【作者】 潘茜；

【导师】 杨向红；

【作者基本信息】 中国医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 前言肾内肾素-血管紧张素系统（renin-angiotensin system,RAS）的局部激活是多种慢性肾脏病变发生发展过程中的主要特点,血管紧张素Ⅱ（AngiotensinⅡ,AngⅡ）作为该系统的效应因子在机体血容量、血流动力学及内环境稳态的调节中发挥主导作用。已有研究表明肾内局部升高的AngⅡ水平与高血压、糖尿病时肾脏病变的发生发展关系密切。研究发现肾内局部升高的AngⅡ不仅可以导致球内高压,同时还具有生长因子和炎症因子的作用,致使肾小球发生硬化病变。肾小球内皮细胞（GlomerularEndothelial Cells,GECs）又称肾脏微血管内皮细胞,是肾小球滤过屏障的重要组成部分,承受着远高于一般毛细血管的血压,是血液内的致病因子以及血液动力学变化易损伤的靶细胞,受损激活后可分泌单核细胞趋化因子（monocytechemoattractant protein-1,MCP-1）等多种炎症因子。目前有关AngⅡ是否会诱导肾小球内皮细胞发生炎症反应及其对增殖和凋亡作用方面的研究,尚未见报道。血管活性肽AngⅡ可激活多条细胞内信号通路,刺激细胞内活性氧基团（reactive oxygen species,ROS）的生成。细胞内ROS的主要来源是通过还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶系统的活化,使活性氧基团ROS生成增加。p47phox蛋白是NADPH氧化酶的亚基,对NADPH氧化酶的活化起到重要作用。ROS作为极其重要的细胞内信使,与多条信号通路的活化相关。核因子-κB（Nuclear factor-κB,NF-κB）的活化,可上调多种相关炎性因子的表达。p38MAPK信号通路的激活,不仅在炎症、应激反应中具有重要作用,还参与细胞的存活、分化和凋亡等过程。AngⅡ的作用主要通过AT1和AT2两种类型受体所介导,在成人肾脏组织中,AT1受体分布广泛,而AT2受体分布很少。AngⅡ在心血管系统中的作用主要通过AT1受体介导。目前有关AngⅡ对血管内皮细胞结构和功能影响的研究愈来愈受到重视,本实验主要从炎症、增殖和凋亡方面探讨AngⅡ对大鼠肾小球内皮细胞的作用及其相关机制,以及AT1受体拮抗剂替米沙坦对其作用的影响。实验方法1、大鼠肾小球内皮细胞的分离培养与鉴定SD胎大鼠（20-30g）无菌条件下取双肾,剥去被膜,眼科剪剪下肾皮质部分于培养皿中,剪碎后经尼龙筛网滤过,采用胰蛋白酶消化法进行原代细胞培养。第3-5代细胞用于实验。细胞的鉴定采用形态学观察法和免疫荧光法检测第Ⅷ因子相关抗原。2、Western Blot法检测RGECs的MCP-1、p47phox蛋白表达收集各组细胞,经RIPA裂解液提取细胞总蛋白,以Lowry法检测总蛋白浓度。电泳后,将蛋白转印到PVDF膜上,5%脱脂奶粉室温封闭2 h,分别加入MCP-1、p47phox一抗（1:200）4℃孵育过夜,二抗（1:2000）37℃孵育2 h,DAB显色,观察结果。扫描PVDF膜并对其进行灰度值测定,对比各组间蛋白表达的差异,以β-actin作为对照。3、RT-PCR法检测AT1受体mRNA水平应用Trizol提取各组细胞总RNA,紫外分光光度计测定RNA的A260/A280比值,琼脂糖凝胶电泳证实所提RNA的完整性。RT和PCR反应按照说明书进行操作,PCR产物5μL用2%琼脂糖凝胶电泳进行检测,采用凝胶成像分析系统进行半定量分析,紫外灯下观察结果并拍照。4、细胞内活性氧ROS的测定采用2’,7’二氯荧光黄双乙酸盐（DCFH-DA）荧光染色检测ROS。DCFH-DA本身没有荧光,但可以自由穿过细胞膜,进入细胞内后,细胞内的ROS可以氧化无荧光的DCFH生成有荧光的DCF。将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10^-5mol/L)作用不同时间（0 min、5 min、10 min、15 min、20min）后,用无血清的培养基清洗细胞两次,加入DCFH-DA（1:1000稀释）到24孔板中,37℃孵育箱中孵育20 min,荧光共聚焦显微镜观察细胞内ROS的荧光强度（激发波长488 nm,发射波长525 nm）。5、免疫荧光法检测NF-κB活化将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10^-5mol/L)作用不同时间（0 min、15 min、30 min、45 min）后,细胞用4%多聚甲醛固定10min,PBS洗后,滴加0.1%Triton—100“打孔”,室温10min;PBS洗;5%BSA封闭10 min,弃去不洗,滴一抗（1:300）,4℃孵育过夜;PBS洗,滴加FITC标记二抗（1:100）,室温1h,PBS洗,90%甘油封片。荧光显微镜下观察。6、MTT法检测大鼠GECs的增殖率GECs以2×10³个/孔的密度接种至96孔板,80%融合后无血清培养液静置12h,分组继续培养。24h后每孔加入MTT溶液（5g/L）20μl,继续孵育4 h,终止培养。吸弃孔中的培养上清,每孔加入150μl DMSO,振荡10 min,酶联免疫检测分析仪测定各组吸光度（OD值）,并计算细胞增值率。7、AV/PI法流式细胞仪检测大鼠GECs的凋亡率GECs以5×10⁵个/瓶传代至25mL的培养瓶中,待融合后无血清培养液静置12h,分组继续培养24 h后,消化收集细胞,将其吹打成单细胞悬液,离心弃上清,用PBS漂洗,再离心,再漂洗后以100目筛网过滤,然后再离心加入细胞凋亡检测AV-PI（磷脂酰丝氨酸结合蛋白联合碘化丙啶双标记）工作液0.5ml,终浓度为10mg/L,室温避光30min后,进行流式细胞仪检测,计数凋亡细胞率。