烫伤脓毒症大鼠肾脏核转录因子-κB活化与肾损伤关系的研究

目的研究烫伤脓毒症大鼠肾脏核转录因子-κB (NF-κB)活化与肾损伤的关系.方法采用30%总体表面积Ⅲ度烫伤加内毒素攻击制备烫伤脓毒症大鼠模型.54只Wistar大鼠随机分为正常对照组、烫伤脓毒症1、2、6、12和24 h组,烫伤脓毒症1、2和6 h+NF-κB抑制剂吡咯烷二硫基甲酸酯(PDTC)组.采用凝胶电泳迁移率改变分析法(EMSA)检测肾组织NF-κB活性;采用酶联免疫吸附法检测血浆及肾组织中肿瘤坏死因子-α(TNF-α)含量的变化;采用自动生化分析仪检测血肌酐(SCr)和尿素氮(BUN)含量.结果肾组织NF-κB活性于烫伤脓毒症后1 h明显增强并达到高峰(P<0.01),PDTC可显著降低烫伤脓毒症后1 h NF-κB的活性.烫伤脓毒症后1 h和2 h血浆及肾组织中TNF-α水平均明显增高(P均<0.01),PDTC可显著降低伤后血浆TNF-α水平(P均<0.01),对肾组织中TNF-α水平影响不明显.烫伤脓毒症后BUN及SCr含量均明显增高(P均<0.01),PDTC对BUN和SCr含量均无显著影响.结论 NF-κB抑制剂可降低烫伤脓毒症大鼠肾组织NFκB活性,但对肾脏功能无明显保护作用.     

内毒素耐受及其与核转录因子-kB的关系

内毒素多次刺激单核／巨噬细胞后可产生内毒素耐受现象，其机制主要为内毒素信号转导功能障碍导致核转录因子-kB（NF-kB）转位受损。创伤、脓毒症、内毒素血症和全身炎症反应综合征（SIRS）患者均具有内毒索耐受特征。内毒素耐受现象应用于临床可能会降低脓毒症和SIRS患者的病死率。     

大黄对脓毒症大鼠核因子-kB活化的抑制作用

目的:探讨脓毒症时肠道组织肿瘤坏死因子-α(TNF-α)、单核细胞趋化蛋白-1(MCP-1)及核因子-κB(NF-κB)活性的相关性,揭示大黄治疗脓毒症的有效机制.方法:采用盲肠结扎穿孔术制备大鼠脓毒症模型.分别在术后1、3、6、12、24、48 h活杀大鼠取肠黏膜组织,用酶联免疫吸附法测TNF-α、MCP-1的含量,用凝胶电泳迁移法测NF-κB的活性.在脓毒症模型基础上加用大黄治疗,分别于治疗后12、24、48、72 h活杀大鼠,用同样的方法检测TNF-α、MCP-1含量及NF-κB活性. 结果:脓毒症大鼠肠黏膜NF-κB活性及TNF-α、MCP-1含量均较相应对照组明显升高(P均<0.01);大黄治疗后能明显抑制升高的NF-κB活性和TNF-α、MCP-1含量(P均<0.05).结论:TNF-α、MCP-1是脓毒症早期激活的细胞因子,其释放与NF-κB活性密切相关;大黄可通过抑制NF-κB活性、减少炎症细胞因子释放而达到抑制炎症反应的作用.     

泛素-蛋白酶体途径对烫伤脓毒症大鼠肠道炎症反应与屏障功能的作用研究

目的研究泛素-蛋白酶体途径对烫伤脓毒症大鼠肠组织核转录因子-κB（NF-κB）活化、肿瘤坏死因子-α（TNF-α）表达以及血浆二胺氧化酶（DAO）活性的作用。方法采用30％总体表面积（TBSA）Ⅲ度烫伤合并内毒素攻击大鼠为模型模拟临床烫伤哝毒症。60只Wistar大鼠随机分为正常对照组、烫伤哝毒症模型组、泛素-蛋白酶体抑制剂N-乙酰亮氨酰亮氨酰正亮氨酸（ALLN）组、NF-κB抑制剂吡咯烷二硫基甲酸酯（PDTC）组。采用凝胶电泳迁移率改变分析法（EMSA）分析肠组织NF—κB活性；采用酶联免疫吸附法（ELISA）检测肠组织TNF-α含量；采用分光光度法检测血浆DAO活性。结果各组肠组织NF—κB活性于伤后1h均明显增强，并达到高峰（P均〈0．01）．之后呈现下降趋势；两种抑制剂均可显著降低伤后1h和2hNF-κB的活性。ALLN可明显降低伤后1h肠组织中TNF—α含量（P〈0.01）。两种抑制剂对伤后1h和2h血浆I）AO活性均无明显影响。结论早期使用泛素-蛋白酶体抑制剂可降低烫伤脓毒症大鼠肠组织NF—κB活性．降低肠组织中炎症反应．但对肠组织屏障功能无保护作用。     

