ROCK抑制剂法舒地尔对脂多糖致大鼠肺微血管内皮细胞损伤的保护作用及机制研究
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摘要
细菌性脓毒症及其伴随的严重的并发症是临床常见的、威胁生命的疾病。而所有这些并发症的一个共同点就是内皮细胞损伤和功能障碍。肺微血管内皮细胞（PMVECs）是肺组织的重要实质细胞，在肺损伤过程中既是首位受损伤的靶细胞，又是活跃的炎症细胞和效应细胞，在疾病发生发展中起关键作用。因此，深入探讨PMVECs损伤机制在肺循环疾病的研究中至关重要。
感染尤其是革兰氏阴性菌脓毒症是急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）等肺循环疾病的主要病因。细菌脂多糖（LPS）作为革兰阴性杆菌外膜上的一种糖蛋白，在体内可引起强烈的炎症反应，直接或间接损伤PMVECs，在感染性ALI/ARDS的病理过程中起关键作用。炎性刺激下PMVECs损伤的调控机制涉及复杂的信号转导途径。研究发现，在ALI/ARDS、肺动脉高压、肺癌等疾病的发生发展中均有RhoA/Rho激酶（ROCK）信号通路的参与。RhoA/ROCK通路作为体内普遍存在的一条信号通路，在细胞的信号转导过程中起着信号转换器或分子开关的作用，能够诱发肌动蛋白细胞骨架重排、调控基因转录及细胞周期进程等。然而，RhoA/ROCK通路在LPS诱导的大鼠PMVECs损伤中的调节机制以及与其他信号通路的交叉反应尚需进一步研究。因此，本实验以LPS诱导大鼠PMVECs损伤为模型，观察RhoA/ROCK通路在PMVECs损伤中的变化。并通过细胞免疫化学、流式细胞术、激光共聚焦及细胞分子生物学等技术研究ROCK抑制剂法舒地尔对LPS诱导的大鼠PMVECs损伤的影响，深入探讨其下游信号转导机制。第一部分RhoA/ROCK通路参与LPS诱导的大鼠肺微血管内皮细胞损伤
目的：研究RhoA/ROCK通路在LPS诱导的大鼠PMVECs损伤中的变化及可能机制。
方法：组织贴块法原代培养大鼠PMVECs，Ⅷ因子相关抗原免疫细胞化学染色及透射电镜观察细胞超微结构鉴定大鼠PMVECs；MTT及乳酸脱氢酶（LDH）法测定PMVECs细胞活力；RT-PCR检测RhoA、ROCK1mRNA的表达；Western blot检测MYPT1磷酸化水平。
结果：1PMVECs体外培养及鉴定
采用组织贴块法并加以改良成功培养出大鼠PMVECs，去除组织块继续培养3~5天后，基本形成细胞单层，汇合呈典型的铺路鹅卵石状排列。VIII因子相关抗原免疫细胞化学染色表明，约95%的细胞表现为阳性。电镜结果表明：多数细胞均可观察到质膜突起，即微绒毛；在细胞浆中还观察到W-P小体；另外有大量的质膜小泡（又称吞饮小泡）存在于细胞浆内，上述结构符合典型的内皮细胞超微结构特征。结合我们的取材部位为肺外边缘组织，因此可以证实分离培养的细胞的确为肺微血管内皮细胞。2LPS对大鼠PMVECs细胞生长的影响及法舒地尔的干预作用
MTT实验结果表明：与对照组（OD值为0.95±0.12）相比，0.01、0.1、1μg/ml LPS对大鼠PMVECs生长没有明显影响；而10、100μg/ml LPS处理组OD值分别降为0.75±0.19（P <0.05）和0.50±0.12（P <0.01），细胞生长明显抑制。10μg/ml LPS能明显造成PMVECs损伤，但又不至于导致损伤过重，仍留有足够数量的细胞进行后续实验，因此，初步选定用10μg/ml LPS制作PMVECs损伤模型。10μg/ml LPS作用6h和12h时对细胞生长没有明显影响，作用24h时OD值为0.73±0.19，与LPS作用0h（OD值为0.93±0.15）相比，PMVECs生长明显受到抑制（P <0.05），在48h和72h时，仍处于较低水平。25，50μM法舒地尔预处理对LPS作用24h导致的细胞损伤有不同程度的改善（与LPS组相比，P <0.05）。3LPS对大鼠PMVECs LDH活性的影响及法舒地尔的干预作用
对照组上清液中LDH活性为127.6±17.3U/L，0.1、1、10、100μg/mlLPS作用24h均不同程度地引起LDH的活性升高，分别升至155.1±23.4U/L（P <0.05）、162.3±34.3U/L（P <0.05）、189.1±25.1U/L（P <0.01）及194.7±22.7U/L（P <0.01）。10μg/ml LPS作用6h时LDH活性明显升至139.7±19.2U/L，与LPS作用0h（LDH活性114.1±21.1U/L）相比差异显著（P <0.05），随着作用时间延长，LDH活性持续增高，说明PMVECs受损越来越严重。不同浓度（10，25，50μM）法舒地尔预处理组上清液中LDH活性均有不同程度地下降（P <0.05或P <0.01），进一步说明法舒地尔在一定程度上减少了LDH的释放，改善了LPS诱导的PMVECs损伤。4LPS诱导的PMVECs损伤中RhoA、ROCK1mRNA表达的变化
与对照组（LPS0min）相比，10μg/ml LPS作用15min即开始明显诱导大鼠PMVECs中RhoA mRNA的表达（P <0.05），作用5min即明显诱导ROCK1mRNA的表达（P <0.05）。随着作用时间的延长，RhoA、ROCK1mRNA的表达逐渐升高，RhoA mRNA的表达在LPS作用60min时达到最高（P <0.01），ROCK1mRNA的表达在LPS作用15min和60min时出现两次表达高峰（P <0.01）。随后，RhoA、ROCK1mRNA的表达虽有所下降，但直到240min仍显著高于对照组（LPS0min）（P <0.01）。表明RhoA/ROCK信号通路可能参与了LPS诱导的大鼠PMVECs损伤。5LPS对PMVECs中MYPT1磷酸化水平的影响及法舒地尔的干预作用
ROCK活化后，进一步磷酸化其下游底物MYPT1，因此，MYPT1磷酸化水平的高低反映了ROCK的活化程度。Western blot结果显示，10μg/ml LPS在作用5min即明显诱导了大鼠PMVECs中p-MYPT1的表达（P <0.05），且在15min时MYPT1磷酸化水平达到最高（P <0.01）。随后，p-MYPT1的表达虽有不同程度地下降，但直到120min仍显著高于对照组（LPS0min）（P <0.01）。法舒地尔10、25、50μM预处理不同程度地降低LPS作用15min诱导的p-MYPT1的表达（P <0.05或P <0.01）。
结论：本实验成功分离和培养了原代大鼠PMVECs。LPS明显诱导了大鼠PMVECs损伤，RhoA/ROCK信号通路参与了LPS诱导的大鼠PMVECs损伤。
第二部分法舒地尔对LPS致大鼠PMVECs凋亡、骨架重排的影响及机制研究
目的：探讨ROCK抑制剂法舒地尔对LPS致大鼠PMVECs凋亡及骨架重排的影响及作用机制。
方法：AnnexinV/PI及Hoechst33258荧光染色法检测大鼠PMVECs凋亡；F-actin免疫荧光染色观察PMVECs骨架结构的改变；Western blot检测ERK1/2、JNK1/2/3、p38的磷酸化水平以及Bax、Bcl-2凋亡相关蛋白的表达。
结果：
1法舒地尔对LPS诱导的大鼠PMVECs凋亡的影响
1.1LPS作用不同时间对大鼠PMVECs凋亡的影响
结果显示：10μg/ml LPS作用6h对凋亡没有明显影响。与对照组（LPS0h，早期和晚期凋亡率分别为3.1±0.7%和1.0±0.3%）相比，LPS作用12、24、48、72h各时间点的早期凋亡率分别增至15.1±3.8%（P <0.01），24.3±3.6%（P <0.01），34.8±4.5%（P <0.01）和43.2±8.2%（P <0.01）。晚期凋亡率分别增至2.1±0.9%（P <0.05），9.6±2.1%（P <0.01），12.2±4.7%（P <0.01），29.1±7.4%（P <0.01），均有不同程度的增加，说明LPS明显诱导了大鼠PMVECs凋亡。
1.2法舒地尔明显降低了LPS诱导的大鼠PMVECs凋亡
与对照组（早期和晚期凋亡率分别为2.1±0.7%和3.0±0.7%）相比，LPS作用24h早期和晚期凋亡率分别增至37.8±8.1%（P <0.01）和10.7±2.6%（P <0.01）。10,25和50μM法舒地尔预处理明显降低了LPS诱导的PMVECs凋亡，早期凋亡率分别降至27.7±4.6%（P <0.05），21.8±5.9%（P <0.01）和18.6±3.9%（P <0.01）；而晚期凋亡率分别降至7.5±1.3%（P <0.05），6.4±1.3%（P <0.05）和5.3±1.4%（P <0.01），与LPS组相比差异显著，说明法舒地尔明显干预了LPS诱导的大鼠PMVECs凋亡。
1.3法舒地尔改善了LPS诱导的大鼠PMVECs凋亡形态的改变
采用Hoechst33258染色观察了PMVECs凋亡的形态学改变。结果可见，正常对照组细胞核呈较规整的圆形或椭圆形，边缘整齐，呈弥漫均匀的低强度蓝色荧光，无明显的调亡细胞；10μg/ml LPS处理24h后，细胞核致密浓染，核固缩，呈月牙形，并呈高亮度荧光，部分核染色质高度凝聚、边缘化，表明LPS明显诱导了PMVECs凋亡。10，25和50μM法舒地尔干预后核浓染、固缩等具有明显凋亡形态的细胞与LPS组相比均有不同程度的减少，进一步说明法舒地尔能明显干预LPS诱导的大鼠PMVECs凋亡。
1.4法舒地尔对凋亡相关蛋白Bax和Bcl-2表达的影响
与对照组相比，10μg/ml LPS作用24h明显上调了Bax的蛋白表达（P <0.01），同时也显著降低了Bcl-2的蛋白表达（P <0.01）。而不同浓度（10，25，50μM）的法舒地尔预处理明显逆转了LPS诱导的Bax和Bcl-2的表达失衡情况，不同程度的抑制了Bax的表达，升高了Bcl-2的表达（P <0.05或P <0.01），表明法舒地尔可能通过调节抗凋亡蛋白和促凋亡蛋白的表达抑制LPS诱导的大鼠PMVECs凋亡。
2法舒地尔对PMVECs骨架蛋白F-actin分布的影响
