胰高血糖素样肽1对脂多糖诱导的血管内皮细胞炎性反应的影响

目的：探讨胰高血糖素样肽1（glucagon like peptide 1,GLP-1）对脂多糖（1ipopolysaccharide,LPS）诱导的血管内皮细胞（VEC）炎性反应的影响。方法：以体外培养的人动脉VEC为研究模型,将细胞分为四组（对照组、LPS刺激组、LPS±GLP-1组、GLP-1组）,Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红（HE）染色观察细胞间连接的形态特征,用示踪剂Rhodamine Bisothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素（IL）-6和IL-8的变化。结果：GLP-1（100nM）可减少LPS（1μg／mL）刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示：GLP-1可明显降低LPS刺激引起的VEC通透性增加[由（2．57±0．19）×10^-5cm／s降至（2．10±0．18）×10^-5cm／s,P〈0．05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由（42130±6522）pg／ml降至（27478±5096）pg／ml和（18376±1561）pg／ml降至（14414±927）pg／ml,均P〈0．05]。结论：GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加．改善LPS诱导的内皮细胞炎性损伤。     

细菌脂多糖诱导人单核细胞株对细菌脂蛋白的耐受性及其与肌动蛋白骨架关系

细菌脂多糖(LPS)可诱导宿主对LPS的耐受,但对细菌脂蛋白(BLP)是否存在交叉耐受,目前报道不一。采用人单核细胞株(THP-1),建立小剂量LPS诱导THP-1对LPS耐受的细胞模型;观察细胞肌动蛋白骨架、炎症因子TNF-α、IL-1β、IL-6的浓度及NF-κB的DNA结合活力的变化情况;探讨BLP交叉耐受及细胞骨架在其中的作用。结果显示,THP-1细胞经小剂量(10ng/ml)LPS、大剂量(100ng/ml)LPS或BLP刺激后,细胞形态严重变形,肌动蛋白重组,细胞周边肌动蛋白丝带消失,出现明显的肌动蛋白收缩团块及伪足,细胞核内NF-κB的DNA结合活性显著升高,培养上清液中炎症因子(TNF-α、IL-1β及IL-6)的释放显著增加;而小剂量LPS预刺激12h后,再用大剂量的LPS或BLP刺激6h,上述指标明显改善;采用细胞骨架肌动蛋白聚集破坏剂鬼笔环肽预处理后的THP-1细胞,可取消由小剂量LPS诱导的自身耐受及对BLP的交叉耐受;可见,细菌LPS、BLP(100ng/ml)可诱导THP-1细胞肌动蛋白骨架的改变,激活NF-κB信号通路,诱导炎性细胞因子TNF-α、IL-1、IL-6过度释放,激活宿主炎症细胞的炎症反应;而小剂量LPS预刺激后可诱导出THP-1细胞对LPS的自身耐受和对BLP的交叉耐受;细胞骨架肌动蛋白参与了小剂量LPS诱导THP-1细胞对LPS自身耐受和对BLP交叉耐受的形成。     

Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。     

Apelin-13对LPS诱导人脐静脉内皮细胞屏障损伤的干预作用*

目的: 探讨Apelin-13对LPS诱导人脐静脉内皮细胞(HUVECs)屏障损伤的影响。方法: 将体外培养的HUVECs分为4组:正常对照组、LPS组、Apelin-13+LPS组、Apelin-13组。用5 μg/ml LPS作用细胞24 h, 复制屏障功能受损模型。1 μmol/L Apelin-13提前30 min给予,再给予LPS作用24 h,确定Apelin-13的影响。通过CCK8法检测Apelin-13对细胞活力的影响;Western blot检测血管内皮细胞钙粘蛋白(VE-cadherin)、纤维状肌动蛋白(F-actin)表达变化;免疫荧光检测VE-cadherin、F-actin的表达变化及核转录因子Kappa B(NF-κB p65)入核情况。结果: 与正常对照组相比,单独给予Apelin-13对细胞活力无明显影响。与正常对照组相比,LPS组细胞活力明显下降(P＜0.01),VE-cadherin蛋白表达下降(P＜0.01)、F-actin蛋白表达升高(P＜0.05),NF-κB p65入核明显增加。与LPS组相比,Apelin-13明显增加细胞活力(P＜0.01),VE-cadherin蛋白表达增加(P＜0.05)、F-actin蛋白表达下降(P＜0.01),蛋白NF-κB p65入核下降明显。结论: Apelin-13可减轻LPS诱导的人脐静脉内皮细胞损伤及屏障受损,其机制可能与抑制炎症相关。     

