K_(ATP)通道在动脉粥样硬化斑块易损性中的作用及分子机制研究
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摘要
研究背景
动脉粥样硬化(atherosclerosis, AS)是严重危害人类健康的疾病,其中易损斑块是急性冠脉综合征(acute coronary syndrome, ACS)以及脑血管疾病的基础病变,易损斑块发病机制和治疗靶点的探索成为当今医学发展的重大挑战。动脉粥样硬化斑块的主要细胞成分包括巨噬细胞、平滑肌细胞和内皮细胞。易损斑块的病理改变包括平滑肌细胞和胶原纤维含量减少,有大的脂质核,斑块内大量的巨噬细胞和淋巴细胞等炎性细胞浸润。
越来越多的研究表明离子通道与血管功能密切相关,在动脉粥样硬化的发生发展过程中发挥重要作用。已有研究报道,内皮细胞钙离子通道、钾离子通道和氯离子通道在剪切力应激和炎症反应中具有调节作用；TRPC通道介导的血管平滑肌细胞、内皮细胞、单核/巨噬细胞和血小板功能失调参与了动脉粥样硬化的病理过程。由此可见,离子通道在动脉粥样硬化中的作用不容忽视。而且,钾离子通道是膜电位的重要组成部分,在维持细胞电稳态中具有重要作用,可以调节细胞内钙离子的浓度进而发挥对细胞功能的调节作用。
三磷酸腺苷(Adenosine triphosphate, ATP)敏感的钾通道(KATP)是由四个孔道形成亚基-Kir6亚基和四个调节亚基-SUR亚基组成的八聚体蛋白。对细胞内腺苷酸变化敏感,是重要的代谢感受器,偶联细胞内代谢状态细胞膜电活动。近年来,KATP通道在糖尿病和心血管疾病中同时发挥的重要作用逐渐得到认可,作为几类不同药物的作用靶点,成为研究活跃的领域。现有的研究主要集中在以下几个方面：心脏功能的调节,包括缺血再灌注、心肌梗死、心肌重构、心衰、缺血预适应和应激的环境中心肌细胞KATP通道的调节作用；血管平滑肌细胞的KATP通道研究也很丰富,包括各种内源性和外源性血管舒缩物质的调节作用以及在高血压和感染的环境中的表达和功能的改变；同样的,血管内皮细胞的KATP通道也越来越受到重视,包括在内皮素和NO释放中的调节作用研究。但是,对于KATP通道在动脉粥样硬化中的作用却没有直接的报道。
因此,本研究提出如下假说：KATP通道在动脉粥样硬化病变的病理生理过程中发挥重要作用,KATP通道可能成为治疗动脉粥样硬化的新靶点。
目的
1.建立动脉粥样硬化病变发展的动物模型；
2.观察斑块平滑肌细胞、巨噬细胞、胶原纤维和脂质表达,评测斑块易损性；
3.探讨KATP通道药物体内干预对动脉粥样硬化病变发展的影响。
方法
本研究以ApoE-/-小鼠80只为研究对象,9周龄始予高脂饮食,10周龄时(25-30g)行右侧颈动脉套管术；8周后,高分辨率小动物显微超声检测颈动脉斑块病变,随后,小鼠随机分为空白对照组,转染重组腺病毒载体Ad5-CMV.lacZ对照组和转染重组腺病毒载体Ad5-CMV.p53实验组,后两组分为转染后1天组、4天组和14天组,转染后4天组和14天组又分为格列苯脲(Glibenclamide)干预组和非干预组,给予相应处理。检测下列指标：
1.血生化分析：血液标本收集,检测血糖(glucose, Glu)、谷丙转氨酶(glutamic pyruvic transaminase, GPT)谷草转氨酶(glutamic oxaloacetic transaminase, GOT)、甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)含量；
2. ApoE-/-小鼠取材,病理组织学检测：实验动物按照实验分组,分别于转染后0天、1天、4天和14天时处死,留取组织用于病理染色及分子生物学实验。苏木素-伊红(hematoxylin and eosin, HE)染色用以检测组织结构变化,油红O(Oil-red O)染色用以检测组织l脂质含量,天狼猩红(Sirius-red)染色用以检测组织胶原纤维含量,p53、SMC-α actin、MOMA-2的免疫组织化学染色分别用以检测组织p53的表达、平滑肌细胞和巨噬细胞的含量；
3.计算斑块易损指数：易损指数=(MOMA-2阳性面积/斑块面积+脂质面积/斑块面积)/(SMC-α actin阳性面积/斑块面积+胶原纤维面积/斑块面积)。结果
1.格列苯脲干预对ApoE-/-小鼠血生化及肝脏组织的影响
格列苯脲灌胃后小鼠血糖、甘油三酯、总胆固醇、转氨酶水平以及肝脏组织结构没有明显变化(P>0.05)。
2.ApoE-/-小鼠颈动脉套管手术及p53转染效率检测
小鼠显微超声示右侧颈动脉套管及套管近心端斑块形成；转染后1天、4天、14天,p53表达在Ad5-CMV.p53转染组比Ad5-CMV.lacZ组明显增多(P<0.05)。
3.ApoE-/-小鼠颈动脉粥样硬化斑块平滑肌细胞及胶原纤维染色
斑块平滑肌细胞含量与胶原纤维含量变化趋势基本一致,在p53转染4天和14天组明显少于相应时间点的lacZ转染组(P<0.05)。
4.ApoE-/-小鼠颈动脉粥样硬化斑块巨噬细胞及脂质染色
斑块巨噬细胞含量与脂质含量变化趋势一致,在转染后4天和14天时,p53组显著高于相应时间点的lacZ组(P<0.05)。
5.ApoE-/-小鼠格列苯脲体内干预稳定颈动脉粥样硬化斑块
格列苯脲体内干预14天,在p53转染组巨噬细胞含量、脂质含量明显降低(P<0.05),斑块胶原成分显著增加(P<0.05),尽管平滑肌细胞含量无明显变化(P>0.05),但是斑块病变改善显著。
6.斑块易损指数计算
在转染后4天和14天时,p53组易损指数比lacZ显著升高,而且时间点愈后p53转染组的易损指数升高愈显著(P<0.05)；在p53转染14天组,格列苯脲体内干预使易损指数明显下降(P<0.05)。
结论
1. ApoE-/-小鼠高脂饮食、颈动脉套管术后局部转染Ad5-CMV.p53诱导斑块易损指数增加；
2.格列苯脲体内干预对Ad5-CMV.p53转染ApoE-/-小鼠血.生化及肝组织结构无明显影响；
3. ApoE-/-小鼠格列笨脲体内干预改善p53转染诱导的颈动脉粥样硬化斑块病变。
研究背景
动脉粥样硬化(atherosclerosis,AS)与细胞膜离子通道关系密切。K+电流是细胞膜电位的主要组成部分,对于细胞功能至关重要。三磷酸腺苷(Adenosine triphosphate,ATP)敏感的钾通道(KATP)是细胞膜离子通道的重要组成部分,通过调节细胞内外钾离子浓度,在维持细胞膜电位、调节其他生物离子进出细胞和调市细胞功能中发挥不可替代的作用,而且是联系细胞电活动与细胞代谢的桥梁。缺血、缺氧、代谢性酸中毒等导致细胞内ADP/ATP升高时,KATP通道开放,细胞膜超极化,电压依赖性钙通道(voltage-dependent calcium channels,VDCCs)关闭,钙内流减少,细胞内[Ca2+]降低,介导缺血-再灌注损伤及心力衰竭中的心肌保护等作用。
KATP,通道是一种异源八聚体复合物,四个Kir6.X亚基构成孔道形成亚基转运钾离子,四个SURs亚基组成调节亚基,根据细胞内ATP水平调节通道活性。Kir6.X存在Kir6.1和Kir6.2两个亚型；磺脲类受体SURs(sulfonylurea receptors)有SUR1和SUR2两种亚型,后者有两种主要的剪接体SUR2A和SUR2B,连接Kir6亚基构成功能性的K/ATP通道。KATP通道在多种组织农达,承载了重要的细胞功能,并且KATP通道的亚基组成具有组织特异性,在同时表达Kir6.1.Kir6.2亚基的细胞系,亚基常常以不同比例组成异源多聚体；相应地,不同组织中的KATP通道有不同的生理特性,也是特异性药物试剂作用于特定组织KATP通道以调节不同细胞功能的重要生理基础。例如,心肌细胞主要表达SUR2A/Kir6.2亚型,在缺血缺氧及其他应激的环境中发挥保护心肌细胞的重要作用；胰腺p细胞主要表达SUR1/Kir6.2亚型,可以调节胰岛素的分泌；SUR2B/Kir6.1主要在血管平滑肌细胞和内皮细胞表达,在调节血管张力、血管的舒缩和内皮保护中发挥重要作用。KATP通道是重要的代谢感受器,而动脉粥样硬化及炎症反应与细胞代谢密切相关。
因此,本研究提出如下假说：KATP通道在动脉粥样硬化斑块的平滑肌细胞、内皮细胞和／或巨噬细胞中表达,与动脉粥样硬化斑块的易损性关系密切。
目的
1.检测KATP通道SUR1、SUR2A、SUR2B亚基和Kir6.1、Kir6.2亚基在动脉粥样硬化斑块的表达和分布；
2.分析KATP通道亚基表达量和AS斑块成分含量、易损指数的相关性；
3.探讨AS斑块平滑肌细胞、内皮细胞和巨噬细胞中KATP通道在病变发展过程中的作用。
方法
本研究以50只ApoE-/-小鼠为研究对象,9周龄始予高脂饮食,10周龄时(25-30g)行右侧颈动脉套管术；8周后,高分辨率小动物显微超声检测颈动脉斑块病变,随后,小鼠随机分为空白对照组,转染重组腺病毒载体Ad5-CMV.lacZ对照组和转染重组腺病毒载体Ad5-CMV.p53实验组,后两组分为转染后1天组、4天组和14天组,给予相应处理,分别于相应时间点处死小鼠,留取取标本,检测下列指标：
1.病理组织学染色：HE染色检测组织形态结构的改变、油红O染色检测标本脂质含量、天狼猩红染色检测标本胶原含量；
2. SMC-a actin和MOMA-2免疫组织化学染色：检测AS斑块平滑肌细胞和巨噬细胞含量；
3. KATP通道亚基免疫组织化学染色：检测AS斑块KATP通道亚基SUR1、 SUR2A、SUR2B、Kir6.1和Kir6.2含量,观察各亚基在斑块平滑肌细胞、内皮细胞和巨噬细胞的表达分布情况；
4.相关性分析：计算分析AS斑块成分含量、易损指数和KATP通道亚基SUR1、SUR2A、SUR2B、Kir6.1、Kir6.2表达的相关性；
5.AS相关的KATP通道亚基和相应细胞的免疫荧光双标染色：观察AS斑块细胞和主要KATP通道亚型的共同定位表达；
