二氯乙酸钠对氧糖剥夺损伤的BV2细胞的保护作用及其机制研究

目的 研究二氯乙酸钠（dichloroacetate,DCA）对氧糖剥夺（oxygen glucose deprivation,OGD）损伤模型中小鼠小胶质细胞（BV2细胞）的保护作用,并探讨其作用机制。方法 将BV2细胞分为3组：对照组、OGD组、DCA治疗组,通过OGD 4 h建立损伤模型。CCK-8和流式细胞仪检测细胞凋亡及ROS和NO的表达,Western blot检测NF-κB通路相关蛋白表达水平。结果 CCK-8及流式细胞检测结果表明,DCA可显著降低OGD诱导的BV2细胞凋亡,并且减少OGD后细胞中活性氧（ROS）和一氧化氮（NO）的表达（P<0.05）。Western blot结果显示,DCA可显著影响OGD损伤介导的BV2细胞JNK、I-κB和NF-κB蛋白表达水平（P<0.05）。结论 DCA对OGD损伤的BV2细胞具有保护作用,其机制与抗凋亡、抗氧化和抗炎作用有关。     

艾拉莫德对脂多糖激活大鼠肺泡巨噬细胞系TNFα产生及NF-κB活性的抑制作用

目的研究非甾体抗炎药艾拉莫德(T-614)对脂多糖(LPS)刺激的大鼠肺泡巨噬细胞系(NR8383)前炎症反应因子TNFα基因表达、蛋白合成的影响及其对核因子κB(NF-κB)的作用。方法体外培养NR8383经T-614(13.4，26.7及53.4 μmol·L^-1)处理，LPS刺激后应用酶联免疫吸附法(ELISA)检测细胞上清液中TNFα的水平，半定量逆转录聚合酶链式反应(RT-PCR)检测TNFα mRNA水平，ELISA法检测NF-κB的活性。结果T-614对LPS诱导的NR8383细胞TNFα mRNA水平和蛋白水平的上调有显著抑制作用，对NF-κB的转录活性也有抑制作用。结论 T-614可能通过抑制LPS诱导的NR8383的NF-κB活性而降低TNFα的产生。     

丁苯酞对糖氧剥夺条件下人脑微血管内皮细胞炎症反应的调节作用

目的 研究丁苯酞调节糖氧剥夺（oxygen-glucose deprivation,OGD）条件下人脑微血管内皮细胞（human brainmicrovascular endothelial cells,HBMEC）炎症反应的机制。方法 采用OGD方法处理HBMEC,将细胞分为对照组、模型组和丁苯酞低、中、高剂量组。其中对照组为正常培养的HBMEC,模型组为OGD处理的细胞,低剂量组为OGD+5 μmol·L^-1的丁苯酞,中剂量组为OGD+10 μmol·L^-1的丁苯酞,高剂量组为OGD+20 μmol·L^-1的丁苯酞。采用流式细胞术检测细胞凋亡水平,CCK-8法检测细胞活力的变化,Western-Blot法检测细胞中HMGB1、TLR4、NF-κB （p-P65）、caspase-1和procaspase-1的表达水平,免疫酶联法检测上清中白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）,白细胞介素6（IL-6）的分泌水平。实时荧光定量PCR检测HMGB1、TLR4、NF-κB（P65）、caspase-1、IL-1β的mRNA表达。结果 OGD处理可以诱导HBMEC活力下调和HBMEC细胞凋亡,且具有时间依赖性,相比对照组具有显著性差异（P<0.05）;而丁苯酞干预后,HBMEC细胞活力比模型组显著上调（P<0.05）,细胞凋亡率比模型组显著下降（P<0.05）。OGD处理可以导致HBMEC中HMGB1-TLR4-NF-κB信号的激活,诱导炎症因子caspase-1、IL-1β的表达上调,相比对照组具有显著性差异（P<0.05）;而丁苯酞干预可以显著下调HMGB1-TLR4-NF-κB信号的表达,下调caspase-1、IL-1β的表达。结论 丁苯酞可能通过抑制HMGB1-TLR4-NF-κB信号的表达调节OGD下HBMEC细胞炎症反应。     

