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.     

异丙酚对IL-1β诱导人脐静脉内皮细胞通透性增高的影响

目的 评价异丙酚对IL-1β诱导人脐静脉内皮细胞(HUVECs)通透性增高的影响.方法 原代分离培养HUVECs并用免疫磁珠法进行纯化.免疫荧光法检测内皮细胞VE-钙粘素的表达.使用Transwell系统检测HUVECs单层通透性,以每孔2× 105个内皮细胞接种于12孔Transwell系统嵌套膜上层,待生长至汇合后3d时以两种方式干预细胞.采用随机数字表法,将细胞分为6组(n=36):对照组不作任何处理,其余5组分别以1、2、5、10和20 ng/ml IL-1β干预24h;采用随机数字表法,将细胞分为5组(n=30):对照组不作任何处理,其余4组分别先以0、10、50和100 μnol/L异丙酚预处理30 min,再以10 ng/ml IL-1β干预24h.采用随机数字表法,将细胞分为3组(n=18):对照组不作任何处理,其余2组分别以50 μmol/L异丙酚处理30 min,再以10 ng/ml IL-1β干预24h或30 min.采用Western blot法检测HUVECs occludin蛋白、磷酸化p38丝裂原活化蛋白激酶(p38 MAPK)及p-p38 MAPK的表达水平.结果 与对照组比较,5、10和20 ng/ml IL-1β呈浓度依赖性地增加HUVECs通透性(P＜0.05或0.01).10、50和100 μnol/L异丙酚可呈浓度依赖性地抑制IL-1β诱导HUVECs通透性增加(P＜0.01).IL-1β可下调HUVECs occludin蛋白表达,激活p38 MAPK信号通路,异丙酚可抑制IL-1β诱导的HUVECs occludin蛋白表达下调和p38 MAPK信号通路激活(P＜0.01).结论 异丙酚可减轻IL-1β诱导HUVECs通透性增高,与抑制p38 MAPK信号通路激活有关.     

异丙酚对脂多糖诱导大鼠腹腔巨噬细胞Toll样受体-4 mRNA表达的影响

目的 观察异丙酚对脂多糖（LPS）诱导大鼠腹腔巨噬细胞Toll样受体-4（TLR-4）mRNA表达的影响，探讨异丙酚抑制LPS诱导白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF—α）产生的机制。方法 雄性Wistar大鼠32只，处死后分离腹腔巨噬细胞，随机分为4组（n=8）：A组（阴性对照组）；B组LPS（终浓度为1μg／ml）／加入巨噬细胞中；C组LPS（终浓度为1μg／ml）＋异丙酚（终浓度为1μg／ml）加入巨噬细胞中；D组LPS（终浓度为1μg／ml）＋异丙酚（终浓度为5μg／ml）加入巨噬细胞中。细胞培养12h后。用ELISA方法检测培养上清液中IL-6、TNF-α的浓度，用RT-PCR方法检测TLR-4mRNA的表达水平。结果 与A组相比，B组IL-6、TNF-α和TLR-4m RNA水平均增加，C组IL-6、TLR-4m RNA水平升高（P〈0．01）；与B组相比，C组、D组IL-6、TNF-α和TLR-4m RNA水平降低（P〈0．05或〈0．01）。结论 异丙酚通过下调TLR-4m RNA的表达水平，从而一定程度上抑制了LPS诱导大鼠腹腔巨噬细胞，TNF-α和IL-6的产生。     

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.     

异丙酚对人血管内皮细胞诱生型一氧化氮合成酶生成的影响

作为临床常用的静脉麻醉药 ,异丙酚 (ＰＦ)引起血压下降的循环抑制作用已经受到重视 ,但机理尚不清楚。迄今为止 ,尚未见到关于ＰＦ对人血管内皮细胞(ＥＣ)一氧化氮合成酶 (ＮＯＳ)作用的报道。本文研究ＰＦ对ＥＣ的诱生型ＮＯＳ(ｉＮＯＳ)生成的影响 ,探讨其引起血压下降的可能机制。资料与方法按文献方法进行人脐带静脉血管ＥＣ培养及鉴定[1] 。将第三代ＥＣ种植在2 4孔培养板内的玻片上培养至融合状态 ,分为实验组和对照组。弃掉原培养液 ,对照组加入ＲＰＭⅠ 16 40培养液 ,实验组在此基础上依所加ＰＦ(Ｚｅｎｅｃｅ公司 )浓度 (分别为 …     

