在内毒素性休克中21-氨基类固醇对枯否细胞核因子-κB激活的作用

21-氨基类固醇(u-74389G)是对氧自由基诱发的和铁催化的类脂过氧化作用的强力抑制剂,也是一非糖类皮质激素,它可减轻内毒素性休克鼠的肝损害,抑制肝内核因子-κB激活而延长其生存时间。作者进行实验研究以观察21-氨基类固醇能否通过抑制肝枯否细胞的核因子(NF)-κB激活而阻止前炎性基因的上调。取200～300g雄性Wistar鼠作实验,用胶原酶灌注和链霉蛋白酶消化法提纯鼠枯否细胞。加入脂多糖后,分别测定肿瘤坏死因子(TNF)-α、白介素(IL)-6、TNF-αmRNA、NF-κB和IκB蛋白,TNF-α和IL-6用酶联免疫吸附法(ELISA)分析,TNF-αmRNA…     

核因子-κB特异性的RNA干扰腺病毒的构建及其对枯否细胞内毒素反应的影响

目的 构建针对大鼠核因子(NF)-κB的腺病毒,采用RNA干扰方法观察其对内毒素诱导的大鼠肝枯否细胞(KC)NF-κB抑制和肿瘤坏死因子(TNF)-α、白细胞介素(IL)-6释放的影响.方法 合成双链寡核苷酸siNF-κB克隆人pShuttleH1载体,取0.5μg线性化后的pShuttleH1-siNF-κB电转化BJ5183感受态细菌获取重组腺病毒骨架质粒;取线性化的重组质粒4μg利用Lipo-fectamineTM2000转染骨架质粒至HEK 293细胞获取重组腺病毒Ad-siNF-κB;TCID50法测定病毒滴度;腺病毒以MOI=10感染原代培养大鼠肝枯否细胞,观察对内毒素诱导的NF-κB抑制和TNF-α、IL-6释放的影响.结果 目的 基因成功克隆入腺病毒,病毒滴度为5.32×109pfu/ml.转染腺病毒Ad-siNF-κB后的内毒素诱导的肝KC及NF-κB mRNA及TNF-α、IL-6的表达均明显减弱.结论 成功构建表达NF-κB siRNA的腺病毒,具有良好的抑制NF-κB和TNF-α、IL-6的作用.     

急性坏死性胰腺炎肠黏膜NFκB介导的细胞因子过度表达及生长激素的作用

目的　本研究旨在动态观察急性坏死性胰腺炎 (ANP)大鼠肠黏膜核因子κB(NFκB)及其介导的细胞因子 ,包括肿瘤坏死因子α(TNFα)、白介素 1(IL 1β)、诱导型一氧化氮合成酶 (iNOS)、细胞间黏附分子 (ICAM 1)和单核细胞趋化蛋白 (MCP 1)mRNA的变化 ,探讨其在ANP并发肠道衰竭发生中的作用 ;并观察生长激素 (GH)对NFκB活化及细胞因子表达的下调作用。方法　SD大鼠72只 ,随机分为三组 :假手术组 (SO) ;ANP组 ;ANP +GH组。GH治疗组术后皮下注射GH溶液(0 75U/kg体重 ) ,非治疗组则注射等容积生理盐水作对照。大鼠胆胰管内逆行注射 5 %牛磺胆酸钠溶液制备ANP模型。提取肠组织RNA ,逆转录聚合酶链反应 (RT PCR)研究术后 6,12 ,2 4h肠黏膜TNFα、IL 1β、iNOS、ICAM 1、MCP 1mRNA表达。术后 3 ,6,12 ,2 4h免疫组化法检测肠黏膜NFκB的活化情况。结果　ANP组大鼠肠黏膜TNFα、IL 1β、iNOS、ICAM 1和MCP 1mRNA表达较SO组增高 ,其中TNFα和IL 1βmRNA表达于术后 6h达峰值 ,且较SO组差异显著 (TNFα :1 0 3± 0 17vs0 3 4± 0 0 7;IL 1β :0 91± 0 0 8vs 0 3 9± 0 0 6,P <0 0 5 ) ;iNOS和ICAM 1mRNA表达于术后 12h达峰值 ,较SO组差异显著 (iNOS :0 62± 0 10vs 0 0 4± 0 0 2 ;ICAM 1:1 48± 0 3 3v     