6. KATP通道亚基实时定量逆转录聚合酶连反应(RT-PCR)定量分析：RT-PCR检测ApoE-/-小鼠颈动脉KATP通道亚基SUR1、SUR2A、SUR2B、Kir6.1和Kir6.2的(?)nRNA表达。
结果
1.AS斑块KATP通道亚基免疫组织化学染色结果
SUR2A和Kir6.2亚基阳性染色主要积聚在粥样硬化斑块肩部和脂质条纹等巨噬细胞聚集的部位,定量分析表明二者变化趋势基本一致,在p53转染4天组表达最高,显著高于lacZ转染4天组(P<0.05); SUR1亚基阳性染色在巨噬细胞密集的斑块肩部和脂质条纹区域,以及血管内膜内皮细胞表现突出,定量分析显示,SUR1亚基表达在p53转染4天组增长最为显著,明显多于lacZ转染4天组(P<0.05); Kir6.1亚基阳性染色在斑块肩部及脂质条纹巨噬细胞聚集的区域、血管内皮细胞、血管中膜及纤维帽平滑肌细胞较丰富的区域均有表现,在转染后1天和4天,p53处理组Kir6.1亚基表达均高于lacZ处理组(P<0.05)；SUR2B亚基阳性染色主要分布在血管中膜及斑块纤维帽平滑肌细胞较集中的部位和血管内皮细胞,SUR2B亚基表达在p53转染4天组明显减少,显著少于lacZ转染4天组(P<0.05)。
2. KATP通道亚基mRNA表达结果
小鼠颈动脉RT-PCR结果显示,KATP通道SUR2A、Kir6.2和SUR1亚基mRNA表达在p53转染4天组增加最为显著,明显高于lacZ转染4天组(P<0.05);Kir6.1亚基在p53转染1天组表达高于lacZ转染1天组(P<0.05); SUR2B亚基在空白对照组表达水平最高(P<0.05)。
3.AS斑块KATP通道染色与斑块负荷相关性分析结果
KATP通道SUR2A亚基和Kir6.2亚基染色,与斑块易损指数正相关,与斑块巨噬细胞含量成正相关,与斑块平滑肌细胞含量负相关(P<0.05)。
4.AS斑块MOMA-2与SUR2A、Kir6.2共表达
免疫荧光双标染色显示,SUR2A亚基和MOMA-2共同定位表达于斑块内及血管壁粘附浸润的泡沫巨噬细胞,这些部位同样粘附聚集Kir6.2亚基和MOMA-2双染巨噬细胞。
结论
1. KATP通道亚基在AS斑块平滑肌细胞、内皮细胞以及巨噬细胞表达；
2. KATP通道SUR2A、Kir6.2亚基与AS斑块负荷密切相关；
3. KATP通道SUR2A/Kir6.2主要在斑块巨噬细胞表达。
研究背景
动脉粥样硬化斑块的主要细胞成分包括巨噬细胞、平滑肌细胞和内皮细胞。与血管平滑肌细胞和内皮细胞显著不同,巨噬细胞来源于循环中的单核细胞,是免疫系统炎症反应的重要组成部分,单核/巨噬细胞粘附、聚集、迁移和分泌炎症因子的功能贯穿动脉粥样硬化斑块的起始、发展和破裂的全过程。
以往的研究发现,单核/巨噬细胞离子通道在动脉粥样硬化病变中发挥重要作用。单核/巨噬细胞TRPC通道通过调节细胞内钙离子浓度,介导细胞的氧化应激,加剧动脉粥样硬化病变：周围的细胞因子和细胞本身的功能状态能够调节单核细胞电压依赖性内向整流钾通道Kir (voltage-dependent inwardly rectifying potassium channels)和延迟外向整流钾通道Kdr (delayed outwardly rectifying potassium channels)诱导单核细胞的活化和分化；单核/巨噬细胞容量调节性氯离子通道(volume-regulated Cl channels)功能失调,促进巨噬细胞吞噬脂质,形成泡沫细胞。更重要的是,MCP-1和LPC通过调节氯离子通道和钙离子激活的钾通道可以促进单核细胞的迁移。单核细胞的迁移是动脉粥样硬化的始动环节,而大量泡沫细胞的形成是不稳定粥样斑块的重要标志。然而,ATP敏感钾通道(ATP-sensitive K+channels, KATP)在动脉粥样硬化中的作用至今鲜有报道。
KATP,通道是异源八聚体蛋白复合物,由四个内向整流K+通道(inwardly rectifying potassium channel, Kir) Kir6.X亚基形成孔道,四四个ATP结合盒(ATP binding cassette, ABC)家族的磺脲类受体(sulfonylurea receptor, SUR)蛋白组成调节亚基。不同组织表达的KATP通道Kir6.X和SUR亚基不尽相同,Kir6.X包括Kir6.1和Kir6.2亚型,SUR包括SUR1、SUR2A和SUR2B亚型,因而呈现出组织特异性的生理学和病理生理学特征。有研究报道,KATP通道阻断剂格列苯脲能够抑制低氧血症和酸中毒时脂多糖(Lipopolysaccharide, LPS)诱导的促炎性细胞因子的释放。而单核/巨噬细胞的炎症反应在动脉粥样硬化病变的形成、发展以及晚期易损斑块破裂中扮演重要角色。LPS是单核/巨噬细胞的有效激活剂,与Toll样受体(TLRs)结合,主要通过NF-κB和MAPKs信号通路的激活,后者包括p38、JNK和ERK1/2信号转导途径,诱导TNF-α等促炎性细胞因子分泌。
因此,我们提出下列假说：单核/巨噬细胞KATP通道在LPS诱导的细胞炎症反应中发挥作用,从而影响动脉粥样硬化病变的发生、发展；单核/巨噬细胞KATP通道通过NF-κB和MAPKs (ERK1/2、p38和JNK)信号转导通路调节细胞的炎症反应。
目的
1.观察LPS对巨噬细胞KATP通道和TNF-α表达的影响；
2.探讨KATP通道对LPS诱导的巨噬细胞TNF-α表达的调节作用；
3.揭示KATP通道对LPS诱导的巨噬细胞内NF-κB和MAPKs信号通路的影响。
方法
本研究以单核/巨噬细胞系RAW264.7细胞为研究对象,KATP通道开放剂吡那地尔或阻断剂格列苯脲预处理后予LPS刺激,采用实时定量聚合酶连反应(RT-PCR)和蛋白免疫印迹(western blot)等实验方法,检测：1.LPS (1μg/ml)处理RAW264.7细胞不同时间(0、2、4、8、24h)后,检测KATP通道各亚基(SUR1、SUR2A、SUR2B、Kir6.1、Kir6.2)和炎症因子TNF-α表达；
2. KATP通道开放剂(吡那地尔,10μM)或阻断剂(格列苯脲,10μM)预处理后,检测LPS刺激RAW264.7细胞TNF-α的表达,检测不同刺激时间(15min、30min、1h、24h) NF-κB和MAPKs信号通路蛋白的磷酸化改变；
3. NF-κB信号通路抑制剂预处理后,检测KATP通道各亚基的表达改变,检测KATP通道干预对RAW264.7细胞TNF-α表达的影响。
结果
1.LPS诱导RAW264.7细胞KATP通道亚基和TNF-α表达
与空白对照组相比较,KATP通道SUR1、SUR2A、Kir6.1和Kir6.2亚基在LPS (1μg/ml)刺激细胞4小时后表达明显升高(P<0.05),同时,TNF-α表达显著增加(P<0.05),但是各组均未检测到SUR2B亚基mRNA的表达。
2. KATP通道药物干预后RAW264.7细胞TNF-α表达和NF-κB、MAPKs蛋白磷酸化改变
与LPS刺激4小时组相比较,KATP通道阻断剂格列苯脲(10μM)预处理后细胞TNF-α表达降低(P<0.05),开放剂吡那地尔(10μM)预处理后细胞TNF-α表达略增加(P<0.05)。
与LPS刺激15分钟组相比较,格列苯脲(10μ,M)预处理后细胞p65、ERK1/2蛋白磷酸化水平下降(P<0.05),而吡那地尔(10μ,M)预处理对细胞p65、ERK1/2蛋白磷酸化水平影响没有统计学意义(P>0.05)；格列苯脲和吡那地尔对p38蛋白磷酸化水平没有明显改变(P>0.05)；与空白对照组相比较,LPS刺激15分钟、1小时或24小时JNK蛋白磷酸化无显著变化(P>0.05)。
3. KATP通道位于RAW264.7细胞LPS诱导活化的NF-κB信号通路上游
与LPS刺激4小时组相比较,NF-κB信号通路抑制剂PDTC(100μM)和DMFR(100μM)预处理显著降低RAW264.7细胞TNF-α的mRNA表达(P<0.05)；KATP通道阻断剂格列苯脲(10μM)能够增强NF-κB抑制剂的作用(P<0.05),而开放剂吡那地尔(10μM)拮抗NF-κB抑制剂的作用(P<0.05)。
与LPS刺激4小时组相比较,PDTC (100μM)和DMFR (100μM)预处理显著增加了RAW264.7细胞KATP通道SUR1、SUR2A、Kir6.1和Kir6.2亚基的表达(P<0.05)。
结论
1.LPS绣导巨噬细胞KATP通道表达：
2.巨噬细胞KATP通道参与调节LPS诱导的炎症反应；
3.巨噬细胞KATP通道通过NF-κB和ERK1/2信号转导通路调节细胞炎症反应。
Background
Atherosclerosis is one of the most serious diseases detrimental to health and vulnerable plaques have been recognized as chief criminal of acute coronary syndrome (ACS) and cerebrovascular disorders. However, the pathogenesis and approach targets of atherosclerotic plaques are still unclear. The main cellular components in plaques include macrophages, smooth muscle cells and endothelial cells, while the decrease of smooth muscle cells and collagen fibers, and the increase of lipids and macrophages happened in vulnerable plaques.
