关键词： PM2.5   凋亡   炎症   卵巢颗粒细胞   ATF3  

摘要： 目的:PM2.5伴随着呼吸进入血液循环,到达全身各个系统,对人体有着潜在的危害。根据调查显示PM2.5对女性生殖系统可造成损伤,尤其是对卵巢功能造成一定损伤。然而,其潜在的影响机制还不明确。本实验是通过气管滴入PM2.5对大鼠进行造模,研究其卵巢损害;观察不同浓度的PM2.5与人卵巢颗粒细胞系(KGN)共培养后细胞活性变化,对细胞周期与凋亡的影响;提取细胞总RNA进行测序寻找靶标基因,并进行机制研究。初步讨论不同浓度的PM2.5处理KGN细胞可引起细胞活性下降,周期和细胞凋亡率的改变。方法:1.采用30只3周龄大小的雌性SD大鼠,随机分为3个组,对照组、低剂量组、高剂量组,每组10只,每隔3天进行一次气管滴注PM2.5,连续滴注5个月,对照组滴注PBS,低剂量组浓度为35 μg/m3,高剂量组浓度为150 μg/m3,5月末取大鼠卵巢,进行HE染色,在显微镜下进行阅片;2.将不同浓度的PM2.5与KGN共培养后,在不同时间点采用Cell Titer-Glo试剂进行细胞活性测定;***暴露于不同浓度的PM2.5后,分别收取24 h和36h的细胞,使用AnnexinV-FITC染料进行细胞凋亡检测;4.使用不同浓度的PM2.5处理KGN,收取细胞,使用PI染料进行细胞周期检测;5.将不同浓度的PM2.5与KGN共培养后,分别收取24 h和36 h的细胞,使用DCFH-DA染料进行细胞活性氧ROS的检测;***暴露于100 μg/mL的PM2.5后,收集24 h后的对照组与实验组总RNA进行RNA-seq测序,所有样本均在Novogene(HiSeq 4000)平台上测序;7.使用不同浓度的PM2.5处理KGN,收集不同时间点的细胞上清液,用于ELIAS试剂盒检测IL-6和TNFα细胞因子的浓度。结果:1.慢性暴露于PM2.5中可引起SD大鼠卵巢炎症改变和卵泡闭锁,影响卵巢储备功能。***2.5可抑制卵巢颗粒细胞活性,呈浓度与时间依赖性改变;***2.5可诱导卵巢颗粒细胞周期阻滞于S期,且浓度越大,S期阻滞越明显。***2.5可促进卵巢颗粒细胞发生凋亡,且浓度越大,凋亡率越明显。***2.5可增加卵巢颗粒细胞炎症因子分泌,IL-6和TNFα呈浓度依赖性改变。***-seq测序数据分析发现343个差异表达基因,其中发现181个基因表达上调,162个基因部分表达被抑制,KEGG通路富集于Apotosis、NF-κB、TNF等信号通路上。***3抑制PM2.5对卵巢颗粒细胞的活性毒性、凋亡发生、炎症的改变及氧化应激水平的作用。结论:1.经气管滴注PM2.5可诱导SD大鼠卵巢发生炎症反应,卵泡闭锁增多。2.暴露于PM2.5的卵巢颗粒细胞系(KGN)可引起细胞活力下降,细胞凋亡增加,细胞周期阻滞于S期,并触发炎症与氧化应激反应。***2.5通过激活转录因子ATF3,在体外抑制卵巢颗粒细胞细胞凋亡水平,减轻炎症和氧化应激反应。

