关键词： 结缔组织病   肺间质病变   吡非尼酮   TGF-β1   ATF3  

摘要： 研究背景弥漫性结缔组织病(connective tissue diseases,CTDs)是一类由自身免疫异常所致机体损伤的疾病总称,这类疾病几乎可累及到机体的每个器官,肺脏是最常受累的器官之一。其中,肺间质病变(Interstitial lung disease,ILD)是危害最大的并发症,可导致肺通气功能受损甚至呼吸功能衰竭,严重影响患者寿命,迄今为止,CTD-ILD的发病机制尚未完全阐明,缺乏根治性治疗方法。因此,本文拟通过研究新型药物吡非尼酮在体内及体外实验及临床治疗中的抗纤维作用,阐明ILD的部分发生机制及治疗对策,为肺间质病变治疗提供新的证据和思路。研究目的1.观察吡非尼酮治疗CTD-ILD的临床疗效和不良反应。2.了解吡非尼酮对博来霉素诱导肺纤维化小鼠的抗纤维化作用。3.阐明吡非尼酮对HLFs增殖、迁移侵袭、分泌功能和表型转化的影响及其机制。研究方法纳入确诊为CTD-ILD的患者,根据患者本人意愿用或不用吡非尼酮进行12个月治疗观察。在0月、3月、6月和12月时随访进行实验室检查、肺功能测试、6分钟步行距离和HRCT评分,吡非尼酮用药组(PFD)和对照组(CTR)间比较采用协方差分析,其中各组间不同时间点的比较采用重复测量数据的方差分析。8周龄雄性C57BL/6小鼠建立博来霉素诱导鼠肺纤维化模型,采用直视下气管内注射博来霉素或生理盐水,分别给予吡非尼酮或相应体积的溶剂灌胃治疗,均在第28天处死后取肺脏组织,随后进行H&E染色、Masson染色和组织免疫荧光染色观察肺组织病理改变和α-SMA、ATF3的表达。手术获取正常肺组织(经病理证实)进行分离原代人肺成纤维细胞(HLFs),CT引导下经皮肺穿刺活检获取CTD-ILD肺脏病变组织。MTT法检测细胞存活率,EdU法检测细胞的增殖能力,Transwell实验检测细胞的迁移和侵袭能力,细胞免疫荧光检测α-SMA和ATF3,免疫印迹法检测α-SMA、ATF3和p-Smad3的蛋白水平,实时定量PCR检测α-SMA、ATF3和Ⅰ、Ⅲ型胶原蛋白的mRNA表达水平。应用慢病毒载体携带ATF3shRNA或对照shRNA感染体外培养的HLFs并进行验证。研究结果PFD组与CTR组相比,PFD组患者在第12月时FVC、TLC和DLco均得到显著改善(p值分别为0.044、0.017和0.001),而在第6月DLco明显增加(p值为0.033)。在随访期间与初始时间点相比,PFD组TLC在第3月即开始有明显改善,随后维持相同水平(p值分别为0.046、0.030和0.056),FVC和DLco在第6月开始有明显改善(p<0.05)。用吡非尼酮治疗3月后PFD组影像炎症评分均明显低于对照组(p值分别为0.021、0.010和0.036)。在随访期间PFD组炎症评分逐渐降低,而对照组则未观察到这种变化。吡非尼酮用药期间,患者最常出现的不良反应为乏力、呼吸困难、咳嗽、皮疹和消化不良,在临床对症处理下可减轻或消失。在博来霉素诱导小鼠肺间质纤维化模型中,预灌胃吡非尼酮可减少小鼠肺部结构破坏程度,降低胶原沉积,改善肺部纤维化。造模组小鼠肺组织中α-SMA的表达明显高于正常组和吡非尼酮干预组小鼠。CTD-ILD肺组织和造模组小鼠肺组织ATF3的表达明显高于正常组,然而吡非尼酮能降低ATF3在肺组织的表达。在TGF-β1诱导下HLFs的增殖、迁移侵袭能力和分泌功能能力显著增强,p-Smad3和ATF3的蛋白和mRNA表达水平明显高于对照组。吡非尼酮预处理HLFs能有效地抑制TGF-β1诱导的增殖、迁移侵袭能力、分泌功能和运动功能,下调p-Smad3和ATF3的蛋白和mRNA表达水平。ATF3敲低的HLFs其对TGF-β1刺激后的增殖、迁移侵袭能力和α-SMA和p-Smad3的蛋白水平均下降,其反应能力均明显弱于正常的HLFs。研究结论吡非尼酮能改善CTD-ILD患者肺功能结果和影像学评分,减少博来霉素肺纤维化鼠模型肺结构破坏、胶原沉积和纤维化程度,其治疗作用可能是通过抑制ATF3/Smad3通路从而减少HLFs表型转换来实现的。

摘要： Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-alpha, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-kappa B and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia.

