生物喋呤合成酶抑制剂对烫伤大鼠金葡菌脓毒症的保护效应及机制

目的　探讨生物喋呤合成限速酶抑制剂 - 2 ,4 二胺 6 羟基嘧啶 (DAHP)对金黄色葡萄球菌 (简称金葡菌 )脓毒症的保护效应及机制。　方法　 5 6只Wistar大鼠随机分为正常对照组、2 0 %TBSAⅢ度烫伤对照组、烫伤后金葡菌感染组和DAHP拮抗组。无菌留取大鼠心、肝、肺、肾组织检测三磷酸鸟苷环水解酶I(GTP CHI)、诱生型一氧化氮合酶 (iNOS)及肿瘤坏死因子α (TNFα )基因表达 ,同时测定组织中四氢生物喋呤 (BH4)和一氧化氮 (NO)的水平。　结果　烫伤后金葡菌感染可导致组织GTP CHI基因表达广泛上调、BH4合成显著增加。同时 ,组织iNOSmRNA表达和NO水平亦明显升高 ,其中肝、肺改变尤为显著。给予DAHP不仅可显著抑制各组织GTP CHImRNA表达和BH4的产生 (P<0 .0 5～ 0 .0 1) ,iNOSmRNA表达和NO的生成亦明显受抑 ,同时TNFα表达也明显降低。此外 ,DAHP拮抗组动物 6h死亡率有所降低 (分别为 2 5 .0 %和 5 5 .6 % ,P =0 .0 8)。　结论　DAHP早期干预可在一定程度上改善革兰阳性菌脓毒症动物的预后 ,其机制可能与DAHP抑制体内BH4和NO的产生有关。     

生物喋呤对烫伤脓毒症大鼠肝组织丝裂原激活蛋白激酶p38活化的影响

目的：探讨烫伤脓毒症时生物喋呤对肝组织丝裂原激活蛋白激酶p38(p38 MAPK)活化的影响及其病理生理意义。方法：采用大鼠20％TBSAⅢ度烫伤复合金黄色葡萄球菌攻击模型，动物随机分为正常对照组、烫伤对照组、烫伤脓毒症组和生物喋呤合成抑制剂（DAHP）治疗组。采用Western blot技术检测肝组织p38 MAPK的磷酸化状态。结果：正常肝组织磷酸化p38MAPK蛋白水平很低。烫伤脓毒症后2h，肝组织p38MAPK磷酸化水平即明显升高，且持续至伤后24h。生物喋呤合成抑制剂早期干预则可显著降低肝组织p38 MAPK磷酸化水平。结果：烫伤脓毒症时生物喋呤与肝组织p38 MAPK活化密切相关。     

脓毒症大鼠生物喋呤的组织分布特点和意义

目的　探讨腹腔感染致脓毒症时重要器官生物喋呤及其合成限速酶基因表达的改变和病理生理意义。方法　腹腔感染致脓毒症模型采用盲肠结扎穿孔法(CLP),用反相高效液相分析法和逆转录-聚合酶链反应方法测定24只大鼠肝、肺、肾等组织生物喋呤含量及三磷酸鸟苷环水解酶I(GTP-CHI)mRNA的表达。结果　脓毒症大鼠2h时肝、肺、肾组织生物喋呤含量显著增多［分别为(4.18±0.16)、(2.71±0.32)、(2.45±0.27)ng/g蛋白］,同时不同组织GTP-CHImRNA表达亦明显增强,各脏器功能均表现不同程度地损害(P＜0.05)。相关分析显示,肝、肺组织生物喋呤与反映相应脏器功能的指标呈高度正相关(分别为r=0.7916,P＜0.001和r=0.8004,P＜0.001)。结论　生物喋呤参与了腹腔感染所致脓毒症的发生、发展过程。     