正常对照组可见少量的肌动蛋白纤维丝，主要分布在细胞周边，且线条完整连续。10μg/ml LPS处理后，胞浆F-actin纤维丝明显增多，大多沿细胞呈纵轴排列。细胞周边的F-actin逐渐断裂消失，胞浆中出现密集的束状应力纤维。且随着LPS作用时间的延长，F-actin在细胞内杂乱无章、弥散分布，甚至细胞间失去正常连接结构。而在预处理了ROCK抑制剂法舒地尔（10，25，50μM）的细胞中，LPS诱导的PMVECs应力纤维形成和细胞骨架形态改变的现象均被不同程度地抑制，F-actin表达及荧光强度也明显减弱，说明法舒地尔能明显抑制LPS诱导的PMVECs骨架结构的改变。
3法舒地尔对LPS诱导的大鼠PMVECs中MAPKs活化的影响
LPS（10μg/ml）在作用15min时即开始明显诱导大鼠PMVECs中ERK1/2磷酸化水平明显升高，且在60min时ERK1/2磷酸化水平达到最高。相同实验条件下，预先加入不同浓度的法舒地尔预处理，并没有改变LPS诱导的ERK1/2磷酸化水平的升高，说明在LPS诱导的大鼠PMVECs凋亡中ERK1/2并不是RhoA/ROCK的下游信号。另外，从JNK1/2/3及p38MAPKs的活化程度来看，LPS（10μg/ml）在作用5min时即开始明显诱导大鼠PMVECs中JNK1/2/3及p38磷酸化水平增高，且在30min时二者的磷酸化水平均达到最高。相同实验条件下，预先加入不同浓度（10，25，50μM）的法舒地尔预处理，均不同程度地抑制了LPS作用30min诱导的JNK1/2/3及p38磷酸化水平的增高（P <0.05或P <0.01）。说明JNK1/2/3及p38可能作为RhoA/ROCK的下游信号分子参与了LPS诱导的大鼠PMVECs凋亡。
4JNK和p38MAPKs的活化参与了LPS诱导的大鼠PMVECs凋亡
为进一步阐明JNK和p38的活化参与了LPS诱导的大鼠PMVECs凋亡。我们在接下来的实验中观察了SB203580（p38抑制剂）以及SP600125（JNK抑制剂）对LPS诱导的大鼠PMVECs凋亡及凋亡相关蛋白表达的影响。SB203580（10μM）、SP600125（20μM）预处理分别明显抑制了LPS诱导的p38和JNK1/2/3磷酸化水平的增高（P <0.01），说明SP600125和SB203580能分别起到抑制JNK1/2/3和p38MAPKs活化的作用。凋亡检测结果表明，SB203580和SP600125预处理明显抑制了LPS诱导的PMVECs凋亡，早期和晚期凋亡率有不同程度下降（P <0.05或P <0.01）。同时，SB203580及SP600125预处理还明显逆转了LPS诱导的凋亡相关蛋白Bax和Bcl-2的表达失衡情况，抑制了Bax的表达，升高了Bcl-2的表达（P <0.01），进一步说明JNK和p38的活化参与了LPS诱导的大鼠PMVECs凋亡。
结论：JNK1/2/3和p38MAPKs作为ROCK的下游信号分子参与了LPS诱导的大鼠PMVECs凋亡。法舒地尔作为唯一在临床应用的ROCK抑制剂，明显干预了LPS诱导的凋亡、骨架蛋白重排等细胞损伤，这一保护作用与其抑制ROCK通路及其下游JNK1/2/3和p38MAPKs信号分子活化有关。
第三部分法舒地尔对LPS诱导的大鼠PMVECs炎性因子表达的影响及机制研究
目的：研究法舒地尔对LPS诱导的大鼠PMVECs炎性因子表达的影响，并从氧化损伤角度出发探讨其作用机制。
方法：RT-PCR检测IL-6、TNF-α及MCP-1mRNA的表达；ELISA法检测上清液中IL-6和MCP-1的含量；激光共聚焦检测PMVECs中ROS的产生；试剂盒测定PMVECs中SOD、GSH-PX活力及MDA含量；Westernblot法测定PMVECs全蛋白及核蛋白中NF-κB p65的表达。
结果：
1法舒地尔对PMVECs中IL-6、TNF-α及MCP-1mRNA表达的影响与对照组（LPS0h）相比，10μg/ml LPS在作用3h时IL-6和MCP-1mRNA的表达即开始明显升高（P <0.01），6h时MCP-1mRNA表达水平达到最高，12h时IL-6mRNA表达水平达到最高，一直到48h，IL-6和MCP-1mRNA仍处于高表达水平（P <0.01）。10、25、50μM的法舒地尔预处理，均明显抑制了LPS作用12h诱导的IL-6mRNA的表达（P <0.01），也不同程度地抑制了LPS作用6h诱导的MCP-1mRNA的表达（P <0.05或P<0.01）。然而，LPS对TNF-α mRNA的表达没有明显影响。
2法舒地尔对LPS诱导大鼠PMVECs分泌IL-6及MCP-1的影响
与对照组（LPS0h, IL-6和MCP-1的分泌量分别为475±76pg/ml、1.082±0.389μg/ml）相比，LPS在作用3h时IL-6蛋白分泌量即开始明显升高（P<0.05），12h时分泌量达最高（1098±81pg/ml，P <0.01），一直到48h仍处于较高水平（P <0.01）。而MCP-1分泌量在LPS作用3、6、12h分别升至1.988±0.017μg/ml（P <0.05）、2.026±0.066μg/ml（P <0.05）、2.038±0.072μg/ml（P <0.05）。而且，LPS处理不同时间组与相应各时间点的对照组相比，IL-6和MCP-1蛋白分泌量均明显升高（P <0.05或P <0.01）。10、25、50μM的法舒地尔预处理，均不同程度地降低了LPS作用不同时间点诱导的IL-6和MCP-1的蛋白分泌水平（P <0.05或P <0.01）。
3LPS诱导的大鼠PMVECs中ROS的生成及法舒地尔对此的影响
与对照组（LPS0h）相比，10μg/ml的LPS作用从3h到48h，荧光强度均不同程度地增强（P <0.01），表明细胞内活性氧生成量明显升高，其中LPS处理6h时荧光强度达最高水平，12h时仍维持在接近于最高水平，随后各时间点（LPS处理24h和48h）荧光强度有所减弱，但仍明显高于对照组（P <0.01）。与LPS作用6h组相比，10，25，50μM的法舒地尔预处理组，细胞内荧光强度均明显降低（P <0.01），说明法舒地尔明显抑制了LPS诱导的PMVECs内ROS的生成。
4法舒地尔对PMVECs中SOD、GSH-PX活性和MDA含量的影响
与对照组（LPS0h）相比，10μg/ml LPS作用3、6、12、24、48h各时间点组，SOD活性均表现出不同程度的下降（P <0.05或P <0.01），其中，LPS作用12h组SOD活性降至最低。而GSH-PX活性在LPS作用6h时即开始有所下降（P <0.05），LPS作用12、24和48h时GSH-PX活性均处于较低水平（P <0.01）。相反，MDA的含量随着LPS作用时间的延长明显升高，在LPS作用6h时即开始明显升高（P <0.01），12h时达最高值，一直到48h，仍处于较高水平。而10，25，50μM法舒地尔预处理均不同程度地逆转了LPS作用12h诱导的PMVECs中SOD和GSH-PX活性的降低（P <0.05或P<0.01）；同时也不同程度地降低了MDA的含量（P <0.05或P <0.01）。
5法舒地尔对LPS作用下大鼠PMVECs中NF-κB p65核转位的影响
与对照组（LPS0h）相比，在LPS作用不同时间点，全蛋白中NF-κB p65的蛋白表达没有明显变化。而PMVECs细胞核内NF-κB p65的蛋白表达水平在LPS作用3h即明显升高（P <0.01），12h时表达水平最高，直到48h仍有较高的表达水平（P <0.01）。10，25，50μM法舒地尔预处理均明显降低了细胞核内NF-κB p65的蛋白表达（P <0.01），推测法舒地尔可能通过抑制NF-κB p65核转位从而降低了LPS诱导的炎性因子的表达。6N-acetylcysteine对LPS作用下大鼠PMVECs中IL-6、MCP-1mRNA表达及NF-kB p65核转位的影响
5、10mM N-acetylcysteine均不同程度地降低了LPS作用12h诱导的细胞核内NF-kB p65的蛋白表达（P <0.05或P <0.01），对LPS诱导的NF-kBp65核转位有一定的抑制作用。同时，5、10mM N-acetylcysteine还明显降低了LPS诱导的IL-6和MCP-1mRNA的表达（P <0.05或P <0.01）。
结论：（1）LPS作用下大鼠PMVECs中ROS的生成增多，炎性因子IL-6和MCP-1的表达升高。ROS抑制剂N-acetylcysteine明显抑制了LPS诱导的NF-κB核转位以及炎性因子的表达，表明ROS信号参与了LPS诱导的PMVECs炎性损伤。（2）法舒地尔抑制了LPS诱导的ROS的生成，逆转了LPS诱导的MDA水平的升高以及SOD和GSH-PX活性的下降，还降低了NF-κB核转位以及炎性因子IL-6和MCP-1的表达，说明法舒地尔通过其抗氧化作用干预了LPS诱导的大鼠PMVECs炎性损伤。
Bacterial sepsis and its more severe complications are clinically commonand potentially lethal diseases. The common denominator for all of thesecomplications is injury or dysfunction of pulmonary microvascular endothelialcells (PMVECs). PMVECs are not only the primary targets of injury, but alsothe active and effective inflammatory cells, which play an important role inthe development of diseases. So, it is crucial in the study of pulmonarycirculation diseases to further study the mechanisms of PMVEC injury.