关键词： 神经病理性疼痛   DRG   mRNA测序   CB1受体   CB2受体   ATF3   NPY  

摘要： 研究背景和目的:神经病理性疼痛(Neuropathic pain,NP)是由躯体感觉神经系统的损伤或疾病直接导致的疼痛,其临床表现为自发性疼痛、痛觉过敏、痛觉超敏和感觉异常等。内源性大麻素在痛觉调节中发挥重要作用,其镇痛作用的发挥主要是通过大麻素受体实现的。大麻素1型受体(Type 1 cannabinoid receptors,CB1受体)是一种Gi/o蛋白偶联受体,主要位于神经元的突触前膜,参与内源性大麻素介导的镇痛作用。CB1受体激动剂能否缓解周围神经损伤引起的神经病理性疼痛目前仍有争议,同时其产生的中枢神经系统副作用也限制了它的临床应用。因此将CB1受体作为神经病理性疼痛的镇痛靶点并不理想。本研究旨在寻找背根神经节(Dorsal root ganglion,DRG)中CB1受体调控的下游致痛基因,探究CB1受体及相关分子在神经病理性疼痛中的作用机制,为神经病理性疼痛的临床治疗提供潜在靶点和理论证据。方法:(1)建立神经病理性疼痛动物模型——坐骨神经慢性缩窄性损伤模型(Chronic constriction injury,CCI),并进行痛行为学检测。对CCI模型大鼠L46 DRG进行RNA测序(RNA sequencing,RNA-seq),采用q PCR和免疫荧光技术(Immunofluorescence,IF)验证差异基因(Differentially expressed genes,DEGs)。(2)腹腔注射CB1受体激动剂或拮抗剂,观察激活或拮抗CB1受体对正常小鼠及CCI模型小鼠痛行为学的影响。利用Advillin-Cre ERT2小鼠和CB1fl/fl小鼠杂交产生CB1 advillin-Cre ERT2小鼠(即初级感觉神经元中CB1受体特异性敲除小鼠,以下简称CB1 c KO小鼠)。检测CB1受体敲除前后及CCI造模后CB1 c KO小鼠的痛行为学变化。对TM诱导14天、28天以及CCI造模后的CB1 c KO小鼠的DRG组织进行m RNA测序,采用q PCR和IF对CB1受体相关分子如大麻素2型受体(Type 2 cannabinoid receptors,CB2受体)、激活转录因子3(Activating transcription factor 3,ATF3)和神经肽Y(Neuropeptide Y,NPY)等进行验证。(3)应用CB2受体拮抗剂AM630、NPY sh RNA和ATF3 sh RNA分别对DRG上CB2受体、NPY、ATF3进行干预,检测其对痛行为学的影响。采用q PCR和IF技术检测DRG中CB2受体、GFAP、S100a8、S100a9、Csf1r、NPY和ATF3 m RNA及蛋白水平的变化。结果:(1)CCI造模可诱导SD大鼠和C57/BL6小鼠产生长期持续的神经病理性疼痛。RNA测序、q PCR及IF证实DRG中CB1受体在CCI造模后显著下调。(2)拮抗CB1受体可诱发正常小鼠的生理性痛觉过敏,并进一步加重CCI诱导的神经病理性疼痛,而激活CB1受体不能改善神经病理性疼痛。同时,DRG初级感觉神经元中CB1受体敲除会诱发生理性痛觉过敏并加重神经病理性疼痛。结合大、小鼠的测序结果,最终筛选出3个与CB1受体相关的分子靶点:CB2受体、NPY和ATF3。(3)DRG中的CB2受体在野生小鼠和CB1 c KO小鼠CCI造模后显著升高。拮抗CB2受体显著缓解CB1 c KO小鼠CCI造模前后的机械痛觉超敏和热痛过敏,并抑制CCI造模引起的DRG中GFAP、Csf1r、S100a8和S100a9 m RNA的上调。拮抗CB2受体也可以改善野生CCI模型小鼠的神经病理性疼痛,但激活CB2受体无镇痛作用。(4)NPY和ATF3的表达在CB1 c KO以及CCI造模后均显著升高。CCI造模后,ATF3在DRG神经元的细胞核中显著增多,并与NPY存在共定位。沉默ATF3和NPY均能挽救CB1 c KO以及野生型小鼠CCI造模导致的机械痛觉超敏。细胞实验及在体实验均显示NPY受到ATF3的正向调控。结论:(1)初级感觉神经元中CB1受体参与生理条件下基础痛阈的维持,并在神经病理性疼痛中发挥镇痛作用。(2)CB2受体、ATF3和NPY是CB1受体调控的致痛靶点。(3)在神经病理痛的维持期,CB2受体的激活促进疼痛的发展,拮抗CB2受体可缓解疼痛。(4)初级感觉神经元中CB1受体通过靶向抑制ATF3/NPY通路发挥镇痛作用。