细胞外信号调节激酶在内毒素休克大鼠生物喋呤诱生中的作用机制探讨

目的观察细胞外信号调节激酶（ERK）通路抑制剂对生物喋呤（BH4）和一氧化氮（NO）表达及核因子-kB（NF-kB）活化的影响,探讨内毒素休克时ERK信号通路与NF-kB的交汇作用及其对BH4诱生NO的调控机制。方法采用内毒素休克模型,60只大鼠随机分为正常对照组（n=8）、内毒素休克组（n=32）和ERK抑制剂PD98059拮抗组（n=20）。留取动物肝、肺、肾组织进行NF-kB活性分析以及三磷酸鸟苷环水解酶I（GTP—CHⅠ）、诱生型一氧化氮合酶（iNOS）基因表达的检测,并测定组织及血浆中BH4、NO水平。结果内毒素攻击可导致动物肝、肺、肾组织GTP-CHⅠ基因表达和BH4水平明显升高,至伤后24h仍持续于较高水平;与之相应,组织iNOS基因表达和NO水平亦明显升高;各组织NF-kB迅速活化,并于2h达峰值。采用PD98059处理后,内毒素休克动物肾组织GTP—CHⅠ mRNA表达明显受抑,肝、肺组织GTP—CHⅠmRNA表达仅呈现降低趋势;血浆及肝、肾组织中BH4水平12h显著降低;同样,各组织iNOS mRNA表达及NO水平早期亦显著降低。此外,PD98059处理组动物肝组织2～6h、肺组织2h、24h和肾组织24h时相点NF-KB活性显著降低。结论内毒素休克时抑制ERK通路,能部分下调BH4和NO表达与NF-kB的活化,表明ERK与NF-kB通路间可能存在交汇作用,共同参与了BH4诱生NO的调控作用。     

高迁移率族-1 蛋白在烫伤后金葡菌脓毒症中的改变与意义

目的 探讨高迁移率族－1（HMG－1）蛋白在烫伤后金黄色葡萄球菌（简称金葡菌)脓毒症中的变化规律及其调控机制。方法 采用大鼠20％体表面积Ⅲ度烫伤复合金葡菌攻击所致脓毒症模型。70只动物随机分为正常对照组（n=10）、烫伤对照组（n＝10）和烫伤后金葡菌感染组（n＝50）,留取肝、肺组织检测HMG－1及脂多糖结合蛋白（LBP）mRNA表达 ,同时测定组织中金葡菌肠毒素B（SEB）和内毒素含量。结果 烫伤后金葡菌感染可导致动物肝、肺组织HMG－1基因表达明显升高,于伤后6－12h达峰值（P＜0．05－0．01）,至24h仍持续于较高水平。相关分析显示,肝、肺组织LBP基因表达与相应脏器HMG－1mRNA表达呈显著正相关（分别为r＝0．800,P＝0．031和r＝0．942,P＝0．002）,但内毒素与之无明显相关性。结论 烫伤后金葡菌感染可导致动物体内HMG－1基因表达上调,后者作为“晚期炎症介质”可能参与了脓毒症的发病过程。     

内毒素休克大鼠组织生物喋呤及其合成限速酶基因表达的改变

目的 了解内毒素休克时重要器官生物喋呤及其合成限速酶基因表达的改变和病理生理意义。方法 采用大鼠内毒素休克模型,检测肝,肾,肠等组织生物喋呤含量,三磷酸鸟苷环水解酶Ｉ（ＧＴＰ－ＣＨＩ）ｍＲＮＡ表达及器官功能指标等。并观察内毒素拮抗剂－重组杀菌／通透性增加蛋白（ｒＢＰＩ２１）的防治效果。结果 内毒素休克动物肝,肾,肠等组织生物喋呤含量明显增多,伤后８小时升高幅度尤为显著（Ｐ〈０．０５,Ｐ〈０．０１０     