性别差异对脓毒症大鼠肝脏核因子-κB活化的影响

目的探讨性别差异对脓毒症大鼠肝脏组织核因子-kB(NF-kB)活化的影响。方法取40只清洁级Wistar大鼠,雌雄各半,采用腹腔注射内毒素(LPS)5mg/kg制作脓毒症大鼠模型,利用凝胶阻滞迁移分析方法(EMSA)检测肝脏组织NF-kB的活化情况,同时检测大鼠血清中丙氨酸氨基转移酶(ALT),肿瘤坏死因子-α(TNF-α)以及雌二醇(E2)含量。结果正常雌雄性大鼠肝脏组织有NF-kB的微弱活化,同时血清中TNF-α及ALT含量较少,各项指标在两组大鼠间差异无显著性(P＞0．05);脓毒症状态下各项指标明显升高,但是雌性大鼠各项指标的变化明显低于雄性(P＜0．01);相关分析表明,雌雄性脓毒症大鼠肝脏组织NF-kB的活化以及血清中TNF-α,ALT含量均与相应组别大鼠血清中E2含量呈显著负相关(P＜0．05)。结论脓毒症大鼠肝脏组织NF-kB的活化及血清中TNF-α,ALT含量存在性别差异,内源性雌激素可能介导了对雌性脓毒症大鼠肝脏的保护作用。     

核转录因子-kB1基因多态性与早发冠心病的相关性研究

目的探讨核转录因子家族中NF-Kb1基因-94ins/delATTG 的多态性与我国早发冠心病（早发CHD）的相关性。方法对早发冠心病（病例组）92例和非冠心病（对照组）94例患者,采用苯酚-氯仿法提取DNA,聚合酶链反应-限制性片段长度多态性方法（PCR-RFLP）分析NF-kappaB1基因多态性。结果两组病例del/del型纯合子,ins/ins型纯合子,del/ins型杂合子基因型频率及ins、del等位基因频率差异无统计学意义。结论 NF-kappaB1基因多态性与我国早发冠心病的易感性可能无关。     

核因子κB在黄芩苷对脓毒症大鼠肾脏保护中的作用

目的：探讨核因子κB（NF-κB）在黄芩苷对脓毒症大鼠肾脏保护中的作用及其机制。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（每组各24只）,每组大鼠再随机分为CLP术后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注射黄芩苷120mg/kg,共2mL液体,假手术组和模型组则腹腔注射等量0.9%氯化钠液。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;检测血清肌酐水平和用免疫组织化学法测定肾组织肿瘤坏死因子α（TNF-α）、NF-κB在肾组织内的水平。结果：与假手术组相比,模型组大鼠血清肌酐浓度及肾组织TNF-α、NF-κB水平均显著升高（P〈0.05）,术后3h即开始升高并持续至术后24h;术后24h肾组织HE染色显示肾小管区大量炎症细胞浸润。与模型组相比,黄芩苷干预组术后3h、6h和24h血清肌酐浓度及肾组织TNF-α、NF-κB水平均显著降低（P〈0.05）;肾脏组织的炎症病理改变也显著减轻。结论：脓毒症大鼠早期血清肌酐升高与肾组织中TNF-α、NF-κB水平升高有关;脓毒症早期应用黄芩苷治疗对脓毒症大鼠肾脏保护作用的机制可能与降低肾组织NF-κB水平,减轻炎性反应有关。     

地塞米松对脓毒症大鼠心肌保护作用的研究

目的观察脓毒症大鼠心脏功能、心肌力学的改变并探讨地塞米松对心脏的保护作用及其机制。方法将96只Wistar大鼠随机分为对照组(32只)、脂多糖(LPS)组(32只,静脉注射LPS4mg/kg)及LPS+地塞米松组(32只,注射LPS的同时静脉注射地塞米松2mg/kg)。各组动物均于实验前(0h)及给药后2、4和6h采血(每个时间点8只)。经右颈总动脉左心室内插管,监测各组动物左心室功能变化并检测血清肿瘤坏死因子α(TNFα)、血浆肌钙蛋白T(TnT)含量。结果1脓毒症状态下TnT显著升高;应用地塞米松后TnT明显降低〔给药后6h为(1.76±0.57)μg/L比(0.70±0.36)μg/L,P<0.01〕。2脓毒症状态下左心室收缩峰压(LVPP)、左心室压力上升/下降最大速率(±dp/dtmax)均有不同程度变化,左心室舒张末期压(LVEDP)升高;应用地塞米松后可明显改善各项指标。3脓毒症状态下TNFα浓度明显升高,应用地塞米松可显著降低TNF浓度〔给药后2h为(11.22±2.38)pmol/L比(7.62±3.21)pmol/L,P<0.01〕。结论脓毒症状态下心肌可发生明显损伤;TNFα为脓毒症状态下心肌损害因素;应用地塞米松可明显减轻内毒素对心肌的损伤。     