关键词： ATF3   Glioma   PHGDH   Serine synthesis   TFCP2  

摘要： Glioma is one of the most common malignancies. De novo serine synthesis promotes glioma progression and therapeutic resistance. Therefore, clarifying the regulatory mechanism of serine synthesis is of great significance for glioma therapy. In this study, we found that the expression of TFCP2 was upregulated in glioma and that TFCP2 promoted glioma cell growth and sphere formation. Knockdown of TFCP2 expression inhibited glioma cell growth, sphere formation and tumorigenicity in nude mice. In terms of its molecular mechanism, TFCP2 was found to interact with ATF3 to cooperatively regulate the de novo synthesis of serine. Knockdown of TFCP2 expression significantly inhibited the binding of ATF3 to the promoter of PHGDH (a rate-limiting enzyme in the serine synthesis process). In conclusion, our studies proved that TFCP2 jointly regulates the de novo synthesis of serine through interaction with ATF3, thus promoting glioma progression. This study suggests that TFCP2 is a potential target for glioma therapy. © 2022 The Authors

关键词： 原发性胆汁性胆管炎   激活转录因子3(ATF3)   信号转导抑制因子-3(SOCS3)   免疫  

摘要： [目 的]本课题拟探讨原发性胆汁性胆管炎（primary biliary cholangitis,PBC）患者肝脏组织中激活转录因子 3（activating transcription factor 3,ATF3）和信号转导抑制因子-3（suppressor of cytokine signalling-3,SOCS3）的表达及其与PBC疾病进展的相关性;探讨ATF3与SOCS3在PBC发病中的可能作用。[方 法]收集昆明医科大学第二附属医院经病理确诊为PBC患者的肝脏穿刺组织共13例,其中早期患者（Ⅰ期:胆管炎期、Ⅱ期:汇管区周围炎期）6例,晚期患者（Ⅲ期:进行性肝纤维化期、Ⅳ期:肝硬化期）7例。免疫组织化学染色检测PBC患者肝穿组织中ATF3和SOCS3的表达,双重免疫荧光染色检测PBC患者肝组织中ATF3与CD68、CD4、CD8共定位情况,观察ATF3和SOCS3在PBC肝组织中的表达,分析ATF3和SOCS3的表达与PBC疾病进展的相关性。体外培养人单核细胞THP-1,并利用佛波酯将其分化为巨噬细胞,以1ug/ml 脂多糖（lipopolysaccharide,LPS）分别刺激 THP-1 和 THP-1 来源巨噬细胞构建体外炎症模型;LPS刺激后利用RT-qPCR和Western-blot分别检测THP-1和巨噬细胞中ATF3、SOCS3的表达;以携带ATF3-shRNA的慢病毒转染THP-1细胞,采用RT-qPCR和ELISA检测巨噬细胞中IL-6、TNF-α、IL-10的表达变化,Western-blot检测巨噬细胞中SOCS3的蛋白质表达变化;染色质免疫共沉淀（CHIP）检测巨噬细胞中ATF3与SOCS3启动子结合情况;ATF3抑制后在LPS刺激下Western-blot检测SOCS3/p-STAT3通路蛋白STAT3、p-STAT3表达情况。[结 果]PBC患者肝穿组织免疫组化染色结果示ATF3阳性表达于炎性浸润部位,且随疾病进展表现出降低趋势;双重免疫荧光染色结果示ATF3与CD68、CD4、CD8共定位,在kupffer细胞、CD4+T淋巴细胞、CD8+T淋巴细胞中阳性表达;SOCS3免疫组化染色结果示SOCS3在PBC患者肝组织炎性浸润部位阳性表达,且早期较晚期显著。在THP-1细胞中,LPS显著诱导ATF3和SOCS3的表达,差异有统计学意义（P＜0.05）,而在THP-1来源巨噬细胞中,ATF3和SOCS3表现出降低趋势（P＜0.05）;ATF3-shRNA抑制ATF3基因表达后,ATF3的mRNA和蛋白质表达显著降低（P＜0.05）;与阴性对照组相比,LPS诱导下 ATF3-shRNA 组中 IL-6、TNF-α、IL-10 表达显著升高（P＜0.05）;ATF3下调后Western-blot检测显示巨噬细胞中SOCS3蛋白表达降低（P＜0.05）,CHIP显示ATF3与SOCS3启动子结合;在LPS刺激下,与阴性对照组相比,ATF3-shRNA 组 p-STAT3 活化增强。[结 论]ATF3和SOCS3在PBC患者肝脏组织炎性浸润部位高表达,均表现出早期高于晚期的趋势;在THP-1来源巨噬细胞中,LPS刺激下ATF3起负性调节炎症因子的作用,ATF3可能通过SOCS3/p-STAT3信号通路调节免疫反应参与PBC的发生发展。

