Cyclic GMP-Dependent Protein Kinases

环 GMP 依赖性蛋白激酶
  • 文章类型: Journal Article
    顶丛寄生虫平衡增殖,持久性,并在它们的后生寄主中传播。AGC激酶,比如PKG,PKA,和PDK1直向序列SPARK,整合环境信号,在复制生命阶段和活动生命阶段之间切换寄生虫。最近的研究已经对丝锥PKG和PKA下游的途径进行了分类;然而,对AGC激酶信号级联的全球整合知之甚少。这里,与无偏倚蛋白质组学相结合的条件遗传学表明,SPARK与elongin样蛋白复合物可调节弓形虫模型中PKA和PKG的稳定性。在PKG和PKA下调后,产生归因于SPARK耗竭的缺陷。缺乏SPARK的寄生虫分化为慢性感染形式,这可能是由于球虫特异性PKA直系同源物的活性降低所致。这项工作描述了AGC激酶的信号拓扑,这些激酶共同控制了这一重要寄生虫家族的无性周期内的过渡。
    Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites.
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  • 文章类型: Journal Article
    丹参酮IIA(TSA),丹参干根茎的主要脂溶性成分,已经被证明可以诱导血管舒张。然而,潜在机制尚不清楚.本研究旨在探讨TSA对离体阻力小动脉血管舒张功能的影响。血管肌电图显示,内皮剥脱显着降低了TSA的血管舒张作用。阻断瞬时受体电位香草酸4(TRPV4)通道可防止TSA诱导的血管舒张。全细胞膜片钳分析显示,在TSA处理后,通过TRPV4通道的电流在内皮细胞(EC)中增加。这归因于TRPV4蛋白降解减少以及其表达增加。TRPV4抑制剂HC-067047降低了一氧化氮(NO)的产生和TSA诱导的内皮型一氧化氮合酶(eNOS)的表达。此外,它增加了环磷酸鸟苷(cGMP)和蛋白激酶G(PKG)的产生。目前的结果表明,TSA诱导内皮依赖性血管舒张,它由TRPV4-NO-PKG信号通路介导。这些发现凸显了TSA的潜力,一种在中药中称为丹参的化合物,未来的心血管治疗策略。
    Tanshinone IIA (TSA), the main lipo-soluble component from the dried rhizome of Salvia miltiorrhiza, has been shown to induce vasodilation. However, the underlying mechanisms remains unclear. This study aimed to investigate the effect of TSA on the vasodilation of small resistant arteries ex vivo. Vascular myography revealed that endothelial denudation reduced significantly the vasodilatory effect of TSA. Blocking transient receptor potential vanilloid 4 (TRPV4) channels prevented TSA-induced vasodilation. Whole-cell patch-clamp analysis revealed that the current passing through TRPV4 channels increased after TSA treatment in endothelial cells (ECs). This was attributed to reduced TRPV4 protein degradation along with its increased expression. The TRPV4 inhibitor HC-067047 lowed nitric oxide (NO) production and TSA-induced expression of endothelial nitric oxide synthase (eNOS). Moreover, it increased the production of cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG). The present results indicate that TSA induces endothelium-dependent vasodilation, which is mediated by the TRPV4-NO-PKG signaling pathway. These findings highlight the potential of TSA, a compound known in traditional Chinese medicine as Danshen (Salvia miltiorrhiza), for future cardiovascular therapeutic strategies.