Numerous studies have indicated that ion channels are related to vascular functions tightly and play an important role in the progression of atherosclerosis. It has been reported that the calcium channels, potassium channels and chloride channels in endothelial cells regulated the shear stress and inflammation; the disorders of vascular smooth muscle cells, endothelial cells, monocytes/macrophages and platelets induced by transient receptor potential canonical channels (TRPC) participated in the pathogenesis of atherosclerosis. So. the role of ion channels in atherosclerosis should never be neglected. Potassium channels contribute to the membrane potential, preserve the cellular electrical homeostasis and could affect the activities of other ions to regulate the cellular state.
ATP-sensitive potassium channels (KATP channels) are heterooctameric complexes composed of Kir6.1or Kir6.2as pore forming subunits, and SUR1, SUR2A or SUR2B as regulatory subunits. KATP channels are sensitive to the change of intracellular ATP as important metabolic sensors, coupling metabolic state with electrical activity of cells. Recently, the role of KATP channels in both of diabetes and cardiovascular diseases has attracted more and more attention. The studies mainly concentrate on the following aspects:the regulation of cardiac functions, under ischemia-reperfusion, myocardial infarction, myocardial remolding, heart failure, ischemic preconditioning and stress; vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors; regulation the effect of endothelium-derived relaxing factor (EDRF), such as nitric oxide (NO), hydrogen sulfide (H2S) and endothelin-1(ET-1). However, the role of KATP channels in atherosclerosis hasn't been investigated directly.
Therefore, we put forward the hypothesis that KATP channels play an important role in the pathogenesis of atherosclerosis and represent the potential therapeutic target of atherosclerosis.
Objective
1. To establish animal models of progressive atherosclerosis;
2. To evaluate the content of smooth muscle cells, macrophages, collagen fibers and lipids in plaques, and the vulnerable index of plaques;
3. To investigate the interventional effects of modulators targeting to KATP channels on the progression of atherosclerosis.
Methods
We applied eighty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, with or without intervention of glibenclamide in vivo, followed by a series of experiments.
1. Blood biochemical analysis:collect blood samples to detect the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma.
2. Tissue harvesting and morphological analysis:the mice were sacrificed0day,1day,4days or14days after transduction according to the assignment, respectively, and the tissue samples were carefully excised for pathological staining and molecular biological experiments. Hematoxylin and eosin (HE)staining was used to show the tissue structure, Oil-red O staining was used to detect the content of lipids, sirius-red staining was used to detect the content of collagen fibers, immunohistochemical staining of p53, SMC-a actin and MOMA-2was used to detect the content of p53protein, smooth muscle cells and macrophages, respectively.
3. Calculation of vulnerable index:vulnerable index=(MOMA-2positive staining area/plaque area+lipids staining area/plaque area)/(SMC-a actin positive staining area/plaque area+collagen fibers staining area/plaque area).
Results
1. The effects of glibenclamide intervention on blood biochemistry and livers in ApoE-/-mice
There were no substantial changes in the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma, and hepatic structures of ApoE-/-mice after intragastric administration of glibenclamide in our experiment (all P>0.05).
2. Identification of carotid collar placement and transduction of p53
The right carotid arteries proximal to collars showed atherosclerotic plaques by ultrasound. Compared with that of control groups, the staining of p53was significantly increased in Ad5-CMV.p53-treated groups, including1,4,14-day subgroups, and the difference increased over time (all P<0.05).
3. The staining of smooth muscle cells and collagen fibers in atherosclerotic plaques
Compared to Ad5-CMV.lacZ-treated groups, the content of smooth muscle cells and collagen fibers in atherosclerotic plaques was decreased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).
4. The staining of macrophages and lipids in atherosclerotic plaques
Compared to Ad5-CMV.lacZ-treated groups, the content of macrophages and lipids in atherosclerotic plaques was increased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).
5. Glibenclamide intervention stabilizing atherosclerotic plaques in vivo
In Ad5-CMV.lacZ-treated-14days groups, the content of macrophages and lipids decreased obviously (both P<0.05), and the content of collagen fibers increased (P<0.05), while the content of smooth muscle cells was not different significantly after intervention of glibenclamide for14days in vivo.
6. Calculation of vulnerable index
The vulnerability index (VI), which was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions to estimate the vulnerable possibility of plaques, was higher in p53-treated groups than that in control groups after4days and14days and the gap was widened over time (both P<0.05).
Conclusions
1. Transduction of Ad5-CMV.p53induced increased vulnerable index of plaques in ApoE-/-mice with high fat diet and collar placement;
2. Glibenclamide intervention didn't affect the blood biochemistry and structure of livers obviously in ApoE-/-mice transfected with Ad5-CMV.p53;
3. Glibenclamide ameliorated the progression of atherosclerosis induced by Ad5-CMV.p53transduction in vivo.
Background
Atherosclerosis (AS) is related to membrane ion channels closely. K+current is the primary component of membrane potential and vitally important to cellular functions. ATP sensitive potassium channels (KAtp) could regulate the concentration of K+and preserve membrane potential to modulate activities of other ions and the whole cell, and couple cellular metabolism with membrane potential. When the intracellular ADP/ATP is raised due to ischemia, hypoxia, metabolic acidosis and so on, KATP channels will open, the cell hyperpolarize, voltage-dependent calcium channels (VDCCs) close, calcium influx decrease and intracellular Ca2+reduce to mediate cardiac protection.
KATP channel is a hetero-octamer complex of four pore forming subunits, inwardly rectifying potassium channels Kir6.X, and four regulatory subunits according to intracellular ATP concentration, sulfonylurea receptors (SURs), member of ATP binding cassette (ABC) superfamily. Kir6.X, including Kir6.1and Kir6.2, links with SURs, including SUR1, SUR2A and SUR2B, to constitute functional KATP channels. KATP channels exist in various tissues and carry an important cellular function with tissue specificity. In cells expressing Kir6.1and Kir6.2at the same time, the subunits often comprise heterogeneous polymers in a different proportion. Accordingly, KATP channels in different tissues exhibit different physiologic characteristics and response with specific reagents. For example, SUR2A/Kir6.2 subtype mainly expresses in myocardial cells and shows cardiac protection under conditions of ischemia, hypoxia and other stress; SUR1/Kir6.2subtype mainly expresses in pancreatic P cells to regulate secretion of insulin; SUR2B/Kir6.1mainly expresses in vascular smooth muscle cells and endothelial dcells and participate in vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors. KATP channels are important metabolic sensors, while AS and inflammation are associated with metabolism closely.
Therefore, we put forward the hypothesis that KATP channels express in vascular smooth muscle cells, endothelial cells and macrophages of atherosclerotic plaques, and should be correlated with vulnerability of plaques.
