关键词： 胰腺癌   长链非编码RNA   LncRNA-MTA2TR   MTA2   ATF3   低氧   HIF-1α  

摘要： 【目的】胰腺癌是全世界第七大癌症死亡原因,尽管近年来取得了很多研究和治疗进展,但其5年生存率仍不到5%。缺乏有效的早期检测方法是该疾病患者生存不良的最重要原因之一。广泛的肿瘤异质性,以及肿瘤上皮和微环境内成分之间的相互作用网络是决定胰腺癌预后不良的生物学特征。最近的研究表明,长链非编码RNA(Inc RNAs)在细胞生长、分化、增殖、侵袭和转移中具有多种生物学功能,在许多癌症中起着至关重要的作用,而lnc RNA在胰腺癌低氧微环境中的功能尚不明确。这项研究的目的是阐明低氧环境下lnc RNA-MTA2TR(转移相关蛋白2转录调节因子RNA,AF083120.1)在胰腺癌中发挥的调控作用。【方法】通过对2个胰腺癌病人的胰腺癌(PC)和癌旁的组织(NP)进行高通量测序,分析了在胰腺癌中表达变化有意义的lnc RNA。通过分析,我们选择了MTA2基因附近转录的lnc RNA-MTA2TR作为研究对象。通过实时荧光定量PCR评估了其在40对胰腺癌和癌旁组织及各种胰腺癌细胞中的表达。单分子RNA荧光原位杂交(FISH)和Northern印记实验确定了lnc RNA-MTA2TR在胰腺癌细胞中的核定位和表达。通过构建lnc RNAMTA2TR的小干扰RNA(si RNA)和过表达质粒,下调或上调胰腺癌细胞中的lnc RNAMTA2TR的水平。MTT和集落形成实验用来测定细胞的增值能力,transwell和划痕实验用来检测细胞的侵袭和转移能力。通过异种种植模型,将转染了敲低lnc RNA-MTA2TR的病毒细胞注入裸鼠体内,进一步探究lnc RNA-MTA2TR对细胞增殖和转移能力的影响。为进一步探索lnc RNA-MTA2TR在胰腺癌中的作用机制,使用实时荧光定量PCR和Western blot用来检测MTA2在lnc RNA-MTA2TR上调或下调细胞中m RNA和蛋白质的表达水平。荧光素酶报告实验用来检测lnc RNA-MTA2TR对MTA2启动子活性的影响。RNA免疫共沉淀(RIP)和RNA pulldown实验用来证明lnc RNA-MTA2TR与转录激活因子3(ATF3)的结合。染色质免疫共沉淀(Ch IP)实验用来证明,转录因子ATF3与MTA2的启动子区域结合。