携带IL-10基因的双歧杆菌对 溃疡性结肠炎小鼠肠道屏障功能的影响

目的观察表达IL-10基因的双歧杆菌对溃疡性结肠炎(UC)小鼠肠道屏障功能的影响,进一步探讨转基因双歧杆菌治疗UC的相关机制。方法 25只小鼠分为空白对照组、结肠炎组(UC-blank组)、双歧杆菌治疗组(UC-bacteria组)、空质粒双歧杆菌治疗组(UC-pBBAD/X-bacteria组)和表达IL-10基因双歧杆菌治疗组(UC-BL-hIL-10-bacteria组),每组5只。采用5%DSS诱导建立UC小鼠模型,利用前期研究已成功筛选出的可稳定表达hIL-10蛋白的BL-hIL-10菌株对小鼠进行治疗。采用HE染色评估小鼠结肠炎症情况;采用荧光定量PCR检测小鼠结肠组织Claudin1、2、4、5、7和8的表达水平;计算小鼠DAI。结果 (1)BL-hIL-10菌株可以降低UC小鼠DAI,减轻UC小鼠结肠组织炎症程度。(2)BL-hIL-10菌株能下调UC小鼠结肠组织Claudin2表达水平,同时能够上调Claudin1、4、5、7和8的表达水平。(3)UC-BL-hIL-10-bacteria组UC小鼠的治疗效果明显优于UC-bacteria组和UC-pBBAD/Xbacteria组。结论 BL-hIL-10菌株能改善UC小鼠结肠黏膜的通透性,其机制可能与其能上调Claudin1、4、5、7和8的表达水平,同时降低Claudin-2的表达水平有关。     

婴儿双歧杆菌对花生过敏小鼠模型肠道Th2反应的调节

目的研究婴儿型双歧杆菌对花生过敏小鼠肠道Th2型反应的调节作用。方法通过应用花生蛋白诱导肠道的Th2型反应,建立食物过敏小鼠模型。过敏小鼠灌胃给予婴儿型双歧杆菌（ATCC菌或CGMCC0313-2）或不做处理。然后分离小鼠小肠黏膜CD4＋T细胞或DC,另取肠黏膜组织进行石蜡包埋甲苯胺蓝染色肥大细胞计数,HE染色进行嗜酸细胞和单个核细胞计数,流式细胞检测CD4＋T中Th2（CD4＋IL4＋T）细胞和Treg（CD4＋CD25＋Foxp3＋T）比例,另取CD4＋T进行CFSE标记,与DC共培养4d后流式细胞检测CD4＋T增殖反应,收集细胞培养液ELISA检测IL-4、IL-5和IL—13分泌水平。结果过敏组小鼠Th2型细胞数,CD4＋T细胞增殖反应,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显高于对照组（P〈0．01）,而Treg数目低于对照组（P〈0．01）,婴儿双歧杆菌干预后,婴儿双歧杆菌组Th2型细胞数,IL4、IL-5和IL-13水平,肠黏膜中肥大细胞、嗜酸性细胞和单个核细胞数均明显低于过敏组（P〈0．01）,而Treg数目高于过敏组（P〈0．01）。结论口服婴儿型双歧杆菌可以抑制花生过敏导致的肠道Th2型反应。     

双歧杆菌小鼠肠道定植的优化

目的评价自制的小鼠灌肠器灌肠效果,并探讨双歧杆菌定植的优化方式,更好地建立双歧杆菌在结肠的定植模型。方法选用SPF级C57BL小鼠40只,每组10只,随机分成4组：正常对照组,常规灌胃组,市售灌肠组,自制灌肠组。从市面不同的材料中筛选小鼠灌肠管,并设计小鼠专用灌肠器。记录不同灌注方式时隐血试验等一般指标检测。RT-PCR检测4组脾脏TLR2 mRNA表达水平。免疫组化观察颈动脉血管HGF蛋白水平。体外模拟不同pH值检测双歧杆菌定植HT-29细胞的粘附水平。结果自制的灌肠器在小鼠灌肠操作过程中明显优于市售灌肠器;自制灌肠组隐血试验明显低于市售灌肠组（P〈0.01）;RT-PCR结果显示,自制灌肠组效果最强,市售灌肠组次之,常规灌胃组一般,但均高于正常对照组;HGF的免疫组化显示,自制灌肠组表达最强;粘附试验显示双歧杆菌在pH 7.0环境中粘附能力优于pH 4.5。结论双歧杆菌在结肠定植采用灌肠方式优于灌胃方式;自制灌肠器优于市售灌肠器;优化灌肠液,采用自制的灌肠器灌肠同时优化灌肠操作,能够在小鼠结肠建立较好的双歧杆菌定植模型。     

