靶向TRIM29基因的siRNA通过抑制PI3K/AKT信号通路诱导鼻咽癌细胞凋亡

目的:探讨沉默TRIM29基因表达对鼻咽癌细胞凋亡及PI3K/AKT信号通路的影响。方法:将人鼻咽癌5-8F细胞分为空白组、阴性对照(NC)组(转染阴性对照siRNA)和si-TRIM29组(转染TRIM29的特异性siRNA),通过CCK-8法检测si-TRIM29转染5-8F细胞0～96 h的细胞活力。通过流式细胞术及Western blot分别检测si-TRIM29转染5-8F细胞48 h的细胞凋亡率及TRIM29、cleaved caspase-3、cleaved caspase-9、Bcl-2、Bax、t-AKT和p-AKT的蛋白水平。10μmol/L的PI3K/AKT信号通路特异性抑制剂LY294002及si-TRIM29单独或联用处理细胞,细胞分为空白组、LY294002组和LY294002+si-TRIM29组,流式细胞术检测3组细胞凋亡率。结果:转染TRIM29 siRNA的5-8F细胞TRIM29蛋白表达明显低于空白组(P0.05)。和空白组比较,si-TRIM29组细胞活力明显降低,凋亡率明显升高,cleaved caspase-3、cleaved caspase-9和Bax蛋白的水平明显升高,Bcl-2和p-AKT蛋白的水平明显降低(P0.05)。LY294002组的细胞凋亡率高于空白组,而LY294002+si-TRIM29组的细胞凋亡率高于LY294002组(P0.05)。结论:沉默TRIM29基因表达可通过抑制PI3K/AKT信号通路诱导鼻咽癌细胞凋亡。     

PI3K/AKT信号通路的抑制促进B7-H4分子的核转移

目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/AKT)信号通路对负性共刺激分子B7-H4细胞定位的调控作用。方法体外培养稳定转染B7-H4/HEK293细胞,采用PI3K/AKT特异性抑制剂LY294002和/或出核转运抑制剂来普霉素B(LMB)处理细胞之后,免疫荧光技术结合激光共聚焦显微镜检测B7-H4蛋白在B7-H4/HEK293细胞中亚细胞定位的变化;Western blot法检测B7-H4蛋白在细胞膜、细胞质和细胞核表达水平的变化。结果激光共聚焦显微镜观察结果显示,PI3K抑制剂LY294002作用于B7-H4稳定转染的B7-H4/HEK293细胞后,与空白对照组相比,B7-H4发生核转移,加入出核转运抑制剂LMB后,核转移的程度显著增加;Western blot法结果显示,LY294002抑制PI3K/AKT信号通路活性24 h后,B7-H4在细胞膜和细胞质中的定位均显著降低(P0.05),而细胞核中B7-H4的定位显著增加(P0.05)。结论 PI3K/AKT信号通路可抑制B7-H4的核转移。     

PTTG抑制PI3K/AKT信号通路诱导皮肤鳞状细胞癌细胞凋亡的机制

目的探讨垂体肿瘤转化基因(pituitary tumoRtransforming genes,PTTG)对皮肤鳞状细胞癌细胞活力、凋亡及PI3K/AKT信号通路的影响。方法将设计合成的PTTG特异性siRNA(si-PTTG)转染人皮肤鳞癌细胞系A431(si-PTTG组),无干扰作用siRNA(NC组)及未转染的空白细胞(空白组)作为对照组,LY294002作为PI3K/AKT信号通路的抑制剂。通过MTT法、Annexin V-FITC/PI细胞凋亡检测试剂盒及ROS检测试剂盒分别检测细胞活力、凋亡率和ROS含量。Western blot法检测PTTG、AKT、p-AKT、Cyclin D1和Bax蛋白表达。结果si-PTTG转染A431细胞后,PTTG表达明显降低(P<0.05)。与空白组比较,si-PTTG组和LY294002组细胞活力明显降低,凋亡率明显升高,si-PTTG组ROS含量明显升高,p-AKT和Cyclin D1蛋白表达明显降低,Bax蛋白表达明显升高(P<0.05)。A431细胞转染si-PTTG,并用LY294002处理,细胞活力明显低于LY294002组,凋亡率高于LY294002组(P<0.05)。结论沉默PTTG表达可抑制皮肤鳞状细胞癌细胞活力,诱导细胞凋亡,其机制与细胞ROS水平增高及PI3K/AKT通路抑制有关。     

