Nrf2/HO-1在远隔缺血后处理减轻大鼠心肺复苏脑损伤中的作用

目的探讨核因子E2相关因子2(Nrf2)/血红素氧化酶-1 (HO-1)在远隔缺血后处理减轻大鼠心肺复苏脑损伤过程中的表达变化及意义。 方法采用随机数字表法将45只成年雄性SD大鼠分为3组：假手术组(Sham组)、模型组(Control组)、远隔缺血后处理组(RIPost组),每组15只。Control组大鼠建立窒息性心肺复苏脑损伤模型,RIPost组大鼠在自主循环恢复后行肢体远隔缺血后处理。在自主循环恢复后24 h分别采用苏木精-伊红染色、酶联免疫吸附试验检测血清神经元特异性烯醇化酶(NSE),观察脑组织的损伤情况;Western Blotting、免疫组织化学染色、实时荧光定量PCR检测脑组织Nrf2、HO-1蛋白及其mRNA表达情况。 结果与Sham组相比,Control组呈急性缺血性改变,海马区神经元有明显的损伤,血清NSE含量显著升高,差异有统计学意义(P<0.05),而RIPost组病理学改变轻于Control组,血清NSE含量也较Control组明显降低,差异有统计学意义(P<0.05);Control组、RIPost组的Nrf2核蛋白和HO-1蛋白表达均高于Sham组,且RIPost组HO-1蛋白表达显著高于Control组,差异均有统计学意义(P值均小于0.05)。实时荧光定量PCR显示Control组及RIPost组Nrf2 mRNA表达水平差异无统计学意义(P>0.05);RIPost组HO-1 mRNA的表达较Control组升高更显著,差异有统计学意义(P<0.05)。 结论远隔缺血后处理可减轻大鼠心肺复苏脑损伤,其作用机制可能与上调大鼠Nrf2 /HO-1通路有关。     

Nrf2-ARE通路在缺氧/吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤中的作用

 目的：探讨核因子E2相关因子2（Nrf2）-抗氧化反应元件（ARE）通路在缺氧后处理和吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤中的作用。方法：建立成年大鼠心肌细胞缺氧复氧模型,分离、培养心肌细胞,随机分为6组（n=8）：正常组（N组）、模型组（M组）、缺氧后处理组（IPO组）和不同浓度吡那地尔后处理组（P25组、P50组和P100组）。分离后的心肌细胞培养20 h后,除N组继续培养外,其余各组均缺氧45 min,复氧60 min。IPO组缺氧45 min后复氧、缺氧循环3次,每次各5 min,在继续复氧60 min;P25组、P50组和P100组缺氧45 min后分别以25、50和100 μmol/L吡那地尔处理5 min,复氧60 min。采用real-time PCR及Western blotting技术检测复氧末心肌细胞中Nrf2、血红素加氧酶1（HO-1）、NAD(P)H:醌氧化还原酶1（NQO1）和超氧化物歧化酶1（SOD1） mRNA及蛋白表达水平。结果：与N组比较,其它各组与Nrf2、NQO1、SOD1及HO-1的mRNA及蛋白质表达均降低（P＜0.05）;与M组比较,其它各组mRNA及蛋白质表达均显著增高（P＜0.05）;其中IPO组和P50组mRNA转录量显著高于P25组和P100组（P＜0.05）;P25组SOD1 mRNA显著高于P100组（P＜0.05）。IPO组和P50组Nrf2、SOD1和NQO1蛋白质表达显著高于P25和P100组（P＜0.05）;P50组HO-1蛋白质表达量显著高于IPO组、P25组和P100组（P＜0.05）;P25组HO-1和NQO1蛋白质表达显著高于P100组（P＜0.05）。结论：缺氧后处理和吡那地尔后处理可能通过激活Nrf2-ARE通路减轻大鼠心肌细胞缺氧复氧损伤。     

