A Nonradioactive Method for Detecting DNA-binding Activity of Nuclear Transcription Factors

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-κB were labled with DIG by terminal transferase After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8 % nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film The results showed that nuclear proteins binded specifically to the NF-κB consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-κB in PMA group was more than that in PMA+PDTC group It is suggested that detection of NF-κB by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories

Nuclear reprogramming: the zygotic transcription program is established through an“erase-and-rebuild” strategy

卵母细胞显示母亲特定的基因表达式侧面，它被换到当染色质的一个单倍体集合被传递到发展成一个胚胎的受精卵时， zygotic 介绍。位于这抄写 reprogramming 下面的机制当前是未知的。等到当抄写在幼芽的泡卵母细胞被关掉时，一般抄写因素和 transcriptional 管理者的一个范围从染色质被分裂，这里，我们表明那。染色质因素(CF ) 的全球分离破坏在染色质和 CF 之间的物理接触并且在功能的水平导致母亲的抄写节目的檫除。与染色质论坛分开的批评抄写因素和管理者遗体延长了时期，并且立即在专业版以后与染色质成为 re-associated 原子形成。Thisis 由象 DNA 和抄写那样的原子功能的重建跟随了速度集合。我们建议母亲的抄写节目在卵子发生期间被擦掉产生相对天真的染色质， zygotic 抄写节目是在授精以后的重建的重新。这个过程作为“ erase-and-rebuild ”被称为过程，它被用来重设抄写节目，和最可能的其它原子过程也，从母亲的到胚胎的。我们进一步在伴随的纸出现(高 T，等，房间物件 2007；17:135-150 .) 一样的策略也被采用到改编 transcriptional，这在体细胞介绍原子转移和单性生殖，建议这当模特儿在早胚胎开始期间对 transcriptional reprogramming 的所有形式普遍适用。从染色质的 CF 的排水量也为抄写沉默的现象提供解释在期间对 zygotic 转变母亲。

Transcription factors specificity protein and nuclear receptor 4A1 in pancreatic cancer

Specificity protein(Sp)transcription factors(TFs)Sp1,Sp3 and Sp4,and the orphan nuclear receptor 4A1(NR4A1)are highly expressed in pancreatic tumors and Sp1 is a negative prognostic factor for pancreatic cancer patient survival.Results of knockdown and overexpression of Sp1,Sp3 and Sp4 in pancreatic and other cancer lines show that these TFs are individually pro-oncogenic factors and loss of one Sp TF is not compensated by other members.NR4A1 is also a prooncogenic factor and both NR4A1 and Sp TFs exhibit similar functions in pancreatic cancer cells and regulate cell growth,survival,migration and invasion.There is also evidence that Sp TFs and NR4A1 regulate some of the same genes including survivin,epidermal growth factor receptor,PAX3-FOXO1,α5-andα6-integrins,β1-,β3-andβ4-integrins;this is due to NR4A1 acting as a cofactor and mediating NR4A1/Sp1/4-regulated gene expression through GC-rich gene promoter sites.Several studies show that drugs targeting Sp downregulation or NR4A1 antagonists are highly effective inhibitors of Sp/NR4A1-regulated pathways and genes in pancreatic and other cancer cells,and the triterpenoid celastrol is a novel dual-acting agent that targets both Sp TFs and NR4A1.

The heterodimeric structure of heterogeneous nuclear ribonucleoprotein C1/C2 dictates 1,25-dihydroxyvitamin D-directed transcriptional events in osteoblasts

Heterogeneous nuclear ribonucleoprotein （hnRNP） C plays a key role in RNA processing but also exerts a dominant negative effect on responses to 1,25-dihydroxyvitamin D （1,25（OH）2D） by functioning as a vitamin D response element-binding protein （VDRE-BP）. hnRNPC acts a tetramer of hnRNPC1 （huC1） and hnRNPC2 （huC2）, and organization of these subunits is critical to in vivo nucleic acid-binding. Overexpression of either huC1 or huC2 in human osteoblasts is sufficient to confer VDRE-BP suppression of 1,25（OH）2D-mediated transcription. However, huC1 or huC2 alone did not suppress 1,25（OH）2D-induced transcription in mouse osteoblastic cells. By contrast, overexpression of huC1 and huC2 in combination or transfection with a bone-specific polycistronic vector using a ＂self-cleaving＂ 2A peptide to co-express huC1/C2 suppressed 1,25D-mediated induction of osteoblast target gene expression. Structural diversity of hnRNPC between human/NWPs and mouse/rat/rabbit/dog was investigated by analysis of sequence variations within the hnRNP CLZ domain. The predicted loss of distal helical function in hnRNPC from lower species provides an explanation for the altered interaction between huC1/C2 and their mouse counterparts. These data provide new evidence of a role for hnRNPC1/C2 in 1,25（OH）2D-driven gene expression, and further suggest that species-specific tetramerization is a crucial determinant of its actions as a regulator of VDR-directed transactivation.