Infection, especially gram negative sepsis, is a major cause of pulmonarydiseases such as ALI/ARDS. Bacterial lipopolysaccharide (LPS), a kind ofglycoprotein on the outer membrane of gram negative bacilli, can cause astrong inflammatory response in the body, damage the PMVECs directly orindirectly, and play a key role in the pathological process of ALI/ARDS.Complex signaling pathways are involved in the injury of PMVECs inducedby inflammation. Studies suggested that RhoA/ROCK pathway was involvedin the occurrence and development of ALI/ARDS, pulmonary arterialhypertension (PAH) and lung cancer. RhoA/ROCK signaling pathway caninduce the rearrangement of the actin cytoskeleton, regulate the genetranscription and cell cycle progression, and act as the signal converter ormolecular switch in the cell signal transduction. However, further studies arerequired for full understanding of the mechanisms of RhoA/ROCK pathway inthe damage of rat PMVECs induced by LPS, as well as the cross-talk ofRhoA/ROCK pathway with other signaling pathways. So, in the present study,LPS were used to induce the model of PMVEC damage and to investigate thechanges of RhoA/ROCK pathway in the process. Moreover,immunocytochemistry, flow cytometry, confocal laser and molecular cell biology technology were also used to investigate the influence of ROCKinhibitor fasudil on LPS-induced PMVECs damage, and to further discuss themechanisms of downstream signal transduction.
Part1RhoA/ROCK pathway is involved in LPS-induced damage of ratPMVECs
Objective: To study the changes of RhoA/ROCK pathway inLPS-induced damage of rat PMVECs and the mechanism of action.
Methods: The tissue-explant technique was used to culture the primaryrat PMVECs. The identification of rat PMVECs was carried out byimmunocytochemical staining of factor Ⅷrelated antigen and transmissionelectron microscope. The viability of PMVECs was determined by MTT andlactate dehydrogenase (LDH) method. The mRNA expression of RhoA andROCK1was detected by RT-PCR. The phosphorylation of MYPT1wasanalyzed by Western blot.
Results:
1The culture and identification of rat PMVECs
We successfully cultured rat PMVECs by tissue-explant technique withsome modification. After removal of explants, the PMVECs were cultured for3~5days and displayed the shape of paving stone at this moment. The resultsof immunocytochemical staining of factor Ⅷ related antigenshowed thatabout95%of the cells were positive. The results of electron microscopeshowed that the plasma membrane protrusions, also namely microvilli, couldbe observed in most cells, W-P bodies were also observed in the cytoplasm. Inaddition, a large number of plasma membrane vesicles (also called pinocytoticvesicles) were found in the cytoplasm of cells. All the above structures wereconsistent with the typical endothelial cell ultrastructure. Considering that thetissue explants were obtained from the lung edge, it could be confirmed thatthe cultured cells were indeed PMVECs.
2Effect of LPS on the viability of rat PMVECs and the intervention effect offasudil
MTT results suggested that the OD value in control group was0.95± 0.12,0.01,0.1and1μg/ml of LPS had no obvious effect on the viability ofPMVECs, while the OD value in LPS10and100μg/ml groups decreased to0.75±0.19(P <0.05) and0.50±0.12(P <0.01) respectively. LPS10μg/mlcould cause the injury of PMVECs, but sufficient cells were still existed forsubsequent test. So, LPS (10μg/ml) was used to induce the damage ofPMVECs. Compared with the OD value (0.93±0.15) in LPS10μg/ml (0h)group, the OD value did not change obviously at6h and12h after LPStreatment, but decreased significantly at24h (0.73±0.19), and stillmaintained at the low level at48h and72h. Fasudil (25,50μM) improved theLPS-induced injury of PMVECs in different degree (compared with the LPSgroup，P <0.05).
3Effect of LPS on the activity of LDH and the intervention effect of fasudil
The activity of LDH in control group was127.6±17.3U/L, which wasincreased to155.1±23.4U/L (P <0.05),162.3±34.3U/L (P <0.05),189.1±25.1U/L (P <0.01) and194.7±22.7U/L (P <0.01) respectively after thecells had been treated with LPS0.1,1,10and100μg/ml for24h. The LDHrelease began to increase at6h after the cells had been treated with LPS10μg/ml and the activity of LDH increased to139.7±19.2U/L, which wassignificantly higher (P <0.05) than that (114.1±21.1U/L) before LPStreatment. With the time prolongation, the activity of LDH increasedcontinuously, suggesting that PMVECs were damaged increasingly. Fasudil(10,25and50μM) pretreatment significantly decreased the LDH activity inthe supernatant (P <0.05or P <0.01), which suggested that fasudil inhibitedthe release of LDH, reduced the injury of PMVECs induced by LPS.
4The changes of mRNA expression of RhoA and ROCK1in LPS-inducedinjury of rat PMVECs
Compared with the control group (LPS0min), the mRNA expression ofRhoA began to increase at15min after LPS (10μg/ml) treatment (P <0.05),and reached the highest level at60min. While the mRNA expression ofROCK1began to increase after LPS (10μg/ml) treatment for5min (P <0.05),and appeared two peaks at15min and60min respectively (P <0.01). With the prolongation of time, the RhoA and ROCK1mRNA expression slightlydecreased, but still maintained at a higher level than that in the control group(LPS0min) at240min after LPS treatment, suggesting that RhoA/ROCKsignal pathway might be involved in the injury of PMVECs induced by LPS.
5Effect of LPS on the phosphorylation of MYPT1and the intervention effectof fasudil
Activated ROCK can further phosphorylate the downstream substrateMYPT1. Therefore, the phosphorylation level of MYPT1reflects the degreeof activation of ROCK. Western blot analysis showed that the phosphorylationof MYPT1began to increase after LPS (10μg/ml) treatment for5min (P <0.05), and reached the highest level at15min (P <0.01). With theprolongation of time, the expression of p-MYPT1slightly decreased, but stillhigher than that of the control group (LPS0min) at120min after LPStreatment (P <0.01). Fasudil (10,25and50μM) pretreatment significantlydecreased the expression of p-MYPT1induced by LPS treatment for15min(P <0.05or P <0.01).
Conclusion: The primary rat PMVECs were successfully cultured bytissue-explant technique in the present experiment. LPS markedly induced thedamage of rat PMVECs, and RhoA/ROCK signaling pathway was involved inthe process.
Part2Protective effect of fasudil against LPS-induced apoptosis andcytoskeleton rearrangement of rat PMVECs and the underlyingmechanisms
Objective: To study the effects of ROCK inhibitor fasudil onLPS-induced apoptosis and cytoskeleton rearrangement and the underlyingmechanisms.
Methods: The apoptosis of PMVECs was evaluated by AnnexinV/PI andHoechst33258fluorescent staining assay. The change of cytoskeleton wasdetected by F-actin staining. The phosphorylation of ERK1/2, JNK1/2/3, p38and the expression of apoptosis related proteins Bax and Bcl-2were detectedby Western blot.
Results:
1Effect of fasudil on LPS-induced apoptosis of rat PMVECs
1.1Effect of different duration of LPS exposure on apoptosis of rat PMVECs
The results of AnnexinV-FITC staining showed that compared with thecontrol group (LPS0h，the early apoptosis rate was3.1±0.7%and lateapoptosis rate was1.0±0.3%),10μg/ml LPS treatment for6h did not affectthe apoptosis of PMVECs. While the early apoptosis rate of PMVECsincreased to15.1±3.8%(P <0.01),24.3±3.6%(P <0.01),34.8±4.5%(P<0.01) and43.2±8.2%(P <0.01), and the late apoptosis rate increased to2.1±0.9%(P <0.05),9.6±2.1%(P <0.01),12.2±4.7%(P <0.01),29.1±7.4%(P <0.01) respectively at12,24,48and72h after LPS treatment,which suggested that LPS obviously induced the apoptosis of rat PMVECs.
1.2Fasudil decreased LPS-induced apoptosis of PMVECs significantly
Compared with the control group (the early apoptosis rate and lateapoptosis rate were2.1±0.7%and3.0±0.7%respectively), the earlyapoptosis rate increased to37.8±8.1%(P <0.01) and the late apoptosis rateincreased to10.7±2.6%(P <0.01) after LPS treatment for24h, which weredecreased in different degree by fasudil pretreatment. The early apoptosis ratedecreased to27.7±4.6%(P <0.05)，21.8±5.9%(P <0.01) and18.6±3.9%(P<0.01), and the late apoptosis rate decreased to7.5±1.3%(P <0.05)，6.4±1.3%(P <0.05) and5.3±1.4%(P <0.01) respectively after10,25and50μM of fasudil pretreatment, suggesting that fasudil has some interventioneffect on LPS-induced apoptosis injury of rat PMVECs.