免疫荧光和RNA-FISH共染色,用来证明lnc RNA-MTA2TR与ATF3结合。此外,实时荧光定量PCR和RNA-FISH用来检测lnc RNA-MTA2TR在低氧条件下胰腺癌细胞中的表达。Ch IP实验用来证明低氧诱导因子-1α(HIF-1α)与lnc RNA-MTA2TR启动子的结合。荧光素酶报告实验用来检测HIF-1α对lnc RNA-MTA2TR启动子活性的影响。蛋白质免疫共沉淀(Co-IP)用来检测lnc RNA-MTA2TR对HIF-1α与MTA2结合的影响。通过免疫荧光和RNA-FISH共染色,我们检测了组织中lnc RNA-MTA2TR和HIF-1α的表达。Kaplan-Meier分析了lnc RNA-MTA2TR对胰腺癌患者生存率的影响。皮尔森相关分析用来分析胰腺癌患者中lnc RNA-MTA2TR与MTA2表达之间的关系。【结果】芯片结果和实时荧光定量PCR结果显示,lnc RNA-MTA2TR在胰腺癌患者癌组织中呈现高水平表达。体外和体内实验结果显示,敲低lnc RNA-MTA2TR显著抑制胰腺癌细胞的增殖和侵袭能力。机制研究表明,lnc RNA-MTA2TR通过将转录因子ATF3募集到MTA2的启动子区域来促进MTA2启动子的活性,从而上调MTA2的表达。另外,我们发现lnc RNA-MTA2TR在低氧下表达上调。进一步的机制研究发现,HIF-1α可以结合在lnc RNA-MTA2TR启动子的低氧反应元件(HRE)上,从而增强lnc RNA-MTA2TR的启动子活性和转录。我们之前的研究表明,MTA2可以通过脱乙酰作用稳定HIF-1α蛋白,从而进一步激活HIF-1α转录活性。我们的实验结果显示,敲低lnc RNA-MTA2TR,MTA2与HIF-1α结合减少,HIF-1α的蛋白稳定性下降。此外,lnc RNA-MTA2TR可以促进MTA2转录,MTA2与HIF-1α结合增加,HIF-1α的脱乙酰化作用增加,从而促进HIF-1α的蛋白稳定性。我们的临床数据进一步表明,lnc RNA-MTA2TR与MTA2的表达呈正相关,且lnc RNA-MTA2TR表达上调与胰腺癌患者的总体生存率降低有关。【结论】低氧诱导的lnc RNA-MTA2TR将ATF3募集到MTA2启动子并调节MTA2的表达,通过依赖MTA2的脱乙酰基作用增强HIF-1α的蛋白稳定性,从而形成了lnc RNA-MTA2TR与HIF-1α的正反馈环路。本研究表明HIF-1α/lnc

