【作者】 窦环；

【导师】 侯亚义；

【作者基本信息】 南京大学 ， 生物学， 2013， 博士

【摘要】 炎症(Inflammation)是宿主针对体内外各种损伤因子的刺激产生大量炎性介质的防御反应,是一种基本的病理过程。适度的炎症反应对人体是有益的,但是当炎症过程中机体自身的调节能力失调,就会导致炎症的失控,使其发生级联的放大效应,导致更严重的自身损伤。近年来研究发现,炎症与许多疾病的发生发展密切相关,比如类风湿性关节炎、动脉粥样硬化、II型糖尿病、肿瘤等等。Toll样受体(Toll-like receptors,TLRs)是天然免疫系统中一类病原模式识别受体,通过选择性识别病原微生物及其细胞壁的病原体相关分子模式(Pathogen-associated molecular patterns,PAMPs),激活其下游一系列的信号通路,活化天然免疫细胞产生炎性因子和I型干扰素,不仅可以启动天然免疫应答,还可对炎症反应的性质、强度和持续时间进行控制,在炎症反应中发挥重要作用。Toll样受体4(TLR4)是人们最早发现的能够直接介导机体与病原体反应的Toll样受体,广泛表达于多种细胞和组织,通过对G-细菌成分脂多糖(Lipopolysaccharide,LPS,又称为细菌内毒素)的识别,激活NF-κB或MAPKs途径,介导LPS的信号转导,促进细胞因子的合成与释放,引发炎症反应。LPS是引起脓毒症(Sepsis)的主要PAMPs,大量LPS释放与TLR4结合形成复合物后,引发单核/巨噬细胞等免疫细胞过度活化,爆发式释放各种炎性介质,如:NO、TNF-α、IL-6、MCP-1和MIP-1α等,这些因子进而作为内源性介质,通过旁分泌或体循环远程分泌到达靶组织和靶细胞部位从而发挥生物学效应,引起全身性炎症反应。因此,脓毒症的本质是机体炎症反应不断加剧、持续恶化的结果。目前针对脓毒症切实有效的治疗药物不多,抗炎治疗效果极不理想。本课题组前期以巨噬细胞系RAW264.7为研究模型,发现小分子化合物D182对LPS诱导巨噬细胞产生的NO具有显著的抑制作用,提示该化合物可能参与调节巨噬细胞细胞因子的释放,调控炎症反应、减轻炎症性损害,从而可能达到控制脓毒症发生发展的作用。本论文首先研究D182在小鼠脓毒症中的治疗作用;进而利用小鼠巨噬细胞系RAW264.7及小鼠腹腔巨噬细胞为模型,体外探讨D182对LPS/TLR4介导的巨噬细胞炎症反应的调控作用及分子机制;在此基础上利用基因芯片表达谱分析技术研究D182的作用靶点及其网络关系;最后通过定点突变技术,优化双分子荧光互补(biomolecular fuorescence complementation fragment,BiFC)技术参数,为可视化炎症反应中蛋白质相互作用提供研究基础。1、小分子化合物D182能够抑制脓毒症模型小鼠的症状LPS/LR4介导巨噬细胞活化并释放多种效应分子是巨噬细胞发挥功能的重要基础,但是大量LPS释放可引起巨噬细胞过度活化导致脓毒血症,是临床常见并发症。本课题首先研究D182对LPS所致脓毒症小鼠的保护作用。雄性BALB/c小鼠40只,随机分组,腹腔注射25 mg/kg LPS建立脓毒症模型,连续7 d观察小鼠生存数量,经生存率统计分析表明:脓毒症小鼠经D182低剂量(30 mg/kg)给药后与模型组相比,生存率显著高于模型组,具有极显著性差异(P<0.01),D182高剂量给药组(100 mg/kg)也得到类似结果,显著性差异P<0.05。接着,另取雄性BALB/c小鼠40只,LPS造模后12 h,眼眶取血,Elisa检测血清中细胞因子和趋化因子水平;摘取肝脏和肺脏,HE染色,观察病理学改变。结果表明,脓毒症小鼠血清中TNF-α、IL-6、MCP-1和MIP-1α水平明显升高,D182高低剂量组均可显著抑制这些因子的表达,病理学显示:两个剂量组均可不同程度缓解肝、肺组织的炎性浸润、充血、细胞坏死和组织结构病变。初步证实,D182对内毒素LPS攻击所致小鼠脓毒症具有保护作用。脓毒症患者最常见的感染源来自腹腔且易造成腹腔内感染,因此腹腔感染模型是目前最常采用的脓毒症动物模型。鼠类盲肠结扎穿孔(cecalligation and puncture,CLP)可模仿腹腔脓毒症感染的临床过程,引起多菌群感染,最终导致全身炎症反应,是脓毒症研究动物模型的“金标准”。因此,在小鼠CLP模型中,评价D182治疗小鼠脓毒症的效果更能反映其真实作用。参考Rittirsch等报道的CLP模型的标准步骤,我们成功复制小鼠CLP模型,术后3 d内CLP模型小鼠全部死亡,血清中TNF-α、IL-6、MCP-1和MIP-1α等特异性指标均显著升高。与LPS所致脓毒症小鼠模型结果类似,D182可显著提高小鼠生存率,减少炎性因子的产生,逆转肝、肺组织形态学改变。此外,我们对术后12 h和24 h小鼠腹腔灌洗液和外周血进行细菌培养,发现D182给药小鼠的腹腔灌洗液,在术后12 h和24 h时,细菌克隆数量均明显低于模型组,外周血涂板培养细菌克隆数量也有降低的趋势。上述两种动物模型实验结果显示,D182在实验性小鼠脓毒症中发挥重要保护作用。2、小分子化合物D182通过IRAK4抑制TLR4介导的炎症反应LPS通过TLR4强力激活巨噬细胞,可引起细胞因子合成和释放,导致全身性炎症反应发生。体外,我们利用小鼠巨噬细胞系RAW264.7及小鼠腹腔巨噬细胞,经过CCK-8检测,选择50 μM以下剂量处理细胞无明显细胞毒性;接着,利用LPS诱导巨噬细胞产生炎症因子TNF-α、IL-6和MCP-1构建细胞炎症反应模型,结果显示:5～45κM D182对TNF-α、IL-6和MCP-1合成和释放具有不同程度的抑制作用,与体内试验结果一致。由此,我们选择小鼠巨噬细胞系RAW264.7作为模型,进一步研究D182抑制TLR4信号传导通路,诱导炎症反应的作用机制。分析上述结果,我们首先考虑,D182是否影响LPS与细胞膜表面TLR4的结合?