禁食对蛋鸡肝脏腺苷-磷酸激活的蛋白激酶活性的影响

腺苷 -磷酸 (AMP)激活的蛋白激酶 (AMP- acti-vated protein kinase,AMPK)是丝氨酸激酶家族的一员 ,由 AMP和其上游激酶 AMPK激酶所活化 ,对细胞内 AMP/ATP的变化非常敏感 [1] ,被称为真核细胞的“代谢感受器”[2 ]。研究发现在跑步 [3 ,4 ]、电刺激肌肉 [5,6]或禁食应激后 [7] ,大鼠肝脏和肌肉中的AMPK活性升高数倍 ,乙酰辅酶 A羧化酶 (ACC)活性显著下降甚至丧失 ,丙二酸单酰辅酶 A产量降低甚至为零 ,脂肪酸合成受抑。同时 ,AMPK活化后 ,脂肪酸的氧化率显著提高 ,CO2 和酮体生成量明显增加[4 ,8] 。这些均表明 ,AMPK活化…     

二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性

探讨二甲双胍对体外培养的大鼠脊髓星形胶质细胞反应性的作用及机制。方法 使用三因子（TNF-α、IL-1α、C1q）诱导产生反应性星形胶质细胞,RT-qPCR及Westernblot检测补体3（C3）的表达;使用5、10、20 mM浓度的二甲双胍处理,Westernblot检测星形胶质细胞中腺苷酸激活蛋白激酶（AMPK）与信号转导和转录激活因子3（STAT3）磷酸化水平;采用AMPK抑制剂Compound C处理,检测AMPK和STAT3磷酸化水平。结果 形态学及RT-qPCR结果显示：与对照组相比,三因子处理改变了星形胶质细胞形态并显著提高了C3的表达（P＜0.05）,表明成功建立反应性星形胶质细胞模型。RT-qPCR结果及Westernblot结果显示：与对照组相比,二甲双胍对C3表达有明显的抑制作用,呈现浓度依赖性（P＜0.05）。在诱导星形胶质细胞反应性过程中,与对照组相比,AMPK的磷酸化水平显著降低（P＜0.05）,使用不同浓度二甲双胍处理后,AMPK的磷酸化水平显著增加且呈浓度依赖性（P＜0.05）,同时信号传导及STAT3的磷酸化水平显著降低且呈浓度依赖性（P＜0.05）。AMPK抑制剂Compound C处理后显著抑制了二甲双胍导致的AMPK活性升高及STAT3活性降低,同时抑制了C3表达下调（P＜0.05）。结论 二甲双胍通过AMPK/STAT3信号通路抑制星形胶质细胞的反应性     

GJB2通过AMPK/mTOR信号通路促进宫颈癌细胞恶性增殖

目的筛选宫颈癌中具有临床意义的潜在药物靶点并研究此靶点蛋白在宫颈癌发生发展中的功能和潜在分子机制。方法收集2018年7月至2019年7月济南市人民医院妇科行宫颈癌手术的30例宫颈癌患者肿瘤组织标本及癌旁组织标本,通过临床数据库分别找到在宫颈癌中高表达的基因(tumor/normal>2)以及在宫颈癌中具有显著临床预后相关性的基因(pValue<0.05),将这两部分基因进行重合分析找到具有显著临床意义的靶基因,进一步通过热图分析找到在宫颈癌中相比正常组织表达差异最明显的前4个基因,通过高表达预后差原则筛选到目的靶基因;通过siRNA介导敲低目的基因的表达,利用细胞增殖实验和划痕迁移实验验证目的基因在宫颈癌发生发展中的重要性;最后通过分析与目的基因共表达基因参与的信号通路,探索目的基因在宫颈癌中发挥功能的潜在分子机制,进一步通过蛋白免疫印迹实验进行验证。结果共有78个基因在宫颈癌中高表达且具有显著临床预后相关性;GJB2在宫颈癌中显著高表达,且高表达的患者具有不良预后;敲低GJB2抑制宫颈癌细胞的生长增殖和迁移;敲低GJB2降低AMPK的磷酸化,升高mTOR的磷酸化,可能通过AMPK/mTOR信号通路参与宫颈癌的发生发展。结论GJB2高表达可能通过抑制AMPK的磷酸化从而升高mTOR的磷酸化参与AMPK/mTOR信号通路促进宫颈癌细胞生长增殖和迁移。     

Vascular Effects of the Polyphenolic Nutraceutical Supplement Taurisolo®: Focus on the Protection of the Endothelial Function

