炎症介质通过延迟嗜中性粒细胞凋亡促进内皮细胞的损伤

本研究采用分离纯化的人外周血NTP,以LPS／TNF激活后5：1和10：1与内皮细胞共同培养或隔离培养,加或不加10ng/ml IL-6、10％（v/v）烧伤血清,24h后观察NTP凋亡对体外培养内皮细胞损伤程度的影响及其关系。结果表明激活的NTP对内皮细胞没有明显损伤,但是加入IL－6和烧伤血清后,NTP凋亡延迟,内皮细胞受到损伤,表现为发生坏死,并且NTP对内皮细胞的损伤需要两种细胞的直接接触。     

大鼠双下肢骨折合并失血对心肌细胞损伤的作用及其机制

目的：探讨双下肢骨折创伤失血反应可否诱导心肌细胞发生凋亡反应,为深入研究骨折创伤后心肌损伤机制奠定基础。方法：SD大鼠20只,随机分为对照组及创伤组（n=10）,制备双下肢骨折创伤失血模型;原代心肌细胞培养复制创伤模型。ELISA检测血清IL-2、IL-6、IL-10、TNF-α水平;心肌组织HE染色、Tunel试验观察心肌受损、凋亡;Western blot及RT-PCR检测心肌组织凋亡调控基因Bcl-2/Bax表达变化。结果：创伤后血清炎性因子时间依赖性改变,IL-2（8 h）、IL-6、IL-10（4 h）、TNF-α（1 h）达到峰值,随后逐步回落;心肌HE染色发现心肌细胞肿大,排列紊乱,炎细胞浸润;Tunel试验证实大量核染成棕褐色的心肌细胞,凋亡指数增加（P<0.05）;Western blot及RT-PCR检测表明,无论在体及心肌细胞培养中,促凋亡基因Bax表达上调（P<0.05）,而抑凋亡基因Bcl-2表达下调（P<0.05）。结论：大鼠双下肢骨折创伤失血反应通过诱导心肌细胞凋亡进而造成心肌损伤。     

趋化因子CXCL10在心肌细胞及巨噬细胞中的表达机制

目的探讨心肌缺血-再灌注损伤中趋化因子CXCL10的产生机制。方法分别用LPS、H2O2、Ca2+载体A23187刺激原代培养的心肌细胞、骨髓来源的巨噬细胞或二者混合培养的共培养系统后,ELISA检测培养基上清中的趋化因子CXCL10和促炎性细胞因子IL-1β、IL-6、TNF-α的含量,观察其表达动力学。结果①大剂量(10μg/mL)的LPS刺激心肌细胞主要产生趋化因子CXCL10;刺激骨髓来源巨噬细胞主要产生促炎性细胞因子IL-1β、IL-6、TNF-α。②H2 O2、Ca2+通道激活剂并不能使产生趋化因子CXCL10或IL-1β、IL-6、TNF-α这些促炎性细胞因子。③骨髓来源的巨噬细胞促进心肌细胞表达趋化因子CXCL10;心肌细胞促进骨髓来源的巨噬细胞表达IL-6、TNF-α,但抑制IL-1β的表达。结论心肌细胞是心肌缺血-再灌注损伤中CXCL10潜在的细胞来源;CXCL10的表达,主要依赖于TLR4的激活。     