人参皂苷Rg1对小鼠脑缺血再灌注后脑组织损伤及Nrf2/HO-1途径的影响

 目的：观察人参皂苷Rg1对小鼠脑缺血再灌注后脑组织损伤的影响,并从核因子E2相关因子2（Nrf2）/血红素加氧酶1（HO-1）信号通路研究其作用机制。方法：C57BL/6小鼠随机分组,连续给药3 d,末次给药1 h后,结扎双侧颈总动脉造成脑缺血20 min,再灌注24 h。以具有抗氧化应激损伤作用、治疗缺血性脑血管病有效的药物依达拉奉作为阳性对照药。取脑组织行海马CA1区神经细胞病理学测定,RT-PCR法测定Nrf2和HO-1 mRNA表达,Western bloting测定脑组织胞核、胞浆Nrf2和全细胞HO-1蛋白含量。结果：(1)缺血再灌注24 h后,神经细胞出现病理性损伤,细胞存活率显著降低。人参皂苷Rg1和依达拉奉可使神经细胞损伤明显减轻,细胞存活率显著升高。(2)脑缺血再灌注24 h,脑组织Nrf2 mRNA和HO-1 mRNA表达增强,同时脑组织胞核和胞浆中Nrf2蛋白含量增加,核转位率升高,HO-1蛋白表达增强。人参皂苷Rg1和依达拉奉均能降低脑组织胞浆Nrf2蛋白含量,升高胞核Nrf2含量,使Nrf2核转位率升高,并使脑组织HO-1 mRNA和蛋白表达显著增加。依达拉奉的作用强于人参皂苷Rg1,但两药对脑组织Nrf2 mRNA表达无显著影响。结论：人参皂苷Rg1具有抗脑缺血再灌注损伤的作用,其机制可能与激活Nrf2/HO-1信号途径、促进Nrf2合成和核转位、从而促进下游抗氧化蛋白HO-1的表达有关。     

PI3K/Nrf2通路在内毒素休克兔肾损伤中的作用

 目的: 探讨磷脂酰肌醇3-激酶/核因子E2相关因子2(PI3K/Nrf2)信号通路在内毒素休克兔急性肾损伤中的作用。方法: 健康清洁级雄性新西兰大白兔50只,随机分为5组(每组10只):对照组(C组)、内毒素休克模型组(L组)、渥曼青霉素+内毒素休克组(WL组)、渥曼青霉素组(W组)和二甲基亚砜组(D组)。W组、WL组经耳缘静脉注射渥曼青霉素0.6 mg/kg,D组注射二甲基亚砜0.08 mL/kg,C组和L组注射等容量生理盐水。30 min后,L组和WL组静脉注射脂多糖5 mg/kg(溶于2 mL生理盐水),C组、W组和D组注射等容量生理盐水。注射脂多糖或生理盐水后6 h处死兔,取肾组织进行肾损伤评分(HSK),测定血尿素氮(BUN)、肌酐(Cr)和尿α1-微球蛋白(α1-MG)浓度,检测肾组织MDA含量及SOD活性,检测肾组织Nrf2和HO-1的mRNA总Akt蛋白、p-Akt蛋白、总Nrf2蛋白、p-Nrf2蛋白、核Nrf2蛋白和HO-1蛋白水平。结果: 与C组、W组及D组比较,L组和WL组HSK、BUN、Cr、α1-MG及MDA含量升高,SOD活性降低,肾组织Nrf2和HO-1的mRNA、p-Akt蛋白、Nrf2总蛋白、p-Nrf2蛋白、Nrf2核蛋白及HO-1蛋白的水平上调(P<0.05)。C组、W组和D组之间上述指标差异无统计学显著性。与L组比较,WL组HSK、BUN、Cr、α1-MG及MDA含量升高,SOD活性降低,肾组织Nrf2和HO-1的mRNA、p-Akt蛋白、Nrf2总蛋白、p-Nrf2蛋白、Nrf2核蛋白及HO-1蛋白的水平降低(P<0.05)。结论: PI3K/Nrf2通路激活是内毒素休克诱发兔急性肾损伤时机体的适应性调节反应机制之一。     