枯否细胞NF-κB 激活对胆源性内毒素血症大鼠肝部分切除后肝细胞再生的影响

目的探讨胆源性内毒素血症(BE)大鼠肝部分切除(PH)后枯否细胞(KCs)核因子-κB(NF-κB))激活对肝细胞再生的影响.方法将Wistar大鼠分为4组(每组72只)N-PH组(正常大鼠70%PH组);BE-PH组(BE大鼠70%PH组);BE-PH 白细胞介素(IL)-10治疗组;BE-PH治疗对照组.检测70%PH后0、1、6、24、48、72h KCs NF-κB 激活、KCs肿瘤坏死因子α(TNFα)mRNA、IL-1βmRNA和IL-6 mRNA表达以及肝细胞溴脱氧核苷尿嘧啶(BrdU)标记.结果 BE-PH组KCs NF-κB活性高于N-PH组(P<0.001),KCs TNFαmRNA、IL-1βmRNA及IL-6 mRNA表达亦明显高于N-PH组,而肝细胞BrdU高峰标记指数(38.82±9.79)低于N-PH组(64.37±13.69)(P<0.01);BE-PHIL-10组KCs NF-κB 活性低于BE-PH组(P<0.01),KCs TNFα、IL-1β及IL-6 mRNA表达减少,而肝细胞BrdU高峰标记指数高于BE-PH组(P<0.05).结论 BE-PH后KCs NF-κB 高水平激活导致KCs TNFαmRNA、IL-1βmRNA及IL-6 mRNA表达增高,从而抑制肝细胞再生,适当调控KCs NF-κB 活性能促进BE-PH后肝细胞再生.     

Kupffer细胞CD14及Toll样受体4介导大鼠肝移植缺血再灌注损伤的机制

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14和Toll样受体4(TLR4)的表达及其参与缺血再灌注损伤的机制。方法建立肝移植缺血再灌注模型,并分为正常对照组、缺血再灌注组、抗CD14抗体组,每组均为10只大鼠。分离培养大鼠肝移植缺血再灌注后的Kupffer细胞。检测Kupffer细胞CD14及TLR4的mRNA、蛋白表达、核转录因子κB(NFκB)活性以及培养上清TNFα的分泌量。结果再灌注后Kupffer细胞CD14及TLR4的mRNA和蛋白表达明显高于正常对照组(P<001),再灌注后核转录因子κB活性、培养上清TNFα表达量明显高于对照组(P<001)。用抗CD14抗体后NFκB活性,TNFα表达量明显下降(与再灌注组相比,P<005),但仍然高于对照组(P<001)。结论缺血再灌注后肠道内毒素(脂多糖)能够上调Kupffer细胞CD14及TLR4的表达,激活NFκB,启动细胞因子的转录和分泌,但除CD14和TLR4以外的其他信号途径参与了缺血再灌注损伤。     