关键词： bacteraemia   blood transcriptome analysis   febrile neutropenia   immune profiling   paediatric cancer   RNAseq   DIFFERENTIAL EXPRESSION ANALYSIS   SEPSIS   FEVER   ATF3   ACTIVATION   REGULATOR   MODEL  

摘要： Objectives. Febrile neutropenia (FN) is a major cause of treatment disruption and unplanned hospitalization in childhood cancer patients. This study investigated the transcriptome of peripheral blood mononuclear cells (PBMCs) in children with cancer and FN to identify potential predictors of serious infection. Methods. Whole-genome transcriptional profiling was conducted on PBMCs collected during episodes of FN in children with cancer at presentation to the hospital (Day 1; n = 73) and within 8-24 h (Day 2; n = 28) after admission. Differentially expressed genes as well as gene pathways that correlated with clinical outcomes were defined for different infectious outcomes. Results. Global differences in gene expression associated with specific immune responses in children with FN and documented infection, compared to episodes without documented infection, were identified at admission. These differences resolved over the subsequent 8-24 h. Distinct gene signatures specific for bacteraemia were identified both at admission and on Day 2. Differences in gene signatures between episodes with bacteraemia and episodes with bacterial infection, viral infection and clinically defined infection were also observed. Only subtle differences in gene expression profiles between non-bloodstream bacterial and viral infections were identified. Conclusion. Blood transcriptome immune profiling analysis during FN episodes may inform monitoring and aid in defining adequate treatment for different infectious aetiologies in children with cancer.

关键词： ATF3   TGF-81   NFATC2   ACTIVATING TRANSCRIPTION FACTOR-3   TRANSFORMING GROWTH-FACTOR-BETA-1   NUCLEAR FACTOR   T-CELLS   C-JUN   COLLAGENASE-3   DIMERIZATION   METASTASIS   PROMOTER   TARGET  

摘要： Activating transcription factor 3 (ATF3), an inducible stress gene, is stimulated by transforming growth factor-beta1 (TGF-81) in a protracted and relentless manner in human mammary cancer cells (hBC cells; MDA-MB231). The molecular mechanism behind this stable expression of ATF3 via TGF-81 in MDA-MB231 cells is unknown. This study found that TGF-81 stimulated the expression of the nuclear factor of activated T Cells 2 (NFATC2) in MDA-MB231 cells and provided evidence of its interaction with ATF3. The functional characterization of NFATC2 in association with ATF3 was determined by silencing of NFATC2 using siRNA. Knock-down of NFATC2 decreased the expression of both ATF3 and its target gene MMP13 (matrix metalloproteinase 13, a critical invasive gene) in hBC cells. Chromatin immunoprecipitation revealed that TGF-81 promoted NFATC2 binding and NFATC2-ATF3 complex binding at the MMP13 promoter region, whereas silencing of NFATC2 decreased their binding in hBC cells. Thus, we uncovered the mechanism of interaction between NFATC2 and ATF3 regulated by TGF-81, and NFATC2 acted as a pivotal factor in providing ATF3 stability and further drove MMP13 transcription. Targeting NFATC2 and blocking its association with ATF3 could therefore help to slow the pro-gression of breast cancer.

