Cannabinoid antagonist AM 281 reduces mortality rate and neurologic dysfunction after cecal ligation and puncture in rats

OBJECTIVES: The purpose of this study was to examine whether anandamide, an endogenous cannabinoid receptor ligand, is involved in the pathogenesis of septic encephalopathy. DESIGN: Prospective, controlled study. SUBJECTS: Male Wistar rats (7 wks old) were randomly divided into four groups as follows: group 1, control (0.5 mL of saline injected subcutaneously); group 2, sham (surgical abdominal incision and suturing were performed, but ligation and puncture of the cecum were omitted); group 3, cecal ligation and puncture (CLP); group 4, CLP + AM 281 ([N-morpholin-4-yl]-5-[2,4-yl]-5-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide) as the cannabinoid receptor antagonist (1 mg/kg intraperitoneally). INTERVENTIONS: Sepsis was induced by CLP under pentobarbital anesthesia (10 mg/kg intraperitoneally) with 1% isoflurane. A 2-Fr high-fidelity micromanometer catheter was inserted into the left ventricle via the right carotid artery to assess hemodynamics. Each of the rats was neurologically assessed at 30 mins and 12, 24, and 48 hrs after the treatment. The cytoplasmic levels of caspase-3 in the hippocampi were assayed before surgery and at 30 mins and 24 and 48 hrs after surgery using Western blotting techniques. To examine the effects of AM 281 on neurologic function and mortality rate, we set another control group treated solely with AM 281. Selective inducible nitric oxide synthase inhibitor, L-N6-(1-iminoethyl)-lysine (4 mg/kg), was injected intraperitoneally immediately after CLP to produce the CLP + L-N6-(1-iminoethyl)-lysine group to exclude the influence of depressed hemodynamics on neurologic impairment. MEASUREMENTS AND MAIN RESULTS: It was found that administration of AM 281 could prevent the hemodynamic changes induced by sepsis. Reflex responses, including the pinna, corneal, paw or tail flexion, and righting reflexes, and the escape response significantly decreased in the CLP and CLP + L-N6-(1-iminoethyl)-lysine groups at 48 hrs after the surgery. In contrast, no changes in these reflex responses were found between the CLP + AM 281 and control and sham groups. In addition, no effects of the administration of AM 281 on neurologic function and mortality rate in the control group were found. Tissue caspase-3 levels were elevated at 48 hrs after CLP in the CLP alone group (means +/- sd: control, 3.9 +/- 0.4; sham, 4.2 +/- 0.4; CLP, 7.1 +/- 1.0 [p < .01]; CLP + AM 281, 4.0 +/- 0.5 densitometric units). In addition, administration of AM 281 also decreased the mortality rate (p < .05). CONCLUSIONS: Administration of AM 281 prevented the hemodynamic changes and development of neurologic dysfunction occurring in association with septic shock, and could decrease the mortality rate in experimentally induced septic shock in rats. Although further studies are necessary to determine whether endogenous cannabinoids cause septic encephalopathy in rats directly or via their effects on systemic hemodynamics, the beneficial effects of AM 281 on these rats might have significant therapeutic implications in cases of septic encephalopathy.     

Sepsis-induced cholestasis, steatosis, hepatocellular injury, and impaired hepatocellular regeneration are enhanced in interleukin-6 -/- mice

OBJECTIVE: Hepatic dysfunction is an important but poorly understood component of sepsis. In severe sepsis, liver dysfunction is characterized by cholestasis, steatosis, hepatocellular injury, impaired regeneration, a decreased response to the cytokine interleukin-6, and high mortality. To determine whether loss of interleukin-6 activity caused hepatic dysfunction and mortality, we induced sepsis in wild-type (interleukin-6 +/+) and interleukin-6 knockout (interleukin-6 -/-) mice. We hypothesized that sepsis in interleukin-6 -/- mice would increase cholestasis, steatosis, hepatocellular injury, and mortality and impair hepatocyte regeneration. DESIGN: Randomized prospective experimental study. SETTING: University medical laboratory. SUBJECTS: Male adolescent C57Bl6 interleukin-6 +/+ and interleukin-6 -/- mice. INTERVENTIONS: Mild sepsis was induced using cecal ligation and single puncture (CLP). Severe, lethal sepsis was induced using cecal ligation and double puncture (2CLP). Some mice received recombinant human interleukin-6 at the time of CLP/2CLP. All animals were fluid resuscitated at the time of surgery and every 24 hrs thereafter. In survival cohorts, mortality at 16, 24, 48, and 72 hrs was recorded. In separate cohorts, surviving animals were killed at 24 and 48 hrs, and liver tissue was harvested. A separate cohort of mice received bromodeoxyuridine for detection of regeneration. MEASUREMENTS AND MAIN RESULTS: 2CLP was 100% fatal within the first 12 hrs in interleukin-6 -/- mice. Mortality from 2CLP in interleukin-6 +/+ mice before 24 hrs was nil but was 90% by 72 hrs. At 72 hrs, CLP was 40% fatal in interleukin-6 +/+ mice but 90% in interleukin-6 -/- mice. CLP induced cholestasis, steatosis, and hepatocellular injury in interleukin-6 -/-, but not interleukin-6 +/+, mice. Regeneration was absent following CLP in interleukin-6 -/- animals but occurred in interleukin-6 +/+ mice. Early administration of recombinant human interleukin-6 did not reverse abnormalities in interleukin-6 -/- mice. CONCLUSIONS: The absence of interleukin-6 is an important determinant of hepatic dysfunction and mortality in sepsis.     