联合应用甘氨酸和甲强龙对失血性休克大鼠肝脏枯否细胞的影响

目的探讨失血性休克大鼠肝脏枯否细胞的功能变化以及联合应用甘氨酸和甲强龙对枯否细胞的影响。方法50只大鼠随机分成假休克组（仅进行手术操作但不放血诱导休克）、休克组、甘氨酸组、甲强龙组和联合治疗组（甘氨酸＋甲强龙）,每组10只。大鼠经动脉放血,造成失血性休克,随后用自体血和生理盐水回输进行复苏。复苏后2h,行肝脏枯否细胞的分离和培养,细胞培养24h后分别用1、10、100和1000ng/ml的脂多糖（LPS）刺激,测定细胞内Ca^2＋和肿瘤坏死因子α（TNF-α）水平。结果枯否细胞内Ca^2＋在20min左右开始大幅度增高,大约27min达到高峰,同时细胞内Ca^2＋浓度增加呈现LPS剂量依赖性。联合治疗组细胞内Ca^2＋浓度和TNF-a含量明显低于休克组以及低于甘氨酸、甲强龙治疗组,差异具有统计学意义（P〈0．005）。结论联合应用甘氨酸和甲强龙比单一制剂更有效抑制失血性休克后枯否细胞内Ca^2＋升高、抑制枯否细胞的激活,抑制TNF-α的过度产生和机体炎症反应。     

氯化钆抑制枯否细胞对大鼠肝脏缺血再灌注损伤的影响

目的探讨抑制枯否细胞对大鼠肝脏缺血再灌注损伤的影响。方法制作部分肝脏缺血再灌注大鼠模型80只,实验组注射氯化钆,对照组注射生理盐水,检测两组大鼠缺血前、再灌注后5min、1和6h血压、心率的变化,血清转氨酶(AST)、肿瘤坏死因子α(TNFα)和白细胞介素1(IL1)的水平及肝组织超微结构的改变。结果实验组再灌注6h血清TNFα和IL1为(0.475±0.069)μg/L和(0.221±0.056)μg/L,显著低于对照组的(0.831±0.167)μg/L和(0.335±0.127)μg/L(P<0.05),两组血压、心率和AST变化的差异也有统计学意义(P<0.05),实验组大鼠肝脏超微结构的损伤程度轻于对照组。结论抑制枯否细胞活化可减轻肝脏缺血再灌注损伤,枯否细胞在肝脏缺血再灌注损伤中的作用很重要。     

核因子κB圈套寡核苷酸减轻大鼠移植肝缺血/再灌注损伤的作用和机制

目的探讨抑制枯否（Kupffer）细胞核因子κB（Nuclearfactor-kappaB，NF-κB）活性对减轻大鼠移植肝缺血／再灌注损伤（IRI）的作用和机制。方法建立大鼠肝移植缺血／再灌注损伤模型。实验分正常对照组、缺血／再灌注组和圈套寡核苷酸组，每组均为8只大鼠。圈套寡核苷酸组于移植术前2d经供者尾静脉注入120μg脂质体包裹的NF-κB圈套寡核苷酸。移植再灌注后2h，取各组受者移植肝分离枯否细胞。凝胶迁移变动分析法（EMSA）检测枯否细胞NF-κB蛋白结合活性，逆转录聚合酶链法（RT—PCR）观察枯否细胞肿瘤坏死因子α（TNF—α）和白细胞介素6（IL-6）mRNA的表达，同时观察肝组织病理及肝功能变化。结果缺血／再灌注组移植肝再灌注后2h，枯否细胞NF-κB活性及TNF-α、IL-6 mRNA表达量较对照组明显升高（P〈0．01）。光镜下肝细胞大量变性、坏死，伴有肝血窦明显淤血，血清丙氨酸转氨酶（ALT）和胆红素总量（TBIL）较对照组明显升高（P〈0．01）。相反，圈套寡核苷酸组枯否细胞NF-κB活性及细胞因子mRNA表达与缺血／再灌注组相比明显下降（P〈0．01），移植肝未见明显病理组织学改变，肝功能明显改善。结论NF-κB圈套寡核苷酸能高效抑制枯否细胞NF-κB活性，并抑制其下游有害细胞因子的产生，从而减轻缺血／再灌注损伤对移植肝的打击和损害。     