有研究表明,LPS诱导细胞活化的信号转导的启动环节(LPS与CD14/TLR4/MD-2复合物的结合)发生在细胞膜上,LPS不需进入胞内即可启动细胞的活化,合成和释放炎性因子。由此,FITC-LPS(2 mg/ml)孵育RAW264.7细胞1 h和2 h,运用流式细胞术检测细胞表面FITC%,D182处理细胞后,FITC-LPS在细胞膜表面结合无统计学差异,阳性药物多粘菌素B则表现出50%以上的抑制效果。为验证上述结果,我们分离小鼠腹腔巨噬细胞重复上述实验,流式细胞术结果显示,小鼠腹腔巨噬细胞经D182处理后不影响LPS在其表面的结合。因此,我们排除了 D182通过影响LPS与TLR4的识别介导下游炎症介质产生的可能性。文献证实,TLR4识别LPS并与之结合,首先启动MyD88依赖途径,TIRAP通过TIR-TIR结构域聚合招募MyD88接头分子形成TIRAP-MyD88复合物,MyD88再经过DD-DD结构域相互作用募集IRAKs家族(IRAK4和IRAK 1),IRAK的磷酸化又依次激活了 TRAF6和TAK1,通过激活NF-κB和MAPK途径;MyD88非依赖途径(也称作TRIF途径)则在TLR4内吞入胞,TIRAP-MyD88复合物解离后,进入初级内体的TLR4同样通过TIR-TIR结构域聚合招募TRIF,通过TRAF6激活IRF3途径;最终导致炎症介质和I型干扰素的产生。由于前炎性细胞因子的产生主要依赖于MyD88依赖途径,我们选择TLR4、MyD88、IRAK4和 TRAF6 四 个 关 键 分 子,利 用 蛋 白 数 据 库(http://www.rcsb.org/pdb/home/home.do),提取晶体结构信息,采用计算机分子模拟计算的方法,尝试将小分子化合物D182与他们分别进行分子对接(Molecular Docking),比对结晶配体的作用模式,结果表明:D182与IRAK4的活性口袋中心存在结合,且两者之间分子结合力为31.07。为进一步研究D182与IRAK4相互作用的可能性,我们采用计算机辅助分子动力学模拟和自由能分析的方法,进行了 8 ns的MD模拟,计算结果说明D182与IRAK4具有结合模式,D182位于ILE22、MET29、VAL37、ALA48、VAL80、TYR96、TYR98、MET99、GLY102和LEU152形成的活性口袋内,通过疏水键这些氨基酸结合,产生空间位阻,其中D182与MET99残基空间距离仅2.14 A;自由能分析表明,体系的结合自由能为60 kal/mol,分解自由能为不同能量项发现D182与IRAK4两个分子之间的范德华作用是主要的驱动力。上述理论计算结果提示,D182与IRAK4之间存在相互结合的可能性。为验证IRAK4蛋白是否与小分子化合D182存在结合作用,我们将IRAK4蛋白偶联到Biacore T200到芯片上进行表面等离子共振分析,结果表明D182与IRAK4具有较强的结合活性,即使在IRAK4的浓度仅0.8μM时,RU值所仍能反映出结合能力,亲和常数为3.103E-6 M,结果说明D182与IRAK4具有结合活性。在此基础上,D182预处理小鼠巨噬细胞系RAW264.7 2 h后,LPS(100 ng/ml)刺激不同时间,Western Blot检测细胞中p-IRAK4、总IRAK4、IRAK1和TRAF6的蛋白表达,结果显示,LPS单独刺激细胞15 min,p-IRAK4升高,总IRAK4不变,IRAK1降解,2 h后TRAF6降解;D182预处理细胞可阻断LPS诱导的IRAK4磷酸化和IRAK1降解,且呈剂量依赖效应,对总IRAK4无影响。重复上述实验,D182对LPS诱导的TRAF6降解具有逆转作用。综合上述结果,D182通过与IRAK4结合,抑制IRAK4的磷酸化,阻断IRAK1和TRAF6的降解,提示IRAK4可能是D182阻断TLR4信号通路的关键靶点。研究表明,在TLRs信号通路中,IRAK4分子联系着上下游的信号转导,在TLR4介导的NF-κB和MAPKs炎症信号通路中的作用更为关键。因此,我们继续研究D182对NF-κB和MAPKs通路的调节作用。我们利用15 μM D182预处理RAW细胞,Western Blot检测显示,LPS刺激15～60 min时,D182处理组p-IκBα和p-P65的蛋白表达减少,IκBα蛋白降解被阻断;细胞免疫荧光染色表明,D182可明显抑制P65从细胞浆向细胞核内转移;NF-κB荧光素酶报告基因检发现,D182也可抑制NF-κB转录活化。结果提示,D182可阻断IRAK4下游NF-κB通路活化。重复上述实验处理,Western Blot检测发现D182对MAPKs通路中P38磷酸化有明显的抑制作用,45 μM处理组对AP-1转录活化有显著抑制作用。NF-κB和MAPKs通路的活化,导致活化的NF-κB和AP-1进入细胞核内结合到TNF-α、IL-6和MCP-1启动子区,启动这些炎性因子的转录表达。RT-qPCR检测结果显示,15和45 D182预处理巨噬细胞均可抑制LPS诱导TNF-α、IL-6和MCP-1表达升高(P<0.05)。综上,D182抑制TLR4介导的炎症反应的分子机制是:D182通过直接与IRAK4结合,抑制IRAK4磷酸化,阻断IRAK1和TRAF6的降解,从而抑制通路下游NF-κB和P38活化,导致核转录因子NF-κB和AP-1转录活性降低,最终TNF-α、IL-6和MCP-1等炎性因子合成和释放减少,调控TLR4介导的小鼠巨噬细胞炎症反应。3、利用基因芯片探索小分子化合物D182作用的多靶点及其网络调控关系研究表明在分子水平,药物小分子产生疾病治疗作用甚至毒副作用,是一个多基因调控,多阶段、多步骤的复杂生物学过程,涉及多种基因的协同作用,若只是孤立的分析某个基因在药物治疗中的作用已经难以适应研究的需要。伴随着人类基因组计划,基因芯片技术以其高通量、高效率、低消耗的特点,广泛应用于疾病相关分子机制研究。