Antibiotics delay but do not prevent bacteremia and lung injury in murine sepsis

OBJECTIVES: To investigate the effect of antibiotics on infection, lung injury, and mortality rate in polymicrobial sepsis and to determine whether an association exists between infection and lung injury and mortality rate. To circumvent the effect of antibiotics on cultures, we used polymerase chain reaction to detect bacteria. DESIGN: Prospective, randomized, controlled laboratory trial. SETTING: University research laboratory. SUBJECTS: C57/BL6 mice. INTERVENTIONS: Mice underwent cecal ligation and puncture without antibiotics (CLP) or with imipenem (CLP + Abx). MEASUREMENTS AND MAIN RESULTS: CLP resulted in 50% mortality rate at 48 hrs and 100% mortality rate at 84 hrs. Antibiotics delayed these time points to 72 and 120 hrs, respectively. Lung injury occurred before mortality in both groups. Polymerase chain reaction detected bacteria in the blood and lungs of all CLP mice by 24 hrs. Antibiotics delayed but did not prevent infection in CLP + Abx mice. Serum tumor necrosis factor-alpha and lung endotoxin were elevated to similar concentrations in both CLP and CLP + Abx mice. CONCLUSIONS: In this model of sepsis, antibiotics delay but do not prevent acute lung injury and mortality. Even in the presence of antibiotics, acute lung injury is strongly associated with bacteremia and bacteria within the lungs.     

脓毒症大鼠肺组织c-FLIP的变化及乌司他丁对c-FLIP的影响

目的 观察脓毒症大鼠的肺组织中c-FLIP的变化情况,并探讨乌司他丁对c-FLIP水平的影响.方法 将30只雄性Sprague-Dawley大鼠随机(随机数字法)分为假手术组(Sham组)、脓毒症模型组(CLP组)及乌司他丁治疗组(UTI组),每组10只.Sham组不结扎穿刺盲肠,余同CLP组;CLP组采用肓肠结扎并穿刺法造模;UTI组造模后立即经阴茎静脉注射乌司他丁注射液(50000 U/kg).12 h后处死大鼠并采取肺组织,光镜下观察形态学变化;Western-Blot法检测c-FLIP蛋白表达,RT-PCR法检测c-FLIP基因表达.应用SPSS 11.5进行统计学分析,多组比较采用方差分析,两两比较采用LSD-t检验.结果 脓毒症组光镜下肺泡腔内中性粒细胞和大量红细胞渗出,c-FLIP蛋白和基因表达较假手术组显著降低(P＜0.05);用乌司他丁治疗后,光镜下病理改变减轻,c-FLIP蛋白和基因表达较脓毒症组显著升高(P＜0.05).结论 脓毒症中c-FLIP在肺组织中的表达下降,提示肺组织的细胞凋亡增加.乌司他丁能上调脓毒症大鼠肺组织中c-FLIP的表达.     