核转录因子-kB、表皮生长因子受体在食管鳞状细胞癌组织中的表达及意义

目的:研究核转录因子-kB[NF-kB(p65)]、表皮生长因子受体(EGFR)在食管鳞状细胞癌组织中的表达及其在食管鳞癌侵袭发展中的作用及可能机制.方法:应用免疫组织化学方法检测40例食管鳞癌患者癌组织及40例正常对照组织中NF-kB(p65)、EGFR的表达.结果:NF-kB(p65)、EGFR在食管鳞癌和正常对照组表达阳性率分别为87.5%/90% vs 42.5%/25%,P＜0.05;在早期和中晚期食管癌表达阳性率NF-kB(p65)为55.6% vs 96.8%,P＜0.01,EGFR为77.8% vs 93.5%,P＞0.05;它们在食管鳞癌无转移和转移组表达阳性率为42.9%/57.1% vs 97%/97%,P＜0.05.结论:NF-kB(p65)、EGFR在食管鳞癌组织中表达增高,可能与肿瘤发生发展相关,EGFR可能是通过激活NF-kB途径来实现的.     

粒细胞-巨噬细胞集落刺激因子对失血性休克诱导急性肺损伤小鼠器官组织核转录因子-κB活化的影响

目的探讨特异性抗粒细胞-巨噬细胞集落刺激因子（GM—CSF）的抗体（22E9）和地塞米松（DEX）对失血性休克诱导急性肺损伤（ALI）小鼠肾、肝、心、肺组织核转录因子-κB（NF-κB）活化的影响，为失血性休克继发的多器官损伤防治提供理论依据。方法C57BL／6雄性小鼠20只，用心脏穿刺致失血性休克诱导ALI小鼠模型。制模前经鼻分别滴入磷酸盐缓冲液（PBS，PCG组）、PBS＋1μg 22E9（HS1组）、PBS＋10pg22E9（HS10组）、PBS＋20μgDEX（DEX组）进行干预，阴性空白对照组（NCG组）经鼻滴入PBS后仅予心脏穿刺。休克4h时采用凝胶电泳迁移率改变分析法（EMSA）检测肺、心、肝、肾组织NF—κB活性；用酶联免疫吸附法（ELISA）检测肺、心组织肿瘤坏死因子-α（TNF—α）含量。结果与PCG组比较，高、低剂量22E9均可显著抑制肝、心、肺组织NF—κB活性，且低剂量的抑制效果比高剂量明显，同时可增加肾组织NF—κB活性（P均〈0．05）。DEX可增强肾、肝组织NF—κB活性（P均〈0．05），但与PCG组比较，DEX对肾、肝、肺组织NF—κB活性未见明显的抑制作用。22E9干预可显著抑制心、肺组织TNF—α含量的升高，DEX仅能有效抑制心组织TNF—α含量（P均〈0．05）。结论22E9能显著抑制失血性休克诱导ALI小鼠多器官组织NF-κB的活化及炎症反应，减少失血性休克引起的多器官损伤；DEX的抑制作用不显著。     

血必净对急性肺损伤兔核因子-κB活性变化的研究

目的观察血必净注射液对脂质糖(LPS)诱导的急性肺损伤(ALI)兔肺组织炎症相关细胞因子的影响,探讨其发挥抗炎作用的细胞信号调控转导机制。方法新西兰兔80只随机分为第一组(空白对照组)、第二组(单纯急性肺损伤组,股静脉注射LPS)、第三组(单纯股静脉注射血必净)、第四组(血必净联合LPS治疗)。观察病理形态和ALI生物标志物变化,并用放射免疫法(RIA)测定肺组织白细胞介素(IL)-6、IL-8、肿瘤坏死因子(TNF-α)水平,凝脉电泳迁延率法(EMSA)加计算机图象分析技术测白细胞兔核因子-κB(NF-κB)活性。结果与单纯急性肺损伤组比较,血必净可减轻兔肺组织的炎性病变,显著降低IL-6、IL-8、TNF-α、NF-κB含量,组间比较差异具有统计学意义(P<0.01)。结论中药制剂血必净注射液通过抑制核因子-κB活性而阻断炎症因子IL-6、IL-8、TNF-α的合成和释放。     