关键词： 山羊   5-HT   全转录组测序分析   竞争内源性RNA   乳腺上皮细胞   钙代谢  

摘要： 围产期奶山羊由于胎儿生长、产道收缩和形成初乳需要消耗大量钙离子,因此血液中钙离子浓度迅速降低,使围产期的奶山羊极易发生低血钙症,进而引发产后瘫痪、乳腺炎、酮病、胎衣不下等疾病。5-羟色胺(5-hydorxytyrptamine,5-HT)可以动员骨骼中的钙释放到血液中,进而平衡围产期奶山羊体内的钙稳态。乳腺上皮细胞的钙代谢和凋亡对奶山羊的泌乳性能有至关重要的影响,但5-HT调控山羊乳腺上皮细胞钙代谢和凋亡的分子机制尚不明确。本研究通过给围产期奶山羊注射5-羟色氨酸(5-HTP,5-hydroxytryptophan),提高奶山羊体内的5-HT浓度,对分娩当天的血液进行全转录组测序分析,筛选出受5-HT浓度正调控并且与钙代谢和细胞凋亡相关的基因,并在山羊乳腺上皮细胞中进行功能验证,研究获得主要结果如下:1.围产期注射5-HTP奶山羊的血液全转录组分析通过在预产期前7天每天给奶山羊按体重(0.8 mg/kg)静脉注射5-HTP(处理组)或生理盐水(对照组),采集分娩当天血液样本进行全转录组测序分析,筛选到受5-HT调控的差异表达的基因999个、LncRNA 967个、Circ RNA 95个、micro RNA 87个。通过功能注释和富集分析发现这些差异表达基因和转录本主要影响钙代谢、脂代谢、免疫反应、细胞增殖和细胞凋亡等生理过程。根据竞争内源性RNA(Competition Endogeno us RNA,ce RNA)作用机制,利用Cytoscape3.6.1构建了LncRNA/Circ RNA-micro RNAm RNA调控网络,并对网络中的差异表达基因作富集分析,结果发现有3个与钙代谢相关的基因,分别是Toll样受体9(Toll Like Receptor 9,TLR9)、缝隙连接蛋白γ2(Ga p Junction Protein Gamma 2,GJC2)和连接素1(Junctophilin1,JPH1)。具体的调控关系为LncRNA MSTRG.101405.3和MSTRG.63774.3都可以分别与JPH1和GJC2竞争性地结合micro RNA NC＿030814.1＿19917,进而调控JPH1和GJC2的表达,LncRNA MST RG.1354.2可以TLR9竞争性地结合micro RNA NC＿030825.1＿46027,进而调控TLR9的表达。***3对山羊乳腺上皮细胞钙离子浓度和细胞凋亡的影响研究围产期注射5-HTP奶山羊的血液转录组分析得到受5-HT调控的差异表达基因999个,结合分析GEO数据库中注射5-HTP的小鼠乳腺转录组测序数据,筛选出受5-HT浓度正调控的ATF3基因,并在山羊乳腺上皮细胞中对其功能进行验证。用5-HTP处理山羊乳腺上皮细胞后,RT-q PCR检测ATF3和钙代谢相关基因的表达变化,荧光染料检测细胞内钙离子浓度变化。结果发现5-HTP处理山羊乳腺上皮细胞后ATF3的表达量升高(p<0.001),这与小鼠和山羊的转录组测序分析结果一致。5-HTP处理山羊乳腺上皮细胞后细胞内的钙离子浓度降低(p<0.05),细胞内与钙代谢相关的基因表达也发生变化,其中SPCA1的表达升高(p<0.001),SPCA2和SERCA2的表达降低(p<0.001)。山羊乳腺上皮细胞中过表达/干扰ATF3后,检测细胞内钙离子浓度变化和凋亡情况,发现ATF3可以降低细胞内的钙离子浓度,并促进山羊乳腺上皮细胞凋亡。以上研究表明,奶山羊产前注射5-HTP使体内血液中5-HT浓度升高,分娩当天血液全转录组测序分析发现5-HT主要影响钙代谢、脂代谢、细胞增殖、细胞凋亡、免疫反应等生理过程。在ce RNA调控网络中发现TLR9、GJC2和JPH1基因与钙代谢相关,在离体培养的山羊乳腺上皮细胞中,5-HT可通过促进ATF3的表达降低细胞内的钙离子浓度,并促进山羊乳腺上皮细胞凋亡。