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    目的:使用一氧化氮(NO)供体可以极大地受益于心血管疾病(CVD)的治疗。这项研究旨在研究NONONO2P的作用机制,该机制有助于在肠系膜动脉中观察到的反应。假设NONO2P与硝普钠(SNP)和NO具有相似的药理作用。
    方法:对雄性Wistar大鼠实施安乐死以分离肠系膜上动脉以进行等距张力记录。使用DAF-FM/DA染料测量NO水平,使用cGMP-ELISA试剂盒测定环磷酸鸟苷(cGMP)水平。
    结果:NONO2P表现出与SNP相似的最大功效。NO(NO•)清除剂(PTIO;100μM和羟钴胺素;30μM)和硝酰基阴离子(NO-)清除剂(L-半胱氨酸;3mM)的自由基降低了NONO2P促进的弛豫。特异性可溶性鸟苷酸环化酶(sGC)抑制剂(ODQ;10μM)的存在几乎消除了血管舒张。cGMP依赖性蛋白激酶(PKG)抑制(KT5823;1μM)减弱NONO2P松弛作用。通过阻断内向整流K通道(Kir),血管松弛反应显着减弱,电压操作K+通道(KV),大电导Ca2+激活K+通道(BKCa)。环吡嗪酸(10μM)减弱了NONO2P诱导的弛豫,表明肌浆网Ca2-ATPase(SERCA)激活参与了这种松弛。此外,NONO2P增加内皮细胞中的NO水平和cGMP产生。
    结论:NONO2P诱导血管舒张的幅度与SNP相同,释放NO•和NO-。它的血管舒张作用涉及sGC,PKG,K+通道打开,和SERCA激活,提示其作为心血管疾病治疗选择的潜力。
    OBJECTIVE: The treatment of cardiovascular diseases (CVD) could greatly benefit from using nitric oxide (NO) donors. This study aimed to investigate the mechanisms of action of NONO2P that contribute to the observed responses in the mesenteric artery. The hypothesis was that NONO2P would have similar pharmacological actions to sodium nitroprusside (SNP) and NO.
    METHODS: Male Wistar rats were euthanized to isolate the superior mesenteric artery for isometric tension recordings. NO levels were measured using the DAF-FM/DA dye, and cyclic guanosine monophosphate (cGMP) levels were determined using a cGMP-ELISA Kit.
    RESULTS: NONO2P presented a similar maximum efficacy to SNP. The free radical of NO (NO•) scavengers (PTIO; 100 μM and hydroxocobalamin; 30 μM) and nitroxyl anion (NO-) scavenger (L-cysteine; 3 mM) decreased relaxations promoted by NONO2P. The presence of the specific soluble guanylyl cyclase (sGC) inhibitor (ODQ; 10 μM) nearly abolished the vasorelaxation. The cGMP-dependent protein kinase (PKG) inhibition (KT5823; 1 μM) attenuated the NONO2P relaxant effect. The vasorelaxant response was significantly attenuated by blocking inward rectifying K+ channels (Kir), voltage-operated K+ channels (KV), and large conductance Ca2+-activated K+ channels (BKCa). NONO2P-induced relaxation was attenuated by cyclopiazonic acid (10 μM), indicating that sarcoplasmic reticulum Ca2+-ATPase (SERCA) activation is involved in this relaxation. Moreover, NONO2P increased NO levels in endothelial cells and cGMP production.
    CONCLUSIONS: NONO2P induces vasorelaxation with the same magnitude as SNP, releasing NO• and NO-. Its vasorelaxant effect involves sGC, PKG, K+ channels opening, and SERCA activation, suggesting its potential as a therapeutic option for CVD.
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  • 文章类型: Journal Article
    背景:管腔抗原的侵入和由物理上皮屏障破坏引起的异常免疫应答是溃疡性结肠炎(UC)的关键特征。受损的上皮功能的恢复对于维持粘膜稳态和疾病静止至关重要。目前UC的治疗主要集中在抑制炎症。然而,随着时间的推移,大多数患者对治疗没有反应或出现继发性耐药性,强调需要开发新的UC治疗靶点。我们的研究旨在从真武汤中确定一种新的改良草药配方的潜在目标,即CDD-2103,它在治疗慢性结肠炎中显示出有希望的疗效。
    方法:使用组织损伤的体外和离体模型检查了CDD-2103对上皮屏障功能的影响,以及慢性结肠炎C57BL/6小鼠模型。在用CDD-2103处理后,采用转录组分析来描述结肠组织中的基因表达变化。
    结果:我们的体内实验表明,CDD-2103剂量依赖性地降低了慢性结肠炎小鼠的疾病严重程度。CDD-2103的功效由杯状细胞损失的减少和紧密连接蛋白完整性的增强介导。机械上,CDD-2103通过激活可溶性鸟苷环化酶(sGC)介导的环磷酸鸟苷(cGMP)/PKG信号级联,抑制上皮细胞凋亡和紧密连接蛋白分解。分子对接分析显示CDD-2103衍生分子对sGC配体的强烈识别,保证进一步调查。
    结论:我们的研究揭示了一种新型制剂CDD-2103,通过激活sGC调节的cGMP/PKG信号来恢复肠屏障功能。此外,我们的研究结果表明,靶向sGC可以成为UC治疗中促进黏膜愈合的有效方法.
