Severe gunshot injuries in a porcine model: impact on central markers of innate immunity

Background: The mechanisms behind lipopolysaccharide (LPS) tolerance remain obscure. LPS signals through Toll‐like receptor 4 (TLR4) and severe trauma/haemorrhage may influence binding and signalling through this receptor, e.g. by changing membrane expression or by releasing endogenous ligands like High Mobility Group Box 1 (HMGB1). The aim of this study was to examine these relations further in a porcine model with standardized trauma. Methods: Nine anaesthetized pigs sustained one gunshot through the femur and one pistol shot through the upper abdomen. Blood was sampled before and 90 min after shooting. The samples were stimulated for 4 h with LPS 10 ng/ml or an equivalent amount of normal saline. The leucocyte response was evaluated by measuring the tumour necrosis factor‐α (TNF‐α) and CXC ligand 8 (CXCL8) in the supernatant. Flow cytometry was used to measure the surface expression of TLR4 on CD14+ monocytes. HMGB1 concentrations were measured in the plasma. Results: Trauma and treatment caused a significant decline in the LPS‐stimulated concentrations of TNF‐α [4.53 ± 0.24 pg/ml (ln) at 0 min, 3.54 ± 0.35 pg/ml (ln) at 90 min, P=0.026], but did not modify the release of CXCL8. Monocyte TLR4 expression was unchanged. Plasma HMGB1 increased significantly [<0.92 vs. 3.02 ± 0.19 ng/ml (ln), P<0.001]. The concentrations of TNF‐α and CXCL8 did not correlate with TLR4 expression or HMGB1 concentrations. Conclusion: The results suggest that trauma‐induced LPS tolerance is not primarily regulated by TLR4 expression on circulating CD14+ monocytes or by the release of HMGB1 from damaged tissues.     

Propofol attenuates hypoxia-induced apoptosis in alveolar epithelial type II cells through down-regulating hypoxia-inducible factor-1α

To investigate the protective effect of propofol against hypoxia-induced apoptosis in alveolar epithelial type II (ATII) cells and to explore whether hypoxia-inducible factor-1α (HIF-1α) is involved in this process. Primary cultured rat ATII cells were randomly assigned to one of the following four groups, namely, Group C: treated under normoxia (21% O(2)), Group P(20): treated with propofol (20 μM) under normoxia (21% O(2)), Group H: treated under hypoxia (5% O(2)), and Group P(20)-H: pre-treated with propofol (20 μM) before hypoxia exposure (5% O(2)). Apoptosis in ATII cells was detected by Annexin V-FITC binding using FACScan. Expressions of HIF-1α and Bnip3L mRNA and protein in ATII cells were examined by quantitative real-time polymerase chain reaction and Western blotting analysis, respectively. Hypoxia exposure (Group H) significantly increased HIF-1α protein expression (P<0.01 vs. Group C) and significantly promoted apoptosis in ATII cells (P<0.01 vs. Group C). Expression of Bnip3L, a target gene of HIF-1α, was also significantly increased at both mRNA and protein levels in response to hypoxia (P<0.01 vs. Group C). Pretreatment with propofol (20 μM, Group P(20)-H) significantly decreased HIF-1α protein expression (P<0.01 vs. Group H) and significantly inhibited apoptosis in ATII cells (P<0.01 vs. Group H), accompanied by decreased expression of Bnip3L at both mRNA and protein levels (P<0.01 vs. Group H). Propofol (20 μM) can attenuate hypoxia-induced apoptosis in ATII cells and inhibit HIF-1α-hypoxia responsive element (HRE) axis involving Bnip3L, which may partly mediate the cytoprotective effects of propofol.     