关键词： CHO细胞   CRISPR/Cas9   ATF3   CK2α   稳定细胞株  

摘要： 背景和目的:中国仓鼠卵巢细胞(CHO)是重组蛋白、单克隆抗体等生物大分子药物生产的主要宿主细胞之一,尤其适合需要复杂翻译后修饰的分子类型,例如需糖基化的细胞因子、各类全长单克隆抗体等。然而,与大肠杆菌、酵母等表达系统相比,以CHO细胞为代表的哺乳动物细胞表达系统的培养成本较高,导致产品价格昂贵、病人难以负担。因此,提高CHO工程细胞株的生长性能及外源蛋白表达能力,对降低药物成本、进而提高市场可及性具有重要意义。以往研究证实,培养基和培养条件优化、筛选外源基因整合位点等方式能够有效提高CHO的生长及表达水平,而近年来,随着高通量测序、高效基因编辑等新兴技术等的涌现,使得功能基因筛选及CHO细胞的精确基因编辑成为可能。本实验室前期进行了CHO细胞的DNA甲基转移酶3A(DNMT3a)敲除,在此基础上通过RNA-seq发现转录激活因子3(ATF3)基因受DNMT3a敲除的影响出现转录水平上调,可能与CHO细胞的生长、代谢及表达水平的变化相关。另一方面,本实验室采用规律成簇的间隔短回文重复/规律成簇的间隔短回文重复结合蛋白9(CRISPR/Cas9)建立了CHO细胞的基因敲除及外源基因整合技术,并筛选到C12orf35、HPRT等稳定性较好的外源基因整合位点,可用于基因定点整合的稳定细胞株建立。本研究中,我们结合前期研究及文献检索,选取具有细胞生长代谢调节功能的ATF3及酪蛋白激酶Ⅱ的α亚基(CK2α)两个基因,深入研究其过表达对CHO细胞的作用。方法:为了探究ATF3和CK2α两个基因对CHO细胞增殖和外源蛋白表达的作用,首先我们构建了瞬时表达ATF3和CK2α的质粒,并将这两种质粒各自转染到红色荧光蛋白(mCherry)稳定表达的CHO细胞株KCSG6中,通过转染后1～5天细胞计数、培养结束时m Cherry蛋白的表达水平检测,来初步评价ATF3和CK2α基因过表达对CHO细胞生长及蛋白表达的影响。为了更严谨地研究ATF3和CK2α两个基因的功能,我们采用CRISPR/Cas9介导的稳定细胞株构建技术,选取HPRT作为整合位点,选取向导RNA(sgRNA)序列,并设计和构建定点整合的Donor质粒ATF3-Donor、CK2α-Donor和ATF3+CK2α-Donor。在KCSG6细胞株上通过电转染、加压筛选以及5’/3’junction PCR的鉴定,获得相应的稳转细胞池,并通过有限稀释法铺板,对得到的单克隆细胞进行5’/3’junction PCR和OUT-OUT PCR的鉴定和测序等鉴定,挑选KCSG6细胞株背景下的ATF3、CK2α以及ATF3+CK2α过表达的细胞株。通过对所筛选到的稳定细胞株进行插入基因表达验证、生长特性以及模式蛋白表达水平检测,并进一步探究插入基因对CHO细胞凋亡和相关信号通路的影响,系统地研究ATF3和CK2α两个基因对CHO细胞的作用和机制。结果:通过瞬时转染ATF3和CK2α质粒进入KCSG6细胞,我们发现ATF3和CK2α对细胞增殖及蛋白表达均具有促进趋势。经过CRISPR/Cas9基因编辑技术将ATF3基因、CK2α基因以及ATF3+CK2α基因成功插入KCSG6细胞株的HPRT位点,最终成功得到3株稳转细胞株:ATF3-8、CK2α-3、AC-2。通过对这三株细胞进行相关性质的探究,证明目的基因定点整合到HPRT位点,并得到成功表达,且ATF3和CK2α对CHO细胞增殖具有促进作用,CK2α对mCherry总蛋白的产量具有促进作用。通过对比这3株细胞株与对照细胞株的细胞凋亡,我们发现AC-2细胞株的ATF3+CK2α过表达具有抑制细胞凋亡的作用,而单独的ATF3或CK2α基因无明显作用;通过对这3株细胞株的信号通路进行初步探究,发现相对于空白细胞,这3株细胞的ERK信号通路水平上调。其中,对DNMT3a敲除的CHO细胞进一步研究发现,ATF3基因的启动子区胞嘧啶-磷酸-鸟嘌呤(CpG)甲基化水平受DNMT3a调控,这对理解ATF3的调控机制具有一定的启示。综上,本研究证实ATF3基因以及CK2α基因过表达对CHO细胞增殖具有促进作用且CK2α对于细胞总蛋白表达具有促进作用,提示该细胞改造策略具有重要意义。本研究中我们还获得了ATF3及CK2α基因过表达的CHO稳定细胞株,可结合细胞培养工艺优化等进行更深入的研究,不断提高CHO细胞培养和蛋白生产水平。