丁基苯酞对原代培养胎大鼠皮层神经细胞外液NO及胞浆内cGMP水平的影响

用分光光度法和放射免疫分析法分别检测NO及cGMP水平,同时观察l-丁基苯酞(l-NBP)和d-丁基苯酞(d-NBP)对原代培养的胎大鼠皮层神经细胞外液NO及胞浆内cGMP水平的影响。结果表明,d-NBP(0.1～100μmol·L^-1)对低糖低氧条件下或含N-甲基-D-门冬氨酸(NMDA)或含KCl的培养基中皮层神经细胞外液NO及细胞内cGMP水平明显升高,而l-NBP(0.1～100μmol·L^-1)则能明显降低NO和cGMP水平。提示:d-NBP和l-NBP对低糖低氧,NMDA或KCl诱导的NO释放和cGMP生成有相反的作用。     

Exendin-4体外通过抑制NF-κB-iNOS-NO信号减轻氧化应激诱导的小鼠MIN6胰岛β细胞凋亡

探讨胰高血糖素样肽-1受体激动剂Exendin-4(Ex-4)在氧化损伤诱导胰岛β细胞凋亡中的保护作用。培养的MIN6胰岛β细胞，通过AO-EB染色观察细胞凋亡形态，Annexin-V-PI染色流式技术测定凋亡率，Griess法检测细胞内一氧化氮水平，Western blotting检测胞浆iNOS蛋白、胞浆及胞核核因子-κBp65(NF-κBp65)蛋白表达水平。Ex-4可抑制叔丁基过氧化氢(t-BHP)诱导的β细胞凋亡，Ex-4(100 nmol·L^-1)预处理较单独t-BHP处理，其凋亡率减少约67%(P<0.001)。Ex-4同时减少NO水平的增高，并抑制t-BHP诱导的β细胞NF-κBp65活化及iNOS蛋白表达水平。Ex-4可能通过抑制细胞内NF-κB活化、胞浆iNOS表达来抑制NO水平，最终减轻氧化损伤诱导的β细胞凋亡。     

抗炎药物对HEK293细胞NF-κB活化的调节作用

目的探讨不同作用机制的抗炎药物对内源性和TNFα诱导的NF-κB的调节作用。方法利用荧光素酶报告基因测定法检测细胞内NF-κB活化水平，MTT法测定细胞增殖，PI染色-流式细胞仪测定细胞凋亡。结果 一定浓度的美洛昔康和地塞米松能够明显降低HEK293细胞内源性和10 ng·mL^-1 TNFα诱导的NF-κB活化，1×10^-9 mol·L^-1氢化考的松分别明显增加和降低这两种活化，而吲哚美辛对内源性活化无明显影响。4种药物对HEK293细胞增殖均无明显作用。地塞米松单独或联合TNFα、吲哚美辛联合TNFα能够引起细胞凋亡。结论不同类型的抗炎药物对内源性和TNFα诱导的NF-κB活化影响不同。提示NF-κB活化调节可能成为药物作用机理研究的有效靶点之一。     

氟哌啶醇对大鼠离体海马脑片和原代神经元缺糖/缺氧和N-甲基-D-天冬氨酸损伤的保护作用

目的观察氟哌啶醇对大鼠离体海马脑片和原代神经元的缺糖/缺氧(OGD)和N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)损伤的潜在保护作用及其机制。方法海马脑片OGD以无葡萄糖的人工脑脊液中通95% N₂+5% CO₂诱导。通过测定TTC染色后形成的红色产物来分析脑片活性。结果氟哌啶醇(1和10 μmol·L^-1)抑制OGD损伤,抑制率分别为17.7%和25%,而D₂多巴胺受体拮抗剂多潘立酮无此作用。NMDA也能显著降低海马脑片及原代神经元的活性,而氟哌啶醇可抑制这一损伤作用。结论氟哌啶醇对大鼠离体海马脑片OGD和原代神经元NMDA损伤有保护作用。     