异丙酚对肝移植大鼠肝窦内皮细胞凋亡的影响

目的研究异丙酚对肝移植大鼠肝窦内皮细胞（SEC）凋亡的影响。方法24只健康雄性SD大鼠建立大鼠原位肝移植模型,随机分为3组（n=8）。Ⅰ组（对照组）移植肝再灌注前30min腹腔注射生理盐水15ml/kg；Ⅱ组和Ⅲ组移植肝再灌注前30min分别腹腔注射异丙酚100mg/kg和50mg/ kg。移植肝再灌注6h后取下腔静脉血,测定血浆谷丙转氨酶（ALT）、谷草转氨酶（AST）活性,TUNEL法测定移植肝SEC凋亡,Western blot法测定肝组织Bcl-2及Bax蛋白表达。结果再灌注6h后,与Ⅰ组比较,Ⅱ组、Ⅲ组大鼠血浆ALT、AST活性降低,凋亡SEC减少,Bcl-2蛋白表达上调,Bax蛋白表达下调（P＜0．01）；与Ⅱ组比较,Ⅲ组大鼠血浆ALT、AST活性增高,凋亡SEC增多,Bax蛋白表达上调（P＜0．01）,Bcl-2蛋白表达下调（P＜0．05）。结论异丙酚可剂量依赖性地抑制大鼠移植肝早期再灌注肝窦内皮细胞凋亡,其机制与上调Bcl-2蛋白表达、下调Bax蛋白表达有关。     

异丙酚对人脐静脉内皮细胞上细胞间粘附分子-1表达的影响

目的 观察异丙酚对人脐静脉内皮细胞(HUVECs)上细胞间粘附分子-1(ICAM-1)表达及对中性粒细胞(PMN)在内皮细胞上粘附数的影响。方法利用人脐带体外培养获得人脐静脉内皮细胞,将其随机分为空白组、异丙酚组、内毒素组、异丙酚 内毒索组。用流式细胞仪检测人脐静脉内皮细胞上ICAM-1的表达量。利用PMN分离液从人全血中分离出PMN。在光镜下计数PMN在内皮细胞上的粘附数。结果 4 μg/ml异丙酚对人脐静脉内皮细胞上ICAM-1的表达和对PMN在内皮细胞上的粘附数均无显著影响(P>0.05);40μg/ml异丙酚对人脐静脉内皮细胞上ICAM-1的表达和对PMN在内皮细胞上的粘附数均有抑制作用(P<0.01)。结论40μg/ml异丙酚对PMN与内皮细胞之间的过度粘附有抑制作用,从而可能对机体有一定的保护作用。     

内毒素对脐静脉内皮细胞分泌功能的影响

目的 探讨内毒素对脐静脉内皮细胞分泌功能的影响。方法 选择体外培养的人脐静脉血管内皮细胞（HUVEC）为研究对象，分别用内毒素，L－左旋精氨酸和硝基左旋精氨酸进行处理，并检测上清液中内皮素－1（ET－1）和一氧化氮（NO）含量。结果 内毒素使ET－1和NO含量增加；NO则使ET－1的含量降低。结论 内毒素能导致人脐静脉内皮细胞损伤，使ET－1和NO的合成和释放增加；NO则抑制ET－1的合成和释放。     

内毒素对脐静脉内皮细胞分泌功能的影响

目的　探讨内毒素对脐静脉内皮细胞分泌功能的影响。方法　选择体外培养的人脐静脉血管内皮细胞 (HUVEC)为研究对象 ,分别用内毒素、L -左旋精氨酸和硝基左旋精氨酸进行处理 ,并检测上清液中内皮素 - 1(ET - 1)和一氧化氮 (NO)含量。结果　内毒素使ET - 1和NO含量增加 ;NO则使ET - 1的含量降低。结论　内毒素能导致人脐静脉内皮细胞损伤 ,使ET - 1和NO的合成和释放增加 ;NO则抑制ET - 1的合成和释放     

异丙酚预先给药对TNF-α诱导人脐静脉血管内皮细胞ICAM-1表达的影响

肿瘤坏死因子-α（TNF-α）是一种重要的炎性介质,也是重要的免疫调节因子,是炎性反应的早期启动因子,可诱导血管内皮细胞上细胞间黏附分子-1（ICAM-1）等黏附分子表达上调,ICAM-1通过识别炎性细胞上相应的配体来介导炎性细胞向局部组织募集,促进炎性反应的发生。异丙酚是广泛用于临床的静脉麻醉药,可通过抑制炎性反应减轻细胞损伤,但其机制有待进一步研究。本研究拟评价异丙酚预先给药对TNF-α诱导人脐静脉血管内皮细胞ICAM-1表达的影响。     