罗格列酮预处理对凝血酶激活的小胶质细胞PPARγ、Nrf2及HO-1表达的影响

 目的: 采用凝血酶激活新生大鼠神经胶质细胞,观察罗格列酮预处理对小胶质细胞过氧化物酶体增殖物活化受体γ(PPARγ)、核因子E2相关因子2(Nrf-2)及血红素加氧酶-1(HO-1)表达的影响。方法: 用新生SD大鼠的脑组织,体外培养原代小胶质细胞14 d左右分离收集细胞,分为:正常对照组、凝血酶刺激组、罗格列酮干预组(罗格列酮+凝血酶组)和维甲酸干预组(维甲酸+凝血酶组)进行实验。分别采用免疫组化染色、real-time PCR和Western blot检测PPARγ、Nrf2和HO-1的表达并进行统计分析。结果: 免疫组化染色显示,与对照组比较,刺激组、罗格列酮+凝血酶组及维甲酸+凝血酶组的PPARγ、Nrf2和HO-1染色细胞数均增多。Real-time PCR结果显示罗格列酮+凝血酶组PPARγ、Nrf2及HO-1的mRNA表达均显著高于刺激组、对照组及维甲酸+凝血酶组(P<0.01),维甲酸+凝血酶组Nrf2及HO-1的mRNA表达均较刺激组和罗格列酮+凝血酶组降低(P<0.01)。Western blot结果显示,罗格列酮+凝血酶组PPARγ、Nrf2及HO-1的蛋白表达也明显高于刺激组、对照组及维甲酸+凝血酶组(P<0.01),维甲酸+凝血酶组Nrf2及HO-1的蛋白表达均较刺激组和罗格列酮+凝血酶组降低(P<0.01)。结论: 罗格列酮预处理后可增加凝血酶激活的小胶质细胞PPARγ、Nrf2及HO-1的表达,通过维甲酸预处理抑制Nrf2的表达后,其下游基因HO-1表达也受影响,说明PPARγ抗氧化作用可能是通过Nrf2调控下游基因实现的。     

党参多糖介导Nrf2通路对缺氧缺血性脑损伤的抗氧化和神经保护作用

目的　研究党参多糖对缺氧缺血性脑损伤的抗氧化和神经保护作用及其机制。 方法　采用Rice法建立HIBI模型。假手术组和模型组灌胃给予生理盐水，模型加药组灌胃给予党参多糖。分别进行神经功能学评分，观察脑积水量，脑组织病理学改变，神经元细胞凋亡情况，脑组织脂质过氧化物水平，抗氧化和神经保护相关蛋白表达水平。 结果　党参多糖能显著改善模型大鼠神经功能、脑水肿（P<0.01）和病理改变，降低细胞凋亡率（P<0.01）和Bax表达（P<0.01），降低LDH和MDA含量（P<0.01）；同时，上调Bcl-2表达（P<0.01）和SOD活性（P<0.01），增加bFGF、BDNF、PSD95、SYP、Nrf2和HO-1表达（P<0.01）。 结论　党参多糖对缺氧缺血性脑损伤具有抗氧化和神经保护作用，可能与介导Nrf2信号通路相关。     

党参多糖介导Nrf2通路对缺氧缺血性脑损伤的抗氧化和神经保护作用

目的　研究党参多糖对缺氧缺血性脑损伤的抗氧化和神经保护作用及其机制。 方法　采用Rice法建立HIBI模型。假手术组和模型组灌胃给予生理盐水,模型加药组灌胃给予党参多糖。分别进行神经功能学评分,观察脑积水量,脑组织病理学改变,神经元细胞凋亡情况,脑组织脂质过氧化物水平,抗氧化和神经保护相关蛋白表达水平。 结果　党参多糖能显著改善模型大鼠神经功能、脑水肿（P<0.01）和病理改变,降低细胞凋亡率（P<0.01）和Bax表达（P<0.01）,降低LDH和MDA含量（P<0.01）;同时,上调Bcl-2表达（P<0.01）和SOD活性（P<0.01）,增加bFGF、BDNF、PSD95、SYP、Nrf2和HO-1表达（P<0.01）。 结论　党参多糖对缺氧缺血性脑损伤具有抗氧化和神经保护作用,可能与介导Nrf2信号通路相关。     

Toll-like receptor 2 contributes to glial cell activation and heme oxygenase-1 expression in traumatic brain injury