异丙酚对LPS诱导中性粒细胞Toll样受体2和Toll样受体4表达的影响

目的 探讨异丙酚对内毒素(LPS)诱导中性粒细胞Toll样受体2(TLR2)和TLR4表达的影响.方法 健康志愿者6名,年龄20～35岁,各采集外周静脉血样50 ml,加入20 U/ml肝素抗凝,分离提纯中性粒细胞,制备细胞悬液,然后随机分为6组,每组6皿:对照组(C组)不给予任何药物,置于37℃、5%CO_2培养箱中培养12 h;异丙酚脂质溶剂intralipid组(I组)、异丙酚组(P组)和LPS组(L组):分别加入intralipid(终浓度为5 μg/ml)、异丙酚(终浓度为5 μg/ml)或LPS(终浓度为1μg/ml),置于37℃、5%CO_2培养箱中孵育12 h;intralipid+LPS组(IL组)和异丙酚+LPS组(PL组)先分别加入intralipid(终浓度为5 μg/ml)或异丙酚(终浓度为5 μg/ml)后,置于37℃、5%CO_2培养箱中孵育20 min,然后加入LPS(终浓度为1μg/ml),置于37℃、5%CO_2培养箱孵育12 h.采用流式细胞仪测定中性粒细胞膜TLR2和TLR4的表达;采用荧光定量PGR检测中性粒细胞膜TLR2 mRNA和TLR4 mRNA的表达;采用ELISA法测定培养上清液TNF-α和IL-8的浓度.结果 与C组比较,I组和P组TLB2和TLR4的表达、1NF-α和IL-8L-8的浓度差异无统计学意义(P>0.05),L组和IL组TLR2和TLR4的表达上调,L组TNF-α和IL-8L-8的浓度升高,IL组IL-8浓度升高(P<0.05);与L组比较,IL组TLR2和TLR4的表达、TNF-α和IL-8L-8的浓度差异无统计学意义(P>0.05),PL组TLR2和TLR4的表达下调,TNF-α和IL-8L-8的浓度降低(P<0.05).各组TLR2 mRNA和TLR4 mRNA的表达差异无统计学意义(P>0.05).结论 异丙酚可下调LPS诱导的中性粒细胞TLR2和TLR4的表达,从而抑制炎性反应.     

异丙酚对内毒素诱导的大鼠肺泡Ⅱ型上皮细胞Toll样受体4表达水平的影响

目的 探讨异丙酚对内毒素诱导的大鼠肺泡Ⅱ型上皮细胞Toll样受体4(TLR4)表达水平的影响.方法 SPF级雄性Wistar大鼠,体重180～250 g,8～9周龄,原代培养大鼠肺泡Ⅱ型上皮细胞,经鉴定后随机分为5组,每组18孔,对照组(C组):不给予任何药物,继续培养3 h;LPS组:加入LPS,终浓度1μg/ml,孵育3 h;异丙酚组(P1～3组):同时加入LPS(终浓度1μg/nd)和终浓度分别为25、50、100 μmol/L的异丙酚,孵育3 h.孵育结束后测定肺泡Ⅱ型上皮细胞TLR4 mRNA、TLR4蛋白表达和肿瘤坏死因子α(TNF-α)的释放量.结果 与C组比较,LPS组和P1组TLR4 mRNA及其蛋白表达上调(P<0.05),P2组和P3组差异无统计学意义(P>0.05);与LPS组比较,P2组和P3组TLB4 mRNA和其蛋白表达下调,TNF-α释放量降低(P<0.05或0.01),P,组上述指标差异无统计学意义(P>0.05);P2组与P3组上述指标差异无统计学意义(P>0.05).结论 异丙酚可抑制LPS诱导的大鼠肺泡Ⅱ型上皮细胞TLR4 mRNA及其蛋白表达上调,且呈浓度依赖性,这可能是其抑制肺局部炎性反应的机制.     

异丙酚预先给药对内毒素诱导大鼠肾小球血管内皮细胞通透性升高的影响

目的 探讨异丙酚预先给药对脂多糖(LPS)诱导大鼠肾小球血管内皮细胞通透性升高的影响.方法 分离、培养SD大鼠肾小球血管内皮细胞,以1×106/ml的密度接种于24孔培养板(200 μl/孔)和transwell小室(100 μl/室),采用随机数字表法,将其随机分为6组(n=10),正常对照组(C组)不作任何处理;脂肪乳对照组(I 组)加入10%脂肪乳4 μg/ml;异丙酚组(P组)加入异丙酚4μg/ml;LPS组(L组):加入LPS 10μg/ml;LPS+脂肪乳组(L+I组)加入10%脂肪乳4 μg/ml及LPS 10μg/ml;LPS+异丙酚组(L+P组)加入异丙酚4μg/ml 及LPS 10 μg/ml.于加入LPS前30 min加入脂肪乳或异丙酚,药物的浓度均为终浓度.加入LPS后6 h,收集细胞,采用逆转录-聚合酶链反应测定血管内皮细胞生长因子(VEGF)mRNA表达水平;收集上清液,采用酶联免疫吸附法测定VEGF浓度;测定血管内皮细胞通透性.结果 与C组比较,L组、L+I组和L+P组VEGF mRNA表达上调,上清液中VEGF浓度和血管内皮细胞通透性增加(P＜0.05),而I组和P组上述指标差异无统计学意义(P＞O.05).与L组比较,L+P组VEGF mRNA表达下凋,上清液中VEGF浓度和血管内皮细胞通透性降低(P＜0.05),L+I组上述指标差异无统计学意义(P＞0.05).上清液中VEGF浓度与血管内皮细胞通透性呈正相关(r=0.833,P＜0.05).结论 异丙酚预先给药可抑制LPS诱导大鼠肾小球血管内皮细胞通透性升高,其机制与下调VEGF表达有关.