ECT2通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响

目的 探讨上皮细胞转化序列2(ECT2)通过调控PI3K/AKT信号通路对胰腺癌细胞恶性行为、糖酵解及TH细胞分化的影响。方法 RT-qRCR检测人脐静脉内皮细胞系HUVEC及胰腺癌细胞中ECT2 mRNA表达，将胰腺癌Panc-1细胞株分为胰腺癌组（Panc-1细胞株正常培养）、NC组（Panc-1细胞株转染ECT2阴性对照）、ECT2 siRNA组（Panc-1细胞株转染ECT2 siRNA）、抑制剂组（Panc-1细胞株转染加入PI3K/AKT抑制剂LY294002,ECT2 siRNA+抑制剂组（Panc-1细胞株转染ECT2 siRNA加入PI3K/AKT抑制剂LY294002），采用RT-qRCR各组细胞中ECT2 mRNA表达；Transwell法检测各组Panc-1细胞侵袭能力；划痕实验检测迁移；流式细胞仪检测各组细胞IFN-γ及IL-4表达；免疫印迹分别检测糖酵解代表蛋白及PI3K/AKT表达。结果 胰腺癌组、NC组、ECT2 siRNA组、抑制剂组及ECT2 siRNA+抑制剂组的ECT2 mRNA比较分别为1.00±0.00、0.95±0.03、0.41±0.08...     

Rho相关激酶抑制剂Y27632对小鼠腔前卵泡体外发育的作用

目的 探讨Rho相关激酶（ROCK）抑制剂Y27632在小鼠腔前卵泡体外发育中的作用。 方法 取出生12.5 d的小鼠卵巢,机械法收集单枚腔前卵泡,采用超低吸附96孔板模拟3D环境对其进行培养。设置对照组和含有Y27632的实验组,通过形态学观察、卵泡直径的变化、成熟卵泡的数量、成熟卵母细胞纺锤体的变化等来评估卵泡的整体发育情况。采用Real-time PCR技术检测颗粒细胞中卵泡刺激素受体（FSHR）、卵母细胞中骨形态发生蛋白15（BMP15）、生长分化因子9（GDF9）等以及凋亡相关基因（Bad、Bax和Caspase-3）的表达情况,同时,用细胞存活染料检测卵泡发育过程中颗粒细胞的凋亡情况。 结果 ROCK抑制剂Y27632对卵泡直径增长和基本发育指标（存活数、成腔率和成熟率）无明显影响（P>0.05）,但对照组卵母细胞成熟后纺锤体组装出现异常。培养8 d后,与对照组相比,实验组颗粒细胞特异性基因FSHR上调;卵母细胞特异性基因BMP15和GDF9上调,而叉头框O3（FoxO3)下调;凋亡基因Bax、Bad和Caspase3表达下调,实验组卵泡内的颗粒细胞凋亡明显少于对照组。 结论 小鼠卵泡体外发育过程中,ROCK抑制剂Y27632能够抑制颗粒细胞的凋亡,防止卵母细胞纺锤体组装异常,进而提高卵泡体外发育质量。     