它能够在同一时间内,通过少量标本,实现生物基因信息的大规模检测,通过比较正常、疾病和治疗状态下的基因转录及其表达的差异,寻找和发现与该疾病治疗相关的候选基因,有可能揭示疾病治疗过程中的多个作用环节,同时也能为疾病的诊断和治疗提供分子生物学方面的证据。本研究首次应用基因表达谱芯片技术研究D182药物调控炎症反应前后基因表达情况,旨在较全面了解药物处理过程中基因表达差异,并寻找其中的关键靶点基因,一定程度上揭示D182对炎症反应的调节机制,为筛选控制炎症免疫性疾病新药提供可靠理论依据。实验分为三个组:正常对照组(C)、LPS模型组(L)和D182(15 μM)治疗组(D),每组各3个生物学重复,将各分组的RAW264.7细胞单独抽提总RNA,纯化后分别制成cRNA探针,通过与包括44,000个转录本的Agilent小鼠基因表达谱芯片杂交,经过信号检测、微阵列图像分析、获得标准化原始数据。首先,我们使用RVM算法分别对9个标准化数据进行3组差异基因筛选,3组比较分别是L vs C、D vs C,D vs L,其中筛选标准为:Fold-change>2.0 或<0.5,P-value<0.05,FDR<0.05。将3组的差异基因L vs C、D vs C,D vs L进行合并,获得总的差异基因列表,共差异基探针为4986个,差异基因为4570个。对4570个基因进行聚类分析,得到Heatmap,C、L和D分别形成3个独立的组,差异基因组内重复性很好。其次,对并集中4570个差异基因进行功能分析,分析全部的差异基因在整个实验中影响的GO功能:在生物学功能方面,差异基因主要表现为天然免疫反应、炎症反应、抗病毒反应等;在细胞运动相关影响因子方面,差异基因主要表现为细胞因子、趋化因子活性,跨膜信号转换等;在细胞组分方面,主要表现为胞外基质、细胞骨架、染色体等。同时,我们对差异基因进行Pathway分析,主要集中在细胞因子-细胞因子受体相互作用通路、NF-κB信号通路、细胞粘附分子相关通路、病毒、细菌感染通路等。由于我们比较关注显著性生物学功能,那么对显著性功能做进一步的分析是非常有必要的,于是,我们提取显著性功能中的所有基因,对并集基因GO分析中的显著性GO(显著性水平0.05)中所有提取出的基因,对其进行聚类分析,截取在L组高表达和低表达,独立展示,这些基因在整个实验当中与三个分组的趋势成正相关或者负相关,对其的分析可能获得与疾病和药物治疗直接相关的基因。结果显示,疾病组高表达的基因数为20个,疾病组低表达的基因为67个。然后,将并集GO聚类分析中疾病组上调和下调的基因全体(87个基因)提出,对其进行共表达分析,获得在这些基因中的关键基因或者核心基因:Mtor、B4galt1、Jmjd6、Dock2、Fzd4、Mdm4、IRAK4、Ngf、Defa-rs1、Vegfa、Rps6ka4(MSK2)、Ajap1 和 C1rb。其中,IRAK4 与我们体内外研究结果一致。最后,对并集基因做功能分析GO-Analysis,对显著性的功能做取显著水平为0.05的GO条目中的所有基因,共有781个基因,对这些基因进行图形化展示,构建基因间的相互作用关系网络。以IRAK4为例,我们找到其上游有Tlrap、Nos2、Tnfrs1a、MyD88、Tlr9等基因对其产生影响,下游调控Ll1rap、L11b、Nfkb1等基因。提示我们,781个基因通过13个关键基因形成调控网络,参与D182抑制巨噬细胞炎症反应,其具体机制有待进一步研究。以上这些发现,D182通过多环节、多靶点调控炎症反应,为筛选、创制新型抗炎药物提供了新的理论依据。4、为了炎症反应中蛋白质相互作用的可视化,优化BiFC技术信/噪比的参数包括脓毒症在内,许多炎症性疾病的发病规律与临床意义的研究取得一定进展,相关抗炎治疗的临床试验并未能取得预期的效果;或仅仅是在理论上认为有效,在临床实践中其结果有待进一步验证。这可能是由于机体的炎症反应为网络样作用,但针对直接单一抗炎作用靶点其作用当然较为有限。基因芯片技术可以从转录组层面研究疾病发生发展过程中基因网络变化模式,但是这并不能反映蛋白水平的真实变化情况。因此,从蛋白质水平,研究蛋白质相互作用和翻译后修饰,更有利于全面认识病理变化过程中生物调控的机制。近年来,双分子荧光互补(bimolecular fluorescence complementation,BiFC)分析技术能够在活细胞生理环境中原位显示蛋白质相互作用,逐渐引起研究者的关注。该技术能够巧妙地将荧光蛋白分子的两个互补片段分别与目标蛋白进行融合表达,如果荧光蛋白恢复其荧光活性则表明两目标蛋白发生了相互作用。BiFC作为一种新近发展的用于蛋白质间相互作用研究的方法,其本身还存在一些局限,本研究通过定点突变技术,比较互补片段间的BiFC荧光信号,提高信/噪比,优化参数,为研究炎症反应中蛋白质相互作用的可视化提供技术基础。Venus荧光蛋白互补片段之间可能发生不依赖蛋白质间相互作用的自发结合而产生背景荧光,产生较高信/噪比,干扰BiFC结果判断。根据文献报道和前期研究,我们选择V150L、V150A、I152L、L201V、L207V五个突变位点,分别与Venus两个荧光蛋白的互补片段VN155和VC155构建VN155(V150L)、VN155(V150A)、VN155(I152L)、VN155(V150A,I152L)、VC155(L201V)、VC155(L207V)和 VC155(L201V,L207V)质粒,检测 VN155/VC155 互补片段直接产生的荧光强度,Western Blot检测突变蛋白表达水平。结果显示,以上突变位点蛋白表达量均一,自发结合产生的荧光信号均显著低于WT型Venus蛋白互补片段。接着,已知bJun/bFos形成的异源二聚体可作为BiFC阳性对照,bJun/Δ bFos因无法相互作用,可作为BiFC阴性对照,因此将bJun和bFos或Δ bFos分别与VN和VC突变片段进行融合,产生的bJun-VN155和bFos-VC155或ΔbFos-VC155质粒,分别共转染COS-1细胞,24 h观察bJun-VN155/bFos-VC155,bJun-VN155/ΔbFos-VC155产生的BiFC信号,经过图像采集分析,数据统计,结果显示:bJun-VN155(I152L)/bFos-VC155,bJun-VN15/bFos-VC155(L207V)产生较强的BiFC信号,且信/噪比与WT相比提高3～5倍。最后,基于Venus,我们制备VN210和VC210另外两个荧光互补片段,分别与bJun和bFos或ΔbFos融合后,产生的BiFC信号与VN155/VC155进行比较,发现 bJun-VN210/bFos-VC210 在 COS-1 细胞内荧光强度低于 bJun-VN155/bFos-VC155,且两组之间信/噪比无统计学差异(P=0.200)。以上结果提示,VN155(I152L)和VC155(L207V)突变荧光片段可以提高BiFC检测的信/噪比。综上所述,本研究首次证明D182能够有效抑制LPS和CLP诱导致小鼠脓毒症模型的症状,显著提高存活率;首次发现D182通过直接与IRAK4结合,抑制IRAK4磷酸化,阻断IRAK1和TRAF6的降解,从而抑制通路下游NF-κB和P38活化,导致核转录因子NF-κB和AP-1转录活性降低,最终TNF-α、IL-6和MCP-1等炎性因子合成和释放减少,调控TLR4介导的小鼠巨噬细胞炎症反应;初次利用基因芯片表达谱深度分析,发现D182调控巨噬细胞炎症反应涉及781个基因及其通过Mtor、Dock、IRAK4、MSK2等13个关键基因形成调控网络;建立了可以提高BiFC检测的信/噪比的Venus荧光蛋白的VN155(I152L)和VC155(L207V)突变荧光片段,为炎症反应中蛋白质相互作用的可视化奠定了研究基础。更多还原

【Abstract】 Inflammation is a host response to traumatic,infectious,post-ischaemic,toxic or autoimmune injury that leads to the release of a large amount of inflammatory mediators.Excess production of these inflammatory mediators is involved in many diseases including rheumatoid arthritis,asthma,pulmonary fibrosis,type 2 diabetes mellitus,atherosclerosis,and cancer.Toll-like receptors(TLRs)are a type of pattern recognition receptors implicated in the defense system against pathogens.They recognize pathogen-associated molecular patterns(PAMPs)molecules traumatic,infectious,post-ischaemic,toxic or autoimmune injury,which are broadly shared by pathogens but distinguishable from host molecules.All TLRs activate through different signal pathways,inducing the production of proinflammatory cytokines or eliciting a type I interferon response.Although full activation of TLR responses is necessary for the elimination of inflammtion to invade pathogens,excessive inflammatory activation through TLR signaling may promote the development of immuno-pathological conditions such as infectious disease and autoimmune disease.One of the TLRs,TLR4,engaged by LPS found on Gram-negative bacteria,orchestrates the immunity response on certain monocytes and macrophages.It is the only known TLR able to activate both MyD8 8-dependent induction of genes encoding inflammatory molecules and TRIF-dependent production of type I interferon.When in excess,LPS/TLR4 signaling leads to serious systemic disorders with a high mortality rate,for example,during sepsis,septic shock,or systemic inflammatory response syndrome.Sepsis remains a critical problem,with significant mortality and morbidity despite intense efforts to find effective therapies.The prevailing theory of sepsis