Endotoxin tolerance from lipopolysaccharide pretreatment induces nuclear factor-kappaB alterations not present in C3H/HeJ mice

BACKGROUND: Lipopolysaccharide (LPS) activation of macrophage (MO) cytokine secretion requires activation and translocation of nuclear factor-kappaB (NF-kappaB). Endotoxin tolerance induced in LPS-responsive C3H/HeN MOs by LPS pretreatment results in decreased tumor necrosis factor (TNF) secretion and altered NF-kappaB activation. C3H/HeJ MOs have a genetic defect that renders them tolerant to LPS activation. We hypothesized that the alterations of NF-kappaB activation seen with LPS tolerance in HeN MOs would be present in HeJ mice. METHODS: MOs from C3H/HeJ and C3H/HeN mice were cultured with +/- 10 ng/mL LPS pretreatment for 24 hours and then stimulated with 1 to 1,000 ng/mL LPS. Activation of NF-kappaB was assayed by gel shift using a 32P-labeled specific oligonucleotide 30 minutes after LPS activation. TNF secretion 6 hours after LPS stimulation was measured by bioassay. RESULTS: LPS stimulation activated NF-kappaB in both HeN and HeJ MOs. We observed decreased NF-kappaB activation and a characteristic mobility shift in endotoxin-tolerant MOs from HeN mice that were not present in HeJ MOs. In contrast with the results in HeN mice, LPS pretreatment did not induce any alterations in NF-kappaB activation in HeJ MOs. LPS-stimulated TNF secretion was decreased in HeN MOs after LPS pretreatment. There was no change in TNF secretion in HeJ MOs, but, overall, TNF secretion by these cells was much less than that seen in HeN cells. CONCLUSION: MOs from C3H/HeN mice rendered LPS-tolerant by low-dose LPS pretreatment have alterations in activation of NF-kappaB not present in LPS-hyporesponsive C3H/HeJ mice.     

Induction of CD14 in tubular epithelial cells during kidney disease

Analysis of gene expression in a mouse model of unilateral ureteral obstruction (UUO) revealed significant induction of CD14 mRNA in kidneys with obstructed ureters. Immunocytochemical analysis indicated that CD14 was upregulated in tubular epithelial cells and this upregulation was not attributable to infiltration of the kidneys by mononuclear cells. This induction of CD14 mRNA was found to occur in BALB/C, C57BL/6, C3H/HeN, and C3H/HeJ mice during UUO. Ischemia/reperfusion of kidneys also induced CD14 mRNA. Mice lacking either of the tumor necrosis factor-alpha receptor (TNFR) genes were also studied; the induction of CD14 was blunted in TNFR 1-knockout mice but not in TNFR2-knockout mice. Apoptosis of tubular cells in lipopolysaccharide-resistant CH3/HeJ mice was significantly (P: < 0. 05) less than that in lipopolysaccharide-responsive CH3/HeN mice during UUO. These results suggest that CD14 is acutely induced in tubular epithelial cells in two mouse models of renal injury. This induction is regulated by tumor necrosis factor-alpha, through TNFR1. CD14 may participate in the apoptosis of tubular epithelial cells on a more chronic basis by activating a pathway that is absent or deficient in C3H/HeJ mice.     

Macrophage antigen presentation and interleukin 1 production after cecal ligation and puncture in C3H/HeN and C3H/HeJ mice

