体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的:研究体外大鼠骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells,BMSCs)在缺血缺氧条件下发生凋亡的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞(BMSCs)在缺血缺氧条件下培养,通过Annexin V/PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组(无缺血缺氧)与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K(Phosphoinosi-tide-3kinase)/Akt(ProteinkinaseB,PKB)信号通路被激活(P<0.05);同时缺血缺氧组与缺血缺氧+PI3K/Akt抑制剂(LY294002)组比较,缺血缺氧+PI3K/Akt抑制剂(LY294002)组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制(P<0.05)。结论:PI3K/Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

体外缺血缺氧条件下大鼠骨髓间充质干细胞凋亡发生的机制研究

目的：研究体外大鼠骨髓间充质干细胞（Bone marrow-derived mesenchymal stem cells, BMSCs）在缺血缺氧条件下发生凋亡的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,骨髓间充质干细胞（BMSCs）在缺血缺氧条件下培养,通过Annexin V／PI双染细胞凋亡检测比较不同组别细胞的凋亡率和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞培养成功。②对照组（无缺血缺氧）与缺血缺氧组比较,缺血缺氧组的凋亡率显著性增加,而通过磷酸化Akt的表达量显著性增加提示PI3K（Phosphoinositide-3kinase）／Akt（ProteinkinaseB,PKB）信号通路被激活（P〈0．05）;同时缺血缺氧组与缺血缺氧＋PI3K／Akt抑制剂（LY294002）组比较,缺血缺氧＋PI3K／Akt抑制剂（LY294002）组的凋亡率显著降低,而通过磷酸化Akt的表达量显著减少提示PI3K/Akt信号通路被抑制（P〈0．05）。结论：PI3K／Akt信号通路对体外缺血缺氧条件下培养的骨髓间充质干细胞凋亡发生有关键性作用。     

PI3K/Akt 通路在电针预处理诱导脑缺血耐受中的机制研究

目的:通过观察电针预处理对磷脂酰肌醇3激酶/蛋白质丝氨酸苏氨酸激酶(PI3K/Akt)通路的变化以及该通路抑制剂对电针预处理的脑保护的影响,探讨电针预处理诱导脑缺血耐受的可能机制。方法:线栓法单侧阻断大脑中动脉120min,再灌注24h制备大鼠大脑局灶性缺血再灌注(I/R)模型;Western Blot检测Akt磷酸化水平的变化;侧脑室注射PI3K/Akt通路抑制剂LY294002;神经行为学评分(Garcia标准)及TTC染色检测脑梗死体积比评价脑损伤程度。结果:电针预处理使大鼠神经行为学评分增高,脑梗死体积比降低(P<0.05);可上调Akt磷酸化水平,I/R2h达高峰(P<0.05)。侧脑室注射PI3K/Akt抑制剂LY294002,拮抗电针预处理的脑保护作用(P<0.05)。结论:电针预处理增加Ak(tSer473)磷酸化水平,在缺血再灌注早期上调PI3K/Akt通路可能是诱导大鼠脑缺血耐受的产生的主要机制。     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

葡萄籽原花青素通过PI3K/Akt信号通路抑制氧化应激诱导的H9c2心肌细胞凋亡

为了探讨葡萄籽原花青素(grape seed proanthocyanidin, GSP)对心肌细胞的保护作用及机制,通过CCK-8法评估细胞活力,采用Western-blot分析评估GSP对凋亡相关蛋白质(cleaved caspase-3、Bax和Bcl-2)和PI3K/Akt通路相关蛋白质(p-PI3K、PI3K、p-Akt和Akt)表达水平的影响,并使用TUNEL染色和Hoechst 33258染色评估H9c2心肌细胞凋亡情况。结果显示, GSP可以抑制H₂O₂诱导的H9c2心肌细胞的细胞毒性和凋亡,使促凋亡蛋白cleaved caspase-3和Bax表达下降,并使抗凋亡蛋白Bcl-2表达水平升高; GSP作用于H9c2细胞后, PI3K和Akt的磷酸化水平增加,使PI3K/Akt信号通路激活。实验结果初步表明, GSP可抑制氧化应激诱导的H9c2心肌细胞凋亡,其作用机制可能与激活PI3K/Akt信号通路有关。     

RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌细胞中的相关性研究

目的探讨肿瘤转移相关因子RhoGDI2与PI3K/Akt/mTOR信号通路在肺癌侵袭转移过程中的作用及相关机制。方法利用PI3K/Akt/mTOR信号通路上特异性的抑制剂,采用MTT法,伤口愈合实验及侵袭实验观察不同浓度药物对肺癌95D细胞生长侵袭转移能力的影响,通过Western Blot方法观察RhoGDI2蛋白水平的变化。结果PI3K抑制剂LY294002及mTOR抑制剂Rapamycin都能抑制肺癌细胞95D的侵袭转移能力,联合应用抑制作用更强。PI3K抑制剂LY294002处理组RhoGDI2蛋白的表达量增加,且随浓度增加RhoGDI2蛋白表达也增加。mTOR抑制剂Rapamycin组,在低浓度时增加RhoGDI2蛋白的表达,但增大Rapamycin的浓度,RhoGDI2蛋白的表达反而降低。低浓度LY294002组和Rapa-mycin组联合应用可以明显增加RhoGDI2蛋白的表达。结论PI3K/Akt/mTOR信号通路中Akt的活化与RhoGDI2密切相关,RhoGDI2可能直接或间接通过与Akt的相互作用参与调节肺癌的侵袭转移的过程。     

PI3K/AKT信号转导通路与人舌鳞癌细胞TCA8113顺铂耐药的相关性研究

目的:探讨PI3K特异性抑制剂LY294002逆转顺铂耐药口腔鳞癌细胞TCA8113/CDDP的可行性。方法:采用间歇性加药,逐步递增CDDP药量,体外连续诱导培养TCA8113/CDDP细胞;用不同浓度的LY294002和顺铂处理TCA8113和TCA8113/CDDP细胞;MTT法观察对细胞增殖的影响,Western印迹分析LY294002作用前后p-Akt、Akt、PI3K蛋白的表达。结果:建立了舌鳞癌耐药细胞TCA8113/CDDP,耐药指数为7.7;MTT实验显示LY294002对TCA8113和TCA8113/CDDP细胞的抑制作用与浓度及作用时间呈正相关;LY294002联合顺铂对2种细胞的抑制作用比单用顺铂效果好;PI3K、Akt、p-AKT蛋白表达明显降低,其中TCA8113/CDDP细胞中PI3K、AKT、p-AKT蛋白的表达比TCA8113细胞明显增多(P0.05)。结论:LY294002能增加耐药口腔鳞癌顺铂化疗的敏感性。     

高糖和LY294002干预影响足细胞内Ⅳ型胶原表达

探讨高糖和PI3K/Akt通路对足细胞内Ⅳ型胶原(Col Ⅳ)表达的影响。体外培养小鼠足细胞,给予高糖(30mmol/L)处理后,分别于0,12,24,48h收集细胞,采用免疫细胞化学染色法和Western blot技术检测Col Ⅳ的表达;Western blot技术检测Akt的活化及LY294002对Col Ⅳ表达的抑制效应。结果表明,高糖诱导足细胞内Col Ⅳ蛋白表达增多,24h明显,各时间点与高糖刺激前相比均有统计学差异(P<0.05);高糖激活Akt蛋白磷酸化,p-Akt随刺激时间延长表达增多。PI3K/Akt通路抑制剂LY294002孵育细胞24h后,可减弱高糖诱导的足细胞内Col Ⅳ的表达(P<0.05)。因此,高糖可能通过激活PI3K/Akt通路上调足细胞内Ⅳ型胶原表达。     

PPAR-α对心肌肥大的调控作用及与PI3K/Akt/mTOR通路的关系

目的:研究过氧化物酶体增殖物激活受体-α(PPAR-α)对心肌细胞肥大的调控作用及其与PI3K/Akt/m TOR通路的关系。方法:异丙肾上腺素(ISO)诱导心肌细胞肥大;Leica图像分析软件测量心肌细胞表面积;qRT-PCR方法检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)、PPAR-αmRNA表达;Western blot检测Akt、哺乳动物雷帕霉素靶蛋白(m TOR)、P70S6K蛋白表达;PPAR-αRNAi抑制PPAR-α的表达。结果:1心肌细胞肥大时,PPAR-α表达显著下降;非诺贝特(Feno)可上调PPAR-α表达,抑制心肌细胞肥大;Feno对心肌细胞肥大的抑制效应可被PPAR-αRNAi所逆转;2Feno能明显抑制ISO诱导的心肌细胞p-Akt、p-m TOR和p-p70S6K蛋白表达的增加;Feno的上述作用可被PPAR-αRNAi所逆转;3PI3K抑制剂LY294002(LY)或m TOR抑制剂雷帕霉素(RAPA)均能明显抑制心肌细胞肥大,LY或RAPA抑制心肌细胞肥大的效应均可被PPAR-αRNAi所取消。结论:PPAR-α通过负性调控抑制心肌细胞肥大。PPAR-α抑制心肌细胞肥大的作用可能与其抑制PI3K/Akt/m TOR信号通路有关。     