丁苯酞抑制原代大鼠海马神经元氧糖剥夺/复氧诱导的凋亡

目的:观察丁苯酞对氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)诱导的SD大鼠原代培养海马神经元损伤的保护作用。方法:原代培养新生24 h内SD乳鼠海马神经元,建立氧糖剥夺30 min/复氧8 h海马神经元损伤模型。实验分为正常对照组、OGD/R模型组、丁苯酞低、中、高剂量组(0.1,1,10μmol·L-1)。采用Hoechest33258染色测定细胞凋亡,免疫细胞化学检测各组细胞中Bcl-2蛋白的表达情况。结果:与OGD/R模型组比较,各丁苯酞给药物组神经元凋亡率均不同程度降低(P<0.01),且随药物浓度升高而降低;Bcl-2蛋白表达不同程度地增高(P<0.01),且随药物浓度升高而升高。结论:丁苯酞能抑制OGD/R诱导的海马神经元细胞凋亡,其可能通过增加神经元内Bcl-2凋亡抑制蛋白的表达而抑制凋亡,进而保护神经元。     

丁基苯酞对原代培养的大鼠皮层神经细胞外液6-keto-PGF1α和TXB2及其比值的影响

目的旨在观察丁基苯酞(NBP)对神经细胞培养液中6-酮-PGF_1α和TXB₂含量及其比值的影响。用放射免疫方法,结果发现神经细胞在低糖低氧5h或低糖低氧5h/恢复糖氧3h条件下,d-,l-和dl-NBP(0.1～100μmol·L^-1)能够剂量依赖性升高细胞外液中的6-酮-PGF_1α含量,降低TXB₂水平,从而使6-酮-PGF_1α与TXB₂比值升高。而阿司匹林仅在小剂量(0.1,1μmol·L^-1)时能升高6-酮 PGF^1α与TXB^{2比值,大剂量(10,100μmol·L-1)时无影响。提示:NBP对6-酮-PGF_1α/TXB₂比值的升高可能与其增加局部脑血流和改善缺血性脑损伤有关。}     

内源性一氧化氮介导脂质胞壁酸预适应对人冠脉内皮细胞再复氧损伤的作用

目的探讨脂质胞壁酸(LTA)诱导的延迟预适应对内皮细胞再复氧(H/R)损伤的作用，以及内源性一氧化氮(NO)参与保护机制的作用。方法采用培养的人冠状动脉内皮细胞(HCAECs)在缺氧条件下培养2 h，然后在常氧条件下复氧培养4 h，模拟缺血/再灌注损伤的模型。HCAECs在缺氧前24 h预先在含LTA(30或300 μg·L^-1)的培养基中培养4 h。用台盼蓝排斥法和培养基中乳酸脱氢酶(LDH)含量来评价内皮细胞损伤程度，比色法检测培养基中一氧化氮(NO)含量。并用RT-PCR检测LTA预适应后(2-24) h HCAECs eNOS mRNA的表达。结果LTA预适应能显著减少台盼蓝排斥实验中的细胞死亡百分比，降低复氧末细胞培养液中LDH的含量。LTA预适应亦能显著增加HCAECs复氧末培养液中NO含量。LTA预适应的效应可被非选择性NOS抑制剂L-单甲基精氨酸(L-NMMA,100 μmol·L^-1)所取消。在LTA预适应后2和4 h，HCAECs的eNOS mRNA表达明显增加。结论 LTA诱导的延迟预适应能显著减少HCAECs 再复氧所致的细胞损伤和功能紊乱，eNOS产生的NO启动并介导了LTA保护内皮细胞的作用。     

N-[3,4-dimethoxycinnamoyl]-anthranilic acid (tranilast) suppresses microglial inducible nitric oxide synthase (iNOS) expression and activity induced by interferon-gamma (IFN-gamma).