Traumatic brain injury is accompanied by glial cell activation around the site of the injury. In this study, we investigated the role of toll-like receptor 2 (TLR2) in glial cell activation using a stab-wound injury (SWI) model with TLR2 knock-out mice. Penetration of a normal mouse brain with a 26-G needle using a stereotaxic instrument resulted in an 18- and 4-fold upregulation of GFAP and CD11b mRNA, respectively, along the needle track in the injury area. However, in the TLR2 knock-out mice, the induced expression of these genes was reduced by 70% and 40%, respectively. Likewise, there was a reduction in the area of activated glial cells detected by immunohistochemistry and the glial cells had a less-activated morphology in the TLR2 knock-out mice. In addition, the expression of the heme oxygenase-1 (HO-1) gene, a glia-expressing wound-responsive gene, was reduced after SWI in TLR2 knock-out mice. Taken together, these data argue that TLR2 contributes to the glial cell activation and HO-1 gene expression associated with traumatic brain injury.     

香烟烟雾提取物通过蛋白激酶C-核因子相关因子2调节大鼠气道上皮细胞血红素加氧酶1表达

 目的：探讨蛋白激酶C（PKC）-红系衍生的核因子相关因子2 (Nrf2)对香烟烟雾提取物（CSE）诱导的大鼠气道上皮细胞血红素加氧酶1（HO-1）表达的影响。方法：通过CSE刺激雄性SD大鼠气道上皮细胞,使用PKC抑制剂RO318220和Nrf2 siRNA,将细胞分为对照组、CSE 3 h组、RO318220组、Nrf2 siRNA组和RO318220+Nrf2 siRNA组,用Western blotting法分别检测HO-1、Nrf2和p-PKC蛋白表达,免疫细胞化学法观察HO-1蛋白表达,逆转录－聚合酶链反应（RT-PCR）法检测HO-1 mRNA表达,免疫荧光法检测Nrf2蛋白定位,测定HO-1活性。结果：暴露CSE 3 h后,Nrf2蛋白主要表达在胞核, 胞核蛋白表达增强,p-PKC蛋白、HO-1的mRNA和蛋白高表达,HO-1活性增强。预先给予RO318220,PKC蛋白、Nrf2胞浆和胞核蛋白、HO-1的mRNA和蛋白表达均明显减弱,HO-1活性显著降低。预先用siRNA沉默Nrf2,胞浆和胞核的 Nrf2蛋白表达均减弱,HO-1活性、mRNA和蛋白水平明显降低。RO318220联合Nrf2 siRNA处理后,PKC蛋白、Nrf2胞浆和胞核蛋白、HO-1 mRNA和蛋白表达均明显降低,HO-1活性明显降低。结论：CSE通过PKC激活Nrf2,诱导Nrf2核转位,从而上调HO-1的表达水平。     

基于Nrf2通路探讨阿里红三萜酸减轻脂多糖诱导的小鼠急性肺损伤的作用

目的:探讨阿里红总三萜酸(Fomes officinalis Ames triterpenic acid,FOTa)对脂多糖(lipopolysac?charide,LPS)诱导的小鼠急性肺损伤(acute lung injury,ALI)的预防作用及核因子E2相关因子2(nuclear factor E2-relat...     

Glutamine decreases intestinal nuclear factor kappa B activity and pro-inflammatory cytokine expression after traumatic brain injury in rats

Objective: To investigate whether glutamine supplementation modulates intestinal nuclear factor kappa B (NF-κB) activity and pro-inflammatory cytokine expression after traumatic brain injury (TBI) in rats. Materials and methods: Right parietal cortical contusion in male rats was made by the weight-dropping method. After trauma, the rats were randomly given chow alone or glutamine mixed chow for 5 d. Gut samples were extracted at 5 d postinjury. We measured NF-κB binding activity by electrophoretic mobility shift assay; NF-κB subunits p50 and p65 expression by immunohistochemistry; the concentrations of interleukin-1β, tumor necrosis factor-α and interleukin-6 by enzyme-linked immunosorbent assay; intestinal mucosal morphological changes by histopathological study and electron microscopy; and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Results: Administration of glutamine following TBI could decrease NF-κB binding activity, NF-κB p65 protein expression and concentrations of pro-inflammatory cytokines in the gut. TBI-induced damage of gut structure was ameliorated after glutamine supplementation. Conclusion: The results of the present study suggest that the therapeutic benefit of post-TBI glutamine supplementation might be due to its inhibitory effects on intestinal NF-κB activation and pro-inflammatory cytokine expression. Received 14 May 2007; returned for revision 9 July 2007; accepted by I. Ahnfeld-R?nne 16 August 2007     