PI3K通路抑制剂LY294002对人脑胶质瘤细胞株U87细胞衰老和凋亡的影响

目的:PI3K/Akt信号通路是与胶质瘤发生发展密切相关的核心通路之一,LY294002是该通路的特异性抑制剂。本研究通过探讨PI3K通路抑制剂LY294002对U87胶质瘤细胞系细胞衰老及凋亡的影响,从而为胶质瘤患者治疗的新策略奠定理论基础。方法:将体外培养的人脑胶质瘤U87细胞株分为DMSO处理的对照组和LY294002(100μM)处理的实验组,采用β-半乳糖苷酶染色和流式细胞术的方法,分别检测并比较两组肿瘤细胞衰老和凋亡的情况。结果:LY294002处理组U87胶质瘤细胞的衰老指数(32.20±4.46%)显著高于DMSO对照组(3.40±1.61%,t=6.254,P0.001)。另外,与DMSO对照组相比,凋亡蛋白caspase-3mRNA的表达在LY294002处理组胶质瘤细胞中显著上调(t=8.923,P0.05)。LY294002处理组肿瘤细胞的凋亡指数(80.10±4.832%)明显高于DMSO对照组(4.260±1.073%,t=8.923,P0.05)。结论:LY294002既能够诱导肿瘤细胞衰老,又能够诱导肿瘤细胞凋亡,然而其诱导胶质瘤细胞凋亡的能力占据主导地位,为其发挥抗胶质瘤效应的主要途径。另外,在LY294002的持续作用下,部分衰老的肿瘤细胞或许会发生凋亡。这些结论为为临床增强胶质瘤患者的联合化疗奠定了理论基础。     

脂联素后处理对大鼠心肌缺血/再灌注损伤的影响及ADP/PI3K/Akt信号通路的作用

目的：探讨脂联素（ADP）后处理对大鼠心肌缺血/再灌注损伤（MIRI）的影响以及脂联素/磷脂酰肌醇-3激酶/蛋白激酶B （ADP/PI3K/Akt）通路的作用。方法：SD大鼠麻醉后气管插管连接呼吸机,开胸暴露心肌,在左心耳和肺动脉圆锥之间用带线圆针对冠脉左前降支（LAD）穿线,LAD结扎断流30 min后松线再灌注120 min,建立大鼠MIRI模型。大鼠随机分为以下5组（n=12）：①假手术组（Sham组）：LAD仅穿线不结扎;② MIRI组;③ADP后处理组（ADP组）：LAD断流10 min时静注ADP继续断流20 min,然后再灌注120 min;④ADP+LY294002组：LAD断流10 min时静注ADP和LY294002,其余同ADP组;⑤LY294002组：LAD断流10 min时静注LY294002,其余同ADP组。各组取血检测LDH和cTnI含量,取左心室心肌测定PI3k、Akt、p-Akt、ADPmRNA、ADPR1mRNA和PI3KmRNA表达。结果：与Sham组比较,MIRI组血浆LDH和cTnI均明显升高（P<0.05）;和MIRI组相比,ADP组心肌损伤指标明显下降（P<0.05）,而应用LY294002的两组心肌损伤比ADP组加重（P<0.05）。ADP组心肌PI3K、p-Akt、ADPmRNA、ADPR1mRNA和PI3kmRNA表达比MIRI组升高（P<0.05）,应用LY294002两组上述5个指标比MIRI组降低（P<0.05）。结论：ADP后处理对大鼠MIRI有保护作用,ADP/PI3K/Akt通路参与了以上作用。     

Overexpression of microRNA‐138 alleviates human coronary artery endothelial cell injury and inflammatory response by inhibiting the PI3K/Akt/eNOS pathway