Preservation of vascular endothelium integrity and functionality represents an unmet medical need. Indeed, endothelial dysfunction leads to decreased nitric oxide biosynthesis, which is prodromic of hypertension and hypercoagulability. In this panorama, the nutraceutical supplement Taurisolo^®, a polyphenolic extract from Aglianico cultivar grape, rich in catechin and procyanidins, was evaluated as a vasoprotective, vasorelaxing, anti-hypertensive and anti-coagulant agent in: cell lines, isolated vessels, in vivo models of chronic hypertension and hypercoagulability, and in clinical tests of endothelial reactivity. Taurisolo^® demonstrated to fully protect vascular cell viability from oxidative stimulus at 100 µg/mL and evoke vasorelaxing effects (Emax = 80.6% ± 1.9 and pEC50 = 1.19 ± 0.03) by activation of the Sirtuins-AMPK-pathway. Moreover, Taurisolo^®, chronically administered at 20 mg/Kg/die in in vivo experiments, inhibited the onset of cardiac hypertrophy (heart weight/rat weight = 3.96 ± 0.09 vs. 4.30 ± 0.03), hypercoagulability (decrease of fibrinogen vs. control: p < 0.01) and hypertension (mean of Psys: 200 ± 2 vs. control 234 ± 2 mmHg) and improved endothelial function (Emax = 88.9% ± 1.5 vs. control 59.6% ± 3.6; flow-mediated dilation in healthy volunteers after 400 mg twice daily for 8 weeks vs. baseline: p = 0.019). In conclusion, Taurisolo^® preserves the vascular function against ox-inflamm-ageing process and the consequent cardiovascular accidents.     

TWIST2通过调控FGF21介导的AMPK/mTOR通路诱导卵巢癌细胞氧化应激和凋亡

目的探究TWIST2对卵巢癌细胞氧化应激和凋亡的作用及相关机制。方法收集2018年6月至2019年10月在山东省妇幼保健院进行治疗的30例卵巢癌患者的癌组织和癌旁组织,将pcDNA3.1-TWIST2质粒(TWIST2组)和pcDNA3.1阴性对照质粒(阴性对照组)分别转染卵巢癌细胞(SK-OV-3和HO-8910),未转染质粒组为空白对照组。通过实时荧光定量PCR(qRT-PCR)和免疫印迹(Western blot)实验检测组织和细胞中TWIST2 mRNA和蛋白质的表达量;细胞计数试剂盒CCK-8检测TWIST2组、阴性对照组和空白对照组的细胞活力;Western blot实验检测3组的凋亡相关蛋白caspase-3、Bax和Bcl-2,AMPK/mTOR通路相关蛋白FGF21、p-AMPK和p-mTOR的蛋白表达水平;试剂盒检测3组活性氧(ROS)基因、ATP的水平和超氧化物歧化酶(SOD)的酶活性。通过检测不同处理组的细胞凋亡率和ROS水平,研究TWIST2与FGF21的相互作用。结果TWIST2在卵巢癌组织和卵巢癌细胞中的表达量显著低于癌旁组织和正常卵巢上皮细胞的表达量(P<0.05)。与空白对照组和阴性对照组相比,TWIST2组的细胞活力显著降低,细胞凋亡率显著提高(P<0.05);ROS的水平显著升高,SOD酶活力、ATP水平、NAD~+/NADH水平显著降低(P<0.05)。TWIST2可以抑制FGF21、p-AMPK、p-mTOR蛋白的表达(P<0.05);在TWIST2组中加入FGF21或AMPK激动剂时,p-AMPK、p-mTOR蛋白表达水平显著升高,卵巢癌细胞的凋亡率和ROS水平显著降低(P<0.05),TWIST2与FGF21的表达量呈负相关(r=-0.6254,P<0.01)。结论TWIST2能够促进卵巢癌细胞氧化应激和凋亡,其作用机制是通过调控FGF21介导的AMPK/mTOR通路来实现的。     

Buformin suppresses proliferation and invasion via AMPK/S6 pathway in cervical cancer and synergizes with paclitaxel