携带IL-10基因的双歧杆菌对 感染后肠易激综合征的治疗作用

目的探讨转基因双歧杆菌对感染后肠易激综合征(PI-IBS)小鼠的治疗作用。方法 50只成年雄性SD小鼠,随机选取10只作为空白对照组(NC组),其余40只PI-IBS模型小鼠随机分为IBS组(IBS组)、双歧杆菌治疗组(BL组)、空质粒双歧杆菌治疗组(BL-0组)和BL-hIL-10治疗组(每组10只)。NC组和IBS组每天予0.2mL PBS灌胃1次;其余三组每天灌胃溶有经0.2%L-Arb诱导24h后的BL、BL-0、BL-hIL-10菌液(双歧杆菌浓度达5×108个/mL)的PBS液0.2mL,每天1次。所有小鼠干预7d后处死,然后行血液及结肠组织学检验。结果 (1)通过评估小鼠疾病活动指数、小鼠脾脏指数和结肠组织HE染色病理学变化,证明口服BL-hIL-10能显著减轻PI-IBS小鼠的炎症症状;(2)BL-hIL-10能升高IL-10比例并降低IL-12的比例,维持Th平衡。结论口服转基因双歧杆菌可以缓解PI-IBS小鼠的炎症反应,其机制可能是通过调节Th平衡来实现。     

携带IL-10基因的双歧杆菌对溃疡性结肠炎模型小鼠血浆及结肠组织NO和VEGF-A表达影响

目的观察携带IL-10基因的双歧杆菌对溃疡性结肠炎(UC)小鼠血浆及结肠组织一氧化氮(NO)与血管内皮生长因子A(VEGF-A)表达的影响,进一步探讨携带IL-10基因的双歧杆菌治疗UC的相关机制。方法筛选出能稳定表达具有生物活性hIL-10蛋白的BL-hIL-10菌株。用5%DSS诱导UC小鼠模型,50只小鼠分为正常对照组、UC模型组、BL治疗组、BL0治疗组和BL-hIL-10治疗组,每组10只。计算小鼠DAI、HE染色评估结肠组织病理学变化;ELISA法测小鼠血浆及结肠组织IL-13、VEGF-A和NO的含量。结果 (1)BL-hIL-10可以降低UC小鼠DAI,减轻UC小鼠结肠组织炎症程度。(2)BL-hIL-10能降低UC小鼠血浆和结肠组织NO、VEGF-A和IL-13水平。结论口服BL-hIL-10对UC小鼠的治疗作用可能与抑制NO产生及下调VEGF-A表达有关。     

马齿苋多糖对溃疡性结肠炎小鼠肠道菌群及血内毒素的影响

目的应用马齿苋多糖对溃疡性结肠炎小鼠进行调整治疗,达到从微生态学角度防治溃疡性结肠炎。方法应用DSS制备溃疡性结肠炎小鼠模型,随机分成2组：正常对照组、模型组,造模成功后模型组再分为自然恢复组、马齿苋多糖治疗组。分别于造模后、给药7 d后处死小鼠,进行肠道菌群检测、血内毒素含量测定。结果 DSS造模后模型组小鼠肠道菌群失调,外周血内毒素含量升高。马齿苋多糖治疗7 d后治疗组小鼠肠道双歧杆菌和乳酸杆菌数量明显上升,外周血内毒素含量明显下降。结论马齿苋多糖可以提高双歧杆菌和乳酸杆菌数量,降低外周血内毒素含量,调节肠道微生态失调,对溃疡性结肠炎发挥了一定的治疗作用。     

马齿苋多糖对溃疡性结肠炎小鼠肠黏膜sIgA及病理表现的影响

目的探讨马齿苋多糖对溃疡性结肠炎小鼠肠黏膜sIgA及病理表现的影响。方法应用硫酸葡聚糖钠(DSS)制备溃疡性结肠炎小鼠模型,随机分成两组:正常对照组、模型组,造模成功后模型组再分为自然恢复组、马齿苋多糖治疗组。分别于造模后、给药7d后处死小鼠,进行肠道菌群、肠黏膜sIgA及结肠组织病理学检测。结果 DSS造模后模型组小鼠肠道菌群失调、肠黏膜sIgA含量下降、结肠组织有病理改变。马齿苋多糖治疗7d后治疗组小鼠肠道双歧杆菌和乳酸杆菌数量明显上升,肠黏膜sIgA含量上升、结肠组织病理改变减轻。结论马齿苋多糖可以提高双歧杆菌和乳酸杆菌数量,提高肠黏膜sIgA含量,改善结肠组织病理变化,对溃疡性结肠炎发挥了一定的治疗作用。     