Abstract：

Objective To investigate the influence of propofol pretreatment on the increased glomerular endothelial cell permeability induced by lipopolysaccharide (LPS) in rats.Methods Glomerular endothelial cells isolated from SD rats were cultured in 24-well plates(200 μl/well) and transwell filters (100 μl/filter) at 1×106/ml and assigned into 6 groups (n=10 each):control group (group C) , introlipid group (group I), propofol group (group P) , LPS group (group L), LPS+introlipid group (group L+I) and LPS+propofol group (group L +P). In group I, 10% introlipid 4 μg/ml was added. In group P, 4 μg/ml propofol was added. In group L, 10 μg/ml LPS was added. In group L+I, 10% introlipid 4 fig/ml combined with 10 μg/ml LPS was added. In group L+ P, 4 μg/ml propofol combined with LPS 10 μg/ml was added. Introlipid or propofol was added 30 min before the administration of LPS and the corresponding concentrations mentioned above were all final concentrations.After 6 h incubation with LPS, the cells were collected for measurement of vascular endothelial growth factor (VEGF) mRNA expression using RT-PCR. The supernatant was collected for determination of the VEGF concentration by ELJSA. The endothelial cell permeability was determined. Results Compared with group C, the expression of VEGF mRNA was up-regulated and the VEGF concentration and endothelial cell permeability were significantly increased in L, L+I and L + P groups (P＜0.05 ) ,but no significant change was found in the parameters mentioned above in I and P groups (P＞0.05). Compared with group L, the expression of VEGF mRNA was downregulated and the VEGF concentration and endothelial cell permeability were significantly decreased in L+P group (P＜0.05), but no significant change was found in the parameters mentioned above in group L+I(P＞0.05). A positive correlation existed between the concentration of VEGF and the permeability of endothelial cells(r= 0.833,P＜0.05).Conclusion Propofol pretreatment can decrease the increased glomerular endothelial cell permeability induced by LPS probably through down-regulation of VEGF expression.     

右美托咪啶对脂多糖诱导大鼠外周血单核细胞Toll样受体4mRNA表达的影响

目的 探讨右美托咪啶对脂多糖(LPS)诱导大鼠外周血单核细胞Toll样受体4(TLR4)mRNA表达的影响.方法 健康雄性Wistar大鼠40只,取外周血分离培养单核细胞,采用随机数字表法,将其随机分为5组(n=8),A组:阴性对照;B组:单核细胞中加入LPS(终浓度为1μg/ml);C组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为0.5 ng/ml);D组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为5.0 ng/ml);E组:单核细胞中加入LPS(终浓度为1μg/ml)+右美托咪啶(终浓度为50.0 ng/ml).孵育24 h后,收集上清液,采用ELISA法测定TNF-α、IL-1β和IL-6的浓度,采用RT-PCR法测定TLR4 mRNA的表达.结果 与A组比较,B组TNF-α、IL-1p、IL-6的浓度升高,TLR4 mRNA表达上调(P＜0.01);与B组比较,C组、D组和E组TNF-α、IL-1β、IL-6的浓度降低,TLR4 mRNA表达下调(P＜0.05或0.01);与C组比较,D组和E组TNF-α、IL-1β、IL-6的浓度降低(P＜0.01),TLR4 mRNA表达差异无统计学意义(P＞0.05);D组和E组各指标比较差异无统计学意义(P＞0.05).结论 右美托咪啶可通过下调TLR4 mRNA表达,抑制TLR4的合成,从而抑制LPS诱导大鼠外周血单核细胞TNF-α、IL-1β和IL-6的生成与释放.

