京尼平对人脐静脉内皮细胞胞吐的抑制作用与其分子机制
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摘要
血管内皮细胞位于血管内腔表面,是血管壁与血液之间的分界屏障细胞。它不仅是生物学上的结构与功能屏障,还是高度活跃的代谢库,能合成多种血管活性物质。血管内皮细胞的损伤和功能异常与多种疾病的发生相关或为始动环节,尤其在心脑血管性疾病发病过程中起到重要作用。在血管内皮细胞功能异常和损伤发生过程中,血管内皮细胞胞吐是最早发生的反应。血管内皮细胞受到炎症刺激时,它通过胞吐释放大量炎症性因子,诱导大量的白细胞、血小板粘附到血管内皮表面,加剧组织炎症与血栓的形成。内皮细胞胞吐已经成为研究血管功能异常和损伤发生机制的一个重要方面。
内皮细胞内贮存大量的颗粒,它们包含前炎症因子与前凝血因子,例如：P-选择素(P-selectin)、E-选择素(E-selectin)、血管内皮粘附分子-1(VCAM-1)和血管假性血友病因子(VWF)等,这些分子与细胞粘附、炎症及止血紧密相关。P-选择素介导中性粒细胞在活化内皮细胞上的滚动,尤其在炎症过程的早期(数分钟)甚为重要,在炎症晚期同其他选择素分子协同发挥作用。P-选择素还参与血小板和某些T细胞亚群的沿血管壁的滚动的过程。E-选择素主要介导白细胞(中性粒细胞、单核细胞和CD4+记忆性T细胞)在内皮细胞表面最初的滞留和滚动,以及随后迁移到炎症的组织。血管内皮粘附分子-1主要介导淋巴细胞、单核细胞嗜酸细胞和内皮细胞的粘附。它特异地与白细胞(除中性粒细胞外)所产生的整合素家族的粘附因子VLA-4结合,介导细胞粘附和信号传递,在细胞分化、炎症反应、动脉粥样硬化等多种疾病病理生理过程中发挥作用。血管假性血友病因子介导血小板粘附与聚集,启动血栓形成,特异性地反映血管内皮细胞功能的异常。许多促分泌剂都能刺激内皮细胞胞吐,例如：凝血酶,组胺,白三烯,补体等。根据胞吐是由钙介导还是CAMP介导,促分泌剂分为两类。目前研究最广的是凝血酶,它是一种钙调促分泌剂,它通过活化钙调蛋白升高细胞内钙离子浓度,导致快速的胞吐。凝血酶与位于内皮细胞表面的受体结合刺激血管内皮细胞释放P-selectin、E-selectin和VCAM-1,这一系列反应不仅推动凝血过程和血小板黏附,而且促进中性粒细胞、单核细胞和淋巴细胞在内皮下聚集,进一步使内皮细胞活化,刺激内皮收缩、通透性增加、黏附分子表达增加、白细胞滚动和穿透力加强。凝血酶刺激内皮细胞已经成为研究血管内皮损伤机制的一个重要模型。
内皮细胞的每个运输小泡的胞吐都经历装载,出芽,移位,对接,诱发,膜融合,及再循环这几个阶段。几套蛋白调节小泡的转运,包括Rab家族成员,N-乙基顺丁烯二酰亚胺敏感性的融合蛋白(NSF),可溶性的NSF附着蛋白,SNAP受体蛋白(SNAREs), Sec/Munc家族。NSF是调节胞吐的重要分子。它是由六个相同的亚基组成的六聚体,每个亚基的分子量约85kD,各含三个结构域,分别命名为N、D1、D2结构域。NSF只有在三个结构域协同作用时才能最大限度地发挥自己的功能,其中N结构域主要与SNARE复合物结合,D1的ATP酶活性是NSF的主要功能,D2主要参与多聚体的形成,球形的N结构域环绕于环的外侧基底部。一氧化氮主要通过巯基亚硝基化NSF的D1结构域的C264位的半胱氨酸残基,阻止NSF与SNAREs的解离,从而阻止胞吐。因此增加一氧化氮的产生就可抑制内皮细胞的胞吐,从而抑制细胞粘附,减缓血管炎症与血栓形成在内皮细胞。在内皮细胞,一氧化氮主要由内皮型一氧化氮合酶催化精氨酸合成一氧化氮。内皮型一氧化氮合酶的活化主要由磷脂酰肌醇3激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路调控。磷脂酰肌醇3激酶被激活后,在细胞膜上生成第二信使磷脂酰肌醇4,5二磷酸(PIPa), PIPa与细胞内含有PH结构域的信号蛋白Akt和磷酸肌醇依赖性蛋白激酶结合,促使磷酸肌醇依赖性蛋白激酶磷酸化Akt蛋白的Ser308导致Akt的活化,Akt激活内皮型一氧化氮合酶,导致NO的产生。此外,内皮细胞粘附性增加,血管炎症发生也与炎症因子诱导的粘附分子大量表达密切相关。核因子-kappa B (NF-κB)信号通路调控许多粘附分子的表达。粘附分子VCAM-1和E-selectin基因的启动子区域存在NF-κB的结合位点。NF-κB是一种普遍存在于各种细胞中的核转录因子。NF-κB由两类亚基形成同源或异源二聚体。一类亚基包括p65(也称RelA)、RelB和C-Rel;另一类亚基包括p50和p52。最常见的NF-κB亚基组成形式为p65/p50或p65/p65。在正常状态下,NF-κB在胞质中与它的抑制因子-κB(IκB)结合在一起,失去转录活性。在炎症因子如凝血酶激活存在的情况下,IκB-a会在Ser32和Ser36被磷酸化,随后被泛素-蛋白酶体途径降解。NF-κB和IκB-a解聚后,其核定位序列被暴露,从而被转运到细胞核内,在核内与目的基因的启动子区域结合,促进NF-κB依赖的基因转录。
京尼平是桅子果实中的主要成分之一栀子苷在肠和肝中的水解产物,是栀子苷的苷元。栀子是我国的一种传统中药,具有泻火除烦,清热利尿,凉血散瘀的功效,被用于治疗热病心烦、火毒疮疡、外治扭挫伤痛、消渴目赤等症,在中医处方中应用甚广。栀子含有多种化学成分,药理实验表明,以栀子苷为代表的烯醚萜类成分为栀子的主要活性成分,它具有利胆、镇痛、保肝、抗癌、抗炎等作用。近年来研究发现栀子苷在体内的主要活性产物是京尼平。京尼平具有明显的抗炎与抗血栓作用。但作用机制尚不清楚,然而内皮细胞胞吐与抗炎和抗血栓有着密切的联系,提示我们京尼平可能具有抑制内皮细胞胞吐作用。
基于上述背景我们确定研究京尼平对人脐带静脉内皮细胞(human umbilical vein endothelail cell, HUVEC)胞吐的作用及其机制。我们首先观察京尼平对内皮细胞的增殖作用,以确定下一步实验京尼平的合适剂量,用于研究京尼平对凝血酶诱导的人脐静脉内皮细胞胞吐的抑制作用。然后在细胞水平研究京尼平对凝血酶诱导的人脐静脉内皮细细胞与单核细胞的粘附抑制作用,以及在动物整体水平研究京尼平对小鼠尾静脉出血时间的影响。最后从一氧化氮的产生,磷脂酰肌醇3激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路和核因子-kappa B信号通路的作用来研究京尼平抑制胞吐的分子机制。
研究主要方法和结果如下：
一、0.5-16μg/ml京尼平不影响人脐静脉内皮细胞的增殖
从新鲜胎盘分离出脐带,然后提取人脐静脉内皮细胞原代培养,采用Cell counting kit-8法评价0.5、1、4、8、16μg/ml的京尼平处理内皮细胞12、24和48 h后的细胞增殖的影响。SPSS 13.0统计软件分析表明0.5、1、4、8、16μg/ml的京尼平处理内皮细胞12、24和48 h不影响人脐静脉内皮细胞的增殖。
二、京尼平抑制凝血酶诱导的人脐静脉内皮细胞胞吐
培养在6孔板中的人脐静脉内皮细胞经不同浓度的京尼平预处理各自不同时间后,再用1U/ml的凝血酶处理30分钟,释放到人脐静脉内皮细胞培养液中人VWF采用酶联免疫吸附方法(ELISA)测定；培养在24孔板中的人脐静脉内皮细胞经不同浓度的京尼平预处理各自不同时间后,再用1U/ml的凝血酶处理30分钟。释放到人脐静脉内皮细胞表面的P-selectin、E-selectin和VCAM-1采用表面ELISA检测。结果表明1-16μg/ml剂量范围内京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞对VWF、P-selectin、E-selectin和VCAM-1的胞吐,抑制作用呈剂量依赖性。抑制作用在京尼平预处理1小时后出现,并呈现明显的时间依赖性。
三、京尼平抑制凝血酶诱导的人脐静脉内皮细胞粘附分子mRNA和蛋白表达
前面结果表明京尼平抑制凝血酶诱导的人脐静脉内皮细胞对内皮细胞粘附分子的胞吐,京尼平是否影响凝血酶诱导的人脐静脉内皮细胞粘附分子mRNA和蛋白表达,接下来我们采用实时定量荧光PCR技术和Western blot技术来验证。不同浓度的京尼平预处理人脐静脉内皮细胞1小时,然后用凝血酶刺激30分钟,接着检测内皮细胞VCAM-1、E-selectin mRNA和蛋白表达。结果表明,4-16μg/ml剂量范围内京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞VCAM-1、E-selectin mRNA和蛋白的表达,抑制作用呈剂量依赖性。
四、京尼平抑制凝血酶诱导的人脐静脉内皮细胞与单核细胞粘附
前面实验表明京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞对粘附分子的胞吐和粘附分子mRNA表达。在炎症性因子的刺激下,内皮细胞释放大量粘附分子,导致白细胞包括中性粒细胞、单核细胞粘附到内皮表面,进一步加剧炎症与凝血。京尼平是否能从功能上抑制凝血酶诱导的内皮细胞与单核细胞的粘附?因此我们从细胞水平来研究京尼平对人脐静脉内皮细胞与单核细胞粘附的影响。我们采用BCECF AM荧光探针装载人单核白血病细胞系THP-1细胞,然后将装载了荧光探针THP-1细胞加入到依次用京尼平、凝血酶处理过的HUVECs细胞培养孔共孵育。由于凝血酶的刺激,HUVECs释放大量粘附分子,导致贴壁生长的HUVECs粘附大量荧光标记的人单核白血病细胞系THP-1细胞,未粘附的细胞用PBS洗去,荧光显微镜下可观察到粘附在HUVECs上的单核白血病细胞系THP-1细胞,裂解细胞后用荧光分光光度计检测荧光强度。比较凝血酶处理组与对照组；比较京尼平处理组与凝血酶处理组荧光强度,考察京尼平对凝血酶诱导的人脐静脉内皮细胞与单核细胞粘附的抑制作用。荧光显微镜下观察发现,对照组：少量THP-1细胞粘附于人脐静脉内皮细胞；凝血酶组：1U/ml的凝血酶处理人脐静脉内皮细胞30分钟,粘附于人脐静脉内皮细胞的THP-1细胞明显增加；处理组：4、8μg/ml京尼平预处理人脐静脉内皮细胞1小时明显抑制THP-1细胞粘附于凝血酶诱导的人脐静脉内皮细胞。荧光分光光度计检测结果：与对照组比较凝血酶处理显著性增加细胞裂解液的荧光强度,表明粘附于凝血酶处理的HUVECs的THP-1细胞数量明显增多；4、8μg/ml京尼平预处理人脐静脉内皮细胞1小时明显抑制凝血酶刺激的荧光强度的增加,表明京尼平预处理明显抑制凝血酶诱导的人脐静脉内皮细胞与THP-1细胞的粘附。
五、京尼平延长小鼠尾静脉出血时间
前面在细胞水平已经证实京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞的胞吐和粘附分子mRNA的表达,并且在细胞水平也证实京尼平具有抑制内皮细胞与单核细胞的粘附作用。在动物体内整体水平下京尼平抑制粘附作用如何呢?因此我们研究了京尼平对小鼠尾静脉出血时间的作用,以此进一步考察京尼平抑制胞吐的作用。我们采用断尾法研究京尼平对小鼠尾静脉出血时间的影响。对照组小鼠尾静脉出血时间约为6分钟。5,12.5,25,50mg/kg剂量的京尼平处理小鼠1小时后,小鼠尾静脉出血时间为：8,10,16,18分钟。当剂量增加到100mg/kg时,小鼠尾静脉出血时间超过20分钟。实验表明12.5,25,50,100mg/kg剂量京尼平明显延长小鼠尾静脉出血时间。不同时间点对照组小鼠尾静脉出血时间约为6分钟。25 mg/kg剂量的京尼平处理小鼠0.5小时后,尾静脉出血时间约为9分钟,与对照组比较明显延长。最大出血时间出现在给药1小时后。给药1.5小时后出血时间逐渐恢复到对照组水平。
六、京尼平诱导人脐静脉内皮细胞一氧化氮的产生
大量研究表明一氧化氮是抑制细胞胞吐的重要介质。在炎症因子的作用下内皮细胞减少一氧化氮的产生,减少了对胞吐的抑制作用,结果粘附分子、促炎性因子大量释放到胞外,使炎症加剧、血栓成。因此恢复内皮细胞一氧化氮的产生能力,可有效防止在炎症过程中过度发生的内皮细胞的胞吐。先前实验在分子、细胞、动物水平证实了京尼平明显抑制凝血酶诱导的内皮胞吐。这种抑制作用是否与京尼平恢复内皮细胞一氧化氮的产生有关还不清楚。因此我们研究了京尼平对人脐静脉内皮细胞一氧化氮产生的作用。采用比色法检测细胞培养液上清中的一氧化氮的量。研究发现：对照组一氧化氮的产生约为80mM。1,4,8,16μg/ml的京尼平处理人脐静脉内皮细胞1小时明显增加一氧化氮的产生。4μg/ml京尼平处理人脐静脉内皮细胞1,1.5,2小时明显增加一氧化氮的产生。结果表明京尼平剂量依赖性和时间依赖性的增加了人脐静脉内皮细胞一氧化氮的产生。为了证实京尼平诱导的一氧化氮的产生介导了京尼平对人脐静脉内皮细胞胞吐的抑制作用,我们研究了L-NAME(一氧化氮产生抑制剂)对京尼平诱导的人脐静脉内皮细胞VWF胞吐的作用。实验结果表明0.5,1.0 mM的L-NAME明显逆转了京尼平对凝血诱导的人脐静脉内皮细胞VWF胞吐的抑制作用。研究证实了京尼平诱导的一氧化氮的产生介导了京尼平对人脐静脉内皮细胞胞吐的抑制作用。