    BACKGROUND: The invasion of luminal antigens and an aberrant immune response resulting from a disrupted physical epithelial barrier are the key characteristics of ulcerative colitis (UC). The restoration of damaged epithelial function is crucial for maintaining mucosal homeostasis and disease quiescence. Current therapies for UC primarily focus on suppressing inflammation. However, most patients fail to respond to therapy or develop secondary resistance over time, emphasizing the need to develop novel therapeutic targets for UC. Our study aimed to identify the potential targets of a novel modified herbal formula from the Zhen Wu Decoction, namely CDD-2103, which has demonstrated promising efficacy in treating chronic colitis.
    METHODS: The effect of CDD-2103 on epithelial barrier function was examined using in vitro and ex vivo models of tissue injury, as well as a chronic colitis C57BL/6 mouse model. Transcriptomic analysis was employed to profile gene expression changes in colonic tissues following treatment with CDD-2103.
    RESULTS: Our in vivo experiments demonstrated that CDD-2103 dose-dependently reduced disease severity in mice with chronic colitis. The efficacy of CDD-2103 was mediated by a reduction in goblet cell loss and the enhancement of tight junction protein integrity. Mechanistically, CDD-2103 suppressed epithelial cell apoptosis and tight junction protein breakdown by activating the soluble guanynyl cyclase (sGC)-mediated cyclic guanosine monophosphate (cGMP)/PKG signaling cascade. Molecular docking analysis revealed strong sGC ligand recognition by the CDD-2103-derived molecules, warranting further investigation.
    CONCLUSIONS: Our study revealed a novel formulation CDD-2103 that restores intestinal barrier function through the activation of sGC-regulated cGMP/PKG signaling. Furthermore, our findings suggest that targeting sGC can be an effective approach for promoting mucosal healing in the management of UC.
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  • 文章类型: Journal Article
    cAMP(EPAC)直接激活的交换蛋白与心脏前心律失常信号通路有关,包括肌浆网的自发性舒张Ca2渗漏和分离的心室心肌细胞中动作电位持续时间(APD)的增加。急性EPAC激活后AP的延长主要是由于复极化稳态K电流(IKSS)的降低,但所涉及的机制尚不清楚。本研究旨在评估EPAC1和2在IKSS降低中的作用,并研究潜在的信号通路。在新鲜分离的大鼠心室肌细胞中,使用膜片钳技术的全细胞配置记录AP和K电流。EPAC1和2分别被8-CPTAM(10μmol/L)药理激活,并被R-Ce3F4和ESI-05抑制。EPAC1和EPAC2的抑制显著降低了8-CPTAM对APD和IKSS的作用,表明两种EPAC同工型都参与这些作用。出乎意料的是,AIP或KN-93抑制CaMKII,细胞内BAPTA螯合Ca2+,不影响对8-CPTAM的反应。然而,PLC/PKC和NOS/PKG通路的抑制部分阻止了8-CPTAM依赖性IKSS的降低。最后,PKC和PKG的累积抑制阻断了8-CPTAM效应,这表明这两个参与者沿着平行的途径工作,在EPAC激活时调节IKSS。根据这些发现,我们认为EPAC1和2通过通过PLC/PKC和NOS/PKG途径抑制K电流参与APD的延长。这可能具有病理学意义,因为EPAC在诸如心脏肥大的疾病中上调。
    The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2\'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.