Abstract：

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of Toll-like receptor 4 (TLR4) mRNA in rat peripheral blood monocytes exposed to lipopolysaccharide ( LPS ). Methods Peripheral blood monocytes isolated from male Wistar rats were seeded in 24-well plate in RPMI 1640 liquid culture medium in CO2 incubator at 37 ℃ and 5% CO2 for 2 h, and were randomly divided into 5 groups ( n = 8 each): group A negative control; group B was exposed to LPS 1 μg/ml and C, D and E groups were exposed to LPS 1 μg/ml + dexmetomidine 0.5, 5.0 and 50.0 ng/ml respectively. The monocytes were then incubated for 24 h. The concentrations of TNF-α, IL-1β and IL-6 in the supernatant of the cultured monocytes were detected by ELISA. The expression of TLR4 mRNA in the monocytes was detected by RT-PCR.Results Exposure to LPS significantly increased the expression of TLR4 mRNA and the concentrations of TNF-α, IL-1β and IL -6 in group B as compared with group A ( P ＜ 0.01 ). Dexmedetomidine attenuated the LPS-induced increase in the expression of TLR 4 mRNA and the concentrations of TNF-α, IL-1β and IL-6 in a dose-dependent manner ( P ＜0.05or 0.01 ). Conclusion Dexmedetomidine can inhibit the synthesis of TLR4 and inhibit the secretion and dilivery of TNF-α, IL-1β and IL-6 by down-regulating the gene expression of TLR4 in rat peripheral blood monocytes exposed to LPS.     

Early time course of altered leukocyte response to lipopolysaccharide and peptidoglycan in porcine gunshot injury

Background: Penetrating injuries are frequently combined with polybacterial soiling. Clearance of the microorganisms depends on the ability to activate immune responses, but post‐traumatic hyporeactivity of immune cells is almost universal. The aim of this study was to map the early time course of this altered leukocyte reactivity, and to compare the reactions to subsequent Gram‐positive or Gram‐negative challenges. Methods: Twelve juvenile pigs sustained two standardized rounds, one through the right femur and one through the left upper abdomen. First aid treatment and acute surgery were started immediately. Blood samples were drawn before trauma and after 10, 30, 60, and 90 min, and thereafter stimulated in ex vivo whole blood for 3 h with lipopolysaccharide (LPS, 10 ng/ml), peptidoglycan (PepG, 1 μg/ml), or an equivalent amount of normal saline. The leukocyte response was evaluated by measurement of tumor necrosis factor (TNF)‐α, interleukin (IL)‐1 β, IL‐6, IL‐8, and IL‐10 in the supernatant. Results: In the post‐traumatic in vivo serum, the concentration of TNF‐α increased steadily (significant after 60 min). A reduced ex vivo reaction to LPS was evident after 10 min, and was statistically significant after 30 min. The lowest levels were reached after 90 min. The ex vivo synthesis of TNF‐α after stimulation with PepG remained unaltered. A similar development was seen for IL‐6. IL‐1 β levels did not change, while IL‐8 increased significantly only after 60 and 90 min. Conclusions: Trauma almost instantaneously reprogrammed circulating leukocytes. As measured with TNF‐α, a profound hyporeactivity to LPS, but not to PepG, was induced. In addition, no global down‐regulation of leukocyte function was found after stimulation with LPS.     

Titanium particles activate toll‐like receptor 4 independently of lipid rafts in RAW264.7 murine macrophages

Adherent pathogen‐associated molecular patterns (PAMPs) act through toll‐like receptor 2 (TLR2) and TLR4 to increase the biological activity of orthopedic wear particles in cell culture and animal models of implant loosening. This study tested whether this is dependent on TLR association with lipid rafts as reported for the response to soluble TLR ligands. For this purpose, RAW264.7 murine macrophages were activated by exposure to titanium particles with adherent PAMPs, soluble lipopolysaccharide (LPS), soluble lipotecichoic acid (LTA), or heat‐killed bacteria that had been extensively washed to remove soluble PAMPs. Lipid rafts were isolated by two independent methods and the location of TLR4 and TLR2 was analyzed by Western blotting. The cognate TLRs associated with lipid rafts when the macrophages were activated with soluble LPS and LTA but not after stimulation with either titanium particles with adherent PAMPs or heat‐killed bacteria. The lipid raft disruptor, methyl‐β‐cyclodextrin, dose‐dependently inhibited TNF‐α release in response to LPS but had no affect on TNF‐α release in response to titanium particles with adherent PAMPs. We conclude, therefore, that titanium particles with adherent PAMPs and heat‐killed bacteria activate TLR2 and TLR4 in macrophages without inducing either TLR to associate with lipid rafts. These results have important implications for the mechanisms of orthopedic implant loosening as well the mechanisms for TLR activation in other inflammatory situations. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:211–217, 2011     