持续静脉泵注利多卡因对脓毒症大鼠急性肺损伤及炎症反应的影响

目的探讨利多卡因对脓毒症大鼠肺损伤及炎症因子表达的影响。 方法采用随机数字表法将60只雄性成年Sprague Dawley大鼠分为假手术组、盲肠结扎穿孔（CLP）组、利多卡因组和乌司他丁组,每组各15只。假手术组大鼠打开腹腔后缝合,其他各组采用CLP法制备脓毒症模型。利多卡因组大鼠在给予10 mg/kg的负荷剂量后,以10 mg·kg^-1·h^-1的剂量通过尾静脉持续泵注利多卡因3h;乌司他丁组大鼠进行CLP的同时,以100 000 U·kg^-1·h^-1的剂量通过尾静脉持续泵注乌司他丁3 h;假手术组和CLP组用等量等渗NaCl溶液代替。于CLP后24 h处死大鼠,采用酶联免疫吸附实验（ELISA）法测定血清中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）及高迁移率族蛋白B1（HMGB1）表达水平;实时荧光定量PCR检测肺组织HMGB1 mRNA表达水平;苏木素-伊红（HE）染色法观察各组大鼠肺组织病理变化。另取40只大鼠（每组10只）用于观察4组大鼠72 h死亡情况。 结果4组大鼠血清中TNF-α、IL-6、HMGB1及HMGB1 mRNA表达水平比较,差异均有统计学意义（F = 189.886、237.952、175.999、179.491,P均< 0.001）。进一步两两比较发现,与假手术组比较,CLP组、利多卡因组和乌司他丁组血清TNF-α、IL-6、HMGB1及HMGB1 mRNA表达水平均显著升高（P均< 0.05）;与CLP组比较,利多卡因组和乌司他丁组血清TNF-α、IL-6、HMGB1及HMGB1 mRNA表达水平均显著降低（P均< 0.05）;与利多卡因组比较,乌司他丁组IL-6表达水平显著升高（P < 0.05）。HE染色结果显示,假手术组大鼠肺泡大小均匀、结构完整,肺泡上皮细胞形态正常;CLP组大鼠肺泡间隔增厚、间质充血水肿、炎症细胞浸润、肺泡塌陷;而利多卡因组和乌司他丁组病理学改变较CLP组均明显减轻,肺组织轻度水肿,肺泡及肺间质出现少量炎症。四组大鼠死亡构成比（0 /10、9/1、4/6、3/7）比较,差异有统计学意义（χ²=17.500,P < 0.001）。CLP组、利多卡因组和乌司他丁组大鼠死亡构成比均较假手术组显著升高（P均<0.008）;利多卡因组和乌司他丁组大鼠死亡构成比均较CLP组显著降低（P均<0.008）;而利多卡因组和乌司他丁组大鼠死亡构成比比较,差异无统计学意义（P > 0.008）。 结论持续静脉泵注利多卡因可以有效降低脓毒症大鼠炎症因子TNF-α、IL-6及HMGB1的表达,抑制肺组织中HMGB1 mRNA表达量,减轻脓毒症对肺组织的损伤,有效提高动物存活率,其减轻脓毒症炎症反应及肺保护作用疗效与乌司他丁相似。     

Hyperbaric oxygen protects from sepsis mortality via an interleukin-10-dependent mechanism

OBJECTIVE: This study was performed to determine whether hyperbaric oxygen (HBO2) therapy is protective in cecal ligation and puncture (CLP)-induced sepsis and if protection is dependent on oxygen dosing. We also wished to determine whether HBO2 affected bacterial clearance or altered macrophage production of interleukin-10 (IL-10)s in the setting of CLP sepsis. Finally, we wished to determine whether the mechanism of HBO2 protection in sepsis was dependent on IL-10 production. DESIGN: Prospective, experimental study. SETTING: University experimental research laboratory. SUBJECTS: C57BL/6 and C57BL/6 IL-10 mice. INTERVENTIONS: Sepsis was induced by CLP. Mice were randomized to receive a 1.5-hr HBO2 treatment at either 1, 2.5, or 3 atmospheres absolute every 12 hrs or HBO2 at 2.5 atmospheres absolute every 24 hrs. Mice were also harvested at 24 hrs for determination of bacterial load and isolation and study of CD11b peritoneal macrophages. MEASUREMENTS AND MAIN RESULTS: Survival was monitored for 100 hrs after CLP +/- HBO2 treatment. HBO2 significantly improved survival when administered at 2.5 atmospheres absolute every 12 hrs. Other treatment schedules were not protective, and treatment at 3.0 atmospheres absolute significantly worsened survival outcome. Bacterial load was significantly reduced in splenic homogenates but not peritoneal fluid at 24 hrs. Macrophages isolated from HBO2-treated mice demonstrated enhanced IL-10 secretion in response to lipopolysaccharide as compared with CLP controls. Mice genetically deficient in IL-10 expression treated with HBO2 at 2.5 atmospheres absolute every 12 hrs were not protected from CLP-induced mortality. CONCLUSION: HBO2 may be protective in CLP sepsis within a window of oxygen dosing. The mechanism of HBO2 protection may be potentially linked in part to expression of IL-10, as peritoneal macrophages demonstrated enhanced IL-10 expression and IL-10 mice were not protected by HBO2 treatment.     