is an unregulated inflammatory response or cytokine storm resulting in organ damage and death.Thus,suppression and/or inhibition of the above-mentioned signaling molecules may have great potential for anti-inflammatory or anticytokine therapies in sepsis.Our group recently found that D182 inhibited the expression of LPS-challenged NO production in murine macrophage cell line RAW264.7.The data has been suggested that D182 could be applied to inflammatory lesions to modulate the inflammatory response and alleviate spesis.In our present study,we demonstrated that D182 shows protective effects in two murine models that D182(ⅰ)efficiently protected the mice from spesis death(ⅱ)attenuated multi-organ inflammatory damage(ⅲ)modulated the production of such proinflammatory cytokines and chemokines as TNF-α,IL-6,MCP-land MIP-la and(ⅳ)decreased the generation of bacteria in peritoneal and blood.Next we selected murine macrophage cell line RAW264.7 and primary peritoneal macrophages to evaluate D182 pharmacological effects and revealed the underling mechanisms.On the production of inflammatory mediators in LPS-induced macrophages,CCK-8 and Elisa tests confirmed that 5-45 μM D182 significantly reduced the production of TNF-α,IL-6 and MCP-l,without any cytotoxic effect on these cells.Using flow cytometric technique,we confirmed that D182 didn’t affect LPS binding to macrophages.Based on TLR4 signal pathway theories,we analyzed TLR4,MyD88,IRAK4,TRAF6 proteins in this pathway by Docking,which is a method predicting the preferred orientation of one molecule to D182 when binds to each other to form a stable complex.we found that D182 could bind to the active center of IRAK4 and the interaction energy between D182 and IRAK4 was 31.07.Then the molecular dynamics simulations and interaction energy calculations were both performed to show theoretically the binding strength of IRAK4(the protein data bank code:2NRY)with the ligands of D182.The total interaction energies,van der Waals energies and electrostatics energies all confirmed that 2NRY…D182 complex interaction energies were so strong.Furthermore,D182 was located in the active loops formed by ILE22,MET29,VAL37,ALA48,VAL80,TYR96,TYR98,MET99,GLY102,and LEU 152.There were hydrophobic interactions between the side chains of these residues and the ligands.The D182…2NRY interaction involved hydrogen bonding interactions between the oxygen in the methoxy group of D182 and the hydrogen in the peptide group of MET99 of 2NRY.The average time evolution of the