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.     

CKS1B promotes cell proliferation and invasion by activating STAT3/PD‐L1 and phosphorylation of Akt signaling in papillary thyroid carcinoma

ObjectiveTo investigate role of GKS1B and its relationship between STAT3/PD‐L1 and p‐Akt in papillary thyroid carcinoma (PTC).MethodsExpression of GKS1B and PD‐L1 was determined in PTC cell lines. GKS1B was overexpressed or knocked down by transfection with overexpression plasmids or si‐CKS1B. STAT3 inhibitor WP1066 was used to suppress STAT3, and PD‐L1 inhibitor Pembrolizumab was used to block PD‐L1. Cell viability and invasion were evaluated by MTT and transwell assay, respectively. The expression of STAT3, p‐STAT3, Akt, and p‐Akt was measured using Western blotting.ResultsBoth protein levels and mRNA levels of CKS1B and PD‐L1 were remarkably up‐regulated in PTC cell lines. Knockdown of CKS1B significantly inhibited cell viability and invasion of PTC cells and suppressed STAT3/PD‐L1 signaling and Akt phosphorylation, while overexpression of CKS1B led to opposite results. Inhibition of STAT3 or PD‐L1 reversed the effects of overexpressed CKS1B on PTC cells.ConclusionThe overexpression of CSK1B could promote cell viability and invasion of PTC cells through activation of STAT3/PD‐L1 signaling and Akt phosphorylation.     

Pentoxifylline modulates p47phox activation and downregulates neutrophil oxidative burst through PKA-dependent and -independent mechanisms

Background and Aim: Pentoxifylline (PTX) has been proven to be an inhibitor of fMLP-induced neutrophil (PMN) oxidative burst and is thought to function by increasing cAMP and Protein kinase A (PKA). We hypothesized that PTX diminishes production of the neutrophil respiratory burst by both PKA-dependent and independent mechanisms.

Material and Methods: Human neutrophils were isolated and stimulated with fMLP (1μM) alone or in combination with PTX (2mM). PMN activation was determined by the cytochrome C reduction method in the presence and absence of p38 MAPK (SB203580), ERK (PD98059), and PKA inhibitors (H89). Western blot analysis of Ras, Raf, p38 MAPK, ERK, and Akt was performed in PMNs exposed to fMLP and PTX. Cell membranes were fractionated to measure membrane-associated p47 phox. Treated cells were imaged using confocal microscopy to examine changes in localization of Akt and p47phox.

Results: PTX produced a decrease in oxidative burst that was diminished but not abrogated by H89 exposure. The reduction in Ras, Raf, and Akt activation seen with PTX was not effected by the presence of H89. The ability of PTX to attenuate phosphorylation of p38 MAPK and ERK was significantly decreased in the presence of H89, suggesting a PKA-dependent mechanisms. Membrane fractions of neutrophils demonstrate that PTX decreased membrane-associated p47phox, thus diminishing the ability to generate oxidative burst. PTX also decreased membrane localization of Akt and p47phox by confocal microscopy.

Conclusions: PTX attenuates activation of signaling molecules involved in activation of p47phox and suppress the subsequent assembly of the NADPH machinery through both PKA-dependent and PKA-independent mechanisms.     

Different regulation of p27 and Akt during cardiomyocyte proliferation and hypertrophy