1. Microglial cells up-regulate inducible nitric oxide synthase (iNOS) expression in response to various pro-inflammatory stimuli including interferon-gamma (IFN-gamma), allowing for the release of nitric oxide (NO). Tranilast (N-[3,4-dimethoxycinnamoyl]-anthranilic acid) is an antiallergic compound with suppressive effects on the activation of monocytes. 2. Here, we show that N9 murine microglial cells express iNOS mRNA and protein and release nitric oxide into the culture medium in response to IFN-gamma (200 u x ml(-1)) as measured by Northern and Western blot analyses and Griess assay. 3. Exposure to non-toxic doses of tranilast (30-300 microM) leads to a concentration-dependent inhibition of IFN-gamma-induced (200 u x ml(-1)) iNOS mRNA and protein expression. This is paralleled by a suppression of NO-release into the cell culture medium. 4. Inhibition of IFN-gamma-induced iNOS mRNA expression by tranilast is paralleled by an inhibition of nuclear factor-kappaB (NF-kappaB) activation and phosphorylation of inhibitory kappaB (IkappaB) as determined by Western blot analyses and NF-kappaB reporter gene assay. 5. These results suggest that tranilast-mediated suppression of microglial iNOS activity induced by IFN-gamma involves the inhibition of NF-kappaB-dependent iNOS mRNA expression.     

Use of brain slices in the study of pathogenic role of inducible nitric oxide synthase in cerebral ischemia-reperfusion.

We have recently demonstrated that inducible nitric oxide synthase (iNOS) is expressed in rat forebrain slices exposed to oxygen and glucose deprivation (OGD). Now, we have found that the expression of iNOS after OGD is time-dependent since 20 min of OGD produces the appearance of iNOS at earlier times than 10 min of OGD. OGD also causes neurotoxicity in this model, as revealed by the increase in excitatory amino acid, neuron specific enolase and lactate dehydrogenase (LDH) efflux to the incubation solution. Finally, the administration of the NMDA receptor antagonist MK-801 (100 nM) inhibits both the expression of iNOS and the release of LDH. Our findings demonstrate that this method may be considered an useful in vitro model of ischemia-reperfusion to determine the therapeutic role of neuroprotective tools.     

脉络宁对原代大鼠海马神经元在氧糖剥夺/复氧中的保护作用

目的:观察脉络宁对氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)诱导的SD大鼠原代培养海马神经元损伤的保护作用及可能机制。方法:原代培养从新生24 h内SD乳鼠取材的海马神经元,建立氧糖剥夺30 min/复氧8 h海马神经元损伤模型。实验分为正常对照组、OGD/R模型组、脉络宁低、中、高剂量组(5,10和20μg.mL-1)。采用Hoechest33258染色测定细胞凋亡,免疫细胞化学检测各组细胞中脑红蛋白(neuroglobin,NGB)及Bcl-2的表达,PCR检测其mRNA的表达情况。结果:与OGD/R模型组比较,各剂量脉络宁组神经元凋亡率均不同程度降低(P<0.01),且随药物浓度的升高而降低;NGB和Bcl-2蛋白表达不同程度地升高(P<0.01),且随药物浓度升高而升高;NGB及Bcl-2蛋白的mRNA亦升高(P<0.01)。结论:脉络宁发挥神经保护作用可能的机制之一是抑制OGD/R诱导的海马神经元细胞凋亡,促进NGB和Bcl-2蛋白的表达。     

Inhibitory effects of epicatechin on interleukin-1beta-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-kappaB activation