烫伤脓毒症大鼠细胞因子信号转导抑制因子基因表达的改变与意义

目的观察烫伤后金黄色葡萄球菌(金葡菌)脓毒症大鼠重要脏器细胞因子信号转导抑制(SOCSs)基因表达的规律及其与细胞因子变化的关系.方法采用大鼠20%体表面积Ⅲ°烫伤后金葡菌攻击致脓毒症模型,检测动物肝、肺组织中SOCS1、SOCS2和SOCS3 mRNA表达,并测定组织中干扰素-γ(INF-γ)水平.结果烫伤后金葡菌感染大鼠肝、肺组织IFN-γ产生均显著增多,分别于伤后0.5 h和6 h达峰值(P＜0.01).同时,动物肺组织SOCS1、SOCS2和SOCS3 mRNA表达均明显上调,其中SOCS2和SOCS3 mRNA表达改变较为迅速,伤后0.5 h即明显高于对照组,2h达峰值.肝组织SOCS1 mRNA表达于伤后2 h明显增强,24 h仍维持于较高水平;而肝脏SOCS2和SOCS3 mRNA表达仅呈现升高趋势.金葡菌肠毒素B单抗早期干预后,随着肺脏IFN-γ产生减少,肺组织SOCS1、SOCS2和SOCS3基因表达亦明显降低.结论烫伤后金葡菌感染可诱导体内SOCSs表达上调,其改变与IFN-γ的"消涨"密切相关,提示它们可能参与了金葡菌脓毒症时炎症反应平衡的调节过程.     

生物喋呤在脓毒症中的作用及其相关信号转导机制的研究进展

许多研究证实，一氧化氮(NO)过度产生在脓毒性休克和多器官功能障碍综合征的发生中具有重要意义，而生物喋呤(主要为5，6，7，8-四氢生物喋呤，tetrahydrohiopterin，BH4)为一氧化氮合酶(NOS)重要的辅因子，调控着细胞内NO的产生，近年研究表明它可能参与了脓毒性休克的病理、生理过程。本文主要介绍BH4的生理及病理作用，以及相关信号转导调控机制在脓毒症中的意义。     

烫伤合并金葡菌感染大鼠组织CD14 mRNA的改变

目的　探讨细菌脂多糖受体CD14在烫伤合并金黄色葡萄球菌 (金葡菌 )感染中的变化规律及其意义。　方法　采用大鼠 2 0 %总体表面积Ⅲ度烫伤合并金葡菌攻击造成脓毒症模型 ,动态检测心、肝、肺、肾等重要器官中CD14mRNA表达的改变 ,同时观察内毒素在动物循环及主要脏器内的分布特点。　结果　烫伤合并金葡菌脓毒症早期 ,各脏器内毒素含量即明显高于正常对照组 ,并于2～ 6h达峰值 ,其中以肝、肺组织内毒素水平升高幅度最为显著 (P <0 .0 5 )。而血浆内毒素水平亦于伤后 2h显著高于正常对照组 (分别为 0 .30 5 6EU/ml和 0 .12 5 0EU/ml,P <0 .0 5 )。与此同时 ,小肠组织中二胺氧化酶的活性明显降低 (P <0 .0 5 )。烫伤合并金葡菌感染后 ,各组织CD14mRNA的表达亦呈不同程度升高 (P <0 .0 5 ) ,其中肺脏改变尤为显著 ,伤后 6、2 4h肺脏CD14mRNA表达分别为正常对照组的 1.80和 1.81倍。　结论　烫伤合并金葡菌攻击可导致内毒素移位和组织CD14mR NA表达不同程度升高 ,CD14基因表达的上调可能与移位内毒素的刺激作用有关。     

诱生型一氧化氮合酶在胆道感染大鼠肝细胞中的表达

目的　了解诱生型一氧化氮合酶 (induciblenitricoxidesynthase ,iNOS)在胆道感染大鼠肝细胞中表达的情况及其规律。方法　制作大鼠胆道感染模型 ,采用还原型辅酶Ⅱ黄递酶组织化学法检测大鼠肝细胞中iNOS的表达。结果　大鼠胆道感染 2h后肝细胞即有iNOS的表达切片积分光度(13 5 8± 0 6 4) ,与对照组切片积分光度 (3 5 9± 0 2 8)相比 ,P <0 0 1。 8h达到峰值切片积分光度(2 9 2 7± 0 90 ) ,2 4h至 48h仍有较高表达切片积分光度分别为 (19 47± 0 6 5 )和 (19 96± 0 78)。结论　胆道感染时肝细胞可持续高效地表达iNOS ,提示胆道感染时肝脏是合成NO的重要器官 ,并可能对胆道感染的转归具有重要影响     