异丙酚对肿瘤坏死因子诱导的内皮细胞凋亡的影响

目的 评价异丙酚对肿瘤坏死因子α(TNF-α)诱导的人脐静脉内皮细胞(HUVECs)凋亡的影响.方法3～4代人脐静脉内皮细胞于融合状态,随机分为5组:①TNF-α组(P0+TNF-α);②12.5μmol/L异丙酚和TNF-α组(P12.5+TNF-α);③25μmol/L异丙酚和TNF-α组(P25+TNF-α);④50μmol/L异内酚和TNF-α组(P50+TNF-α);⑤100μmol/L异丙酚和TNF-α组(P100+TNF-α);TNF-α终末浓度为2000U/ml,各组先加入不同浓度的异丙酚孵育30min后再加入TNF-α共同培养24h。用DNA原位缺口末端标记(TUNEL)技术检测细胞凋亡指数(AI),并用透射电镜观察细胞形态学改变。结果 肿瘤坏死因子组(P0+TNF-α)可见较多凋亡细胞,不同浓度的异丙酚预处理后再加入肿瘤坏死因子的各组(P12。5+TNF-α、P25+TNF-α、P50+TNF-α、P100+TNF-α细胞凋亡指数与肿瘤坏死因子组(P0+TNF-α)比较,均有明显下降(P<0.05或P<0.01),且随异丙酚浓度的升高,AI逐渐减小,内皮细胞损伤明显减轻。结论 临床相关浓度的异丙酚可抑制TNF-α诱导的内皮细胞凋亡。     

丙泊酚对脂多糖引起的Ana-1巨噬细胞炎症损伤的影响

目的研究丙泊酚对脂多糖(LPS)引起的小鼠巨噬细胞Ana-1炎症损伤的影响。方法用脂多糖处理Ana-1细胞作为细胞损伤模型,将培养的Ana-1细胞分成六组:对照组(C组)、LPS处理组(L组)、LPS+丙泊酚10μmol/L处理组(LP1组)、LPS+丙泊酚20μmol/L处理组(LP2组)、LPS+丙泊酚40μmol/L处理组(LP3组)、LPS+丙泊酚80μmol/L处理组(LP4组)。采用MTT法测定细胞的存活率,并测定丙泊酚作用前后IL-1β、IL-6、TNF-α含量。结果 L组Ana-1细胞的存活率明显低于C组,而LP1、LP2、LP3、LP4组可明显提高Ana-1细胞的存活率,且对细胞存活率的提高程度呈剂量递增的效应关系(P0.01);L组IL-1β、IL-6、TNF-α的含量明显高于C组,而LP1、LP2、LP3、LP4组可明显降低损伤细胞IL-1β、IL-6、TNF-α的含量(P0.01),且其降低的程度呈剂量效应关系(P0.01)。在本实验所选择剂量范围内,LP4组的保护效果最佳。结论丙泊酚通过降低炎症损伤来减轻LPS诱导的Ana-1细胞损伤。     

The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells

BACKGROUND: Reperfusion injury is characterized by free radical production and endothelial inflammation. Neutrophils mediate much of the end-organ injury that occurs, requiring P-selectin-mediated neutrophil-endothelial adhesion, and this is associated with decreased endothelial nitric oxide production. Propofol has antioxidant properties in vitro which might abrogate this inflammation. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia and then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg/l or propofol 5 microg/l for 4 h after re-oxygenation and were then examined for P-selectin expression and supernatant nitric oxide concentrations for 24 h. P-selectin was determined by flow cytometry, and culture supernatant nitric oxide was measured as nitrite. RESULTS: In saline-treated cells, a biphasic increase in P-selectin expression was demonstrated at 30 min (P = 0.01) and 4 h (P = 0.023) after re-oxygenation. Propofol and Diprivan prevented these increases in P-selectin expression (P < 0.05). Four hours after re-oxygenation, propofol decreased endothelial nitric oxide production (P = 0.035). CONCLUSION: This is the first study to demonstrate an effect of propofol upon endothelial P-selectin expression. Such an effect may be important in situations of reperfusion injury such as cardiac transplantation and coronary artery bypass surgery. We conclude that propofol attenuates re-oxygenation-induced endothelial inflammation in vitro.     

Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1)

We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO-) donor 3-morpholino sydnonimine (SIN-1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mM SIN-1, with or without 10-3 M propofol (Diprivan). Cytotoxicity, assessed by measuring the release of pre-incorporated 51Cr, increased when the concentration of SIN-1 increased, and was significantly decreased by 10-3 M propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0 mM SIN-1, respectively). Cell protection against 1 mM SIN-1 was tested with 0.03-1.0 mM propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose-dependent manner (r = 0.98; p < 0.001) and was as effective as tyrosine. Finally, using high-performance liquid chromatography, we demonstrated that propofol reacted with ONOO- more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5 mM ONOO- with 1 mM tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5 mM propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO-. The anti-oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage.     