Following cecal ligation and puncture with a 25-gauge needle, endotoxin-sensitive C3H/HeN mice have a 45% mortality compared with no mortality in endotoxin-resistant C2H/HeJ mice. Macrophage production of interleukin 1 and antigen presentation were studied in these two strains of mice following cecal ligation and puncture at 2, 4, 8, 16, and 24 hours and at 2, 4, 6, and 8 days. Splenic macrophages were cultured with a T-helper cell clone (D10.G4.1), and antigen presentation and interleukin 1 production were measured by D10.G4.1 proliferation. Macrophage antigen presentation by C2H/HeJ mice was markedly increased compared with that in C3H/HeN mice at all times after cecal ligation and puncture, most strikingly at 2 days (185m740 cpm for C3H/HeJ mice vs 30,300 for C2H/HeN mice). Macrophage interleukin 1 production was significantly increased in C3H/HeJ mice vs C3H/HeN mice at all times after cecal ligation and puncture (except at 2 days) and was maximal at 8 days (25,000 cpm for C3H/HeJ mice vs 5190 for C3H/HeN mice). These data suggest that the differences in mortality after cecal ligation and puncture between these two strains of mice may relate to a supranormal response of macrophages of C3H/HeJ mice or to an inadequate response of macrophages of C3H/HeN mice.     

Effect of TLR-4 and HO-1 on acute lung injury induced by hemorrhagic shock in mice

Objective： To examine whether TLR-4 has an ettect on hemorrhage induced changes in lung, and to investigate the change of heme oxygenase-1 （HO-1） on acute lung injury （ALl） induced by hemorrhagic shock in mice.
Methods： Forty-eight male mice, including C3H/HeN mice and C3H/HeJ mice, were randomly divided into sham group （n=12）, hemorrhagic shock group with twelve mice in each phase. Blood pressure （BP） was monitored continuously by attaching carotid artery catheter to a strain gauge pressure transducer/ polygraph. Arterial blood samples were taken for blood gas analysis. A mouse model of non-lethal hemorrhagic shock and resuscitation was used to observe pulmonary myeloperoxidase （MPO） activity and wet/dry weight ratio （W/D）. The expression of HO- 1 was observed by means of RT-PCR and immunohistochemistry. IL-6 and IL-10 in lung tissue homogenate were assayed by enzyme-linked immunosorbent assay （ELISA）. The pulmo- nary pathologic changes were observed under electron microscope and light microscope.
Results： Compared with sham group, the expression of HO- 1 in lung tissue was significantly higher in Hem 24 h and Hem 48 h of C3H/HeN mice （P〈0.01）. The expression of HO-1 mRNA and the levels of IL-6, IL-10 and MPO in lung tissue were markedly increased in Hem 24 h （P〈0.01 or P〈0.05）; Compared with C3H/HeN mice, the expression of HO- 1 rnRNA and the levels of IL-6 and IL-10 in C3H/HeJ mice significantly decreased in Hem 24 h and Hem 48 h （P〈0.01 or P〈O.05）, and the W/D, MPO in C3H/HeJ mice were obvi- ously lower in Hem 24 h （P〈0.05）. The injuries of lung tissues after hemorrhagic shock have been demonstrated by histological examination with electron microscope and light microscope.
Conclusions： TLR-4 and HO-1 might modulate the bal- ance of pro- and anti-inflammatory processes in inflamma- tory reaction of hemorrhagic shock-induced ALl, and the activation of Toll-like receptor might induce the transcrip- tion activity of HO- 1, which may play a k     

Outcome following femur fracture and subsequent cecal ligation and puncture in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (C3H/HeJ) mice

This study examined the effect of sepsis following trauma in a reproducible model of sepsis--cecal ligation and puncture (CLP)--in endotoxin-sensitive (C3H/HeN) and endotoxin-resistant (CeH/HeJ) mice. Studies used CLP with a 25-gauge needle at different time intervals following injury, as induced by femur fracture (FF), to determine the effects of sublethal sepsis on survival after trauma. There was a 3% mortality for FF alone in both groups. Mortality in C3H/HeJ mice was not significantly increased over FF alone except when CLP followed FF by 3 days (45%, P less than 0.02, Chi-square). In contrast, C3H/HeN mice had significantly increased mortality rates (75 to 90%, P less than 0.001) versus FF alone at all intervals between FF and CLP. Mortality for FF plus CLP was significantly greater for C3H/HeN compared to C3H/HeJ (P less than 0.001) for all time intervals between FF and CLP. In conclusion, animals exposed to a septic episode following FF had significantly greater mortality than FF animals without a septic challenge. Endotoxin-sensitive mice had significantly higher mortality after CLP and significantly increased mortality when CLP followed FF (regardless of timing) compared to endotoxin-resistant mice.     