The protective effect of N-acetylcysteine on apoptotic lung injury in cecal ligation and puncture-induced sepsis model

Apoptotic loss of parenchymal cells may lead to organ dysfunctions in critically ill patients with septic states. As an antioxidant, the protective effects of N-acetylcysteine (NAC) are documented in many experimental and clinical studies. In this experimental study, we investigated the role of chronically used NAC in septic lung injury on a cecal ligation and puncture (CLP) model. To evaluate this, 30 male Wistar rats were randomly divided into four groups as sham (n = 7), CLP (n = 8), sham + NAC (n = 7) and CLP + NAC (n = 8) groups. NAC was administered 150 mg kg(-1) day through intramuscular route beginning 6 h after the operations and lasting for a period of 1 week. One week later, histopathology and epithelial apoptosis were assessed by hematoxylin-eosin and immunohistochemically by M30 and caspase 3 staining to demonstrate septic lung injury. Additionally, lung tissue myeloperoxidase (MPO) activity, malondialdehyde (MDA), and nitrite/nitrate levels were measured. The MPO activity and MDA levels in lung homogenates were found to be increased in CLP group and the administration of NAC prevented their increase significantly (P < 0.05). However, there were no significant differences among the groups regarding nitrite/nitrate levels. The number of apoptotic cells was significantly lower in CLP+NAC group than CLP group, and this finding was supported by M30 and caspase 3 expression in lung (P < 0.05). Lung histopathology was also protected by NAC in CLP-induced sepsis. In conclusion, the chronic use of NAC inhibited MPO activity and lipid peroxidation, which resulted in reduction of apoptosis in lung in this CLP model. Because lung tissue nitrite/nitrate levels did not change significantly, organs other than the lungs may be responsible for producing the increased nitric oxide during sepsis. The chronic use of NAC needs further investigation for its possible antiapoptotic potential in septic states besides its documented antioxidant and antiinflammatory effects.     

Cognitive impairment in sepsis survivors from cecal ligation and perforation

OBJECTIVE: Critical illness survivors present long-term cognitive impairment, including problems with memory and learning. We evaluated cognitive performance in rats that survived from sepsis induced by cecal ligation and puncture (CLP). DESIGN: Prospective, controlled experiment. SETTING: Animal basic science laboratory. SUBJECTS: Male Wistar rats, weighing 300-350 g. INTERVENTIONS: The rats underwent CLP (sepsis group) with "basic support" (saline at 50 mL/kg immediately and 12 hrs after CLP plus ceftriaxone at 30 mg/kg and clindamycin at 25 mg/kg 6, 12, and 18 hrs after CLP) or sham-operated (control group). MEASUREMENTS AND MAIN RESULTS: Ten days after surgery, the animals underwent three behavioral tasks: a) inhibitory avoidance task; b) habituation to an open field; and c) continuous multiple-trials step-down inhibitory avoidance task (CMSIA). In the habituation to an open-field task, there were no differences in the number of crossings and rearings. The sepsis group showed significantly decreased performance in latency retention compared with the sham group in inhibitory avoidance. Furthermore, when tested by the habituation to an open-field task, the sepsis group did not show any difference between training and test, indicating memory impairment. In the CMSIA, the sepsis group showed a significant increase in the number of training trials required to reach the acquisition criterion. CONCLUSION: Our data provide the first experimental demonstration that survivors from CLP show learning and memory impairment after complete physical recovery from sepsis.     

脓毒症急性肺损伤大鼠肺组织细胞凋亡的研究

目的观察脓毒症急性肺损伤（ALI）大鼠肺组织的细胞凋亡情况。方法24只SPF级SD大鼠采用盲肠结扎穿孔术（CLP）复制脓毒症模型，分别于CLP后0、12、36和72h颈椎脱臼法处死大鼠，取肺组织。行肺组织HE染色，观察病理变化，以透射电镜、TUNEL法检测细胞凋亡。结果HE染色可见，CLP后12、36、72h肺泡间隔增宽，间质充血水肿，肺泡腔变窄，炎症细胞渗出。TUNEL法检测发现，CLP后12、36、72h肺组织内细胞凋亡指数（Al）增加，其中36hAl值最高（P〈0．01）。透射电镜证实细胞凋亡的特征性改变。结论脓毒症大鼠肺组织的Al明显增加，细胞凋亡可能在脓毒症ALI发生、发展中起着重要作用。     