Hepatitis C Virus non-structural 5A abrogates signal transducer and activator of transcription-1 nuclear translocation induced by IFN-α through dephosphorylation

AIM: To study the effect of Hepatitis C virus non- structural 5A (HCV NS5A) on IFNα induced signal transducer and activator of transcription-1 (STAT1) phosphorylation and nuclear translocation. METHODS: Expression of STAT1 Tyr701 phosphorylation at different time points was confirmed by Western blot, and the time point when p-STAT1 expressed most, was taken as the IFN induction time for further studies. Immunocytochemistry was used to confirm the successful transient transfection of NS5A expression plasmid. Immunofluorescene was performed to observe if there was any difference in IFNα-induced STAT1 phosphorylation and nuclear translocation between HCV NS5A-expressed and non-HCV NS5A-expressed cells. Western blot was used to compare the phosphorylated STAT1 protein of the cells. RESULTS: Expression of HCV NS5A was found in the cytoplasm of PCNS5A-transfected Huh7 cells, but not in the PRC/ CMV transfected or non-transfected cells. STAT1 Tyr701 phosphorylation was found strongest in 30 min of IFN induction. STAT1 phosphorylation and nuclear import were much less in the presence of HCV NS5A protein in contrast to PRC/CMV-transfected and non-transfected cells under fluorescent microscopy, which was further confirmed by Western blot. CONCLUSION: HCV NS5A expression plasmid is successfully transfected into Huh7 cells and HCV NS5A protein is expressed in the cytoplasm of the cells. IFN-α is able to induce STAT1 phosphrylation and nuclear translocation, and this effect is inhibited by HCV NS5Aprotein, which might be another possible resistance mechanism to interferon alpha therapy.

Isoflavone Attenuates the Nuclear Transcription Factor Kappa B (NF-<i>κ</i>B) Activation on MPP⁺-Induced Apoptosis of PC12 Cells

Objective: To explore the underlying molecular mechanisms of cellular response to the challenge by 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of PC12 cells, an in vitro cell model for Parkinson’s disease, and the effect of NF-κB activation on the protection of Parkinson’s disease by Isoflavone (I). Methods: PC12 cells were used to establish the cell model of Parkinson’s disease, and are divided into five groups: control group;MPP+ group;I (Isoflavone) + MPP+ group;I group;SN-50 + MPP+ group. The content of NF-κB in PC12 cells was determined by immunocytochemistry;The viability of PC12 cells after treated with cell-permeable NF-κB inhibitor SN-50 and cell viability were measured by MTT assay;the expression levels of NF-κB p65 in cytoplasm and nuclear fractions were evaluated by western blot analysis;the mRNA expression of NF-κB p65 was analyzed by in situ hybridization (ISH). Results: Compared with the control group, the protein of NF-κB p65 both in cytoplasm and in nuclei was significantly higher than in I + MPP+ and MPP+ groups;similarly, the mRNA expression level of NF-κB p65 gene was also significantly higher;moreover, the protein expression of NF-κB p65 was much lower in I group (P + group, the protein of NF-κB p65 was significantly lower in I + MPP+ group, the mRNA expression level of NF-κB p65 gene was also significantly lower, and the protein expression level of NF-κB p65 was much lower in I + MPP+ group (P + group (P > 0.05). Conclusion: NF-κB activation is essential to MPP+-induced apoptosis in PC12 cells;but Isoflavone can inhibit the cell damage to some extent to execute its protective function, which may be involved in nigral neurodegeneration in patients with Parkinson’s disease.