1.3Fasudil improved LPS-induced apoptotic morphological changes of ratPMVECs
From the results of Hoechst33258staining we observed that the nucleusof PMVECs in control group was round or oval with neat edge withhomogeneous and low-intensity blue fluorescence. No obvious apoptotic cellswere found in control group. After treatment with LPS for24h, the nucleus ofPMVECs was condensed and hyperchromatic with high-brightnessfluorescence. Part of nuclear chromatin condensed highly and marginalized, and the number of PMVECs with crescent-shaped nuclei increased, whichsuggested that LPS induced apoptosis of rat PMVECs significantly. Fasudil(10,25and50μM) pretreatment significantly weakened the apoptoticmorphological changes of PMVECs induced by LPS, further illustrating thatfasudil can prevent the apoptosis of rat PMVECs induced by LPS.
1.4Effect of fasudil on the expression of Bax and Bcl-2proteins
Compared with the control group, treatment of PMVECs with10μg/mlLPS increased the protein expression of Bax (P <0.01) and decreased theprotein expression of Bcl-2(P <0.01). Fasudil (10,25and50μM)pretreatment significantly reversed the imbalance of protein expression of Baxand Bcl-2caused by LPS, with the decreased Bax expression and increasedBcl-2expression (P <0.05or P <0.01), which indicated that fasudil mightinhibit LPS-induced apoptosis of rat PMVECs by regulating the expression ofanti-apoptotic and pro-apoptotic proteins.
2Effect of fasudil on the distribution of skeleton protein F-actin in ratPMVECs
There are a few of actin filaments in the control group, which mainlydistributed in the periphery of cells. After treatment with LPS, the cytoplasmicF-actin fibers increased significantly, and most of them arranged along thelongitudinal of PMVECs. The F-actin fibers around the cells graduallydisappeared, and intensive fasciculate stress fibers appeared in the cytoplasm.With the prolongation of time, the arrangement of F-actin became more andmore disorderly and dispersive, and the normal connections between cellswere almost lost. Meanwhile, the formation of stress fiber and themorphological changes of cytoskeleton induced by LPS were inhibited by10,25and50μM of fasudil pretreatment in different degree, and fasudil alsoreduced the fluorescence intensity of F-actin.
3Effect of fasudil on the activation of MAPKs in LPS-treated rat PMVECs
LPS (10μg/ml) caused obvious phosphorylation of ERK1/2at15minafter treatment and with a peak arrived at60min (P <0.01). Whereas,pretreatment with10,25and50μM of fasudil did not inhibit the ERK1/2 phosphorylation, suggesting that ERK1/2was not as the downstream signal ofRhoA/ROCK in LPS-induced apoptotic injury. In addition, thephosphorylation of JNK1/2/3and p38began to increase after LPS treatmentfor5min and with the highest level both arrived at30min, which was allinhibited by fasudil (10,25and50μM) pretreatment (P <0.05or P <0.01),suggesting that JNK1/2/3and p38might act as the downstream signals ofRhoA/ROCK in LPS-induced apoptotic injury.
4The activation of JNK and p38MAPKs participated in the apoptotic injuryinduced by LPS in rat PMVECs
In order to further clarify that JNK and p38were involved inLPS-induced apoptosis of rat PMVECs, we observed the effects of SB203580(p38inhibitor) and SP600125(JNK inhibitor) on apoptosis and the expressionof apoptosis-related proteins induced by LPS. SB203580(10μM) andSP600125(20μM) pretreatment significantly reduced the phosphorylation ofp38and JNK MAPKs respectively, suggesting that SB203580and SP600125can inhibite the activation of p38and JNK MAPKs respectively. MeanwhileSB203580and SP600125pretreatment significantly inhibited the apoptosisinduced by LPS in rat PMVECs, and the early apoptosis rate and lateapoptosis rate were all decreased (P <0.05or P <0.01). SB203580andSP600125pretreatment also reversed the imbalance of the expression of Baxand Bcl-2proteins induced by LPS, with a decreased expression of Bax and anincreased expression of Bcl-2(P <0.01), further suggesting that activation ofJNK and p38participated in the LPS-induced apoptosis of rat PMVECs.
Conclusion: Activation of JNK and p38MAPKs, the downstreamsignaling molecules of RhoA/ROCK signaling pathway, played an importantrole in LPS-induced apoptosis of rat PMVECs. Fasudil, a potent and selectiveinhibitor of ROCK, exerted an anti-apoptotic effect and inhibited thecytoskeletal rearrangement in rat PMVECs, which were mediated by theinhibition of RhoA/ROCK and its downstream JNK and p38MAPKs.
Part3Effects of fasudil on the expression of inflammatory factorsinduced by LPS in rat PMVECs and the underlying mechanisms
Objective: To study the effects of fasudil on the expression ofinflammatory factors induced by LPS in rat PMVECs and the antioxidativemechanisms.
Methods: The mRNA expression of IL-6, TNF-αand MCP-1wasevaluated by RT-PCR; The content of IL-6and MCP-1in supernatant wasdetermined by ELISA assay kit; ROS was measured by confocal microscopywith DCFH-DA staining. The activity of SOD and GSH-PX and content ofMDA were measured using kits supplied by Nanjing JianchengBiotechnological Company. Western blot analysis was used to determine theprotein expression of NF-κB p65in the cytoplasm and nucleus.
Results:
1Effects of fasudil on the mRNA expression of IL-6, TNF-αand MCP-1induced by LPS in rat PMVECs
Compared with the control group (LPS0h), the mRNA expression ofIL-6and MCP-1began to increase after LPS treatment for3h (P <0.01), andmaintained at a higher level until the end of the experiment. The highest levelof mRNA expression of MCP-1arrived at6h, and that of IL-6arrived at12h,which were both reduced by10,25and50μM of fasudil pretreatment (P <0.05or P <0.01). LPS exerted no obvious effect on the expression of TNF-α.
2Effect of fasudil on the secretion of IL-6and MCP-1induced by LPS in ratPMVECs
Compared with the control group (LPS0h, the concentration of IL-6andMCP-1was475±76pg/ml and1.082±0.389μg/ml respectively), thesecretion of IL-6began to increase after LPS treatment for3h (P <0.05), andmaintained at a high level until48h (P <0.01), with the highest level arrivedat12h (1098±81pg/ml, P <0.01). The secretion of MCP-1also increased to1.988±0.017μg/ml (P <0.05),2.026±0.066μg/ml (P <0.05) and2.038±0.072μg/ml (P <0.05) at3,6and12h respectively. Meanwhile, the contentof both IL-6and MCP-1increased at different time points after LPS treatmentcompared with that in the corresponding control group (P <0.05or P <0.01).Fasudil (10,25and50μM) pretreatment decreased the secretion of IL-6and MCP-1induced by LPS at different time points (P <0.05or P <0.01).
3Effect of LPS on the production of ROS in rat PMVECs and the interventioneffect of fasudil
Compared with the control group (LPS0h), the fluorescence intensity ofROS was significantly increased from3h to48h after LPS treatment, andwith the highest level arrived at6h. After that, the fluorescence intensity hadsome decrease at24h and48h after LPS treatment, but still stronger than thatin control group (P <0.01), suggesting that the production of ROS increasedobviously after the cells were treated with LPS. Compared with the LPS (6h)group, pretreatment with fasudil (10,25and50μM) obviously reduced thefluorescence intensity of ROS induced by LPS treatment for6h (P <0.01),which suggested that fasudil significantly inhibited the production of ROSinduced by LPS in rat PMVECs.
4Effect of fasudil on the activity of SOD and GSH-PX and the content ofMDA
Compared with the control group (LPS0h), the activity of SODdecreased significantly from3h to48h after LPS treatment (P <0.05or P <0.01), and the lowest level arrived at12h. The activity of GSH-PX began todecrease at6h (P <0.05), and maintained the low level at12,24and48hafter LPS treatment (P <0.01). On the contrary, the content of MDA began toincrease after LPS treatment for6h (P <0.01), and reached the highest levelat12h (P <0.01), which maintained until48h after LPS treatment. Whilefasudil (10,25and50μM) pretreatment increased the activity of SOD andGSH-PX reduced by LPS treatment for12h (P <0.05or P <0.01), anddecreased the content of MDA in different degree (P <0.05or P <0.01).
5Effect of fasudil on the nuclear translocation of NF-κB p65induced by LPS
Compared with the control group (LPS0h), the expression of NF-κB p65in the whole cell extracts of rat PMVECs showed no obvious changes afterLPS treatment for different time, while that in the nuclear proteins wasobviously increased after LPS treatment for3h (P <0.01). The expression ofNF-κB p65in the nuclear proteins arrived the highest level at12h, and maintained at a high level until48h after LPS treatment (P <0.01).Meanwhile, fasudil (10,25and50μM) pretreatment significantly decreasedthe expression of NF-κB p65in the nuclear proteins of rat PMVECs, whichsuggested it might be through inhibiting the nuclear translocation of NF-κBp65that fasudil reduced the secretion of inflammatory factors.
6Effects of N-acetylcysteine on the mRNA expression of IL-6and MCP-1and the nuclear translocation of NF-κB p65induced by LPS
N-acetylcysteine (5,10mM) pretreatment significantly decreased theexpression of NF-κB p65in the nuclear proteins of rat PMVECs induced byLPS treatment for12h (P <0.05or P <0.01). Meanwhile, the mRNAexpression of IL-6and MCP-1induced by LPS was also reduced byN-acetylcysteine (5,10mM) pretreatment (P <0.05or P <0.01).
Conclusion:(1) N-acetylcysteine, the inhibitor of ROS, obviouslyinhibited the production of ROS and the expression of IL-6and MCP-1induced by LPS in rat PMVECs, which suggested that ROS was involved inLPS-induced inflammatory injury of rat PMVECs.(2) Fasudil inhibited theproduction of ROS. The increased level of MDA and the decreased activity ofSOD and GSH-PX induced by LPS were reversed by fasudil. Meanwhile,fasudil decreased the expression of IL-6and MCP-1by inhibiting the nucleartranslocation of NF-κB p65, suggesting that fasudil ameliorated theinflammatory injury induced by LPS through its antioxidant effect.
