CAT, GSH-Px and SOD in placental tissues. The results showed that the activity of SOD (P < .001), GSH-Px (P < .01) and CAT (P < .01) in placental tissues of patients with PE were significant different from those of normal placental tissues. The expression level of Keap1 in placenta of patients with PE was slightly lower than that of normal placenta. While the expression of Nrf2 in placenta of patients with PE was significantly higher than that of normal placenta. HO-1 expression in placenta of patients with PE was significantly higher than that of normal placenta. These results implicate the importance of Keap-1/Nrf2 pathway in PE.     

Sinapic acid ameliorates D-galactosamine/lipopolysaccharide-induced fulminant hepatitis in rats: Role of nuclear factor erythroid-related factor 2/heme oxygenase-1 pathways

BACKGROUND Sinapic acid(SA)has been shown to have various pharmacological properties such as antioxidant,antifibrotic,anti-inflammatory,and anticancer activities.Its mechanism of action is dependent upon its ability to curb free radical production and protect against oxidative stress-induced tissue injuries.AIM To study the hepatoprotective effects of SA against lipopolysaccharide(LPS)/Dgalactosamine(D-GalN)-induced acute liver failure(ALF)in rats.METHODS Experimental ALF was induced with an intraperitoneal(i.p.)administration of 8μg LPS and 800 mg/kg D-GalN in normal saline.SA was administered orally once daily starting 7 d before LPS/D-GalN treatment.RESULTS Data showed that SA ameliorates acute liver dysfunction,decreases serum levels of alanine transaminase(ALT),and aspartate aminotransferase(AST),as well as malondialdehyde(MDA)and NO levels in ALF model rats.However,pretreatment with SA(20 mg/kg and 40 mg/kg)reduced nuclear factor kappalight-chain-enhancer of activated B cells(NF-κB)activation and levels of inflammatory cytokines(tumor necrosis factor-αand interleukin 6).Also,SA increased the activity of the nuclear factor erythroid-related factor 2/heme oxygenase-1(Nrf2/HO-1)signaling pathway.CONCLUSION In conclusion,SA offers significant protection against LPS/D-GalN-induced ALF in rats by upregulating Nrf2/HO-1 and downregulating NF-κB.     

Induction of HO-1 and redox signaling in endothelial cells by advanced glycation end products: A role for Nrf2 in vascular protection in diabetes

Background and aims

Hyperglycemia and diabetes are associated with increased formation of advanced glycation end products and enhanced oxidative stress, leading to the progression of diabetic vascular disease. We have investigated the mechanisms by which AGE-modified bovine albumin (AGE-BSA) induces reactive oxygen species (ROS) generation, leading to nuclear factor-erythroid 2-related factor (Nrf2) dependent induction of the antioxidant genes heme oxygenase-1 (HO-1) and NADPH:quinone oxidoreductase 1 (NQO1) in bovine aortic endothelial cells.

Methods and results

AGE-BSA (100 μg ml⁻¹, 0-24 h), but not native BSA, elicited time-dependent increases in ROS generation, Nrf2 nuclear translocation and enhanced mRNA and protein expression of HO-1 and NQO1, but not glutathione peroxidase-1. Inhibition of ROS production with the superoxide scavenger Tiron or inhibitors of flavoproteins (diphenylene iodonium) and NADPH oxidase (apocynin), but not eNOS (l-NAME) or mitochondria complex I (rotenone) abrogated HO-1 induction by AGE-BSA. Although AGE-BSA induced rapid phosphorylation of JNK and Akt, only inhibition of JNK abrogated HO-1 expression, implicating the involvement of the JNK signaling pathway in AGEs activation of Nrf2/ARE-linked antioxidant gene expression.

Conclusion

Our findings establish that AGEs activate redox sensitive Nrf2-dependent antioxidant gene expression in bovine aortic endothelial cells, providing an adaptive endogenous defense against oxidative stress in diabetes.     

川芎嗪对哮喘小鼠p38蛋白激酶及类胰蛋白酶表达的影响

目的探讨川芎嗪对哮喘小鼠p38蛋白激酶（p38MAPK）及类胰蛋白酶表达的影响。方法将30只BALB/c小鼠随机分为3组：A组（对照组）、B组（哮喘组）及C组（川芎嗪组）,每组10只,采用鸡卵清蛋白（OVA）与免疫佐剂（氢氧化铝）腹腔注射致敏及用1%OVA生理盐水溶液雾化激发的方法制备哮喘小鼠模型,C组小鼠于每次激发前1 h腹腔注射川芎嗪（80mg/kg/d）,每天1次,持续5 d,A组腹腔注射等量生理盐水致敏及雾化吸入。行HE染色镜下观察各组肺组织病理改变,并采用免疫组织化学染色SP法半定量测定行肺组织中p38MAPK及类胰蛋白酶表达情况。结果 C组小鼠肺组织HE染色病理改变较B组有减轻,且免疫组化结果显示测定C组小鼠肺组织较B组p38MAPK及类胰蛋白酶MOD有降低（P〈0.05）。结论川芎嗪减轻哮喘气道炎症,部分机制可能是通过影响p38蛋白激酶信号通路及肥大细胞活化实现。     

红系衍生核因子相关因子2基因在芬维A胺诱导NB4细胞凋亡中的作用

目的:研究干扰红系衍生核因子相关因子2(NRF-2)基因表达对芬维A胺诱导NB4白血病细胞凋亡的影响,探寻白血病治疗的新思路。方法:采用核转染技术将NRF-2的小干扰RNA(siRNA)转入NB4细胞干扰NRF-2的表达,然后再用芬维A胺(1μmol/L)处理NB4细胞,24、48 h后应用流式细胞术膜联蛋白(Annexin-V)异硫氰酸荧光素(FITC)/碘化丙啶(PI)双标记法检测细胞凋亡情况。结果:1μmol/L芬维A胺能明显诱导NB4细胞凋亡,并呈时间依赖性和药物剂量依赖性,在凋亡发生过程中NRF-2的mRNA和蛋白水平有上升趋势。通过RNA干扰NRF-2表达后,NRF-2的mRNA以及蛋白水平都明显下降,芬维A胺诱导NB4细胞凋亡的效能也随之减弱。流式细胞术分析干扰组细胞凋亡率仅(29.90±2.45)%,明显低于非干扰组的(43.85±14.40)%(P=0.001)。结论:NRF-2在芬维A胺诱导NB4细胞凋亡过程中扮演着重要的角色,提示芬维A胺可能通过氧化应激途径诱导细胞凋亡。通过这样的途径,设计提高白血病细胞的氧化应激能力可能成为靶向治疗白血病的新途径。     

The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI.