水合氯醛保护LPS/D-GalN诱发小鼠致死性肝损伤和zymosan诱发腹膜炎及其机理研究
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摘要
急性尤其是致死性感染或非感染性炎症是临床与基础研究的重要课题,也是新药研发的重要目标。人们在化学药物、植物提取物,包括中草药中不懈地寻找新的先导化合物,同时也发现一些已经被批准的,或长期用于临床其他方面的药物具良好的抗炎活性。近年来,人们发现一些麻醉剂除了止痛及肌肉松弛作用还具有消炎和抗感染的影响,如利多卡因,氯胺酮,异氟醚和戊巴比妥可显著提高小鼠和内毒素休克大鼠存活率,防止或减轻肝脏和肾脏损伤及盲肠穿刺(CLP)所致的感染性腹膜炎。我们在前期研究中毒性休克与过敏性休克的动物实验中偶尔发现作为麻醉剂的水合氯醛影响了休克动物的血液动力学参数。而目前国内外尚无有关对水合氯醛影响急性炎症过程与转归的报道。尤其令我们感兴趣的是,水合氯醛做为一种古老而安全的临床常用的镇静剂与辅助麻醉药物已有百年的应用历史。目前仍在儿科的高热惊厥辅助治疗,在磁共振成像(MRI)和超声心电图的辅助操作中经常使用,并在许多国家被用于动物实验的麻醉。此外,正是因为水合氯醛在临床的应用并未过时,对其药理机制及安全性评价也仍被重视。也许,我们的发现与研究可为使这一古老的药物在抗炎领域展示新的用途。
在本研究中,我们采用了两个经典的动物模型,即脂多糖／D-氨基半乳糖(LPS/D-GalN)诱发的急性致死性肝损伤和酵母多糖诱发的急性非致死性腹膜炎。其中LPS/D-GalN诱发的急性致死性肝损伤模型是一种被广泛使用了20多年的实验动物模型,其机制是导致急性肝功能衰竭而死亡,被认为与人类基于内毒素血症或败血症合并肝衰竭相关,如人类全身炎症反应综合征(SIRS)在非感染状态下可出现类似败血症表现。因此,该模型被用于研究内毒素血症合并急性肝损伤的机理、治疗策略与药物筛选。我们的研究中采用的另一个经典模型是有酵母多糖诱发的腹膜炎,属相对温和的非致死性的急性炎症。在确定LPS/D-GalN模型条件时,我们选择较大剂量的LPS与D-GalN,旨在建立一个比较严重的、存活时间很短的重症肝损伤,即造成一个比较极端的状态以观察水合氯醛的干预效果；而另一个模型是非致死性炎症,以此观察水合氯醛是否对这类更接近于临床多见的非致死性炎性状态具干预作用。
在观察并确定了水合氯醛可干预两种模型动物炎症的一般表现与指标的同时,我们对其作用机制做了初步的探索,其重点放在LPS/D-GalN诱发的急性致死性肝损伤模型。主要包括促炎症细胞因子水平及核因子κappaB (NF-κB)的活性改变。参考Bozza等对临床败血性休克患者的预后相关细胞因子谱的研究,我们选择IL-6,肿瘤坏死因子α(TNF-α)和MCP-I做为检测评价指标。其中,TNF-α已被证明和LPS/D-GalN攻击小鼠后生存率密切相关。MCP-1在急性致死性炎症如败血症中是一个重要的细胞因子,可平衡促炎和抗炎反应。
在明确水合氯醛的体内保护作用的基础上,我们设计了探讨机制的体外实验。具体为用小鼠巨噬细胞系RAW264.7细胞和小鼠腹腔巨噬细胞为研究对象,观察水合氯醛体外作用对这两种细胞NF-κB炎症信号分子激活和炎症因子生成的影响。观察水合氯醛处理是否会引起靶细胞(选择RAW264.7和腹腔巨噬细胞)的凋亡,以及通过什么途径诱导靶细胞的凋亡,以及与体内应用水合氯醛干预所诱发的凋亡是否一致。
第一章水合氯醛对LPS/D-GaIN诱发致死性肝损伤和zymosan诱发腹膜炎小鼠模型的保护作用
基于本研究采用水合氯醛(chloral hydrate, CH)一次性干预模式,在短时间内发挥作用,研究模型之一首先选择较严重的急性重症炎症模型：LPS2630(10μg)/D-GalN(800mg/Kg)诱发的急性致死性肝损伤模型(中位生存时间9.20h)。用CH以不同剂量(80,160和320mg/Kg)在LPS/D-GalN攻击后不同时间点(0,1,3和5h)体内一次性干预,发现CH干预以剂量依赖的方式延长小鼠的生存时间,以160和320mg/Kg组存在显著差异(分别P=0.024,P=0.001)。同一剂量(320mg/kg)在LPS/D-GalN攻击后0,1,和3h干预与对照组比较显著延长小鼠生存时间(分别P=0.002,P=0.004和P=0.002)。干预组3h点MCP-1,IL-6和TNF-a的水平显著低于对照组,而后IL-6和TNF-a在6h点逐渐上升显著高于对照组。干预组ALT在干预后3,6和9h点,均显著低于对照组,而AST9h点显著低于对照组。肝脏充血程度在9h点干预组较对照组显著减轻,HE染色显示两组均有肝脏小叶结构破坏和严重的肝细胞坏死和出血,在6和9h点对照组显著严重于干预组。两组血清皮质酮水平无显著差异,但干预组均低于对照组。而用有效干预剂量CH(320mg/Kg)被证明处理正常小鼠是安全的。
实验结果也证实了CH(320mg/Kg)可有效抑制：zymosan A诱发的急性腹膜炎。表现为干预组腹腔渗出液白细胞总数高峰显著推迟至少16h,在5和8h点细胞数量较对照组显著减少(分别P=0.03,P=0.006)。腹腔渗出液蛋白含量结果类似,高峰值延迟约4h,尤其是在0.5和1h点两组有显著差异急性腹膜炎小鼠诱发后1,2和8h点,TNF-α,MCP-1和IL-6的水平均显著低于对照组。
本章工作证明水合氯醛(320mg/Kg)体内干预对LPS/D-GalN诱发的急性致死性肝损伤和zymosan诱发急性腹膜炎小鼠具有显著的保护作用,可以显著降低和延迟炎症相关因子的产生。
第二章水合氯醛干预对炎症信号转导分子NF-κB抑制的体内外研究
基于CH(320mg/Kg)一次性体内应用可改变LPS/D-GalN诱发致死性肝损伤和zymosan诱发腹膜炎模型小鼠血液中炎症因子TNF-α,IL-6和MCP-1的水平。推测CH干预是否与抑制炎症因子合成的关键上游控制元件NF-κB有关。为证实这一推测设计了以整体成像技术与体外转染细胞系研究NF-κB活性。
体内研究选择NF-κB转基因小鼠NF-κB-RE-RE-luc(Oslo),用LPS/D-GalN诱发急性致死性肝损伤(同前),CH(320mg/Kg)干预显著降低LPS/D-GalN诱发的NF-κB转基因小鼠NF-κB控制下的荧光素酶报告基因的表达。无干预对照组荧光素酶表达在4h点达到高峰,干预组在3,4,5和6h点均显著低于对照组(均为P=0.000)。肝脏,肠道和肺脏为主要受累器官,CH干预能显著降低受累器官荧光素酶表达(分别P=0.002,P=0.026,P=0.015)。
体外细胞模型研究采用转NF-κB荧光素酶报告质粒RAW264.7细胞和小鼠腹腔巨噬细胞。结果表明：CH(0.5和1 mg/ml)干预显著降低由LPS/LTA刺激转染NF-κB荧光素酶报告质粒RAW264.7的荧光素酶报告基因的表达活性。显著抑制LPS/LTAJ刺激腹腔巨噬细胞IL-6和TNF-α的产生。
本章通过体内外即整体与细胞水平研究发现CH干预可以显著抑制由LPS/D-GalN,LPS和LTA刺激引起的炎症信号转导分子NF-κL活性,以及下游炎症因子的生成。
第三章CH诱发单个核细胞凋亡及其机制研究
体外选择CH不同浓度(0.25,1和2 mg/ml)在体外对RAW264.7细胞和腹腔巨噬细胞进行干预。采用观察细胞形态和功能(如吞噬功能)改变,早期凋亡检测,Hochest 33258染色,DNA ladder等多种方法验证是否确实引起了靶细胞的凋亡这一关键环节。
结果显示：CH处理RAW264.7细胞形态由典型的梭形,变圆直至脱落悬浮；PI/FITC染色经流式检测显示CH诱导RAW264.7细胞从早期向晚期凋亡的转换过程,并可被典型的DNA ladder所证实；Hochest 33258染色发现核明显固缩密度增加、凝聚和断裂；而且吞噬能力较正常对照被显著抑制。CH处理小鼠腹腔巨噬细胞具有同样的诱导凋亡效果。此外,CH(320mg/Kg)干预LPS/D-GalN诱发致死性肝损伤小鼠的脾脏单个核细胞凋亡,干预组在2和3h点较对照组显著诱发了脾脏单个核细胞的早期凋亡(分别P=0.001,P=0.000)。
为阐明CH以何种方式诱导靶细胞的凋亡,用RAW264.7细胞为模型在体外研究,同时观察无干预及CH(320mg/Kg)干预的正常小鼠与肝损伤小鼠的脾脏组织切片。发现体外CH(0.5和1mg/ml)处理RAW264.7细胞12和24h均可诱发RAW264.7表达Fas,但不表达FasL;Fas在正常小鼠脾脏少量表达,水合氯醛(320mg/Kg)一次性干预9h后,其脾脏细胞Fas表达显著升高。
本章采用多种方法检测体外RAW264.7细胞和腹腔巨噬细胞,体内模型的小鼠脾脏单个核细胞以及脾脏的凋亡,证明水合氯醛在体外干预可以诱导单个核细胞从早期到晚期的典型凋亡过程,且同时高表达Fas。
统计学处理
所有数据都表示为平均值士标准差(Mean±SD)。小鼠生存时间分析采用Kaplan-Meier分析进行对照组和CH干预组比较,显著性差异用Breslow检验。相同时间点CH干预组和对照组各因素比较使用独立样本t (Independent-student T test)检验。对照组(NS)和水合氯醛干预组(CH)RAW264.7细胞荧光信号relative light unit (RLU)(fold change)比较用one-way ANOVA。处理软件均为SPSS 13.0,P<0.05认为有显著性差异。
结论
研究发现CH(320mg/Kg)干预可以保护由LPS/D-GalN攻击小鼠的急性致死性肝损伤,显著延长其生存时间；且在急性致死性肝损伤诱导后1到3个1进行干预仍然显著有效。CH(320mg/Kg)干预可以减轻zymosan A诱发的急性非致死性腹膜炎小鼠的炎症程度。CH(320mg/Kg)干预可降低和延迟以上2个急性炎症模型小鼠血清促炎细胞因子IL-6,TNF-α, MCP-1水平,降低肝损伤小鼠肝脏病理损伤程度。
采用整体成像技术证实CH(320mg/Kg)干预可以保护NF-κB转基因小鼠,抑制由LPS/D-GalN诱发的NF-κB分子的活性。体外细胞水平研究证实CH(0.5mg/ml)处理转染NF-κB荧光素酶报告质粒的RAW264.7细胞,可抑制由LPS和LTA刺激引起的胞内NF-κB活性。同时CH(0.5和1mg/ml)处理腹腔巨噬细胞,可抑制由LPS和LTA刺激诱发的IL-6和TNF-α的生成。采用多种凋亡检测方法证实CH干预可以诱导RAW264.7细胞和腹腔巨噬细胞从早期到晚期的典型凋亡过程,且凋亡的RAW264.7细胞高表达Fas；水合氯醛(320mg/Kg)一次性干预正常小鼠9h后,其脾脏细胞Fas表达显著升高。
Background
Acute or serious inflammation induced by infection or uninfection has been considered as a very important issue in clinical and the research on pathogenesis and development of new medicines. Scientists are doing great efforts to find new leads of medicine including chemical compounds, plant extracts, even combinatory protein or peptide. In addition, people found that some of the medicines which were approved being used in other fields have good anti-inflammatory activities. Recently, several anesthetics have been found to possess anti-inflammatory and anti-infective effects apart from their functions of pain relief and muscle relaxation. For example, lidocaine, ketamine, isoflurane and pentobarbital can significantly improve the survival of mice and rats with endotoxic shock and protect them against liver and renal injury resulting from cecal ligation and puncture (CLP)-induced septic peritonitis .In the previous work, we occasionally found that chloral hydrate used as an amimanl anesthetic can improve the hemodynamic parameters of mice with endotoxin shock and anaphylactic shock. Therefore, we want to know whether chloral hydrate can function as an inhibitor of inflammation, because there are no any reports about the effect of chloral hydrate on acute inflammation. What made us interested at this issue is that chloral hydrate as a sedative and anesthetic has been used in clinical for over one hundred years, and it is currently used in pediatric manipulations like magnetic resonance imaging (MRI) and echocardiogram on children and in animal experiments at many countries. Furthermore, the evaluation of pharmacologic mechanism and safety of chloral hydrate have been still emphasized in human which suggests that chloral hydrate would not be obsolete even today.
In the present study, we sought to investigate the effects of chloral hydrate on the acute lethal liver injury induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) or on the acute peritonitis, a mild inflammatory response induced by zymosan A, which have been considered as well defined animal models . Especially, LPS/D-GalN-induced acute lethal liver injury in mice is a widely used as one of experimental animal models for more than 20 years, by which the mechanisms of lethal hepatic failure and development of the effective therapeutic strategies against endotoxin challenge have been investigated, because endotoxemia or sepsis is also associated with fulminant hepatic failure in human Moreover, the manifestation of systemic inflammatory response syndrome (SIRS) in the absence of infection may occour the similar syndrome to sepsis Therefore, our work could provide useful evidence used in research of LPS/D-GalN-induced acute lethal liver injury and sepsis. Another classic model used in our work is acute peritonitis induced by zymosan A, a kind of mild non-lethal acute inflammation. The mice with lethal acute liver injury induced by LPS/D-GalN can live a short period of time and caused an extreme serious state in order to observe the protection of chloral hydrate. The protection of chloral hydrate on peritonitis, a mild non-lethal inflammation is more close to clinical.
We also intended to explore the mechanisms of the protective effects against LPS/D-GalN-induced acute lethal liver injury and peritonitis by chloral hydrate, including the profiles of proinflammatory cytokines and the change of activities of nuclear factorκappaB (NF-κB). Assessment and selection of the parameters for present work including levels of IL-6, TNF-a and MCP-1 were according to the work on prognosis-related cytokines in patients with septic shock as reported by Bozza. Among them, TNF-a has been proved to be closed associated with survival of mice challenged with LPS only or LPS/GalN and MCP-1 is an important cytokine, which can balance the proinflammatory and anti-inflammatory reactions in acute lethal inflammation like sepsis.
Based on the protection of chloral hydrate in vivo, we also selected RAW264.7 cell and mouse peritoneal macrophages as models to study how chloral hydrate affects the activity of NF-κB and the production of inflammatory factors.
PartⅠ. Protection of chloral hydrate on LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis in mice
Since chloral hydrate (CH) treatment in this work is once injection and maintaining for short period, we chose a more serious acute inflammation model induced by the intraperitoneal (i.p.)injection of 10μg LPS-2630 (Escherichia coli serotype 2630) plus 800 mg of D-galactosamine hydrochloride (D-GalN, Sigma-Aldrich, St. Louis, MO, USA)×kg-1 of body weight, which median survival time is 9.2 h. The effect with CH treatment at different doses (80,160 and 320 mg/Kg) and different time points (0,1,3 and 5 h after the challenge by LPS/D-GalN) were observed, based on the safety proved by 320 mg/Kg CH on mice in preliminary test.The results showed that CH treatment improved the median survival of mice in a dose-dependent manner, especially that in group which received 160(11.68±2.62 h) and 320 mg/Kg (13.78±1.21 h) was significantly improved compared with controls (9.17±1.10h) (P=0.024 and P=0.001),and CH administered at 0,1 and 3 h after the challenge by LPS/D-GalN significantly improved the median survival of these mice compared with controls(13.70,13.30 and 11.60h vs.9.40h, P=0.002, P=0.004 and P=0.002, respectively). CH treatment attenuated the rises of serum MCP-1,IL-6 and TNF-a levels at 3h after LPS/D-GalN challenge compared with those control group (P=0.001,P=0.009 and P=0.015,respectively). In contrast to the control group, however, the levels of IL-6 and TNF-αunderwent a sharp rise in the treatment group at 6 h after LPS/D-GalN challenge, which was significantly higher than that of the control group. The levels of ALT in mice with LPS/D-GalN-induced acute lethal liver injury remained significantly lower in the CH treatment group than those of the controls at 3,6 and 9 h after challenged with LPS/D-GalN(P=0.023,P=0.003 and P=0.042. respectively),while AST levels were significantly lower than those in the control group at 9 h after challenged with LPS/D-GalN (P=0.007). Gross examination of the liver showed a marked swelling, congestion and hemorrhage in the control group at 9 h after the challenge with LPS/D-GalN, while the liver had only mild congestion in the CH treatment group. And the liver sections stained by H&E showed that LPS/D-GalN-induced necrosis of hepatocytes in both control and CH-treated groups, but more severe liver pathologic changes induced by LPS/D-GalN were occurred in the control group than in the CH-treated group; especially the markedly differences appeared at 9 h after challenge, in which the structure of the liver lobules was destroyed and serious necrosis of hepatocytes and hemorrhage in the control group occurred, while only mild pathologic changes were shown in the CH-treated group.
The treatment with CH (320 mg/Kg) on zymosan-induced peritonitis in mice significantly decreased inflammatory response. The peak of leukocytes count in the peritoneal exudates in mice treated by CH was delayed at least 16h compared to control group. The leukocytes count in the CH treatment group was significantly lower than the control group at 5 and 8 h (P=0.03,P=0.006, respectively). Also the peak of the protein in the peritoneal exudates in the CH treatment group was lowered and delayed 4 h, which also differed between the two groups at 0.5,1 and 8 h after zymosan A challenge.The levels of TNF-α,MCP-1 and IL-6 in the serum of CH treatment group was significantly lowered at 1,2 and 8 h after zymosan A challenge compared to the control group.
In this part, it was found that CH (320 mg/Kg) treatment on acute lethal liver injury of mice induced by LPS/D-GalN and acute peritonitis induced by zymosan A significantly decreased the levels of inflammatory cytokines.
PartⅡ. Chlorate hydrate inhibited the activity of NF-κB in vitro and in vivo
Based on the changes of the levels of TNF-a, IL-6 and MCP-1 by CH treatment in mice with LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis, we intended to detect activity of NF-κB in NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice using in vivo bioluminescence imaging technology and in RAW264.7 cells transfected with NF-κB luciferase plasmid in vitro.
The effect of CH (320 mg/Kg) treatment on the activation of NF-κB was examined in six of the NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice after challenged by LPS/D-GalN. The results suggested that LPS/D-GalN challenge increased the NF-κB activities in mice treated only by NS, which peaked around 4 h after challenged with LPS/D-GalN. CH, however, significantly attenuated the rise of NF-κB activities at 3,4,5 and 6 h time point compared to control group (All P=0.000). Examination of individual organs for NF-κB activities further revealed the highest NF-κB activities in the liver followed intestines and lungs, while the NF-κB activities in these three organs in the control group were significantly higher than those in the CH treatment group(P=0.002, P=0.026,P=0.015, respectively).
RAW264.7 cells transfected with NF-κB luciferase plasmid and peritoneal macrophages were chosen as models in vitro. CH (0.5 and 1 mg/ml) treatment significantly inhibited the activity of NF-κB and decreased the production of IL-6 and TNF-a by peritoneal macrophages after stimulated by LPS/LTA.
In this part, it was found that CH treatment significantly inhibited the activity of NF-κB in mice stimulated by LPS/D-GalN and the production of inflammatory cytokines stimulated by LPS/LTA in vitro and in vivo.
Part III. Chloral hydrate induced the apoptosis of mononuclear cells and its mechanism
The different concentrations of CH (0.25,1 and 2 mg/ml) were used to treat RAW264.7 cells and peritoneal macrophages and the change of cell shape and function (eg, phagocytosis) were observed using detection of early apoptosis, Hochest 33258 staining, DNA ladder and other methods.
The results showed that CH treatment induced the apoptosis of RAW264.7 cells, in which. From the typical spindle shape of cells gradually became to round and finally shedding suspended under the treatment of CH; the treatment with CH (0.5 mg/ml) treated for 2,3,5 h and 7 h significantly induced early apoptosis (P=0.000) and typical DNA ladder of RAW264.7 cells. The photos usting Hochest 33258 staining showed that CH treatment induced the typical nuclear features of apoptosis: nuclear pyknosis and density markedly significantly increased, cohesion and fracture, while NS treatment in control showed nucleus homogeneous blue fluorescence. The phagocytic activity of RAW 264.7 were reduced treated by CH(P=0.000).CH treatment also induced the apoptosis of peritoneal macrophages. Early apoptosis of spleen mononuclear gradually increased in LPS/D-GalN induced liver injury mice and the apoptotic rate of in group with CH (320 mg/Kg) treatment was higher than control group at 2 and 3 h time point(P=0.001 and P=0.000, respectively).
We chose RAW264.7 cells as in vitro model and spleen tissues of normal mice, CH (320 mg/Kg) treated mice challenged by LPS/D-GalN as in vivo model to illustrate the mechanism of CH induced the apoptosis of target cells. The results showed that CH treatment induced the expression of Fas not FasL on RAW264.7 cells, and increased the expression of Fas on spleen tissues of mice receiving CH (320 mg/Kg) for 9 h, compared with normal spleen tissues expressing a less Fas. No significant difference in Fas expression between control group and CH (320 mg/Kg) treatment group of mice under challenge with LPS/D-GalN, but the expression in both group were higher than that in normal mice.
In this part, several methods were used to detect the apoptosis of RAW264.7 cells and peritoneal macrophages in vitro and spleen mononuclear cells and spleen biopsy in vivo, the results showed that the expression of Fas by apoptotic RAW264.7 cells and spleen tissues of normal mice under treatment with CH (320 mg/Kg) significantly higher than normal without CH.
Statistics
All results were expressed as (Mean±SD).The survival rates were estimated using SPSS 13.0 Kaplan-Meier method and tested for statistical significance using Breslow-Gehan-Wilcoxon test. Comparisons between control and CH treated group were performed using Independent-student T test. Comparisons among control and CH treated groups on RLU (relative light unit) were performed using one-way ANOVA. P<0.05 was considered to be significant.
Conclusion
In conclusion, it has been demonstrated that CH can attenuate and delay the inflammatory response of mice with LPS/D-GalN-induced acute lethal liver injuryor peritonitis and improve the survival of mice with LPS/D-GalN-induced acute lethal liver injury. The changed inflammatory response is associated with an inhibiting effect of CH against the increase of NF-κB activities and in the serum levels of the proinflammatory cytokines. It is also worth to emphasize that the anti-inflammatory effect of CH can be exerted even with administration at 1 to 3 h after the challenge of LPS/D-GalN. Moreover, CH treatment also can decrease the activity of NF-κB in RAW264.7 cells transfected with NF-κB luciferase plasmid stimulated by LTA/LPS in vitro, and the production of IL-6 and TNF-αby peritoneal macrophages. In addition, we have found that the expression of Fas on apoptotic RAW264.7 cells and spleen tissues of normal mouse under the treatment with CH (320 mg/Kg) are significantly higher than normal controls.
在本研究中,我们采用了两个经典的动物模型,即脂多糖／D-氨基半乳糖(LPS/D-GalN)诱发的急性致死性肝损伤和酵母多糖诱发的急性非致死性腹膜炎。其中LPS/D-GalN诱发的急性致死性肝损伤模型是一种被广泛使用了20多年的实验动物模型,其机制是导致急性肝功能衰竭而死亡,被认为与人类基于内毒素血症或败血症合并肝衰竭相关,如人类全身炎症反应综合征(SIRS)在非感染状态下可出现类似败血症表现。因此,该模型被用于研究内毒素血症合并急性肝损伤的机理、治疗策略与药物筛选。我们的研究中采用的另一个经典模型是有酵母多糖诱发的腹膜炎,属相对温和的非致死性的急性炎症。在确定LPS/D-GalN模型条件时,我们选择较大剂量的LPS与D-GalN,旨在建立一个比较严重的、存活时间很短的重症肝损伤,即造成一个比较极端的状态以观察水合氯醛的干预效果；而另一个模型是非致死性炎症,以此观察水合氯醛是否对这类更接近于临床多见的非致死性炎性状态具干预作用。
在观察并确定了水合氯醛可干预两种模型动物炎症的一般表现与指标的同时,我们对其作用机制做了初步的探索,其重点放在LPS/D-GalN诱发的急性致死性肝损伤模型。主要包括促炎症细胞因子水平及核因子κappaB (NF-κB)的活性改变。参考Bozza等对临床败血性休克患者的预后相关细胞因子谱的研究,我们选择IL-6,肿瘤坏死因子α(TNF-α)和MCP-I做为检测评价指标。其中,TNF-α已被证明和LPS/D-GalN攻击小鼠后生存率密切相关。MCP-1在急性致死性炎症如败血症中是一个重要的细胞因子,可平衡促炎和抗炎反应。
在明确水合氯醛的体内保护作用的基础上,我们设计了探讨机制的体外实验。具体为用小鼠巨噬细胞系RAW264.7细胞和小鼠腹腔巨噬细胞为研究对象,观察水合氯醛体外作用对这两种细胞NF-κB炎症信号分子激活和炎症因子生成的影响。观察水合氯醛处理是否会引起靶细胞(选择RAW264.7和腹腔巨噬细胞)的凋亡,以及通过什么途径诱导靶细胞的凋亡,以及与体内应用水合氯醛干预所诱发的凋亡是否一致。
第一章水合氯醛对LPS/D-GaIN诱发致死性肝损伤和zymosan诱发腹膜炎小鼠模型的保护作用
基于本研究采用水合氯醛(chloral hydrate, CH)一次性干预模式,在短时间内发挥作用,研究模型之一首先选择较严重的急性重症炎症模型：LPS2630(10μg)/D-GalN(800mg/Kg)诱发的急性致死性肝损伤模型(中位生存时间9.20h)。用CH以不同剂量(80,160和320mg/Kg)在LPS/D-GalN攻击后不同时间点(0,1,3和5h)体内一次性干预,发现CH干预以剂量依赖的方式延长小鼠的生存时间,以160和320mg/Kg组存在显著差异(分别P=0.024,P=0.001)。同一剂量(320mg/kg)在LPS/D-GalN攻击后0,1,和3h干预与对照组比较显著延长小鼠生存时间(分别P=0.002,P=0.004和P=0.002)。干预组3h点MCP-1,IL-6和TNF-a的水平显著低于对照组,而后IL-6和TNF-a在6h点逐渐上升显著高于对照组。干预组ALT在干预后3,6和9h点,均显著低于对照组,而AST9h点显著低于对照组。肝脏充血程度在9h点干预组较对照组显著减轻,HE染色显示两组均有肝脏小叶结构破坏和严重的肝细胞坏死和出血,在6和9h点对照组显著严重于干预组。两组血清皮质酮水平无显著差异,但干预组均低于对照组。而用有效干预剂量CH(320mg/Kg)被证明处理正常小鼠是安全的。
实验结果也证实了CH(320mg/Kg)可有效抑制：zymosan A诱发的急性腹膜炎。表现为干预组腹腔渗出液白细胞总数高峰显著推迟至少16h,在5和8h点细胞数量较对照组显著减少(分别P=0.03,P=0.006)。腹腔渗出液蛋白含量结果类似,高峰值延迟约4h,尤其是在0.5和1h点两组有显著差异急性腹膜炎小鼠诱发后1,2和8h点,TNF-α,MCP-1和IL-6的水平均显著低于对照组。
本章工作证明水合氯醛(320mg/Kg)体内干预对LPS/D-GalN诱发的急性致死性肝损伤和zymosan诱发急性腹膜炎小鼠具有显著的保护作用,可以显著降低和延迟炎症相关因子的产生。
第二章水合氯醛干预对炎症信号转导分子NF-κB抑制的体内外研究
基于CH(320mg/Kg)一次性体内应用可改变LPS/D-GalN诱发致死性肝损伤和zymosan诱发腹膜炎模型小鼠血液中炎症因子TNF-α,IL-6和MCP-1的水平。推测CH干预是否与抑制炎症因子合成的关键上游控制元件NF-κB有关。为证实这一推测设计了以整体成像技术与体外转染细胞系研究NF-κB活性。
体内研究选择NF-κB转基因小鼠NF-κB-RE-RE-luc(Oslo),用LPS/D-GalN诱发急性致死性肝损伤(同前),CH(320mg/Kg)干预显著降低LPS/D-GalN诱发的NF-κB转基因小鼠NF-κB控制下的荧光素酶报告基因的表达。无干预对照组荧光素酶表达在4h点达到高峰,干预组在3,4,5和6h点均显著低于对照组(均为P=0.000)。肝脏,肠道和肺脏为主要受累器官,CH干预能显著降低受累器官荧光素酶表达(分别P=0.002,P=0.026,P=0.015)。
体外细胞模型研究采用转NF-κB荧光素酶报告质粒RAW264.7细胞和小鼠腹腔巨噬细胞。结果表明：CH(0.5和1 mg/ml)干预显著降低由LPS/LTA刺激转染NF-κB荧光素酶报告质粒RAW264.7的荧光素酶报告基因的表达活性。显著抑制LPS/LTAJ刺激腹腔巨噬细胞IL-6和TNF-α的产生。
本章通过体内外即整体与细胞水平研究发现CH干预可以显著抑制由LPS/D-GalN,LPS和LTA刺激引起的炎症信号转导分子NF-κL活性,以及下游炎症因子的生成。
第三章CH诱发单个核细胞凋亡及其机制研究
体外选择CH不同浓度(0.25,1和2 mg/ml)在体外对RAW264.7细胞和腹腔巨噬细胞进行干预。采用观察细胞形态和功能(如吞噬功能)改变,早期凋亡检测,Hochest 33258染色,DNA ladder等多种方法验证是否确实引起了靶细胞的凋亡这一关键环节。
结果显示：CH处理RAW264.7细胞形态由典型的梭形,变圆直至脱落悬浮；PI/FITC染色经流式检测显示CH诱导RAW264.7细胞从早期向晚期凋亡的转换过程,并可被典型的DNA ladder所证实；Hochest 33258染色发现核明显固缩密度增加、凝聚和断裂；而且吞噬能力较正常对照被显著抑制。CH处理小鼠腹腔巨噬细胞具有同样的诱导凋亡效果。此外,CH(320mg/Kg)干预LPS/D-GalN诱发致死性肝损伤小鼠的脾脏单个核细胞凋亡,干预组在2和3h点较对照组显著诱发了脾脏单个核细胞的早期凋亡(分别P=0.001,P=0.000)。
为阐明CH以何种方式诱导靶细胞的凋亡,用RAW264.7细胞为模型在体外研究,同时观察无干预及CH(320mg/Kg)干预的正常小鼠与肝损伤小鼠的脾脏组织切片。发现体外CH(0.5和1mg/ml)处理RAW264.7细胞12和24h均可诱发RAW264.7表达Fas,但不表达FasL;Fas在正常小鼠脾脏少量表达,水合氯醛(320mg/Kg)一次性干预9h后,其脾脏细胞Fas表达显著升高。
本章采用多种方法检测体外RAW264.7细胞和腹腔巨噬细胞,体内模型的小鼠脾脏单个核细胞以及脾脏的凋亡,证明水合氯醛在体外干预可以诱导单个核细胞从早期到晚期的典型凋亡过程,且同时高表达Fas。
统计学处理
所有数据都表示为平均值士标准差(Mean±SD)。小鼠生存时间分析采用Kaplan-Meier分析进行对照组和CH干预组比较,显著性差异用Breslow检验。相同时间点CH干预组和对照组各因素比较使用独立样本t (Independent-student T test)检验。对照组(NS)和水合氯醛干预组(CH)RAW264.7细胞荧光信号relative light unit (RLU)(fold change)比较用one-way ANOVA。处理软件均为SPSS 13.0,P<0.05认为有显著性差异。
结论
研究发现CH(320mg/Kg)干预可以保护由LPS/D-GalN攻击小鼠的急性致死性肝损伤,显著延长其生存时间；且在急性致死性肝损伤诱导后1到3个1进行干预仍然显著有效。CH(320mg/Kg)干预可以减轻zymosan A诱发的急性非致死性腹膜炎小鼠的炎症程度。CH(320mg/Kg)干预可降低和延迟以上2个急性炎症模型小鼠血清促炎细胞因子IL-6,TNF-α, MCP-1水平,降低肝损伤小鼠肝脏病理损伤程度。
采用整体成像技术证实CH(320mg/Kg)干预可以保护NF-κB转基因小鼠,抑制由LPS/D-GalN诱发的NF-κB分子的活性。体外细胞水平研究证实CH(0.5mg/ml)处理转染NF-κB荧光素酶报告质粒的RAW264.7细胞,可抑制由LPS和LTA刺激引起的胞内NF-κB活性。同时CH(0.5和1mg/ml)处理腹腔巨噬细胞,可抑制由LPS和LTA刺激诱发的IL-6和TNF-α的生成。采用多种凋亡检测方法证实CH干预可以诱导RAW264.7细胞和腹腔巨噬细胞从早期到晚期的典型凋亡过程,且凋亡的RAW264.7细胞高表达Fas；水合氯醛(320mg/Kg)一次性干预正常小鼠9h后,其脾脏细胞Fas表达显著升高。
Background