氧化苦参碱预处理对急性心肌梗死大鼠的保护作用与机制

目的:探讨氧化苦参碱(OMT)对冠脉结扎诱导的急性心肌梗死大鼠的保护作用与机制。方法:将SD大鼠随机分为4组:假手术组、假手术+OMT组、心梗模型组,OMT预处理组(ig给予OMT 100 mg/kg)。给药12小时后,结扎冠状动脉左前降支(LAD)复制大鼠急性心肌梗死模型。8小时后,取大鼠心肌组织,通过TUNEL染色观察大鼠心肌细胞损伤及凋亡情况;收集大鼠血清,检测LDH与CK水平,过氧化氢酶(CAT)、超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH)的活力,丙二醛(MDA)含量,ELISA法分析血清中IL-1β、IL-6和TNF-α的水平。结果:与假手术组比较,模型组大鼠的凋亡心肌细胞数明显增加(P0.05),血清CK、LDH水平显著升高(P0.05);同时,血清CAT、SOD与GSH的活性明显降低(P0.001),MDA的含量、IL-1β、IL-6和TNF-α水平显著增加(P0.001)。OMT预处理明显减轻了心肌梗死大鼠心肌细胞的损伤和凋亡,降低了其血清MDA含量,IL-1β、IL-6和TNF-α水平,增加了其CAT、SOD与GSH的活性。结论:氧化苦参碱预处理能够显著减轻心肌梗死大鼠的心肌损伤,这可能与其抗炎、抗凋亡与抗氧化损伤作用有关。     

IL-1β对星形胶质细胞的激活作用

目的研究IL-1β对体外原代培养的星形胶质细胞中的激活及增殖作用,并探讨IL-1β对星形胶质细胞细胞周期的影响.方法将单层培养于盖玻片上的纯化的星形胶质细胞分为4组,分别采取血清培养和血清剥夺培养,加入不同浓度IL-1β,其浓度依次为0ng/ml、1ng/ml、10ng/ml、100ng/ml,作用24小时.采用免疫细胞化学观察GFAP和PCNA的表达.并且采用流式细胞术观察其对星形胶质细胞周期的影响.结果血清培养时不同浓度IL-1β组的星形胶质细胞GFAP表达和细胞指数无明显改变,PCNA表达较对照组明显增强,但是1ng/ml和10ng/ml IL-1β时星形胶质细胞PCNA表达无明显变化.而血清剥夺时不同浓度IL-1β组的星形胶质细胞GFAP和PCNA表达较对照组明显增强,S和G2/M期的细胞指数较对照组增多.结论 IL-1β激活星形胶质细胞,上调GFAP和PCNA的表达,并启动细胞周期进程,促使星形胶质细胞进入增殖周期.这对中枢神经系统损伤和疾病时反应性胶质增生及胶质瘢痕的形成机制起着重要作用.     

胰蛋白酶诱导人脐静脉内皮细胞分泌白细胞介素-8

目的:研究胰蛋白酶对IL-8释放的影响。方法:分离、培养人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)、倒置显微镜观察形态变化,流式细胞术检测内皮细胞标志和蛋白酶活化受体-2(proteinase-activated receptor-2,PAR-2)表达,ELISA检测HUVECs培养上清中IL-8水平。结果:HUVECs表达内皮细胞标志和PAR-2。刺激16 h,1 g/ml胰蛋白酶和100M PAR-2激活肽组HUVECs单层均匀性降低。胰蛋白酶能够显著刺激HUVECs释放IL-8,PAR-2激活肽也诱导IL-8水平升高。蛋白酶抑制剂和PAR-2抑制肽均能够显著抑制胰蛋白酶诱导的IL-8释放。PAR-2激活肽和胰蛋白酶诱导升高的IL-8水平之间成正相关性。结论:胰蛋白酶很可能通过PAR-2激活促进血管内皮细胞释放IL-8。     

肠缺血/再灌注损伤后白介素1-β水平与磷脂酶A2激活的关系

为了探讨肠缺血/再灌注损伤后IL-1β基因表达和蛋白含量变化与磷脂酶A2抑制之间的关系,采用大鼠肠缺血/再灌注损伤模型,在对照组,损伤组和磷脂酶A2抑制剂处理组动物中收集血清,肺灌洗液,腹腔灌洗液及全身重要脏器组织样品,采用放射免疫法测定IL-1β含量,并且RT-PCR法测定肺组织中IL-1β和Ⅱ型PLA2基因表达,结果表明,损伤后6h血清中IL-1β含量明显高于对照组;损伤后1和3h,腹腔注保IL-1β也明显高于对照组;损伤后肝组织中IL-1β水平有明显增加,而肺,肾、肠组织中IL-1β没有明显变化。损伤后肺灌洗液中IL-1β也明显高于对照组水平,肺组织中IL-1βmRNA表达增加,而Ⅱ型PLA2mRNA在损伤后表达反而有所下降,采用磷脂酶A2抑制剂氯喹,环氧化物酶抑制剂消炎痛,血小板活化因子受体阻断剂SR27417后,IL-1β蛋白和基因表达有不同的改变,提示肠缺血/再灌注损伤后一定时间内,肝内IL-1βmRNA表达和血中IL-1β水平明显增高,但是否与磷脂酶A2激活或其代谢产物的释放有关尚需进一步证明。     