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  • 文章类型: Journal Article
    柚皮素(NAR)是一种突出的黄烷酮,已被认为具有促进人牙周膜干细胞(hPDLSC)成骨分化的能力。本研究旨在探讨NAR如何促进hPDLSCs的成骨分化,并评估其在牙槽骨缺损修复中的功效。为此,通过mRNA测序和网络药理学分析,建立了NAR作用的蛋白质-蛋白质相互作用网络。通过逆转录定量和蛋白质印迹评估基因和蛋白质表达水平。采用茜素红和碱性磷酸酶染色观察hPDLSCs的成骨能力,免疫荧光用于检测NAR分子探针和AKT在细胞中的共定位。通过显微计算机断层扫描(micro-CT)评估下颌骨缺损的修复,Masson染色和免疫荧光。此外,计算机模拟对接软件用于确定NAR与靶蛋白的结合亲和力,AKT.结果表明,一氧化氮(NO)-环磷酸鸟苷(cGMP)-蛋白激酶G(PKG)信号通路的激活可以促进hPDLSCs的成骨分化。抑制AKT,内皮型一氧化氮合酶和可溶性鸟苷酸环化酶分别减弱NAR促进hPDLSCs成骨分化的能力。Micro-CT和Masson染色显示,NAR管饲组在缺损部位表现出更多的新骨形成。免疫荧光分析证实了在NAR灌胃组中,Runt相关转录因子2和骨桥蛋白的表达上调。总之,本研究结果表明,NAR通过与AKT结合激活NO‑cGMP‑PKG信号通路,促进hPDLSCs的成骨分化.
    Naringenin (NAR) is a prominent flavanone that has been recognized for its capacity to promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The present study aimed to explore how NAR promotes the osteogenic differentiation of hPDLSCs and to assess its efficacy in repairing alveolar bone defects. For this purpose, a protein‑protein interaction network of NAR action was established by mRNA sequencing and network pharmacological analysis. Gene and protein expression levels were evaluated by reverse transcription‑quantitative and western blotting. Alizarin red and alkaline phosphatase staining were also employed to observe the osteogenic capacity of hPDLSCs, and immunofluorescence was used to examine the co‑localization of NAR molecular probes and AKT in cells. The repair of mandibular defects was assessed by micro‑computed tomography (micro‑CT), Masson staining and immunofluorescence. Additionally, computer simulation docking software was utilized to determine the binding affinity of NAR to the target protein, AKT. The results demonstrated that activation of the nitric oxide (NO)‑cyclic guanosine monophosphate (cGMP)‑protein kinase G (PKG) signaling pathway could promote the osteogenic differentiation of hPDLSCs. Inhibition of AKT, endothelial nitric oxide synthase and soluble guanylate cyclase individually attenuated the ability of NAR to promote the osteogenic differentiation of hPDLSCs. Micro‑CT and Masson staining revealed that the NAR gavage group exhibited more new bone formation at the defect site. Immunofluorescence assays confirmed the upregulated expression of Runt‑related transcription factor 2 and osteopontin in the NAR gavage group. In conclusion, the results of the present study suggested that NAR promotes the osteogenic differentiation of hPDLSCs by activating the NO‑cGMP‑PKG signaling pathway through its binding to AKT.
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  • 文章类型: Journal Article
    cGMP依赖性蛋白激酶(PKG)是疟疾寄生虫中唯一的cGMP传感器,作为一个重要的信号枢纽,管理整个寄生虫生命周期的关键发育过程。尽管PKG在临床相关的无性血液阶段很重要,疟疾PKG监管的许多方面,包括磷酸化的重要性,仍然知之甚少。在这里,我们使用遗传和生化方法来显示减少的cGMP与环核苷酸结合域B的结合不会影响体外激酶活性,但会阻止寄生虫的外出。同样,我们发现激活环中一个关键苏氨酸残基(T695)的磷酸化对于体外激酶活性是可有可无的,但对于体内PKG功能是必需的,T695磷酸化的缺失导致整个寄生虫蛋白质组的异常磷酸化事件,并改变PKG的底物特异性。我们的发现表明,疟原虫PKG受到独特的调节,以转导对疟疾寄生虫发育至关重要的信号。
    The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.