Effects of hypoxia-reoxygenation on isolated liver nonparenchymal cells

To clarify the relationship between Kupffer cells (KC) and hepatocytes in hepatic ischemia-reperfusion injury, isolated liver nonparenchymal cells (NPC), including a large proportion of KC, from C3H/HeN and C3H/HeJ mice were utilized in an in vitro hypoxia-reoxygenation system which enabled precise control of the oxygen concentration in the circumferential air during incubation. The viability of NPC, concentrations of cytokines and superoxides released from NPC, and the effects of antioxidants on hypoxiareoxygenation were investigated. The results were: (1) The deterioration of NPC was slow under hypoxia, but a significant decrease in their viability was observed with reoxygenation. These results were virtually identical in C3H/HeN and C3H/HeJ mice. (2) The concentration of tumor necrosis factor (TNF)-α from NPC of C3H/HeJ was much lower than that from NPC of C3H/HeN, and production of O ₂ ⁻ was lower in the NPC of C3H/HeJ than in C3H/HeN, but changes in the viability of NPCs which were reoxygenated following various intervals of hypoxia were almost identical in both types of mice. (3) Viability after reoxygenation was improved by the addition of catalase or superoxide dismutase (SOD), and further improved with the combined use of catalase and SOD. These results suggested that the changes in KC function after reoxygenation decreased the viability of the cells, due predominantly to the release of reactive oxygen metabolites, not to the release of TNF-α.     

Toll样受体4在失血性休克复苏致小鼠急性肺损伤中的作用

目的 探讨Toll样受体4(TLR4)在失血性休克复苏致小鼠急性肺损伤中的作用.方法 TLR4基因突变型C3H/HeJ小鼠和野生型C3H/HeN小鼠各24只,两种品系小鼠各随机分为2组:假手术组(S组,n=6)、失血性休克复苏组(HSR组,n=18)制备失血性休克复苏模型,并于复苏后6、24、48 h时各取6只小鼠颈动脉放血处死后开胸取肺组织,免疫组织化学法检测p38 MAPK表达水平,酶联免疫吸附法测定白细胞介素(IL)-10和IL-6含量,透射电镜下观察肺组织超微结构.结果 与C3H/HeN小鼠比较,C3H/HeJ小鼠复苏后肺组织p38 MAPK表达下调,IL-6和IL-10含量降低(P<0.05或0.01),病理损伤程度减轻.两种品系小鼠中,与S组比较,HSR组复苏后24 h时肺组织IL-6和IL-10含量增加,复苏后48 h时肺组织IL-6含量增加(P<0.05或0.01);C3H/HeN小鼠中,与S组比较,HSR组复苏后6 h和24 h时肺组织p38 MAPK蛋白表达上调,复苏后48 h时肺组织IL-10含量增加(P<0.01).结论 TLR4参与小鼠失血性休克复苏致急性肺损伤的发生,其机制与激活p38 MAPK信号转导通路有关.     

当归对大鼠心肌缺血/再灌注损伤蛋白激酶C的影响

目的 目的研究当归抗心肌缺血／再灌注损伤的分子生物学机制。方法 结扎／开放左冠状动脉前降支结扎４０ｍｉｎ／开放１２０ｍｉｎ建立心肌缺血／再灌注模型。选用ＳＤ大鼠４０只,随机分成三组：对照组（Ａ组,ｎ＝１０只）,丝线穿过冠状动脉前降支但不结扎;缺血／再灌注组（Ｂ组,ｎ－１５只）,缺血／再灌注前舌静脉注射生理盐水０．８ｍｌ／１００ｇ。采用免疫组化ＳＰ法测定蛋白激酶Ｃ（ＰＫＣ）含量和同位素标记法测定ＰＫ     