Clinicopathologic significance of expression of nuclear factor-kB RelA and its target gene products in gastric cancer patients

AIM:To assess the prognostic significance of nuclear factor-kB (NF-kB) and its target genes in gastric cancer. METHODS:The tumor tissues of 115 patients with gastric cancer were immunohistochemically evaluated using monoclonal antibodies against NF-kB RelA. Preoperative serum levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) were assessed via enzyme-linked immuno-sorbent assay. C-reactive protein (CRP) and serum amyloid A (SAA) were measured via immunotrubidimetry. RESULTS:Positive rate of NF-kB RelA was 42.6%. NF-kB RelA expression in tumor tissues was also related to serum levels of IL-6 (P = 0.044) and CRP (P = 0.010). IL-6, SAA, CRP were related to depth of invasion, VEGF and SAA were correlated with lymph node metastasis. IL-6, VEGF, SAA and CRP were related to the stage. Univariate analysis demonstrated that immunostaining of NF-kB RelA, levels of IL-6, VEGF, SAA were significantly related with both disease free survival and over-all survival (OS). Multivariate analysis verified that NF-kB RelA [hazard ratio (HR): 3.40, P = 0.024] and SAA (HR: 3.39, P = 0.045) were independently associated with OS. CONCLUSION: Increased expression of NF-kB RelA and high levels of serum SAA were associated with poor OS in gastric cancer patients.

NMR assignments and characterization of the DNA-binding domain of Arabidopsis transcription factor WRKY11--Dedicated to Professor Xiuwen Han on the occasion of her 80th birthday

The WRKY proteins are a family of plant-specific transcription factors(TFs)that are widely involved in plant development and anti-stress responses.Arabidopsis WRKY11(AtWRKY11)functions in regulating plant defense against abiotic stress and belongs to the Ild subgroup of WRKY TFs.We herein report the expres sion,purification and preliminary structural characterization of AtWRKY11 DNA-binding domain(DBD)using solution NMR Almost complete backbone chemical shift assignments of AtWRKY11-DBD have been ob-tained.Chemical shift-based secondary structure analysis suggests that AtWRKY11-DBD may exhibit local conformational diferences from the X-ray structure of the C-terminal WRKY domain of AtWRKY1,particularly in the β1 and β5 strands.Our current study provides the basis for further structural and interactional studies.

Prospects for inhibiting the post-transcriptional regulation of gene expression in hepatitis B virus

There is a continuing need for novel antivirals to treat hepatitis B virus(HBV) infection, as it remains a ma-jor health problem worldwide. Ideally new classes of antivirals would target multiple steps in the viral life-cycle. In this review, we consider the steps in which HBV RNAs are processed, exported from the nucleus and translated. These are often overlooked steps in the HBV life-cycle. HBV, like retroviruses, incorporates a number of unusual steps in these processes, which use a combination of viral and host cellular machinery. Some of these unusual steps deserve a closer scrutiny. They may provide alternative targets to existing anti-viral therapies, which are associated with increasing drug resistance. The RNA post-transcriptional regula-tory element identified 20 years ago promotes nucleo-cytoplasmic export of all unspliced HBV RNAs. There is evidence that inhibition of this step is part of the antiviral action of interferon. Similarly, the structured RNA epsilon element situated at the 5' end of the poly-cistronic HBV pregenomic RNA also performs key roles during HBV replication. The pregenomic RNA, which is the template for translation of both the viral core and polymerase proteins, is also encapsidated and used in replication. This complex process, regulated at the epsilon element, also presents an attractive antiviral target. These RNA elements that mediate and regu-late gene expression are highly conserved and could be targeted using novel strategies employing RNAi, miRNAs or aptamers. Such approaches targeting these functionally constrained genomic regions should avoid escape mutations. Therefore understanding these regulatory elements, along with providing potential targets, may also facilitate the development of other new classes of antiviral drugs.