Ang(1-7)和依那普利拉对大鼠严重烧伤早期心脏保护作用的研究

目的:观察Ang(1-7)和依那普利拉对烧伤早期心功能及心肌损伤的保护作用.方法:健康雄性Wistar大鼠24只,随机分为正常组、烧伤组、Ang(1-7)组和依那普利拉组.于烧伤后6H检测血流动力和心肌力学指标(SBP,DBP,LVSP,LVEDP, LVdp/dt-max,-LVdp/dtmax),血清cTnI,及血清和心肌组织中AngⅡ含量变化.结果:ACEi组和Ang(1-7)组SBP,DBP,LVSP, LVdp/dt-max,-LVdp/dtmax较烧伤组均明显增高(P＜0.01),LVEDP和血清cTnI明显降低(P＜0.01),ACEi组血清和心肌中AngⅡ较烧伤组明显降低(P＜0.01).结论:Ang(1-7)和ACEi均能有效的改善大鼠烧伤早期心功能,减轻心肌损伤.     

辛伐他汀对雷帕霉素作用下心肌微血管内皮细胞的影响

目的:研究辛伐他汀(SIM)对雷帕霉素(RAPA)引起的体外心肌微血管内皮细胞(CMECs)损害的保护机制。方法:分离、培养大鼠心肌微血管内皮细胞。经形态学及Dil-ac-LDL吞噬试验进行鉴定后,采用RAPA(100 nM)处理24小时建立CMECs损伤模型,然后加入不同浓度的SIM(0,10-2,10-1,100,101μM)培养24小时后,采用MTT,WST-8及Transwell检测受损后CMECs的增殖和迁移;采用Hoechst 33258及Caspase-3检查各组CMECs的凋亡;采用蛋白免疫印迹(Western blotting)检测Akt/p70 S6K磷酸化的程度;格里斯反应及实时逆转录PCR分别检测一氧化氮(NO)含量及一氧化氮合酶(eNOS)mRNA的表达。结果:①经形态学及Dil-ac-LDL吞噬试验均表明,成功培养CMECs;②低浓度SIM 100μM可显著改善100 nM RAPA对CMECs增殖、迁移和凋亡的影响;③SIM可通过上调PI3K/Akt进而上调p70s6K磷酸化(P0.05或P0.01);④SIM通过PI3K/Akt促进CMECs分泌NO及eNOS mRNA的表达(P0.05或P0.01)。结论:一定浓度下的SIM可改善RAPA作用下CMECs的增殖,迁移和凋亡,这些保护作用可能是通过其激活PI3K/Akt/mTOR/p70S6K信号通路实现的。     

LncRNA SNHG3/miR-423-5p对IL-1β诱导的骨关节炎软骨细胞损伤的影响

该文旨在探讨lncRNA SNHG3/miR-423-5p轴对白细胞介素-1β(IL-1β)诱导的骨关节炎软骨细胞损伤的影响。IL-1β诱导软骨细胞建立细胞损伤模型,将si-NC、si-SNHG3、miR-NC、miR-423-5p mimics分别转染至软骨细胞后加入10μg/L IL-1β处理24 h,将si-SNHG3和anti-miR-NC、si-SNHG3和anti-miR-423-5p分别共转染至软骨细胞后加入10μg/L IL-1β处理24 h; MTT法、流式细胞术分别检测细胞增殖及凋亡情况; ELISA法检测IL-6、IL-8、IL-10的水平;双荧光素酶报告实验检测SNHG3与miR-423-5p的靶向关系; Western blot检测Bax、Bcl-2蛋白表达量。IL-1β诱导的软骨细胞中SNHG3的表达量升高(P<0.05), miR-423-5p的表达量降低(P<0.05);转染si-SNHG3或转染miR-423-5p mimics后细胞增殖抑制率、细胞凋亡率和Bax蛋白水平降低(P<0.05), IL-6、IL-8的水平降低(P<...     

LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum.

In this in vitro study, the influence of serum-concentration, heat inactivation of the serum and the origin of the serum on the responsiveness of cultured human umbilical vein endothelial cells (HUVEC) to immunological challenges was investigated. Addition of human serum during stimulation with 1 microgram/ml bacterial lipopolysaccharide (LPS) increased endothelial cell ELAM-1 expression and interleukin (IL)-6 release five to ten-fold. Full endothelial cell responsiveness to LPS required 10 to 50% human serum and was largely abrogated after heating the serum for 30 minutes at 56 degrees C. Addition of newborn or fetal bovine serum instead of human serum, induced even higher IL-6 release and ELAM-1 expression in response to LPS, whilst heat-inactivation of these serum-batches only moderately decreased endothelial cell responses. Endothelial cell IL-6 release and ELAM-1 expression after stimulation with IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) were less influenced by heat inactivation of the serum and by omission of serum, whilst responses to PMA remained completely unaffected by such modifications in assay media. Finally, we demonstrated that endothelial cell IL-8 release also and ICAM-1 expression in response to LPS and cytokines were increased by addition of human serum, indicating that the use of serum-free assay media, or the use of media enriched with heat-inactivated (HI) human serum interferes with physiological endothelial cell responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and expression kinetics of an endothelial cell activation antigen present in vivo only in acute inflammatory tissues

Endothelial cell activation by endotoxin (LPS), tumor necrosis factor (TNF), Interleukin-1-alpha, beta (IL-1-alpha, beta) and phorbolesters (TPA) results in increased monocyte adhesion. Examination of kinetics of monocyte adhesion shows that the onset of adherence enhancement (AE) is similar in all five agents (about 300% AE at 6 h), while its decrease is delayed in LPS/TNF versus IL-1-alpha, beta/TPA-induced activation (LPS versus IL-1-beta:260% versus 60% at 18 h). Monoclonal antibody (4D10), raised against 24 h LPS-stimulated endothelial cells detects an endothelial cell-specific activation antigen at Mr 81,000 that is induced by LPS, TNF, IL-1-alpha, beta and TPA (within 6 h about 100% positive cells). Decrease in antigen-positive cells is delayed in LPS/TNF versus IL-1-alpha, beta/TPA-induced antigen expression (LPS vs. IL-1-beta: 60% vs. 5% at 24 h). In situ the antigen is not expressed in normal and chronic inflammatory tissues. Acute inflammatory tissues, including contact and atopic dermatitis, psoriasis and periodontitis, however, show endothelial cells staining strongly positive. In contact eczemas at different times after elicitation (0, 6, 24, 72, 96 h), expression of the antigen is first seen after 24 h and is still strong at 96 h. These data indicate that LPS/TNF conduct an endothelial cell activation program in vitro, showing the same prolonged kinetics that is found for endothelial cell activation in the acute inflammatory process in vivo.     

Activation of intrinsic and extrinsic proapoptotic signaling pathways in interleukin-18-mediated human cardiac endothelial cell death