This study aimed to investigate the role of miR‐138 in human coronary artery endothelial cell (HCAEC) injury and inflammatory response and the involvement of the PI3K/Akt/eNOS signalling pathway. Oxidized low‐density lipoprotein (OX‐LDL)‐induced HCAEC injury models were established and assigned to blank, miR‐138 mimic, miR‐138 inhibitor, LY294002 (an inhibitor of the PI3K/Akt/eNOS pathway), miR‐138 inhibitor + LY294002 and negative control (NC) groups. qRT‐PCR and Western blotting were performed to detect the miR‐138, PI3K, Akt and eNOS levels and the protein expressions of PI3K, Akt, eNOS, p‐Akt, p‐eNOS, Bcl‐2, Bax and caspase‐3. ELISAs were employed to measure the expressions of TNF‐α, IL‐4, IL‐6, IL‐8, IL‐10 and nitric oxide (NO) and the activities of lactate dehydrogenase (LDH) and eNOS. MTT and flow cytometry were performed to assess the proliferation and apoptosis of HCAECs. Compared to the blank group, PI3K, Akt and eNOS were down‐regulated in the miR‐138 mimic and LY294002 groups but were up‐regulated in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups showed decreased concentrations of TNF‐α, IL‐6, IL‐8 and NO and reduced activities of LDH and eNOS, while opposite trends were observed in the miR‐138 inhibitor group. The concentrations of IL‐4 and IL‐10 increased in the miR‐138 mimic and LY294002 groups but decreased in the miR‐138 inhibitor group. The miR‐138 mimic and LY294002 groups had significantly decreased cell proliferation and increased cell apoptosis compared to the blank group. These findings indicate that up‐regulation of miR‐138 alleviates HCAEC injury and inflammatory response by inhibiting the PI3K/Akt/eNOS signalling pathway.     

Protective effect of α-mangostin derivatives on hypoxia/reoxygenation-induced apoptosis in H9C2 cells and their mechanism

In this study, 17 α-mangostin Boc amino acid/organic acid ester derivatives 1–17 were synthesized and subjected to cytotoxicity and cell viability screening assays. A hypoxia/reoxygenation model of cardiomyocyte injury was selected and compound 5 was found to have a better protective effect against hypoxia/reoxygenation-induced myocardial injury by prophylactic administration screening. The levels of LDH and CK-MB in extracellular fluid were detected by ELISA; apoptosis was detected by Hoechst3358/PI double staining, Annexin V-FITC/PI double staining and mitochondrial membrane potential; the expression of key proteins in PI3K/Akt signaling pathway was detected by western blot. The result showed that compound 5 was non-toxic and has a significant cytoprotection effect at concentrations of 1 μM and 10 μM, and reduced the levels of LDH and CK-MB in the extracellular fluid. Hoechst 33,258/PI double staining results showed that compound 5 treatment significantly reduced bright blue cell nuclei and had anti-apoptotic effects; flow cytometry results showed that compound 5 improved hypoxia/reoxygenation-induced mitochondrial membrane potential and thus apoptosis. The western blot results showed that compound 5 upregulated the levels of p-PI3K and p-Akt, decreased the expression of cleaved caspase-3, cleaved caspase-9 and increased the Bcl-2/Bax ratio in a concentration-dependent manner. In addition, compound 5 reversed the effect of the LY294002 inhibitor. The present study suggests that compound 5 may serve as a potential PI3K activator and a safe and effective lead compound for the treatment of cardiovascular disease.     

Salidroside Improves Behavioral and Histological Outcomes and Reduces Apoptosis via PI3K/Akt Signaling after Experimental Traumatic Brain Injury

Background

Traumatic brain injury (TBI) induces a complex sequence of apopototic cascades that contribute to secondary tissue damage. The aim of this study was to investigate the effects of salidroside, a phenolic glycoside with potent anti-apoptotic properties, on behavioral and histological outcomes, brain edema, and apoptosis following experimental TBI and the possible involvement of the phosphoinositide 3-kinase/protein kinase B (PI3K)/Akt signaling pathway.