distances(A)between the oxygen in the methoxy group of D182 and the hydrogen in the peptide group of MET99 of 2NRY was 2.14 A.These results indicated that the interaction between 2NRY and D182 was strong.Sequentially,a direct binding assay using surface plasmon resonance ascertained the stoichiometry,binding affinity and kinetics for the interaction of our compound with the binding groove of IRAK4.In the experimental set-up used with the BIAcore T200,sensorgrams and steady state curves indicated that a compound binds to a target protein..In particular,the activities of D182 on IRAK4 in cells were assessed.Immuno blot showed that D182 significantly reduced IRAK4 phosphorylation,blocked the degredation of IRAKI and TRAF6.To further elucidate the molecular mechanisms of D182 inhibition on IRAK4,the activations of NF-κB and MAPKs ’down-stream’ the IRAK4 were investigated.Pre-exposure of RAW264.7 cells to D182 resulted in the rapid inhibitory effect of the LPS-mediated phosphorylation and degradation of IκBα at 15 min,inhibited P65 phosphorylation and translocation into the nucleus.NF-κB luciferase construct clearly inhibited luciferase activity by D182 on LPS-induced NF-κB activation.Although D182-treated macrophages showed diminished activation of P38 compared with LPS-only macrophages,activation of JNK and ERK1/2 was minimally affected.Accordingly,AP-1 luciferase reporter gene assay appeared to be differentially expressed in D182-pertreated cells,and it confirmed that only in high dose treated group(45 κM),D182 significantly impaired AP-1 activation in LPS-stimulated RAW264.7 cells.Last to determine D182 inhibiting IRAK4 activity in MyD88-dependent TLR signaling,murine macrophage cell line RAW264.7 was treated with the TLR4 agonist LPS.mRNA expression for MyD88-dependent genes TNF-α,IL-6 and MCP-1 in LPS-treated macrophages was significantly high compared with the D182 treated cells.Then we analysed whole-genome-widely for the genes that account for the effect of D182 in LPS-stimulated RAW264.7 cells,using a Agilent 4 × 44 K Mouse Whole-Genome oligonucleotide microarray.D182 significantly inhibited LPS-induced NO production and iNOS expression in RAW264.7 cells.The results of the microarray analysis showed that 4,570 genes were differentially expressed between "C"(Control),"L"(LPS-only)and "D"(D 182 pretreated+LPS)groups.To further understand the biological relevance of these gene expression profiles,we carried out bioinformatic analyses to determine the Gene Ontology(GO)terms and pathways most