感染尤其是革兰氏阴性菌脓毒症是急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）等肺循环疾病的主要病因。细菌脂多糖（LPS）作为革兰阴性杆菌外膜上的一种糖蛋白，在体内可引起强烈的炎症反应，直接或间接损伤PMVECs，在感染性ALI/ARDS的病理过程中起关键作用。炎性刺激下PMVECs损伤的调控机制涉及复杂的信号转导途径。研究发现，在ALI/ARDS、肺动脉高压、肺癌等疾病的发生发展中均有RhoA/Rho激酶（ROCK）信号通路的参与。RhoA/ROCK通路作为体内普遍存在的一条信号通路，在细胞的信号转导过程中起着信号转换器或分子开关的作用，能够诱发肌动蛋白细胞骨架重排、调控基因转录及细胞周期进程等。然而，RhoA/ROCK通路在LPS诱导的大鼠PMVECs损伤中的调节机制以及与其他信号通路的交叉反应尚需进一步研究。因此，本实验以LPS诱导大鼠PMVECs损伤为模型，观察RhoA/ROCK通路在PMVECs损伤中的变化。并通过细胞免疫化学、流式细胞术、激光共聚焦及细胞分子生物学等技术研究ROCK抑制剂法舒地尔对LPS诱导的大鼠PMVECs损伤的影响，深入探讨其下游信号转导机制。第一部分RhoA/ROCK通路参与LPS诱导的大鼠肺微血管内皮细胞损伤
目的：研究RhoA/ROCK通路在LPS诱导的大鼠PMVECs损伤中的变化及可能机制。
方法：组织贴块法原代培养大鼠PMVECs，Ⅷ因子相关抗原免疫细胞化学染色及透射电镜观察细胞超微结构鉴定大鼠PMVECs；MTT及乳酸脱氢酶（LDH）法测定PMVECs细胞活力；RT-PCR检测RhoA、ROCK1mRNA的表达；Western blot检测MYPT1磷酸化水平。
结果：1PMVECs体外培养及鉴定
采用组织贴块法并加以改良成功培养出大鼠PMVECs，去除组织块继续培养3~5天后，基本形成细胞单层，汇合呈典型的铺路鹅卵石状排列。VIII因子相关抗原免疫细胞化学染色表明，约95%的细胞表现为阳性。电镜结果表明：多数细胞均可观察到质膜突起，即微绒毛；在细胞浆中还观察到W-P小体；另外有大量的质膜小泡（又称吞饮小泡）存在于细胞浆内，上述结构符合典型的内皮细胞超微结构特征。结合我们的取材部位为肺外边缘组织，因此可以证实分离培养的细胞的确为肺微血管内皮细胞。2LPS对大鼠PMVECs细胞生长的影响及法舒地尔的干预作用
MTT实验结果表明：与对照组（OD值为0.95±0.12）相比，0.01、0.1、1μg/ml LPS对大鼠PMVECs生长没有明显影响；而10、100μg/ml LPS处理组OD值分别降为0.75±0.19（P <0.05）和0.50±0.12（P <0.01），细胞生长明显抑制。10μg/ml LPS能明显造成PMVECs损伤，但又不至于导致损伤过重，仍留有足够数量的细胞进行后续实验，因此，初步选定用10μg/ml LPS制作PMVECs损伤模型。10μg/ml LPS作用6h和12h时对细胞生长没有明显影响，作用24h时OD值为0.73±0.19，与LPS作用0h（OD值为0.93±0.15）相比，PMVECs生长明显受到抑制（P <0.05），在48h和72h时，仍处于较低水平。25，50μM法舒地尔预处理对LPS作用24h导致的细胞损伤有不同程度的改善（与LPS组相比，P <0.05）。3LPS对大鼠PMVECs LDH活性的影响及法舒地尔的干预作用
对照组上清液中LDH活性为127.6±17.3U/L，0.1、1、10、100μg/mlLPS作用24h均不同程度地引起LDH的活性升高，分别升至155.1±23.4U/L（P <0.05）、162.3±34.3U/L（P <0.05）、189.1±25.1U/L（P <0.01）及194.7±22.7U/L（P <0.01）。10μg/ml LPS作用6h时LDH活性明显升至139.7±19.2U/L，与LPS作用0h（LDH活性114.1±21.1U/L）相比差异显著（P <0.05），随着作用时间延长，LDH活性持续增高，说明PMVECs受损越来越严重。不同浓度（10，25，50μM）法舒地尔预处理组上清液中LDH活性均有不同程度地下降（P <0.05或P <0.01），进一步说明法舒地尔在一定程度上减少了LDH的释放，改善了LPS诱导的PMVECs损伤。4LPS诱导的PMVECs损伤中RhoA、ROCK1mRNA表达的变化
与对照组（LPS0min）相比，10μg/ml LPS作用15min即开始明显诱导大鼠PMVECs中RhoA mRNA的表达（P <0.05），作用5min即明显诱导ROCK1mRNA的表达（P <0.05）。随着作用时间的延长，RhoA、ROCK1mRNA的表达逐渐升高，RhoA mRNA的表达在LPS作用60min时达到最高（P <0.01），ROCK1mRNA的表达在LPS作用15min和60min时出现两次表达高峰（P <0.01）。随后，RhoA、ROCK1mRNA的表达虽有所下降，但直到240min仍显著高于对照组（LPS0min）（P <0.01）。表明RhoA/ROCK信号通路可能参与了LPS诱导的大鼠PMVECs损伤。5LPS对PMVECs中MYPT1磷酸化水平的影响及法舒地尔的干预作用
ROCK活化后，进一步磷酸化其下游底物MYPT1，因此，MYPT1磷酸化水平的高低反映了ROCK的活化程度。Western blot结果显示，10μg/ml LPS在作用5min即明显诱导了大鼠PMVECs中p-MYPT1的表达（P <0.05），且在15min时MYPT1磷酸化水平达到最高（P <0.01）。随后，p-MYPT1的表达虽有不同程度地下降，但直到120min仍显著高于对照组（LPS0min）（P <0.01）。法舒地尔10、25、50μM预处理不同程度地降低LPS作用15min诱导的p-MYPT1的表达（P <0.05或P <0.01）。
结论：本实验成功分离和培养了原代大鼠PMVECs。LPS明显诱导了大鼠PMVECs损伤，RhoA/ROCK信号通路参与了LPS诱导的大鼠PMVECs损伤。
第二部分法舒地尔对LPS致大鼠PMVECs凋亡、骨架重排的影响及机制研究
目的：探讨ROCK抑制剂法舒地尔对LPS致大鼠PMVECs凋亡及骨架重排的影响及作用机制。
方法：AnnexinV/PI及Hoechst33258荧光染色法检测大鼠PMVECs凋亡；F-actin免疫荧光染色观察PMVECs骨架结构的改变；Western blot检测ERK1/2、JNK1/2/3、p38的磷酸化水平以及Bax、Bcl-2凋亡相关蛋白的表达。
结果：
1法舒地尔对LPS诱导的大鼠PMVECs凋亡的影响
1.1LPS作用不同时间对大鼠PMVECs凋亡的影响
结果显示：10μg/ml LPS作用6h对凋亡没有明显影响。与对照组（LPS0h，早期和晚期凋亡率分别为3.1±0.7%和1.0±0.3%）相比，LPS作用12、24、48、72h各时间点的早期凋亡率分别增至15.1±3.8%（P <0.01），24.3±3.6%（P <0.01），34.8±4.5%（P <0.01）和43.2±8.2%（P <0.01）。晚期凋亡率分别增至2.1±0.9%（P <0.05），9.6±2.1%（P <0.01），12.2±4.7%（P <0.01），29.1±7.4%（P <0.01），均有不同程度的增加，说明LPS明显诱导了大鼠PMVECs凋亡。
1.2法舒地尔明显降低了LPS诱导的大鼠PMVECs凋亡
与对照组（早期和晚期凋亡率分别为2.1±0.7%和3.0±0.7%）相比，LPS作用24h早期和晚期凋亡率分别增至37.8±8.1%（P <0.01）和10.7±2.6%（P <0.01）。10,25和50μM法舒地尔预处理明显降低了LPS诱导的PMVECs凋亡，早期凋亡率分别降至27.7±4.6%（P <0.05），21.8±5.9%（P <0.01）和18.6±3.9%（P <0.01）；而晚期凋亡率分别降至7.5±1.3%（P <0.05），6.4±1.3%（P <0.05）和5.3±1.4%（P <0.01），与LPS组相比差异显著，说明法舒地尔明显干预了LPS诱导的大鼠PMVECs凋亡。
1.3法舒地尔改善了LPS诱导的大鼠PMVECs凋亡形态的改变
采用Hoechst33258染色观察了PMVECs凋亡的形态学改变。结果可见，正常对照组细胞核呈较规整的圆形或椭圆形，边缘整齐，呈弥漫均匀的低强度蓝色荧光，无明显的调亡细胞；10μg/ml LPS处理24h后，细胞核致密浓染，核固缩，呈月牙形，并呈高亮度荧光，部分核染色质高度凝聚、边缘化，表明LPS明显诱导了PMVECs凋亡。10，25和50μM法舒地尔干预后核浓染、固缩等具有明显凋亡形态的细胞与LPS组相比均有不同程度的减少，进一步说明法舒地尔能明显干预LPS诱导的大鼠PMVECs凋亡。
1.4法舒地尔对凋亡相关蛋白Bax和Bcl-2表达的影响
与对照组相比，10μg/ml LPS作用24h明显上调了Bax的蛋白表达（P <0.01），同时也显著降低了Bcl-2的蛋白表达（P <0.01）。而不同浓度（10，25，50μM）的法舒地尔预处理明显逆转了LPS诱导的Bax和Bcl-2的表达失衡情况，不同程度的抑制了Bax的表达，升高了Bcl-2的表达（P <0.05或P <0.01），表明法舒地尔可能通过调节抗凋亡蛋白和促凋亡蛋白的表达抑制LPS诱导的大鼠PMVECs凋亡。
2法舒地尔对PMVECs骨架蛋白F-actin分布的影响