关键词： ATF3/MAP2K3/p38 MAPK信号通路   活血益气组分配伍中药   能量代谢   心肌梗死   心室重构   炎症  

摘要： 研究目的最新流行病学研究表明,中国地区居民急性心肌梗死(Acute myocardial infarction,AMI)的发病率仍保持上升趋势,且渐趋年轻化。急性心肌梗死后心室重构(Ventricular remodeling,VR)是心肌梗死后心脏出现的一种代偿性、继发性的复杂病理生理过程,涉及多种影响因素。长期持续的心室重构导致心脏结构和功能异常,最终诱发心力衰竭或死亡,严重影响患者的生活质量,是影响心血管病患者死亡率的决定因素之一。因此,抑制或逆转心室重构过程已成为预防和治疗心肌梗死后患者心室重构的关键环节。临床研究表明,急性心肌梗死后心室重构患者基本中医证型为气虚血瘀证,或兼挟其他证型。活血益气中药及其有效组分作为基本方在治疗心室重构方面取得了显著的临床疗效,尤其是有效组分中药,其成分相对明确,药理作用较为清晰,且制剂多样、携带方便,具有广阔的应用前景。国内外研究表明,三七总皂苷(Panaxnotoginsengsaponins,PNS)具有扩张血管、改善微循环、减少心肌耗氧量等药理作用;丹参酮IIA(Tanshinone IIA,Tan IIA)能通过抑制心肌纤维化,减少心肌细胞凋亡和炎症反应,改善心脏神经重构和电生理异常等;人参总皂苷(Total ginsenosides,TGS)显著的耐缺氧作用可增强心肌收缩力,加快脂质代谢和能量转化过程,从而改善AMI后的心功能和血流动力学指标,三者是本院院内制剂活血化瘀方主要组成药物的中药有效成分,单独应用可在不同层面上保护心肌梗死后心血管系统重构,而三者联合使用干预急性心肌梗死后心室重构还未有研究。最新文献研究表明,ATF3/MAP2K3/p38 MAPK信号通路可以通过调节NFκB相关因子的表达影响心肌缺血期的炎症浸润,同时抑制TGF-β1和Collagenl.的表达,降低心肌成纤维细胞功能,保护心肌细胞及其细胞外基质的重构;还能通过减少心肌细胞中Runx1、提高PPARα的表达改善心肌细胞的能量代谢从而防治心肌梗死后心室重构。根据导师的多年临床用药经验和本课题组前期的研究基础,结合网络药理学预测药物-疾病靶点,本研究探索活血益气组分配伍中药PNS、TanⅡA、TGS联合用药,以PNS为君,TanⅡ 和 TGS 分别佐使,通过调节 ATF3、MAP2K3、p38MAPK、TGF-β1、Collagen I、NFκB p65、IκB、Runx1、PPARα 等基因和蛋白的表达,从而激活 ATF3/MAP2K3/p38 MAPK信号通路,发挥防治心肌梗死后心室重构的作用,为将活血益气组分中药开发成防治急性心肌梗死后心室重构的临床新药提供扎实有力的实验依据。研究方法结扎健康的SPF级SD(Sprague Dawley)雄性大鼠心脏冠状动脉左前降支血管,构建心肌梗死动物模型。将造模成功的大鼠随机均分为六组:模型组(MI)、福辛普利组(Fosinopril,FIP,1.2mg/kg·d)、三七总皂苷组(PNS,40mg/kg·d)、丹参酮IIA 组(TanⅡA,70mg/kg·d)、人参总皂苷(TGS,30 mg/kg·d)、组分配伍组(Component compatibility,CC,PNS 40mg/kg-d+Tan IIA 70mg/kg.d+TGS 30mg/kg.d);假手术组(Sham)大鼠在冠状动脉左前降支相同位置只穿线不结扎,作为阳性对照。各给药组以灌胃方式给予相应药物,MI组大鼠和Sham组大鼠给予去离子水,连续给药4周。4周后检测各组大鼠心脏超声后取材,计算各组大鼠心脏质量指数(Heartweight/bodyweight,HW/BW);用HE和Mansson染色法观察大鼠心脏的病理学改变和心肌纤维化程度;用ELISA法检测大鼠血清中心室重构标志物CRP、TNF-α、GDF-15和MMP9/TIMP1水平以及大鼠心肌组织中游离脂肪酸(Free fatty acid,FFA)含量;用高效液相色谱法检测大鼠心肌组织中ATP、ADP、AMP水平;用化学比色法检测大鼠心肌组织中Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶含量;用免疫组织化学法、Western blotting法和RT-PCR法检测大鼠心肌组织 ATF3/MAP2K3/p38 MAPK 信号通路中 ATF3、MAP2K3、p38 MAPK、NFκB p65、TGF-β1、Collagen I、Runx1、PPARα等相关蛋白和基因的表达水平。研究结果1活血益气组分配伍中药对心肌梗死大鼠心脏功能和结构的影响1.1超声心动图检测大鼠心脏,结果显示,与假手术组大鼠相比,模型组大鼠心脏左室收缩末期内径(LVIDs)、左室舒张末期内径(LVIDd)明显升高,左室射血分数(LVEF)和左室缩短分数(LVF