significantly associated with these genes.The main GO categories in biological progress(BP)were related to innate immune response,inflammatory response,immune response,defense response to virus,cell cycle,cell surface receptor signaling pathway,cellular response to interferon-beta,defense response to protozoan,response to virus,positive regulation of cell proliferation,negative regulation of viral genome replication,cellular response to lipopolysaccharide,elevation of cytosolic calcium ion concentration,and defense response to bacterium.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed the enrichment of a variety of processes of D182 physiology represented mostly by immunity pathways,such as TLR signaling pathway,NF-κB signaling pathway,JAK-STAT signaling pathway,cytokine-cytokine receptor interaction,cell adhesion molecules,herpes simplex infection,herpes simplex infection,etc.Such GO and KEGG pathway analyses results were in accordance with our purpose,and then we focused on genes belonging to categories of macrophage-mediated immunity.The system allowed us to pin down the genes related to immune response.Once again,hierarchical clustering was used to focuse on the gene differentially expressed in BP categories.As a result,87 genes could be truly the representatives accounting for the intrinsic susceptibility to LPS and its induced reversal in the RAW cells.This simple dichotomy classification further allowed us to analyze the differentially expressed genes for their gene ontology and coexpression with ease and confidence.Focusing on the gene categories,we identified the major cellular processes involved the reversed susceptibility in the LPS-induced cells.Prominently,these include 13 genes as "key regulatory" genes:Mtor,B4galtl,Jmjd6,Dock2,Fzd4,Mdm4,IRAK4,Ngf,Defa-rsl,Vegfa,Rps6ka4,Ajapl,Clrb.We then mapped all the "key regulatory"genes on KEGG pathways from the database,and a clear signal pathway picture emerged.With D182 pre-treatment,up-reuglated genes,down-reuglated genes and genes highly related to key regulatory genes are all presented in this picture,giving us strong suggestions to find novel regulatory genes and possible signal transduction pathway.So more deep-going work should be done to confirm such hypothesis.Lastly,in an application of bimolecular fluorescence complementation(BiFC)to visualize protein-protein interactions(PPIs)during drug-elicited processes,we compared V150L,V150A,I152L,L201V and L207V mutations in Venus using the interaction between the basic region-leucine