关键词： 急性肺损伤   活化转录因子3   基因芯片   差异基因表达  

摘要： 目的利用基因芯片技术筛选LPS诱导的急性肺损伤小鼠肺组织中与活化转录因子3(acting transcription factor 3,ATF3)相关的差异基因表达,以发现与ATF3可能相互作用的基因。方法采用基因芯片技术筛选野生型C57小鼠和ATF3敲基因小鼠经LPS处理后肺组织中的差异表达基因。进一步采用RT-PCR技术对重点基因的表达进行验证。结果与野生型C57小鼠比较,ATF3敲基因小鼠共筛选出1 692个差异基因,有21个与炎症调控相关的基因表达差异明显,其中,Myo18a、Mest、Gpr124、Rcan2、Tnfsf15、Ltbp3、Cdh2、Ppm1f、Spns2、Rarres2、Cgn、Mmrn2、Crb3、Adamts2、Mrc2、Sh3tc2、Cdk5rap1、Cenpf共18个基因在ATF3敲基因小鼠肺组织中表达明显上调;Padi4、Wfdc82个基因表达明显下调。对重点基因Tnfsf15(又称为TL1A)进一步验证,RT-PCR检测结果显示,表达趋势与基因芯片一致。结论 TL1A可能参与了ATF3对LPS诱导的急性肺损伤的炎症保护作用。

关键词： beta-catenin   noncoding RNA   ASBEL   colorectal tumorigenesis   ATF3  

摘要： Wnt/beta-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that beta-catenin directly activates the transcription of the long noncoding RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. ASBEL interacts with and recruits TCF3 to the activating transcription factor 3 (ATF3) locus, where it represses the expression of ATF3. Furthermore, we demonstrate that ASBEL-TCF3-mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL. Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/beta-catenin-mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/beta-catenin signaling.

关键词： ATF3   Human labour   Inflammation   Infection   Fetal membranes  

摘要： Introduction: Infection and inflammation stimulate pro-inflammatory cytokines, prostaglandins and matrix metalloproteinase (MMP)-9, which play a central role in myometrial contractions and rupture of fetal membranes. In human and mouse immune cells, activating transcription factor 3 (ATF3) is a negative regulator of inflammation. No studies have examined the role of ATF3 in human labour. Methods: Primary amnion cells were used to determine the effect of interleukin (IL)-1 beta and the bacterial product fibroblast-stimulating lipopeptide (fsl-1) on ATF3 expression, and the effect of ATF3 siRNA on pro-labour mediators. ATF3 expression was assessed in fetal membranes from non-labouring and labouring women at term and preterm, and after preterm pre-labour rupture of membranes (PPROM). Results: IL-1 beta and fsl-1 significantly increased ATF3 expression. Silencing ATF3 significantly increased IL-1 beta- or fsl-1-induced expression of pro-inflammatory cytokines (TNF-alpha, IL-1 alpha, IL-1 beta, IL-6) and chemokines (IL-8 and monocyte chemoattractant protein-1 (MCP-1));cyclooxygenase-2 (COX-2) mRNA expression and prostaglandin PGF(2 alpha) release;and MMP-9 expression. ATF3 expression was decreased in fetal membranes with term labour. There was no effect of preterm labour or PPROM on ATF3 expression. Discussion: ATF3 is a negative regulator of inflammation in human fetal membranes;in primary amnion cells, ATF3 expression is induced by IL-1 beta and fsl-1, and ATF3 silencing further exacerbates the inflammatory response when stimulated with these factors. Subsequently, ATF3 expression is decreased in fetal membranes after term labour and with preterm chorioamnionitis, conditions closely associated with inflammation and infection. Our data suggest that ATF3 may play a role in the terminal processes of human labour and delivery. (C) 2016 Elsevier Ltd. All rights reserved.

关键词： cAMP response element-binding protein   glucocorticoid   immunosuppression   liver metabolism   S100 proteins  

摘要： Glucocorticoids (GCs) used as inflammation suppressors have harmful side effects, including induction of hepatic steatosis. The underlying mechanisms of GC-promoted dysregulation of lipid metabolism, however, are not fully understood. GCs could facilitate the accumulation of myeloid-derived suppressor cells (MDSC) in the liver of animals, and the potential role of MDSCs in GC-induced hepatic steatosis was therefore investigated in this study. We demonstrated that granulocytic (G)-MDSC accumulation mediated the effects of GCs on the fatty liver, in which activating transcription factor 3 (ATF3)/S100A9 signaling plays an important role. ATF3-deficient mice developed hepatic steatosis and displayed expansion of G-MDSCs in the liver and multiple immune organs, which shared high similarity with the phenotype observed in GC-treated wild-type littermates. Adoptive transfer of GC-induced or ATF3-deficient G-MDSCs promoted lipid accumulation in the liver, whereas depletion of G-MDSCs alleviated these effects. Mechanistic studies showed that in MDSCs, ATF3 was transrepressed by the GC receptor GR through direct binding to the negative GR-response element. S100A9 is the major transcriptional target of ATF3 in G-MDSCs. Silencing S100A9 clearly alleviated G-MDSCs expansion and hepatic steatosis caused by ATF3 deficiency or GC treatment. Our study uncovers an important role of G-MDSCs in GC-induced hepatic steatosis, in which ATF3 may have potential therapeutic implications.