Acute or serious inflammation induced by infection or uninfection has been considered as a very important issue in clinical and the research on pathogenesis and development of new medicines. Scientists are doing great efforts to find new leads of medicine including chemical compounds, plant extracts, even combinatory protein or peptide. In addition, people found that some of the medicines which were approved being used in other fields have good anti-inflammatory activities. Recently, several anesthetics have been found to possess anti-inflammatory and anti-infective effects apart from their functions of pain relief and muscle relaxation. For example, lidocaine, ketamine, isoflurane and pentobarbital can significantly improve the survival of mice and rats with endotoxic shock and protect them against liver and renal injury resulting from cecal ligation and puncture (CLP)-induced septic peritonitis .In the previous work, we occasionally found that chloral hydrate used as an amimanl anesthetic can improve the hemodynamic parameters of mice with endotoxin shock and anaphylactic shock. Therefore, we want to know whether chloral hydrate can function as an inhibitor of inflammation, because there are no any reports about the effect of chloral hydrate on acute inflammation. What made us interested at this issue is that chloral hydrate as a sedative and anesthetic has been used in clinical for over one hundred years, and it is currently used in pediatric manipulations like magnetic resonance imaging (MRI) and echocardiogram on children and in animal experiments at many countries. Furthermore, the evaluation of pharmacologic mechanism and safety of chloral hydrate have been still emphasized in human which suggests that chloral hydrate would not be obsolete even today.
In the present study, we sought to investigate the effects of chloral hydrate on the acute lethal liver injury induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) or on the acute peritonitis, a mild inflammatory response induced by zymosan A, which have been considered as well defined animal models . Especially, LPS/D-GalN-induced acute lethal liver injury in mice is a widely used as one of experimental animal models for more than 20 years, by which the mechanisms of lethal hepatic failure and development of the effective therapeutic strategies against endotoxin challenge have been investigated, because endotoxemia or sepsis is also associated with fulminant hepatic failure in human Moreover, the manifestation of systemic inflammatory response syndrome (SIRS) in the absence of infection may occour the similar syndrome to sepsis Therefore, our work could provide useful evidence used in research of LPS/D-GalN-induced acute lethal liver injury and sepsis. Another classic model used in our work is acute peritonitis induced by zymosan A, a kind of mild non-lethal acute inflammation. The mice with lethal acute liver injury induced by LPS/D-GalN can live a short period of time and caused an extreme serious state in order to observe the protection of chloral hydrate. The protection of chloral hydrate on peritonitis, a mild non-lethal inflammation is more close to clinical.
We also intended to explore the mechanisms of the protective effects against LPS/D-GalN-induced acute lethal liver injury and peritonitis by chloral hydrate, including the profiles of proinflammatory cytokines and the change of activities of nuclear factorκappaB (NF-κB). Assessment and selection of the parameters for present work including levels of IL-6, TNF-a and MCP-1 were according to the work on prognosis-related cytokines in patients with septic shock as reported by Bozza. Among them, TNF-a has been proved to be closed associated with survival of mice challenged with LPS only or LPS/GalN and MCP-1 is an important cytokine, which can balance the proinflammatory and anti-inflammatory reactions in acute lethal inflammation like sepsis.
Based on the protection of chloral hydrate in vivo, we also selected RAW264.7 cell and mouse peritoneal macrophages as models to study how chloral hydrate affects the activity of NF-κB and the production of inflammatory factors.