关键词： Alcoholic liver disease   RNA-seq   Liver   NMN   ATF3   ERK1/2   Sirtuin  

摘要： Background: Chronic alcohol consumption is a significant cause of liver disease worldwide. Several biochemical mechanisms have been linked to the initiation and progression of alcoholic liver disease (ALD) such as oxidative stress, inflammation, and metabolic dysregulation, including the disruption of NAD+/NADH. Indeed, an ethanol-mediated reduction in hepatic NAD(+) levels is thought to be one factor underlying ethanol-induced steatosis, oxidative stress, steatohepatitis, insulin resistance, and inhibition of gluconeogenesis. Therefore, we applied a NAD(+) boosting supplement to investigate alterations in the pathogenesis of early-stage ALD. Methods: To examine the impact of NAD(+) therapy on the early stages of ALD, we utilized nicotinamide mononucleotide (NMN) at 500 mg/kg intraperitoneal injection every other day, for the duration of a Lieber-DeCarli 6-week chronic ethanol model in mice. Numerous strategies were employed to characterize the effect of NMN therapy, including the integration of RNA-seq, immunoblotting, and metabolomics analysis. Results: Our findings reveal that NMN therapy increased hepatic NAD(+) levels, prevented an ethanol-induced increase in plasma ALT and AST, and changed the expression of 25% of the genes that were modulated by ethanol metabolism. These genes were associated with a number of pathways including the MAPK pathway. Interestingly, our analysis revealed that NMN treatment normalized Erk1/2 signaling and prevented an induction of Atf3 overexpression. Conclusions: These findings reveal previously unreported mechanisms by which NMN supplementation alters hepatic gene expression and protein pathways to impact ethanol hepatotoxicity in an early-stage murine model of ALD. Overall, our data suggest further research is needed to fully characterize treatment paradigms and biochemical implications of NAD(+)-based interventions.

关键词： Mitochondrial oxidative stress   Activating transcription factor 3   Inflammation   Brain microvascular endothelial cells   Lipolysis   Triglyceride-rich lipoproteins   RNA-Seq  

摘要： Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of th

关键词： Diabetes   Drug discovery   Medical research   Obesity   Transcription  

摘要： Billions of people have obesity-related metabolic syndromes such as diabetes and hyperlipidemia. Promoting the browning of white adipose tissue has been suggested as a potential strategy, but a drug still needs to be identified. Here, genetic deletion of activating transcription factor 3 (ATF3(-/-)) in mice under a high-fat diet (HFD) resulted in obesity and insulin resistance, which was abrogated by virus-mediated ATF3 restoration. ST32da, a synthetic ATF3 inducer isolated from Salvia miltiorrhiza, promoted ATF3 expression to downregulate adipokine genes and induce adipocyte browning by suppressing the carbohydrate-responsive element-binding protein-stearoyl-CoA desaturase-1 axis. Furthermore, ST32da increased white adipose tissue browning and reduced lipogenesis in HFD-induced obese mice. The anti-obesity efficacy of oral ST32da administration was similar to that of the clinical drug orlistat. Our study identified the ATF3 inducer ST32da as a promising therapeutic drug for treating diet-induced obesity and related metabolic disorders.