甘氨酸对出血性休克大白鼠生存率的影响

目的　研究甘氨酸对出血性休克大白鼠生存率的影响并探讨机制。方法　大白鼠经动脉放血 ,造成出血性休克 ,随后用自体血和生理盐水回输进行复苏。复苏前大白鼠分成 3组 :假休克组 ,休克组和甘氨酸治疗组。结果　 (1)复苏后 72h ,休克组与甘氨酸治疗组的生存率分别为 2 0 0 %和 77 8% ,差异有显著意义 (P <0 0 5 )。 (2 )复苏后 18h器官取材显示 :休克组大白鼠的肺、肾等组织出现水肿、变性、炎性细胞浸润等 ,甘氨酸治疗组上述的病理改变明显减轻。 (3)复苏后 18h ,肌酸磷酸激酶、肌酐、谷丙转氨酶、谷草转氨酶、碱性磷酸酶检测 ,休克组明显升高 ,而甘氨酸治疗组只有轻度增高 ,差异有非常显著性意义 (P <0 0 1)。 (4)复苏后 2h ,肝脏分离培养的枯否细胞用内毒素刺激后 ,休克组细胞内Ca2 浓度和TNF α产量明显高于甘氨酸治疗组 ,差异有非常显著性意义 (P <0 0 1)。结论　甘氨酸通过抑制枯否细胞内Ca2 浓度升高和抑制TNF α的过度产生 ,降低机体全身炎症反应程度 ,减轻出血性休克大白鼠脏器的损伤和降低病死率。     

肿瘤坏死因子-α、Fas/FasL在急性胰腺炎肝细胞凋亡中的作用

目的 观察大鼠肝枯否细胞(KCs)产生的炎症因子肿瘤坏死因子(TNF)-α、Fas/FasL和核因子(NF)-κB在重症急性胰腺炎(SAP)肝细胞凋亡中的作用,并探讨其与全身炎症反应综合征(SIRS)之间的关系.方法 将50只SD大鼠随机分成3组:(1)假手术对照(SO)组;(2)SAP组;(3)SAP(枯否细胞抑制)组.SAP模型通过胰胆管逆行注射5%牛磺胆酸钠诱导.假手术及造模大鼠均24 h后处死,无菌条件下取肝脏组织,制作石蜡切片,免疫组织化学SP法检测TNF-α、Fas/FasL的表达,原位杂交法检测NF-κB的表达,TUNEL法检测肝细胞凋亡.结果 (1)TNF-α、Fas/FasL和NF-κB在SAP组高表达,阳性率分别为90.0%、85.0%和80.0%,与SO组比较差异均有统计学意义(P<0.05);SAP枯否细胞抑制组它们表达下降,与SAP组比较差异有统计学意义(P<0.05),与SO组比仍然高表达(P<0.05).(2)NF-κB与Fas/FasL及TNF-α的表达均密切相关,相关系数分别为r1=0.78(P<0.05),r2=0.88(P<0.05).(3)肝细胞的凋亡与TNF-α、Fas/FasL和NF-κB的表达呈正相关,相关系数分别为ra=0.72,rb=0.91,rc=0.34,差异均有统计学意义(P<0.05).结论 在SAP时TNF-α能造成肝细胞凋亡并能引起全身炎症反应,NF-κB在SAP时细胞因子基因表达中起中心作用,Fas/FasL在肝细胞凋亡中起重要作用;枯否细胞在SAP时释放炎症因子造成肝损伤引起肝细胞凋亡,抑制枯否细胞能减轻SAP时肝细胞凋亡,从而降低SAP病死率,提高SAP的治愈率.     

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells

BACKGROUND: Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. METHODS: An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay. RESULTS: Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively). CONCLUSIONS: Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.     