七、京尼平活化人脐静脉内皮细胞PI3K-Akt-eNOS信号通路
前面的实验表明京尼平通过增加一氧化氮产生抑制内皮细胞的胞吐。在内皮细胞一氧化氮的产生来自于内皮型一氧化氮合酶(eNOS)的活化。eNOS也位于内皮细胞的细胞膜上,一旦被上游信号分子激活在还原型辅酶II (NADPH)参与下催化L2精氨酸生成内皮细胞衍生性一氧化氮和瓜氨酸。大量研究表明eNOS的活化受到磷脂酰肌醇3-激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路的调控。磷脂酰肌醇3-激酶(PI3K)家族参与多种信号通路,调节细胞的主要功能。它调控下游分子丝氨酸/苏氨酸蛋白激酶Akt的磷酸化,磷酸化的Akt通过磷酸化作用激活eNOS,催化精氨酸合成NO。我们研究发现,与对照组比较,0.5-8μg/ml京尼平处理人脐静脉内皮细胞1小时明显增加内皮细胞中磷酸化的内皮型一氧化氮合酶的量。0.5-8μg/ml京尼平处理人脐静脉内皮细胞1小时并不影响内皮型一氧化氮合酶的总量。与对照组比较,1μg/ml京尼平分别处理人脐静脉内皮细胞1、2小时,磷酸化的内皮型一氧化氮合酶的量明显增加,然而并不影响内皮型一氧化氮合酶的总量。研究表明京尼平剂量依赖性和时间依赖性活化内皮型一氧化氮合酶。另外我们发现,4 u g/ml京尼平处理人脐静脉内皮细胞1小时,与对照组比较,在内皮细胞中磷酸化的丝氨酸/苏氨酸蛋白激酶Akt的量明显增加。4μg/ml京尼平处理人脐静脉内皮细胞1小时并不影响丝氨酸/苏氨酸蛋白激酶Akt的总量。5μM的LY294002磷脂酰肌醇3-激酶抑制剂预处理内皮细胞15分钟,明显抑制京尼平诱导的丝氨酸/苏氨酸蛋白激酶Akt的磷酸化,但不影响丝氨酸/苏氨酸蛋白激酶Akt的总量。我们研究表明京尼平是通过激活PI3K,诱导Akt的磷酸化从而激活eNOS,增加一氧化氮的产生而发挥抑制胞吐的作用。
八、京尼平诱导脐静脉内皮细胞NSF的亚硝基化
NSF是调节胞吐的重要分子,它是是一种寡聚蛋白质,由六个相同的亚基组成的六聚体。每个亚基各含三个结构域,分别命名为N、D1、D2结构域。NSF只有在三个结构域协同作用时才能最大限度地发挥自己的功能,其中N结构域主要与SNARE复合物结合,D1的ATP酶活性是NSF的主要功能,D2主要参与多聚体的形成,球形的N结构域环绕于环的外侧基底部。一氧化氮主要通过巯基亚硝基化NSF的D1结构域的C264位的半胱氨酸残基,阻止NSF与SNAREs的解离,从而阻止胞吐。因此增加一氧化氮的产生就可抑制内皮细胞的胞吐,从而抑制细胞粘附,减缓血管炎症与血栓形成在内皮细胞。然后,我们采用亚硝基生物素转化技术、免疫沉淀技术和Western blot技术研究了京尼平对人脐静脉内皮细胞NSF蛋白巯基亚硝基化的作用。4μg/ml京尼平处理脐静脉内皮细胞1小时,与对照组比较,内皮细胞亚硝基化的NSF量明显增加。1.0mM的L-NAME(一氧化氮产生抑制剂)预处理HUVECs1小时明显逆转了京尼平诱导的NSF的亚硝基化。5μM的LY294002(磷脂酰肌醇3-激酶抑制剂)预处理内皮细胞15分钟,明显逆转了京尼平诱导的NSF的亚硝基化。研究结果表明,京尼平活化PI3K-Akt-eNOS信号通路,增加内皮细胞NO的产生,促进NSF蛋白巯基亚硝基化,抑制内皮细胞胞吐。
九、京尼平抑制凝血酶诱导的人脐静脉内皮细胞NF-kappa B信号通路的活化
前面实验已经证明京尼平抑制凝血酶诱导的粘附分子VCAM-1和E-selectin mRNA的表达,是否这种抑制作用与它抑制NF-κB信号通路有关?我们采用Western blot和细胞免疫荧光技术研究了京尼平对NF-kappa B信号通路的影响。与对照组比较,1U/ml的凝血酶处理人脐静脉内皮细胞30分钟明显增加了人脐静脉内皮细胞胞核NF-κB p65的量；4-16μg/ml京尼平预处理人脐静脉内皮细胞4小时,与凝血酶组比较,人脐静脉内皮细胞胞核NF-κB p65的量明显减少。实验表明在凝血酶的刺激下NF-κB p65由胞浆向细胞核的转运增加,然而京尼平明显抑制了NF-κB p65由胞浆向细胞核的转运。与对照组比较,1U/ml的凝血酶处理人脐静脉内皮细胞30分钟明显减少了人脐静脉内皮细胞胞浆I-κB的量；4-16μg/ml京尼平预处理人脐静脉内皮细胞4小时,与凝血酶组比较,人脐静脉内皮细胞胞浆I-κB的量明显增加。实验表明在凝血酶的刺激下人脐静脉内皮细胞胞浆I-κB降解增加,然而京尼平明显抑制了胞浆I-κB降解。接下来我们采用细胞免疫方法用荧光显微镜观察NF-κB p65由胞浆向胞核的转运,进一步证实京尼平抑制了NF-κB p65由胞浆向细胞核的转运。与对照组比较,在凝血酶处理组人脐静脉内皮细胞胞核红色荧光明显增强,表明凝血酶增加了NF-κB p65由胞浆向细胞核的转运。4μg/ml京尼平预处理人脐静脉内皮细胞4小时明显减少NF-κB p65由胞浆向细胞核的转运。我们研究表明京尼平能抑制凝血酶诱导的人脐静脉内皮细胞NF-κB信号通路的活化,同时减少粘附分子的表达。
根据上述实验结果,本研究的结论如下：
一、首次发现京尼平抑制凝血酶诱导的人脐静脉内皮细胞细胞粘附分子、选择素和血管假性血友病因子的胞吐。
二、京尼平减少凝血酶诱导的人脐静脉内皮细胞与单核细胞的粘附,并且明显延长小鼠尾静脉出血时间。
三、京尼平通过激活PI3K,诱导Akt的磷酸化从而激活eNOS,增加一氧化氮的产生,促进NSF蛋白巯基亚硝基化,从而发挥抑制胞吐的作用。
四、京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞VCAM-1和E-selectin mRNA和蛋白的表达以及细胞核因子-κB信号通路的活化。
综上所述,京尼平通过活化PI3K-Akt-eNOS信号通路,增加一氧化氮的产生,促进NSF蛋白巯基亚硝基化,从而发挥抑制胞吐的作用。同时它也能抑制核因子-κB信号通路从而抑制粘附分子的表达。京尼平通过影响上述两条通路,共同发挥抑制人脐静脉内皮细胞与单核细胞的粘附,以及延长小鼠尾静脉出血时间的作用。本研究为阐明京尼平抗炎和抗血栓的作用与分子机制提供了新的实验依据。
Endothelail cells lining in inner surface of vasculature provide a physical barrier between circulating blood and vessel wall. It is not only a barrier in biological structure and function, but also an active metabolism pool which synthesizes numerous bioactive product. Endothelial cell dyfunction and injury which play important roles in cardio-cerebrovascular disease are tightly associated with initial stage of various diseases. In the development of endothelial cell dyfunction and injury, exocytosis of endothelial cell grannules is one of the earliest responses to injury or stimmulation of vascular issue. When endothelial cells are stimulated, the membranes of Weibel-Palade bodies (WPB) rapidly fuse with the endothelial plasma membrane, releasing their contents into outside of the endothelial cells. The proteins released from WPB exocytosis promote neutrophils and platelet adhesion to vessel walls, and cause vascular inflammation and thrombogensis. Therefore, exocytosis becomes an important aspect for exploring the mechanism of vascualr dysfunction and injury.
WPB mainly stores in endothelial cells and contains pro-inflammatory as well as pro-thrombotic proteins such as P-selectin, E-selectin, vascular endothelial cell adhesion molecule-1(VCAM-1) and von Willebrand factor (VWF). Most of them are related with cell adhesion, inflammation and hemostasis. P-selectin is a cell adhesion molecule (CAM) on the surfaces of activated endothelial cells. It plays an essential role in the initial recruitment of leukocytes to the site of injury in the early stage of inflammation. In the advanced stage, P-selectin mediates adhesion event with other adhesion molecules. P-selectin is also responsible for the rolling phase of the leukocyte adhesion cascade. When endothelial cells are activated by molecules such as thrombin and histamine, P-selectin moves from an internal cell location to the endothelial cell surface. E-selectin, also known as CD62e, is a cell adhesion molecule expressed only on endothelial cells. E-selectin recognizes and binds to sialylated carbohydrates present on the surface proteins of certain leukocytes. These carbohydrates include members of the Lewis X and Lewis. During inflammation, E-selectin recruits leukocytes to the site of injury. The local release of cytokines IL-1 and TNF by damaged cells induces the over-expression of E-selectin on endothelial cells of nearby blood vessels. Leukocytes in the blood expressing the correct ligand will bind with low affinity to E-selectin, causing the leukocytes to roll along the internal surface of the blood vessel. As the inflammatory response progresses, chemokines released by injured tissue enter the blood vessels and activate the rolling leukocytes, which are now able to tightly bind to the endothelial surface and begin making their way into the tissue. The VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or a4β1) of theβ1 subfamily of integrins, and for integrin a4β7. The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. VWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (a-granules of platelets), and subendothelial connective tissue. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites. WPBs are released from endothelial cells in response to a large number of secretagogues such as thrombin, histamine, peptido-leukotrienes, complement components C5a and C5b-9, superoxide anion, vascular endothelial growth factor (VEGF), sphingosine 1-phosphate, ceramide, purine nucleotides, serotonin, epinephrine, and vasopressin. These agonists of exocytosis can be divided into two distinct groups, those that act by elevating intracellular Ca2+ levels and those that act by raising cAMP levels in the cell. Ca2+-raising agonist like thrombin and histamine are expected to induce a much more vigorous response. It is an important model to explore the mechanism of endothelial cell dysfunction and injury in vitro that thrombin induces endothelial cell exocytosis of WPB.
Exocytosis of WPB involves a cycle of budding, docking, priming ,triggering, fusion and recycling. Several sets of proteins mediate exocytosis, including SNAREs, N-ethyl-maleimide-sensitive factor (NSF), Rab, Munc, and other accessory proteins. Soluble NSF attachment protein receptors (SNAREs) are transmembrane proteins that regulate vesicle fusion. Vesicle SNAREs are localized to vesicle membranes, and target SNAREs are localized to target membranes. A set of three SNARE molecules assembles into a stable ternary complex, bringing the vesicle into apposition with a target membrane. SNARE assemble and disassembly funntion importantly in the cycle of vesicle and granule exocytosis. SNARE disassembly is mainly regulated by NSF. NSF regulates vesicle fusion and is inhibited by N-ethyl-maleimide. Each of the three domains of NSF has a specific function. The N-terminal domain of NSF interacts indirectly with SNARE proteins via soluble NSF attachment proteins (SNAP). The D1 domain of NSF hydrolyzes ATP and alters the conformation of the ternary SNARE complex, the D2 domain of NSF that regulates NSF oligomerization. Nitric oxide (NO) is an important molecule that inhibits exocytosis. NO can chemically modify NSF by S-nitrosylating sulfhydryl groups in cysteine residues and prevent disassembly of SNAREs and NSF. Accordingly, elevated production of nitric oxide can inhibit exocytosis. NO is one of the most important molecules synthesized by the endothelial cell. Production of NO results from endothelial NO synthase (eNOS) enzymatic conversion of L-arginine to L-citrulline in the presence of essential cofactors such as FAD, NAD(P)H and tetrahydrobiopterin (BH4). Activation of eNOS is regualated by phosphatidylinositol 3-kinase (PI3 kinase)- serine/threonine protein kinase(Akt) signaling pathway. Akt can phosphorylate eNOS resulting in activation of eNOS. In addition, endothelial dysfunction and injury are closely associated with overexpression of adhesion molecules in endothelial cells. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Under normal condition, NF-κB is sequestrated in cytoplasm bound to inhibitory factor of NF-κB(IκB). When endothelial cells are stimulated by inflammatory mediators such as thrombin, IκB is degraded through complicated upstream molecules. Then NF-κB is released from the NF-κB/IκB complex and translocated from cytoplasm to nucleus, followed by increasing gene expression of cell adhesion molecules such as VCAM-1 and E-selectin. Genipin is a metabolite of geniposide, the major active ingredient of Gardemia jasminoids Ellis fruits, which have long been used in traditional Chinese medicine. This compound has been reported to have anti-inflammatory, anti-diabetic, anti-thrombotic, anti-oxidative, anti-angiogenic and neurotrophic activities. In a variety of animal models, genipin was shown to have anti-inflammatory activities. It significantly inhibited acute inflammation in carrageenan-induced rat paw edema, carrageenan-induced rat air pouch edema and croton oil-induced mouse ear edema models. It also inhibited mouse vascular permeability induced by acetic acid. Genipin may inhibit inflammation in part by inhibiting the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide upon stimulation by lipopolysaccharide/interferon in a murine macrophage cell line, as well as by inhibition of NF-κB activation. However, the molecular mechanisms of anti-inflammatory action of genipin are still not fully understood. Since endothelial exocytosis plays important roles in vascular inflammation, we hypothesized that genipin would exert its anti-inflammation by inhibiting WPB exocytosis from endothelial cells. In this study, we investigated the effects of ginipin on VWF release and P-selecti、E-selectin and VCAM-1 translocation in primary culture of human umbilical vein endothelial cells (HUVECs). We also performed experiments to further examine the mechanisms responsible for the inhibitory effect of endothelial exocytosis induced by this compound.
