【作者】 李翔；

【导师】 文继舫；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 前言:矽肺是由二氧化硅（SiO₂）粉尘引起的、以肺纤维化为主的,呈不可逆性进展的组织破坏性疾病。早期病变以肺间质炎症、肺泡巨噬细胞（AM）聚集及肺泡上皮细胞损伤与修复为主要特征。在参与矽肺纤维化发生、发展的多种细胞中,以活化的AM在纤维化起始及进展过程中的作用最为关键。AM是肺内重要的防御细胞,它不仅可以吞噬外来的有害物质,还可以合成和释放大量具有多种生物活性的细胞因子、前炎症介质、趋化因子及蛋白酶类等,这些因子的生物学作用相互交叉与重叠,并相互影响,从而构成复杂的细胞因子网络,在肺炎症/肺纤维化发生发展过程中发挥极其重要的作用。其中转化生长因子（TGF）-β1和肿瘤坏死因子（TNF）-α是研究最深入、最关键的细胞因子,前者对成纤维细胞有趋化作用和致有丝分裂活性,可刺激细胞外基质和平滑肌肌动蛋白α基因表达,改变成纤维细胞收缩表型、调控肺泡Ⅱ型细胞增生。后者的作用之一是能诱导AM产生TGF-β1,我们前期的实验已经证实SiO₂能诱导二者在RAW264.7细胞中表达明显增高。MAPKs激酶可对多种细胞外刺激起反应而活化,在调节细胞生长、分化和凋亡的信号转导过程中发挥了重要的作用。大部分信号转导通路通过转录因子作用于靶基因。MAPKs家族成员磷酸化常常导致下游核转录因子如NF-KB、AP-1、Erg-1等的活化,从而引起下游因子表达的改变。已有文献报道SiO₂能诱导肺纤维化效应细胞的MAPK和AP-1的活化,因此我们假设MAPKs/AP-1信号通路在SiO₂诱导RAW264.7细胞TNF-α和TGF-β1表达中发挥作用,此研究尚未见文献报道。目的:探讨MAPKs/AP-1信号传导通路在SiO₂诱导RAW264.7细胞TNF-α和TGF-β1表达中的作用。方法:小鼠巨噬细胞（RAW264.7）培养后用100μg/ml的SiO₂刺激至预定的时间。我们利用western blotting检测p-p38激酶,p-Erk,p-JNK以及核蛋白中AP-1（c-jun/c-fos）表达;不同浓度的SB203580和PD98059预处理细胞分别阻断p38激酶和Erk活性,不同浓度的Curcumin（AP-1的阻断剂）预孵育细胞来阻断AP-1活性,C-Jun显性负性突变体（TAM67）被转染进细胞阻断AP-1的活性,AP-1 DNA结合活性用EMSA检测;TNF-α和TGF-β1的mRNA和蛋白表达用RT-PCR和ELISA检测。结果:（1）在RAW264.7细胞中,SiO₂诱导磷酸化的p38激酶和Erk活性增高,均于120min到达高峰,但磷酸化的JNK没有表达。（2）SB203580和PD98059能分别阻断p-p38激酶和p-Erk的活性,并呈剂量依赖性,50μMPD98059能明显抑制SiO₂诱导的TNF-α和TGF-β1的表达,但20μM SB203580没有这个作用。（3）SiO₂刺激3h后AP-1DNA结合活性明显增高,6h恢复到基础水平;PD98059能明显抑制SiO₂诱导的AP-1DNA结合活性,而SB203580没有抑制作用。（4）10μM Curcumin能抑制SiO₂诱导的核蛋白AP-1（c-jun/c-fos）的表达,20μM Curcumin抑制作用最强;20μMCurcumin能明显抑制TNF-α和TGF-β1的表达。（5）TAM67能明显下调SiO₂诱导的AP-1DNA结合活性,并抑制TNF-α和TGF-β1的表达。结论:1.SiO₂可诱导RAW264.7细胞TNF-α和TGF-β1表达。2.SiO₂可活化RAW264.7细胞MAPKs/AP-1信号通路。3.首次发现Erk/AP-1信号通路参与调节了SiO₂诱导RAW264.7细胞产生的TNF-α和TGF-β1表达。4.首次发现Curcumin在SiO₂刺激RAW264.7细胞产生的TNF-α和TGF-β1表达中起抑制作用,为肺纤维化治疗提供了新策略。更多还原

【Abstract】 Backgrond:Silicosis is a sort of destructive lung disease caused by inhalation of crystalline silica,and is characterized by interstitial pulmonary fibrosis.The early pathological processes of silicosis include: interstitial inflammation,assembling of alveolar macrophages（AM）, damage and repair of alveolar epithelial cell.Among the cells participated in pulmonary fibrosis,the activated AM play an important role in the progress of silicosis.Macrophages are the initial source of inflammatory mediators produced in response to silica exposure that include oxygen radicals, leukotrienes,cytokines,and growth factors.These factors interact each other and compose complicated cytokine network which play important roles in pulmonary inflammation and fibrosis.Transforming growth factor-β1（TGF-β1） and tumor necrosis factor-α（TNF-α） are the most key cytokines.TGF-β1 has the function of chemotaxis and mitogen activation on fibroblasts and stimulates the expression of extracellular matrix andα-smooth muscle actin（α-SMA）,which can change fibroblast phenotype and regulate the proliferation of alveolar typeⅡepithelial cells.One