间充质干细胞外泌体对海马星形胶质细胞活化的抑制作用研究

目的探讨间充质干细胞外泌体 (MSC-Exo)对海马星形胶质细胞活化的抑制作用。方法实验通过超速离心法提取脐带MSC-Exo,并使用PKH-26染料标记;MSC-Exo预处理原代海马星形胶质细胞后使用脂多糖 (LPS)诱导细胞活化,并分为对照组、LPS组和LPS+MSC-Exo组,进而行免疫细胞化学检测胶质纤维酸性蛋白(GFAP)、C3的表达、免疫印迹实验检测C3、GFAP的蛋白表达,以及酶联免疫吸附测定炎症因子白介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α的水平,分别检测各组细胞的抗原表达、表型变化和炎症反应。统计采用单因素方差分析 (ANOVA)及最小显著性差异法 (LSD)检验。结果实验提取的MSC-Exo符合外泌体的一般生物学特性,且能迁移到星形胶质细胞胞体和突起。海马星形胶质细胞经LPS诱导后,其标记性抗原GFAP (1.47±0.15比0.95±0.17,P < 0.01)、A1型标记物C3 (1.32±0.23比0.87±0.16,P < 0.05)、IL-1β[(282.90±5.13)pg/ mL比 (252.53±5.51)pg/mL,P < 0.05]、TNF-α[(128.26±1.89) pg/ mL比(111.86±2.84)pg/mL,P < 0.01]和IL-6[(180.95±3.16) pg/ mL比(163.95±3.71)pg/mL,P < 0.05]的表达较正常组明显升高;MSC-Exo预处理后,GFAP(0.97±0.16 比1.47±0.15,P < 0.01)、C3 (0.83±0.12比1.32±0.23,P < 0.01)、IL-1β[(262.20±2.64)pg/mL比(282.90±5.13) pg/mL,P < 0.05]和TNF-α[(120.79±0.42)pg/mL比(128.26±1.89)pg/mL,P < 0.05]的表达较LPS组明显降低,IL-6[(187.25±0.64)pg/mL比(180.95±3.16)pg/mL,P > 0.05]的表达未见明显变化。结论 MSC-Exo对海马星形胶质细胞活化具有明显的抑制作用。     

长链非编码RNA KCNQ1OT1影响脂多糖诱导的血管内皮细胞凋亡及其炎性因子表达的机制

该研究探讨了长链非编码RNA KCNQ1OT1对脂多糖(LPS)诱导的血管内皮细胞(VEC)凋亡和炎性因子表达的影响以及其可能机制。通过体外培养VEC,分别转染KCNQ1OT1过表达载体、miR-223抑制剂或共转染KCNQ1OT1过表达载体与miR-223模拟物后,用1.0 mg/mL LPS干预24 h,然后采用RT-qPCR法检测细胞中KCNQ1OT1和miR-223的表达水平,流式细胞仪检测细胞凋亡,Western blot检测细胞中Bcl-2和Bax蛋白表达,ELISA试剂盒检测细胞培养上清中TNF-α、IL-1和IL-6水平。双荧光素酶报告基因实验验证KCNQ1OT1与miR-223的调控关系。结果显示,LPS可抑制VEC中KCNQ1OT1的表达,而促进miR-223表达;上调KCNQ1OT1或下调miR-223后均可降低LPS诱导的VEC凋亡率、Bax蛋白及TNF-α、IL-1和IL-6表达(P<0.05),而促进Bcl-2蛋白表达(P<0.05)。KCNQ1OT1靶向负调控miR-223表达,上调miR-223则逆转上调KCNQ1OT1对LPS诱导的VEC凋亡及炎性因子表达的抑制作用。这表明,上调KCNQ1OT1抑制LPS诱导的VEC凋亡及炎性因子表达,其作用机制可能与靶向负调控miR-223有关,KCNQ1OT1/miR-223轴可能为血管内皮细胞损伤的治疗提供了新靶点。     