Objective
1. To detect the expression and distribution of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in atherosclerotic plaques;
2. To make correlation analysis between content of KATP subunits and vulnerability of plaques;
3. To evaluate the effect of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages on progression of atherosclerosis.
Methods
We applied fifty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, respectively, followed by a series of experiments.
1. Morphological staining:Hematoxylin and eosin (HE) staining was used to show the tissue structure, sirius-red staining was used to detect the content of collagen fibers and Oil-red O staining was used to detect the content of lipids;
2. SMC-a actin and MOMA-2staining:Immunohistochemical staining of SMC-a actin and MOMA-2was used to detect the content of smooth muscle cells and macrophages, respectively;
3. KATP Subunits staining:Immunohistochemical staining of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in AS plaques to investigate the expression and distribution of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages;
4. Correlation analysis:The content of smooth muscle cells, collagens, macrophages and lipids in plaques was evaluated, the vulnerable index was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions, and then, we anlysed the correlation between AS components, vulnerable index and KATP Subunits;
5. Immunofluorescence:Immunofluorescence double staining was used to detect the co-localization of atherosclerotic cells and KATP Subunits;
6. RT-PCR quantitative analysis of KATP Subunits:The mRNA expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in carotid arteries was detected by RT-PCR.
Results
1. The staining of KATP Subunits in atherosclerotic plaques
Positive staining of SUR2A and Kir6.2subunits mainly accumulated in plaque shoulder and fatty streak with more macrophages, and quantitative analysis showed similar expressional tendency:SUR2A and Kir6.2expression was detected mostly in p53-treated4-day group and was higher than that of lacZ treated-control groups after1day and4days treatment (P<0.05). Positive staining of SUR1subunit mainly accumulated in plaque shoulder and fatty streak, as well as endothelial cells, and quantitative analysis showed that the staining was most markedly in p53-treated4-day group and was higher than that of control group (P<0.05). Positive staining of Kir6.1subunit distributed in plaque shoulder and fatty streak, as well as endothelial cells and smooth muscle cells, and quantitative analysis showed that the staining was higher in p53treated groups after1day and4days compared with that of lacZ treated groups (P<0.05). Positive staining of SUR2B subunit mainly accumulated in vascular smooth muscle cells and endothelial cells, and quantitative analysis showed that the staining was decreased in p53-treated4-day group compared with that of lacZ treated group (P<0.05).
2. The mRNA expression of KATP subunits in carotid arteries
At mRNA level, the expression of SUR2A, Kir6.2and SUR1subunits was higher in p53-treated group than that of control group after4-day treatment (P<0.05); the expression of Kir6.1subunit was higher in p53-treated group than that of control group after1-day treatment (P<0.05); and the largest value of SUR2B transtripts was found in blank control group (P<0.05).
3. The correlation analysis between KATP subunits and plaque burden
The expression of SUR2A and Kir6.2subunits in plaques was positively correlated with vulnerable index and the content of macrophages in plaques, and negatively correlated with the content of smooth muscle cells in plaques (all P<0.05).
4. Co-localization of MOMA-2with SUR2A and Kir6.2subunits in plaques
The result of immunofluorescence double staining showed that MOMA-2and SUR2A, as well as MOMA-2and Kir6.2, co-located at macrophages attached to vascular walls and within plaques.
Conclusions
1. KATP subunits expressed in smooth muscle cells, endothelial cells and macrophages of AS plaques;
2. SUR2A and Kir6.2subunits were associated with plaque burden closely;
3. The subtype of SUR2A/Kir6.2mainly located in macrophages of atherosclerotic plaques.
Background
Atherosclerotic plaques consist of macrophages, smooth muscle cells and endothelial cells. Differentiating from vascular smooth muscle cells and endothelial cells, macrophages derive from circular monocytes and contribute to immune inflammatory response. The activation of monocytes/macrophages is considered as the initiation of atherosclerosis and works through the whole process of atherosclerosis with accumulating, adhesion, migration and producing inflammatory cytokines.
The previous studies have showed that ion channels expressed in monocytes/macrophages played an important role in atherosclerosis. Dysfunction of monocytes/macrophages mediated by TRPC (transient receptor potential canonical) channels was associated with the development of atherosclerosis; the changes of voltage-dependent inwardly rectifying potassium channels (Kir) and delayed outwardly rectifying potassium channels induced by inflammatory cytokines promoted the activation and differentiation of monocytes; disordered volume-regulated Cl channels promoted foam cell formation from macrophages in atherosclerosis; moreover, MCP-1and LPC regulated the chloride channels and calcium activated potassium channels to accelerate the migration of monocytes. The migration of monocytes initiates the process of atherosclerosis and the formation of considerable foam cells designates the vulnerable plaques. Whereas, KATP channels in monocytes/macrophages have drawn relatively a little attention.
KATP channels are octamers of four pore forming subunits-inwardly rectifying potassium channel Kir6.X and four regulatory subunits-a member of ATP binding cassette (ABC) superfamily called sulfonylurea receptor (SUR). The former contains Kir6.1and Kir6.2subtypes, and the latter contains SUR1, SUR2A and SUR2B subtypes. They comprise various subtypes of KATP channels in different tissues with distinct physiologic and pathological properties. It has been reported that the blocker of KATP channels, glibenclamide, could inhibit the release of proinflammatory cytokines induced by Lipopolysaccharide (LPS) in condition of hypoxia and acidosis. The inflammation mediated by monocytes/macrophages play an important role in initiation and progression of atherosclerotic lesions. LPS, a typical bacterial pathogens known as most powerful activators of macrophages, could stimulate monocytes/macrophages to produce proinflammatory cytokines such as TNF-a by bounding with toll like receptors (TLRs) through two major signal transduction pathways:nuclear factor κB (NF-κB) proteins and mitogen-activated protein kinases (MAPKs), the latter including extracellular signal related kinase (ERK1/2), p38MAPK (p38) and c-Jun N-terminal kinase (JNK) pathways.
Therefore, we put forward the hypothesis that KATP channels in monocytes/macrophages play an important role in inflammation to mediate the progression of atherosclerotic lesions and they work through NF-κB/MAPKs signal transduction pathways to regulate the inflammatory response.
Objective
1. To detect the expression of KATP subunits and TNF-a induced by LPS in macrophages;
2. To explore the effect of KATP channels on the expression of TNF-a induced by LPS in macrophages;
3. To investigate the the effect of KATP channels on NF-κB and MAPKs pathways induced by LPS in macrophages.
Methods
We used the cell line of mice monocytes/macrophages, RAW264.7cells, as the object of this study, pretreated with the opener or blocker of KATP channels, pinacidil or glibenclamide, followed by stimulation of LPS. And then we applied real time RT-PCR and western blotting to detect the role and mechanisms of KATP channels in macrophages.
1. RAW264.7cells were treated with LPS (1μg/ml) for0,2,4,8or24h to detect the expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subunits, as well as TNF-a.
2. RAW264.7cells were pretreated with the opener or blocker of KATP channels, pinacidil (10μM) or glibenclamide (10μM), to detect the expression of TNF-a induced by LPS and the phosphorylation of NF-κB and MAPKs signal transduction proteins at various time (15min,30min,1h, and24h).
3. After intervention with inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), the expression of KATP subunits and TNF-a inducted by LPS in macrophages was investigated.
Results
1. LPS induced the expression of KATP subunits and TNF-a in RAW264.7cells
Compared with control group, the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits, as well as TNF-a, was increased in LPS (1μg/ml) treated RAW264.7cells for4h (all P<0.05). However, the expression of SUR2B subunit was not detected.
2. The regulation of KATP channels in expression of TNF-a and phosphorylation of NF-κB and MAPKs signal proteins
The blocker of KATP channels, glibenclamide (10κM). could reduce the expression of TNF-a induced by LPS in RAW264.7cells for4h. while the opener pinacidil (10λM) raised the expression slightly (both P<0.05). Moreover, glibenclamide decreased the phosphorylation of p65and ERK1/2induced by LPS for15min (both P<0.05), while the effect of pinacidil was not obvious (P>0.05).
3. KATP channels located at upstream of NF-κB activated by LPS in RAW264.7 cells
The inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), decreased the expression the TNF-a induced by LPS in RAW264.7cells obviously (P<0.05), and glibenclamide could enhance the effect of NF-κB inhibitors, while pinacidil antagonized their effect (P<0.05).
PDTC (100μM) and DMFR (100μM) increased the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits induced by LPS in RAW264.7cells significantly (P<0.05).