Endothelial cells are the primary targets of circulating immune and inflammatory mediators. We hypothesize that interleukin-18, a proinflammatory cytokine, induces endothelial cell apoptosis. Human cardiac microvascular endothelial cells (HCMEC) were treated with interleukin (IL) 18. mRNA expression was analyzed by ribonuclease protection assay, protein levels by immunoblotting, and cell death by enzyme-linked immunosorbent assay and fluorescence-activated cell sorter analysis. We also investigated the signal transduction pathways involved in IL-18-mediated cell death. Treatment of HCMEC with IL-18 increases 1) NF-kappaB DNA binding activity; 2) induces kappaB-driven luciferase activity; 3) induces IL-1beta and TNF-alpha expression via NF-kappaB activation; 4) inhibits antiapoptotic Bcl-2 and Bcl-X(L); 5) up-regulates proapoptotic Fas, Fas-L, and Bcl-X(S) expression; 6) induces fas and Fas-L promoter activities via NF-kappaB activation; 7) activates caspases-8, -3, -9, and BID; 8) induces cytochrome c release into the cytoplasm; 9) inhibits FLIP; and 10) induces HCME cell death by apoptosis as seen by increased annexin V staining and increased levels of mono- and oligonucleosomal fragmented DNA. Whereas overexpression of Bcl-2 significantly attenuated IL-18-induced endothelial cell apoptosis, Bcl-2/Bcl-X(L) chimeric phosphorothioated 2'-MOE-modified antisense oligonucleotides potentiated the proapoptotic effects of IL-18. Furthermore, caspase-8, IKK-alpha, and NF-kappaB p65 knockdown or dominant negative IkappaB-alpha and dominant negative IkappaB-beta or kinase dead IKK-beta significantly attenuated IL-18-induced HCME cell death. Effects of IL-18 on cell death are direct and are not mediated by intermediaries such as IL-1beta, tumor necrosis factor-alpha, or interferon-gamma. Taken together, our results indicate that IL-18 activates both intrinsic and extrinsic proapoptotic signaling pathways, induces endothelial cell death, and thereby may play a role in myocardial inflammation and injury.     

Adipose-derived mesenchymal stem cells ameliorate the inflammatory reaction in CLP-induced septic acute lung injury rats via sTNFR1

We hypothesized that the adipose-derived mesenchymal stem cells (ADMSCs), which secrete high amounts of soluble molecules, such as soluble tumor necrosis factor receptor 1 (sTNFR1), may ameliorate sepsis-induced acute lung injury (ALI). A total of 120 male adult Sprague–Dawley rats were separated into four groups: the sham control (SC), sepsis induced by cecal ligation and puncture (CLP), CLP–ADMSCs, and CLP–sTNFR1 small interfering RNA (siRNA) groups; CLP groups underwent CLP and then received 1 × 10⁶ ADMSCs with or without knockdown of sTNFR1 intravenously at 1 hr after surgery. Rats were killed at 3, 6, 24, and 48 hr after the SC or CLP procedures. 5-Ethynyl-2′-deoxyuridine-labeled ADMSCs extensively colonized the lungs at 6, 24, and 72 hr after injection. The lung wet/dry (W/D) weight ratios in the CLP group were higher than those in SC group; however, ADMSCs ameliorated the W/D weight ratios following CLP, and this effect was abolished by sTNFR1 siRNA treatment. The levels of serum sTNFR1 and interleukin-10 (IL-10) were higher in the CLP–ADMSCs group and lower in the SC group than in other groups; interestingly, these levels were higher in CLP and CLP–sTNFR1 siRNA groups than in SC group. Tumor necrosis factor-α and IL-6 levels increased significantly after CLP, and ADMSCs could alleviate these changes, but the effect was weakened by sTNFR1 siRNA treatment. The lung cell apoptosis and edema levels were consistent with IL-6 levels among all groups. Therapeutically administered ADMSCs secrete sTNFR1, which most likely protects against ALI in septic rats by ameliorating inflammation and lung edema.     