8、统计学分析采用SPSS17.0统计软件处理,计量资料以均数±标准差（x±s）表示,采用one-way ANOVA分析,组间比较采用LSD检验,P＜0.05为差异有统计学意实验结果1、大鼠肾小球内皮细胞的培养与鉴定原代分离培养肾小球内皮细胞生长5-7天时,呈单层贴壁生长,互不重叠,细胞呈角形或短梭形,外观呈铺路石状,边界清楚。Ⅷ因子相关抗原免疫荧光鉴定,大鼠GECs胞膜和胞浆处呈红色荧光,证实为肾小球内皮细胞。2、AngⅡ对大鼠肾小球内皮细胞MCP-1蛋白表达的影响Western blot检测结果显示,浓度效应组中,正常对照组MCP-1蛋白表达量较低,施加AngⅡ刺激因素后MCP-1表达量明显高于正常对照组,且随着浓度的增加(10^-7-10^-5M)而表达增加,成剂量依赖效应;NADPH氧化酶抑制剂DPI(10^-6mol/L)和AT1受体拮抗剂TEL(10^-6mol/L)抑制AngⅡ的作用。3、AngⅡ对大鼠肾小球内皮细胞p47phox蛋白表达的影响Western blot检测结果显示,与正常对照组相比给予10^-5M AngⅡ刺激因素后,大鼠肾小球内皮细胞内p47phox蛋白表达明显增加,成时间依赖效应。AngⅡ的这一作用效果可分别被TEL(10^-6M)和DPI(10^-6M)所抑制。4、AngⅡ对大鼠肾小球内皮细胞AT₁受体mRNA水平的影响RT-PCR检测结果显示,与正常对照组相比给予10^-5mol/L的AngⅡ刺激因素后,大鼠肾小球内皮细胞AT1受体的mRNA水平明显增加,成时间依赖效应。5、AngⅡ对大鼠肾小球内皮细胞ROS生成的影响DCFH-DA荧光染色检测结果显示,与正常对照组相比,随着作用时间延长AngⅡ作用组细胞内ROS的生成量逐渐增加,15min时ROS生成量达到顶峰,后逐渐减少;加入TEL和DPI后DCFH荧光值降低,ROS生成量明显减少。6、AngⅡ对大鼠肾小球内皮细胞NF-κB活化的影响正常状态无AngⅡ刺激时,NF-κB主要表达在细胞浆中,细胞核内少见表达;给予AngⅡ刺激因素后NF-κB即开始活化,迅速核转移,荧光显微镜显示30 min时NF-κB核内表达达到高峰,后逐渐减弱。药物TEL和DPI抑制NF-κB的核转移。7、AngⅡ对大鼠GECs增殖的影响与对照组相比较,低浓度AngⅡ(10^-8M)作用组,大鼠GECs增殖率增加1.33倍左右,高浓度AngⅡ(10^-6M)作用组,细胞增殖率降低2.3倍左右,分别为1.33±0.03和0.43±0.03（与对照组比较,P＜0.01）。低浓度AngⅡ组和高浓度AngⅡ组之间增殖率有统计学差异。8、AngⅡ对大鼠GECs凋亡的影响与对照组比较,低浓度AngⅡ(10^-8M)组和高浓度AngⅡ(10^-6M)组大鼠GECs凋亡率分别降低和增加50%左右,与高浓度AngⅡ(10^-6M)组比较,AngⅡ+TEL和AngⅡ+SB组细胞凋亡率均下降,但两组间细胞凋亡率无统计学差异。结论1、AngⅡ促进RGECs的MCP-1表达,在一定范围内呈剂量依赖效应;2、AngⅡ上调RGECs的AT1受体mRNA水平,在一定范围内呈时间依赖效应;3、AngⅡ上调RGECs的p47phox蛋白表达,通过NADPH氧化酶依赖的ROS生成途径促进NF-κB的活化;4、AT1受体拮抗剂替米沙坦可抑制AngⅡ的促炎性因子MCP-1表达作用;5、低浓度AngⅡ促进RGECs的增殖,高浓度AngⅡ抑制RGECs增殖;6、低浓度AngⅡ抑制RGECs的凋亡,高浓度AngⅡ促进RGECs凋亡;7、p38MAPK信号通路参与AngⅡ对肾小球内皮细胞凋亡的调控;8、AT₁受体拮抗剂替米沙坦抑制AngⅡ所诱导的RGECs凋亡发生。更多还原

【Abstract】 IntroductionActivation of the intrarenal renin-angiotensin system(RAS) is a characteristic feature in the development and progression of chronic kidney disease.AngiotensinⅡ(AngⅡ),the main effector of RAS,is implicated in the pathogenesis of renal diseases. AngⅡinitiates its effects by interaction with at lease two pharmacologically distinct subtypes of cell-surface receptors,AT1 and AT2,The major functions of AngⅡin cardiovascular system are mediated through AT1 receptor.An intriguing concept has emerged that AngⅡnot only mediates intraglomerular hypertension but also behaves as a pro-inflammatory and growth-stimulating factor contributing to the renal hypertension and sclerosis.AngⅡhas growth-stimulating properties on different renal cell types.However,possible inflammatory effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvascularture have not formally been investigated.Glomerular