核因子κB诱骗寡核苷酸对大鼠肝脏的Kupffer细胞共刺激分子表达的影响

目的 探讨核因子κB(NF-κB)诱骗寡核苷酸(ODN)对大鼠肝脏Kupffer细胞(KC)膜表面共刺激分子表达的影响.方法 取雄性SD大鼠,麻醉后经门静脉插管,以0.5 g/L Ⅳ型胶原酶体外循环灌注消化肝脏,不连续Percoll密度梯度离心分离KC.以人工合成的NF-κB诱骗ODN转染KC,再以终浓度为1 mg/L的脂多糖(LPS)刺激(NF-κB诱骗组),培养8 h后,采用逆转录聚合酶链反应测定KC膜表面CD80、CD40和CD54 mRNA的表达.以不转染NF-κB诱骗ODN、但接受LPS刺激的KC为对照(LPS刺激组),正常大鼠KC为对照组.每组每份标本含KC 1×105个.结果 每个大鼠肝脏可以分离得到KC(3～4)×106个,NF-κB诱骗ODN可以在KC内维持12 d而不被降解.对照组KC膜表面CD80、CD40、CD54 mRNA呈低表达;LPS刺激组KC表面3种共刺激分子表达均较对照组明显升高,分别是对照组的2.53、2.51和2.74倍(P<0.01);NF-κB诱骗组CD80和CD40 mRNA表达明显低于LPS组,仅为LPS组的0.52和0.55倍(P<0.01),而CD54 mRNA表达的差异无统计学意义(P>0.05).结论 在体外,NF-κB诱骗ODN可以高效地抑制KC表面共刺激分子的表达,为活体内应用NF-κB诱骗ODN抑制肝移植后急性排斥反应提供了实验依据.     

p38丝裂原活化蛋白激酶信号转导通路在严重烧伤大鼠枯否细胞肿瘤坏死因子α和白细胞介素1β产生中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)信号转导通路在严重烧伤大鼠枯否细胞(KCs)促炎性细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)产生中的作用。方法 健康成年的雄性SD大鼠32只,随机分为：假烫组;假烫 SB203580组;烧伤对照组;烧伤 SB203580组,每组8只。假烫或烧伤24h后分离出肝脏KCs,培养18h后加入50ng／ml的LPS进行刺激,18h后取上清液,用酶联免疫吸附法(ELISA)测定TNF-α和IL-1β的含量,并收集KCs,实时逆转录聚合酶链反应检测KCs内TNF-α和IL-1β mRNA表达的改变,蛋白印迹(Western blot)法检测KCs中p38MAPK和JNK活性的变化。结果 烧伤大鼠分离出的KCs培养上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达均较假烫组的明显增强,同时KCs中p38 MAPK活性和JNK活性升高,SB203580能显著抑制大鼠KCs上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达和p38MAPK活性的升高,对JNK活性无影响。结论p38MAPK信号转导通路介导了严重烧伤大鼠KCs促炎性细胞因子TNF-α和IL-1β的产生。     

Endotoxin tolerance attenuates liver ischemia/reperfusion injury by down-regulation of interleukin-1 receptor-associated kinase 4 in kupffer cells

Aim

The aim of this study was to study the role of interleukin-1 receptor-associated kinase 4 (IRAK-4) in the formation of endotoxin tolerance (ET) in liver ischemia/reperfusion (I/R) injury.

Methods

Animals were randomly divided into 3 groups: control group, I/R group, and ET group. Liver morphological changes were observed using optical microscopy with hematoxylin eosin (HE) staining. Alanine aminotransferase (ALT) was quantified to measure liver functional injury. The messenger RNA (mRNA) and protein expressions of IRAK-4 in Kupffer cells (KCs) isolated from recipients were detected using real-time polymerase chain reaction (PCR) and Western blot, respectively. The activities of NF-κB and the supernatant levels of tumor necrosis factor-alpha (TNF-α), IL-10 were assayed using enzyme-linked immunosorbent assay (ELISA).