马齿苋多糖对溃疡性结肠炎小鼠肠黏膜细胞因子及肠道菌群的影响

目的观察马齿苋多糖对溃疡性结肠炎小鼠肠黏膜细胞因子及肠道菌群的影响。方法应用硫酸葡聚糖钠(DSS)制备溃疡性结肠炎小鼠模型,随机分成2组:正常对照组、模型组,造模成功后模型组再分为自然恢复组、马齿苋多糖治疗组。分别于造模后、给药7 d后处死小鼠,进行肠黏膜细胞因子测定、肠道菌群检测。结果 DSS造模后模型组小鼠肠黏膜细胞因子TNF-а、IL-6升高,IL-10减少;小鼠肠道菌群失调。马齿苋多糖治疗7 d后治疗组小鼠与模型组小鼠比TNF-α、IL-6下降,IL-10增加;肠道双歧杆菌和乳杆菌数量上升。结论马齿苋多糖可以提高抗炎细胞因子IL-10的水平并降低致炎细胞因子TNF-α、IL-6的水平;可以提高双歧杆菌和乳杆菌数量。马齿苋多糖通过抗炎和降低肠道过度的免疫反应以及调节肠道微生态失调,对溃疡性结肠炎发挥治疗作用。     

双歧三联活菌胶囊对结肠造口术后腹泻患者肠道菌群及肠黏膜屏障功能的影响

目的探讨双歧三联活菌胶囊对结肠造口术后腹泻患者肠道菌群及肠黏膜屏障功能的影响。方法选取外科门诊就诊结肠造口术后腹泻患者82例,随机分为观察组和对照组各41例。两组患者均予以口服补液盐、止泻药等常规治疗。观察组加用双歧三联活菌胶囊温水口服,630mg/次,2次/d,连用4周。于治疗前后观察两组患者肠道菌群数量及肠黏膜屏障功能指标的变化,并比较其临床效果。结果治疗4周后,两组患者双歧杆菌、乳杆菌数量及B/E比值上升,大肠埃希菌和肠球菌数量下降(P0.05),且观察组变化幅度更大;两组患者血清D-乳酸和二胺氧化酶(DAO)水平降低(P0.05或P0.01),且观察组下降幅度更大(P0.05);在总有效率上观察组较对照组更佳(P0.05)。结论双歧三联活菌胶囊治疗结肠造口术后腹泻患者效果较佳,机制与其能调节肠道微生态平衡紊乱,重建肠道微生态平衡,并能保护与修复肠黏膜屏障,减少其通透性,改善肠道功能密切相关。     

双歧杆菌对中国对虾原肌球蛋白致敏小鼠的免疫调控作用

【目的】比较研究婴儿双歧杆菌(Bifidobacterium infantis 13.085)对中国对虾原肌球蛋白致敏BALB/c小鼠预防与治疗过敏反应的差异,探究其对致敏小鼠Treg/Th17细胞平衡及相关细胞因子的影响。【方法】采用硫酸铵盐析及等电点沉淀法纯化中国对虾原肌球蛋白(TM),将中国对虾TM和弗氏佐剂混合液腹腔注射诱发BALB/c小鼠致敏,建立动物过敏模型。将实验小鼠随机分为正常对照组、治疗对照组、双歧杆菌治疗组、预防对照组和双歧杆菌预防组。观察分析小鼠过敏症状(腹泻、肺组织HE染色比较、称重法测定小鼠体重和脾脏脏器系数变化),采用ELISA测定小鼠血清中特异性IgE、IgG2a和组胺的含量,采用流式细胞术测定脾脏T淋巴细胞亚群(Treg、Th17)数量,采用荧光定量PCR测定脾脏中Treg型和Th17型细胞因子和转录因子的表达量。【结果】纯化得到中国对虾原肌球蛋白纯度为84.93%,得率为60.88%。体内试验表明,双歧杆菌治疗组和预防组相比于对照组,腹泻和过敏症状均有明显的缓解;不同时期的双歧杆菌干预均对过敏小鼠肺组织症状有明显的改善作用,且可降低过敏小鼠的脾脏脏器系数。第56天实验周期结束后发现,相比预防对照组和治疗对照组,双歧杆菌预防组和治疗组小鼠血清中特异性IgE和组胺含量显著降低(P0.05),脾脏Treg/Th17比值显著升高(P0.05),Th17型细胞因子IL-17A mRNA表达水平显著降低(P0.01);双歧杆菌治疗组相对于治疗对照组,Treg型细胞因子CD25mRNA表达水平显著升高(P0.01)。此外,双歧杆菌治疗组血清特异性IgE及IL-17A mRNA转录水平显著低于双歧杆菌预防组(P0.05),而Treg/Th17比值及CD25 mRNA转录水平显著高于预防组(P0.05)。【结论】双歧杆菌13.085能有效缓解小鼠过敏症状,且治疗免疫调控效果优于预防效果,其作用可能通过平衡Treg/Th17细胞亚群数量,促进Treg型细胞因子表达而抑制Th17型细胞因子分泌,从而阻断炎性抗体及组胺释放。     