妊娠高血压外周血中促Th2 的细胞因子水平及IL-2/IL-10 平衡 的临床意义

目的:检测妊娠高血压患者外周血中促Th2的分子IL-4、IL-2与IL-10的水平,探讨IL-2/IL-10在妊高症中的临床意义。方法:选择40例未妊娠妇女为对照组,30例正常妊娠妇女为妊娠组,28例妊娠高血压患者为妊娠高血压组,ELISA检测血清中IL-4、IL-2和IL-10的水平。结果:与对照组外周血中IL-4水平(0.53±0.04)pg/ml相比:正常妊娠组IL-4水平升高至(0.91±0.03)pg/ml(P<0.05),妊娠高血压组IL-4水平(0.67±0.35)pg/ml升高但明显低于正常妊娠组(P<0.01)。与对照组外周血中IL-2水平(0.41±0.05)pg/ml相比:正常妊娠组IL-2水平升高至(0.82±0.11)pg/ml(P<0.01);妊娠高血压组IL-2水平高达1.57±0.22(pg/ml)明显高于其它两组(P<0.01)。妊娠高血压组外周血中IL-10水平明显低于正常妊娠组IL-10水平(P<0.01);妊娠高血压组外周血中IL-2/IL-10比值明显高于于对照组及正常妊娠组的比值。结论:妊娠高血压患者外周血中细胞因子IL-2和IL-10分泌异常且诱导Th2细胞产生的IL-4降低,打破Th1/Th2平衡,致使Th1型免疫反应增强,使早孕期滋养细胞受到免疫损伤以致侵入能力下降,导致妊娠期高血压疾病的发生。     

慢性丙型肝炎患者治疗前后血清中IL-18、IFN-γ及IL-4的表达及意义

目的:研究慢性丙型肝炎患者治疗前后血清中白介素-18(IL-18)、干扰素-γ(IFN-γ)及白介素-4(IL-4)的表达情况,探讨IL-18、IFN-γ在及IL-4在慢性丙型肝炎发病中的作用及可能临床意义。方法:酶联免疫吸附法(ELISA)检测20例正常人,42例慢性丙型肝炎患者治疗前后血清中IL-18、IFN-γ在及IL-4水平。结果慢性丙型肝炎患者血清IL-18表达高于健康对照组(380.3±27.2pg/mlvs104.1±10.9pg/ml,P<0.05),血清IFN-γ表达也高于健康对照组(3.2±0.4IU/mlvs1.2±0.2IU/ml,P<0.05),而慢性丙型肝炎患者与健康对照组间IL-4表达无统计学差异(23.8±2.7pg/mlvs23.5±2.9pg/ml,P>0.05)。慢性丙型肝炎患者血清IL-18表达与谷丙转氨酶具有正相关性(r1=0.701,P<0.05);IFN-γ表达与谷丙转氨酶均具有正相关性(r2=0.629,P<0.05)。治疗前慢性丙型肝炎患者应对组血清中IL-18及IFN-γ表达高于无应答组(380.3±27.2pg/mlvs280.1±19.8pg/ml,P<...     

MT2A对脂多糖介导的人肺毛细血管内皮细胞损伤的保护作用

目的:探索不同浓度的金属硫蛋2A(Metallothionein 2A, MT2A)对脂多糖(Lipopolysaccharid, LPS)介导的人肺微血管内皮细胞损伤的保护作用。方法:培养人肺毛细血管内皮细胞株(Human lung microvascular endothelial cells, HPMVECs),经过一定浓度LPS溶液进行刺激后,利用不同浓度的MT2A与对照组共同培养,一段时间后观察炎性介质IL-6、TNF-α释放的量及荧光显微镜观察组HPMVECs骨架形态变化。结果:各组TNF-α浓度均在0 h最低,随之逐渐升高,到6 h达到高峰;从各时间点来看,除0 h各组TNF-α浓度无显著差异外(F=0.717, P=0.549),其余各时间点B1、B2、B3均显著高于A组(均P0.05)。各组中IL-6浓度均在0 h最低,A组在2 h达到高峰,随后逐渐下降;B1组在4 h达到高峰,随之下降;B2、B3组从0 h开始逐渐升高,到6 h达到峰值;从各时间点来看,除0 h各处理因素无显著差异外(F=2.341, P=0.092),其余各时间点B1、B2、B3均低于A组(均P0.05)。A组6 h小时后纤维状肌动蛋白(F-actin)明显解聚,分布明显减少,应力纤维排列紊乱或者消失;B1组、B2组、B3组6 h小时后,与A组相比,F-actin的分布明显较多,应力纤维排列较为整齐。结论:LPS刺激人肺毛细血管内皮细胞有明显损伤效应,加入MT2A后细胞相关炎性因子释放量、细胞骨架损伤情况明显减轻,表明一定浓度的MT2A对LPS介导的肺毛细血管损伤有明显保护作用。     