Methodology/Principal Findings

Mice subjected to controlled cortical impact injury received intraperitoneal salidroside (20, or 50 mg/kg) or vehicle injection 10 min after injury. Behavioral studies, histology analysis and brain water content assessment were performed. Levels of PI3K/Akt signaling-related molecules, apoptosis-related proteins, cytochrome C (CytoC), and Smac/DIABLO were also analyzed. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, was administered to examine the mechanism of protection. The protective effect of salidroside was also investigated in primary cultured neurons subjected to stretch injury. Treatment with 20 mg/kg salidroside_significantly improved functional recovery and reduced brain tissue damage up to post-injury day 28. Salidroside_also significantly reduced neuronal death, apoptosis, and brain edema at day 1. These changes were associated with significant decreases in cleaved caspase-3, CytoC, and Smac/DIABLO at days 1 and 3. Salidroside increased phosphorylation of Akt on Ser473 and the mitochondrial Bcl-2/Bax ratio at day 1, and enhanced phosphorylation of Akt on Thr308 at day 3. This beneficial effect was abolished by pre-injection of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Moreover, delayed administration of salidroside at 3 or 6 h post-injury reduced neuronal damage at day 1. Salidroside treatment also decreased neuronal vulnerability to stretch-induced injury in vitro.

Conclusions/Significance

Post-injury salidroside improved long-term behavioral and histological outcomes and reduced brain edema and apoptosis following TBI, at least partially via the PI3K/Akt signaling pathway.     

PI3K/Akt信号通路在白藜芦醇抗大鼠缺血/再灌注性心律失常中的作用及机制

目的：探讨磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路在白藜芦醇抗缺血/再灌注性心律失常中的作用及机制。方法：48只健康雄性SD大鼠,取心电图正常者随机分为4组（n=10）：假手术（SC组）组、缺血/再灌注（I/R组）组、白藜芦醇处理（Res处理组）组、PI3K抑制剂LY294002（LY294002组）组。建立大鼠在体心肌缺血/再灌注模型,观察各组心律失常的发生情况及左室血流动力学变化,Western blot法测定心肌组织中蛋白激酶B（Akt）、磷酸化蛋白激酶B（p-Akt）、缝隙连接蛋白43（Cx43）蛋白表达水平,以RT-PCR法从转录水平检测Cx43的表达水平。结果：与I/R组相比,Res处理组心律失常的发生率（心律失常评分）明显降低、左室舒缩功能明显升高,同时心肌Akt、Cx43蛋白表达及Cx43mRNA水平也明显升高;使用PI3K抑制剂LY294002后,心肌Akt、Cx43蛋白表达及Cx43mRNA水平下降的同时心律失常的发生率明显升高、左室舒缩功能明显降低。结论：白藜芦醇的抗再灌注性心律失常作用可能是通过激活PI3K/Akt信号通路,改变Cx43活性及分布实现的。     

Cardiomyocyte protective effects of thyroid hormone during hypoxia/reoxygenation injury through activating of IGF-1-mediated PI3K/Akt signalling

Ischaemia/reperfusion (I/R) injury is a common clinical condition that results in apoptosis and oxidative stress injury. Thyroid hormone was previously reported to elicit cardiac myocyte hypertrophy and promote cardiac function after cardiac injury. We used an in vivo mouse model of I/R injury and in vitro primary cardiomyocyte culture assays to investigate the effects of thyroid hormone on cardiomyocytes during hypoxia/reoxygenation (H/R) injury. The results showed that T3 pretreatment in vivo significantly improved left ventricular function after I/R injury. In vitro, T3 pretreatment decreased cell apoptosis rate, inhibited caspase-3 activity and decreased the Bax/Bcl-2 ration induced by H/R injury. T3 pretreatment significantly attenuated the loss of mitochondrial membrane potential. Furthermore, it was observed that T3 diminished the expression of NCX1 protein and decreased SERCA2a protein expression in H/R-induced cardiomyocytes, and T3 prevented intracellular Ca²⁺ increase during H/R injury. Also, T3 increased the expression of IGF-1, and PI3K/Akt signalling in cardiomyocytes under H/R-induced injury, and that the protective effect of T3 against H/R-induced injury was blocked by the PI3K inhibitor LY294002. IGF-1 receptor (IGF-1R) inhibitor GSK1904529A significantly inhibited the expression of IGF-1R and PI3K/Akt signalling. In summary, T3 pretreatment protects cardiomyocytes against H/R-induced injury by activating the IGF-1-mediated PI3K/Akt signalling pathway.     