Conclusions
1. LPS increased the expression of KATP channels in macrophages;
2. KATP channels in macrophages participated in inflammatory response induced by LPS;
3. KATP channels in macrophages regulated inflammation via NF-κB and ERK1/2pathways.
动脉粥样硬化(atherosclerosis, AS)是严重危害人类健康的疾病,其中易损斑块是急性冠脉综合征(acute coronary syndrome, ACS)以及脑血管疾病的基础病变,易损斑块发病机制和治疗靶点的探索成为当今医学发展的重大挑战。动脉粥样硬化斑块的主要细胞成分包括巨噬细胞、平滑肌细胞和内皮细胞。易损斑块的病理改变包括平滑肌细胞和胶原纤维含量减少,有大的脂质核,斑块内大量的巨噬细胞和淋巴细胞等炎性细胞浸润。
越来越多的研究表明离子通道与血管功能密切相关,在动脉粥样硬化的发生发展过程中发挥重要作用。已有研究报道,内皮细胞钙离子通道、钾离子通道和氯离子通道在剪切力应激和炎症反应中具有调节作用；TRPC通道介导的血管平滑肌细胞、内皮细胞、单核/巨噬细胞和血小板功能失调参与了动脉粥样硬化的病理过程。由此可见,离子通道在动脉粥样硬化中的作用不容忽视。而且,钾离子通道是膜电位的重要组成部分,在维持细胞电稳态中具有重要作用,可以调节细胞内钙离子的浓度进而发挥对细胞功能的调节作用。
三磷酸腺苷(Adenosine triphosphate, ATP)敏感的钾通道(KATP)是由四个孔道形成亚基-Kir6亚基和四个调节亚基-SUR亚基组成的八聚体蛋白。对细胞内腺苷酸变化敏感,是重要的代谢感受器,偶联细胞内代谢状态细胞膜电活动。近年来,KATP通道在糖尿病和心血管疾病中同时发挥的重要作用逐渐得到认可,作为几类不同药物的作用靶点,成为研究活跃的领域。现有的研究主要集中在以下几个方面：心脏功能的调节,包括缺血再灌注、心肌梗死、心肌重构、心衰、缺血预适应和应激的环境中心肌细胞KATP通道的调节作用；血管平滑肌细胞的KATP通道研究也很丰富,包括各种内源性和外源性血管舒缩物质的调节作用以及在高血压和感染的环境中的表达和功能的改变；同样的,血管内皮细胞的KATP通道也越来越受到重视,包括在内皮素和NO释放中的调节作用研究。但是,对于KATP通道在动脉粥样硬化中的作用却没有直接的报道。
因此,本研究提出如下假说：KATP通道在动脉粥样硬化病变的病理生理过程中发挥重要作用,KATP通道可能成为治疗动脉粥样硬化的新靶点。
目的
1.建立动脉粥样硬化病变发展的动物模型；
2.观察斑块平滑肌细胞、巨噬细胞、胶原纤维和脂质表达,评测斑块易损性；
3.探讨KATP通道药物体内干预对动脉粥样硬化病变发展的影响。
方法
本研究以ApoE-/-小鼠80只为研究对象,9周龄始予高脂饮食,10周龄时(25-30g)行右侧颈动脉套管术；8周后,高分辨率小动物显微超声检测颈动脉斑块病变,随后,小鼠随机分为空白对照组,转染重组腺病毒载体Ad5-CMV.lacZ对照组和转染重组腺病毒载体Ad5-CMV.p53实验组,后两组分为转染后1天组、4天组和14天组,转染后4天组和14天组又分为格列苯脲(Glibenclamide)干预组和非干预组,给予相应处理。检测下列指标：
1.血生化分析：血液标本收集,检测血糖(glucose, Glu)、谷丙转氨酶(glutamic pyruvic transaminase, GPT)谷草转氨酶(glutamic oxaloacetic transaminase, GOT)、甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)含量；
2. ApoE-/-小鼠取材,病理组织学检测：实验动物按照实验分组,分别于转染后0天、1天、4天和14天时处死,留取组织用于病理染色及分子生物学实验。苏木素-伊红(hematoxylin and eosin, HE)染色用以检测组织结构变化,油红O(Oil-red O)染色用以检测组织l脂质含量,天狼猩红(Sirius-red)染色用以检测组织胶原纤维含量,p53、SMC-α actin、MOMA-2的免疫组织化学染色分别用以检测组织p53的表达、平滑肌细胞和巨噬细胞的含量；
3.计算斑块易损指数：易损指数=(MOMA-2阳性面积/斑块面积+脂质面积/斑块面积)/(SMC-α actin阳性面积/斑块面积+胶原纤维面积/斑块面积)。结果
1.格列苯脲干预对ApoE-/-小鼠血生化及肝脏组织的影响
格列苯脲灌胃后小鼠血糖、甘油三酯、总胆固醇、转氨酶水平以及肝脏组织结构没有明显变化(P>0.05)。
2.ApoE-/-小鼠颈动脉套管手术及p53转染效率检测
小鼠显微超声示右侧颈动脉套管及套管近心端斑块形成；转染后1天、4天、14天,p53表达在Ad5-CMV.p53转染组比Ad5-CMV.lacZ组明显增多(P<0.05)。
3.ApoE-/-小鼠颈动脉粥样硬化斑块平滑肌细胞及胶原纤维染色
斑块平滑肌细胞含量与胶原纤维含量变化趋势基本一致,在p53转染4天和14天组明显少于相应时间点的lacZ转染组(P<0.05)。
4.ApoE-/-小鼠颈动脉粥样硬化斑块巨噬细胞及脂质染色
斑块巨噬细胞含量与脂质含量变化趋势一致,在转染后4天和14天时,p53组显著高于相应时间点的lacZ组(P<0.05)。
5.ApoE-/-小鼠格列苯脲体内干预稳定颈动脉粥样硬化斑块
格列苯脲体内干预14天,在p53转染组巨噬细胞含量、脂质含量明显降低(P<0.05),斑块胶原成分显著增加(P<0.05),尽管平滑肌细胞含量无明显变化(P>0.05),但是斑块病变改善显著。
6.斑块易损指数计算
在转染后4天和14天时,p53组易损指数比lacZ显著升高,而且时间点愈后p53转染组的易损指数升高愈显著(P<0.05)；在p53转染14天组,格列苯脲体内干预使易损指数明显下降(P<0.05)。
结论
1. ApoE-/-小鼠高脂饮食、颈动脉套管术后局部转染Ad5-CMV.p53诱导斑块易损指数增加；
2.格列苯脲体内干预对Ad5-CMV.p53转染ApoE-/-小鼠血.生化及肝组织结构无明显影响；
3. ApoE-/-小鼠格列笨脲体内干预改善p53转染诱导的颈动脉粥样硬化斑块病变。
研究背景
动脉粥样硬化(atherosclerosis,AS)与细胞膜离子通道关系密切。K+电流是细胞膜电位的主要组成部分,对于细胞功能至关重要。三磷酸腺苷(Adenosine triphosphate,ATP)敏感的钾通道(KATP)是细胞膜离子通道的重要组成部分,通过调节细胞内外钾离子浓度,在维持细胞膜电位、调节其他生物离子进出细胞和调市细胞功能中发挥不可替代的作用,而且是联系细胞电活动与细胞代谢的桥梁。缺血、缺氧、代谢性酸中毒等导致细胞内ADP/ATP升高时,KATP通道开放,细胞膜超极化,电压依赖性钙通道(voltage-dependent calcium channels,VDCCs)关闭,钙内流减少,细胞内[Ca2+]降低,介导缺血-再灌注损伤及心力衰竭中的心肌保护等作用。
KATP,通道是一种异源八聚体复合物,四个Kir6.X亚基构成孔道形成亚基转运钾离子,四个SURs亚基组成调节亚基,根据细胞内ATP水平调节通道活性。Kir6.X存在Kir6.1和Kir6.2两个亚型；磺脲类受体SURs(sulfonylurea receptors)有SUR1和SUR2两种亚型,后者有两种主要的剪接体SUR2A和SUR2B,连接Kir6亚基构成功能性的K/ATP通道。KATP通道在多种组织农达,承载了重要的细胞功能,并且KATP通道的亚基组成具有组织特异性,在同时表达Kir6.1.Kir6.2亚基的细胞系,亚基常常以不同比例组成异源多聚体；相应地,不同组织中的KATP通道有不同的生理特性,也是特异性药物试剂作用于特定组织KATP通道以调节不同细胞功能的重要生理基础。例如,心肌细胞主要表达SUR2A/Kir6.2亚型,在缺血缺氧及其他应激的环境中发挥保护心肌细胞的重要作用；胰腺p细胞主要表达SUR1/Kir6.2亚型,可以调节胰岛素的分泌；SUR2B/Kir6.1主要在血管平滑肌细胞和内皮细胞表达,在调节血管张力、血管的舒缩和内皮保护中发挥重要作用。KATP通道是重要的代谢感受器,而动脉粥样硬化及炎症反应与细胞代谢密切相关。
因此,本研究提出如下假说：KATP通道在动脉粥样硬化斑块的平滑肌细胞、内皮细胞和／或巨噬细胞中表达,与动脉粥样硬化斑块的易损性关系密切。
目的
1.检测KATP通道SUR1、SUR2A、SUR2B亚基和Kir6.1、Kir6.2亚基在动脉粥样硬化斑块的表达和分布；
2.分析KATP通道亚基表达量和AS斑块成分含量、易损指数的相关性；
3.探讨AS斑块平滑肌细胞、内皮细胞和巨噬细胞中KATP通道在病变发展过程中的作用。
方法
本研究以50只ApoE-/-小鼠为研究对象,9周龄始予高脂饮食,10周龄时(25-30g)行右侧颈动脉套管术；8周后,高分辨率小动物显微超声检测颈动脉斑块病变,随后,小鼠随机分为空白对照组,转染重组腺病毒载体Ad5-CMV.lacZ对照组和转染重组腺病毒载体Ad5-CMV.p53实验组,后两组分为转染后1天组、4天组和14天组,给予相应处理,分别于相应时间点处死小鼠,留取取标本,检测下列指标：
1.病理组织学染色：HE染色检测组织形态结构的改变、油红O染色检测标本脂质含量、天狼猩红染色检测标本胶原含量；
2. SMC-a actin和MOMA-2免疫组织化学染色：检测AS斑块平滑肌细胞和巨噬细胞含量；
3. KATP通道亚基免疫组织化学染色：检测AS斑块KATP通道亚基SUR1、 SUR2A、SUR2B、Kir6.1和Kir6.2含量,观察各亚基在斑块平滑肌细胞、内皮细胞和巨噬细胞的表达分布情况；
4.相关性分析：计算分析AS斑块成分含量、易损指数和KATP通道亚基SUR1、SUR2A、SUR2B、Kir6.1、Kir6.2表达的相关性；