Role of peroxynitrite anion in renal hypothermic preservation injury

BACKGROUND: Peroxynitrite anions may play a role in normothermic renal ischemia and reperfusion. The purpose of this study was to determine if endogenous peroxynitrite anion is involved in renal preservation injury. METHODS: Experiments were conducted in isolated canine renal tubules and in a canine autotransplant model of hypothermic preservation injury. RESULTS: Isolated renal tubules demonstrated progressive loss of membrane transport function after reperfusion with increasing cold storage times in UW solution as assessed by tetraethylammonium cation transport (TEA). This transport defect was not altered by reperfusion in the presence of WW85, a peroxynitrite decomposition catalyst. Likewise, tubule LDH release was not altered by WW85. Renal tubules did not demonstrate any evidence of peroxynitrite formation after cold storage (0-120 h) or after subsequent reperfusion in vitro as measured by nitrotyrosine adduct formation. Addition of exogenous peroxynitrite (1 mM) directly to freshly isolated renal tubules produced strong nitrotyrosine signals but failed to alter membrane function (TEA uptake). Conversely, SIN-1, a peroxynitrite generator molecule, failed to produce a nitrotyrosine signal in extracted renal tubule proteins but significantly impaired transport function. Finally, function of cold stored canine autografts was not affected by the scavenging of peroxynitrite anions (WW85) before kidney harvest and immediately at reperfusion. Tissue biopsies from cold stored kidney autografts also failed to show evidence of peroxynitrite synthesis either after cold storage (72 h) or after kidney transplantation (60 min reperfusion). CONCLUSIONS: This study concludes that peroxynitrite anions are not formed and are not involved in renal preservation injury.     

丙泊酚对止血带诱导的缺血-再灌注损伤患者血浆循环内皮细胞和血管性假血友病因子的影响

目的 探讨丙泊酚靶控输注(TCI)对止血带引起的下肢缺血-再灌注损伤患者血浆循环内皮细胞(CEC)和血管性假血友病因子(vWF)的影响.方法 30例下肢需止血带术患者随机均分为Pro组和Iso组.Pro组采用丙泊酚TCI维持麻醉,Iso组则吸入异氟醚维持麻醉.分别于术前(缺血前T0)、放气后30 min(T1)、60 min(T2)、90 min(T3)、术后1 d(T4)五个时点抽股静脉血测定血浆CEC和vWW含量.结果 两组血浆CEC和vWV含量在T1～T4时均显著高于T0时(P<0.05).两组T1～T4血浆CEC和vWF比较差异均有统计学意义(P<0.05).结论 丙泊酚TCI可减少止血带引起的下肢缺血-再灌注损伤患者血浆CEC和vWF的含量,对血管内皮细胞具有保护作用.     

丙泊酚对过氧化氢诱导入脐静脉内皮细胞超氧化物歧化酶1表达的影响

目的 探讨丙泊酚对过氧化氢(H2O2)诱导入脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)超氧化物歧化酶1(superoxide dismutase-1,SOD1)表达的影响.方法 将体外培养24孔板中的HUVEC分为3部分:第1部分(分4组,每组6例):①正...     

丙泊酚对过氧化氢诱导入脐静脉内皮细胞超氧化物歧化酶1表达的影响

Objective To investigate the effects of propofol on the expression of superoxide dismutase-1 (SOD1 ) in hydrogen dioxide (H2O2)-mediated human umbilical vein endothelia cells (HUVEC). Methods The HUVEC cultured in 24-well plate was with propofol 12.5, 25, 50, 100 and 200 μmol/L respectively for 30 min before H2O2 sitimulating HUVEC. Part 3 (divided into five groups, n=6): the optimal concentration of propofol was used for pretreatment for 30 min before H2O2 sitimulated HUVEC,HUVEC was cultured for 6, 12, 24, 36 and 48 h accordingly. Western blot was used to determine the dose-depedent and time-dependent effect of propofol on the expression of SOD1 in HUVEC. The HUVEC which cultured in 96-well plate was divided into three groups assay was used to detect the survival rate of HUVEC. Results After the cells were pretreated with hydrogen peroxide (200 μmol/L), the expression of SOD1 in HUVEC decreased dramatically,however, propofol can partially reverse this effect on H2O2 induced SOD1 expression. When HUVEC were pretr eated with 100 μmol/L propofol for 30 min before the treatment of H2O2, 24 h later, the expression of SOD1 reached to its highest level. Conclusion Propofol can protect endothelial cells from the injury of reactive oxygen species through up-regulating the expression of SOD1.