关键词： Forensic pathology   Skin contusion   Vital reaction   ATF3   BTG2  

摘要： The detection of vitality of wounds, especially when the wounds are inflicted very close to the time of death, is one of the most challenging issues in forensic pathology. This study investigated expression levels of ATF3 and BTG2 in mouse and human skin wounds. Protein levels examined by western blot showed that there was no significant change in ATF3 and BTG2 between wounded and intact skins. However, mRNA levels demonstrated higher expression of ATF3 and BTG2 in ante-mortem contused mouse skins, compared with the intact and postmortem contused skins. Increased ATF3 and BTG2 in the level of mRNA could also be detected until 96 h and 48 h after death, respectively. Human wounded skin samples from forensic autopsy cases were also examined. Increased ATF3 mRNA levels were detected until 48 h after autopsy in 5 of 6 cases. However, no differences were observed between wounded and intact skins for BTG2. These findings suggest that the detection of mRNA levels of ATF3, but not BTG2, can be considered as a potential marker for vital reaction of skin contusion. Postmortem human samples should be used in order to validate the availability of markers screened by animal experiment. (C) 2019 Elsevier B.V. All rights reserved.

关键词： Oxaliplatin   Metformin   Neurotoxicity  

摘要： Oxaliplatin is a third-generation platinum drug commonly used as the first line treatment of metastatic colorectal cancer. Oxaliplatin-based anticancer regimens course with dose-limiting neurotoxicity. The pharmacological strategies used to manage such side effect are not totally effective. Metformin is an anti-diabetic drug that is described to negatively modulate painful diabetic neuropathy. Then, this study aimed to assess the effect of metformin in the oxaliplatin-induced peripheral sensory neuropathy in mice. For that purpose, Swiss male mice were injected with oxaliplatin (1, 2 or 4 mg/kg, i.v., twice a week with a total of nine injections) alone or in combination with daily administration of metformin (250 mg/kg, p.o.). Thermal and mechanical nociceptive tests were performed once a week for five weeks. Then, the animals were euthanized on day 35 post-first injection of oxaliplatin and the dorsal root ganglia were harvested for the assessment of c-Fos and ATF3 expressions. Oxaliplatin caused a nociceptive response accompanied by the increased expression of c-Fos and ATF3 in the dorsal root ganglia and spinal cord. In addition, the oxaliplatin-associated nociception was significantly attenuated by metformin (P < 0.05), which also reduced the expression of c-Fos and ATF3 (P < 0.05). Therefore, metformin protected from the peripheral sensory neuropathy induced by oxaliplatin, which was confirmed by the reduction of c-Fos and ATF3 expression, two known neuronal activation and damage markers, respectively.

关键词： Pirfenidone   Activating transcription factor 3 (ATF3)   Rheumatoid arthritis   Interstitial lung disease  

摘要： Objective: Pirfenidone (PFD) is an oral anti-fibrotic drug used for idiopathic pulmonary fibrosis (IPF) therapy. We determined the role of activating transcription factor 3 (ATF3) and the effect of PFD on fibroblast to myofibroblast transition (FMT) in rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Methods: RA-ILD lung specimens were obtained by CT-guided percutaneous transthoracic biopsy. A pulmonary fibrosis mouse model was established by the intratracheal injection of bleomycin. Pathological variation and the expression of alpha-SMA and ATF3 were observed by H&E, Masson and immunofluorescence staining. Primary human lung fibroblasts (pHLFs) were isolated from lung tissues that were pathologically confirmed to be normal by pneumonectomy. Cell viability was detected using an MTT assay. Cell migration and invasion were detected using a Transwell chamber. The protein levels of alpha-SMA, ATF3, Smad3 and p-Smad3 were measured by Western blot. HLFs were infected with lentiviruses expressing ATF3 or scrambled shRNA. Results: ATF3 was dramatically upregulated in lung tissues from both bleomycin-induced mice and patients with RA-ILD compared with controls. The upregulation of ATF3 and the accumulation of collagen in the lung tissues of mice with pulmonary fibrosis were reduced by PFD. PFD significantly inhibited increases in the proliferation, invasion and migration of pHLFs stimulated by TGF-beta 1. Moreover, we observed the inhibitory effect of PFD on FMT via the downregulation of ATF3, which was further confirmed in ATF3 knockdown (KD) pHLFs. Conclusions: This work shows the inhibitory effect of PFD on FMT in pHLFs, which is mediated by the downregulation of ATF3. Our findings suggest that PFD might have therapeutic potential for the treatment of RA-ILD.