【作者】 冯皓；

【导师】 张俊平；

【作者基本信息】 第二军医大学 ， 微生物与生化药学， 2011， 博士

【摘要】 【研究目的】中药商陆系商陆属植物商陆和垂序商陆的干燥根,为历版中国药典收载品种。商陆的水溶性成分中主要是多种具生理活性的商陆皂苷(Esculentoside,简称Es),从中的分离的单体称为商陆皂苷甲(Esculentoside A,简称EsA)。以往的研究发现该物质对体液免疫和非特异性细胞免疫有显著的抑制活性。近年来有研究者采用基因芯片技术比对正常胸腺细胞和经过EsA处理的胸腺细胞的基因表达谱,发现若干表达差异基因,其中包括同炎性介质一氧化氮相关的NO合酶抑制蛋白(protein inhibitor of nitric oxide synthase,PIN) mRNA的表达上调,推测该蛋白的表达上调与EsA抗炎作用机理有关。PIN是含89个氨基酸的多肽,生物进化过程序列高度保守,并且在多种组织中表达。PIN能结合超过15种的蛋白分子和mRNA,这些蛋白功能涉及细胞凋亡、酶活性调控、肿瘤形成、器官发育、胰岛素释放等。据此有人将PIN归类为多功能的枢纽蛋白(hub protein)。近来有关PIN同炎症免疫调控方面的报道引起我们的关注。有研究发现,炎症介质PGE2、TNF-a可诱导神经元和Rat-1细胞系PIN表达。在纤维母细胞中,cAMP水平提高能促使PIN蛋白表达上调从而调控与炎症、凋亡密切相关的蛋白激酶p38信号途径。而在人肥大细胞中,PIN能与nNOS结合调控白三烯的产生。这些结果提示PIN可能作为细胞内的效应蛋白,参与细胞外信号对炎症免疫反应的调控。另外,PIN独特的分子结合特性也引起我们的注意。一系列研究发现,PIN分子是一个紧密结合的二聚体结构(dimer),在二聚体表面的对称位置上存在两个序列完全相同的多肽结合凹槽(groove),凹槽能识别和结合具有(K/R)XTQT或G(I/V)QVD指纹区结构的的靶蛋白。如果结合指纹区(K/R)XTQT或G(I/V)QVD部分氨基酸替换,靶蛋白则不能与PIN结合,这表明(K/R)XTQT或G(I/V)QVD结构具有序列特异性。研究还发现,PIN与靶蛋白结合后可促进这些局部构象异常恢复正常或形成稳定结构。因此PIN蛋白具有结合并调节靶蛋白功能的活性。PIN能与核转录因子抑制蛋白IkB结合,阻止其被IKK磷酸化、负调控NF-kB的转录活性;某些氧化因子使PIN第二位上的半胱氨酸残基脱氢后,通过二硫键形成稳定的PIN二聚体,可以解除PIN对IkB的结合,促进NF-kB转录活性。据此我们设想:是否可能设计具有(K/R)XTQT或G(I/V)QVD结构的短肽分子,导入细胞内与PIN结合,阻断PIN同靶蛋白(比如IkB)的结合,实现对细胞功能的某种调节。商陆在中国用于某些急性炎症疾病的治疗由来已久,我们希望通过该课题的研究对这一传统中药有效成分——商陆皂苷甲(EsA)的作用机理进行阐述,对EsA抗炎活性同PIN蛋白的关系进行分析,对EsA提高PIN蛋白的作用机理进行研究。另外,我们设计具有PIN结合基序的短肽,在细胞水平研究短肽分子对PIN抗炎免疫调节作用,在蛋白功能调节水平上进一步阐明PIN的作用,同时希望发现一种全新的炎症免疫调控方式和新型的抗炎免疫药物的先导分子。【研究方法】(一)PIN参与EsA抗炎免疫效应活性作用机理的研究1、EsA对TNF-a的调控作用:不同浓度EsA预处理RAW264.7细胞后,加入LPS刺激12小时,ELISA检测细胞产生TNF-a的水平变化。EsA作用不同时间后收集细胞,real-time定量PCR检测TNF-a的mRNA表达水平。2、EsA作用RAW264.7细胞,ELISA检测cAMP水平变化,Western blot检测PIN蛋白表达;以cAMP依赖性PKA抑制剂H-89处理细胞,Western blot检测PIN蛋白表达的变化。3、RNA靶向干扰方法建立PIN低表达的RAW264.7细胞;瞬时转化表达载体方法建立PIN高表达RAW264.7细胞。4、LPS刺激PIN低表达细胞和PIN高表达细胞,ELISA检测TNF-a、NO等炎性介质的水平。5、在PIN低表达的RAW264.7细胞中,EsA预处理细胞,加入LPS,real-time定量PCR检测对TNF-a mRNA表达水平。(二)研究PIN结合短肽对IL-8产生的影响,以及对IkB磷酸化的影响1、设计具有(K/R)XTQT或G(I/V)QVD的“结合基序短肽”;改变TQT或QVD氨基酸序列,设计“序列突变型短肽”作为对照,以空载体腺病毒作为空白对照。2、利用AdEasy系统构建表达含有PIN结合基序的短肽,HEK293细胞包装并反复感染,扩增高效表达短肽的腺病毒,浓缩纯化、测定滴度后保存。3、“短肽-腺病毒”转染Jurkat细胞,ELISA检测TNF-a诱导细胞产生IL-8的水平。4、“短肽-腺病毒”转染Jurkat细胞,western blot检测TNF-a作用细胞后IkB磷酸化水平的在5分钟和10分钟时的情况。【结果】第一部分:1、EsA对LPS诱导RAW264.7细胞产生TNF-a具有负调控作用,该抑制活性呈剂量依赖性和时间依赖性。2、EsA能够提高RAW264.7细胞内cAMP水平,该效应与腺苷酸环化酶刺激剂forskolin诱导cAMP的效应相比无显著差异(p>0.05);EsA提高PIN蛋白表达,该效应可被cAMP依赖性PKA抑制剂H-89削弱。3、建立高表达PIN蛋白的RAW264.7细胞;建立siRNA靶向干扰的PIN蛋白低表达细胞。4、PIN高表达RAW264.7细胞LPS诱导炎性介质TNF-a产生和NO释放显著下降(p<0.05);PIN低表达细胞则TNF-a显著上调(p<0.05)、NO显著上调(p<0.05)。5、同转化scramble序列的RAW264.7细胞相比,EsA对于PIN低表达的RAW264.7细胞的TNF-a的负调控作用显著削弱(p<0.01)。提示该抑制活性是PIN依赖性的。第二部分:1、利用AdEasy腺病毒表达载体,表达含有相关序列的“短肽-腺病毒”。通过扩增获得高滴度病毒上清。2、病毒感染Jurkat细胞,发现“结合基序短肽-腺病毒”能促进IL-8产生,呈剂量依赖性,“序列突变短肽-腺病毒”和空载体腺病毒无明显活性;3、“结合基序短肽-腺病毒”作用Jurkat细胞后,IkB磷酸化水平无明显变化。【结论】(一) EsA对免疫介质TNF-a具有负调控作用;EsA通过cAMP-PKA途径上调PIN蛋白; EsA免疫调控作用是PIN依赖性的。(二)含有PIN结合基序的人工短肽对细胞因子IL-8具有正调控作用,但并非通过提高IkB磷酸化途径起作用。更多还原

【Abstract】 【Background and Objective】Esculentoside A (EsA) is the major saponin isolated from the Chinese herb Phytolacca esculenta. It has been showed that EsA possessed comprehensive pharmacological effects in the study during the last thirty years. It is supposed that EsA might have potency on treatment of inflammation. The possible mechanisms were investigated through anti-inflammation and immunoregulation after the positive results being found. EsA inhibits over-reactive macrophage by decreasing