高氧抑制LPS/ATP诱导的骨髓源性巨噬细胞的焦亡

目的:研究不同浓度的氧气在LPS/ATP诱导的骨髓源性巨噬细胞焦亡中的作用。方法:提取C57BL/6小鼠的骨髓源性巨噬细胞,用1μg/ml脂多糖(LPS)刺激细胞24 h,用5 mM三磷酸腺苷(ATP)刺激细胞4 h,酶联免疫吸附测定法(ELISA)检测细胞培养上清液中IL-1β水平的变化。用5 mM ATP刺激细胞后,给予细胞40%、60%和100%的氧气处理1.5 h,ELISA检测细胞培养上清液中IL-1β水平的变化。结果:1μg/mL LPS和5 mM ATP先后刺激下,骨髓源性巨噬细胞培养上清液中IL-1β的水平明显升高(P0.001),用caspase-1特异性抑制剂AC-YVAD-CMK刺激骨髓源性巨噬细胞后IL-1β水平明显降低(P0.001)。5 mM ATP刺激之后给予细胞不同浓度的氧气干预1.5 h后,细胞培养上清液中IL-1β的水平明显下降。结论:高氧抑制LPS/ATP诱导的骨髓源性巨噬细胞的焦亡。     

Integrin ανβ3 modulates lipopolysaccharide-induced hyperpermeability in cardiac microvascular endothelial cells

ABSTRACT

Previous studies suggest an association of cardiac microvascular endothelial cells (CMECs) hyperpermeability with sepsis-related cardiac injury. Our results showed that CMECs permeability was dependent upon concentration and time of lipopolysaccharides (LPS) stimulation. Integrin ανβ3 expression decreased after LPS stimulation. Pretreatment with anti-integrin ανβ3 antibody enhanced LPS-induced hyperpermeability. Upregulation of integrin ανβ3 decreased LPS-induced hyperpermeability. F-actin remodeling was enhanced after LPS stimulation and was inhibited by up-regulation of integrin ανβ3. Inhibition of Src or Rac1 reduced CMECs permeability after LPS stimulation, but there were no differences in the phosphorylation of Src and Rac1 when over-expressing or blocking integrin β3. After pretreatment with Src or Rac1 inhibitor, no significant difference was found in the expression of integrin ανβ3 in LPS-induced CMECs. These finding suggested that integrin ανβ3 overexpression decreased LPS-stimulated CMECS permeability by inhibition of cytoskeletal remodeling, but the mechanism might not be mediated via Src/Rac1 signaling.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway

Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.     

Lipopolysaccharide activated phosphatidylcholine-specific phospholipase C and induced IL-8 and MCP-1 production in vascular endothelial cells

Secretion of proinflammatory cytokines by lipopolysaccharide (LPS) activated vascular endothelial cells (VECs) contributes substantially to the pathogenesis of several inflammatory diseases such as atherosclerosis and septic shock. However, the mechanisms involved in this process are not well understood. Here, we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in LPS-induced IL-8 and MCP-1 production in VECs. The results showed that LPS elevated the level of PC-PLC and the production of IL-8 and MCP-1 in Human umbilical vein vascular endothelial cells (HUVECs). Blocking the function of PC-PLC by exploiting the neutralization antibody of PC-PLC or tricyclodecan-9-yl-xanthogenate (D609), an inhibitor of PC-PLC, significantly inhibited LPS-induced production of IL-8 and MCP-1 in HUVECs. Furthermore, the in vivo experimental results showed that the levels of PC-PLC, IL-8, and MCP-1 in the aortic endothelium and serum were increased in mice injected with LPS. The increased levels of these molecules were also inhibited by the treatment with D609. The data suggested that blocking PC-PLC function significantly inhibited LPS-induced IL-8 and MCP-1 production in cultured HUVECs and in vivo. PC-PLC might be a potential target for therapy in inflammation associated-diseases such as atherosclerosis.     