Secoemestrin C Ameliorates Psoriasis-like Skin Inflammation in Mice by Suppressing the TNF-α/NF-κB Signaling Pathway

Objective Secoemestrin C(SC),an epitetrathiodioxopiperazine isolated from Aspergillus nidulans,has been previously reported to have immunomodulatory and hepatoprotective effects against acute autoimmune hepatitis.However,the effect of SC on regulating the inflammation and its underlying mechanisms in the pathogenesis of psoriasis remain unclear.This study aimed to evaluate the effects of SC on inflammatory dermatosis both in vitro and in vivo.Methods In vitro,HaCaT cells were induced with tumor necrosis factor-alpha(TNF-α,10 ng/mL)to establish an inflammatory injury model,and the expression of nuclear transcription factor-κB(NF-κB)pathway components was measured using qRT-PCR and Western blotting.An in vivo mouse model of imiquimod(IMQ)-induced psoriasis-like skin inflammation was used to evaluate the effectiveness of SC in alleviating psoriasis.Results SC significantly blocked the activation of NF-κB signaling in TNF-α-stimulated HaCaT cells.In addition,systemic and local administration of SC improved psoriatic dermatitis in the IMQ-induced mouse model.SC reduced skin scale and significantly inhibited the secretion of inflammatory factors in skin lesions.Conclusion The protective effect of SC against psoriatic-associated inflammation reveals its potential therapeutic value for treating psoriasis.

大豆GmNF-YA13互作蛋白的筛选及鉴定

大豆GmNF-YA13蛋白是一个核转录因子Y(NF-Y),在干旱和高盐响应过程中均发挥重要作用。为研究其抗旱和耐盐的作用机理,寻找GmNF-YA13的互作蛋白,构建pGBKT7-GmNF-YA13诱饵载体,采用酵母双杂交筛选大豆酵母文库,并进行X-α-gal染色验证。结果显示:酵母双杂交获得85个阳性克隆,测序分析后得到36个候选的互作蛋白。功能预测显示互作蛋白主要参与生长发育、胁迫响应、能量代谢、转录调控和信号转导等生物过程。选择GmUVR8、GmCML41、GmFbox13和GmFBA与诱饵pGBKT7-GmNF-YA13进行一对一验证,只有GmFBA能与GmNF-YA13发生相互作用,预示GmNF-YA13功能的发挥需要GmFBA的参与。该结果可为NF-YA抗逆分子网络的研究提供基础。

五味子乙素通过TLR4/NF-κB信号通路对急性胰腺炎大鼠肺部损伤的影响

目的:探讨五味子乙素通过Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路对急性胰腺炎(AP)大鼠肺部损伤的影响。方法:取SD大鼠,通过胆胰管内逆行注射5%牛磺胆酸钠方法诱导建立AP肺损伤模型,经随机数表法分为模型组、五味子乙素组、TLR4过表达载体组、TLR4空载组、五味子乙素+TLR4过表达载体组,每组12只大鼠,再取12只SD大鼠仅翻动肠管不注射5%牛磺胆酸钠,作为假手术组。以药物分别干预大鼠后,检测各组大鼠肺功能及各组大鼠腹水量与肺组织湿重/干重(W/D);HE染色检测各组大鼠肺组织病理形态并评分;检测各组大鼠动脉血气;全自动生化分析仪检测大鼠血清淀粉酶,ELISA检测炎症细胞因子IL-6、IL-18水平;蛋白免疫印迹法检测肺组织TLR4/NF-κB通路蛋白表达;免疫组织化学染色检测肺组织TLR4蛋白表达。结果:与假手术组相比,模型组大鼠肺组织出现病理损伤改变,模型组大鼠MV、PEF、PaO_(2)、OI显著降低(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平显著升高(P<0.05)。与模型组、五味子乙素+TLR4过表达载体组分别相比,五味子乙素组大鼠肺组织病理损伤改变程度均减轻,MV、PEF、PaO_(2)、OI均升高(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平均降低(P<0.05);TLR4过表达载体组大鼠肺组织病理损伤改变程度均加重,MV、PEF、PaO_(2)、OI均降低(P<0.05),Ri、腹水量与W/D、PaCO_(2)、Holfbauer评分、血清淀粉酶、IL-6与IL-18水平、肺组织TLR4阳性细胞比例、TLR4与MYD88蛋白表达、p-NF-κB p65/NF-κB p65水平均升高(P<0.05)。与模型组相比,TLR4空载组大鼠各指标差异无统计学意义(P>0.05)。结论:五味子乙素可通过下调TLR4/NF-κB信号通路,抑制炎症,减轻AP大鼠肺部损伤,修复肺功能。