CD14的表达及其在库普弗细胞激活中的意义

目的　探讨LPS对库普弗细胞 (KC)CD14表达的影响及CD14在LPS激活KC中的意义。　方法　在分离培养大鼠KC的基础上 ,应用免疫组织化学染色、RT PCR等方法分别测定CD14表达的变化、培养KC中上清中TNFα、IL 6和NO浓度。　结果　 (1)不同浓度的LPS使KC中CD14mRNA的表达及其蛋白合成明显增加 ,其表达量与LPS浓度呈剂量依赖性相关 ;(2 )同一浓度的LPS可使KC中CD14mRNA的表达及其蛋白合成明显增加 ,且在 3～ 6h左右达到高峰 ;(3)LPS刺激KC后产生的活性介质能明显上调新培养KC中CD14mRNA的表达及其蛋白合成 ;(4)在血清存在的情况下加入抗CD14单抗或在无血清的情况下单独加入LPS ,可明显降低KCTNFα、IL 6和NO的释放。而后者如果同时加入LBP ,则可明显上调培养KC中的TNFα、IL 6和NO浓度。　结论　 (1)LPS及其刺激KC后产生的活性介质与CD14mRNA的表达及其蛋白合成密切相关 ,并推测在实验 1～ 3h的CD14表达的增强可能主要由LPS引起 ,而此后CD14表达的进一步增强可能与KC释放的细胞因子密切相关 ;(2 )低浓度LPS对KC的激活是CD14依赖的。     

Serine/threonine kinase, Cot/Tpl2, regulates renal cell apoptosis in ischaemia/reperfusion injury

Aim: Cot/Tpl2, a serine/threonine (Ser/Thr) protein kinase, has been classified as a member of the mitogen‐activated protein kinase (MAPK) family, and is known to have a pleiotropic role. Many studies have reported the involvement of Cot/Tpl2, mainly as a member of the Toll‐like receptor (TLR) 4 signalling pathway in lipopolysaccharide (LPS)‐induced tumor necrosis factor‐α (TNF‐α) production. At the same time, it is also related to the caspase‐dependent apoptotic pathway. Thus, the role of Cot/Tpl2 in ischaemia/reperfusion injury (IRI) in which TNF‐α and apoptosis are the major pathogenetic factors was studied. Methods: IRI was induced in wild type (Cot/Tpl2^+/+) mice and in Cot/Tpl2‐deficient (Cot/Tpl2^?/?) mice. The extent of tubular injury and renal function were studied. TNF‐α production, neutrophil infiltration and apoptosis were also compared between the two groups. Results: Cot/Tpl2^?/? mice had preserved renal function compared with wild type mice in IRI. Although Cot/Tpl2 was phosphorylated in IRI and in the cultured tubular epithelial cells (TEC) after stimulation with LPS and hydrogen peroxide, there were no significant differences in terms of TNF‐α production, neutrophil infiltration or MAPK activation between Cot/Tpl2^+/+ and Cot/Tpl2^?/? mice. In contrast, Cot/Tpl2^?/? mice showed obviously reduced terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling positive cells and cleaved caspase‐3 positive cells. Furthermore, Cot/Tpl2‐deficient TECs demonstrated significantly less caspase‐3 activation after hydrogen peroxide stimulation with comparable caspase‐9 activation to wild type TEC. Conclusion: Cot/Tpl2 did not function as a member of MAPK family, but as a promoter of apoptosis in IRI. These results suggest that Cot/Tpl2 could be a possible therapeutic target in IRI.     

肿瘤坏死因子α、干扰素γ刺激对肺泡巨噬细胞表面主要模式识别受体表达的影响

目的观察脓毒症主要相关细胞因子肿瘤坏死因子α(TNFα)和干扰素γ(IFNγ)对巨噬细胞表面主要模式识别受体(PRRs)表达的影响。方法分离培养小鼠肺泡巨噬细胞,用TNFα、IFNγ(终浓度均为20ng/ml)分别刺激细胞3h、6h、12h,通过逆转录聚合酶链反应和免疫组织化学检测细胞内PRRs,包括白细胞分化抗原14(CD14)、Toll样受体4(TLR4)、清道夫受体(SR)、细菌脂蛋白受体TLR2和细菌DNA受体TLR9mRNA表达及其蛋白表达。结果TNFα和IFNγ表现为不同程度地上调与炎细胞激活作用有关的PRRs(CD14、TLR2、TLR9)(P<0.05),下调与炎细胞防御作用有关的SR(P<0.05),而对TLR4基因及蛋白表达无显著刺激作用(P>0.05)。结论效应细胞释放的促炎因子TNFα、IFNγ能从基因转录和蛋白水平上对细胞表面PRRs产生明显的反馈调控作用,对于促进失控性炎症反应的发生具有一定病理生理意义。     