the expression of NO and down-regulation of phagocytic. The regulatory effects of EsA on inflammation cytokines including IL-1、PGE2、PAF、TNF-a . EsA possess the the pro-apoptosis effect on thymocyte stimulate by ConA. The effect of EsA on gene expression profile changes in cells was investigated using microarray in 2002 by Xiao et al. It was found that EsA could regulate the expression of gene of protein inhibitor of nitric oxide synthase (PIN), tissue inhibitor of metalloproteinase (TIMP), GSK3βand PIAS1. EsA also reconstructs the balance of cytokines by regulation of IL-10, IL-12, TNF-a, TGF-b and regulate the immunological function by increasing the expression of immunophilin (FKBP1a, Cyclophilin).In 1996 Jaffrey and Snyder identified protein inhibitor of nNOS (PIN), in rat brain, which interacts with the NH2-terminal part of the enzyme. Binding of PIN to nNOS prevents homodimerization of the enzyme resulting in an inhibition of NO production. PIN is an ubiquitous protein expressed in rats and humans to an extent depending on the tissue studied. PIN is largely present in testis; moderately in different brain areas, kidney, and ventilary muscles; and faintly in other tissues. Furthermore, PIN has also been identified as the light chain of cytoplasmic and flagellar dyneins and myosin V, two cytoskeletal components involved in the traffic of intracellular organelles and flagellar movements. In addition, PIN is also able to interact with other partners as diverse as Bim (a proapoptotic factor of the Bcl-2 family), IκB (the cytoplasmic inhibitor of nuclear factor-κB), DAP1α(an N-methyl-D-aspartate scaffolding protein), NRF-1 (nuclear respiratory factor-1), Swallow (an mRNA localization protein), and gephyrin (aγ-aminobutyric acid [GABA] or glycine scaffolding protein), suggesting an interplay of PIN in a number of biological functions.Mapping of protein binding interactions with PIN has led to the identification of two consensus motifs, (K/R)XTQT (found primarily in viral proteins that bind PIN) and GIQVD (found primarily in cellular proteins that bind PIN). X-ray diffraction and NMR spectroscopy studies have revealed that these short motifs stabilize interactions with the PIN dimer by binding inside the PIN intermonomer groove, while biochemical studies have confirmed that this conserved motif mediates association between PIN and its protein partners. Additionally, pepscan techniques have demonstrated the ability of short peptides containing these consensus motifs to bind recombinant PIN in vitro. Due to the identification of consensus amino acid motifs that bind PIN, this subunit is considered an attractive target for regulation of PIN-binding protein via a short peptide.Previous studies showed that EsA increased the expression of PIN mRNA. In this study we explored the essential role of PIN in the response of LPS-inducted RAW264.7 cell and the regulation of PIN involved in the anti-inflammation effect of EsA; in addition, we designed the peptide with the consensus motifs of (K/R)XTQT or GIQVD , to invest its regulation to phosphorylation of IkB and production of IL-8 in Jurkat cell stimulated by TNF-a.