正常对照组可见少量的肌动蛋白纤维丝，主要分布在细胞周边，且线条完整连续。10μg/ml LPS处理后，胞浆F-actin纤维丝明显增多，大多沿细胞呈纵轴排列。细胞周边的F-actin逐渐断裂消失，胞浆中出现密集的束状应力纤维。且随着LPS作用时间的延长，F-actin在细胞内杂乱无章、弥散分布，甚至细胞间失去正常连接结构。而在预处理了ROCK抑制剂法舒地尔（10，25，50μM）的细胞中，LPS诱导的PMVECs应力纤维形成和细胞骨架形态改变的现象均被不同程度地抑制，F-actin表达及荧光强度也明显减弱，说明法舒地尔能明显抑制LPS诱导的PMVECs骨架结构的改变。
3法舒地尔对LPS诱导的大鼠PMVECs中MAPKs活化的影响
LPS（10μg/ml）在作用15min时即开始明显诱导大鼠PMVECs中ERK1/2磷酸化水平明显升高，且在60min时ERK1/2磷酸化水平达到最高。相同实验条件下，预先加入不同浓度的法舒地尔预处理，并没有改变LPS诱导的ERK1/2磷酸化水平的升高，说明在LPS诱导的大鼠PMVECs凋亡中ERK1/2并不是RhoA/ROCK的下游信号。另外，从JNK1/2/3及p38MAPKs的活化程度来看，LPS（10μg/ml）在作用5min时即开始明显诱导大鼠PMVECs中JNK1/2/3及p38磷酸化水平增高，且在30min时二者的磷酸化水平均达到最高。相同实验条件下，预先加入不同浓度（10，25，50μM）的法舒地尔预处理，均不同程度地抑制了LPS作用30min诱导的JNK1/2/3及p38磷酸化水平的增高（P <0.05或P <0.01）。说明JNK1/2/3及p38可能作为RhoA/ROCK的下游信号分子参与了LPS诱导的大鼠PMVECs凋亡。
4JNK和p38MAPKs的活化参与了LPS诱导的大鼠PMVECs凋亡
为进一步阐明JNK和p38的活化参与了LPS诱导的大鼠PMVECs凋亡。我们在接下来的实验中观察了SB203580（p38抑制剂）以及SP600125（JNK抑制剂）对LPS诱导的大鼠PMVECs凋亡及凋亡相关蛋白表达的影响。SB203580（10μM）、SP600125（20μM）预处理分别明显抑制了LPS诱导的p38和JNK1/2/3磷酸化水平的增高（P <0.01），说明SP600125和SB203580能分别起到抑制JNK1/2/3和p38MAPKs活化的作用。凋亡检测结果表明，SB203580和SP600125预处理明显抑制了LPS诱导的PMVECs凋亡，早期和晚期凋亡率有不同程度下降（P <0.05或P <0.01）。同时，SB203580及SP600125预处理还明显逆转了LPS诱导的凋亡相关蛋白Bax和Bcl-2的表达失衡情况，抑制了Bax的表达，升高了Bcl-2的表达（P <0.01），进一步说明JNK和p38的活化参与了LPS诱导的大鼠PMVECs凋亡。
结论：JNK1/2/3和p38MAPKs作为ROCK的下游信号分子参与了LPS诱导的大鼠PMVECs凋亡。法舒地尔作为唯一在临床应用的ROCK抑制剂，明显干预了LPS诱导的凋亡、骨架蛋白重排等细胞损伤，这一保护作用与其抑制ROCK通路及其下游JNK1/2/3和p38MAPKs信号分子活化有关。
第三部分法舒地尔对LPS诱导的大鼠PMVECs炎性因子表达的影响及机制研究
目的：研究法舒地尔对LPS诱导的大鼠PMVECs炎性因子表达的影响，并从氧化损伤角度出发探讨其作用机制。
方法：RT-PCR检测IL-6、TNF-α及MCP-1mRNA的表达；ELISA法检测上清液中IL-6和MCP-1的含量；激光共聚焦检测PMVECs中ROS的产生；试剂盒测定PMVECs中SOD、GSH-PX活力及MDA含量；Westernblot法测定PMVECs全蛋白及核蛋白中NF-κB p65的表达。
结果：
1法舒地尔对PMVECs中IL-6、TNF-α及MCP-1mRNA表达的影响与对照组（LPS0h）相比，10μg/ml LPS在作用3h时IL-6和MCP-1mRNA的表达即开始明显升高（P <0.01），6h时MCP-1mRNA表达水平达到最高，12h时IL-6mRNA表达水平达到最高，一直到48h，IL-6和MCP-1mRNA仍处于高表达水平（P <0.01）。10、25、50μM的法舒地尔预处理，均明显抑制了LPS作用12h诱导的IL-6mRNA的表达（P <0.01），也不同程度地抑制了LPS作用6h诱导的MCP-1mRNA的表达（P <0.05或P<0.01）。然而，LPS对TNF-α mRNA的表达没有明显影响。
2法舒地尔对LPS诱导大鼠PMVECs分泌IL-6及MCP-1的影响
与对照组（LPS0h, IL-6和MCP-1的分泌量分别为475±76pg/ml、1.082±0.389μg/ml）相比，LPS在作用3h时IL-6蛋白分泌量即开始明显升高（P<0.05），12h时分泌量达最高（1098±81pg/ml，P <0.01），一直到48h仍处于较高水平（P <0.01）。而MCP-1分泌量在LPS作用3、6、12h分别升至1.988±0.017μg/ml（P <0.05）、2.026±0.066μg/ml（P <0.05）、2.038±0.072μg/ml（P <0.05）。而且，LPS处理不同时间组与相应各时间点的对照组相比，IL-6和MCP-1蛋白分泌量均明显升高（P <0.05或P <0.01）。10、25、50μM的法舒地尔预处理，均不同程度地降低了LPS作用不同时间点诱导的IL-6和MCP-1的蛋白分泌水平（P <0.05或P <0.01）。
3LPS诱导的大鼠PMVECs中ROS的生成及法舒地尔对此的影响
与对照组（LPS0h）相比，10μg/ml的LPS作用从3h到48h，荧光强度均不同程度地增强（P <0.01），表明细胞内活性氧生成量明显升高，其中LPS处理6h时荧光强度达最高水平，12h时仍维持在接近于最高水平，随后各时间点（LPS处理24h和48h）荧光强度有所减弱，但仍明显高于对照组（P <0.01）。与LPS作用6h组相比，10，25，50μM的法舒地尔预处理组，细胞内荧光强度均明显降低（P <0.01），说明法舒地尔明显抑制了LPS诱导的PMVECs内ROS的生成。
4法舒地尔对PMVECs中SOD、GSH-PX活性和MDA含量的影响
与对照组（LPS0h）相比，10μg/ml LPS作用3、6、12、24、48h各时间点组，SOD活性均表现出不同程度的下降（P <0.05或P <0.01），其中，LPS作用12h组SOD活性降至最低。而GSH-PX活性在LPS作用6h时即开始有所下降（P <0.05），LPS作用12、24和48h时GSH-PX活性均处于较低水平（P <0.01）。相反，MDA的含量随着LPS作用时间的延长明显升高，在LPS作用6h时即开始明显升高（P <0.01），12h时达最高值，一直到48h，仍处于较高水平。而10，25，50μM法舒地尔预处理均不同程度地逆转了LPS作用12h诱导的PMVECs中SOD和GSH-PX活性的降低（P <0.05或P<0.01）；同时也不同程度地降低了MDA的含量（P <0.05或P <0.01）。
5法舒地尔对LPS作用下大鼠PMVECs中NF-κB p65核转位的影响
与对照组（LPS0h）相比，在LPS作用不同时间点，全蛋白中NF-κB p65的蛋白表达没有明显变化。而PMVECs细胞核内NF-κB p65的蛋白表达水平在LPS作用3h即明显升高（P <0.01），12h时表达水平最高，直到48h仍有较高的表达水平（P <0.01）。10，25，50μM法舒地尔预处理均明显降低了细胞核内NF-κB p65的蛋白表达（P <0.01），推测法舒地尔可能通过抑制NF-κB p65核转位从而降低了LPS诱导的炎性因子的表达。6N-acetylcysteine对LPS作用下大鼠PMVECs中IL-6、MCP-1mRNA表达及NF-kB p65核转位的影响
5、10mM N-acetylcysteine均不同程度地降低了LPS作用12h诱导的细胞核内NF-kB p65的蛋白表达（P <0.05或P <0.01），对LPS诱导的NF-kBp65核转位有一定的抑制作用。同时，5、10mM N-acetylcysteine还明显降低了LPS诱导的IL-6和MCP-1mRNA的表达（P <0.05或P <0.01）。
结论：（1）LPS作用下大鼠PMVECs中ROS的生成增多，炎性因子IL-6和MCP-1的表达升高。ROS抑制剂N-acetylcysteine明显抑制了LPS诱导的NF-κB核转位以及炎性因子的表达，表明ROS信号参与了LPS诱导的PMVECs炎性损伤。（2）法舒地尔抑制了LPS诱导的ROS的生成，逆转了LPS诱导的MDA水平的升高以及SOD和GSH-PX活性的下降，还降低了NF-κB核转位以及炎性因子IL-6和MCP-1的表达，说明法舒地尔通过其抗氧化作用干预了LPS诱导的大鼠PMVECs炎性损伤。
Bacterial sepsis and its more severe complications are clinically commonand potentially lethal diseases. The common denominator for all of thesecomplications is injury or dysfunction of pulmonary microvascular endothelialcells (PMVECs). PMVECs are not only the primary targets of injury, but alsothe active and effective inflammatory cells, which play an important role inthe development of diseases. So, it is crucial in the study of pulmonarycirculation diseases to further study the mechanisms of PMVEC injury.