姜黄素抑制NF-κB信号通路缓解氧化应激对成骨分化的损害发挥抗骨质疏松作用

目的 探讨姜黄素抑制氧化应激对成骨分化损害的机制及以剂量依赖的方式发挥抗骨质疏松的作用。方法 采用细胞氧化应激模型,加入不同浓度的姜黄素,测定骨形成指标,并检测参与的潜在信号通路。同时,用姜黄素处理小鼠去卵巢(ovariectomized, OVX)骨质疏松动物模型来证实其抗骨质疏松的作用。结果 体外实验发现,低浓度姜黄素(1~10μmol·L^(-1))促进成骨细胞增殖,提高骨形成碱性磷酸酶(alkaline phosphatase, ALP)活性,逆转氧化应激导致的成骨钙沉积下降,降低了核因子kappa-B配体的受体激动剂(RANKL)和白介素-6 (IL-6)的表达。体内实验结果显示,姜黄素(5 mg·kg^(-1))给药后部分逆转了OVX小鼠血液中丙二醛(malondialdehyde, MDA)和谷胱甘肽(glutathione, GSH)活性的比例、降低高骨代谢、增加骨密度(bone density, BMD)、改善了骨小梁的微结构,但高浓度姜黄素无氧化应激保护作用。结论 姜黄素可以减轻氧化应激的骨形成损害,NF-κB信号通路是主要参与通路,姜黄素可能是预防骨质疏松症的理想药物。

NEAT1、miR-27a-3p在阿尔茨海默病患者血清和脑脊液中的表达关系

目的:探究长链非编码RNA核富含丰富的转录本1(long chain non-coding RNA nuclear-enriched abundant transcript 1, LncRNA NEAT1)、miR-27a-3p在阿尔茨海默病(Alzheimer disease, AD)患者血清和脑脊液中的表达关系及意义。方法:选择2019年10月至2021年9月天津市第五中心医院神经内科收治的AD患者66例作为病例组,根据临床痴呆评定量表(clinical dementia rating, CDR)评分分为轻度组(≤1分,n=41)与中重度组(>1分,n=25);另取同期门诊其他就诊患者血清和脑脊液标本66例志愿者作为对照组。收集所有受试者的一般资料并评估认知程度,采用实时荧光定量PCR检测血清和脑脊液miR-27a-3p、NEAT1表达水平,酶联免疫吸附试验检测脑脊液β-淀粉样前体蛋白裂解酶1(β-site amyloid precursor protein cleaving enzyme 1,BACE1)、β淀粉样蛋白(amyloid β,Aβ)40和Aβ42水平,采用Spearman法分析血清miR-27a-3p、NEAT1水平与简易精神状态检测量表的相关性,采用Pearson法分析血清miR-27a-3p、NEAT1水平与Aβ沉积平均标准摄取值比率(standardized uptake value ratio, SUVR)及脑脊液miR-27a-3p、NEAT1、BACE1、Aβ42、Aβ40水平的相关性。结果:MMSE评分[21 (17,25),9(7,11)vs. 27 (21,34)]、MoCA评分[17 (12,21),10 (7,13)vs. 27 (21,31)]、血清miR-27a-3p水平(0.55±0.13,0.46±0.06 vs. 0.97±0.22)、脑脊液miR-27a-3p(0.48±0.10,0.35±0.10 vs. 1.03±0.31)、Aβ42水平[(303.55±36.77) ng/L,(231.45±34.14) ng/L vs.(499.99±53.63) ng/L]及Aβ42/Aβ40比值(0.030±0.008, 0.022±0.007 vs. 0.048±0.010)轻度组、中重度组AD患者均低于对照组(P均<0.05),且中重度组AD患者较轻度组低(P均<0.05);血清NEAT1水平(2.31±0.64,3.13±0.76 vs. 1.05±0.20)、SUVR(1.50±0.29,1.76±0.52 vs. 0.74±0.15)及脑脊液NEAT1(3.51±1.24,4.30±1.65 vs. 1.01±0.23)、BACE1水平[(55.78±5.98)μg/L,(72.32±16.08)μg/L vs.(21.39±3.73)μg/L]轻度组、中重度组AD患者均高于对照组(P均<0.05),且中重度组AD患者较轻度组高(P均<0.05)。AD患者血清NEAT1水平与SUVR及脑脊液NEAT1、BACE1呈正相关(r=0.350,0.606,0.341,P<0.05),与MMSE评分、MoCA评分呈负相关(r=-0.473,-0.482,P均<0.05);血清miR-27a-3p水平与脑脊液miR-27a-3p水平、MMSE评分、MoCA评分呈正相关(r=0.695,0.424,0.412,P<0.05),与SUVR及脑脊液BACE1水平呈负相关(r=-0.521、-0.447,P均<0.05)。结论:NEAT1、miR-27a-3p在AD患者血清及脑脊液中表达趋势具有一致性,NEAT1水平均升高,miR-27a-3p水平均降低,二者水平呈负相关,与AD患者脑中Aβ沉积程度有关,并参与AD的病情进展。