爱灵生乐（2036）螺旋藻双歧胶囊对抗生素相关性腹泻小鼠的治疗作用

目的采用盐酸林克霉素诱发小鼠相关性腹泻模型,观察螺旋藻、婴儿型双歧杆菌及其混合物爱灵生乐（2036）螺旋藻双歧胶囊（胶囊内容物,含婴儿型双歧杆菌、螺旋藻,下同）对抗生素相关性腹泻小鼠的治疗作用。方法经口投予盐酸林克霉素0.15 g/（d.鼠）,连续3 d,然后经口分别给予螺旋藻、双歧杆菌及爱灵生乐（2036）螺旋藻双歧胶囊1.66 g/（kg.b.w）,连续5 d。结果经口投予螺旋藻、婴儿型双歧杆菌及爱灵生乐（2036）螺旋藻双歧胶囊1.66 g/（kg.b.w）,连续5 d,能有效改善盐酸林克霉素诱发的小鼠相关性腹泻症状,特别是给予爱灵生乐（2036）螺旋藻双歧胶囊组,可有效治疗盐酸林克霉素诱发小鼠相关性腹泻。结论螺旋藻与双歧杆菌复合制剂对抗生素相关性腹泻有明确治疗作用。     

蛋白酶激活受体2(PAR-2)激动剂对肥大细胞释放类胰蛋白酶的影响

研究反肉桂酰-亮-异亮-甘-精-亮-鸟-[酰胺]（tc-LIGRLO),一种PAR-2激动剂,对肥大细胞类胰蛋白酶释放的影响。结果显示,经过15min的培养,tc-LIGRLO可引起比基础分泌量增加1倍以上的类胰蛋白酶释放,作用强度超过抗IgE抗体和钙离子导入剂（calcium ionophore A23187,CI),而反PAR-2激动剂-反肉桂酰-鸟-亮-精-甘-异亮-亮-[酰胺]（tc-OLRGIL)无此作用,培养时间延长到30min时对tc-LIGRLO的作用无明显影响,其时间关系曲线表明,tc-LIGRLO的作用从1min开始,3min后达高峰,结果表明,PAR-2激动剂tc-LIGRLO是一种高效类胰蛋白酶释放刺激剂,在肥大细胞上可能有PAR-2存在。     

脂磷壁酸的提取工艺及免疫功能的研究

目的确定婴儿双歧杆菌脂磷壁酸（LTA）的最佳提取工艺及其免疫调节作用。方法通过对不同方法提取婴儿双歧杆菌LTA的测定及对植瘤小鼠淋巴细胞的转化来探讨LTA对小鼠细胞免疫的调节作用。结果采用脱脂后水法提取效果最好,与对照组和全菌组相比,LTA使淋巴细胞转化增加。结论婴儿双歧杆菌及其LTA均有免疫功能,但LTA的效果要优于婴儿双歧杆菌。     

Gastrointestinal dysfunction induced by early weaning is attenuated by delayed weaning and mast cell blockade in pigs