棕榈酸与脂多糖协同促进主动脉平滑肌细胞MCP-1的表达

目的研究棕榈酸及脂多糖(LPS)对主动脉平滑肌细胞MCP-1表达的影响及可能的机制。方法使用棕榈酸和(或)LPS对大鼠胸主动脉平滑肌细胞(A7r5)处理24h后,应用RT-qPCR、ELISA法检测MCP-1表达及应用RT-qPCR和Western Blotting法检测toll样受体4 (TLR4)表达情况;细胞在TLR4抑制剂(TAK-242)预处理1 h后加人棕榈酸和(或)LPS处理24h,检测MCP-1表达情况。结果使用棕榈酸和(或)LPS处理的A7r5细胞,其MCP-1 mRNA表达水平和蛋白质分泌增加,LPS增强由棕榈酸诱导的MCP-1的表达;棕榈酸和LPS均可上调TLR4蛋白质表达,两者同时作用于A7r5细胞时,TLR4蛋白表达水平进一步升高;T AK242 (5μM)处理A7r5细胞后,棕榈酸及LPS诱导的MCP-1蛋白分泌减少。结论棕榈酸与LPS通过TLR4协同促进主动脉平滑肌细胞MCP-1表达,参与动脉粥样硬化病变过程。     

Kupffer细胞CD14及Toll样受体4介导大鼠肝移植缺血再灌注损伤的机制

目的研究大鼠肝移植缺血再灌注后Kupffer细胞CD14和Toll样受体4(TLR4)的表达及其参与缺血再灌注损伤的机制。方法建立肝移植缺血再灌注模型,并分为正常对照组、缺血再灌注组、抗CD14抗体组,每组均为10只大鼠。分离培养大鼠肝移植缺血再灌注后的Kupffer细胞。检测Kupffer细胞CD14及TLR4的mRNA、蛋白表达、核转录因子κB(NFκB)活性以及培养上清TNFα的分泌量。结果再灌注后Kupffer细胞CD14及TLR4的mRNA和蛋白表达明显高于正常对照组(P<001),再灌注后核转录因子κB活性、培养上清TNFα表达量明显高于对照组(P<001)。用抗CD14抗体后NFκB活性,TNFα表达量明显下降(与再灌注组相比,P<005),但仍然高于对照组(P<001)。结论缺血再灌注后肠道内毒素(脂多糖)能够上调Kupffer细胞CD14及TLR4的表达,激活NFκB,启动细胞因子的转录和分泌,但除CD14和TLR4以外的其他信号途径参与了缺血再灌注损伤。     

Role of Toll-like receptor 4 for the pathogenesis of acute lung injury in Gram-negative sepsis

BACKGROUND AND OBJECTIVE: Proinflammatory cytokines as well as nitric oxide (NO) play a major role in mediating the response to lipopolysaccharide (LPS). The present study tested the hypothesis that LPS induces proinflammatory cytokines in the lung via the Toll-like receptor 4 (TLR4)/CD14 signalling cascade. METHODS: Control mice and TLR4-deficient (TLR4-D) mice were used to test TLR4-mediated effects of LPS. Both strains received either Escherichia coli LPS (20 mg kg-1 intraperitoneal) or saline and their lungs were collected at different time points. Pulmonary nuclear factor kappaB (NFkappaB) activation was investigated with electromobility shift assay. mRNA expression of inflammatory mediators and their corresponding receptors were detected with Ribonuclease Protection Assay. Protein expression was detected by ELISA and western blotting. Inducible NO synthase (iNOS) expression was monitored by RT-PCR and iNOS activity by conversion of l-arginine to citrulline. Immune cells were sampled by bronchoalveolar lavage (BAL) and classified. RESULTS: LPS application induced CD14-, but not TLR4 protein expression in control mice. Activation of pulmonary NFkappaB was observed within 60 min in control, but not in TLR4-D mice. Six hours of LPS administration induced a significant increase in pulmonary tumour necrosis factor alpha-, interleukin-1beta- and interleukin-6 mRNA and protein expression in control mice compared to TLR4-D mice. Furthermore, LPS induced a significantly higher increase of the iNOS expression and catalytic activity in control mice than in TLR4-D mice. BAL revealed an increase in total cell count in all LPS treated mice. CONCLUSION: Our findings suggest that TLR4 plays a key role for regulating the expression of relevant cytokines within the lung during endotoxic shock.     