Toll样受体4对失血性休克小鼠肺组织血红素加氧酶和诱导型一氧化氮合酶的影响

目的 观察Toll样受体4(TLR4)对失血性休克小鼠所致急性肺损伤(ALI)中肺组织血红素加氧酶-1(HO-1)和诱导型一氧化氮合酶(iNOS)的影响.方法 TLR4基因突变型小鼠C3H/HeJ和野生型小鼠C3H/HeN(48只),随机分为假手术组和失血性休克(6、24、48 h)组.采用小鼠失血性休克致急性肺损伤模型,观察血气分析,HO-1和iNOS蛋白表达,白细胞介素-6(IL-6)水平,肺组织肺湿/干重比和病理形态学改变.结果 与假手术组比较,C3H/HeN和C3H/HeJ小鼠失血休克后24 h肺组织HO-1蛋白强阳性表达,IL-6含量明显增加为365.38±48.26和300.89±39.34;6 h肺组织iNOS蛋白强阳性表达(P＜0.01).与C3H/HeN小鼠比较,C3H/HeJ小鼠失血休克后24 h肺组织HO-1、IL-6含量和W/D明显降低(P＜0.05);6 h肺组织iNOS蛋白显著减少为0.049±0.013.病理学检查显示失血休克后各时点肺组织损伤程度较假手术组明显加重.结论 TLR4在失血性休克后ALI过程中被激活,通过影响HO-1和iNOS的表达参与了失血性休克致急性肺损伤的过程.     

Cardioprotective effects of the serine protease inhibitor aprotinin after regional ischemia and reperfusion on the beating heart

OBJECTIVE: Early coronary reperfusion of the ischemic myocardium is a desired therapeutic goal to preserve myocardium. However, reperfusion itself contributes to an additional myocardial injury (ie, reperfusion injury), which has been attributed to neutrophil infiltration with subsequent release of proteases and oxygen-derived radicals. We studied the effects of the serine protease inhibitor aprotinin (Trasylol) on myocardial ischemia and reperfusion in a rat model. METHODS: The effects of aprotinin (5000 and 20,000 U/kg) were examined in vivo in a rat model of regional myocardial ischemia (20 minutes) and long-term reperfusion (24 hours). Cardioprotecive effects were determined by means of measurement of creatine kinase and myeloperoxidase activity within the myocardium, as well as histochemical analysis. RESULTS: Aprotinin (20,000 U/kg) administrated 2 minutes before reperfusion significantly attenuated myocardial injury expressed as creatine kinase washout compared with that seen in vehicle-treated rats (65 +/- 25 vs 585 +/- 98 creatine kinase difference in units per 100 mg, P <.01). Administration of 5000 U/kg of the protease inhibitor resulted in partial inhibition of myocardial reperfusion injury. Moreover, cardiac myeloperoxidase activity in the ischemic myocardium, a marker of neutrophil accumulation, was significantly reduced after aprotinin treatment. Histologic analysis of the reperfused myocardium demonstrated reduced polymorphonuclear leukocyte infiltration and reduced tissue injury. Furthermore, aprotinin treatment resulted in decreased induction of cardiac myocyte apoptosis compared with that seen in vehicle-treated rats. CONCLUSIONS: Inhibition of serine proteases with aprotinin appears to be an effective means of preserving ischemic myocardium from reperfusion injury, even after 24 hours of reperfusion. Aprotinin might exert cardioprotection through inhibition of polymorphonuclear leukocyte-induced myocardial injury and inhibition of reperfusion-induced apoptosis of cardiac myocytes.     