子宫内膜癌组织NF-κB、STAT3蛋白对子宫内膜癌的诊断及预后价值意义分析

目的探讨与分析子宫内膜癌(EC)组织核因子κB(NF-κB)、信号转导与转录激活因子3(STAT3)蛋白对子宫内膜癌的诊断及预后价值意义。方法选择2019年9月—2022年11月石河子大学第一附属医院收治的88例子宫内膜癌患者作为研究对象,取所有患者术中切除的新鲜子宫内膜癌肿瘤组织标本(肿瘤组)和癌旁正常子宫内膜组织(癌旁组),采用免疫组化法检测NF-κB、STAT3蛋白表达阳性率,调查患者的病理特征、随访预后并进行相关性分析。结果肿瘤组NF-κB、STAT3蛋白表达阳性率显著高于癌旁组,差异具有统计学意义(P<0.05)。在88例患者中,不同年龄、临床分期、分化程度、肌层浸润、淋巴结转移患者的NF-κB、STAT3蛋白表达阳性率比较,差异具有统计学意义(P<0.05)。所有患者随访到2023年6月1日,生存患者NF-κB、STAT3蛋白表达阳性率明显低于死亡患者,差异具有统计学意义(P<0.05)。Cox回归分析显示NF-κB、STAT3蛋白表达阳性率为影响预后中位生存时间的重要因素,差异有统计学意义(P<0.05)。结论子宫内膜癌组织多伴随有NF-κB、STAT3蛋白的高表达,其表达水平与患者的临床病理特征和预后生存情况都存在相关性。

银杏二萜内酯葡胺在超时间窗AIS中的治疗效果及对患者血清TLR4/NF-κB信号通路因子水平的影响

目的观察银杏二萜内酯葡胺(GDLG)治疗超时间窗急性缺血性脑卒中(AIS)的效果,并探讨其对患者血清Toll样受体4(TLR4)/核转录因子-κB(NF-κB)信号通路因子水平的影响。方法选取2020年1月至2023年1月郑州大学第一附属医院收治的170例超时间窗AIS患者作为研究对象,采用电脑随机数表法分为对照组和研究组各85例。对照组患者给予常规治疗,研究组患者在常规治疗基础上给予GDLG治疗,均持续治疗两周。比较两组患者的治疗效果、治疗前、治疗1周、2周后脑血流灌注指标[脑血容量(CBV)、脑血流量(CBF)、平均通过时间(MTT)]、血清神经损伤标志物[基质金属蛋白酶抑制物-1(TIMP-1)、神经元特异性烯醇化酶(NSE)、半乳糖凝集素-3(Galectin-3)]、TLR4/NF-κB信号通路因子(TLR4、NF-κB)水平、神经功能、日常生活能力,统计比较两组治疗期间不良反应及治疗后第3个月预后情况。结果研究组患者的治疗总有效率为95.29%,明显高于对照组的84.71%,差异有统计学意义(P<0.05);治疗1周、2周后,研究组患者的CBF分别为(82.69±12.54)mL/100 g、(85.17±12.36)mL/100 g,明显高于对照组的(77.01±11.86)mL/100 g、79.24±12.10)mL/100 g,CBV分别为(6.84±1.22)mL/(100 g·min)、(7.01±1.28)mL/(100 g·min),明显高于对照组的(6.05±1.17)mL/(100 g·min)、(6.19±1.23)mL/(100 g·min),MTT分别为(9.16±1.73)s、(8.92±1.65)s,明显低于对照组的(10.02±1.85)s、(9.84±1.71)s,差异均有统计学意义(P<0.05);治疗1周、2周后,研究组患者的血清NSE水平分别为(13.75±2.81)μg/L、(12.24±2.59)μg/L,明显低于对照组的(16.29±3.68)μg/L、(15.36±3.42)μg/L,TIMP-1水平分别为(72.26±16.73)ng/mL、(68.19±14.84)ng/mL,明显低于对照组的(83.51±18.20)ng/mL、(79.81±15.72)ng/mL,Galectin-3水平分别为(4.12±1.04)ng/mL、(3.75±0.96)ng/mL,明显低于对照组的(5.03±1.08)ng/mL、(4.69±1.02)ng/mL,差异均有统计学意义(P<0.05);治疗1周、2周后,研究组患者的血清TLR4水平分别为(2.61±0.78)ng/mL、(2.39±0.74)ng/mL,明显低于对照组的(3.42±0.85)ng/mL、(3.25±0.81)ng/mL,NF-κB水平分别为(108.69±21.36)ng/mL、(98.74±18.65)ng/mL,明显低于对照组的(121.54±22.06)ng/mL、(110.23±20.67)ng/mL,差异均具有统计学意义(P<0.05);治疗1周、2周后,研究组患者的美国国立卫生院卒中量表(NIHSS)评分分别为(10.19±2.18)分、(9.21±2.04)分,明显低于对照组的(44.26±4.05)分、(62.51±4.36)分,改良Barthel指数(MBI)评分分别为(47.39±4.28)分、(65.40±4.71)分,明显高于对照组的(44.26±4.05)分、(62.51±4.36)分,差异均有统计学意义(P<0.05);治疗期间,研究组患者的不良反应总发生率为10.59%,略高于对照组的7.06%,但差异无统计学意义(P>0.05);治疗后第3个月,研究组患者的预后良好率为64.71%,明显高于对照组的48.24%,差异有统计学意义(P<0.05)。结论超时间窗AIS患者在常规治疗基础上联合GDLG治疗能明显提高治疗效果,且能更有效下调血清TLR4/NF-κB信号通路因子水平。