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  • 文章类型: Journal Article
    骨质疏松性骨折是长期糖皮质激素治疗的主要并发症。糖皮质激素短暂增加骨吸收,但它们主要抑制骨形成并诱导骨细胞凋亡,导致骨质流失。目前对糖皮质激素诱导的骨质疏松症的治疗主要旨在减少骨吸收,因此是不够的。我们先前表明,通过NO/cGMP/蛋白激酶G途径的信号传导在骨骼稳态中起关键作用。这里,我们表明,药理PKG激活与鸟苷酸环化酶-1激活剂Cinaciguat或表达一个组成型活性,突变PKG2R242Q恢复增殖,分化,和暴露于地塞米松的原代小鼠成骨细胞的存活率。Cinaciguat处理野生型小鼠或在转基因小鼠中成骨细胞特异性表达PKG2R242Q可预防地塞米松诱导的皮质骨质量和强度损失。cinaciguat和PKG2R242Q表达的这些作用是由于保留的骨形成参数和骨细胞存活。PKG2效应的基础似乎是通过Wnt/β-catenin信号的恢复,它被糖皮质激素抑制,但对增殖至关重要,分化,和成骨细胞谱系细胞的存活。Cinaciguat减少破骨细胞的地塞米松激活,但这在PKG2R242Q转基因小鼠中没有发生,提示在骨保护中的次要作用。我们建议现有的PKG靶向药物可以代表预防糖皮质激素诱导的骨质疏松症的新治疗方法。
    Osteoporotic fractures are a major complication of long-term glucocorticoid therapy. Glucocorticoids transiently increase bone resorption, but they predominantly inhibit bone formation and induce osteocyte apoptosis, leading to bone loss. Current treatments of glucocorticoid-induced osteoporosis aim mainly at reducing bone resorption and are, therefore, inadequate. We previously showed that signaling via the NO/cGMP/protein kinase G pathway plays a key role in skeletal homeostasis. Here, we show that pharmacological PKG activation with the guanylyl cyclase-1 activator cinaciguat or expression of a constitutively active, mutant PKG2R242Q restored proliferation, differentiation, and survival of primary mouse osteoblasts exposed to dexamethasone. Cinaciguat treatment of WT mice or osteoblast-specific expression of PKG2R242Q in transgenic mice prevented dexamethasone-induced loss of cortical bone mass and strength. These effects of cinaciguat and PKG2R242Q expression were due to preserved bone formation parameters and osteocyte survival. The basis for PKG2\'s effects appeared to be through recovery of Wnt/β-catenin signaling, which was suppressed by glucocorticoids but critical for proliferation, differentiation, and survival of osteoblast-lineage cells. Cinaciguat reduced dexamethasone activation of osteoclasts, but this did not occur in the PKG2R242Q transgenic mice, suggesting a minor role in osteoprotection. We propose that existing PKG-targeting drugs could represent a novel therapeutic approach to prevent glucocorticoid-induced osteoporosis.
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  • 文章类型: Journal Article
    一氧化氮(NO)通过各种机制促进血管生成;然而,在缺血性疾病中NO的有效传播尚不清楚。在这里,我们在动物后肢缺血模型中测试了释放NO的纳米纤维是否能调节治疗性血管生成.用释放NO的3-甲基氨基丙基三甲氧基硅烷(MAP3)衍生或对照(即,非NO释放)纳米纤维,将它们涂在伤口上20分钟,每两天三次。通过NO荧光测定法评估从纳米纤维进入组织的NO的量。通过蛋白质印迹分析确定cGMP依赖性蛋白激酶(PKG)的活性。使用激光多普勒成像在诱导缺血后2、4和14天测量灌注比。在第4天,用F4/80和明胶酶谱进行免疫组织化学(IHC)。在第14天进行CD31的IHC。为了确定释放NO的纳米纤维的血管生成潜力,主动脉环外植体用MAP3或对照纤维处理20分钟,并且在6天后检查芽的长度。根据LDPI(激光多普勒灌注图像)比率或CD31毛细血管密度测量,MAP3纳米纤维组中缺血后肢的血管生成得到改善;此外,加合物肌肉中硝酸盐/亚硝酸盐的总浓度增加。巨噬细胞浸润数量和基质金属蛋白酶-9(MMP-9)活性降低。血管扩张剂刺激磷蛋白(VASP),PKG的主要底物之一,MAP3组磷酸化增加。MAP3纳米纤维或NO供体SNAP(s-亚硝基-n-乙酰基青霉胺)处理的主动脉外植体在离体主动脉环测定中显示出芽增强,KT5823是一种有效的PKG抑制剂。这些发现表明,新型释放NO的纳米纤维,MAP3激活PKG并促进对后肢缺血的治疗性血管生成。
    Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.