The effect of 2,4-diamino-6-hydroxy-pyrimidine on postburn Staphylococcus aureus sepsis in rats

GTP-cyclohydrolase I (GTP-CHI) is the first and rate-limiting enzyme for the de novo biosynthesis of biopterin. The present study was to observe the effect of 2,4-diamino-6-hydroxy-pyrimidine (DAHP),an inhibtor of GTP-CHI, on the development of postburn Staphylococcus aureus sepsis. Methods: 56 male Wistar rats were randomly divided into four groups as follows: normal control group (n= 10), scald control group(n= 10),pos tburn sepsis group (n= 20) and DA HP treatment group (n= 16). In the scald control group, rats were subjected to a 20% total body surface area (TBSA) Ⅲ° scald injury, then sacrificed at 24 hrs. In the postburn sepsis group (n=20), rats were inflicted with 20% TBSA Ⅲ° scald followed by Staphylococcus aureus challenge, and they were further divided into 2 and 6 hrs groups. In the DAHP treatment group (n= 16), animals were intraperitoneally injected with a dose of 1g/kg DAHP prior to Staphylococcus aureus challenge, and then further divided into 2, 6 hrs groups. Tissue samples from liver, kidneys, lungs and heart were collected to determine GTP-CHI, inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) mRNA expression. Meanwhile, biopterin and nitric oxide (NO) levels in these tissues were also measured. Results: After the scald injury followed by Staphylococcus aureus challenge, GTP-CHI mRNA expression and biopterin levels significantly elevated in various tissues such as liver, heart, kidneys and lungs, so did the values of iNOS mRNA expression and NO formation (P＜0.01). Pretreatment with DAHP could significantly reduce GTP-CHI/biopterin induction (P＜0. 05～0. 01), and the up-regulation of iNOS/NO was also suppressed. Furthermore, DAHP administration could also inhibit the gene expression of TNF-α. 2 hrs after septic challenge, TNF-α mRNA expression in liver, kidneys and lungs in DAHP-treated group were 35.7%, 37.3% and 33.0% of those in postburn septic group, respectively. Additionally, in animals without DAHP treatment, the 6-hour mortality was 55.6% (20/36), while it was only 25.0% in DAHP-treated animals (4/16, P=0. 08). Conclusions: Early treatment with DAHP might be a potential strategy to prevent the development of postburn Staphylococcal sepsis, which appears to be associated with down-regulation of biopterin and NO formation by DAHP.     

Significance of biopterin induction in rats with postburn Staphylococcus aureus sepsis

Objective: 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 (S.aureus) sepsis to observe the time course and tissue distribution of biopterin in postburn S. aureus infection, and to investigate its potential role in the pathogenesis of gram-positive sepsis. Wistar rats were inflicted with a 20% total body surface area (TBSA) full-chickness scald injury followed by S. aureus challenge, then guanosine triphosphatecyclohydrolase I (GTP-CHI) mRNA expression and biopterin levels in liver, kidneys, lungs and heart were determined at 0. 5, 2, 6, 12 and 24 hours after S. aureus challenge. We found that after S. aureus challenge, GTP-CHI gene expressions and biopterin levels were markedly up-regulated in various tissues, and remained at high values up to 24 hours (P＜ 0. 05-0.01). Meanwhile, the organ function indexes, including serum alanine amimotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), MB isoenzyme of creatine kinase (CK-MB), levels and pulmonary myeloperoxidase (MPO) activities significantly increased at 24 hours postburn, and the multiple organ dysfunction was aggravated by S. aureus challenge. Moreover, it was shown that cardiac GTP-CHI mRNA expression and renal BH4levels were positively correlated with CK-MB and Cr (r=0. 892, P=0. 0012 and r=0. 9423,P=0.0015, respectively). Conclusion: These results suggested that thermal injury combined with S. aureus challenge could induce de novo biosynthesis of biopterin, which acts as the most important cofactor of iNOS, might play a role in the development of multiple organ dysfunction syndrome secondary to postburn sepsis.     