5.AS相关的KATP通道亚基和相应细胞的免疫荧光双标染色：观察AS斑块细胞和主要KATP通道亚型的共同定位表达；
6. KATP通道亚基实时定量逆转录聚合酶连反应(RT-PCR)定量分析：RT-PCR检测ApoE-/-小鼠颈动脉KATP通道亚基SUR1、SUR2A、SUR2B、Kir6.1和Kir6.2的(?)nRNA表达。
结果
1.AS斑块KATP通道亚基免疫组织化学染色结果
SUR2A和Kir6.2亚基阳性染色主要积聚在粥样硬化斑块肩部和脂质条纹等巨噬细胞聚集的部位,定量分析表明二者变化趋势基本一致,在p53转染4天组表达最高,显著高于lacZ转染4天组(P<0.05); SUR1亚基阳性染色在巨噬细胞密集的斑块肩部和脂质条纹区域,以及血管内膜内皮细胞表现突出,定量分析显示,SUR1亚基表达在p53转染4天组增长最为显著,明显多于lacZ转染4天组(P<0.05); Kir6.1亚基阳性染色在斑块肩部及脂质条纹巨噬细胞聚集的区域、血管内皮细胞、血管中膜及纤维帽平滑肌细胞较丰富的区域均有表现,在转染后1天和4天,p53处理组Kir6.1亚基表达均高于lacZ处理组(P<0.05)；SUR2B亚基阳性染色主要分布在血管中膜及斑块纤维帽平滑肌细胞较集中的部位和血管内皮细胞,SUR2B亚基表达在p53转染4天组明显减少,显著少于lacZ转染4天组(P<0.05)。
2. KATP通道亚基mRNA表达结果
小鼠颈动脉RT-PCR结果显示,KATP通道SUR2A、Kir6.2和SUR1亚基mRNA表达在p53转染4天组增加最为显著,明显高于lacZ转染4天组(P<0.05);Kir6.1亚基在p53转染1天组表达高于lacZ转染1天组(P<0.05); SUR2B亚基在空白对照组表达水平最高(P<0.05)。
3.AS斑块KATP通道染色与斑块负荷相关性分析结果
KATP通道SUR2A亚基和Kir6.2亚基染色,与斑块易损指数正相关,与斑块巨噬细胞含量成正相关,与斑块平滑肌细胞含量负相关(P<0.05)。
4.AS斑块MOMA-2与SUR2A、Kir6.2共表达
免疫荧光双标染色显示,SUR2A亚基和MOMA-2共同定位表达于斑块内及血管壁粘附浸润的泡沫巨噬细胞,这些部位同样粘附聚集Kir6.2亚基和MOMA-2双染巨噬细胞。
结论
1. KATP通道亚基在AS斑块平滑肌细胞、内皮细胞以及巨噬细胞表达；
2. KATP通道SUR2A、Kir6.2亚基与AS斑块负荷密切相关；
3. KATP通道SUR2A/Kir6.2主要在斑块巨噬细胞表达。
研究背景
动脉粥样硬化斑块的主要细胞成分包括巨噬细胞、平滑肌细胞和内皮细胞。与血管平滑肌细胞和内皮细胞显著不同,巨噬细胞来源于循环中的单核细胞,是免疫系统炎症反应的重要组成部分,单核/巨噬细胞粘附、聚集、迁移和分泌炎症因子的功能贯穿动脉粥样硬化斑块的起始、发展和破裂的全过程。
以往的研究发现,单核/巨噬细胞离子通道在动脉粥样硬化病变中发挥重要作用。单核/巨噬细胞TRPC通道通过调节细胞内钙离子浓度,介导细胞的氧化应激,加剧动脉粥样硬化病变：周围的细胞因子和细胞本身的功能状态能够调节单核细胞电压依赖性内向整流钾通道Kir (voltage-dependent inwardly rectifying potassium channels)和延迟外向整流钾通道Kdr (delayed outwardly rectifying potassium channels)诱导单核细胞的活化和分化；单核/巨噬细胞容量调节性氯离子通道(volume-regulated Cl channels)功能失调,促进巨噬细胞吞噬脂质,形成泡沫细胞。更重要的是,MCP-1和LPC通过调节氯离子通道和钙离子激活的钾通道可以促进单核细胞的迁移。单核细胞的迁移是动脉粥样硬化的始动环节,而大量泡沫细胞的形成是不稳定粥样斑块的重要标志。然而,ATP敏感钾通道(ATP-sensitive K+channels, KATP)在动脉粥样硬化中的作用至今鲜有报道。
KATP,通道是异源八聚体蛋白复合物,由四个内向整流K+通道(inwardly rectifying potassium channel, Kir) Kir6.X亚基形成孔道,四四个ATP结合盒(ATP binding cassette, ABC)家族的磺脲类受体(sulfonylurea receptor, SUR)蛋白组成调节亚基。不同组织表达的KATP通道Kir6.X和SUR亚基不尽相同,Kir6.X包括Kir6.1和Kir6.2亚型,SUR包括SUR1、SUR2A和SUR2B亚型,因而呈现出组织特异性的生理学和病理生理学特征。有研究报道,KATP通道阻断剂格列苯脲能够抑制低氧血症和酸中毒时脂多糖(Lipopolysaccharide, LPS)诱导的促炎性细胞因子的释放。而单核/巨噬细胞的炎症反应在动脉粥样硬化病变的形成、发展以及晚期易损斑块破裂中扮演重要角色。LPS是单核/巨噬细胞的有效激活剂,与Toll样受体(TLRs)结合,主要通过NF-κB和MAPKs信号通路的激活,后者包括p38、JNK和ERK1/2信号转导途径,诱导TNF-α等促炎性细胞因子分泌。
因此,我们提出下列假说：单核/巨噬细胞KATP通道在LPS诱导的细胞炎症反应中发挥作用,从而影响动脉粥样硬化病变的发生、发展；单核/巨噬细胞KATP通道通过NF-κB和MAPKs (ERK1/2、p38和JNK)信号转导通路调节细胞的炎症反应。
目的
1.观察LPS对巨噬细胞KATP通道和TNF-α表达的影响；
2.探讨KATP通道对LPS诱导的巨噬细胞TNF-α表达的调节作用；
3.揭示KATP通道对LPS诱导的巨噬细胞内NF-κB和MAPKs信号通路的影响。
方法
本研究以单核/巨噬细胞系RAW264.7细胞为研究对象,KATP通道开放剂吡那地尔或阻断剂格列苯脲预处理后予LPS刺激,采用实时定量聚合酶连反应(RT-PCR)和蛋白免疫印迹(western blot)等实验方法,检测：1.LPS (1μg/ml)处理RAW264.7细胞不同时间(0、2、4、8、24h)后,检测KATP通道各亚基(SUR1、SUR2A、SUR2B、Kir6.1、Kir6.2)和炎症因子TNF-α表达；
2. KATP通道开放剂(吡那地尔,10μM)或阻断剂(格列苯脲,10μM)预处理后,检测LPS刺激RAW264.7细胞TNF-α的表达,检测不同刺激时间(15min、30min、1h、24h) NF-κB和MAPKs信号通路蛋白的磷酸化改变；
3. NF-κB信号通路抑制剂预处理后,检测KATP通道各亚基的表达改变,检测KATP通道干预对RAW264.7细胞TNF-α表达的影响。
结果
1.LPS诱导RAW264.7细胞KATP通道亚基和TNF-α表达
与空白对照组相比较,KATP通道SUR1、SUR2A、Kir6.1和Kir6.2亚基在LPS (1μg/ml)刺激细胞4小时后表达明显升高(P<0.05),同时,TNF-α表达显著增加(P<0.05),但是各组均未检测到SUR2B亚基mRNA的表达。
2. KATP通道药物干预后RAW264.7细胞TNF-α表达和NF-κB、MAPKs蛋白磷酸化改变
与LPS刺激4小时组相比较,KATP通道阻断剂格列苯脲(10μM)预处理后细胞TNF-α表达降低(P<0.05),开放剂吡那地尔(10μM)预处理后细胞TNF-α表达略增加(P<0.05)。
与LPS刺激15分钟组相比较,格列苯脲(10μ,M)预处理后细胞p65、ERK1/2蛋白磷酸化水平下降(P<0.05),而吡那地尔(10μ,M)预处理对细胞p65、ERK1/2蛋白磷酸化水平影响没有统计学意义(P>0.05)；格列苯脲和吡那地尔对p38蛋白磷酸化水平没有明显改变(P>0.05)；与空白对照组相比较,LPS刺激15分钟、1小时或24小时JNK蛋白磷酸化无显著变化(P>0.05)。
3. KATP通道位于RAW264.7细胞LPS诱导活化的NF-κB信号通路上游
与LPS刺激4小时组相比较,NF-κB信号通路抑制剂PDTC(100μM)和DMFR(100μM)预处理显著降低RAW264.7细胞TNF-α的mRNA表达(P<0.05)；KATP通道阻断剂格列苯脲(10μM)能够增强NF-κB抑制剂的作用(P<0.05),而开放剂吡那地尔(10μM)拮抗NF-κB抑制剂的作用(P<0.05)。
与LPS刺激4小时组相比较,PDTC (100μM)和DMFR (100μM)预处理显著增加了RAW264.7细胞KATP通道SUR1、SUR2A、Kir6.1和Kir6.2亚基的表达(P<0.05)。
结论
1.LPS绣导巨噬细胞KATP通道表达：
2.巨噬细胞KATP通道参与调节LPS诱导的炎症反应；
3.巨噬细胞KATP通道通过NF-κB和ERK1/2信号转导通路调节细胞炎症反应。
Background
Atherosclerosis is one of the most serious diseases detrimental to health and vulnerable plaques have been recognized as chief criminal of acute coronary syndrome (ACS) and cerebrovascular disorders. However, the pathogenesis and approach targets of atherosclerotic plaques are still unclear. The main cellular components in plaques include macrophages, smooth muscle cells and endothelial cells, while the decrease of smooth muscle cells and collagen fibers, and the increase of lipids and macrophages happened in vulnerable plaques.