炎症因子、生长因子以及凋亡因子在压疮慢性难愈合性创面中的表达及作用

目的：探讨炎症反应、生长因子及凋亡因子在压疮慢性创面中的表达及作用。方法：选取2013年10月至2015年7月河南大学第一附属医院收治的患者,其中临床Ⅲ、Ⅳ期压疮患者共20例,急性创面10例,正常皮肤组织6例。通过HE染色观察不同创面组织的形态学特征;免疫组织化学法检测组织中细胞凋亡因子Caspase-3的分布规律;荧光定量PCR法定量分析IL-1β、IL-6、TNF-α、VEGF、bFGF及其受体KDR、FGFR1基因水平的变化特征。结果：Ⅲ、Ⅳ期压疮创面中可见炎性细胞浸润;凋亡信号因子caspase-3在压疮组中的表达高于其他两组,差异有统计学意义;IL-1β、IL-6、TNF-α表达高于急性创面组和正常皮肤组;VEGF和bFGF生长因子及其受体KDR和bFGFR1表达分别低于对照组。结论：炎症因子和凋亡因子在压疮慢性创面中持续长时间的高表达、生长因子及其受体显著的低表达可能是压疮慢性难愈合性创面形成的机制和难以彻底治愈的重要因素。     

Homocysteine-thiolactone induces caspase-independent vascular endothelial cell death with apoptotic features

Objective. Cell death is generally classified into two large categories: apoptosis, which represents active, physiological programmed cell death, and necrosis, which represents passive cell death without underlying regulatory mechanisms. Apoptosis plays an important role in tissue homeostasis and its role in endothelium integrity can be influenced by the functional status of endothelial cells. Homocysteine, a sulfated amino-acid product of methionine demethylation, is an independent risk factor for vascular disease (arterial and venous thombosis). Our goal was to investigate the thiol-derivatives effect on the endothelial cell apoptosis. Methods. Three parameters were measured: mitochondrial membrane potential using DiOC₆(3) as the probe, DEVDase activation, and phosphatidylserine exposure on the cell surface with fluorosceinated annexin V labeling which allows apoptosis to be distinguished from necrosis. Results. Homocysteine-thiolactone induced endothelial cell apoptosis in a concentration-dependent manner (range: 50–200 M), independently of the caspase pathway. Only homocysteine-thiolactone, among the thiol derivatives tested, induced apoptosis. Apoptosis was not influenced by the serum concentration in culture medium, suggesting that the observed apoptotic process could occur in vivo. None of the inhibitors used (e.g., leupeptin, fumosinin Bl, catalase, or z-VAD-fmk) was able to prevent homocysteine-induced apoptosis of vascular endothelial cells. Conclusion. The apoptosis of vascular endothelial cells induced by high concentration of homocysteine-thiolactone might be one step atherosclerotic cardiovascular disease, and contribute to its complication.     

Electron microscopic analysis of glucose-induced endothelial damage in primary culture: possible mechanism and prevention

We previously reported that high glucose treated cultured endothelial cells (ECs) showed intercellular gaps by transmission electron microscopy (TEM). These gaps were abrogated with insulin and/or heparin treatment. Our aims were to assess the severity of injury in ECs treated with high glucose for variable duration, and to further study the protective effects of insulin and/or heparin. Cells were also treated with L-buthionine sulfoximine (BSO), a glutathione inhibitor, to help understand the mechanism of high glucose injury. Primary porcine ECs were treated with high glucose (30 mM) for 2, 6 or 10 days; and glucose plus insulin (1 U/ml), glucose plus heparin (5 microg/ml), glucose plus insulin plus heparin for 6 days. ECs were treated with BSO (0.001-0.05 mM) for 2 days. Pellets from trypsinized cells were processed for TEM. High glucose treatment revealed apoptosis or necrosis showing variable cell size, abnormal nuclei, condensation of nuclear chromatin, few mitochondria, cell membrane disruption and needle-shaped structures. Changes increased with duration of exposure. In high glucose plus heparin or insulin treated cultures at least one-half of the cells appeared normal. Most ECs were intact when treated with high glucose plus insulin plus heparin. BSO treatment showed dose-dependent changes with low doses showing apoptosis whereas higher doses revealed necrosis similar to high glucose treatment for 6 or 10 days. High glucose-induced EC injury increased with duration of exposure. These data demonstrate that high glucose injury resembles that of BSO treatment, suggesting that glutathione depletion may be involved in EC injury. Insulin and/or heparin protect against high glucose-induced injury.