磨损颗粒刺激巨噬细胞引起人工关节松动的作用机制

目的探讨来源于人外周血的单核／巨噬细胞对直径1μm以下超高分子聚乙烯（UHMWPE）磨损颗粒的反应及其机制。方法从因无菌性人工髋关节松动而需要行翻修手术的患者假体周围组织中提取UHMWPE磨损颗粒。从10名健康志愿者分别抽取50ml外周血，梯度离心获得外周血中的单核／巨噬细胞。将细胞分为6组：A：单核／巨噬细胞＋UHMWPE磨损颗粒；B：单核／巨噬细胞＋UHMWPE磨损颗粒＋100μM Rotenone；C：单核／巨噬细胞＋UHMWPE磨损颗粒＋10μM U0126；D：单核／巨噬细胞＋UHMWPE磨损颗粒＋10ng／ml Cerivastatin；E：单核／巨噬细胞；F：单核／巨噬细胞＋脂多糖（LPS）。检测各组细胞的TNFα表达。结果UHMWPE磨损颗粒明显刺激单核／巨噬细胞分泌TNFα。Rotenone、U0126和Cerivastatin均可抑制UHMWPE磨损颗粒刺激单核／巨噬细胞分泌TNFα，且以U0126明显（P〈0．01）。结论UHMWPE磨损颗粒刺激单核／巨噬细胞产生TNFα可通过NF—κB和MAPK通路，但以MAPK通路为主。     

异丙酚对内毒素诱导人脐静脉内皮细胞过氧亚硝基阴离子生成的影响

目的 探讨异丙酚对内毒素诱导人脐静脉内皮细胞过氧亚硝基阴离子(ONOO-)生成的影响.方法 培养至活细胞计数大于95%的人脐静脉内皮细胞,随机分为7组(n=5),对照组(C组)不给予任何处理;LOS0.1组、LPS1组和LPS10组分别加入内毒素(LPS)至终浓度为0.1、1和10 μg/ml,于37℃5%CO2培养箱中孵育6 h;P4+LPS10组和P40+LPS10组预先加入异丙酚至终浓度为4、40μg/ml,I40+LPS10组预先加入脂质溶剂Introlipid至终浓度为40 μg/ml,于37℃ 5%CO2培养箱中孵育30 min,再分别加入LPS至终浓度为10μg/ml,于培养箱中继续孵育6 h.孵育6 h时,测定细胞活力和乳酸脱氢酶(LDH)释放率;采用免疫组化法和Western blot法测定硝基酪氨酸蛋白(NT)表达.结果 与C组比较,其余各组细胞活力降低,内皮细胞NT表达上调,LPS1组、LPS10组、I40+LPS10组、P4+LPS10组和P40+LPS10组LDH释放率升高(P<0.01);与LPS0.1组比较,LPS1组细胞活力、LDH释放率和内皮细胞NT表达差异无统计学意义(P>0.05),LPS10组细胞活力降低,LDH释放率升高,内皮细胞NT表达上调(P<0.01);与LPS10组比较,I40+LPS10组细胞活力、LDH释放率和内皮细胞NT表达差异无统计学意义(P>0.05),P4+LPS10组和P40+LPS10组细胞活力升高,LDH释放率降低,内皮细胞NT表达下调(P<0.01).结论 异丙酚可通过抑制ONOO'-的生成,减轻内毒素诱导人脐静脉内皮细胞损伤.     

