Resveratrol Inhibits the Invasion of Glioblastoma-Initiating Cells via Down-Regulation of the PI3K/Akt/NF-κB Signaling Pathway

Invasion and metastasis of glioblastoma-initiating cells (GICs) are thought to be responsible for the progression and recurrence of glioblastoma multiforme (GBM). A safe drug that can be applied during the rest period of temozolomide (TMZ) maintenance cycles would greatly improve the prognosis of GBM patients by inhibiting GIC invasion. Resveratrol (RES) is a natural compound that exhibits anti-invasion properties in multiple tumor cell lines. The current study aimed to evaluate whether RES can inhibit GIC invasion in vitro and in vivo. GICs were identified using CD133 and Nestin immunofluorescence staining and tumorigenesis in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. Invasive behaviors, including the adhesion, invasion and migration of GICs, were determined by tumor invasive assays in vitro and in vivo. The activity of matrix metalloproteinases (MMPs) was measured by the gelatin zymography assay. Western blotting analysis and immunofluorescence staining were used to determine the expression of signaling effectors in GICs. We demonstrated that RES suppressed the adhesion, invasion and migration of GICs in vitro and in vivo. Moreover, we proved that RES inhibited the invasion of GICs via the inhibition of PI3K/Akt/NF-κB signal transduction and the subsequent suppression of MMP-2 expression.     

RISK信号通路在1-磷酸鞘氨醇后适应减轻心肌细胞缺氧/复氧损伤中的作用

目的研究再灌注损伤挽救激酶(reperfusion injury salvage kinase,RISK)信号通路在1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)后适应减轻H9c2心肌细胞缺氧/复氧损伤中的作用。方法将H9c2细胞随机分为7组,即(1)正常对照组(C);(2)缺氧/复氧组(H/R);(3)S1P组;(4)S1P+LY294002组(S1P+LY);(5)LY组;(6)S1P+PD98059组(S1P+PD);(7)PD组。采用MTT法测定各组细胞存活率;比色法测定丙二醛(MDA)含量、总超氧化物歧化酶(TSOD)和锰超氧化物歧化酶(Mn-SOD)活力;采用激光共聚焦显微镜技术检测细胞内游离钙离子浓度变化;流式细胞仪检测心肌细胞凋亡率;Western blot法测定Akt和ERK1/2蛋白的磷酸化水平。结果与H/R组比较,S1P组可明显增加H9c2细胞缺氧/复氧损伤后细胞存活率及凋亡率,增加TSOD及Mn-SOD活力,降低MDA含量,降低钙离子浓度,增加Akt和ERK1/2磷酸化水平,而PI3K/Akt信号通路阻断剂LY294002或ERK1/2阻断剂PD98059可阻断S1P对H9c2的上述作用。结论 S1P能够减轻H9c2细胞缺氧/复氧损伤,加入PI3K/Akt抑制剂LY294002和ERK1/2抑制剂PD98059均使S1P的保护作用被取消,表明S1P通过RISK信号通路发挥抗H9c2细胞缺氧/复氧损伤作用。     