莱菔硫烷对糖尿病大鼠视网膜神经细胞的保护作用

目的:研究莱菔硫烷(SF)对糖尿病大鼠视网膜神经细胞的保护效应,并初步探讨其作用机制。方法:通过一次性腹腔注射链脲佐菌素的方法制备糖尿病大鼠模型;通过测定活性氧簇(ROS)的生成、TUNEL法检测视网膜细胞的凋亡和计数存活的视网膜神经节细胞(RGCs)等方法作为指标观察SF对糖尿病大鼠视网膜神经细胞的保护效应;以免疫组织化学染色和Western blot法检测视网膜核因子E2相关因子2(Nrf2)的核转移和血红素加氧酶1(HO-1)的表达变化。结果:SF能抑制糖尿病大鼠视网膜ROS的生成,抑制视网膜神经细胞的凋亡并增加糖尿病大鼠视网膜RGCs的存活数量;同时SF还可促进Nrf2的激活及HO-1蛋白表达;使用HO-1抑制剂锌原卟啉可明显减弱SF对糖尿病大鼠视网膜RGCs凋亡的抑制作用。结论:SF可能通过激活Nrf2/HO-1抗氧化通路改善糖尿病大鼠视网膜氧化应激状态,减少视网膜神经细胞凋亡,减轻糖尿病大鼠的视网膜损伤。     

Effect of APOE polymorphisms on early responses to traumatic brain injury

To investigate the relationship between apolipoprotein E (APOE) polymorphisms and the severity of traumatic brain injury (TBI) in acute stage in the cohort of mainland Chinese patients. We prospectively identified admissions to the two neurosurgical departments for head injury. A total of 110 subjects with TBI (80 males and 30 females, with mean age of 43.87 years) were enrolled from December 2003 to May 2004, and demographic and clinical data were collected. Venous blood was collected from patients with TBI on admission to determine the APOE genotype polymorphisms. The APOE genotyping was performed by means of PCR-RFLP. The deterioration of patients’ condition in acute stage (<7 days after TBI) was judged by either of following criteria: decrease of GCS, increase in hematoma volume or delayed hematoma both detected by repeated CT scanning. χ²-test and logistic regression analyses were done by SPSS. The distributions of APOE genotypes and alleles matched Hardy–Weinberg law. In 110 Chinese patients, 19 subjects presented with deteriorated clinical condition after hospitalization, and seven of 17 patients with APOE ?4 (41.2%) had a deteriorated condition which was significantly different from those without APOE ?4 (12 of 93 patients, 12.9%, P = 0.01). However, neither the presence of ?2 nor of ?3 was significantly different from those absent of it (P > 0.05). Logistic regression analyses showed that APOE ?4 was a risk factor (OR = 4.836, P = 0.011, 95% CI 1.443–16.208) to predispose to clinical deterioration after adjusting for patient age, sex, smoking or not, alcohol-drinking or not, injury severity, injury mechanisms, treatments, and pattern of TBI. This finding suggests that the patients with APOE ?4 predispose to clinical deterioration in acute phase after TBI and APOE polymorphisms play a role in early responses to TBI.     

创伤性脑损伤后大鼠外周血CD8的变化

目的:探讨外周血CD8在创伤性脑损伤后的变化特点。方法:改良Feeney's自由落体打击法制备创伤性脑损伤模型;ELISA检测大鼠血清中神经元特异性烯醇化酶(NSE)和CD8的含量,双变量分析两者的相关性;Western blot法检测大鼠血液淋巴细胞FasL蛋白的表达。结果:颅脑外伤后大鼠血清CD8升高滞后于NSE。大鼠伤后1 d血清NSE水平即显著升高,伤后3 d达到峰值,随后逐渐下降;大鼠血清CD8在伤后3 d开始升高,峰值出现在伤后7 d,随后逐渐下降。统计分析显示伤后1 d、3 d、7 d的血清的NSE与伤后3 d、7 d、14 d的血清CD8含量变化呈现正相关。但伤后不同时间大鼠血液淋巴细胞FasL蛋白表达的差异均无统计学显著性。结论:创伤性脑损伤后释放入血的NSE刺激机体免疫应答,引起外周血CD8增多,推测其可能与创伤性脑损伤后继发性损伤的免疫应答有关。     