金黄色葡萄球菌肠毒素B单克隆抗体对烫伤脓毒症大鼠急性肺损伤的影响

目的探讨金黄色葡萄球菌(简称金葡菌)肠毒素B(SEB)单克隆抗体(单抗)对烫伤脓毒症大鼠急性肺损伤的保护作用。方法雄性Wistar大鼠56只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=20)和SEB单克隆抗体(单抗)拮抗组(n=16)。测定肺组织SEB水平、髓过氧化物酶(MPO)活性、肿瘤坏死因子(TNF)-α和干扰素(IFN)-γ表达的改变。结果烫伤后金葡菌脓毒症动物肺脏SEB含量明显升高,伤后2、6h分别为66.85ng/g组织和92.46ng/g组织,与正常对照组(14.26ng/g组织)和烫伤对照组(17.32ng/g组织)相比均为P＜0.01;同时,肺组织MPO活性显著增强,峰值可达7.39U/g组织,与正常对照组(2.09U/g组织)相比P＜0.05。与之相应,肺组织MPO活性显著增强(P<0.05)。同时,局部组织IFN-γ和TNF-α基因及其蛋白质表达明显上调(P<0.05),并与肺脏SEB含量呈高度正相关(分别为r=0.9207、P=0.0033和r=0.8142、P=0.0258)。SEB单抗早期干预可有效降低肺组织中SEB含量,并显著抑制IFN-γ和TNF-α的产生,肺脏病理改变亦明显减轻。结论SEB单抗干预可抑制IFN-γ和TNF-α等炎症介质的产生,从而显著减轻烫伤后金葡菌对机体的损害。     

The effect of Staphylococcus aureus on apoptosis of cultured human osteoblasts

Objective: To investigate the effect of Staphylococcus aureus (S. aureus) on cultured human osteoblast apoptosis and the corresponding mode of action. Methods: Transmission electron microscopy (TEM), assessment of DNA laddering, and flow cytometry assays were used to investigate human osteoblast apoptosis following infection with S. aureus. Results: TEM examination and DNA laddering assessment indicated that S. aureus can induce cultured human osteoblast apoptosis. Flow cytometry assays showed that human osteoblast apoptosis occurs in a dose‐dependent manner following infection with S. aureus. In addition, compared with under co‐culture conditions, inhibition of invasion by S. aureus resulted in a 64.62% reduction in the percentage of early apoptotic cells (P < 0.01); 7.09% ± 1.21% of human osteoblasts under indirect co‐culture with S. aureus at a multiplicity of infection of 250 showed an early apoptotic profile compared with uninfected controls(P < 0.01). Conclusions: S. aureus induces cultured human osteoblast apoptosis in a dose‐dependent manner. Intracellular S. aureus is mainly responsible for cultured human osteoblast apoptosis following infection; secreted soluble factor(s) of S. aureus playing a minor role in this process.     