Methods and results
1. Treatment with 0.5-16μg/mL genipin had no effect on HUVEC viability Primary human umbilical vein endothelial cell (HUVEC) was isolated from human umbilical cord and cultured The cell viability assessment was performed using Cell Counting Kit-8. HUVECs were treated with 0.5-16μg/mL genipin for 12,24 and 48 h. The results showed that genipin treatment had no effect on HUVEC viability.
2. Genipin inhibits thrombin-induced exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECs
HUVECs were pretreated with increasing concentrations of genipin for various times, and then stimulated with 1 U/mL thrombin for 30 min. VWF concentration released in the cell supernatants was measured using VWF ELISA Kit. The amount of P-selectin, E-selectin and VCAM-1 translocation on the cell surface was using cell surface ELISA. Results showed that genipin blocked the exocytosis of P-selectin, E-selectin, VCAM-1 and VWF in dose-dependent manner, with a maximal effect achieved at 8μg/mL. The significant inhibition of genipin was seen within 1 hour of pretreatment and increased in time-dependent way.
3.Genipin inhibits mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs induced by thrombin
HUVECs were pretreated with increasing concentrations of genipin for various lengths of time and then stimulated with thrombin for 30 min. The mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs was evaluated by real-time RT-PCR and western blot. The results showed that thrombin significantly increased mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs. However, pretreatment with 4-16μg/ml genipin inhibited mRNA and protein expression of VCAM-1 and E-selectin induced by thrombin in dose-dependent manner.
4. Genipin inhibits THP-1 cells adhesion to HUVECs induced by thrombin
HUVECs were pretreated with 8μg/mL genipin for 1 h and then stimulated with thrombin for 30 min. Fluorescence-labeled THP-1 cells were added to thrombin-stimulated HUVECs and co-cultured for 1 h. After removal of the non-adhesion cells, the remaining adhesion cells were photographed with fluorescence microscopy. The cells were lysed and measured using a spectrofluorometer. Results showed that, after stimulation with thrombine for30 min, adhesion of monocytes to HUVECs significantly increased (P<0.01 compared to normal treatment). When we pretreated HUVECs with 4 and 8μg/mL genipin for 1 h, this compound was found to markedly decrease monocyte adhesion to HUVECs induced by thrombin. (P<0.01 compared to thrombin treatment alone).
5. Genipin prolongs bleeding time in mice
Since platelet adherence to the vessel wall is mediated by the VWF, P-selectin, VCAM-1 and E-selectin,we predicted that genipin-induced inhibition of endothelial exocytosis would decrease platelet adherence and prolong the bleeding time. Thus, we measured the effect of genipin on the bleeding time in mice to obtain functional evidence that genipin inhibits HUVEC exocytosis in vivo. Anesthetized mice were injected intravenously with various concentrations of genipin or PBS. At various times after drug injection, the distal tip of tail was amputated, and bleeding time was measured. Treatment with PBS had no effect on the bleeding time, with a bleeding time of approximately 6 min. In contrast, genipin dramatically prolonged the bleeding time in dose- dependent manner. At the doses of 5,12.5,25 and 50mg/kg, the bleeding times were 8,10,16 and 18min, respectively. When the dose of the drug increased to 100mg/kg, the bleeding time was more than 20 min. The genipin-induced maximum bleeding time occurred at the 1 hour after the drug treatment, and then the prolonged bleeding time returned to normal level after 1.5 hours of genipin treatment.
6. NO mediates genipin-induced inhibition of HUVEC exocytosis
NO is an important molecule that inhibits exocytosis, we next examine the ability of genipin to stimulate NO production. We treated HUVECs with various genipin concentrations and with different duration of genipin pretreatment, and then measured the NO level in the cell supernatants. Our results showed that genipin activated HUVEC synthesis of NO in a dose- and time-dependent way.
In order to determine whether or not genipin-induced NO production is involved in the exocytosis inhibition by this compound, we examined the effect of L-NAME, an inhibitor of NOS, on genipin-induced exocytosis inhibition. HUVECs were pretreated with various concentrations of L-NAME for 1 h, and then genipin was added for 2 h. The cells were stimulated with thrombin (1U/mL) and VWF level in the supernatants was measured. Our results showed that L-NAME reversed the inhibitory effects of genipin on endothelial exocytosis, suggesting that genipin-induced NO production is involved in its inhibition of endothelial exocytosis.
7.Genipin activates PI3K-Akt- eNOS signaling pathway in HUVECs
Previous study showed that genipin inhibited thrombin-induced exocytosis in HUVECs through production of NO. Production of NO resulting from activation of eNOS is regualated by PI3K-Akt- eNOS signaling pathway. Phosphorylation of eNOS by Akt resulted in NO production. Therefore, we investigated the effect of genipin on eNOS activation in order to explore the mechanisms responsible for the inhibitory effect of genipin-induced endothelial exocytosis. HUVECs were treated with different concentrations of genipin or control for various times, and cell lysates were immunoblotted for phospho-eNOS (Ser-1177) or eNOS. Genipin increased phosphorylation level of eNOS in dose and time-dependent manner, but had no effect on the total amount of eNOS within 2 h after drug treatment. These results demonstrated that genipin stimulated eNOS phosphorylation and activated eNOS. We used a pharmacological approach to explore the role of PI3K in genipin activation of eNOS. The LY294002, an inhibitor of PI3K, diminished the ability of genipin to activate phosphorylation of Akt and eNOS. Taken together, these data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, and that this pathway mediates genipin inhibition of exocytosis.
8. Genipin promotes S-nitrosylation of NSF in HUVECs
NO S-nitrosylates sulfhydryl groups of NSF in cysteine residues and prevents disassembly of SNAREs and NSF, leading to decrease of exocytosis. In order to explore effect of genipin on S-nitrosylation of NSF, we used biotin-switch technique (BST), immunoprecipitation and Western blot to determine S-nitrosylation of NSF in HUVECs. Results showed that treatment with 4μg/mL genipin for 1h markedly increased the S-nitrosylation of NSF in HUVECs compared with control group. However, pretreatment with L-NAME(an inhibitor of NOS) or LY294002(an inhibitor of PI3K) inhibited the S-nitrosylation of NSF induced by genipin. All data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, increases NO production and promotes S-nitrosylation of NSF in HUVECs.
9.Genipin inhibited thrombin-induced NF-κB signaling pathway activation in HUVECs
In addition, endothelial dysfunction and injury is closely assocated with overexpression of adhesion molecules in endothelial cells. Previous study showed that genipin inhibited the gene expression of VCAM-1 and E-selectin. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Therefore, in this study we explored the effect of genipin on thrombin-induced NF-κB signaling pathway activation using western blotting and immunocytochemistry. Results showed that thrombin significantly increased NF-κB p65 expression in the nuclear extracts compared to normal treatment. In contrast, pretreatment with genipin 8-16μg/mL for 1 h markedly inhibited thrombin-induced increase of nuclear NF-κB p65 in dose-dependent manner. Furthermore, thrombin significantly decreased the IκB expression levels in cytoplasm. However, pretreatment with 8-16μg/mL genipin for
1 h markedly inhibited thrombin-induced decrease of IκB expression levels in dose-dependent manner.
To confirm the consistency to the result of western blotting, we observed the translocation of NF-κB p65 by using immuocytochemistry.Results showed that thrombin caused NF-κB p65 translocation into nucleus compared to normal treatment. However, pretreatment with 4μg/mL genipin inhibited the translocation of NF-κB p65 from cytoplasm to nucleus. These results were consistent with the result of western blotting.
Take together, the conclusions of present study are as follow
1. Genipin inhibited exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECs induce by thrombin.
2. Genipin decreased THP-1 cells adhesion to HUVECs induced by thrombin and prolonged the blood time of mice tail vein.
3. Genipin activated of PI3K-Akt-eNOS signaling pathway, increases NO production, and induces S-nitrosylation of NSF in HUVECs.
4. Genipin inhibited thrombin-induced HUVEC NF-κB signaling pathway activation, which in turn inhibited the gene and protein expression of adhesion molecules such as VCAM-1 and E-selectin.
In conclusion, excessive endothelial exocytosis may contribute to the pathophysiology of inflammation and thrombosis. Genipin, a novel inhibitor of endothelial exocytosis found in this study, may target acute inflammatory events and suppress vascular and endothelial cell inflammatory activation. This compound may represent a new treatment for inflammation and/or thrombosis in which excess exocytosis plays a pathophysiological role.