丰宫并殖吸虫感染大鼠后诱导肺组织细胞自噬的初探

目的通过检测蛋白激酶B (Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路相关因子的表达,探讨丰宫并殖吸虫感染大鼠是否可导致肺组织细胞发生自噬。方法 40只SD大鼠随机分为健康对照组,感染后3、 7、 14 d组,每组10只,感染组每鼠腹壁皮下注射6条丰宫并殖吸虫后尾蚴,分别在感染后3、 7、14 d取各组大鼠血清和肺组织,ELISA检测血清中IL-1、 IL-6水平。取肺组织用于透射电镜观察自噬体,HE染色观察肺组织病理学改变,蛋白质免疫印迹(Western blotting)和免疫组化检测Akt、雷帕霉素靶蛋白(mTOR)、程序性死亡受体-1 (Beclin 1)及微管相关蛋白轻链3 (LC3Ⅱ)相关因子的蛋白表达。采用SPSS 19.0软件对数据进行统计学分析。结果ELISA检测结果显示,感染后3、 7、 14 d组血清中IL-1水平分别为(1 558.0±123.6)、(1 511.0±213.1)和(1 448.0±176.8) pg/ml,均高于对照组的(1 222.0±112.8) pg/ml (P <0.05);感染后3、 7 d组IL-6水平分别为(1 481.0±197.9)、(1 423.0±210.0) pg/ml,均高于对照组的(1 221.0±138.9) pg/ml (P <0.05)。透射电镜观察在不同的感染阶段,肺组织中线粒体均出现自噬现象。大鼠组织肺病理学检测结果显示,各感染组细胞排列紊乱,肺泡结构遭到不同程度的破坏。各感染组Akt蛋白表达水平与对照组比较,差异无统计学意义(P> 0.05);感染后3、 7 d组磷酸化蛋白激酶B (p-Aktser473)蛋白表达水平为(1.288±0.109)、(1.619±0.132),均高于对照组(0.733±0.135)(P <0.01);感染后3、 7、 14 d组磷酸化雷帕霉素靶蛋白(p-m TORser2448)蛋白表达水平分别为(1.574±0.278)、(2.384±0.125)和(1.808±0.121),均高于对照组(1.260±0.087)(P <0.05);感染后3 d组mTOR、 Beclin 1蛋白表达水平分别为(1.714±0.217)和(2.736±0.333),均高于对照组(1.345±0.067)和(1.974±0.225)(P <0.01);感染后14 d组LC3Ⅱ蛋白表达(1.938±0.191)高于对照组(1.401±0.200)(P <0.01)。免疫组化分析结果显示,对照组肺组织细胞呈蓝色,阳性呈棕黄色,各因子阳性定位于细胞膜和细胞浆。各感染组Akt、 mTOR与对照组相比,肺组织细胞中棕黄色显色不明显,A450值与对照组比较差异无统计学意义(P> 0.05);感染后3、 7、 14 d组p-Aktser473、 p-mTORser2448及Beclin 1与对照组相比,棕黄色显色加深明显,A450值分别为(0.104±0.010)、(0.143±0.022)、(0.088±0.013),(0.100±0.007)、(0.151±0.006)、(0.120±0.012)和(0.129±0.005)、(0.047±0.004)、(0.050±0.005),均高于相应对照组(0.032±0.001)、(0.065±0.002)和(0.031±0.001)(P <0.05);感染后3、 14 d组LC3Ⅱ与对照组比较棕黄色明显加深,A450值分别为(0.056±0.006)、(0.120±0.007),均高于对照组(0.042±0.004)(P <0.05)。结论丰宫并殖吸虫感染大鼠所致的肺损伤,炎症反应可诱导肺组织细胞发生自噬,该自噬作用可通过检测Akt/mTOR信号通路相关因子的表达来初步实现。     

PI3K/Akt/mTOR信号通路对大鼠肾小球系膜细胞增殖的调控作用探讨

目的:探讨磷脂酰肌醇-3(PI3K)/蛋白激酶(Akt)/哺乳动物雷帕霉素靶蛋白(m TOR)信号通路对大鼠肾小球系膜细胞(MC)增殖的调控作用。方法:体外培养大鼠MC,随机分为正常对照组、表皮生长因子(EGF)(10ng/m L)组、PI3K抑制剂LY294002组(2μg/m L)、EGF联合LY294002组。采用四甲基偶氮唑蓝(MTT)法检测4组MC的增殖,流式细胞术检测MC周期,免疫荧光法检测系膜细胞m TOR的表达。结果:与正常对照组相比,EGF组能显著促进大鼠MC增殖,G0/G1期细胞减少,S期及G2/M期细胞增加,且m TOR表达增加;LY294002组大鼠MC增殖受抑制,G0/G1期细胞增加,S期及G2/M期细胞减少,且m TOR表达降低;与EGF组比较,EGF联合LY294002干预组MC增殖受抑制,G0/G1期细胞增多,S期及G2/M期细胞减少,且m TOR表达降低。结论:m TOR信号通路参与了大鼠肾小球MC增殖的调控,抑制该信号通路的活化可显著抑制MC的增殖和MC周期的进程。     

Stimulation of mTORC2 by integrin αIIbβ3 is required for PI3Kβ-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen

Abstract

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The β isoform of PI3K is implicated in integrin αIIbβ3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbβ3 outside-in signaling PI3Kβ-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbβ3 is PI3Kβ-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/β phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kβ-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbβ3 is regulated by PI3Kβ but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbβ3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kβ-regulated platelet spreading on fibrinogen.     