细胞外信号调节激酶抑制剂对烫伤脓毒症大鼠肿瘤坏死因子-α表达的影响及意义

目的　观察细胞外信号调节激酶 (ERK)抑制剂对烧伤后金黄色葡萄球菌 (金葡菌 )脓毒症动物组织肿瘤坏死因子 (TNF) α表达及多器官功能损害的影响。方法　采用SD大鼠 2 0 %总体表面积Ⅲ度烫伤后金葡菌攻击所致脓毒症模型 ,34只动物随机分为正常对照组 (n =6 )、烫伤对照组 (n=6 )、烫伤后金葡菌感染组 (n =12 )和ERK抑制剂AG12 6拮抗组 (n =10 ) ,检测动物肝、肾、肺组织中ERK磷酸化和TNF α基因 /蛋白表达的改变。结果　烫伤脓毒症后 0 5～ 2 0h肝、肺、肾组织ERK均呈现不同程度的活化 ,其中 2 0h分别为正常对照组的 1 94倍 (P <0 0 5 )、2 86倍 (P <0 0 1)、1 4 1倍。AG12 6拮抗组肺组织磷酸化ERK水平在 2 0h下降 70 6 % (P <0 0 1) ,而肝、肾组织其磷酸化水平不同时相点几乎完全抑制 ,同时各组织中TNF α基因及蛋白表达水平明显下调 (P<0 0 5或 0 0 1)。与烫伤脓毒症组相比 ,AG12 6拮抗组 2 0h肝、肾功能指标明显改善 ,肺组织髓过氧化物酶活性下降 4 0 3% (P <0 0 5 )。结论　ERK信号通路参与了严重烧伤后金葡菌感染所致炎症反应与急性组织损伤的病理过程 ,针对该环节进行早期干预可有效缓解多器官功能异常改变。     

The promotional effect of bone wax on experimental Staphylococcus aureus osteomyelitis

The consequences of using surgical bone wax are not well studied. We evaluated the infection-promoting potential of sterile bone wax in a rat model of chronic Staphylococcus aureus osteomyelitis. The addition of bone wax greatly reduced the quantitative bacterial inoculum (log colony-forming units) required to establish chronic osteomyelitis in 50% and 100% of challenged animals. The 50% infection rate was reduced from log 6.9 to 2.6 and the 100% infection rate from 8.2 to 4.4, respectively (p less than 0.015, t test for parallelism). Separate experiments were done 10 to 30 minutes after inoculation with only log 6.4 staphylococci. Tibiae of animals that received bone wax yielded more organisms than those that did not (log 2.76 +/- 0.68 versus 1.72 +/- 0.94, p less than 0.01). At 24 hours quantitative colony counts were not significantly different whether animals received wax or not (log 5.02 +/- 0.42 versus 4.43 +/- 0.65, p greater than 0.09). These studies suggest that the routine surgical use of bone wax should be reassessed.     

Experimental study of topical uses of para-chloro-methyl-xylenol in burns: therapeutic effect against Staphylococcus aureus

The therapeutic effect of para-chloro-methyl-xylenol (PCMX), a disinfectant of phenol compound, against Staphylococcus aureus infection in burns is evaluated in the present article. The MIC of PCMX is 50 micrograms/ml. Experimental study was done on deep burn wounds of white rabbit. Bacterial stock of Staphylococcus aureus was inoculated on the wound surface 15 min after the burn. Five hours later, cream base, and creams of 1% silver sulfadiazine (Ag-SD) and 5% PCMX were topically applied followed by dressing change once a day. Biopsy was performed on the third postburn day. Samples of subeschar tissue were sent for bacterial count and pathological examination. The average bacterial count per gram of subeschar tissue of cream base, Ag-SD cream, and PCMX cream groups was 4.69 X 10(8), 3.05 X 10(6), zero, respectively. Gross inspection of the wound surface showed dry and intact in PCMX cream group, while in the other two groups autolysis of the eschar were seen. Microscopic examination of the pathological sections indicated a generalized lesion with degeneration and necrosis of the epithelium and the dermis, subcutaneous edema, infiltration of inflammatory cells, and degeneration of myofascia. The lesion seen in PCMX group was mild. While those of the other two groups were severe with the amount distribution of Staphylococcus aureus and more severe in the cream base group. Results of the study demonstrated that PCMX is an effective antimicrobial agent against Staphylococcus aureus. Its better therapeutic effect might be due to a higher concentration of PCMX cream than that reported in the literature was used in the present study.