关键词： 结直肠癌干细胞   活化转录因子3   自我更新   上皮间质转化   免疫抑制因子  

摘要： 目的研究敲减活化转录因子3(ATF3)对结直肠癌肿瘤干细胞(CRC-CSCs)自我更新能力与上皮间质转化的影响,并检测免疫抑制因子分泌的变化。方法培养人结直肠癌细胞系HCT116,通过流式细胞术分选CD133+表达的CRC-CSCs细胞,转染sh-ATF3慢病毒载体至CRC-CSCs细胞作为实验组(sh-ATF3组),以转染sh-NC慢病毒载体作为阴性对照组(sh-NC组),正常培养的CRC-CSCs细胞作为对照组。实时荧光定量PCR(qRT-PCR)检测ATF3相对表达量,CCK-8法检测细胞活性变化,成球实验检测细胞生长更新能力,平板克隆形成实验检测细胞克隆形成能力,免疫荧光染色和蛋白质免疫印迹(Western Blot)法检测E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达,酶联免疫吸附法(ELISA法)检测细胞分泌免疫抑制因子TGF-β1、VEGF、IL-6、IL-10的含量变化,Western Blot检测自杀相关因子(Fas)、Fas配体(FasL)蛋白表达水平。结果对照组比较,sh-ATF3组CRC-CSCs细胞中ATF3 mRNA相对表达量下降(P<0.05),转染后48 h和72 h细胞活性降低(P<0.05),细胞成球能力与克隆形成能力均下降(P<0.01),细胞中E-cadherin蛋白表达较高而Vimentin蛋白表达较低(P<0.05),同时,细胞上清液中TGF-β1、VEGF、IL-10的含量均明显下降(P<0.01),Fas蛋白表达升高,FasL蛋白表达下降(P<0.05)。结论敲减ATF3可抑制结直肠癌肿瘤干细胞自我更新与上皮间质转化,下调免疫抑制因子TGF-β1、VEGF、IL-10表达,并且上调Fas蛋白,下调FasL蛋白的表达。

关键词： 胶质母细胞瘤   替莫唑胺   自噬   转录激活因子3   14-3-3ζ  

摘要： 目的:探讨在替莫唑胺(temozolomide,TMZ)治疗敏感与耐药的胶质母细胞瘤中转录激活因子3 (activatingt ranscription factor3,ATF3)、14-3-3ζ和beclin-1的表达水平变化及其与TMZ耐药产生的相关意义。方法:采用RT-PCR及免疫组织化学法检测并比较同一患者TMZ治疗前及耐药时胶质母细胞瘤组织内ATF3、14-3-3ζ和beclin-1基因及蛋白质表达水平。取30μg/m LTMZ培养的U87细胞为实验组,普通溶剂培养的U87细胞为对照组,培养72h,MTT及annexin V-别藻蓝素(allophycoc yanin,APC)单染法分别检测并比较肿瘤细胞的生长抑制率、凋亡率,RT-PCR及蛋白质印迹检测并比较细胞内ATF3、14-3-3ζ和beclin-1基因及蛋白质表达水平。结果:TMZ耐药时胶质母细胞瘤组织中ATF3、14-3-3ζ和beclin-1表达水平均高于TMZ治疗前(P<0.05);TMZ敏感的U87细胞培养72 h肿瘤细胞生长抑制率和凋亡率均增加(P<0.05),细胞内ATF3、14-3-3ζ和beclin-1表达水平均低于对照组(P<0.05);三者呈同向表达。结论:在TMZ用药过程中,胶质母细胞瘤内ATF3激活可能上调14-3-3ζ的表达,进而启动并使自噬增强,导致TMZ耐药产生。