zipper(bZIP)domains of Jun(bJun)and Fos(bFos)in the nucleus as a model system.I152L,V150L and V150A mutations were introduced to Venus N-terminal(VN)respectively.And V150A and I152L double mutation in VN155 at the same time.To Venus C-terminal(VC)fragment,L201V,L207V and the double mutations were introduced repectively.Each mutation fragment was coexpressed with wild-type VN or VC fragment in COS-1 cells to examine their self-assembly.The fluorescence intensity derived from the self-assembly was signifcantly reduced with all these mutations,while L207V mutation in VC fragment had the higher intensity of fluorescence than I152L in VN.It suggested that these substitutions could decrease the affinity between the N-and C-terminal fragments,which may lead to increasing the S/N ratio between proteins fused to the fragments.We also examined the BiFC signals of the Venus variants.We used transcription factors,bJun and bFos,known to form a hetero dimer,as positive control,and bJun and AbFos,lacking the carboxy-terminal half of bFos bZIP,as negative control for the PPIs in BiFC assay.The results suggested that point mutations of I152L and L207V effeciently reduced the non-specific fluorescent intensity and the S/N ratios of these two variants were much higher than wild type.The third we compared the two pairs of N-and C-terminal fragments VN155/VC155 and VN210/VC210,using the same model system.And VN210/VC210 fragment pair could not produce better S/N ratio improvement in BiFC assay.In summary,D182 rescued mice from lethal sepsis.Its mechanism might involve a down-regulation of inflammatory mediators production via IRAK4.Thus,D182 could be a useful therapeutic candidate for the treatment of sepsis and other inflammatory disorders.Microarray assay revealed that D182 possessing anti-inflammatory properties was involved in the regulation of immunity and defense related genes that act as key mediators in various inflammatory processes.Moreover in the same cell model,we first compared reported mutations that could specifically reduce the spontaneous self-assembly of the two nonfluorescent fragments in the Venus-based BiFC assay.Here we identified that I152L and L207V effeciently reduced the non-specific fluorescent intensity and the S/N ratios of these two variants were much higher than wild-type.Besides,VN210/VC210 fragment pair could not produce better S/N ratio improvement in BiFC assay.It might help to clarify our understanding of drug action and enhance the productivity of drug-discovery research.更多还原