小鼠全肝缺血再灌注时肺泡巨噬细胞TLR2的激活与肺损伤的机制

目的：探讨肺泡巨噬细胞Toll样受体2（TLR2）的激活机制及其在肝脏缺血再灌注（HIR）中肺损伤的意义。方法：用野生型小鼠C3h/Heouj和TLR4缺失小鼠C3h/Hej建立HIR动物模型。于再灌注1，6，12h后经支气管肺泡灌洗液获取肺泡巨噬细胞，采用荧光定量PCR方法检测TLR2/4mRNA的表达。同时检测支气管肺泡灌洗液中内毒素及肿瘤坏死因子（TNF）的水平，肺组织湿干重比值，肺组织髓过氧化物酶的浓度，并进行肺组织学评分。结果：C3h/Heouj组HIR缺血再灌后各时点肺泡巨噬细胞TLR2/4mRNA表达升高，TLR2mRNA表达持续升高，TLR4mRNA6h达到最高值。同时C3h/Heouj组HIR后支气管肺泡灌洗液中TNF水平明显升高，肺损伤加重，肺组织湿干重比值持续升高，肺组织髓过氧化物酶持续增加(P<0.05)。C3h/Hej组HIR后TLR2mRNA表达仅轻度升高，且支气管肺泡灌洗液中TNF水平低于C3h/Heouj组(P<0.05)，肺损伤轻于C3h/Heouj组(P<0.05)。结论：HIR可致肺泡巨噬细胞表面TLR4的激活，可上调TLR2的表达，从而可加重HIR时的肺损伤。     

Administration of interleukin-1 receptor antagonist ameliorates renal ischemia-reperfusion injury.

Interleukin (IL)-1 is a major contributor to inflammation and apoptosis during ischemia/reperfusion (I/R) injury. Its deleterious effects are primarily mediated by the activation of nuclear factor-kappaB (NF-kappaB). Receptor-binding and signaling of IL-1 can be blocked by the IL-1 receptor antagonist (IL-1ra). The aim of our study was to characterize effects and mechanisms of IL-1ra administration on inflammation, apoptosis, and infiltration in renal I/R injury. Renal ischemia was induced in Lewis rats by clamping of the left renal artery for 45 min. Kidneys were removed for histological and molecular analysis 24 h or 5 days after reperfusion. IL-1ra ameliorated I/R induced renal injury and inflammation. Furthermore, the number of apoptotic tubular cells was lower in IL-1ra-treated animals 24 h after ischemia, which was paralleled by a Bax/Bcl-2 mRNA ratio towards anti-apoptotic effects. IL-1ra reduced the expression of monocyte chemoattractant protein-1 (MCP-1) mRNA at 24 h and 5 days and that of intracellular adhesion molecule-1 (ICAM-1) expression at 24 h in the ischemic reperfused kidneys. Our results indicate that IL-1ra treatment ameliorates renal I/R injury and this protective effect might be mediated by reduced induction of NF-kappaB mediated MCP-1, ICAM-1, and a decreased ratio between Bax and Bcl-2 mRNA expression.     

吸入麻醉药预处理对心肌缺血"第二保护窗"的作用机制

Volatile anesthetic preconditioning can elicit acute and delayed (also called as "second window") myocardial protection,and the latter may be more clinicaUy significant in decreasing the risk of myocardial ischemia/reperfusion injury for patientswith cardiovascular disease.The "second window" protection might be achieved by stimulating myocardium to trigger adenosine and nitric oxide release,passing through protein kinase C and nuclear factor-κB signal transduction pathways,activating the ATP-sensirive potassium channel and reactive oxygen species as the final effectors,presenting a delayed myocardial protective effect.This review summarizes the latest studies about volatile anesthetic preconditioning-induced "second window" protection.     

吸入麻醉药预处理对心肌缺血“第二保护窗”的作用机制

吸入麻醉药预处理对心肌缺血侑罐注损伤具有急性期和“第二保护窗”两个时间段的保护作用。“第二保护窗”起效缓慢而持久,临床上有充分的时间在手术前给予,能更方便有效地预防围术期心肌缺血的并发症。它可诱导一些触发因子如腺苷、一氧化氮的产生,通过信号转导通路蛋白激酶C、核因子-κB等,作用于ATP敏感性钾通道及活性氧族等终末效应离子通道或保护蛋白而发挥迟发性心肌保护。现就近年来关于吸入麻醉药预处理对心肌“第二保护窗”的作用机制作一综述。     

The portosystemic shunt protects liver against ischemic reperfusion injury.