Sex differences in XIAP cleavage after traumatic brain injury in the rat

Sex influences histological and behavioral outcomes following traumatic brain injury (TBI), but the underlying sex-dependent pathomechanisms regulating outcome measures remain poorly defined. Here, we investigated the TBI-induced regulation of the X-linked inhibitor of apoptosis protein (XIAP) that, in addition to suppressing cell death by inhibition of caspases, is involved in signaling cascades, including immune regulation and cell migration. Since estrogen has been shown to have anti-apoptotic properties, we specifically examined sex differences and the influence of estrogen on XIAP processing after TBI. Sprague–Dawley male (TBI-M), female (TBI-F), ovariectomized female (TBI-OVX) and ovariectomized females supplemented with estrogen (TBI-OVX + EST) were subjected to moderate (1.7–2.2 atm) fluid percussion (FP) injury. Animals were sacrificed 24 h after FP injury; cortical tissue (ipsilateral and contralateral) was dissected and analyzed for XIAP processing by immunoblot analysis (n = 6–7/group) or confocal microscopy (n = 2–3/group). Significant differences in XIAP cleavage products in the ipsilateral cortex were found between groups (p < 0.03). Post hoc analysis showed an increase in XIAP processing in both TBI-F and TBI-OVX + EST compared to TBI-M and TBI-OVX (p < 0.05), indicating that more XIAP is cleaved following injury in intact females and TBI-OVX + EST than in TBI-M and TBI-OVX groups. Co-localization of XIAP within neurons also demonstrated sex-dependent changes. Based on these data, it appears that the processing of XIAP after injury is different between males and females and may be influenced by exogenous estrogen treatment.     

FGF-21和Nrf2对博莱霉素诱导的小鼠肺纤维化的影响

目的:研究成纤维细胞生长因子21(FGF-21)对博莱霉素(BLM)诱导的小鼠肺纤维化炎症应答及氧化应激的影响,并探讨其抗肺纤维化的作用机制。方法:建立BLM诱导的小鼠肺纤维化的模型,40只小鼠随机分为对照组、BLM组及FGF-21(1、2及5 mg/kg)+BLM组。Western blot检测I型胶原蛋白(collagen I)、纤连蛋白(fibronectin)和核因子E2相关因子2(Nrf2)蛋白表达水平。DCFH-DA染色检测活性氧簇(ROS)的生成。ELISA用于测定肺组织炎症因子的表达。试剂盒检测各组小鼠肺组织中的丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、谷胱甘肽过氧化物酶(GPx)活性和羟脯氨酸(HYP)含量。结果:FGF-21处理显著降低BLM诱导的肺组织炎症介质肿瘤坏死因子α、白细胞介素1β和白细胞介素6的表达水平,减少ROS及MDA的含量,并增加抗氧化酶系统SOD和GPx的活性(P 0. 05)。同时,FGF-21下调BLM诱导的collagen I和fibronectin,并减少TGF-β1及HYP的含量。Nrf2沉默能够逆转FGF-21的抗纤维化作用。结论:FGF-21通过激活Nrf2信号抑制炎症应答进程,减轻氧化损伤,减少细胞外基质沉积,从而缓解BLM诱导的肺纤维化。这可能为肺间质纤维化治疗提供新的靶点。     

脑外伤小鼠海马水通道蛋白-9表达的变化

目的探讨脑外伤小鼠海马水通道蛋白-9(aquaporin 9,AQP9)表达的变化。方法40只Balb/c小鼠随机分为假手术组(10只)和脑外伤组(30只)。脑外伤组依据脑外伤的不同时间点再分6 h、1 d、3 d三个小组,每组10只。免疫组化和Western blot检测各组小鼠海马AQP9蛋白的表达。RT-PCR方法检测各组小鼠海马AQP9 mRNA的表达变化。结果免疫组化和Western blot结果发现,脑外伤组小鼠海马AQP9蛋白表达量明显高于假手术组(P<0.05);RT-PCR结果也发现,脑外伤组小鼠海马AQP9 mRNA表达量明显高于假手术组(P<0.05)。结论脑外伤小鼠海马AQP9表达明显升高。