Results

Endotoxin preconditioning improved hepatic tissue injury as indicated by morphological analysis, whereas serum ALT levels were significantly decreased at various times (P < .05); concurrently, the expression of IRAK-4 and TNF-α in KCs was down-regulated (P < .05) and the secretion of IL-10 was enhanced (P < .05); NF-κB DNA-binding activity of KCs was also significantly inhibited by endotoxin preconditioning (P < .05).

Conclusion

Endotoxin preconditioning attenuated the liver I/R injury caused by transplantation. The expression of IRAK-4 in KCs may play an important role in the formation of ET.     

镧对内毒素/脂多糖诱导的巨噬细胞核因子κB活化的抑制作用

目的 了解稀土元素镧对内毒素／脂多糖（LPS）诱导的小鼠腹腔巨噬细胞（Mφ）核因子KB（NF-κB）活化的影响,并探讨其机制。方法分离培养小鼠腹腔Mφ,分为：空白对照组,无刺激因素：LPS组,用1μg／mlLPS刺激30min;镧离子（La^3＋）组,用2,5μmol／L La^3＋刺激30min;La^3＋＋LPS组,先后用2,5-μmol／L La^3＋、1μg／ml LPS各刺激30min;La^3＋／L PS组,2．5μmol／L La^3＋刺激30min后,洗涤细胞,再加入1μg／ml LPS刺激30min。采用细胞免疫荧光染色法观察NF-κB在各组细胞中的分布与表达;酶联免疫吸附测定（ELISA）法检测胞核中NF-κB／p65蛋白活性;蛋白质印迹法检测细胞核内NF-κB／p65蛋白及胞质中核因子抑制蛋白α（IκBα）的表达量。ELISA法测定各组细胞培养上清液中肿瘤坏死因子α（TNF-α）的含量。结果 （1）免疫荧光染色显示,空白对照组、La^3＋组、La^3＋＋LPS组和La^3＋／LPS组M小绿色荧光多分布于胞质,胞核荧光强度分别为42±7、73±30、48±11和67±19;LPS组绿色荧光集中于胞核,荧光强度为116±14,明显高于其余4组（P〈0.01）。（2）胞核NF-κB／p65蛋白活性：LPS组吸光度值为0．435±0．066,与空白对照组（0．048±0．027）、La^3＋组（0．062±0．049）、La^3＋＋LPS组（0.066±0.031）、La^3＋／LPS组（0．108±0.017）比较．明显偏高（P〈0．01）。（3）LPS组M由胞核NF-κB／p65蛋白表达水平明显高于其余4组,胞质IκBα蛋白表达水平明显低于其余4组。（4）TNF-α含量：La^3＋组培养上清液中TNF-α含量低于试剂盒检测下限（25pg／m1）及空白对照组（P〈0.05）,La^3＋＋LPS组和La^3＋／LPS组低于LPS组（P〈0．01）,但高于空白对照组。结论 LPS可激活小鼠腹腔M由NF-κB／p65核移位,并使胞核中NF-κB／p65的表达量和活性升高、胞质IκBα蛋白表达下调,导致TNF-α分泌增多。镧可抑制上述效应。     

脂质体介导NF-κB圈套寡核苷酸体外转染Kupffer细胞的实验研究

目的 研究核因子κB(NF-κB)圈套寡核苷酸(decoy oligodeoxyribonucleotide,ODN)体外借助阳离子脂质体转染大鼠Kupffer细胞(KCs)的效率,并观察其对KCs活化的抑制作用.方法 24只Wistar大鼠分为3组,每组8只.(1)对照组:分离培养正常大鼠KCs;(2)LPs组:KCs培养液中加入终浓度为1 ms/L的LPS作为刺激因素;(3)NF-κB圈套寡核苷酸组:先用阳离子脂质体(lipofectamine)将人工合成的NF-κB圈套寡核苷酸转染KCs(4μg/1×105个KCs),观察转染效果,再用1 mg/L LPS刺激,观察其对KCs活化的抑制作用,包括KCs的吞噬功能、NF-κB易位,以及膜表面分子CD4O mRNA的表达.结果 在LPS刺激下,KCs呈高度活化状态,吞噬功能增强,NF-κB向细胞核移位,细胞膜表面共刺激分子CD40 mRNA呈高表达状态,与对照组相比差异有统计学意义(t=4.01,P<0.01).在阳离子脂质体介导下,人工合成的NF-κB圈套ODN可以高效转染KCs,有效地抑制NF-κB活化,降低其下游基因的表达,与LPS组相比差异有统计学意义(t=4.89,P<0.01).结论 在脂质体介导下,NF-κB圈套寡核苷酸可以高效转染KCs,并有效抑制KCs的活化.     