Buformin is an old anti-diabetic agent and manifests potent anti-tumor activities in several malignancies. In the present study, we aimed to explore the functions of buformin in human cervical cancer. As our data shown, buformin exhibited significant anti-proliferative effects in a dose-dependent manner in 4 cervical cancer cell lines. Compared to the control, buformin notably suppressed colony formation and increased ROS production in C33A, Hcc94 and SiHa cells. Flow cytometric analysis showed that buformin induced marked cell cycle arrest but only resulted in mild apoptosis. The invasion of C33A and SiHa cells sharply declined with buformin treatment. Consistently, western blotting showed that buformin activated AMPK and suppressed S6, cyclin D1, CDK4, and MMP9. Moreover, we found that buformin enhanced glucose uptake and LDH activity, increased lactate level, while decreased ATP production in cervical cancer cells. In addition, low doses of buformin synergized with routine chemotherapeutic drugs (such as paclitaxel, cisplatin, and 5-FU) to achieve more significant anti-tumor effects. In vivo, a single use of buformin exerted moderate anti-tumor effects, and the combination with buformin and paclitaxel exhibited even greater suppressive effects. Buformin also consistently showed synergistic effects with paclitaxel in treating primary cultures of cervical cancer cells. Take together, we are the first to demonstrate that buformin suppresses cellular proliferation and invasion through the AMPK/S6 signaling pathway, which arrests cell cycle and inhibits cellular invasion. Buformin also could synergize with routine chemotherapies, producing much more powerful anti-tumor effects. With these findings, we strongly support buformin as a potent choice for treating cervical cancer, especially in combination with routine chemotherapy.     

Dissecting the role of AMP-activated protein kinase in human diseases

AMP-activated protein kinase (AMPK), known as a sensor and a master of cellular energy balance, integrates various regulatory signals including anabolic and catabolic metabolic processes. Accompanying the application of genetic methods and a plethora of AMPK agonists, rapid progress has identified AMPK as an attractive therapeutic target for several human diseases, such as cancer, type 2 diabetes, atherosclerosis, myocardial ischemia/reperfusion injury and neurodegenerative disease. The role of AMPK in metabolic and energetic modulation both at the intracellular and whole body levels has been reviewed elsewhere. In the present review, we summarize and update the paradoxical role of AMPK implicated in the diseases mentioned above and put forward the challenge encountered. Thus it will be expected to provide important clues for exploring rational methods of intervention in human diseases.     

负荷渐增式训练对老年小鼠骨骼肌卫星细胞AMPK磷酸化的影响

目的 探讨负荷渐增式训练对老年小鼠骨骼肌卫星细胞腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）磷酸化的影响。方法 实验小鼠分为 3 组：青年对照组（YC组，n=12）、老年对照组（OC组，n=12）与老年运动组（OT组，n=12）。OT组进行负荷渐增式训练，流式细胞分选技术分离CD45-/CD31-/Sca1-/VCAM(CD106)+细胞群体，分选细胞通过desmin、Myod肌原性染色以及成肌分化诱导培养进行肌卫星细胞鉴定，免疫组化结合Western blotting方法检测肌卫星细胞p-AMPK水平。结果 YC组骨骼肌卫星细胞AMPK及p-AMPK表达水平显著高于OC组（P<0.05）；OT组与OC组AMPK表达无明显变化（Ｐ>0.05），而OT组p-AMPK表达水平显著高于OC组（P<0.05）。结论 负荷渐增式训练可促进老年小鼠骨骼肌卫星细胞AMPK磷酸化，改善老年小鼠骨骼肌能量代谢。     

DW14006 as a direct AMPKα1 activator improves pathology of AD model mice by regulating microglial phagocytosis and neuroinflammation

Alzheimer’s disease (AD) is a progressively neurodegenerative disease with typical hallmarks of amyloid β (Aβ) plaque accumulation, neurofibrillary tangle (NFT) formation and neuronal death extension. In AD brain, activated microglia phagocytose Aβ and neuronal debris, but also aggravate inflammation stress by releasing inflammatory factors and cytotoxins. Improving microglia on Aβ catabolism and neuroinflammatory intervention is thus believed to be a promising therapeutic strategy for AD. AMP-activated protein kinase (AMPK) is highly expressed in microglia with AMPKα1 being tightly implicated in neuroinflammatory events. Since indirect AMPKα1 activators may cause side effects with undesired intracellular AMP/ATP ratio, we focused on direct AMPKα1 activator study by exploring its potential function in ameliorating AD-like pathology of AD model mice. Here, we reported that direct AMPKα1 activator DW14006 (2-(3-(7-chloro-6-(2′-hydroxy-[1,1′-biphenyl]-4-yl)-2-oxo-1,2-dihydroquinolin-3-yl)phenyl)acetic acid) effectively improved learning and memory impairments of APP/PS1 mice, and the underlying mechanisms have been intensively investigated. DW14006 reduced amyloid plaque deposition by promoting microglial o-Aβ₄₂ phagocytosis and ameliorated innate immune response by polarizing microglia to an anti-inflammatory phenotype. It selectively enhanced microglial phagocytosis of o-Aβ₄₂ by upgrading scavenger receptor CD36 through AMPKα1/PPARγ/CD36 signaling and suppressed inflammation by AMPKα1/IκB/NFκB signaling. Together, our work has detailed the crosstalk between AMPKα1 and microglia in AD model mice, and highlighted the potential of DW14006 in the treatment of AD.