异丙酚预先给药对BV-2细胞缺氧复氧时TLR4表达的影响

目的 评价异丙酚对BV-2细胞缺氧复氧时Toll样受体4(TLR4)表达的影响.方法 小鼠小胶质细胞(BV-2细胞)于6孔培养板中培养4～6 d后随机分为4组(n=4),正常对照组(C组)不给予任何处理;缺氧复氧组(A/R组)缺氧3 h、复氧12 h;缺氧复氧+25μmol/L异丙酚组(P25组)和缺氧复氧+100μmol/L异丙酚组(P100组)于缺氧前30 min加入异丙酚,终浓度分别为25、100μmol/L.于复氧12 h时收集细胞,测定TLR4 mRNA、NF-κB mRNA和TLR4蛋白水平;收集细胞上清液,测定TNF-α水平.结果 与C组比较,A/R组、P25组和P100组TLR4 mRNA、NF-κB mRNA、TLR4蛋白和TNF-α水平均升高(P<0.01);与MR组比较,P25组和P100组TLR4 mRNA、NF-κB mBNA、TLB4蛋白和TNF-α水平均下降(P<0.01);与P25组比较,P100组TLR4 mRNA、NF-κB mRNA、TLR4蛋白和TNF-α水平均降低(P<0.01).结论 异丙酚25、100 μmol/L预先给药可抑制BV-2细胞缺氧复氧时TLR4 mRNA表达上调.     

脂多糖对小鼠肺成纤维细胞活化的影响

目的 评价脂多糖(LPS)对小鼠肺成纤维细胞活化的影响.方法 原代培养的小鼠肺成纤维细胞,接种于96孔培养板,采用随机数字表法,将其随机分为2组,正常对照组(C组,外=6)不作任何处理,LPS组(n=24)加入LPS 1μg/ml,分别于LPS孵育3、6、24、72 h时取6孔(C组于培养72 h时),收集细胞,采用实时PCR法测定Ⅰ型前胶原mRNA、α-平滑肌肌动蛋白(α-SMA)mRNA、Toll样受体4(TLR4)mRNA和整合素β1 mRNA的表达水平.结果 与C组比较,LPS组LPS孵育3、6和24 h时Ⅰ型前胶原mRNA、α-SMA mRNA、TLR4 mRNA和整合素β1 mRNA的表达水平差异无统计学意义(P＞0.05),LPS孵育72 h时上述指标表达水平上调(P＜0.05).结论 在急性肺损伤的早期LPS一方面可直接活化小鼠肺成纤维细胞,导致肺纤维化;另一方面可上调TLR4和整合素β1的表达,增加细胞对LPS的反应性,从而加速小鼠肺成纤维细胞的活化,促进肺纤维化.

Abstract：

Objective To investigate the effect of lipopolysaccharide (LPS) on the activation of mouse lung fibroblasts. Methods Primary cultured mouse lung fibroblasts were incubated in 96 well plates and randomly divided into 2 groups: control group ( group C, n = 6) and LPS group ( n = 24). The fibroblasts were cultured for 72 h in group C. LPS 1 μg/ml was added and then the fibroblasts were incubated for 72 h in group LPS. The expression of type Ⅰ procollagen mRNA, α-smooth muscle actin (α-SMA) mRNA, Toll-like receptor 4 (TLR4)mRNA and integrin β1 mRNA was determined using real-time PCR at 3, 6, 24 and 72 h of incubation (6 wells at each time point). Results Compared with group C, there was no significant change in the expression of type Ⅰ procollagen mRNA, α-SMA mRNA, TLR4 mRNA and integrin β1 mRNA at 3, 6 and 24 h of incubation ( P ＞0.05), but the parameters mentioned above were significantly up-regulated at 72 h of incubation in group LPS ( P ＜ 0.05). Conclusion In the early acute lung injury, LPS leads to pulmonary fibrosis through activating lung fibroblasts directly, and also accelerates the activation of lung fibroblasts and promotes the process of pulmonary fibrosis through up-regulating the expression of TLR4 and integrin β1 in mice.     

丙泊酚与咪达唑仑对离体细胞内毒素损伤后炎性反应的影响

目的 探讨丙泊酚与咪达唑仑对离体细胞内毒素损伤后炎性反应的影响.方法 将内毒素诱导血清分为A、B、C、D4组:A组:对照组;B组:丙泊酚+咪达唑仑组,加入丙泊酚和咪达唑仑配制成含有两药(各)10μmol/L;C组:咪达唑仑组含咪达唑仑10μmol/L;D组:丙泊酚组含丙泊酚10μmol/L.分别在给药前(T0)、给药后即刻(T1)、80min(T2)、120min(T3)、180min(T4)时间点采用化学发光法进行测定肿瘤坏死因子α(TNF-α)的浓度.结果 T0-T2时,各组间血清TNF活性无统计学差异,T3-T4时各试验组TNF活性均明显低于对照组(3121.0±157.8和1720.0±902.0),各试验组之间差异无统计学意义.结论 丙泊酚与咪达唑仑能较好的抑制离体细胞内毒素损伤后炎性因子的释放.