PartⅠ. Protection of chloral hydrate on LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis in mice
Since chloral hydrate (CH) treatment in this work is once injection and maintaining for short period, we chose a more serious acute inflammation model induced by the intraperitoneal (i.p.)injection of 10μg LPS-2630 (Escherichia coli serotype 2630) plus 800 mg of D-galactosamine hydrochloride (D-GalN, Sigma-Aldrich, St. Louis, MO, USA)×kg-1 of body weight, which median survival time is 9.2 h. The effect with CH treatment at different doses (80,160 and 320 mg/Kg) and different time points (0,1,3 and 5 h after the challenge by LPS/D-GalN) were observed, based on the safety proved by 320 mg/Kg CH on mice in preliminary test.The results showed that CH treatment improved the median survival of mice in a dose-dependent manner, especially that in group which received 160(11.68±2.62 h) and 320 mg/Kg (13.78±1.21 h) was significantly improved compared with controls (9.17±1.10h) (P=0.024 and P=0.001),and CH administered at 0,1 and 3 h after the challenge by LPS/D-GalN significantly improved the median survival of these mice compared with controls(13.70,13.30 and 11.60h vs.9.40h, P=0.002, P=0.004 and P=0.002, respectively). CH treatment attenuated the rises of serum MCP-1,IL-6 and TNF-a levels at 3h after LPS/D-GalN challenge compared with those control group (P=0.001,P=0.009 and P=0.015,respectively). In contrast to the control group, however, the levels of IL-6 and TNF-αunderwent a sharp rise in the treatment group at 6 h after LPS/D-GalN challenge, which was significantly higher than that of the control group. The levels of ALT in mice with LPS/D-GalN-induced acute lethal liver injury remained significantly lower in the CH treatment group than those of the controls at 3,6 and 9 h after challenged with LPS/D-GalN(P=0.023,P=0.003 and P=0.042. respectively),while AST levels were significantly lower than those in the control group at 9 h after challenged with LPS/D-GalN (P=0.007). Gross examination of the liver showed a marked swelling, congestion and hemorrhage in the control group at 9 h after the challenge with LPS/D-GalN, while the liver had only mild congestion in the CH treatment group. And the liver sections stained by H&E showed that LPS/D-GalN-induced necrosis of hepatocytes in both control and CH-treated groups, but more severe liver pathologic changes induced by LPS/D-GalN were occurred in the control group than in the CH-treated group; especially the markedly differences appeared at 9 h after challenge, in which the structure of the liver lobules was destroyed and serious necrosis of hepatocytes and hemorrhage in the control group occurred, while only mild pathologic changes were shown in the CH-treated group.
The treatment with CH (320 mg/Kg) on zymosan-induced peritonitis in mice significantly decreased inflammatory response. The peak of leukocytes count in the peritoneal exudates in mice treated by CH was delayed at least 16h compared to control group. The leukocytes count in the CH treatment group was significantly lower than the control group at 5 and 8 h (P=0.03,P=0.006, respectively). Also the peak of the protein in the peritoneal exudates in the CH treatment group was lowered and delayed 4 h, which also differed between the two groups at 0.5,1 and 8 h after zymosan A challenge.The levels of TNF-α,MCP-1 and IL-6 in the serum of CH treatment group was significantly lowered at 1,2 and 8 h after zymosan A challenge compared to the control group.
In this part, it was found that CH (320 mg/Kg) treatment on acute lethal liver injury of mice induced by LPS/D-GalN and acute peritonitis induced by zymosan A significantly decreased the levels of inflammatory cytokines.
PartⅡ. Chlorate hydrate inhibited the activity of NF-κB in vitro and in vivo
Based on the changes of the levels of TNF-a, IL-6 and MCP-1 by CH treatment in mice with LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis, we intended to detect activity of NF-κB in NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice using in vivo bioluminescence imaging technology and in RAW264.7 cells transfected with NF-κB luciferase plasmid in vitro.
The effect of CH (320 mg/Kg) treatment on the activation of NF-κB was examined in six of the NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice after challenged by LPS/D-GalN. The results suggested that LPS/D-GalN challenge increased the NF-κB activities in mice treated only by NS, which peaked around 4 h after challenged with LPS/D-GalN. CH, however, significantly attenuated the rise of NF-κB activities at 3,4,5 and 6 h time point compared to control group (All P=0.000). Examination of individual organs for NF-κB activities further revealed the highest NF-κB activities in the liver followed intestines and lungs, while the NF-κB activities in these three organs in the control group were significantly higher than those in the CH treatment group(P=0.002, P=0.026,P=0.015, respectively).
RAW264.7 cells transfected with NF-κB luciferase plasmid and peritoneal macrophages were chosen as models in vitro. CH (0.5 and 1 mg/ml) treatment significantly inhibited the activity of NF-κB and decreased the production of IL-6 and TNF-a by peritoneal macrophages after stimulated by LPS/LTA.
In this part, it was found that CH treatment significantly inhibited the activity of NF-κB in mice stimulated by LPS/D-GalN and the production of inflammatory cytokines stimulated by LPS/LTA in vitro and in vivo.
Part III. Chloral hydrate induced the apoptosis of mononuclear cells and its mechanism
The different concentrations of CH (0.25,1 and 2 mg/ml) were used to treat RAW264.7 cells and peritoneal macrophages and the change of cell shape and function (eg, phagocytosis) were observed using detection of early apoptosis, Hochest 33258 staining, DNA ladder and other methods.
The results showed that CH treatment induced the apoptosis of RAW264.7 cells, in which. From the typical spindle shape of cells gradually became to round and finally shedding suspended under the treatment of CH; the treatment with CH (0.5 mg/ml) treated for 2,3,5 h and 7 h significantly induced early apoptosis (P=0.000) and typical DNA ladder of RAW264.7 cells. The photos usting Hochest 33258 staining showed that CH treatment induced the typical nuclear features of apoptosis: nuclear pyknosis and density markedly significantly increased, cohesion and fracture, while NS treatment in control showed nucleus homogeneous blue fluorescence. The phagocytic activity of RAW 264.7 were reduced treated by CH(P=0.000).CH treatment also induced the apoptosis of peritoneal macrophages. Early apoptosis of spleen mononuclear gradually increased in LPS/D-GalN induced liver injury mice and the apoptotic rate of in group with CH (320 mg/Kg) treatment was higher than control group at 2 and 3 h time point(P=0.001 and P=0.000, respectively).
We chose RAW264.7 cells as in vitro model and spleen tissues of normal mice, CH (320 mg/Kg) treated mice challenged by LPS/D-GalN as in vivo model to illustrate the mechanism of CH induced the apoptosis of target cells. The results showed that CH treatment induced the expression of Fas not FasL on RAW264.7 cells, and increased the expression of Fas on spleen tissues of mice receiving CH (320 mg/Kg) for 9 h, compared with normal spleen tissues expressing a less Fas. No significant difference in Fas expression between control group and CH (320 mg/Kg) treatment group of mice under challenge with LPS/D-GalN, but the expression in both group were higher than that in normal mice.
In this part, several methods were used to detect the apoptosis of RAW264.7 cells and peritoneal macrophages in vitro and spleen mononuclear cells and spleen biopsy in vivo, the results showed that the expression of Fas by apoptotic RAW264.7 cells and spleen tissues of normal mice under treatment with CH (320 mg/Kg) significantly higher than normal without CH.