miR-301a的上调通过PTEN/PI3K/AKT信号轴诱导巨噬细胞M1极化促进动脉瘤的进展

目的:研究miR-301a通过PTEN/PI3K/AKT信号轴调节巨噬细胞M1极化加速血管紧张素Ⅱ(AngⅡ)诱导的腹动脉瘤(AAA)进展的机制。方法:体内实验中,8~10周龄ApoE^(-/-)雄性小鼠分为对照组、模型组、miR-301a过表达组,输注生理盐水(saline)为对照组(n=15),通过血管紧张素Ⅱ(AngⅡ)诱导建成AAA模型(n=45);THP-1细胞随机分为对照组、AngⅡ+agomir NC组、AngⅡ+miR-301a agomir组和AngⅡ+miR-301a agomir+LY(LY294002)组;使用生物信息学、ELISA、Western blot、qRT-PCR和双荧光素酶实验研究miR-301a调控巨噬细胞极化促动脉瘤进展的机制。结果:与对照组相比,AAA模型组的miR-301a表达上调,主动脉直径增粗,miR-301a的过表达显著增加了动脉直径;TargetScan7.2数据库预测miR-301a与PTEN 3’-UTR之间存在结合位点,双荧光素酶报告基因分析证实PTEN是THP-1巨噬细胞中miR-301a的直接靶标;GO和KEGG通路富集分析显示PI3K/AKT/和NF-κB信号的通路可能是巨噬细胞向M1极化的关键通路;体内、体外实验证明miR-301a通过PI3K/AKT/NF-κB信号通路调控巨噬细胞向M1表型极化,上调基质金属蛋白酶和炎症因子的表达,促进动脉瘤进展。此外,miR-301a联合MMP-9可作为预测动脉瘤破裂的非侵入性生物标志物。结论:miR-301a通过PTEN/PI3K/AKT途径促进巨噬细胞的M1极化,从而加速动脉瘤的进展,在AAA的疾病进展中发挥了关键作用。

缺血性脑卒中后促进转录因子EB核转位改善神经元自噬流障碍的分子机制

脑卒中(cerebral stroke)是由脑动脉出血或梗塞引起的急性脑血管病,约80%的脑卒中临床病例为缺血性脑卒中。自噬流障碍是导致神经元缺血性损伤的重要致病因素,而如何改善自噬流障碍从而减轻缺血性脑损伤的策略仍需深入探究。研究表明,转录因子EB(transcription factor EB,TFEB)是自噬-溶酶体信号通路的关键调节分子。TFEB的活性由其磷酸化水平决定,磷酸化的TFEB通过与14-3-3黏附蛋白结合存留于胞质,当其去磷酸化后则快速向核内转位,进而上调“协同溶酶体表达与调控(coordinated lysosomal expression and regulation,CLEAR)”信号,促进溶酶体生成及自噬相关基因转录,从而增强自噬流。本文重点就TFEB核转位改善缺血性脑卒中神经元自噬流障碍的分子机制进行详细阐述,旨在为脑卒中治疗及脑缺血病理研究提供参考。