Infection, especially gram negative sepsis, is a major cause of pulmonarydiseases such as ALI/ARDS. Bacterial lipopolysaccharide (LPS), a kind ofglycoprotein on the outer membrane of gram negative bacilli, can cause astrong inflammatory response in the body, damage the PMVECs directly orindirectly, and play a key role in the pathological process of ALI/ARDS.Complex signaling pathways are involved in the injury of PMVECs inducedby inflammation. Studies suggested that RhoA/ROCK pathway was involvedin the occurrence and development of ALI/ARDS, pulmonary arterialhypertension (PAH) and lung cancer. RhoA/ROCK signaling pathway caninduce the rearrangement of the actin cytoskeleton, regulate the genetranscription and cell cycle progression, and act as the signal converter ormolecular switch in the cell signal transduction. However, further studies arerequired for full understanding of the mechanisms of RhoA/ROCK pathway inthe damage of rat PMVECs induced by LPS, as well as the cross-talk ofRhoA/ROCK pathway with other signaling pathways. So, in the present study,LPS were used to induce the model of PMVEC damage and to investigate thechanges of RhoA/ROCK pathway in the process. Moreover,immunocytochemistry, flow cytometry, confocal laser and molecular cell biology technology were also used to investigate the influence of ROCKinhibitor fasudil on LPS-induced PMVECs damage, and to further discuss themechanisms of downstream signal transduction.
Part1RhoA/ROCK pathway is involved in LPS-induced damage of ratPMVECs
Objective: To study the changes of RhoA/ROCK pathway inLPS-induced damage of rat PMVECs and the mechanism of action.
Methods: The tissue-explant technique was used to culture the primaryrat PMVECs. The identification of rat PMVECs was carried out byimmunocytochemical staining of factor Ⅷrelated antigen and transmissionelectron microscope. The viability of PMVECs was determined by MTT andlactate dehydrogenase (LDH) method. The mRNA expression of RhoA andROCK1was detected by RT-PCR. The phosphorylation of MYPT1wasanalyzed by Western blot.
Results:
1The culture and identification of rat PMVECs
We successfully cultured rat PMVECs by tissue-explant technique withsome modification. After removal of explants, the PMVECs were cultured for3~5days and displayed the shape of paving stone at this moment. The resultsof immunocytochemical staining of factor Ⅷ related antigenshowed thatabout95%of the cells were positive. The results of electron microscopeshowed that the plasma membrane protrusions, also namely microvilli, couldbe observed in most cells, W-P bodies were also observed in the cytoplasm. Inaddition, a large number of plasma membrane vesicles (also called pinocytoticvesicles) were found in the cytoplasm of cells. All the above structures wereconsistent with the typical endothelial cell ultrastructure. Considering that thetissue explants were obtained from the lung edge, it could be confirmed thatthe cultured cells were indeed PMVECs.
2Effect of LPS on the viability of rat PMVECs and the intervention effect offasudil
MTT results suggested that the OD value in control group was0.95± 0.12,0.01,0.1and1μg/ml of LPS had no obvious effect on the viability ofPMVECs, while the OD value in LPS10and100μg/ml groups decreased to0.75±0.19(P <0.05) and0.50±0.12(P <0.01) respectively. LPS10μg/mlcould cause the injury of PMVECs, but sufficient cells were still existed forsubsequent test. So, LPS (10μg/ml) was used to induce the damage ofPMVECs. Compared with the OD value (0.93±0.15) in LPS10μg/ml (0h)group, the OD value did not change obviously at6h and12h after LPStreatment, but decreased significantly at24h (0.73±0.19), and stillmaintained at the low level at48h and72h. Fasudil (25,50μM) improved theLPS-induced injury of PMVECs in different degree (compared with the LPSgroup，P <0.05).
3Effect of LPS on the activity of LDH and the intervention effect of fasudil
The activity of LDH in control group was127.6±17.3U/L, which wasincreased to155.1±23.4U/L (P <0.05),162.3±34.3U/L (P <0.05),189.1±25.1U/L (P <0.01) and194.7±22.7U/L (P <0.01) respectively after thecells had been treated with LPS0.1,1,10and100μg/ml for24h. The LDHrelease began to increase at6h after the cells had been treated with LPS10μg/ml and the activity of LDH increased to139.7±19.2U/L, which wassignificantly higher (P <0.05) than that (114.1±21.1U/L) before LPStreatment. With the time prolongation, the activity of LDH increasedcontinuously, suggesting that PMVECs were damaged increasingly. Fasudil(10,25and50μM) pretreatment significantly decreased the LDH activity inthe supernatant (P <0.05or P <0.01), which suggested that fasudil inhibitedthe release of LDH, reduced the injury of PMVECs induced by LPS.
4The changes of mRNA expression of RhoA and ROCK1in LPS-inducedinjury of rat PMVECs
Compared with the control group (LPS0min), the mRNA expression ofRhoA began to increase at15min after LPS (10μg/ml) treatment (P <0.05),and reached the highest level at60min. While the mRNA expression ofROCK1began to increase after LPS (10μg/ml) treatment for5min (P <0.05),and appeared two peaks at15min and60min respectively (P <0.01). With the prolongation of time, the RhoA and ROCK1mRNA expression slightlydecreased, but still maintained at a higher level than that in the control group(LPS0min) at240min after LPS treatment, suggesting that RhoA/ROCKsignal pathway might be involved in the injury of PMVECs induced by LPS.
5Effect of LPS on the phosphorylation of MYPT1and the intervention effectof fasudil
Activated ROCK can further phosphorylate the downstream substrateMYPT1. Therefore, the phosphorylation level of MYPT1reflects the degreeof activation of ROCK. Western blot analysis showed that the phosphorylationof MYPT1began to increase after LPS (10μg/ml) treatment for5min (P <0.05), and reached the highest level at15min (P <0.01). With theprolongation of time, the expression of p-MYPT1slightly decreased, but stillhigher than that of the control group (LPS0min) at120min after LPStreatment (P <0.01). Fasudil (10,25and50μM) pretreatment significantlydecreased the expression of p-MYPT1induced by LPS treatment for15min(P <0.05or P <0.01).
Conclusion: The primary rat PMVECs were successfully cultured bytissue-explant technique in the present experiment. LPS markedly induced thedamage of rat PMVECs, and RhoA/ROCK signaling pathway was involved inthe process.
Part2Protective effect of fasudil against LPS-induced apoptosis andcytoskeleton rearrangement of rat PMVECs and the underlyingmechanisms
Objective: To study the effects of ROCK inhibitor fasudil onLPS-induced apoptosis and cytoskeleton rearrangement and the underlyingmechanisms.
Methods: The apoptosis of PMVECs was evaluated by AnnexinV/PI andHoechst33258fluorescent staining assay. The change of cytoskeleton wasdetected by F-actin staining. The phosphorylation of ERK1/2, JNK1/2/3, p38and the expression of apoptosis related proteins Bax and Bcl-2were detectedby Western blot.
Results:
1Effect of fasudil on LPS-induced apoptosis of rat PMVECs
1.1Effect of different duration of LPS exposure on apoptosis of rat PMVECs
The results of AnnexinV-FITC staining showed that compared with thecontrol group (LPS0h，the early apoptosis rate was3.1±0.7%and lateapoptosis rate was1.0±0.3%),10μg/ml LPS treatment for6h did not affectthe apoptosis of PMVECs. While the early apoptosis rate of PMVECsincreased to15.1±3.8%(P <0.01),24.3±3.6%(P <0.01),34.8±4.5%(P<0.01) and43.2±8.2%(P <0.01), and the late apoptosis rate increased to2.1±0.9%(P <0.05),9.6±2.1%(P <0.01),12.2±4.7%(P <0.01),29.1±7.4%(P <0.01) respectively at12,24,48and72h after LPS treatment,which suggested that LPS obviously induced the apoptosis of rat PMVECs.
1.2Fasudil decreased LPS-induced apoptosis of PMVECs significantly
Compared with the control group (the early apoptosis rate and lateapoptosis rate were2.1±0.7%and3.0±0.7%respectively), the earlyapoptosis rate increased to37.8±8.1%(P <0.01) and the late apoptosis rateincreased to10.7±2.6%(P <0.01) after LPS treatment for24h, which weredecreased in different degree by fasudil pretreatment. The early apoptosis ratedecreased to27.7±4.6%(P <0.05)，21.8±5.9%(P <0.01) and18.6±3.9%(P<0.01), and the late apoptosis rate decreased to7.5±1.3%(P <0.05)，6.4±1.3%(P <0.05) and5.3±1.4%(P <0.01) respectively after10,25and50μM of fasudil pretreatment, suggesting that fasudil has some interventioneffect on LPS-induced apoptosis injury of rat PMVECs.
1.3Fasudil improved LPS-induced apoptotic morphological changes of ratPMVECs
From the results of Hoechst33258staining we observed that the nucleusof PMVECs in control group was round or oval with neat edge withhomogeneous and low-intensity blue fluorescence. No obvious apoptotic cellswere found in control group. After treatment with LPS for24h, the nucleus ofPMVECs was condensed and hyperchromatic with high-brightnessfluorescence. Part of nuclear chromatin condensed highly and marginalized, and the number of PMVECs with crescent-shaped nuclei increased, whichsuggested that LPS induced apoptosis of rat PMVECs significantly. Fasudil(10,25and50μM) pretreatment significantly weakened the apoptoticmorphological changes of PMVECs induced by LPS, further illustrating thatfasudil can prevent the apoptosis of rat PMVECs induced by LPS.