还原型谷胱甘肽对大鼠脓毒症肺损伤外周血淋巴细胞凋亡率及TNF-α、IL-6水平的影响

目的观察还原型谷胱甘肽对大鼠脓毒症肺损伤外周血淋巴细胞凋亡率及血浆细胞因子TNF-α、IL-6水平的影响，探讨还原型谷胱甘肽对大鼠脓毒症肺损伤的保护作用及其机制。方法应用盲肠结扎穿孔（CLP）法复制大鼠脓毒症肺损伤模型。将清洁级雄性SD大鼠112只，随机分成假手术组（Sham）、脓毒症肺损伤组（ALI）、还原型谷胱甘肽治疗组（GSH）、左旋氧氟沙星治疗组（LEV），每组再分为3、6、12、24h等4个亚组，每个亚组n=7。观察大鼠脓毒症肺损伤肺组织的病理形态学改变，并检测外周血淋巴细胞凋亡率及血浆TNF-α、IL-6水平的变化。结果在脓毒症肺损伤组及左旋氧氟沙星治疗组淋巴细胞凋亡率较假手术组及GSH治疗组明显升高（P〈0．05）；在脓毒症肺损伤组血浆TNF-α水平在CLP术后6h出现升高，较GSH治疗组升高明显（P〈0．01）。脓毒症肺损伤组大鼠血浆IL-6水平于CLP术后3h升高，GSH治疗组CLP术后3h血浆IL-6水平较脓毒症肺损伤组低（P〈0．05）。脓毒症肺损伤组大鼠病理显示明显肺损伤，GSH治疗组大鼠肺损伤程度明显减轻。结论还原型谷胱甘肽能显著抑制外周血淋巴细胞凋亡及血浆TNF-α和IL-6表达水平，对大鼠脓毒症急性肺损伤具有明显的保护作用。     

Alteration in diaphragmatic contractility during septic peritonitis in rats: effect of polyethylene glycol-absorbed superoxide dismutase

OBJECTIVES: To assess the alterations in diaphragmatic contractility measured in vitro during experimental septic peritonitis and to evaluate the effect of polyethylene glycol-absorbed superoxide dismutase (PEG-SOD) on the alterations in contractility. DESIGN: Prospective, randomized, controlled animal trial. SETTING: Research laboratory. SUBJECTS: A total of 321 male Wistar rats, weighing 250-300 g. INTERVENTIONS: Rats were treated with cecal ligation and puncture (CLP). In the first experiment, diaphragmatic contractility was measured at 4, 10, 12, and 16 hrs after CLP. In the second experiment, PEG-SOD (4,000 units/kg) was administered intraperitoneally, and then diaphragmatic contractility was measured at 10 and 16 hrs after CLP. Levels of lipid peroxides and antioxidant enzymes in the diaphragm tissue were measured at 10 and 16 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: In experiment 1, diaphragmatic twitch characteristics and force-frequency relationships were determined at 4, 10, 12, and 16 hrs after CLP. In experiment 2, the effects of administration of PEG-SOD on twitch characteristics and force-frequency relationships were determined at 10 and 16 hrs after CLP. The levels of diaphragmatic thiobarbituric acid reactive substances and superoxide dismutase (SOD) and glutathione peroxidase activities were measured at 10 and 16 hrs after CLP. Twitch tension and force-frequency curves were significantly lower in the CLP groups than in the sham-operated group. Administration of PEG-SOD attenuated the reduction in twitch tension and the downward shift of force-frequency curves after CLP. Diaphragmatic levels of thiobarbituric acid reactive substances increased after CLP. However, the administration of PEG-SOD prevented increases in levels of diaphragmatic thiobarbituric acid reactive substances after CLP. Diaphragmatic SOD activity, but not glutathione peroxidase activity, was increased after CLP. CONCLUSIONS: Intra-abdominal sepsis (CLP) induced a marked reduction in diaphragmatic contractility, but PEG-SOD attenuated this reduction. Therefore, we conclude that oxygen-derived free radicals play an important role in the alterations in diaphragmatic contractility during intra-abdominal sepsis.     

脓毒症大鼠脾脏淋巴细胞凋亡的实验研究

目的 观察脓毒症大鼠脾脏T、B淋巴细胞的数量变化及细胞凋亡情况,探讨脓毒症时免疫失衡的机制.方法 健康雄性Wistar大鼠80只,按随机数字表法分为假手术组(30只)、模型组(50只).采用盲肠结扎穿孔术(CLP)制备脓毒症大鼠模型,于制模后6、12、24、48、96 h活杀动物取脾脏,行苏木素-伊红(HE)染色,光镜下观察脾脏组织病理变化;采用免疫组化法检测脾脏CD4~+、CD8~+T淋巴细胞和B淋巴细胞以及Bax、Bcl-2蛋白表达;采用原位末端缺刻标记法(TUNEL)检测脾脏细胞凋亡指数.结果 光镜下观察模型大鼠脾脏白髓逐渐萎缩,淋巴小结结构破坏.制模6、12、24、48、96 h,模型组大鼠脾脏CD4~+T淋巴细胞数、B淋巴细胞数、Bcl-2蛋白表达均较假手术组明显降低(P均<0.01),CD8~+T淋巴细胞数与假手术组比较差异无统计学意义(P均>0.05),细胞凋亡指数及Bax蛋白表达均较假手术组显著增加(P均<0.01).相关分析表明:Bcl-2蛋白表达与细胞凋亡指数呈负相关(r=0.659,P<0.01),而Bax蛋白表达与细胞凋亡指数呈正相关(r=0.522,P<0.01).结论 脓毒症早期免疫功能处于紊乱状态,表现为脾脏CD4~+T淋巴细胞数、B淋巴细胞数减少,细胞凋亡显著增加;Bax、Bcl-2在脓毒症脾细胞凋亡中发挥了关键作用.     