Statistics
All results were expressed as (Mean±SD).The survival rates were estimated using SPSS 13.0 Kaplan-Meier method and tested for statistical significance using Breslow-Gehan-Wilcoxon test. Comparisons between control and CH treated group were performed using Independent-student T test. Comparisons among control and CH treated groups on RLU (relative light unit) were performed using one-way ANOVA. P<0.05 was considered to be significant.
Conclusion
In conclusion, it has been demonstrated that CH can attenuate and delay the inflammatory response of mice with LPS/D-GalN-induced acute lethal liver injuryor peritonitis and improve the survival of mice with LPS/D-GalN-induced acute lethal liver injury. The changed inflammatory response is associated with an inhibiting effect of CH against the increase of NF-κB activities and in the serum levels of the proinflammatory cytokines. It is also worth to emphasize that the anti-inflammatory effect of CH can be exerted even with administration at 1 to 3 h after the challenge of LPS/D-GalN. Moreover, CH treatment also can decrease the activity of NF-κB in RAW264.7 cells transfected with NF-κB luciferase plasmid stimulated by LTA/LPS in vitro, and the production of IL-6 and TNF-αby peritoneal macrophages. In addition, we have found that the expression of Fas on apoptotic RAW264.7 cells and spleen tissues of normal mouse under the treatment with CH (320 mg/Kg) are significantly higher than normal controls.
引文
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[25]Bozza F A, Salluh J I, Japiassu A M, et al. Cytokine profiles as markers of disease severity in sepsis:a multiplex analysis.[J].Crit Care,2007,11(2):R49.
[26]Silverstein R. D-galactosamine lethality model:scope and limitations.[J].J Endotoxin Res,2004,10(3):147-162.
[27]Gomes R N, Figueiredo R T, Bozza F A, et al. Increased susceptibility to septic and endotoxic shock in monocyte chemoattractant protein 1/cc chemokine ligand 2-deficient mice correlates with reduced interleukin 10 and enhanced macrophage migration inhibitory factor production.[J].Shock,2006, 26(5):457-463.
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[29]Galley H F, Dimatteo M A, Webster N R. Immunomodulation by anaesthetic, sedative and analgesic agents:does it matter?[J].Intensive Care Med,2000,26 (3):267-274.
[30]Hotchkiss R S, Karl I E. The pathophysiology and treatment of sepsis.[J].N Engl J Med,2003,348(2):138-150.
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[32]Inoue K, Takano H, Sato H, et al. Protective role of urinary trypsin inhibitor in lung expression of proinflammatory cytokines accompanied by lethal liver injury in mice.[J].Immunopharmacol Immunotoxicol,2009,31(3):446-450.
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[34]Zhang S H, Liao C X, Zhang C X, et al. [Establishment of a mouse model of biliary obstruction and its dynamic observations][J].Nan Fang Yi Ke Da Xue Xue Bao,2008,28(9):1579-1581.
[35]Chen J C, Ng C J, Chiu T F, et al. Altered neutrophil apoptosis activity is reversed by melatonin in liver ischemia-reperfusion.[J].J Pineal Res,2003,34(4):260-264.
[36]Kolaczkowska E, Arnold B, Opdenakker G. Gelatinase B/MMP-9 as an inflammatory marker enzyme in mouse zymosan peritonitis:comparison of phase-specific and cell-specific production by mast cells, macrophages and neutrophils.[J].Immunobiology,2008,213(2):109-124.
[37]Kolaczkowska E, Lelito M, Kozakiewicz E, et al. Resident peritoneal leukocytes are important sources of MMP-9 during zymosan peritonitis: superior contribution of macrophages over mast cells.[J].Immunol Lett.2007, 113(2):99-106.
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[41]Murphy S L. Deaths:final data for 1998.[J].Natl Vital Stat Rep,2000,48(11):1-105.
[42]Gong X, Luo F L, Zhang L, et al. Tetrandrine attenuates lipopolysaccharide-induced fulminant hepatic failure in D-galactosamine-sensitized mice.[J].Int Immunopharmacol,2010,10(3):357-363.
[43]Ikeda T, Abe K, Kuroda N, et al. The inhibition of apoptosis by glycyrrhizin in hepatic injury induced by injection of lipopolysaccharide/D-galactosamine in mice.[J].Arch Histol Cytol,2008,71(3):163-178.
[44]Zhang L, Li H Z, Gong X, et al. Protective effects of Asiaticoside on acute liver injury induced by lipopolysaccharide/D-galactosamine in mice.[J]. Phytomedicine,2010.
[45]Wilhelm E A, Jesse C R, Nogueira C W. Protective effect of p-methoxyl-diphenyl diselenide in lethal acute liver failure induced by lipopolysaccharide and D-galactosamine in mice.[J].Fundam Clin Pharmacol,2009,23(6):727-734.
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[47]Pignatelli D, Magalhaes M M, Magalhaes M C. Direct effects of stress on adrenocortical function.[J].Horm Metab Res,1998,30(6-7):464-474.
[48]Siegel H S, Latimer J W. Interaction of high temperature and Salmonella pullorum antigen concentration on serum agglutinin and corticosteroid responses in white rock chickens.[J].Poult Sci,1984,63(12):2483-2491.
[49]Wu Y, Zhou B P. TNF-alpha/NF-kappaB/Snail pathway in cancer cell migration and invasion.[J]. Br J Cancer,2010,102(4):639-644.
[50]Meeus M, Mistiaen W, Lambrecht L, et al. Immunological similarities between cancer and chronic fatigue syndrome:the common link to fatigue?[J]. Anticancer Res,2009,29(11):4717-4726.
[51]Lin Y, Bai L, Chen W, et al. The NF-kappaB activation pathways, emerging molecular targets for cancer prevention and therapy.[J].Expert Opin Ther Targets,2010,14(1):45-55.
[52]Li Q, Verma I M. NF-kappaB regulation in the immune system.[J].Nat Rev Immunol,2002,2(10):725-734.
[53]Carlsen H, Moskaug J O, Fromm S H, et al. In vivo imaging of NF-kappa B activity.[J].J Immunol,2002,168(3):1441-1446.
[54]Luker K E, Luker G D. Applications of bioluminescence imaging to antiviral research and therapy:multiple luciferase enzymes and quantitation.[J]. Antiviral Res,2008,78(3):179-187.
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