羧甲司坦通过巯基上调HDAC2 抑制气道炎症

目的:探究羧甲司坦(carbocysteine,S-CMC)在气道炎症中对组蛋白去乙酰化酶2(histone deacetylase2,HDAC2)表达的调控作用和机制。方法:建立脂多糖(lipopolysaccharide,LPS)诱导大鼠肺泡巨噬细胞(NR8383)炎症模型、短期烟熏Sprague Dawely(SD)大鼠气道炎症模型,采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测炎症因子白细胞介素6(interleukin-6,IL-6)和白细胞介素8(interleukin-8,IL-8)的水平,蛋白免疫印迹(Western blotting)及免疫组化染色检测HDAC2的表达。结果:与对照组相比,模型组NR8383细胞中HDAC2的表达明显降低至对照组的0.47±0.11倍,细胞上清IL-6、IL-8水平明显升高,分别为157.6±15.0 pg/m L、378.0±17.9 pg/m L;模型组SD大鼠肺组织中HDAC2的表达明显降低到对照组的0.42±0.12倍,气道灌洗液(bronchoalveolar lavage fluid,BALF)中IL-6、IL-8分别为162.2±51.4 pg/m L、331.4±62.7 pg/m L,炎症因子水平明显升高。而与模型组比较,经S-CMC处理后细胞中HDAC2表达明显上调至对照组的1.23±0.05倍,细胞上清中IL-6为92.3±4.3 pg/m L,IL-8为300.7±17.7 pg/m L,炎症因子水平降低;肺组织中HDAC2为对照组的0.78±0.10倍,表达水平明显升高,BALF中IL-6、IL-8水平分别为100.6±32.7 pg/m L,185.0±50.4 pg/m L(P0.05)炎症因子明显降低。组蛋白去乙酰化酶抑制剂曲古抑霉素A(trichostatin,TSA)能够抑制NR8383细胞中HDAC2的表达至对照组的0.19±0.06倍,增加IL-6(197.0±42.6 pg/m L)、IL-8(567.0±97.4 pg/m L)水平,该作用可以被S-CMC所逆转(P0.05)。另外,加入巯基供体二硫苏糖醇(dithiothreitol,DTT)可增强S-CMC上调HDAC2表达,降低IL-6、IL-8的作用,而巯基耗竭剂丁硫氨酸亚砜亚胺(buthionine-sulfoximine,BSO)可减弱S-CMC的作用(P0.05)。进一步表明S-CMC调控HDAC2的过程与巯基相关。结论:S-CMC可通过巯基上调HDAC2的表达抑制气道炎症。     

Lipopolysaccharide induces autophagy through BIRC2 in human umbilical vein endothelial cells

Lipopolysaccharide (LPS), as an important proinflammatory agent, targets the endothelium. However, almost all in vitro experiments of the effect of LPS on vascular endothelial cells (VECs) were performed under an artificially decreased concentration of serum that was not enough to maintain the cell growth for a long time. The mechanism underlying LPS action on VECs cultured in a nutrient‐rich condition is not clear. To address this question and mimic the in vivo condition, we investigated the effect of LPS on VEC autophagy, which is involved in numerous physiological processes. The effect of LPS on microtubule‐associated protein 1 light chain 3 (LC3) distribution, LC3‐II accumulation and p62 degradation showed that LPS effectively induced autophagy in VECs cultured in the presence of 20% serum. To understand the mechanism by which LPS triggers the cell autophagy, we first investigated the effects of LPS on the expression of BIRC2 (cIAP1), a well‐known apoptosis inhibitor, and on the kinase activity of mammalian target of rapamycin (mTOR) and nuclear translocation of p53. LPS increased BIRC2 expression in a dose‐ and time‐dependent manner and elevated the intranuclear level of p53 but had no effect on the mTOR pathway when it triggered VEC autophagy. Furthermore, knockdown of BIRC2 by RNA interference inhibited the autophagy and the translocation of p53 to nuclei induced by LPS. These data suggest a novel role for BIRC2 in LPS‐induced autophagy in VECs. J. Cell. Physiol. 225: 174–179, 2010. © 2010 Wiley‐Liss, Inc.     