蛋白酶体抑制剂在烫伤脓毒症大鼠肺脏炎症反应中的作用

目的研究干预泛素-蛋白酶体途径对烫伤脓毒症大鼠肺脏核因子-(?)B(NF-(?)B)活化、肿瘤坏死因子-α的产生以及髓过氧化物酶(MPO)活性的影响。方法采用30%TBSAⅢ度烫伤加内毒素攻击大鼠为模型模拟临床烫伤脓毒症,72只Wistar大鼠随机分为正常对照组、烫伤脓毒症组、烫伤脓毒症 蛋白酶体抑制剂N-Acetyl-leucinyl-leucinyl-norleucinal(ALLN)组、烫伤脓毒症 NF-(?)B抑制剂吡咯烷二硫基甲酸酯(Pyrrolidine Dithiocarbamate．PDTC)组,采用凝胶电泳迁移率改变分析法(EMSA)分析肺脏NF-(?)B活性,采用酶联免疫吸附试验检测肺脏TNF-α的变化,采用分光光度法检测肺组织髓过氧化物酶(MPO)的活性。结果肺组织NF-(?)B活性于伤后1h明显增强达到高峰(P＜0．01),伤后2h仍保持较高的活化水平,之后呈逐渐下降趋势。PDTC可明显降低其在伤后1h和2h的活性(P＜0．01),而ALLN可明显降低其在伤后1h的活性(P＜0．01)。PDTC两种抑制剂均可明显降低伤后2h肺组织TNF-α产生,明显降低伤后2h和6h肺组织MPO的活力(P＜0．01)。结论蛋白酶体抑制剂可降低烫伤脓毒症大鼠肺组织NF-(?)B的活性。降低肺组织的炎症反应。     

脓毒症患者肾损伤程度与肾损伤分子-1表达水平的相关性研究

目的 探讨脓毒症患者肾损伤程度与尿液肾损伤分子-1(Kim-1)表达水平的相关关系,为脓毒症急性肾损伤(AKI)的早期诊断和预防提供简便、可靠的循证医学依据.方法 65例入住ICU的脓毒症患者,根据肾损伤程度,按AKIN诊断和分期标准,分为非AKI组(A组,n=36),轻度AKI组(B组,n=13),中重度AKI组(C组,n=16).另外选取20例健康体检者作为健康对照组(D组,n=20).采集患者静脉血和尿液,用ELISA法检测尿Kim-1表达水平,酶法测定血肌酐(Scr)水平,并计算肌酐清除率(Ccr),分析Kim-1表达水平与肾损伤程度之间的关系;绘制受试者工作特征曲线(ROC),计算曲线下面积(AUC),评价Kim-1对脓毒症AKI的诊断价值.结果 与A组及D组比较,B、C两组尿Kim-1水平均有明显升高(P均<0.05);其中C组较B组升高更明显(P<0.01);各组Kim-1的表达水平与Ccr水平呈负相关(r=-0.792);Kim-1的AUC为0.814(P<0.01).结论 脓毒症AKI患者尿液Kim-1的表达水平较正常人或脓毒症非AKI患者显著增加,其增加幅度与AKI的严重程度呈显著正相关,Kim-1可以作为检测脓毒症AKI的敏感指标.     

Significance of biopterin induction in rats with postburn Staphylococcus aureus sepsis

It has been demonstrated that biopterin, an essential cofactor of nitric oxide synthase (NOS), plays an important role in the pathogenesis of endotoxin-induced shock, yet its biological significance in gram-positive sepsis remains unclear. In this study, we adopted a rat model of postburn Staphylococcus aureus sepsis to investigate the potential role of biopterin in the pathogenesis of gram-positive sepsis. Wistar rats were inflicted with a 20% total body surface area (TBSA) full-thickness scald injury followed by S. aureus challenge, and then guanosine triphosphate-cyclohydrolase I (GTP-CHI) mRNA expression and biopterin levels in liver, kidneys, lungs, and heart were determined. We found that after S. aureus challenge, GTP-CHI gene expressions and biopterin levels were markedly upregulated in various tissues. Meanwhile, multiple organ dysfunction was induced by S. aureus challenge. It was shown that cardiac GTP-CHI mRNA expression and renal BH(4) levels were positively correlated with MB isoenzyme of creatine kinase (CK-MB) and creatinine (r = 0.892, P = 0.0012 and r = 0.9423, P = 0.0015, respectively). These results suggested that thermal injury combined with S. aureus challenge could induce de novo biosynthesis of biopterin, which might play a role in the development of multiple organ dysfunction syndrome secondary to postburn sepsis.     