关键词： 子宫颈鳞癌   鳞状细胞癌抗原   激活转录因子3   相关性  

摘要： 目的：研究子宫颈鳞癌患者血清SCC-Ag 及组织ATF3表达与临床分期及肿瘤直径的相关性，为临床治疗提供参考。方法选取我院2014年1月～2016年6月收治的81例宫颈癌患者为观察对象，检测血清SCC-Ag和组织ATF3表达水平，并采用Logistic回归性分析来分析血清SCC-Ag 及组织ATF3的表达与子宫颈鳞癌临床分期及肿瘤直径的相关性。结果患者血清SCC-Ag及组织ATF3检测显示，临床分期从玉到郁期阳性率逐渐递增；肿瘤直径小于或等于4cm的患者SCC-Ag及ATF3表达阳性率明显小于肿瘤直径大于4cm的患者；Logistic回归性分析显示，SCC-Ag及ATF3升高均是子宫颈鳞癌的危险因素。结论血清SCC-Ag、组织ATF3表达均与子宫颈鳞癌的癌变程度相关，患者从玉期到郁期SCC-Ag和ATF3水平均呈上升趋势；肿瘤直径越大，SCC-Ag和ATF3水平越高，联合监测有助于评估患者病情。

关键词： Pterostilbene   ATF3   Apoptosis   Colon cancer   Chemoprevention   ER stress  

摘要： Pterostilbene (Ptero), a major active compound of blue berries, is a natural antioxidant phytochemical which has a strong potential for cancer prevention and intervention. However, it has not been shown the detailed anti-cancer mechanism of action for Ptero which has not fully elucidated. In present study, we report that Ptero treatment in human colon cancer cells inhibits the cell cycle via the suppression of Cyclin D1 expression. This inhibition of Cyclin D1 was associated with changes in the TCF4 expression and activation of PARP cleavage. The induction of apoptotic cells by Ptero was observed by Hoechst staining. We also observed that Ptero promotes to delay S phase into G2/M phase cell cycle transition in colon cancer cells. Analysis of the gene expression profiles by western blotting revealed that Ptero increased the expression of GRP78 protein related to the unfolded protein response. This induction was associated with the activation of the eIF2 alpha-ATF3 cascade of endoplasmic reticulum (ER) stress. Moreover, our result showed that p38MAPK is essential factor for Ptero-induced ATF3 expression. The current study shows that Ptero stimulates GPR78-eIF2 alpha-ATF3 cascade and subsequently induces apoptosis. Our results therefore provide new insights into the molecular mechanisms of Ptero-induced cell death in human colon cancer cells and suggest Ptero might be a potential chemotherapeutic agent which is able to intervene and prevent the human colon cancer. (C) 2016 Elsevier Ltd. All rights reserved.

关键词： FORKHEAD transcription factors   PROLACTIN   ENDOMETRIUM   EMBRYO transplantation   STROMAL cells   OVEREXPRESSION (Genetics)   IMMUNOFLUORESCENCE  

关键词： Experimental allergic encephalomyelitis   Demyelination   ATF3   Dorsal root ganglion   Spinal cord   Myelin oligodendrocyte glycoprotein   Pertussis toxin  

摘要： Experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis produced by immunization with myelin oligodendrocyte glycoprotein (MOG) and adjuvants, results from profound T-cell mediated CNS demyelination. EAE is characterized by progressive, ascending motor dysfunction and symptoms of ongoing pain and hypersensitivity, in some cases preceding or concomitant with the motor deficits. In this regard, the EAE model mimics major features of multiple sclerosis, where a central neuropathic pain state is common. Although the latter condition is presumed to arise from a CNS loss of inhibitory controls secondary to the demyelination, dysfunction of sensory neurons may also contribute. Based on our previous studies that demonstrated the utility of monitoring expression of activating transcription factor 3 (ATF3), a sensitive marker of injured sensory neurons, here we followed both ATF3 and CD4 + T cells invasion of sensory ganglia (as well as the CNS) at different stages of the EAE model. We found that ATF3 is induced in peripheral sensory ganglia and brainstem well before the appearance of motor deficits. Unexpectedly, the ATF3 induction always preceded T cell infiltration, typically in adjacent, but non-overlapping regions. Surprisingly, control administration of the pertussis toxin and/or Complete Freund's adjuvants, without MOG, induced ATF3 in sensory neurons. In contrast, T cell infiltration only occurred with MOG. Taken together, our results suggest that the clinical manifestations in the EAE result not only from central demyelination but also from neuronal stress and subsequent pathophysiology of sensory neurons. (C) 2016 Published by Elsevier Inc.