阿奇霉素对胃癌细胞增殖、凋亡及炎症因子表达水平的影响

目的探讨阿奇霉素对人胃癌细胞(AGS细胞)上清液中炎症因子表达、增殖和凋亡的影响及核转录因子-κB(NF-κB)信号通路的调控作用。方法体外培养AGS细胞,将其分为对照组(不进行干预)和不同水平(12.5、25.0、50.0、100.0μg/mL)阿奇霉素组,干预24 h,筛选阿奇霉素最适水平用于后续实验。细胞分组:对照组、阿奇霉素组(50.0μg/mL阿奇霉素)、阳性药物组(50.0μg/mL 5-氟尿嘧啶)、抑制剂组(50.0μg/mL阿奇霉素+1.0μmol/L NF-κB通路抑制剂BAY11-7082)和激活剂组(50.0μg/mL阿奇霉素+1.0μmol/L NF-κB通路激动剂Prostratin),干预24 h。采用细胞计数试剂盒-8(CCK-8)检测细胞活力;采用酶联免疫吸附试验(ELISA)测定细胞上清液中的炎症因子[白细胞介素(IL)-10及IL-1β]水平;采用5-乙炔基-2′脱氧尿嘧啶核苷(EdU)测定细胞增殖率;采用Hoechst33258染色试剂盒测定细胞凋亡率;采用蛋白免疫印迹(WB)法测定增殖细胞核抗原(PCNA)、半胱氨酸蛋白酶-3(Caspase-3)及NF-κB通路相关蛋白表达水平。结果用CCK-8检测AGS细胞活力,根据实验结果选择50.0μg/mL阿奇霉素用于后续实验。与对照组比较,阿奇霉素组和阳性药物组AGS细胞上清液中IL-1β水平、细胞增殖率、PCNA表达水平、磷酸化(p)NF-κB p65/NF-κB p65和p-IκBα/IκBα明显降低(P<0.05),IL-10水平、细胞凋亡率和Caspase-3表达水平明显升高(P<0.05);与阿奇霉素组比较,抑制剂组中BAY11-7082的出现增强了阿奇霉素对AGS细胞的作用(P<0.05),激活剂组中Prostratin的出现则削弱了阿奇霉素对AGS细胞的作用(P<0.05)。结论阿奇霉素能抑制AGS细胞的炎症和增殖,并诱导其凋亡,其作用机制可能与阻滞NF-κB通路信号转导有关。

NF-κB介导VEGF-A促进食管鳞状细胞癌血管生长和细胞增殖的实验研究

目的:探讨NF-κB、VEGF-A在食管鳞状细胞癌(ESCC)患者癌组织中的表达情况及NF-κB与VEGF-A在促进食管鳞癌血管生成和细胞增殖的关系。方法:收集32例行食管癌切除术患者食管鳞癌组织及癌旁组织标本,蛋白印迹法(Western blot)检测癌及癌旁组织中NF-κB、VEGF-A的蛋白表达情况;Pearson相关系数分析NF-κB与VEGF-A的相关性。在体外实验中过表达和敲低食管鳞癌细胞中的NF-κB后,通过CCK-8、平板克隆实验、流式细胞术检测NF-κB对肿瘤细胞恶性表型的影响。Western blot检测不同NF-κB表达水平对VEGF-A及VEGF-R2的表达水平的变化。结果:NF-κB、VEGF-A在癌组织中高表达,在癌旁组织中低表达。过表达和敲低NF-κB对细胞的增殖分别有促进和抑制作用、对细胞的凋亡有抑制和促进作用、对细胞周期的G1/S转换有促进和抑制作用。过表达和敲低NF-κB后VEGF-A、VEGF-R2的表达也随之升高和降低。结论:ESCC组织中的NF-κB与VEGF-A表达高于癌旁组织,二者正相关,且NF-κB通过过表达VEGF-A对ESCC血管生成和细胞增殖具有正向促进作用。