Role of interferon-gamma in lung inflammation following cecal ligation and puncture in rats.

Interferon-gamma (IFN-gamma) has been implicated in the mortality of animal models of endotoxemia. On the other hand, the specific role of IFN-gamma in the development of organ inflammation in a model of polymicrobial sepsis has not been elucidated. In this study, we hypothesized that IFN-gamma plays an important role in lung inflammation after cecal ligation and puncture (CLP). To verify this hypothesis, lung tissue was removed 5 h after CLP or from sham controls. The mRNA expression (by RT-PCR) of IFN-gamma was increased in lung homogenates of CLP rats compared to sham controls. Using immunohistochemistry, we show for the first time the increased presence of IFN-gamma staining cells in the lung following CLP. Only very small amounts of positive staining for IFN-gamma was observed in lungs of sham controls. The presence of IFN-gamma in the lung 5 h after CLP correlated with a twofold increases in lung superoxide generation and MPO activity (index of neutrophil sequestration). Plasma and lung nitrite levels (breakdown product of nitric oxide) were also significantly increased in CLP rats. IFN-gamma antibody (1.2 mg/kg, i.v.) administered immediately after CLP significantly decreased lung superoxide levels to levels similar to the sham controls without affecting MPO activity, or lung or plasma nitrite levels. These results provide evidence that IFN-gamma may contribute to lung inflammation 5 h following CLP via increased production of superoxide.     

高迁移率族蛋白B1 / Toll样受体4信号通路在脓毒症大鼠致急性肺损伤中的作用研究

目的探讨高迁移率族蛋白B1（HMGB1） / Toll样受体4（TLR4）信号通路对脓毒症导致的急性肺损伤大鼠的影响。 方法将60只清洁级雄性Sprague Dawley大鼠分为假手术组、脓毒症组和实验组,每组各20只。假手术组大鼠麻醉后开腹翻动肠道,随即关腹;脓毒症组和实验组行盲肠结扎穿孔（CLP）术后0.5 h于尾静脉分别注射等渗NaCl溶液（4 mL / kg）及抗HMGB1单克隆抗体（2 mg / kg）。各组分别取10只用于观察大鼠CLP建模后7 d存活情况,其余大鼠于造模后24 h处死并留取肺组织标本。计算各组大鼠的肺损伤Smith评分,并比较各组大鼠HMGB1和TLR4阳性蛋白在肺组织中的表达水平。采用酶联免疫吸附测定检测各组大鼠肺泡灌洗液（BALF）中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、HMGB1和TLR4水平,计算每个巨噬细胞内微球蛋白数以比较各组大鼠肺泡巨噬细胞（AM）的吞噬功能,并采用Western-blotting法测定AM内HMGB1、TLR4蛋白表达水平。 结果假手术组大鼠建模后7 d全部存活,脓毒症组大鼠仅3只存活,实验组大鼠5只存活。各组大鼠间存活情况的比较,差异有统计学意义（ χ² = 10.833,P = 0.004）;且假手术组大鼠的存活情况明显优于脓毒症组与实验组大鼠（P均< 0.017）,而脓毒症组与实验组大鼠间存活情况比较,差异无统计学意义（P = 0.120）。假手术组、脓毒症组及实验组大鼠间肺组织损伤评分[（2.20 ± 0.27）、（8.20 ± 1.27）、（4.25 ± 2.21）分,F = 56.432,P < 0.001],肺组织HMGB1阳性蛋白[（10.4 ± 1.5）、（34.4 ± 5.0）、（26.6 ± 6.9）,F = 35.203,P = 0.003]、TLR4阳性蛋白[（10.6 ± 2.1）、（48.0 ± 5.8）、（38.2 ± 5.3）,F = 103.414,P = 0.002],BALF中TNF-α[（19 ± 4）、（45 ± 4）、（35 ± 4）μg / L,F = 2.749,P < 0.001]、IL-6[（56 ± 19）、（86 ± 15）、（70 ± 19）μg / L,F = 4.648,P = 0.001]、HMGB1[（41 ± 18）、（70 ± 15）、（56 ± 12）μg / L,F = 7.254,P = 0.002]、TLR4[（20.9 ± 1.8）、（51.2 ± 1.6）、（49.8 ± 2.6）μg / L,F = 3.978,P = 0.035],AM的吞噬功能[（21.8 ± 2.7）、（3.1 ± 1.9）、（12.6 ± 2.2）个,F = 32.821,P = 0.001]及AM中HMGB1蛋白[（11 ± 3）、（40 ± 15）、（24 ± 13）,F = 7.253,P < 0.001]、TLR4蛋白[（0.9 ± 0.4）、（1.2 ± 0.6）、（1.1 ± 0.4）,F = 3.984,P = 0.028]的比较,差异均有统计学意义。进一步两两比较发现,脓毒症组与实验组大鼠肺组织损伤评分,肺组织HMGB1阳性蛋白、TLR阳性蛋白表达水平,BALF中TNF-α、IL-6、HMGB1水平及AM中HMGB1蛋白表达水平均明显高于假手术组大鼠,且脓毒症组大鼠更高（P均< 0.05）;脓毒症组及实验组大鼠AM的吞噬功能均显著低于假手术组,且脓毒症组更低（P均< 0.05）;脓毒症组及实验组大鼠BALF中TLR4水平及AM中TLR4蛋白表达水平均显著高于假手术组（P均< 0.05）,但脓毒症组与实验组间上述指标的比较,差异均无统计学意义（P均> 0.05）。 结论通过抑制HMGB1 / TLR4信号通路可以缓解脓毒症致肺损伤大鼠肺组织的炎症损伤,抑制炎症介质过度释放,并增强AM的细胞吞噬功能。     