endothelial cells(GECs),being exposed to the bloodstream,are the target of many different factors,that may alter the functional state of these cells and induce them to release inflammatory cytokines.Inflammatory processes involve both the synthesis of inflammatory cytokines, such as monocyte chemoattractant protein-1(MCP-1),and the activation of their distinct signaling cascades.Recent findings suggest that AngⅡactivates intracellular signaling processes,including the polyol pathway and generation of reactive oxygen species(ROS).In cellular systems,a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.Activation of NAD(P)H oxidase system requires the participation of p47phox,one of the NAD(P)H oxidase subunits,which plays a central role in the scenario of NAD(P)H oxidase activation.10Activation of nuclear factor -ΚB(NF-ΚB) is involved in the expression of pro-inflammatory genes.11 In this study,we investigated wether AngⅡactivates the NF-ΚB cascade by ROS and whether the ROS-dependent mechanism may be involved in AngⅡ-induced MCP-1 formation.Further,we tested whether telmisartan,an AT1 receptor blocker,and DPI,an inhibitor of NAD(P)H oxidase,modulate endothelial inflammation and oxidative cell damage induced by AngⅡ-dependent stimuli in cultured rat glomerular endothelial cells.MethodsWe isolated and characterized primary cultures in rat glomerular endothelial cells(GECs).We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B (NF-ΚB).Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AngⅡon rat GECs.We study the effect of AngⅡon the propagation and apoptosis of RGECs by the methods of MTT and AV/PI.ResultsWe found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B(NF-ΚB).And the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AugⅡon rat GECs.DiscussionThe present study demonstrates that AngⅡ-induced ROS generation in rat GECs depends on the P47phox subunit of NADPH Oxidase.ROS are additionally required for AngⅡ-induced activation of NF-ΚB.Blockade of the NADPH Oxidase by its inhibitor DPI significantly abolished AngⅡ-induced MCP-1 formation,indicating that NF-ΚB when activated by ROS,participates in the AngⅡ-induced MCP-1 production.It’s now well accepted that AngⅡmay function as a potent pro-inflammatory mediator and be implicated in the pathogenesis of chronic renal disease.13,14GECs are at the interface between blood and adjacent cell population and play a crucial role in preserving the kidney function.The monocyte-endothelium interaction