of the TNF-α’s function is to induce AM produce TGF-β1.Our previous studies have proved that SiO₂ can induce overexpression of TGF-β1 and TNF-αin RAW264.7 cells.Mitogen-activated protein kinase（MAPKs） are activated in response to a variety of extracellular stimuli and play complex roles in the regulation of different fundamental cellular processes,such as cell proliferation,differentiation and apoptosis.Signal transduction pathways affecting target genes are via nuclear transcription factors.The phosphorylation of MAPKs leads to the activation of transcription factors（NF- K B,AP-1,Erg-1）,which regulate the expression of downstream target genes.Some studies have demonstrated that SiO₂ can induce activation of MAPK and AP-1,so we hypothesis that MAPKs/ activator protein-1（AP-1） play important roles in the silica-induced TNF-αand TGF-β1 expression in macrophages,which remains to be determined.Objective:The present study was to investigate the role of MAPKs/ AP-1 signaling pathways in silica-induced TNF-αand TGF-β1 expression in macrophages.Methods:Murine macrophage cells（RAW264.7） were cultured and then stimulated with 100μg/ml SiO₂ for indicated times.We use Western blotting to assay AP-1（c-jun/c-fos） in nuclear protein and the phosphorylation of MAPKs（p38 kinase,Erk and JNK）.SB203580, PD98059 and Curcumin were added into medium to prevent the activiy of p38 kinase,Erk and AP-1,respectively.C-Jun dominant negative mutant（TAM67） was transfected into cells to prevent AP-1 activity.AP-1 DNA binding activity was measured by electrophoretic mobility shift assay（EMSA）.The expression of TNF-αand TGF-β1 were detected by RT-PCR and ELISA.Results:（1） SiO₂ activated p38 kinase and Erk,but not JNK in RAW264.7 cells.（2） Erk inhibitor（PD98059） and p38 kinase inhibitor （SB203580） inhibited silica-induced Erk and p38 kinase activity, respectively.The induction of TGF-β1 and TNF-αby SiO₂ was suppressed by 50μM PD98059,but not 20μM SB203580.（3） AP-1 DNA binding activity induced by SiO₂ increased after 3h and decreased after 6h.AP-1 DNA binding activity induced by SiO₂ was abolished by PD98059,not by SB203580.（4） AP-1 inhibitor Curcumin inhibited silica-induced AP-1（c-jun/c-fos） expression in nuclear protein.Curcumin downregulated the expression of TGF-β1 and TNF-αinduced by silica. （5）TAM67 inhibited the silica-induced AP-1 DNA binding activity, TNF-αand TGF-β1 expression.Conclusion:SiO₂ induced TGF-β1 and TNF-αexpression in RAW264.7 cells. SiO₂ induced the activation of MAPKs/AP-1 pathways in RAW264.7 cells.Our study is the first report to demonstrate that Erk/AP-1 signal pathways play important roles in silica-induced expression of TGF-β1 and TNF-αin RAW264.7 cells.Our study is the first report to Curcumin inhibited the expression of TGF-β1 and TNF-αin RAW264.7 cells.It provides new therapeutic options for silicosis.更多还原