Acute kidney injury in sepsis

Acute kidney injury (AKI) and sepsis carry consensus definitions. The simultaneous presence of both identifies septic AKI. Septic AKI is the most common AKI syndrome in ICU and accounts for approximately half of all such AKI. Its pathophysiology remains poorly understood, but animal models and lack of histological changes suggest that, at least initially, septic AKI may be a functional phenomenon with combined microvascular shunting and tubular cell stress. The diagnosis remains based on clinical assessment and measurement of urinary output and serum creatinine. However, multiple biomarkers and especially cell cycle arrest biomarkers are gaining acceptance. Prevention of septic AKI remains based on the treatment of sepsis and on early resuscitation. Such resuscitation relies on the judicious use of both fluids and vasoactive drugs. In particular, there is strong evidence that starch-containing fluids are nephrotoxic and decrease renal function and suggestive evidence that chloride-rich fluid may also adversely affect renal function. Vasoactive drugs have variable effects on renal function in septic AKI. At this time, norepinephrine is the dominant agent, but vasopressin may also have a role. Despite supportive therapies, renal function may be temporarily or completely lost. In such patients, renal replacement therapy (RRT) becomes necessary. The optimal intensity of this therapy has been established, while the timing of when to commence RRT is now a focus of investigation. If sepsis resolves, the majority of patients recover renal function. Yet, even a single episode of septic AKI is associated with increased subsequent risk of chronic kidney disease.     

The potential role of Staphylococcal enterotoxin B in rats with postburn Staphylococcus aureus sepsis

Staphylococcal enterotoxin B (SEB) is an important member of the superantigen family, which exerts a number of pathological effects in the human, as well as susceptible animals. The present study was conducted to observe the time course and tissue distribution of SEB in postburn Staphylococcus aureus infection; meanwhile, the relationship between SEB and multiple organ dysfunction was also studied. Eighty-six male Wistar rats were randomly divided into four groups as follows: normal control group (n = 10); scald control group (n = 10); postburn sepsis group (n = 50) in which rats inflicted with 20% total body surface area (TBSA) III degrees scald followed by SEB-producing S. aureus challenge were further divided into 0.5-, 2-, 6-, 12-, and 24-h subgroups, with 10 rats in each subgroup; and SEB monoclonal antibody (MAb) treatment group (n = 16) in which a dose of 4 mg/kg SEB MAb was given intravenously just before S. aureus challenge, and the rats were further divided into 2- and 6-h subgroups. It was found that after thermal injury combined with S. aureus infection, SEB was widely distributed to the liver, kidneys, lungs, and heart, exacerbating the pathophysiology of multiple organ dysfunction induced by postburn sepsis. At the same time, the gene and protein expressions of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were also markedly upregulated in various tissues. Early treatment with SEB-specific MAb-MAb2D(1)-could markedly decrease SEB levels in plasma as well as in various tissues, and could significantly reduce the 6-h mortality rate (17.64% [3/17] vs. 55.6% [20/36], P = 0.02). These data suggested that neutralization of SEB is effective in ameliorating S. aureus sepsis and subsequent multiple organ damage, which might be attributed to its inhibitory effect on inflammatory mediator formation.     

生物喋呤对烫伤脓毒症大鼠一氧化氮合成的影响

目的：探讨生物喋呤BH4在金黄色葡萄球菌（简称金葡菌）脓毒症中的生物学效应，阐明BH4对一氧化氮（NO）诱生的调控作用。方法：76只Wistar大鼠随机分为正常对照组（n=10)、烫伤对照组（n=10)、烫伤后金葡菌感染组（n=40)和羟基嘧啶（DAHP）拮抗组（n=16)。无菌留取动物肝、肺组织采用RT－PCR方法检测三磷酸鸟苷环水解酶I（GTP－CHI）、诱生型一氧化氮合酶（iNOS)基因表达，同时测定组织中BH4和NO的水平。结果：烫伤后金葡菌感染组动物肝、肺组织中GTP－CHI基因表达明显上调，BH4产生显著增加，iNOS mRNA表达和NO的水平亦明显升高，DAHP组GTP－CHI基因表达上调和BH4合成NO的产生亦明显下降。结论：烫伤后金葡菌感染可诱导体内BH4的合成，BH4在基因和蛋白水平调控着iNOS所介导的NO大量生成，从而对金葡菌脓毒症的病理过程起促进作用。