Postnatal cardiomyocytes normally grow by hypertrophy but show a limited proliferate response to certain stimuli. Although the proliferative capacity declines shortly after birth, neonatal cardiomyocytes can grow both by hypertrophy and by proliferation. Therefore, we have used neonatal cardiomyocytes to investigate the molecular differences between hypertrophic and proliferative growth of cardiomyocytes. Stimulation of neonatal cardiomyocytes with angiotensin II mainly induced hypertrophy, whereas PDGF only had a minor effect on the size of the myocytes. In contrast, PDGF induced significant proliferation in the cardiomyocyte cultures whereas angiotensin II treatment only resulted in a small increase in the number of cells. Measurement of cyclin D-dependent kinase specific phosphorylation of pRb by immunohistochemistry showed that, both stimuli activate the G1 phase of the cell cycle. By western blotting we found that PDGF-induced proliferation correlates with activation of Akt, inactivation of GSK-3β and downregulation of the cyclin-dependent kinase inhibitor p27, whereas angiotensin II only had a small effect on Akt, GSK-3β and p27. Our data support the hypothesis that, the hypertrophic and proliferative responses are both activated by G1 cell cycle molecules. The difference between the two responses appears to be that high amounts of p27 are present during hypertrophic growth, whereas proliferation involves downregulation of p27 and GSK-3β activity and upregulation of Akt.     

CNTN-1 promotes docetaxel resistance and epithelial-to-mesenchymal transition via the PI3K/Akt signaling pathway in prostate cancer

IntroductionTherapy options for prostate cancer (PCa) typically are centered on docetaxel-based chemotherapy but are limited by the effects of multi-drug resistance. Recent advances have illustrated a role of contactin-1 (CNTN-1) in tumor chemoresistance, while the function and mechanism of CNTN-1 in the resistance of docetaxel in prostate cancer have not yet been elucidated.Material and methodsDocetaxel (Dox)-resistant PCa cell lines of PC3 (PC3-DR) and DU145 (DU145-DR) were established, and short hairpin RNA (shRNA) constructs targeting CNTN-1 were generated to analyze the effect of knockdown of CNTN-1 on PCa progression. Cell Counting Kit-8 (CCK-8), flow cytometry, wound-healing, transwell and western blotting analysis were used to analyze cell proliferation, apoptosis, migration, invasion and related protein expression levels, respectively.ResultsKnockdown of CNTN-1 in PC3-DR and DU145-DR cells attenuated cell proliferation, migration, invasion, EMT phenotype, and drug resistance, and increased cell apoptosis further reduced the tumorigenic phenotype. Knockdown of CNTN-1 resulted in an anti-tumor effect in the xenograft tumor model, and decreased activity of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway both in vitro and in vivo.ConclusionsThe results of the present study suggest that downregulation of CNTN-1 may be an important mechanism to reverse chemoresistance in Dox-resistant PCa progression, thus shedding light on the development of novel anti-tumor therapeutics for the treatment of PCa.     

染料木素通过TIPE 2负性调控Akt活性促进脂多糖活化的巨噬细胞凋亡

目的探究染料木素(genistein,GEN)对脂多糖(lipopolysaccharide,LPS)活化的RAW264.7细胞凋亡的影响及其可能的药理学作用机制。方法GEN预孵育RAW264.7细胞或慢病毒介导的肿瘤坏死因子α诱导蛋白8样分子2(tumor necrosis factor-α-induced protein 8-like 2,TIPE 2)过表达细胞2 h,再与LPS共孵育24 h,采用CCK 8试剂盒检测细胞活力,Annexin V-FITC/PI试剂盒检测细胞凋亡水平,qRT-PCR检测TNF-α、IL-6、caspase-8、caspase-3和TIPE 2 mRNA,Western blot检测iNOS、COX-2、caspase-8、caspase-3、TIPE 2、Akt和p-Akt蛋白表达。结果LPS促进RAW264.7细胞TNF-α、IL-6、iNOS、COX-2合成;GEN抑制LPS活化的RAW264.7细胞活力,凋亡细胞增多,并上调caspase-8、caspase-3、TIPE 2 mRNA及蛋白表达;TIPE 2过表达上调活化RAW264.7细胞caspase-8、caspase-3 mRNA及蛋白表达,减少Akt磷酸化,且与GEN具有协同作用。结论GEN可能通过上调TIPE 2抑制Akt活性,激活外源性凋亡途径,促进LPS活化的RAW264.7细胞凋亡。