Sepsis-induced lung injury in rats increases alveolar epithelial vulnerability to stretch

OBJECTIVE: Previous in vitro models have shown that cellular deformation causes dose-dependent injury and death in healthy rat alveolar epithelial cells (AECs). We compared the viability of AECs from septic rats with those from nonseptic rats after 1 hr of cyclic equibiaxial stretch. We hypothesized that sepsis would increase stretch-induced cell death. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Thirty-seven male Sprague-Dawley rats weighing 240-260 g. INTERVENTIONS: Anesthetized rats were subjected to cecal ligation and double puncture (2CLP) or sham laparotomy without cecal ligation or puncture (sham). After 24 or 48 hrs, AECs were isolated, seeded in custom wells, and maintained in culture for 48 hrs before study. AECs were stretched cyclically (15/min) to a 0%, 12%, 25%, or 37% change in surface area (DeltaSA) for 1 hr. Cell viability, phenotypic markers, and nuclear factor-kappaB intracellular localization were assessed using fluorescent immunocytochemistry. MEASUREMENTS AND MAIN RESULTS: Phase and fluorescent images were evaluated for all studies. Response to stretch was the same at 24 and 48 hrs after 2CLP. Relative to sham, 2CLP significantly increased cell death at 25 and 37% DeltaSA (p<.003, analysis of variance). Relative to sham, 2CLP did not alter expression of type I or type II phenotypic markers. Nuclear factor-kappaB within the nuclear compartment was observed after 2CLP in unstretched cells and after 1 hr of cyclic stretch at 37% DeltaSA. In sham, nuclear factor-kappaB within the nuclear compartment was seen only after stretch. CONCLUSIONS: AECs isolated from septic rats are more vulnerable to mechanical deformation injury than AECs from nonseptic animals.     

高迁移率族蛋白B1表达水平与大鼠脓毒症严重程度及预后关系的实验研究

目的探讨高迁移率族蛋白B1（HMGB1）与脓毒症严重程度及预后间的关系，寻找致死性脓毒症的可靠监测及治疗指标。方法90只SPF级雄性SD大鼠（体重250～300g）被随机分为假手术组（A组）、盲肠结扎穿孔术（CLP）组（B组）及丙酮酸乙酯（EP）治疗组（C组），每组30只，分别施以假手术（A组）或CLP（B组和C组）。术后立即腹腔注射生理盐水（Ns）2ml（A组和B组）或EP2ml（130mg／kg，C组），12h重复1次。以术后0、6、12、24、48和72h为观察点，观察各组大鼠术后活动、进食、竖毛、腹泻、眼球凹陷、呼吸等情况；活杀并观察腹腔肠管扩张、充血、腹水、病灶包裹、肺表面充血等情况。另取20只大鼠以B、C两组同样的模型及处理方式观察生存时间。采用逆转录-聚合酶链反应（RT—PCR）技术检测肝组织HMGBl mRNA表达水平，酶联免疫吸附法（ELISA）检测血浆白细胞介素-1β（IL-1β）、IL-6及肿瘤坏死因子-α（TNF-α）水平；绘制各组生存曲线，并进行相关分析。结果B组大鼠术后脓毒症表现最强，C组明显减轻。而A组没有相关表现或表现轻微。B组血浆IL-1β、IL-6和TNF-α水平于术后6h显著升高，至12h已明显下降；而C组上升的幅度明显低于B组，但明显高于A组。B组肝脏HMGBl mRNA表达水平在12h开始升高，24h达高峰，并维持至48h后开始下降，且HMGBl表达水平与IL-6水平呈正相关（r=0．91）。C组生存时间较B组明显延长（P〈0．01），HMGBl表达水平与脓毒症严重程度及动物生存率显著相关。结论脓毒症晚期释放的HMGBl是脓毒症致死效应的关键炎症介质，其表达水平与实验动物的脓毒症严重程度及预后高度相关。     