Cytokines that are released by infiltrating inflammatory cells around the pancreatic islets are involved in the pathogenesis of type 1 diabetes mellitus. Specifically, interleukin-1beta (IL-1beta) stimulates inducible nitric oxide synthase (iNOS) expression and nitric oxide overproduction, leading to the beta-cell damage. In activating this pathway, nuclear factor-kappaB (NF-kappaB) plays a crucial role, and many of the IL-1beta-sensitive genes contain NF-kappaB binding sites in their promoter regions. We have recently shown that epicatechin, which is a flavonoid, had a protective effect on pancreatic beta-cells in both streptozotocin-treated rats and islets. In the present study, the effects of epicatechin on IL-1beta-induced beta-cell damage were examined. RINm5F cells and islets were pretreated with epicatechin and next incubated with IL-1beta. The released nitrite, iNOS protein and mRNA expression levels were then measured. IkappaBalpha protein, nuclear translocation of NF-kappaB, and NF-kappaB DNA binding activity were also determined. Following the transient transfection of an iNOS promoter into the cells, the iNOS promoter activity was measured. ATP- or D-glucose-induced insulin release was measured in RINm5F cells and islets, respectively. Epicatechin significantly reduced IL-1beta-induced nitrite production, iNOS protein and mRNA expressions, and it also inhibited IL-1beta-induced IkappaBalpha protein degradation, NF-kappaB activation, and iNOS promoter activity. Epicatechin partly restored the IL-1beta-induced inhibition of insulin release. These results suggest that epicatechin inhibits the IL-1beta-induced iNOS expression by down-regulating NF-kappaB activation, and protecting beta-cells from IL-1beta.     

Protective effects of costunolide on mouse brain slice injury induced by oxygen glucose deprivation/reoxygenation

OBJECTIVE To investigate the protective effects and mechanisms of costunolide against mousebrain slice injury induced by oxygen-glucose deprivation/reoxygenation(OGD/R). METHODS Mouse brain slice injury was induced by OGD/R in vitro, and the degree ofinjury was evaluated by measuring the release of lactate dehydrogenase(LDH) and 2,3,5-triphenyltetrazolium chloride(TTC) staining. Western blotting was used to analyze the expression of Bax, Bcl-2, Cyt-c, caspase-9, caspase-7 and caspase-3. RESULTS Compared with OGD/R, 1, 5, and 10 μmol·L~(-1) costunolide decreased the LDH levels, increased the TTC staining intensity, inhibited Bax, Cyt-c, caspase-9, caspase-7, caspase-3 expression levels, and enhanced Bcl-2 expression level. CONCLUSION Costunolide has latent neuroprotective activities by the regulation of apoptosis via the mitochondrial apoptosis pathway.     

Attenuation of cerebral ischemia-reperfusion injury by specific activation of Kv7/KCNQ/Mchannels

OBJECTIVE To test any neuroprotective effect of KCNQ channel openers on ischemic injury in mice. METHODS Mouse model of ischemic-reperfusion(I/R) was established by transient middle cerebral artery occlusion(t MCAO). Neurologic deficit scores and infarct volume measurement were assessed. Open field, accelerating rotarod and grip strength were performed for assessment of neurological behaviors. The in vitro model of oxygenglucose deprivation(OGD)-induced injury in mouse primary culture of cortical neurons was used, and cell viability was determined by measurement of release of lactate dehydrogenase(LDH) and using Cell Counting Kit-8(CCK-8) assay after OGD injury. Whole-cel clamp recordings of cortical neuronswere carried out before and after OGD injury. RESULTS The t MCAO caused predictable and consistently sized necrotic brain lesions and behavioral deficits. Injection of KCNQ channel openersretagabine(10 mg·kg-1) or SCR2862(1 mg·kg-1) into tail vein 30 min before surgery significantly decreased neurological scores and brain infarct size in male mice(8-10 weeks)at 24 h after t MCAO. Kv7 activation improved the track length and speed of mice in open field test, and increased the latency to fallon rotarod test. The grip strength of affected side forelimb was also enhanced in mice treated with KCNQ channel openers. Furthermore, neurons incubated with KCNQ channel openers 1 h before OGD exposure showed an increased cell vitality and decreased release of LDH when subjected to OGD injuryfor 20 min.OGD exposure for 5 min caused a significant depolarization of resting membrane potential and action potential threshold in cortical neurons. Electrophysiological recordings revealed that Kv7 opener retigabine can suppress neuronal excitability by reducing the resting membrane potential, decreasing firing frequency and prolonging the action potential latency. CONCLUSION An obvious improvement in the reduction of brain injury and was observed in mouse model of ischemic injury after administration of KCNQ channel openers attenuates brain ischemic injury and also improves functional outcome. Activation of Kv7 channel function also protects cortical neurons against OGD-induced injury. Mechanistical y, KCNQ channel openers suppress neuronal hyperexcitability after OGD and brain ischemia injury.     