【Methods】Part one : PIN involving in the anti-inflammation effect of EsA1. The potency of EsA on TNF-a stimulated by LPS in RAW264.7 cells:a) Ascending concentration of EsA affected on RAW264.7 cells pre-stimulated by LPS (1ug/ml)b) Different working-time of EsA affected on RAW264.7 cells pre-stimulated by LPS (1ug/ml)2. The potency of EsA on cAMP levels in RAW264.7 cells.3. The effect of EsA on expression of PIN in RAW264.7 cells in a cAMP-PKA depended-pathway: the protein expression was determined by western blot assay.4. Regulatory role of PIN to inflammation responsesa) Using siRNA targeting PIN and PIN-expressing vector, RAW264.7 cells PIN up- and down- expressing was established. Western blot assay was performed to examine the expression level of PIN.b) TNF-a and NO levels were valued in PIN up-expressed cell or down- expressed cell, respectively.5. PIN involving in the anti-inflammation effect of EsA: expression of TNF-a mRNA was tested by real-time PCR in PIN-interfered RAW264.7 cells. Part two: Effect of the peptides with PIN-binding motif in Jurkat cells1. Ad-peptide and null adenovirus (AdGFP) packaged in 293 cells respectively. The adenoviruses were stored after purification by cesium chloride (CsCl) gradient centrifugation and determination of the viral titers.2. Production of IL-8 was tested in supernant of Jurkat cells transfected with Ad-peptide or AdGFP. ELISA assay was carried out after an additional 4-hour stimulation of TNF-a.3. Phosphorylation level of IkB in Jurkat cells was estimated by western bolting. Jurkat cells were transfected with Ad-peptide or AdGFP followed with an additional 5minues or 10minues’stimulation of TNF-a.【Results】Part one. PIN involving in the anti-inflammation effect of EsA1. Dose-response relationship of EsA to RAW 264.7 cells pre-stimulated with LPS was explored. Concentration of 100uM was selected as a favorable working concentration.2. Time-respones relationship of EsA to RAW 264.7 cells pre-stimulated with LPS was explored as well. 3 hour was the perfect time-window to test the mRNA expression of TNF-a.3. The promotion effect of EsA on cAMP compared with that of forskolin in RAW264.7 cell. There was no difference between them. (p>0.05)4. TNF-a decreased or increased significantly in PIN up-expressed cell or down- expressed cell, respectively; (p<0.05); NO releasing pattern was similar with that of TNF-a in relevant cells.5. The regulatory effect of EsA on TNF-a expression was attenuated significantly in PIN-interfered cells compared with control ones(p<0.05). It was shown that the regulatory effect of EsA to inflammation was PIN-dependant.Part two. Inhibitory effect of the peptide with PIN-binding motif on phosphorylation of IkB and production of IL-8 in Jurkat cells1. The PIN-binding peptide, PIN-binding mutant peptide and control marking protein (GFP) in replication-deficient recombinant adenoviruses were established.2. Administration of Ad-PIN-binding-peptide promoted the production of IL-8 in Jurkat cells significantly, comparing with that of mutant peptide or control. (p<0.05) 3. None of Ad-peptides effected the phosphorylation of IkB.【Conclusion】1. EsA negatively regulated TNF-a in LPS-inducting macrophages. EsA up-regulates the cAMP to promote the transcription of PIN; PIN involved in the down-regulator effect of EsA; the regulatory effect of EsA on inflammation was PIN-dependant.2. PIN-binding peptide promoted the production of IL-8 yet has no effction to phosphorylation of IkB in Jurkat cell.更多还原