1.4Effect of fasudil on the expression of Bax and Bcl-2proteins
Compared with the control group, treatment of PMVECs with10μg/mlLPS increased the protein expression of Bax (P <0.01) and decreased theprotein expression of Bcl-2(P <0.01). Fasudil (10,25and50μM)pretreatment significantly reversed the imbalance of protein expression of Baxand Bcl-2caused by LPS, with the decreased Bax expression and increasedBcl-2expression (P <0.05or P <0.01), which indicated that fasudil mightinhibit LPS-induced apoptosis of rat PMVECs by regulating the expression ofanti-apoptotic and pro-apoptotic proteins.
2Effect of fasudil on the distribution of skeleton protein F-actin in ratPMVECs
There are a few of actin filaments in the control group, which mainlydistributed in the periphery of cells. After treatment with LPS, the cytoplasmicF-actin fibers increased significantly, and most of them arranged along thelongitudinal of PMVECs. The F-actin fibers around the cells graduallydisappeared, and intensive fasciculate stress fibers appeared in the cytoplasm.With the prolongation of time, the arrangement of F-actin became more andmore disorderly and dispersive, and the normal connections between cellswere almost lost. Meanwhile, the formation of stress fiber and themorphological changes of cytoskeleton induced by LPS were inhibited by10,25and50μM of fasudil pretreatment in different degree, and fasudil alsoreduced the fluorescence intensity of F-actin.
3Effect of fasudil on the activation of MAPKs in LPS-treated rat PMVECs
LPS (10μg/ml) caused obvious phosphorylation of ERK1/2at15minafter treatment and with a peak arrived at60min (P <0.01). Whereas,pretreatment with10,25and50μM of fasudil did not inhibit the ERK1/2 phosphorylation, suggesting that ERK1/2was not as the downstream signal ofRhoA/ROCK in LPS-induced apoptotic injury. In addition, thephosphorylation of JNK1/2/3and p38began to increase after LPS treatmentfor5min and with the highest level both arrived at30min, which was allinhibited by fasudil (10,25and50μM) pretreatment (P <0.05or P <0.01),suggesting that JNK1/2/3and p38might act as the downstream signals ofRhoA/ROCK in LPS-induced apoptotic injury.
4The activation of JNK and p38MAPKs participated in the apoptotic injuryinduced by LPS in rat PMVECs
In order to further clarify that JNK and p38were involved inLPS-induced apoptosis of rat PMVECs, we observed the effects of SB203580(p38inhibitor) and SP600125(JNK inhibitor) on apoptosis and the expressionof apoptosis-related proteins induced by LPS. SB203580(10μM) andSP600125(20μM) pretreatment significantly reduced the phosphorylation ofp38and JNK MAPKs respectively, suggesting that SB203580and SP600125can inhibite the activation of p38and JNK MAPKs respectively. MeanwhileSB203580and SP600125pretreatment significantly inhibited the apoptosisinduced by LPS in rat PMVECs, and the early apoptosis rate and lateapoptosis rate were all decreased (P <0.05or P <0.01). SB203580andSP600125pretreatment also reversed the imbalance of the expression of Baxand Bcl-2proteins induced by LPS, with a decreased expression of Bax and anincreased expression of Bcl-2(P <0.01), further suggesting that activation ofJNK and p38participated in the LPS-induced apoptosis of rat PMVECs.
Conclusion: Activation of JNK and p38MAPKs, the downstreamsignaling molecules of RhoA/ROCK signaling pathway, played an importantrole in LPS-induced apoptosis of rat PMVECs. Fasudil, a potent and selectiveinhibitor of ROCK, exerted an anti-apoptotic effect and inhibited thecytoskeletal rearrangement in rat PMVECs, which were mediated by theinhibition of RhoA/ROCK and its downstream JNK and p38MAPKs.
Part3Effects of fasudil on the expression of inflammatory factorsinduced by LPS in rat PMVECs and the underlying mechanisms
Objective: To study the effects of fasudil on the expression ofinflammatory factors induced by LPS in rat PMVECs and the antioxidativemechanisms.
Methods: The mRNA expression of IL-6, TNF-αand MCP-1wasevaluated by RT-PCR; The content of IL-6and MCP-1in supernatant wasdetermined by ELISA assay kit; ROS was measured by confocal microscopywith DCFH-DA staining. The activity of SOD and GSH-PX and content ofMDA were measured using kits supplied by Nanjing JianchengBiotechnological Company. Western blot analysis was used to determine theprotein expression of NF-κB p65in the cytoplasm and nucleus.
Results:
1Effects of fasudil on the mRNA expression of IL-6, TNF-αand MCP-1induced by LPS in rat PMVECs
Compared with the control group (LPS0h), the mRNA expression ofIL-6and MCP-1began to increase after LPS treatment for3h (P <0.01), andmaintained at a higher level until the end of the experiment. The highest levelof mRNA expression of MCP-1arrived at6h, and that of IL-6arrived at12h,which were both reduced by10,25and50μM of fasudil pretreatment (P <0.05or P <0.01). LPS exerted no obvious effect on the expression of TNF-α.
2Effect of fasudil on the secretion of IL-6and MCP-1induced by LPS in ratPMVECs
Compared with the control group (LPS0h, the concentration of IL-6andMCP-1was475±76pg/ml and1.082±0.389μg/ml respectively), thesecretion of IL-6began to increase after LPS treatment for3h (P <0.05), andmaintained at a high level until48h (P <0.01), with the highest level arrivedat12h (1098±81pg/ml, P <0.01). The secretion of MCP-1also increased to1.988±0.017μg/ml (P <0.05),2.026±0.066μg/ml (P <0.05) and2.038±0.072μg/ml (P <0.05) at3,6and12h respectively. Meanwhile, the contentof both IL-6and MCP-1increased at different time points after LPS treatmentcompared with that in the corresponding control group (P <0.05or P <0.01).Fasudil (10,25and50μM) pretreatment decreased the secretion of IL-6and MCP-1induced by LPS at different time points (P <0.05or P <0.01).
3Effect of LPS on the production of ROS in rat PMVECs and the interventioneffect of fasudil
Compared with the control group (LPS0h), the fluorescence intensity ofROS was significantly increased from3h to48h after LPS treatment, andwith the highest level arrived at6h. After that, the fluorescence intensity hadsome decrease at24h and48h after LPS treatment, but still stronger than thatin control group (P <0.01), suggesting that the production of ROS increasedobviously after the cells were treated with LPS. Compared with the LPS (6h)group, pretreatment with fasudil (10,25and50μM) obviously reduced thefluorescence intensity of ROS induced by LPS treatment for6h (P <0.01),which suggested that fasudil significantly inhibited the production of ROSinduced by LPS in rat PMVECs.
4Effect of fasudil on the activity of SOD and GSH-PX and the content ofMDA
Compared with the control group (LPS0h), the activity of SODdecreased significantly from3h to48h after LPS treatment (P <0.05or P <0.01), and the lowest level arrived at12h. The activity of GSH-PX began todecrease at6h (P <0.05), and maintained the low level at12,24and48hafter LPS treatment (P <0.01). On the contrary, the content of MDA began toincrease after LPS treatment for6h (P <0.01), and reached the highest levelat12h (P <0.01), which maintained until48h after LPS treatment. Whilefasudil (10,25and50μM) pretreatment increased the activity of SOD andGSH-PX reduced by LPS treatment for12h (P <0.05or P <0.01), anddecreased the content of MDA in different degree (P <0.05or P <0.01).
5Effect of fasudil on the nuclear translocation of NF-κB p65induced by LPS
Compared with the control group (LPS0h), the expression of NF-κB p65in the whole cell extracts of rat PMVECs showed no obvious changes afterLPS treatment for different time, while that in the nuclear proteins wasobviously increased after LPS treatment for3h (P <0.01). The expression ofNF-κB p65in the nuclear proteins arrived the highest level at12h, and maintained at a high level until48h after LPS treatment (P <0.01).Meanwhile, fasudil (10,25and50μM) pretreatment significantly decreasedthe expression of NF-κB p65in the nuclear proteins of rat PMVECs, whichsuggested it might be through inhibiting the nuclear translocation of NF-κBp65that fasudil reduced the secretion of inflammatory factors.
6Effects of N-acetylcysteine on the mRNA expression of IL-6and MCP-1and the nuclear translocation of NF-κB p65induced by LPS
N-acetylcysteine (5,10mM) pretreatment significantly decreased theexpression of NF-κB p65in the nuclear proteins of rat PMVECs induced byLPS treatment for12h (P <0.05or P <0.01). Meanwhile, the mRNAexpression of IL-6and MCP-1induced by LPS was also reduced byN-acetylcysteine (5,10mM) pretreatment (P <0.05or P <0.01).
Conclusion:(1) N-acetylcysteine, the inhibitor of ROS, obviouslyinhibited the production of ROS and the expression of IL-6and MCP-1induced by LPS in rat PMVECs, which suggested that ROS was involved inLPS-induced inflammatory injury of rat PMVECs.(2) Fasudil inhibited theproduction of ROS. The increased level of MDA and the decreased activity ofSOD and GSH-PX induced by LPS were reversed by fasudil. Meanwhile,fasudil decreased the expression of IL-6and MCP-1by inhibiting the nucleartranslocation of NF-κB p65, suggesting that fasudil ameliorated theinflammatory injury induced by LPS through its antioxidant effect.
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