瑞舒伐他汀通过调控UCP2-SIRT3通路减轻脑缺血/再灌注对神经元线粒体的损伤

目的探讨瑞舒伐他汀(rosuvastatin,RS)通过UCP2-SIRT3信号通路在脑缺血/再灌注(CIR)神经元线粒体损伤中的作用及其机制。方法建立SH-SY5Y细胞的脑梗死再灌注模型(OGD/R),给予不同浓度RS(40和2.5μmol·L^-1)分别处理,观察两组中细胞增殖和凋亡的变化及UCP2和SIRT3分子的表达;构建UCP2沉默细胞系,研究不同浓度RS对UCP2沉默前后细胞形态和线粒体膜电位的影响、以及SIRT3分子、线粒体外膜异位酶20(TOMM20)和线粒体合成相关蛋白(Drp1、Opa1和PGC1)的表达变化。结果RS能提高OGD/R细胞的存活率、抑制细胞凋亡、改变细胞形态、稳定细胞线粒体膜电位;增加OGD/R细胞中UCP2、SIRT3分子和TOMM20蛋白表达,并诱导Drp1和Opa1 mRNA的表达,抑制PGC1 mRNA的表达;沉默UCP2后能明显降低OGD/R细胞的存活率及TOMM20蛋白的表达,降低Drp1和Opa1 mRNA的表达,使PGC1 mRNA的表达增加。结论RS通过调控UCP2-SIRT3通路减轻CIR对神经元线粒体的损伤,发挥神经细胞保护作用。     

Preliminary exploration on anti-inflammatory mechanism of Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) in vitro

Corilagin (beta-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) is a novel member of the tannin family which has been discovered from many medicinal plants and has been confirmed in many pharmacological activities. However, the purified Corilagin that was used in experiment is rare, and the anti-inflammatory mechanism of Corilagin has not been investigated clearly. This study is to explore the inner anti-inflammatory mechanism of Corilagin. Inflammatory cellular model was established by lipopolysaccharide (LPS) interfering on RAW264.7 cell line. Levels of TNF-alpha, IL-1beta, IL-6, NO and IL-10 in supernatant, mRNA expression of TNF-alpha, COX-2, iNOS and HO-1, protein expression of COX-2 and HO-1, translocation of NF-kappaB were assayed by ELISA or Griess method, real-time quantitative PCR, western blot and immunocytochemistry method, respectively. As a result, Corilagin could significantly reduce production of pro-inflammatory cytokines and mediators TNF-alpha, IL-1beta, IL-6, NO (iNOS) and COX-2 on both protein and gene level by blocking NF-kappaB nuclear translocation. Meanwhile Corilagin could notably promote release of anti-inflammatory factor HO-1 on both protein and gene level, but suppress the release of IL-10. In conclusion, the anti-inflammatory effects of Corilagin are attributed to the suppression of pro-inflammatory cytokines and mediators by blocking NF-kappaB activation. Corilagin also can promote HO-1 production to induce regression of inflammation but can inhibit IL-10 production like Dexamethasone. Corilagin possesses a potential anti-inflammatory effect by not only abating inflammatory impairment but also promoting regression of inflammation and has a good prospect to be used in many inflammation-related diseases.