Numerous studies have indicated that ion channels are related to vascular functions tightly and play an important role in the progression of atherosclerosis. It has been reported that the calcium channels, potassium channels and chloride channels in endothelial cells regulated the shear stress and inflammation; the disorders of vascular smooth muscle cells, endothelial cells, monocytes/macrophages and platelets induced by transient receptor potential canonical channels (TRPC) participated in the pathogenesis of atherosclerosis. So. the role of ion channels in atherosclerosis should never be neglected. Potassium channels contribute to the membrane potential, preserve the cellular electrical homeostasis and could affect the activities of other ions to regulate the cellular state.
ATP-sensitive potassium channels (KATP channels) are heterooctameric complexes composed of Kir6.1or Kir6.2as pore forming subunits, and SUR1, SUR2A or SUR2B as regulatory subunits. KATP channels are sensitive to the change of intracellular ATP as important metabolic sensors, coupling metabolic state with electrical activity of cells. Recently, the role of KATP channels in both of diabetes and cardiovascular diseases has attracted more and more attention. The studies mainly concentrate on the following aspects:the regulation of cardiac functions, under ischemia-reperfusion, myocardial infarction, myocardial remolding, heart failure, ischemic preconditioning and stress; vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors; regulation the effect of endothelium-derived relaxing factor (EDRF), such as nitric oxide (NO), hydrogen sulfide (H2S) and endothelin-1(ET-1). However, the role of KATP channels in atherosclerosis hasn't been investigated directly.
Therefore, we put forward the hypothesis that KATP channels play an important role in the pathogenesis of atherosclerosis and represent the potential therapeutic target of atherosclerosis.
Objective
1. To establish animal models of progressive atherosclerosis;
2. To evaluate the content of smooth muscle cells, macrophages, collagen fibers and lipids in plaques, and the vulnerable index of plaques;
3. To investigate the interventional effects of modulators targeting to KATP channels on the progression of atherosclerosis.
Methods
We applied eighty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, with or without intervention of glibenclamide in vivo, followed by a series of experiments.
1. Blood biochemical analysis:collect blood samples to detect the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma.
2. Tissue harvesting and morphological analysis:the mice were sacrificed0day,1day,4days or14days after transduction according to the assignment, respectively, and the tissue samples were carefully excised for pathological staining and molecular biological experiments. Hematoxylin and eosin (HE)staining was used to show the tissue structure, Oil-red O staining was used to detect the content of lipids, sirius-red staining was used to detect the content of collagen fibers, immunohistochemical staining of p53, SMC-a actin and MOMA-2was used to detect the content of p53protein, smooth muscle cells and macrophages, respectively.
3. Calculation of vulnerable index:vulnerable index=(MOMA-2positive staining area/plaque area+lipids staining area/plaque area)/(SMC-a actin positive staining area/plaque area+collagen fibers staining area/plaque area).
Results
1. The effects of glibenclamide intervention on blood biochemistry and livers in ApoE-/-mice
There were no substantial changes in the concentration of glucose (Glu), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), triglyceride (TG) and total cholesterol (TC) in plasma, and hepatic structures of ApoE-/-mice after intragastric administration of glibenclamide in our experiment (all P>0.05).
2. Identification of carotid collar placement and transduction of p53
The right carotid arteries proximal to collars showed atherosclerotic plaques by ultrasound. Compared with that of control groups, the staining of p53was significantly increased in Ad5-CMV.p53-treated groups, including1,4,14-day subgroups, and the difference increased over time (all P<0.05).
3. The staining of smooth muscle cells and collagen fibers in atherosclerotic plaques
Compared to Ad5-CMV.lacZ-treated groups, the content of smooth muscle cells and collagen fibers in atherosclerotic plaques was decreased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).
4. The staining of macrophages and lipids in atherosclerotic plaques
Compared to Ad5-CMV.lacZ-treated groups, the content of macrophages and lipids in atherosclerotic plaques was increased by Ad5-CMV.p53transduction after4days and14days (both P<0.05).
5. Glibenclamide intervention stabilizing atherosclerotic plaques in vivo
In Ad5-CMV.lacZ-treated-14days groups, the content of macrophages and lipids decreased obviously (both P<0.05), and the content of collagen fibers increased (P<0.05), while the content of smooth muscle cells was not different significantly after intervention of glibenclamide for14days in vivo.
6. Calculation of vulnerable index
The vulnerability index (VI), which was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions to estimate the vulnerable possibility of plaques, was higher in p53-treated groups than that in control groups after4days and14days and the gap was widened over time (both P<0.05).
Conclusions
1. Transduction of Ad5-CMV.p53induced increased vulnerable index of plaques in ApoE-/-mice with high fat diet and collar placement;
2. Glibenclamide intervention didn't affect the blood biochemistry and structure of livers obviously in ApoE-/-mice transfected with Ad5-CMV.p53;
3. Glibenclamide ameliorated the progression of atherosclerosis induced by Ad5-CMV.p53transduction in vivo.
Background
Atherosclerosis (AS) is related to membrane ion channels closely. K+current is the primary component of membrane potential and vitally important to cellular functions. ATP sensitive potassium channels (KAtp) could regulate the concentration of K+and preserve membrane potential to modulate activities of other ions and the whole cell, and couple cellular metabolism with membrane potential. When the intracellular ADP/ATP is raised due to ischemia, hypoxia, metabolic acidosis and so on, KATP channels will open, the cell hyperpolarize, voltage-dependent calcium channels (VDCCs) close, calcium influx decrease and intracellular Ca2+reduce to mediate cardiac protection.
KATP channel is a hetero-octamer complex of four pore forming subunits, inwardly rectifying potassium channels Kir6.X, and four regulatory subunits according to intracellular ATP concentration, sulfonylurea receptors (SURs), member of ATP binding cassette (ABC) superfamily. Kir6.X, including Kir6.1and Kir6.2, links with SURs, including SUR1, SUR2A and SUR2B, to constitute functional KATP channels. KATP channels exist in various tissues and carry an important cellular function with tissue specificity. In cells expressing Kir6.1and Kir6.2at the same time, the subunits often comprise heterogeneous polymers in a different proportion. Accordingly, KATP channels in different tissues exhibit different physiologic characteristics and response with specific reagents. For example, SUR2A/Kir6.2 subtype mainly expresses in myocardial cells and shows cardiac protection under conditions of ischemia, hypoxia and other stress; SUR1/Kir6.2subtype mainly expresses in pancreatic P cells to regulate secretion of insulin; SUR2B/Kir6.1mainly expresses in vascular smooth muscle cells and endothelial dcells and participate in vascular response to various pharmacological or endogenous vasodilators and vasoconstrictors. KATP channels are important metabolic sensors, while AS and inflammation are associated with metabolism closely.
Therefore, we put forward the hypothesis that KATP channels express in vascular smooth muscle cells, endothelial cells and macrophages of atherosclerotic plaques, and should be correlated with vulnerability of plaques.