辛伐他汀对冠心病患者内皮祖细胞增殖的影响及其机制初步探讨

目的:观察辛伐他汀对冠心病患者外周血内皮祖细胞(EPCs)增殖的影响并初步探讨其机制。方法:冠心病患者28例,随机分为辛伐他汀组和对照组,前者给予辛伐他汀40mg/d口服,治疗前及治疗后2、4周抽取外周血,采用密度梯度离心法获取单个核细胞,培养7d后对贴壁的细胞进行分析。以激光共聚焦显微镜鉴定为FITC标记的荆豆凝集素Ⅰ和Dil(一种亲脂性碳化青荧光染料)标记的乙酰化低密度脂蛋白双染色阳性细胞为EPCs。计数法比较二组EPCs数目,并对病人血浆低密度脂蛋白-胆固醇(LDL-C)水平和EPCs数进行相关分析。另取10例冠心病患者血培养EPCs,加入辛伐他汀、PI3K/Akt信号转导通路抑制剂Wortmannin后观察对其增殖的影响。结果:辛伐他汀组病人治疗后2周、4周外周血中EPCs明显上升(P<0.01),其变化与LDL-C变化无相关性(P>0.05),体外实验辛伐他汀 Wortmannin组的血EPC数目较辛伐他汀组明显下降(P<0.01)。结论:辛伐他汀能刺激EPCs的增殖,这种作用能被Wortmannin阻断。故辛伐他汀作用可能与激活PI3K/Akt信号转导通路有关。     

Polygalasaponin F inhibits neuronal apoptosis induced by oxygen‐glucose deprivation and reoxygenation through the PI3K/Akt pathway

Cerebral ischaemia is a common cerebrovascular disease and often induces neuronal apoptosis, leading to brain damage. Polygalasaponin F (PGSF) is one of the components in Polygala japonica Houtt, and it is a triterpenoid saponin monomer. This research focused on anti‐apoptotic effect of PGSF during oxygen‐glucose deprivation and reoxygenation (OGD/R) injury in rat adrenal pheochromocytoma cells (PC12) and primary rat cortical neurons. OGD/R treatment reduced viability of PC12 cells and primary neurons. This reduced viability was prevented by PGSF, as shown by MTT assay. OGD/R insult decreased expression of Bcl‐2/Bax both in PC12 cells and primary neurons but elevated levels of caspase‐3 in primary neurons. However, PGSF may up‐regulate expression of Bcl‐2/Bax and down‐regulate caspase‐3 in these particular cells. Furthermore, Bcl‐2/Bax and the ratio between phosphorylated Akt and total Akt were decreased in PC12 cells treated with OGD/R, and both were increased by PGSF. Moreover, increase in the ratios of Bcl‐2/Bax and phosphorylated Akt/total Akt in PC12 cells was suppressed by phosphatidylinositol 3‐kinase (PI3K) inhibitor. Data suggest PGSF might prevent OGD/R‐induced injury via activation of PI3K/Akt signalling. The ability of PGSF to block the effects of OGD/R appears to involve regulation of Bcl‐2, Bax and caspase‐3, which are related to apoptosis.     

CKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia

BackgroundChemokine-like factor 1 (CKLF1) is a chemokine increased significantly in ischemic brain poststroke. It shows chemotaxis effects on various immune cells, but the mechanisms of CKLF1 migrating neutrophils are poorly understood. Recent studies have provided evidence that CC chemokine receptor 5 (CCR5), a receptor of CKLF1, is involved in ischemic stroke.PurposesTo investigate the effects of HIF-1α guided AAV in ischemic brain, investigating the outcome of stroke, and examining the involvement of CKLF1/CCR5 axis in recruitment of neutrophils.ResultsHIF-1α guided AAV knocked down CKLF1 in ischemic area and alleviated brain damage of rats. CKLF1 migrated neutrophils through CCR5, worsening inflammatory responses. Akt/GSK-3β pathway may involve in CKLF1/CCR5 axis guided neutrophils chemotaxis.ConclusionsCKLF1/CCR5 axis is involved in neutrophils migration of rats with transient cerebral ischemia. CKLF1/CCR5 axis may be a useful target for stroke therapy.     

Upregulation of Sirt1 by tyrosol suppresses apoptosis and inflammation and modulates extracellular matrix remodeling in interleukin-1β-stimulated human nucleus pulposus cells through activation of PI3K/Akt pathway

Intervertebral disc degeneration (IDD) is the major pathogenesis of lower back pain. Tyrosol is a polyphenolic compound that exhibits anti-oxidant, anti-apoptotic, and anti-inflammatory effects. Herein, we explored the effects and mechanisms of tyrosol on IDD progression in interleukin (IL)-1β-stimulated human nucleus pulposus cells (HNPCs). Cell viability and apoptosis were detected by CCK-8 and flow cytometry analysis, respectively. The production of tumor necrosis factor-α (TNF-α), IL-6, nitric oxide (NO), and prostaglandin E2 (PGE2) was examined to evaluate inflammation. The mRNA expression of matrix metalloproteinases (MMPs) (MMP-3/9/13), collagen type II, SRY-related high mobility group box 9 (SOX-9), and aggrecan was measured by qRT-PCR. Protein levels of silent information regulator 2 homolog 1 (Sirt1), phosphorylated protein kinase B (p-Akt), Akt, collagen type II, SOX-9, and aggrecan were determined by western blot. Results showed that tyrosol attenuated IL-1β-induced viability reduction, apoptosis, and caspase-3/7 activity in HNPCs. The increase in the production of TNF-α, IL-6, NO, and PGE2 in IL-1β-treated HNPCs was abolished by tyrosol treatment. Tyrosol treatment reversed IL-1β-induced upregulation of MMP-3, MMP-9, and MMP-13, and downregulation of collagen II, SOX-9, and aggrecan in HNPCs. Additionally, tyrosol treatment activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in IL-1β-stimulated HNPCs. Sirt1 was upregulated by tyrosol, and Sirt1 silencing inhibited Akt phosphorylation in HNPCs. Sirt1 knockdown attenuated the effects of tyrosol on IL-1β-induced apoptosis, inflammation, and ECM remodeling in HNPCs. In summary, upregulation of Sirt1 by tyrosol suppressed apoptosis and inflammation and regulated ECM remodeling in IL-1β-stimulated HNPCs through activation of PI3K/Akt pathway.     

Emodin ameliorates ovalbumin-induced airway remodeling in mice by suppressing airway smooth muscle cells proliferation

Increased number of airway smooth muscle cells (ASMCs) is a characteristic of airway remodeling in asthma. In this study we investigated whether emodin alleviated airway remodeling in a murine asthma model and reduced the proliferation of ASMCs in vitro. We provided in vivo evidence suggesting that intraperitoneal injection of emodin (20 mg/kg) 1 h prior to OVA challenge apparently alleviated the thickness of airway smooth muscle, the mass of alpha-smooth muscle actin (α-SMA), collagen deposition, epithelial damage, goblet cell hyperplasia, airway inflammation and airway hyperresponsiveness (AHR) in lung tissue. Meanwhile, we found that emodin suppressed the activation of the Akt pathway in lung tissue of allergic mouse models. Additionally, we found that emodin inhibited cellular proliferation and Akt activation in a dose-dependent manner in vitro. Furthermore, LY294002, an inhibitor for PI3K, abrogated serum-induced phosphorylation of Akt, and decreased the proliferation of ASMCs. These findings indicated that emodin alleviated ASMCs proliferation by inhibiting PI3K/Akt pathway in vivo and in vitro, which may provide a potential therapeutic option for airway smooth muscle remodeling in asthma.