Our previous work has demonstrated that weaning at 19 days of age has deleterious effects on mucosal barrier function in piglet intestine that are mediated through peripheral CRF receptor signaling pathways. The objectives of the present study were to assess the impact of piglet age on weaning-associated intestinal dysfunction and to determine the role that mast cells play in weaning-induced breakdown of mucosal barrier function. Nursing Yorkshire-cross piglets were either weaned at 19 days of age (early-weaned, n = 8) or 28 days of age (late-weaned, n = 8) and housed in nursery pens. Twenty-four hours postweaning, segments of midjejunum and ascending colon from piglets within each weaning age group were harvested and mounted on Ussing chambers for measurements of transepithelial electrical resistance and serosal-to-mucosal [(3)H]mannitol fluxes. Early weaning resulted in reductions in transepithelial electrical resistance and increases in mucosal permeability to [(3)H]mannitol in the jejunum and colon (P < 0.01). In contrast, postweaning reductions in intestinal barrier function were not observed in piglets weaned at 28 days of age. Early-weaned piglet intestinal mucosa had increased expression of CRF receptor 1 protein, increased mucosal mast cell tryptase levels, and evidence of enhanced mast cell degranulation compared with late-weaned intestinal mucosa. Pretreatment of piglets with the mast cell stabilizer drug cromolyn, injected intraperitoneally 30 min prior to weaning, abolished the early-weaning-induced intestinal barrier disturbances. Our results indicate that early-weaning stress induces mucosal dysfunction mediated by intestinal mast cell activation and can be prevented by delaying weaning.     

CFTR基因在小鼠肠道组织中的差异表达

目的研究肠道组织CFTR基因表达与分泌性腹泻发生的关系。方法选取KM小鼠24只,雌雄各半,随机分为3组（每组8只）：对照组经小鼠腹腔注射0.2 mL生理盐水,实验组小鼠经腹腔注射LPS[6 mg/（kg·bw）]分别作用1 h、8 h,于注射后通过小鼠精神状态、肠道组织形态学判定分泌性腹泻模型的建立,利用荧光定量PCR法检测各段肠道组织CFTR基因的表达。结果 LPS成功诱导小鼠发生了分泌性腹泻;CFTR基因在小鼠十二指肠、空肠、回肠和结肠组织中均有不同的表达丰度,以结肠最高,但各段肠道间差异不显著;与对照组相比,LPS上调了十二指肠、空肠和回肠CFTR基因的转录,下调了结肠CFTR基因的转录。结论提示肠道组织CFTR基因转录水平的上调与LPS诱导分泌性腹泻的发生密切相关,且在各肠段发挥的作用不同,其中空肠在氯离子（Cl-）分泌中发挥主要作用,结肠的作用最弱。     

Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.     

Localization of Protease-Activated Receptors -1 and -2 in Human Mast Cells: Indications for an Amplified Mast Cell Degranulation Cascade

Protease-activated receptors (PARs) belong to a family of G-coupled seven transmembrane receptors that are activated by a proteolytic cleavage of their N-termini. Recent studies suggest the involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2) activators in mast cell de-granulation in various physiological and pathophysiological processes in inflammatory responses. Although PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have been associated with mast cell activation, PAR-1 and PAR-2 have not been localized within these cells. We describe here the localization of PAR-1 and PAR-2 in mast cells from various normal human tissues using im-munohistochemical and double immunofluorescence techniques. The presence of these receptors on the membrane may explain the actions of accessible extracellular thrombin and tryptase for mast cell activation. In addition to the membrane labeling, these receptors are also localized on the membrane of the intracellular tryptase-positive granules, which may function to sustain further mast cell degranulation upon exocytosis. The localization of these two receptors in mast cells suggests a novel mechanism for controlling mast cell activation through regulation of PARI and PAR-2.     

Tryptase promotes human monocyte-derived macrophage foam cell formation by suppressing LXRα activation

Accumulated mast cells in atherosclerotic plaques secrete a high level of tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. However, the role of tryptase in the lipid metabolism of macrophages remains to be defined. In the present study, we found that the addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. Tryptase promoting foam cell formation was also observed by transmission electron microscope. These effects were resisted by APC366, a selective inhibitor of mast cell tryptase. Tryptase dramatically resisted 22RHC induced activation of LXRα protein expression, which can be reversed by SAM-11 (a PAR-2-specific neutralizing antibody) and reduced LXRα, ABCG1, ABCA1 and SREBP-1c mRNA levels and ABCG1 protein level, which were all blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation to activate LXRα, ABCG1 protein expression, and mRNA levels of LXRα and its target genes in both THP-1-derived macrophages and primary human monocyte-derived macrophages. In primary macrophages that were first transfected with PAR-2 siRNA and then treated with tryptase, both the ABCG1 protein level and mRNA levels of LXRα and ABCG1 were higher than those in the control siRNA-treated cells. Taken together, our data clarified the PAR-2 expression of human macrophages and suggested that tryptase might promote lipid accumulation in macrophages and foam cell formation by suppressing LXRα activation via PAR-2/LXRα/LXRα target genes signaling pathway. This investigation sheds a new light on the role of tryptase in foam cell formation and pathogenesis of atherosclerosis.     

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.