Ketamine suppresses endotoxin-induced NF-kappaB activation and cytokines production in the intestine

BACKGROUND: Ketamine has been advocated for anesthesia in endotoxemic and other severely ill patients because it is a cardiovascular stimulant. However, ketamine also suppresses serum levels of endotoxin-induced tumor necrosis factor-alpha, and reduces mortality in mice in endotoxin shock. Our study was designed to investigate the protective effect of ketamine on the endotoxin-induced proinflammatory cytokines and nuclear factor kappa B (NF-kappaB) activation in vivo. METHODS: Adult male Wistar rats were randomly divided into six groups: saline controls; rats challenged with endotoxin (5 mg kg(-1)) and treated with saline; challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (0.5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (5 mg kg(-1)); challenged with endotoxin (5 mg kg(-1)) and treated with ketamine (50 mg kg(-1)); and saline injected and treated with ketamine (50 mg kg(-1)). TNF-alpha, IL-6 and NF-kappaB were investigated in the tissues of the intestine (jejunum) after 1, 4 and 6 h. RESULTS: Endotoxin caused transient production of TNF-alpha and IL-6 and activation of NF-kappaB in the intestine at peak times of 1, 4 and 1 h, respectively. Ketamine 0.5 mg kg(-1) suppressed endotoxin-induced TNF-alpha elevation and inhibited NF-kappaB activation in the intestine; a dose of 5 mg kg(-1) was required to inhibit IL-6. CONCLUSION: Ketamine suppresses the production of proinflammatory cytokines such as TNF-alpha and IL-6 in the intestine, possibly via inhibition of NF-kappaB.     

Role of Kupffer cells in lung injury in rats administered endotoxin 1

The purpose of this study was to investigate the regulation of lung macrophages (Muvarphis) by Kupffer cells (KCs) in lung injury caused by endotoxemia. Phenotypic differences in tissue Muvarphis were also investigated. Muvarphis were isolated from gadolinium chloride (GdCl(3))- or saline-treated rats 2 h after saline or lipopolysaccharide (LPS) administration. Furthermore, rats were given GdCl(3) 24 h prior to LPS administration, and survival rate was assessed for 24 h. Moreover, lung edema was assessed 9 h after LPS injection. Expression of inflammatory mediators was measured in the liver and lung. KCs were divided into three subpopulations based on size and phagocytosis. The expression of TNF-alpha and MIP-2 was greater in the small KCs and lung Muvarphis, while the expression of IL-6, IL-10, and MCP-1 was greater in the large and intermediate KCs. GdCl(3) eliminated ED2-positive large KCs and did not have any effect on the lung Muvarphis. The number of ED1-positive KCs increased significantly in both organs after LPS challenge and was reduced by GdCl(3). The population of ED2-positive KCs did not change following LPS administration. GdCl(3) completely prevented increases in lung microvascular permeability and mortality after LPS infusion. After LPS administration, expression of TNF-alpha and IL-6 increased rapidly and then decreased gradually in both organs. GdCl(3) inhibited these increases in the liver significantly and enhanced the expression of MCP-1 and IL-10 in the lung 9 h after LPS administration. Thus, the heterogeneous response of KCs to endotoxin leads to production of certain cytokines and chemokines that affect lung function.