Quercetin Attenuates Cell Apoptosis in Focal Cerebral Ischemia Rat Brain Via Activation of BDNF–TrkB–PI3K/Akt Signaling Pathway

Many studies have demonstrated that apoptosis play an important role in cerebral ischemic pathogenesis and may represent a target for treatment. Neuroprotective effect of quercetin has been shown in a variety of brain injury models including ischemia/reperfusion. It is not clear whether BDNF?CTrkB?CPI3K/Akt signaling pathway mediates the neuroprotection of quercetin, though there has been some reports on the quercetin increased brain-derived neurotrophic factor (BDNF) level in brain injury models. We therefore first examined the neurological function, infarct volume and cell apoptosis in quercetin treated middle cerebral artery occlusion (MCAO) rats. Then the protein expression of BDNF, cleaved caspase-3 and p-Akt were evaluated in either the absence or presence of PI3K inhibitor (LY294002) or tropomyosin receptor kinase B (TrkB) receptor antagonist (K252a) by immunohistochemistry staining and western blotting. Quercetin significantly improved neurological function, while it decreased the infarct volume and the number of TdT mediated dUTP nick end labeling positive cells in MCAO rats. The protein expression of BDNF, TrkB and p-Akt also increased in the quercetin treated rats. However, treatment with LY294002 or K252a reversed the quercetin-induced increase of BDNF and p-Akt proteins and decrease of cleaved caspase-3 protein in focal cerebral ischemia rats. These results demonstrate that quercetin can decrease cell apoptosis in the focal cerebral ischemia rat brain and the mechanism may be related to the activation of BDNF?CTrkB?CPI3K/Akt signaling pathway.     

Hypoxia/Reoxygenation Stimulates Proliferation Through PKC-Dependent Activation of ERK and Akt in Mouse Neural Progenitor Cells

Cerebral ischemia increases neural progenitor cell proliferation and neurogenesis. However, the precise molecular mechanism is poorly understood. The present study was undertaken to determine roles of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/Akt and their signaling pathways in neural progenitor cells exposed to hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion. Neural progenitor cells were isolated from postnatal mouse brain. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of MEK, but not by LY294002, a specific inhibitor of PI3K, whereas the Akt activation was blocked by LY294002, but not by U0126. Reoxygenation following 4-h hypoxia stimulated cell proliferation, which was dependent on ERK and Akt activation. Inhibitors of growth factor receptor (AG1478) and Src (PP2) and the antioxidant N-acetylcysteine did not affect activation of ERK and Akt, while the Ras and Raf inhibitors inhibited activation of ERK, but not Akt. PKC inhibitors inhibited both ERK and Akt activation. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt survival signaling pathways through a PKC-dependent mechanism. These pathways may be responsible for the repair process during ischemia/reperfusion.     

Over-expression of microRNA-494 up-regulates hypoxia-inducible factor-1 alpha expression via PI3K/Akt pathway and protects against hypoxia-induced apoptosis

Background

Hypoxia-inducible factor-1 alpha (HIF-1α) is one of the key regulators of hypoxia/ischemia. MicroRNA-494 (miR-494) had cardioprotective effects against ischemia/reperfusion (I/R)-induced injury, but its functional relationship with HIF-1α was unknown. This study was undertaken to determine if miR-494 was involved in the induction of HIF-1α.

Results

Quantitative RT-PCR showed that miR-494 was up-regulated to peak after 4 hours of hypoxia in human liver cell line L02. To investigate the role of miR-494, cells were transfected with miR-494 mimic or miR-negative control, followed by incubation under normoxia or hypoxia. Our results indicated that overexpression of miR-494 significantly induced the expression of p-Akt, HIF-1α and HO-1 determined by qRT-PCR and western blot under normoxia and hypoxia, compared to negative control (p < 0.05). While {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 treatment markedly abolished miR-494-inducing Akt activation, HIF-1α and HO-1 increase under both normoxic and hypoxic conditions (p < 0.05). Moreover, apoptosis detection using Annexin V indicated that overexpression of miR-494 significantly decreased hypoxia-induced apoptosis in L02 cells, compared to control (p < 0.05). MiR-494 overexpression also decreased caspase-3/7 activity by 1.27-fold under hypoxia in L02 cells.

Conclusions

Overexpression of miR-494 upregulated HIF-1α expression through activating PI3K/Akt pathway under both normoxia and hypoxia, and had protective effects against hypoxia-induced apoptosis in L02 cells. Thus, these findings suggested that miR-494 might be a target of therapy for hepatic hypoxia/ischemia injury.