BACKGROUND: The goal of this study was to characterize the importance of splanchnic viscera in liver ischemic reperfusion injury and to enhance the tolerance of liver to warm ischemia injury with portosystemic shunt. METHODS: The hepatic blood flow of male Sprague Dawley rats was subjected to 45, 60, 120, and 150 min liver warm ischemia with or without portosystemic shunt (splenic-caval shunt). The production of tumor necrosis factor a (TNFa), nuclear factor-kappaB activation, inducible NO synthase (iNOS) expression, and apoptosis were examined. RESULTS: A total of 67% of rats with 45 min liver warm ischemia (n=6) and 100% of rats with 60 min liver warm ischemia (n=6) died within 1 day. However, all rats with 120 min (n=8) liver warm ischemia in splenic-caval shunt group survived for over 1 day, 6/8 for over 3 days, and 5/8 for over 5 days without significant histological changes of the liver. Serum tumor necrosis factor levels in liver warm ischemic rats were increased, This increase was significantly reversed after portosystemic shunt. After challenge with lipopolysaccharide (1 mg/kg, p.v.), naive rats survived for over 5 days (n=4) with the peak value of rat tumor necrosis factor (240 pg/ml) at 90 min. In contrast, all rats died within one day (n=5) with the peak value of rat tumor necrosis factor a (465 pg/ml) at 45 min after administration of lipopolysaccharide in the rats with liver warm ischemia plus splenic-caval shunt. iNOS expression and nuclear factor-kappaB activation were very strongly increased in the hepatocytes after liver warm ischemia with portosystemic shunt, compared with liver ischemia without portosytemic shunt. CONCLUSIONS: We conclude that the splanchnic viscera can contribute to liver ischemic reperfusion injury. Portosystemic shunt enhances the tolerance of liver to warm ischemia through the protective role of iNOS and nuclear factor-kappaB (NF-kappaB).     

The evolution of ischemic spinal cord injury in function, cytoarchitecture, and inflammation and the effects of adenosine A2A receptor activation

OBJECTIVE: Spinal cord ischemia/reperfusion injury involves multiple factors that may be modulated by adenosine A 2A receptor activation. This study defines injury progression in terms of function, cytoarchitecture, and inflammation and assesses whether adenosine A 2A receptor activation by ATL-146e limits injury progression. METHODS: Mature swine were divided into 3 groups: sham thoracotomy, IR (30 minutes of ischemia followed by reperfusion), and ATL (ischemia/reperfusion with ATL-146e administration for the first 3 hours of reperfusion). Subgroups were killed at 0, 3, 6, 12, 24, and 48 hours after reperfusion. Function was followed up with Tarlov scores. Spinal cord tissue was evaluated for neuronal viability, microtubule-associated protein-2 immunohistochemistry, and neutrophil sequestration (myeloperoxidase assay). Spinal cord tissue, cerebrospinal fluid, and serum were evaluated for tumor necrosis factor-alpha by enzyme-linked immunosorbent assay. RESULTS: Function was significantly impaired at 24, 36, and 48 hours in the IR group compared with the sham and ATL groups ( P < .05). Neuronal viability and microtubule-associated protein-2 staining were significantly preserved in the sham and ATL groups compared with the IR group at 24 and 48 hours ( P < .05). Spinal cord myeloperoxidase levels were significantly higher in the IR group than in the sham and ATL groups at 24 and 48 hours. Although negligible in serum and cerebrospinal fluid, tumor necrosis factor-alpha levels in the spinal cord peaked significantly higher in the IR group compared with the sham and ATL groups at 6 and 24 hours ( P < .05). CONCLUSIONS: Spinal cord ischemia/reperfusion induced changes in neutrophil sequestration, microtubule-associated protein-2 expression, and neuronal viability within 24 hours of reperfusion. Spinal cord tumor necrosis factor-alpha increased significantly by 6 to 12 hours after reperfusion. Adenosine A 2A receptor activation attenuates spinal cord inflammation, which may be critical for the preservation of neuronal function and cytoarchitecture after ischemia/reperfusion.