黄芩苷对脓毒症大鼠早期肾功能损伤的保护作用

目的：通过观察黄芩苷对脓毒症大鼠血清中性粒细胞明胶酶相关载脂蛋白（NGAL）浓度的影响,探讨黄芩苷对脓毒症大鼠肾功能损伤的早期保护作用。方法：采用盲肠结扎穿孔术（CLP）制备SD大鼠脓毒症模型。实验大鼠随机分成假手术组、模型组、黄芩苷干预组（每纽各24只）,每组大鼠再随机分为0h、3h、6h、24h4个亚组（每组各6只）。黄芩苷组在造模后立即经腹腔给予黄芩苷120mg／kg共2mL液体,假手术组和模型组则给予等量％0．9氯化钠液腹腔注射。每组大鼠在4个时间点（CLP术后0h、3h、6h、24h）采集血标本和肾组织标本。HE染色观察肾组织病理改变;检测血清NGAL和肌酐浓度和用免疫组织化学法测定肾组织肿瘤坏死因子“（TNF-α）水平。结果：与假手术组相比,模型组大鼠血清NGAL和肌酐浓度显著升高（P〈0．05）,术后3h开始升高,术后24h继续升高;肾组织TNF-α水平也显著升高（P〈0．05）,术后3h开始升高,术后24h仍升高;术后24h肾组织HE染色显示肾组织内大量炎症细胞浸润。与模型组相比,黄芩苷干预组术后3h、6h和24h血清NGAL和肌酐浓度以及肾组织TNF-α水平均显著降低（P〈0．05）,肾脏组织的炎症病理改变也明显减轻。结论：黄芩苷对脓毒症大鼠早期肾功能损伤有保护作用。     

The activity of tissue factor pathway inhibitor in experimental models of superantigen-induced shock and polymicrobial intra-abdominal sepsis

OBJECTIVES: To study recombinant human tissue factor pathway inhibitor (rhTFPI) in a superantigen-induced shock model and in a cecal ligation and puncture (CLP) model of peritonitis in mice. DESIGN: Prospective, randomized, experimental study. SETTING: An experimental animal research laboratory. SUBJECTS: Eighty BALB/c mice for the superantigen model, and 56 BALB/c mice for the CLP model. INTERVENTIONS: In the superantigen-induced shock model, animals received rhTFPI (350 mg/kg) subcutaneously every 12 hrs (n = 30) or saline control (n = 30) for 60 hrs after staphylococcal enterotoxin B (SEB; 10 microg iv) and a sublethal dose of E. coli 0111:B4 lipopolysaccharide (LPS; 75 microg ip). Control groups received SEB alone (n = 10) and LPS alone (n = 10). In the CLP model, rhTFPI or saline was given every 8 hrs for 48 hrs by using a 21-gauge needle (n = 9) or 23-gauge needle (n = 14) for CLP. A sham surgery control group (n = 10) was also included. MEASUREMENTS AND MAIN RESULTS: There was 0% mortality in the SEB and LPS control groups. The mortality rate was 64% in the saline control group that received both SEB and LPS (19 of 30), whereas the rhTFPI- treated animals had a mortality rate of 20% (6 of 30; p < .01). The rhTFPI-treated group had significantly lower interleukin-6 levels (61.8 +/- 41 pg/mL vs. 285 +/- 63 pg/mL; p < .05) than the control group but no differences in tumor necrosis factor-alpha or interferon-gamma levels. In the CLP experiment, rhTFPI-treated animals did not have any survival advantage over the control group after the large-bore (21-gauge) needle puncture. The rhTFPI group had significantly improved 7-day mortality rate after CLP with the small-bore needle (23-gauge; 21.4% [rhTFPI] vs. 71.4% [control], p < .01). Plasma LPS, interleukin-6, interferon-gamma, and tumor necrosis factor-alpha levels were unchanged by rhTFPI treatment, but significantly reduced LPS (p = .006) and IFNgamma (p = .001) levels were found in the peritoneal fluid. CONCLUSIONS: Tissue factor pathway inhibitor significantly improves the mortality rate in models of superantigen-induced shock and polymicrobial intra-abdominal infection, supporting its potential use in clinical trials for septic shock.