内皮细胞内贮存大量的颗粒,它们包含前炎症因子与前凝血因子,例如：P-选择素(P-selectin)、E-选择素(E-selectin)、血管内皮粘附分子-1(VCAM-1)和血管假性血友病因子(VWF)等,这些分子与细胞粘附、炎症及止血紧密相关。P-选择素介导中性粒细胞在活化内皮细胞上的滚动,尤其在炎症过程的早期(数分钟)甚为重要,在炎症晚期同其他选择素分子协同发挥作用。P-选择素还参与血小板和某些T细胞亚群的沿血管壁的滚动的过程。E-选择素主要介导白细胞(中性粒细胞、单核细胞和CD4+记忆性T细胞)在内皮细胞表面最初的滞留和滚动,以及随后迁移到炎症的组织。血管内皮粘附分子-1主要介导淋巴细胞、单核细胞嗜酸细胞和内皮细胞的粘附。它特异地与白细胞(除中性粒细胞外)所产生的整合素家族的粘附因子VLA-4结合,介导细胞粘附和信号传递,在细胞分化、炎症反应、动脉粥样硬化等多种疾病病理生理过程中发挥作用。血管假性血友病因子介导血小板粘附与聚集,启动血栓形成,特异性地反映血管内皮细胞功能的异常。许多促分泌剂都能刺激内皮细胞胞吐,例如：凝血酶,组胺,白三烯,补体等。根据胞吐是由钙介导还是CAMP介导,促分泌剂分为两类。目前研究最广的是凝血酶,它是一种钙调促分泌剂,它通过活化钙调蛋白升高细胞内钙离子浓度,导致快速的胞吐。凝血酶与位于内皮细胞表面的受体结合刺激血管内皮细胞释放P-selectin、E-selectin和VCAM-1,这一系列反应不仅推动凝血过程和血小板黏附,而且促进中性粒细胞、单核细胞和淋巴细胞在内皮下聚集,进一步使内皮细胞活化,刺激内皮收缩、通透性增加、黏附分子表达增加、白细胞滚动和穿透力加强。凝血酶刺激内皮细胞已经成为研究血管内皮损伤机制的一个重要模型。
内皮细胞的每个运输小泡的胞吐都经历装载,出芽,移位,对接,诱发,膜融合,及再循环这几个阶段。几套蛋白调节小泡的转运,包括Rab家族成员,N-乙基顺丁烯二酰亚胺敏感性的融合蛋白(NSF),可溶性的NSF附着蛋白,SNAP受体蛋白(SNAREs), Sec/Munc家族。NSF是调节胞吐的重要分子。它是由六个相同的亚基组成的六聚体,每个亚基的分子量约85kD,各含三个结构域,分别命名为N、D1、D2结构域。NSF只有在三个结构域协同作用时才能最大限度地发挥自己的功能,其中N结构域主要与SNARE复合物结合,D1的ATP酶活性是NSF的主要功能,D2主要参与多聚体的形成,球形的N结构域环绕于环的外侧基底部。一氧化氮主要通过巯基亚硝基化NSF的D1结构域的C264位的半胱氨酸残基,阻止NSF与SNAREs的解离,从而阻止胞吐。因此增加一氧化氮的产生就可抑制内皮细胞的胞吐,从而抑制细胞粘附,减缓血管炎症与血栓形成在内皮细胞。在内皮细胞,一氧化氮主要由内皮型一氧化氮合酶催化精氨酸合成一氧化氮。内皮型一氧化氮合酶的活化主要由磷脂酰肌醇3激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路调控。磷脂酰肌醇3激酶被激活后,在细胞膜上生成第二信使磷脂酰肌醇4,5二磷酸(PIPa), PIPa与细胞内含有PH结构域的信号蛋白Akt和磷酸肌醇依赖性蛋白激酶结合,促使磷酸肌醇依赖性蛋白激酶磷酸化Akt蛋白的Ser308导致Akt的活化,Akt激活内皮型一氧化氮合酶,导致NO的产生。此外,内皮细胞粘附性增加,血管炎症发生也与炎症因子诱导的粘附分子大量表达密切相关。核因子-kappa B (NF-κB)信号通路调控许多粘附分子的表达。粘附分子VCAM-1和E-selectin基因的启动子区域存在NF-κB的结合位点。NF-κB是一种普遍存在于各种细胞中的核转录因子。NF-κB由两类亚基形成同源或异源二聚体。一类亚基包括p65(也称RelA)、RelB和C-Rel;另一类亚基包括p50和p52。最常见的NF-κB亚基组成形式为p65/p50或p65/p65。在正常状态下,NF-κB在胞质中与它的抑制因子-κB(IκB)结合在一起,失去转录活性。在炎症因子如凝血酶激活存在的情况下,IκB-a会在Ser32和Ser36被磷酸化,随后被泛素-蛋白酶体途径降解。NF-κB和IκB-a解聚后,其核定位序列被暴露,从而被转运到细胞核内,在核内与目的基因的启动子区域结合,促进NF-κB依赖的基因转录。
京尼平是桅子果实中的主要成分之一栀子苷在肠和肝中的水解产物,是栀子苷的苷元。栀子是我国的一种传统中药,具有泻火除烦,清热利尿,凉血散瘀的功效,被用于治疗热病心烦、火毒疮疡、外治扭挫伤痛、消渴目赤等症,在中医处方中应用甚广。栀子含有多种化学成分,药理实验表明,以栀子苷为代表的烯醚萜类成分为栀子的主要活性成分,它具有利胆、镇痛、保肝、抗癌、抗炎等作用。近年来研究发现栀子苷在体内的主要活性产物是京尼平。京尼平具有明显的抗炎与抗血栓作用。但作用机制尚不清楚,然而内皮细胞胞吐与抗炎和抗血栓有着密切的联系,提示我们京尼平可能具有抑制内皮细胞胞吐作用。
基于上述背景我们确定研究京尼平对人脐带静脉内皮细胞(human umbilical vein endothelail cell, HUVEC)胞吐的作用及其机制。我们首先观察京尼平对内皮细胞的增殖作用,以确定下一步实验京尼平的合适剂量,用于研究京尼平对凝血酶诱导的人脐静脉内皮细胞胞吐的抑制作用。然后在细胞水平研究京尼平对凝血酶诱导的人脐静脉内皮细细胞与单核细胞的粘附抑制作用,以及在动物整体水平研究京尼平对小鼠尾静脉出血时间的影响。最后从一氧化氮的产生,磷脂酰肌醇3激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路和核因子-kappa B信号通路的作用来研究京尼平抑制胞吐的分子机制。
研究主要方法和结果如下：
一、0.5-16μg/ml京尼平不影响人脐静脉内皮细胞的增殖
从新鲜胎盘分离出脐带,然后提取人脐静脉内皮细胞原代培养,采用Cell counting kit-8法评价0.5、1、4、8、16μg/ml的京尼平处理内皮细胞12、24和48 h后的细胞增殖的影响。SPSS 13.0统计软件分析表明0.5、1、4、8、16μg/ml的京尼平处理内皮细胞12、24和48 h不影响人脐静脉内皮细胞的增殖。
二、京尼平抑制凝血酶诱导的人脐静脉内皮细胞胞吐
培养在6孔板中的人脐静脉内皮细胞经不同浓度的京尼平预处理各自不同时间后,再用1U/ml的凝血酶处理30分钟,释放到人脐静脉内皮细胞培养液中人VWF采用酶联免疫吸附方法(ELISA)测定；培养在24孔板中的人脐静脉内皮细胞经不同浓度的京尼平预处理各自不同时间后,再用1U/ml的凝血酶处理30分钟。释放到人脐静脉内皮细胞表面的P-selectin、E-selectin和VCAM-1采用表面ELISA检测。结果表明1-16μg/ml剂量范围内京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞对VWF、P-selectin、E-selectin和VCAM-1的胞吐,抑制作用呈剂量依赖性。抑制作用在京尼平预处理1小时后出现,并呈现明显的时间依赖性。
三、京尼平抑制凝血酶诱导的人脐静脉内皮细胞粘附分子mRNA和蛋白表达
前面结果表明京尼平抑制凝血酶诱导的人脐静脉内皮细胞对内皮细胞粘附分子的胞吐,京尼平是否影响凝血酶诱导的人脐静脉内皮细胞粘附分子mRNA和蛋白表达,接下来我们采用实时定量荧光PCR技术和Western blot技术来验证。不同浓度的京尼平预处理人脐静脉内皮细胞1小时,然后用凝血酶刺激30分钟,接着检测内皮细胞VCAM-1、E-selectin mRNA和蛋白表达。结果表明,4-16μg/ml剂量范围内京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞VCAM-1、E-selectin mRNA和蛋白的表达,抑制作用呈剂量依赖性。
四、京尼平抑制凝血酶诱导的人脐静脉内皮细胞与单核细胞粘附
前面实验表明京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞对粘附分子的胞吐和粘附分子mRNA表达。在炎症性因子的刺激下,内皮细胞释放大量粘附分子,导致白细胞包括中性粒细胞、单核细胞粘附到内皮表面,进一步加剧炎症与凝血。京尼平是否能从功能上抑制凝血酶诱导的内皮细胞与单核细胞的粘附?因此我们从细胞水平来研究京尼平对人脐静脉内皮细胞与单核细胞粘附的影响。我们采用BCECF AM荧光探针装载人单核白血病细胞系THP-1细胞,然后将装载了荧光探针THP-1细胞加入到依次用京尼平、凝血酶处理过的HUVECs细胞培养孔共孵育。由于凝血酶的刺激,HUVECs释放大量粘附分子,导致贴壁生长的HUVECs粘附大量荧光标记的人单核白血病细胞系THP-1细胞,未粘附的细胞用PBS洗去,荧光显微镜下可观察到粘附在HUVECs上的单核白血病细胞系THP-1细胞,裂解细胞后用荧光分光光度计检测荧光强度。比较凝血酶处理组与对照组；比较京尼平处理组与凝血酶处理组荧光强度,考察京尼平对凝血酶诱导的人脐静脉内皮细胞与单核细胞粘附的抑制作用。荧光显微镜下观察发现,对照组：少量THP-1细胞粘附于人脐静脉内皮细胞；凝血酶组：1U/ml的凝血酶处理人脐静脉内皮细胞30分钟,粘附于人脐静脉内皮细胞的THP-1细胞明显增加；处理组：4、8μg/ml京尼平预处理人脐静脉内皮细胞1小时明显抑制THP-1细胞粘附于凝血酶诱导的人脐静脉内皮细胞。荧光分光光度计检测结果：与对照组比较凝血酶处理显著性增加细胞裂解液的荧光强度,表明粘附于凝血酶处理的HUVECs的THP-1细胞数量明显增多；4、8μg/ml京尼平预处理人脐静脉内皮细胞1小时明显抑制凝血酶刺激的荧光强度的增加,表明京尼平预处理明显抑制凝血酶诱导的人脐静脉内皮细胞与THP-1细胞的粘附。
五、京尼平延长小鼠尾静脉出血时间
前面在细胞水平已经证实京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞的胞吐和粘附分子mRNA的表达,并且在细胞水平也证实京尼平具有抑制内皮细胞与单核细胞的粘附作用。在动物体内整体水平下京尼平抑制粘附作用如何呢?因此我们研究了京尼平对小鼠尾静脉出血时间的作用,以此进一步考察京尼平抑制胞吐的作用。我们采用断尾法研究京尼平对小鼠尾静脉出血时间的影响。对照组小鼠尾静脉出血时间约为6分钟。5,12.5,25,50mg/kg剂量的京尼平处理小鼠1小时后,小鼠尾静脉出血时间为：8,10,16,18分钟。当剂量增加到100mg/kg时,小鼠尾静脉出血时间超过20分钟。实验表明12.5,25,50,100mg/kg剂量京尼平明显延长小鼠尾静脉出血时间。不同时间点对照组小鼠尾静脉出血时间约为6分钟。25 mg/kg剂量的京尼平处理小鼠0.5小时后,尾静脉出血时间约为9分钟,与对照组比较明显延长。最大出血时间出现在给药1小时后。给药1.5小时后出血时间逐渐恢复到对照组水平。
六、京尼平诱导人脐静脉内皮细胞一氧化氮的产生
大量研究表明一氧化氮是抑制细胞胞吐的重要介质。在炎症因子的作用下内皮细胞减少一氧化氮的产生,减少了对胞吐的抑制作用,结果粘附分子、促炎性因子大量释放到胞外,使炎症加剧、血栓成。因此恢复内皮细胞一氧化氮的产生能力,可有效防止在炎症过程中过度发生的内皮细胞的胞吐。先前实验在分子、细胞、动物水平证实了京尼平明显抑制凝血酶诱导的内皮胞吐。这种抑制作用是否与京尼平恢复内皮细胞一氧化氮的产生有关还不清楚。因此我们研究了京尼平对人脐静脉内皮细胞一氧化氮产生的作用。采用比色法检测细胞培养液上清中的一氧化氮的量。研究发现：对照组一氧化氮的产生约为80mM。1,4,8,16μg/ml的京尼平处理人脐静脉内皮细胞1小时明显增加一氧化氮的产生。4μg/ml京尼平处理人脐静脉内皮细胞1,1.5,2小时明显增加一氧化氮的产生。结果表明京尼平剂量依赖性和时间依赖性的增加了人脐静脉内皮细胞一氧化氮的产生。为了证实京尼平诱导的一氧化氮的产生介导了京尼平对人脐静脉内皮细胞胞吐的抑制作用,我们研究了L-NAME(一氧化氮产生抑制剂)对京尼平诱导的人脐静脉内皮细胞VWF胞吐的作用。实验结果表明0.5,1.0 mM的L-NAME明显逆转了京尼平对凝血诱导的人脐静脉内皮细胞VWF胞吐的抑制作用。研究证实了京尼平诱导的一氧化氮的产生介导了京尼平对人脐静脉内皮细胞胞吐的抑制作用。
七、京尼平活化人脐静脉内皮细胞PI3K-Akt-eNOS信号通路