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  • 文章类型: Journal Article
    非小细胞肺癌(NSCLC)是一种总体治愈率和生存率较低的恶性肿瘤。发现异常表达的基因对于开发NSCLC中的新型靶向疗法非常重要。本研究旨在发现NSCLC新的差异表达基因(DEGs)。在来自基因表达综合(GEO)数据库的八个数据集中鉴定NSCLC的DEGs。使用GEPIA数据库分析了SCN4B在LUAD和LUSC中的表达谱和预后意义。LinkedOmics用于鉴定与SCN4B共表达的基因,进一步进行KEGG途径富集分析。将过表达SCN4B的质粒(pcDNA/SCN4B)转染到A549和NCI-H2170细胞中以提高SCN4B的表达。进行MTT和TUNEL测定以评估细胞活力和凋亡。依靠GEO数据库筛选出的DEG,我们发现,SCN4B在LUAD和LUSC中显著下调.我们使用GEPIA数据库证实了SCN4B在NSCLC组织中的下调。SCN4B在LUAD中具有预后价值,但不是LUSC。SCN4B相关基因的KEGG通路富集分析显示cGMP-PKG信号通路可能参与了SCN4B在NSCLC中的作用。SCN4B在A549和NCI-H2170细胞中的过表达抑制细胞活力。此外,SCN4B过表达诱导A549和NCI-H2170细胞凋亡。SCN4B抑制NSCLC细胞中PKG1和p-CREB的表达。此外,SCN4B对肿瘤恶性的抑制作用被PKG激活剂减弱。总之,综合生物信息学分析证明SCN4B在NSCLC中下调并具有预后意义。体外实验研究表明,SCN4B通过抑制cGMP-PKG信号通路调节NSCLC细胞的活力和凋亡。
    Non-small cell lung cancer (NSCLC) is a malignant tumor with low overall cure and survival rates. Uncovering abnormally expressed genes is significantly important for developing novel targeted therapies in NSCLC. This study aimed to discover new differentially expressed genes (DEGs) of NSCLC. The DEGs of NSCLC were identified in eight data sets from Gene Expression Omnibus (GEO) database. The expression profiles and the prognostic significance of SCN4B in LUAD and LUSC were analyzed using GEPIA database. LinkedOmics was used to identify co-expressed genes with SCN4B, which were further subjected to KEGG pathway enrichment analysis. SCN4B-overexpressing plasmid (pcDNA/SCN4B) was transfected into A549 and NCI-H2170 cells to elevate the expression of SCN4B. MTT and TUNEL assays were performed to evaluate cell viability and apoptosis. Relying on the screened DEGs from GEO database, we identified that SCN4B was significantly downregulated in LUAD and LUSC. We confirmed the downregulation of SCN4B in NSCLC tissues using GEPIA database. SCN4B has a prognostic value in LUAD, but not LUSC. KEGG pathway enrichment analysis of SCN4B-related genes showed that cGMP-PKG signaling pathway might be involved in the role of SCN4B in NSCLC. Overexpression of SCN4B in A549 and NCI-H2170 cells inhibited the cell viability. Besides, SCN4B overexpression induced apoptosis of A549 and NCI-H2170 cells. SCN4B inhibited the expression of PKG1 and p-CREB in NSCLC cells. Moreover, the inhibitory effects of SCN4B on tumor malignancy were attenuated by the activator of PKG. In conclusion, integrated bioinformatical analysis proved that SCN4B was downregulated and had a prognostic significance in NSCLC. In vitro experimental studies demonstrated that SCN4B regulated NSCLC cells viability and apoptosis via inhibiting cGMP-PKG signaling pathway.
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