Beneficial effects of ApoA-I on LPS-induced acute lung injury and endotoxemia in mice

High density lipoprotein (HDL) binds lipopolysaccharide (LPS) and neutralizes its toxicity. The aim of our study was to investigate the effects of Apolipoprotein (ApoA-I), the major apolipoprotein of HDL, on LPS-induced acute lung injury (ALI) and endotoxemia. BALB/c mice were challenged with LPS, followed by ApoA-I or saline administration for 24h. The mice were then sacrificed and histopathological analysis of the lung was performed. We found that ApoA-I could attenuate LPS-induced acute lung injury and inflammation. To investigate the mechanisms, we measured tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels in the serum and bronchoalveolar lavage (BAL) fluid and found that ApoA-I could significantly inhibit LPS-induced increases in the IL-1beta and TNF-alpha levels in serum (P<0.05, respectively), as well as in the IL-1beta, TNF-alpha, and IL-6 levels in BAL fluid (P<0.01 and P<0.05, P<0.05, respectively). Moreover, we evaluated the effect of ApoA-I on the mortality of L-929 cells which were attacked by LPS-activated peritoneal macrophages. We found that ApoA-I could significantly inhibit the LPS-induced cell death in a dose-dependent fashion. Furthermore, we investigated in vivo the effects of ApoA-I on the mortality rate and survival time after LPS administration and found that ApoA-I significantly decreased the mortality (P<0.05) and increased the survival time (P<0.05). In summary, the results suggest that ApoA-I could effectively protect against LPS-induced endotoxemia and acute lung damage. The mechanism might be related to inhibition of inflammatory cytokine release from macrophages.     

Both inflammatory and classical lipolytic pathways are involved in lipopolysaccharide-induced lipolysis in human adipocytes

High fat diet-induced endotoxaemia triggers low-grade inflammation and lipid release from adipose tissue. This study aims to unravel the cellular mechanisms leading to the lipopolysaccharide (LPS) effects in human adipocytes. Subcutaneous pre-adipocytes surgically isolated from patients were differentiated into mature adipocytes in vitro. Lipolysis was assessed by measurement of glycerol release and mRNA expression of pro-inflammatory cytokines were evaluated by real-time PCR. Treatment with LPS for 24 h induced a dose-dependent increase in interleukin (IL)-6 and IL-8 mRNA expression. At 1 μg/ml LPS, IL-6 and IL-8 were induced to 19.5 ± 1.8-fold and 662.7 ± 91.5-fold (P < 0.01 vs basal), respectively. From 100 ng/ml to 1 μg/ml, LPS-induced lipolysis increased to a plateau of 3.1-fold above basal level (P < 0.001 vs basal). Co-treatment with inhibitors of inhibitory kappa B kinase kinase beta (IKKβ) or NF-κB inhibited LPS-induced glycerol release. Co-treatment with the protein kinase A (PKA) inhibitor H-89, the lipase inhibitor orlistat or the hormone-sensitive lipase (HSL) inhibitor CAY10499 abolished the lipolytic effects of LPS. Co-treatment with the MAPK inhibitor, U0126 also reduced LPS-induced glycerol release. Inhibition of lipolysis by orlistat or CAY10499 reduced LPS-induced IL-6 and IL-8 mRNA expression. Induction of lipolysis by the synthetic catecholamine isoproterenol or the phosphodiesterase type III inhibitor milrinone did not alter basal IL-6 and IL-8 mRNA expression after 24 treatments whereas these compounds enhanced LPS-induced IL-6 and IL-8 mRNA expression. Both the inflammatory IKKβ/NF-κB pathway and the lipolytic PKA/HSL pathways mediate LPS-induced lipolysis. In turn, LPS-induced lipolysis reinforces the expression of pro-inflammatory cytokines and, thereby, triggers its own lipolytic activity.