前面的实验表明京尼平通过增加一氧化氮产生抑制内皮细胞的胞吐。在内皮细胞一氧化氮的产生来自于内皮型一氧化氮合酶(eNOS)的活化。eNOS也位于内皮细胞的细胞膜上,一旦被上游信号分子激活在还原型辅酶II (NADPH)参与下催化L2精氨酸生成内皮细胞衍生性一氧化氮和瓜氨酸。大量研究表明eNOS的活化受到磷脂酰肌醇3-激酶-丝氨酸/苏氨酸蛋白激酶Akt (PI3K-Akt)信号通路的调控。磷脂酰肌醇3-激酶(PI3K)家族参与多种信号通路,调节细胞的主要功能。它调控下游分子丝氨酸/苏氨酸蛋白激酶Akt的磷酸化,磷酸化的Akt通过磷酸化作用激活eNOS,催化精氨酸合成NO。我们研究发现,与对照组比较,0.5-8μg/ml京尼平处理人脐静脉内皮细胞1小时明显增加内皮细胞中磷酸化的内皮型一氧化氮合酶的量。0.5-8μg/ml京尼平处理人脐静脉内皮细胞1小时并不影响内皮型一氧化氮合酶的总量。与对照组比较,1μg/ml京尼平分别处理人脐静脉内皮细胞1、2小时,磷酸化的内皮型一氧化氮合酶的量明显增加,然而并不影响内皮型一氧化氮合酶的总量。研究表明京尼平剂量依赖性和时间依赖性活化内皮型一氧化氮合酶。另外我们发现,4 u g/ml京尼平处理人脐静脉内皮细胞1小时,与对照组比较,在内皮细胞中磷酸化的丝氨酸/苏氨酸蛋白激酶Akt的量明显增加。4μg/ml京尼平处理人脐静脉内皮细胞1小时并不影响丝氨酸/苏氨酸蛋白激酶Akt的总量。5μM的LY294002磷脂酰肌醇3-激酶抑制剂预处理内皮细胞15分钟,明显抑制京尼平诱导的丝氨酸/苏氨酸蛋白激酶Akt的磷酸化,但不影响丝氨酸/苏氨酸蛋白激酶Akt的总量。我们研究表明京尼平是通过激活PI3K,诱导Akt的磷酸化从而激活eNOS,增加一氧化氮的产生而发挥抑制胞吐的作用。
八、京尼平诱导脐静脉内皮细胞NSF的亚硝基化
NSF是调节胞吐的重要分子,它是是一种寡聚蛋白质,由六个相同的亚基组成的六聚体。每个亚基各含三个结构域,分别命名为N、D1、D2结构域。NSF只有在三个结构域协同作用时才能最大限度地发挥自己的功能,其中N结构域主要与SNARE复合物结合,D1的ATP酶活性是NSF的主要功能,D2主要参与多聚体的形成,球形的N结构域环绕于环的外侧基底部。一氧化氮主要通过巯基亚硝基化NSF的D1结构域的C264位的半胱氨酸残基,阻止NSF与SNAREs的解离,从而阻止胞吐。因此增加一氧化氮的产生就可抑制内皮细胞的胞吐,从而抑制细胞粘附,减缓血管炎症与血栓形成在内皮细胞。然后,我们采用亚硝基生物素转化技术、免疫沉淀技术和Western blot技术研究了京尼平对人脐静脉内皮细胞NSF蛋白巯基亚硝基化的作用。4μg/ml京尼平处理脐静脉内皮细胞1小时,与对照组比较,内皮细胞亚硝基化的NSF量明显增加。1.0mM的L-NAME(一氧化氮产生抑制剂)预处理HUVECs1小时明显逆转了京尼平诱导的NSF的亚硝基化。5μM的LY294002(磷脂酰肌醇3-激酶抑制剂)预处理内皮细胞15分钟,明显逆转了京尼平诱导的NSF的亚硝基化。研究结果表明,京尼平活化PI3K-Akt-eNOS信号通路,增加内皮细胞NO的产生,促进NSF蛋白巯基亚硝基化,抑制内皮细胞胞吐。
九、京尼平抑制凝血酶诱导的人脐静脉内皮细胞NF-kappa B信号通路的活化
前面实验已经证明京尼平抑制凝血酶诱导的粘附分子VCAM-1和E-selectin mRNA的表达,是否这种抑制作用与它抑制NF-κB信号通路有关?我们采用Western blot和细胞免疫荧光技术研究了京尼平对NF-kappa B信号通路的影响。与对照组比较,1U/ml的凝血酶处理人脐静脉内皮细胞30分钟明显增加了人脐静脉内皮细胞胞核NF-κB p65的量；4-16μg/ml京尼平预处理人脐静脉内皮细胞4小时,与凝血酶组比较,人脐静脉内皮细胞胞核NF-κB p65的量明显减少。实验表明在凝血酶的刺激下NF-κB p65由胞浆向细胞核的转运增加,然而京尼平明显抑制了NF-κB p65由胞浆向细胞核的转运。与对照组比较,1U/ml的凝血酶处理人脐静脉内皮细胞30分钟明显减少了人脐静脉内皮细胞胞浆I-κB的量；4-16μg/ml京尼平预处理人脐静脉内皮细胞4小时,与凝血酶组比较,人脐静脉内皮细胞胞浆I-κB的量明显增加。实验表明在凝血酶的刺激下人脐静脉内皮细胞胞浆I-κB降解增加,然而京尼平明显抑制了胞浆I-κB降解。接下来我们采用细胞免疫方法用荧光显微镜观察NF-κB p65由胞浆向胞核的转运,进一步证实京尼平抑制了NF-κB p65由胞浆向细胞核的转运。与对照组比较,在凝血酶处理组人脐静脉内皮细胞胞核红色荧光明显增强,表明凝血酶增加了NF-κB p65由胞浆向细胞核的转运。4μg/ml京尼平预处理人脐静脉内皮细胞4小时明显减少NF-κB p65由胞浆向细胞核的转运。我们研究表明京尼平能抑制凝血酶诱导的人脐静脉内皮细胞NF-κB信号通路的活化,同时减少粘附分子的表达。
根据上述实验结果,本研究的结论如下：
一、首次发现京尼平抑制凝血酶诱导的人脐静脉内皮细胞细胞粘附分子、选择素和血管假性血友病因子的胞吐。
二、京尼平减少凝血酶诱导的人脐静脉内皮细胞与单核细胞的粘附,并且明显延长小鼠尾静脉出血时间。
三、京尼平通过激活PI3K,诱导Akt的磷酸化从而激活eNOS,增加一氧化氮的产生,促进NSF蛋白巯基亚硝基化,从而发挥抑制胞吐的作用。
四、京尼平明显抑制凝血酶诱导的人脐静脉内皮细胞VCAM-1和E-selectin mRNA和蛋白的表达以及细胞核因子-κB信号通路的活化。
综上所述,京尼平通过活化PI3K-Akt-eNOS信号通路,增加一氧化氮的产生,促进NSF蛋白巯基亚硝基化,从而发挥抑制胞吐的作用。同时它也能抑制核因子-κB信号通路从而抑制粘附分子的表达。京尼平通过影响上述两条通路,共同发挥抑制人脐静脉内皮细胞与单核细胞的粘附,以及延长小鼠尾静脉出血时间的作用。本研究为阐明京尼平抗炎和抗血栓的作用与分子机制提供了新的实验依据。
Endothelail cells lining in inner surface of vasculature provide a physical barrier between circulating blood and vessel wall. It is not only a barrier in biological structure and function, but also an active metabolism pool which synthesizes numerous bioactive product. Endothelial cell dyfunction and injury which play important roles in cardio-cerebrovascular disease are tightly associated with initial stage of various diseases. In the development of endothelial cell dyfunction and injury, exocytosis of endothelial cell grannules is one of the earliest responses to injury or stimmulation of vascular issue. When endothelial cells are stimulated, the membranes of Weibel-Palade bodies (WPB) rapidly fuse with the endothelial plasma membrane, releasing their contents into outside of the endothelial cells. The proteins released from WPB exocytosis promote neutrophils and platelet adhesion to vessel walls, and cause vascular inflammation and thrombogensis. Therefore, exocytosis becomes an important aspect for exploring the mechanism of vascualr dysfunction and injury.
WPB mainly stores in endothelial cells and contains pro-inflammatory as well as pro-thrombotic proteins such as P-selectin, E-selectin, vascular endothelial cell adhesion molecule-1(VCAM-1) and von Willebrand factor (VWF). Most of them are related with cell adhesion, inflammation and hemostasis. P-selectin is a cell adhesion molecule (CAM) on the surfaces of activated endothelial cells. It plays an essential role in the initial recruitment of leukocytes to the site of injury in the early stage of inflammation. In the advanced stage, P-selectin mediates adhesion event with other adhesion molecules. P-selectin is also responsible for the rolling phase of the leukocyte adhesion cascade. When endothelial cells are activated by molecules such as thrombin and histamine, P-selectin moves from an internal cell location to the endothelial cell surface. E-selectin, also known as CD62e, is a cell adhesion molecule expressed only on endothelial cells. E-selectin recognizes and binds to sialylated carbohydrates present on the surface proteins of certain leukocytes. These carbohydrates include members of the Lewis X and Lewis. During inflammation, E-selectin recruits leukocytes to the site of injury. The local release of cytokines IL-1 and TNF by damaged cells induces the over-expression of E-selectin on endothelial cells of nearby blood vessels. Leukocytes in the blood expressing the correct ligand will bind with low affinity to E-selectin, causing the leukocytes to roll along the internal surface of the blood vessel. As the inflammatory response progresses, chemokines released by injured tissue enter the blood vessels and activate the rolling leukocytes, which are now able to tightly bind to the endothelial surface and begin making their way into the tissue. The VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or a4β1) of theβ1 subfamily of integrins, and for integrin a4β7. The VCAM-1 protein mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. VWF is a large multimeric glycoprotein present in blood plasma and produced constitutively in endothelium (in the Weibel-Palade bodies), megakaryocytes (a-granules of platelets), and subendothelial connective tissue. Its primary function is binding to other proteins, particularly Factor VIII and it is important in platelet adhesion to wound sites. WPBs are released from endothelial cells in response to a large number of secretagogues such as thrombin, histamine, peptido-leukotrienes, complement components C5a and C5b-9, superoxide anion, vascular endothelial growth factor (VEGF), sphingosine 1-phosphate, ceramide, purine nucleotides, serotonin, epinephrine, and vasopressin. These agonists of exocytosis can be divided into two distinct groups, those that act by elevating intracellular Ca2+ levels and those that act by raising cAMP levels in the cell. Ca2+-raising agonist like thrombin and histamine are expected to induce a much more vigorous response. It is an important model to explore the mechanism of endothelial cell dysfunction and injury in vitro that thrombin induces endothelial cell exocytosis of WPB.
Exocytosis of WPB involves a cycle of budding, docking, priming ,triggering, fusion and recycling. Several sets of proteins mediate exocytosis, including SNAREs, N-ethyl-maleimide-sensitive factor (NSF), Rab, Munc, and other accessory proteins. Soluble NSF attachment protein receptors (SNAREs) are transmembrane proteins that regulate vesicle fusion. Vesicle SNAREs are localized to vesicle membranes, and target SNAREs are localized to target membranes. A set of three SNARE molecules assembles into a stable ternary complex, bringing the vesicle into apposition with a target membrane. SNARE assemble and disassembly funntion importantly in the cycle of vesicle and granule exocytosis. SNARE disassembly is mainly regulated by NSF. NSF regulates vesicle fusion and is inhibited by N-ethyl-maleimide. Each of the three domains of NSF has a specific function. The N-terminal domain of NSF interacts indirectly with SNARE proteins via soluble NSF attachment proteins (SNAP). The D1 domain of NSF hydrolyzes ATP and alters the conformation of the ternary SNARE complex, the D2 domain of NSF that regulates NSF oligomerization. Nitric oxide (NO) is an important molecule that inhibits exocytosis. NO can chemically modify NSF by S-nitrosylating sulfhydryl groups in cysteine residues and prevent disassembly of SNAREs and NSF. Accordingly, elevated production of nitric oxide can inhibit exocytosis. NO is one of the most important molecules synthesized by the endothelial cell. Production of NO results from endothelial NO synthase (eNOS) enzymatic conversion of L-arginine to L-citrulline in the presence of essential cofactors such as FAD, NAD(P)H and tetrahydrobiopterin (BH4). Activation of eNOS is regualated by phosphatidylinositol 3-kinase (PI3 kinase)- serine/threonine protein kinase(Akt) signaling pathway. Akt can phosphorylate eNOS resulting in activation of eNOS. In addition, endothelial dysfunction and injury are closely associated with overexpression of adhesion molecules in endothelial cells. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Under normal condition, NF-κB is sequestrated in cytoplasm bound to inhibitory factor of NF-κB(IκB). When endothelial cells are stimulated by inflammatory mediators such as thrombin, IκB is degraded through complicated upstream molecules. Then NF-κB is released from the NF-κB/IκB complex and translocated from cytoplasm to nucleus, followed by increasing gene expression of cell adhesion molecules such as VCAM-1 and E-selectin. Genipin is a metabolite of geniposide, the major active ingredient of Gardemia jasminoids Ellis fruits, which have long been used in traditional Chinese medicine. This compound has been reported to have anti-inflammatory, anti-diabetic, anti-thrombotic, anti-oxidative, anti-angiogenic and neurotrophic activities. In a variety of animal models, genipin was shown to have anti-inflammatory activities. It significantly inhibited acute inflammation in carrageenan-induced rat paw edema, carrageenan-induced rat air pouch edema and croton oil-induced mouse ear edema models. It also inhibited mouse vascular permeability induced by acetic acid. Genipin may inhibit inflammation in part by inhibiting the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide upon stimulation by lipopolysaccharide/interferon in a murine macrophage cell line, as well as by inhibition of NF-κB activation. However, the molecular mechanisms of anti-inflammatory action of genipin are still not fully understood. Since endothelial exocytosis plays important roles in vascular inflammation, we hypothesized that genipin would exert its anti-inflammation by inhibiting WPB exocytosis from endothelial cells. In this study, we investigated the effects of ginipin on VWF release and P-selecti、E-selectin and VCAM-1 translocation in primary culture of human umbilical vein endothelial cells (HUVECs). We also performed experiments to further examine the mechanisms responsible for the inhibitory effect of endothelial exocytosis induced by this compound.