induced by AngⅡmay contribute to the initiation of vascular inflammation.15 MCP-1,when expressed at the plasma membrane of endothelial cells,can mediate the initial capture of monocytes.16 Our results suggest that AngⅡcan induce MCP-1 formation in rat GECs and promote the monocyte adhesion to endothelial cells.AngⅡacts via AT1 and AT2 receptors.Both receptor subtypes have been found in rat GECs.17 AngⅡexerts most of its already well-defined physiologic and pathophysiologic actions through AT1 receptors.18 Consistent with this concept,our results showed that the AngⅡeffect on MCP-1 formation was inhibited by the selective AT1 receptor antagonist telmisartan.In mammalian cells,a major source of ROS derives from the membrane-bound NADPH Oxidase system,which exists in nonphagocytic cells of the vascular wall,for example,fibroblast,vascular smooth muscle cells and endothelial cells.19 Increased ROS generation can induce cell inflammation.20,21 The present study isolated rat glomerular endothelial cells and demonstrated that the expression of p47phox protein, one of the NADPH Oxidase subunits,was up-regulated by AngⅡtreatment in accordance with the increased ROS generation.Moreover,blockade of NADPH Oxidase by DPI reduced the P47phox protein synthesis and ROS generation.Nuclear factor-ΚB normally exists in the cytoplasm in an inactive form bound to the inhibitory protein IΚB.Upon cellular activation,IΚB is rapidly degraded prior to the translocation of NF-ΚB into the nucleus and its subsequent activation,resulting in the transcriptional regulation of target genes encoding pro-inflammatory cytokines.NF-ΚB consists of two subunits,p50 and p65,with p65 containing a transcription domain.11,22 We here studied the possible involvement of NF-ΚB activation in AngⅡ-induced MCP-1 production in rat GECs.In the present study,AngⅡstimulation increased the translocation of NF-ΚB P65 subunit to the nucleus,which was reduced by DPI.The observation suggests that AngⅡ-induced NF-ΚB activation is partially mediated by ROS.In summary,we demonstrated that AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.Our finding may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with the chronic renal disease.Conclusions1 AngⅡincreases the expression of MCP- 1 and AT1mRNA;2 AngⅡincreases the expression of p47phox in a certain ranges showing a time-dependent manner.3 AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.4 the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.更多还原