Objective
1. To detect the expression and distribution of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in atherosclerotic plaques;
2. To make correlation analysis between content of KATP subunits and vulnerability of plaques;
3. To evaluate the effect of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages on progression of atherosclerosis.
Methods
We applied fifty ApoE-/-mice to study, with high-fat diet at9-week old and with right common carotid artery-collar placement at10-week old (about25-30g);8weeks later, right carotid plaques were detected by ultrasound biomicroscopic imaging system; and then we randomly divided the mice into Ad5-CMV.p53-treated group, Ad5-CMV.lacZ-treated group and blank control group and further divided the interventional group into the following three subgroups:1-day,4-day and14-day groups, respectively, followed by a series of experiments.
1. Morphological staining:Hematoxylin and eosin (HE) staining was used to show the tissue structure, sirius-red staining was used to detect the content of collagen fibers and Oil-red O staining was used to detect the content of lipids;
2. SMC-a actin and MOMA-2staining:Immunohistochemical staining of SMC-a actin and MOMA-2was used to detect the content of smooth muscle cells and macrophages, respectively;
3. KATP Subunits staining:Immunohistochemical staining of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in AS plaques to investigate the expression and distribution of KATP channels in vascular smooth muscle cells, endothelial cells and macrophages;
4. Correlation analysis:The content of smooth muscle cells, collagens, macrophages and lipids in plaques was evaluated, the vulnerable index was calculated as the ratio of macrophages and lipids content to SMCs and collagens content in atherosclerotic lesions, and then, we anlysed the correlation between AS components, vulnerable index and KATP Subunits;
5. Immunofluorescence:Immunofluorescence double staining was used to detect the co-localization of atherosclerotic cells and KATP Subunits;
6. RT-PCR quantitative analysis of KATP Subunits:The mRNA expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subnuits in carotid arteries was detected by RT-PCR.
Results
1. The staining of KATP Subunits in atherosclerotic plaques
Positive staining of SUR2A and Kir6.2subunits mainly accumulated in plaque shoulder and fatty streak with more macrophages, and quantitative analysis showed similar expressional tendency:SUR2A and Kir6.2expression was detected mostly in p53-treated4-day group and was higher than that of lacZ treated-control groups after1day and4days treatment (P<0.05). Positive staining of SUR1subunit mainly accumulated in plaque shoulder and fatty streak, as well as endothelial cells, and quantitative analysis showed that the staining was most markedly in p53-treated4-day group and was higher than that of control group (P<0.05). Positive staining of Kir6.1subunit distributed in plaque shoulder and fatty streak, as well as endothelial cells and smooth muscle cells, and quantitative analysis showed that the staining was higher in p53treated groups after1day and4days compared with that of lacZ treated groups (P<0.05). Positive staining of SUR2B subunit mainly accumulated in vascular smooth muscle cells and endothelial cells, and quantitative analysis showed that the staining was decreased in p53-treated4-day group compared with that of lacZ treated group (P<0.05).
2. The mRNA expression of KATP subunits in carotid arteries
At mRNA level, the expression of SUR2A, Kir6.2and SUR1subunits was higher in p53-treated group than that of control group after4-day treatment (P<0.05); the expression of Kir6.1subunit was higher in p53-treated group than that of control group after1-day treatment (P<0.05); and the largest value of SUR2B transtripts was found in blank control group (P<0.05).
3. The correlation analysis between KATP subunits and plaque burden
The expression of SUR2A and Kir6.2subunits in plaques was positively correlated with vulnerable index and the content of macrophages in plaques, and negatively correlated with the content of smooth muscle cells in plaques (all P<0.05).
4. Co-localization of MOMA-2with SUR2A and Kir6.2subunits in plaques
The result of immunofluorescence double staining showed that MOMA-2and SUR2A, as well as MOMA-2and Kir6.2, co-located at macrophages attached to vascular walls and within plaques.
Conclusions
1. KATP subunits expressed in smooth muscle cells, endothelial cells and macrophages of AS plaques;
2. SUR2A and Kir6.2subunits were associated with plaque burden closely;
3. The subtype of SUR2A/Kir6.2mainly located in macrophages of atherosclerotic plaques.
Background
Atherosclerotic plaques consist of macrophages, smooth muscle cells and endothelial cells. Differentiating from vascular smooth muscle cells and endothelial cells, macrophages derive from circular monocytes and contribute to immune inflammatory response. The activation of monocytes/macrophages is considered as the initiation of atherosclerosis and works through the whole process of atherosclerosis with accumulating, adhesion, migration and producing inflammatory cytokines.
The previous studies have showed that ion channels expressed in monocytes/macrophages played an important role in atherosclerosis. Dysfunction of monocytes/macrophages mediated by TRPC (transient receptor potential canonical) channels was associated with the development of atherosclerosis; the changes of voltage-dependent inwardly rectifying potassium channels (Kir) and delayed outwardly rectifying potassium channels induced by inflammatory cytokines promoted the activation and differentiation of monocytes; disordered volume-regulated Cl channels promoted foam cell formation from macrophages in atherosclerosis; moreover, MCP-1and LPC regulated the chloride channels and calcium activated potassium channels to accelerate the migration of monocytes. The migration of monocytes initiates the process of atherosclerosis and the formation of considerable foam cells designates the vulnerable plaques. Whereas, KATP channels in monocytes/macrophages have drawn relatively a little attention.
KATP channels are octamers of four pore forming subunits-inwardly rectifying potassium channel Kir6.X and four regulatory subunits-a member of ATP binding cassette (ABC) superfamily called sulfonylurea receptor (SUR). The former contains Kir6.1and Kir6.2subtypes, and the latter contains SUR1, SUR2A and SUR2B subtypes. They comprise various subtypes of KATP channels in different tissues with distinct physiologic and pathological properties. It has been reported that the blocker of KATP channels, glibenclamide, could inhibit the release of proinflammatory cytokines induced by Lipopolysaccharide (LPS) in condition of hypoxia and acidosis. The inflammation mediated by monocytes/macrophages play an important role in initiation and progression of atherosclerotic lesions. LPS, a typical bacterial pathogens known as most powerful activators of macrophages, could stimulate monocytes/macrophages to produce proinflammatory cytokines such as TNF-a by bounding with toll like receptors (TLRs) through two major signal transduction pathways:nuclear factor κB (NF-κB) proteins and mitogen-activated protein kinases (MAPKs), the latter including extracellular signal related kinase (ERK1/2), p38MAPK (p38) and c-Jun N-terminal kinase (JNK) pathways.
Therefore, we put forward the hypothesis that KATP channels in monocytes/macrophages play an important role in inflammation to mediate the progression of atherosclerotic lesions and they work through NF-κB/MAPKs signal transduction pathways to regulate the inflammatory response.
Objective
1. To detect the expression of KATP subunits and TNF-a induced by LPS in macrophages;
2. To explore the effect of KATP channels on the expression of TNF-a induced by LPS in macrophages;
3. To investigate the the effect of KATP channels on NF-κB and MAPKs pathways induced by LPS in macrophages.
Methods
We used the cell line of mice monocytes/macrophages, RAW264.7cells, as the object of this study, pretreated with the opener or blocker of KATP channels, pinacidil or glibenclamide, followed by stimulation of LPS. And then we applied real time RT-PCR and western blotting to detect the role and mechanisms of KATP channels in macrophages.
1. RAW264.7cells were treated with LPS (1μg/ml) for0,2,4,8or24h to detect the expression of SUR1, SUR2A, SUR2B, Kir6.1and Kir6.2subunits, as well as TNF-a.
2. RAW264.7cells were pretreated with the opener or blocker of KATP channels, pinacidil (10μM) or glibenclamide (10μM), to detect the expression of TNF-a induced by LPS and the phosphorylation of NF-κB and MAPKs signal transduction proteins at various time (15min,30min,1h, and24h).
3. After intervention with inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), the expression of KATP subunits and TNF-a inducted by LPS in macrophages was investigated.
Results
1. LPS induced the expression of KATP subunits and TNF-a in RAW264.7cells
Compared with control group, the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits, as well as TNF-a, was increased in LPS (1μg/ml) treated RAW264.7cells for4h (all P<0.05). However, the expression of SUR2B subunit was not detected.
2. The regulation of KATP channels in expression of TNF-a and phosphorylation of NF-κB and MAPKs signal proteins
The blocker of KATP channels, glibenclamide (10κM). could reduce the expression of TNF-a induced by LPS in RAW264.7cells for4h. while the opener pinacidil (10λM) raised the expression slightly (both P<0.05). Moreover, glibenclamide decreased the phosphorylation of p65and ERK1/2induced by LPS for15min (both P<0.05), while the effect of pinacidil was not obvious (P>0.05).
3. KATP channels located at upstream of NF-κB activated by LPS in RAW264.7 cells
The inhibitors of NF-κB, PDTC (100μM) or DMFR (100μM), decreased the expression the TNF-a induced by LPS in RAW264.7cells obviously (P<0.05), and glibenclamide could enhance the effect of NF-κB inhibitors, while pinacidil antagonized their effect (P<0.05).
PDTC (100μM) and DMFR (100μM) increased the expression of SUR1, SUR2A, Kir6.1and Kir6.2subunits induced by LPS in RAW264.7cells significantly (P<0.05).
Conclusions
1. LPS increased the expression of KATP channels in macrophages;
2. KATP channels in macrophages participated in inflammatory response induced by LPS;
3. KATP channels in macrophages regulated inflammation via NF-κB and ERK1/2pathways.
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