Methods and results
1. Treatment with 0.5-16μg/mL genipin had no effect on HUVEC viability Primary human umbilical vein endothelial cell (HUVEC) was isolated from human umbilical cord and cultured The cell viability assessment was performed using Cell Counting Kit-8. HUVECs were treated with 0.5-16μg/mL genipin for 12,24 and 48 h. The results showed that genipin treatment had no effect on HUVEC viability.
2. Genipin inhibits thrombin-induced exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECs
HUVECs were pretreated with increasing concentrations of genipin for various times, and then stimulated with 1 U/mL thrombin for 30 min. VWF concentration released in the cell supernatants was measured using VWF ELISA Kit. The amount of P-selectin, E-selectin and VCAM-1 translocation on the cell surface was using cell surface ELISA. Results showed that genipin blocked the exocytosis of P-selectin, E-selectin, VCAM-1 and VWF in dose-dependent manner, with a maximal effect achieved at 8μg/mL. The significant inhibition of genipin was seen within 1 hour of pretreatment and increased in time-dependent way.
3.Genipin inhibits mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs induced by thrombin
HUVECs were pretreated with increasing concentrations of genipin for various lengths of time and then stimulated with thrombin for 30 min. The mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs was evaluated by real-time RT-PCR and western blot. The results showed that thrombin significantly increased mRNA and protein expression of VCAM-1 and E-selectin in the HUVECs. However, pretreatment with 4-16μg/ml genipin inhibited mRNA and protein expression of VCAM-1 and E-selectin induced by thrombin in dose-dependent manner.
4. Genipin inhibits THP-1 cells adhesion to HUVECs induced by thrombin
HUVECs were pretreated with 8μg/mL genipin for 1 h and then stimulated with thrombin for 30 min. Fluorescence-labeled THP-1 cells were added to thrombin-stimulated HUVECs and co-cultured for 1 h. After removal of the non-adhesion cells, the remaining adhesion cells were photographed with fluorescence microscopy. The cells were lysed and measured using a spectrofluorometer. Results showed that, after stimulation with thrombine for30 min, adhesion of monocytes to HUVECs significantly increased (P<0.01 compared to normal treatment). When we pretreated HUVECs with 4 and 8μg/mL genipin for 1 h, this compound was found to markedly decrease monocyte adhesion to HUVECs induced by thrombin. (P<0.01 compared to thrombin treatment alone).
5. Genipin prolongs bleeding time in mice
Since platelet adherence to the vessel wall is mediated by the VWF, P-selectin, VCAM-1 and E-selectin,we predicted that genipin-induced inhibition of endothelial exocytosis would decrease platelet adherence and prolong the bleeding time. Thus, we measured the effect of genipin on the bleeding time in mice to obtain functional evidence that genipin inhibits HUVEC exocytosis in vivo. Anesthetized mice were injected intravenously with various concentrations of genipin or PBS. At various times after drug injection, the distal tip of tail was amputated, and bleeding time was measured. Treatment with PBS had no effect on the bleeding time, with a bleeding time of approximately 6 min. In contrast, genipin dramatically prolonged the bleeding time in dose- dependent manner. At the doses of 5,12.5,25 and 50mg/kg, the bleeding times were 8,10,16 and 18min, respectively. When the dose of the drug increased to 100mg/kg, the bleeding time was more than 20 min. The genipin-induced maximum bleeding time occurred at the 1 hour after the drug treatment, and then the prolonged bleeding time returned to normal level after 1.5 hours of genipin treatment.
6. NO mediates genipin-induced inhibition of HUVEC exocytosis
NO is an important molecule that inhibits exocytosis, we next examine the ability of genipin to stimulate NO production. We treated HUVECs with various genipin concentrations and with different duration of genipin pretreatment, and then measured the NO level in the cell supernatants. Our results showed that genipin activated HUVEC synthesis of NO in a dose- and time-dependent way.
In order to determine whether or not genipin-induced NO production is involved in the exocytosis inhibition by this compound, we examined the effect of L-NAME, an inhibitor of NOS, on genipin-induced exocytosis inhibition. HUVECs were pretreated with various concentrations of L-NAME for 1 h, and then genipin was added for 2 h. The cells were stimulated with thrombin (1U/mL) and VWF level in the supernatants was measured. Our results showed that L-NAME reversed the inhibitory effects of genipin on endothelial exocytosis, suggesting that genipin-induced NO production is involved in its inhibition of endothelial exocytosis.
7.Genipin activates PI3K-Akt- eNOS signaling pathway in HUVECs
Previous study showed that genipin inhibited thrombin-induced exocytosis in HUVECs through production of NO. Production of NO resulting from activation of eNOS is regualated by PI3K-Akt- eNOS signaling pathway. Phosphorylation of eNOS by Akt resulted in NO production. Therefore, we investigated the effect of genipin on eNOS activation in order to explore the mechanisms responsible for the inhibitory effect of genipin-induced endothelial exocytosis. HUVECs were treated with different concentrations of genipin or control for various times, and cell lysates were immunoblotted for phospho-eNOS (Ser-1177) or eNOS. Genipin increased phosphorylation level of eNOS in dose and time-dependent manner, but had no effect on the total amount of eNOS within 2 h after drug treatment. These results demonstrated that genipin stimulated eNOS phosphorylation and activated eNOS. We used a pharmacological approach to explore the role of PI3K in genipin activation of eNOS. The LY294002, an inhibitor of PI3K, diminished the ability of genipin to activate phosphorylation of Akt and eNOS. Taken together, these data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, and that this pathway mediates genipin inhibition of exocytosis.
8. Genipin promotes S-nitrosylation of NSF in HUVECs
NO S-nitrosylates sulfhydryl groups of NSF in cysteine residues and prevents disassembly of SNAREs and NSF, leading to decrease of exocytosis. In order to explore effect of genipin on S-nitrosylation of NSF, we used biotin-switch technique (BST), immunoprecipitation and Western blot to determine S-nitrosylation of NSF in HUVECs. Results showed that treatment with 4μg/mL genipin for 1h markedly increased the S-nitrosylation of NSF in HUVECs compared with control group. However, pretreatment with L-NAME(an inhibitor of NOS) or LY294002(an inhibitor of PI3K) inhibited the S-nitrosylation of NSF induced by genipin. All data suggest that genipin activates a pathway that includes PI3K, Akt, and eNOS, increases NO production and promotes S-nitrosylation of NSF in HUVECs.
9.Genipin inhibited thrombin-induced NF-κB signaling pathway activation in HUVECs
In addition, endothelial dysfunction and injury is closely assocated with overexpression of adhesion molecules in endothelial cells. Previous study showed that genipin inhibited the gene expression of VCAM-1 and E-selectin. Expression of adhesion molecules is regulated by nuclear factor -κB (NF-κB) signaling pathway. These two genes of VCAM-1 and E-selectin have putative binding sites for NF-κB at their promoter regions to activate gene expression. Therefore, in this study we explored the effect of genipin on thrombin-induced NF-κB signaling pathway activation using western blotting and immunocytochemistry. Results showed that thrombin significantly increased NF-κB p65 expression in the nuclear extracts compared to normal treatment. In contrast, pretreatment with genipin 8-16μg/mL for 1 h markedly inhibited thrombin-induced increase of nuclear NF-κB p65 in dose-dependent manner. Furthermore, thrombin significantly decreased the IκB expression levels in cytoplasm. However, pretreatment with 8-16μg/mL genipin for
1 h markedly inhibited thrombin-induced decrease of IκB expression levels in dose-dependent manner.
To confirm the consistency to the result of western blotting, we observed the translocation of NF-κB p65 by using immuocytochemistry.Results showed that thrombin caused NF-κB p65 translocation into nucleus compared to normal treatment. However, pretreatment with 4μg/mL genipin inhibited the translocation of NF-κB p65 from cytoplasm to nucleus. These results were consistent with the result of western blotting.
Take together, the conclusions of present study are as follow
1. Genipin inhibited exocytosis of P-selectin, E-selectin, VCAM-1 and VWF from HUVECs induce by thrombin.
2. Genipin decreased THP-1 cells adhesion to HUVECs induced by thrombin and prolonged the blood time of mice tail vein.
3. Genipin activated of PI3K-Akt-eNOS signaling pathway, increases NO production, and induces S-nitrosylation of NSF in HUVECs.
4. Genipin inhibited thrombin-induced HUVEC NF-κB signaling pathway activation, which in turn inhibited the gene and protein expression of adhesion molecules such as VCAM-1 and E-selectin.
In conclusion, excessive endothelial exocytosis may contribute to the pathophysiology of inflammation and thrombosis. Genipin, a novel inhibitor of endothelial exocytosis found in this study, may target acute inflammatory events and suppress vascular and endothelial cell inflammatory activation. This compound may represent a new treatment for inflammation and/or thrombosis in which excess exocytosis plays a pathophysiological role.
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