关键词： Pelvic organ prolapse   Vaginal fibroblasts   Oxidation stress   Extracellular matrix   MMPs/TIMPs   p38/Nrf2 pathway   ACTIVATING TRANSCRIPTION FACTOR-3   LIGAMENT FIBROBLASTS   PREMENOPAUSAL WOMEN   CONNECTIVE-TISSUE   EXPRESSION   EPIDEMIOLOGY   PATHOGENESIS   INFLAMMATION   MECHANISMS   PROTEINS  

摘要： Pelvic organ prolapse (POP) is a common disorder in women, and it is characterized by weakening of pelvic supportive structure with extracellular matrix (ECM) degradation. Activating transcription factor 3 (ATF3) was upregulated in anterior vaginal wall tissues of POP patients. We hypothesized that upregulation of ATF3 might contribute to POP development. This study aims to unveil the role of ATF3 in the pathogenesis of POP using a H2O2-induced in vitro model. Vaginal fibroblasts were isolated from woman with POP-Q stage greater than II and asymptomatic women with normal pelvic floor support. Knockdown of ATF3 enhanced cell viability and decreased cell apoptosis. Flow cytometry and immunnofluorescence showed that ATF3 deficiency inhibited H2O2-induced ROS production and the expression of 8 OHdG and 4-HNE. Western blot and Real-time PCR analysis revealed that ATF3 deficiency attenuated ECM component degradation (increasing collagen I, collagen III and elastin) and MMPs/TIMPs imbalance (decreasing MMP2 and MMP9 and increasing TIMP2). Moreover, knockdown of ATF3 induced the activation of p38/Nrf2/HO-1 signaling pathway. Further treatment with p38 inhibitor SB203580 abolished the protection of ATF3 deficiency against H2O2-induced cell damage, which was reverted by Nrf2 activator TBHQ. Thus, ATF3 likely contributes to POP progression by inducing cell apoptosis, oxidative stress and ECM degradation via regulating p38/Nrf2 pathway, which provides a potential therapeutic target for POP.

关键词： Gastric cancer   ATF3   Cisplatin resistance   Ferroptosis   Nrf2   Keap1 signaling   ACTIVATING TRANSCRIPTION FACTOR-3   CELL-DEATH  

摘要： Background Currently, resistance against cisplatin (DDP) is a frequent problem for the success of advanced gastric carcinoma (GC) chemotherapy. Here, we sought to investigate the function of activating transcription factor 3 (ATF3) n GC chemoresistance. Methods Expression of ATF3 was determined in GC cell lines (MNK45, SGC7901, and BGC823) and cisplatin (DDP)-resistant cells (SGC7901/DDP and BGC823/DDP). Biological informatics was performed to analyze ATF3 expression and prognosis in GC patients. Cisplatin resistance was evaluated. Ferroptosis was detected after ATF3 transfection of cells. The underlying molecular mechanism was also investigated. Results Transcripts of ATF3 were decreased in GC cells and GC tissues. Kaplan-Meier plotter analysis revealed that ATF3 expression was positively related to the overall survival of GC patients. In particular, lower levels of ATF3 were observed in cisplatin-resistant SGC7901/DDP and BGC823/DDP relative to their parental cells. Notably, ATF3 elevation sensitized cisplatin-resistant cells to cisplatin. Mechanically, compared with parental cells, SGC7901/DDP and BGC823/DDP cells exhibited lower ferroptosis evident by lower ROS, MDA and lipid peroxidation and higher intracellular GSH levels. However, ATF3 elevated ferroptosis in SGC7901/DDP and BGC823/DDP cells. Intriguingly, ATF3 overexpression together with ferroptosis activator erastin or RSL3 treatment further enhanced ferroptosis and cisplatin resistance; however, the ferroptosis suppressor liproxstatin-1 reversed the function of ATF3 in ferroptosis and cisplatin resistance. Additionally, cisplatin-resistant cells exhibited stronger activation of Nrf2/Keap1/xCT signaling relative to parental cells, which was restrained by ATF3 up-regulation. Importantly, restoring Nrf2 signaling overturned ATF3-mediated ferroptosis and cisplatin resistance. Conclusion ATF3 may sensitize GC cells to cisplatin by induction of ferroptosis via blocking Nrf2/Keap1/xCT signaling, supporting a pro