RAD51相关蛋白1在宫颈癌组织中的表达及通过TGF-β/Smad 通路 参与调控人宫颈癌细胞的增殖和凋亡*

目的：探讨RAD51相关蛋白1（RAD51AP1）通过调节转化生长因子β（TGF-β）/Smad 信号通路对人宫颈 癌细胞（SiHa）增殖和凋亡的影响。方法：收集2018 年7 月至2021 年3 月间南阳市中心医院51 例宫颈癌组织与 癌旁组织标本,免疫组织化学法检测宫颈癌组织RAD51AP1蛋白表达。筛选RAD51AP1高表达宫颈癌细胞系, 体外培养SiHa 细胞,分为对照组、si-RAD51AP1 组（ 转染si-RAD51AP1 质粒）、si-NC 组（ 转染si-NC 质粒）、 抑制剂组（TGF-β/Smad 通路抑制剂LY2109761）、si-RAD51AP1+ 激活剂组（ 转染si-RAD51AP1 质粒+TGF-β/ Smad 通路激活剂人重组TGF-β1）。采用MTT法与平板克隆形成实验检测各组SiHa 细胞增殖;流式细胞术检测 细胞凋亡;实时荧光定量PCR法检测细胞中RAD51AP1、TGF-β1 mRNA 水平;免疫印迹检测细胞中RAD51AP1 蛋白、TGF-β/Smad 通路相关蛋白及增殖、凋亡相关蛋白表达。结果：RAD51AP1在宫颈癌组织与SiHa 细胞系中 均显著高表达;抑制RAD51AP1表达或抑制TGF-β/Smad 通路后,细胞增殖活性、细胞克隆形成率及cyclin D1、 TGF-β1、p-SMAD2、p-SMAD3 蛋白表达水平显著减低,细胞凋亡率、Bax、caspase-3、cleaved caspase 3 蛋白 表达水平显著升高,而抑制RAD51AP1表达基础上使用TGF-β/Smad 通路激活剂后,可部分逆转上述变化。结 论：RAD51AP1在宫颈癌中表达上调,抑制RAD51AP1表达可通过抑制TGF-β/Smad 通路而抑制宫颈癌细胞增殖, 促进细胞凋亡。     

PTEN调控大鼠原始卵泡启动和生长

目的探讨PTEN对大鼠原始卵泡激活和生长的调控作用和机制。方法 2日龄大鼠卵巢在Waymouth培养系统中培养0、4和8 d,用HE染色检测慢病毒PTEN siRNA的沉默效率及其对原始卵泡生长启动情况。同步用实时定量聚合酶链反应(RT-PCR)和免疫组织化学染色检测PTEN mRNA和蛋白在卵泡生长中的表达和动态变化;用慢病毒PTEN-shRNA沉默卵巢中PTEN表达后,观察对原始卵泡启动生长及雌二醇和孕酮分泌的影响;此外,利用PI3K抑制剂LY294002探讨了PTEN在原始卵泡形成过程中的可能信号通路。结果 PTEN mRNA和蛋白在原始卵泡中均有表达,随着原始卵泡的发育,其表达水平降低(P0.01);经PTEN-shRNA慢病毒转染后,荧光成像显示慢病毒在体外已成功转染到卵巢组织,HE染色显示原始卵泡数量减少(P0.01),表明PTEN参与了原始卵泡的起始。结论 PTEN可通过PI3K信号通路和调节雌、孕激素分泌调控原始卵泡发育。它在原始卵泡的发育启动中起着重要的作用。     

PI3K/Akt通路介导TRPV1抑制离体小鼠心脏缺血再灌注后的细胞凋亡

目的研究瞬时受体电位香草酸亚型1(TRPV1)对离体小鼠心脏缺血/再灌注(I/R)后心肌细胞凋亡的影响及与PI3K/Akt通路的关系。方法 TRPV1基因敲除(TRPV1~(-/-))和野生型(WT)小鼠各54只,均各随机分为假手术组(sham)、缺血再灌注组(I/R)和LY294002处理组(LY)。建立Langendorff离体心脏灌注模型,检测心功能;灌注结束后,TTC染色法检测心肌梗死面积;TUNEL法检测心肌细胞凋亡;Western blot检测Bcl-2、Bax、Akt和p-Akt的蛋白表达水平。结果 I/R后,TRPV1~(-/-)小鼠较WT小鼠的LVDP明显降低(P0.001),LVEDP明显升高(P0.001),同时心肌梗死面积和心肌凋亡细胞明显增加(P0.01),Bcl-2/Bax和p-Akt表达水平下降(P0.001)。LY294002阻断PI3K后,与相应的I/R组相比,WT小鼠心肌梗死面积明显扩大(P0.001),心肌凋亡细胞明显增加(P0.01),Bcl-2/Bax和p-Akt蛋白表达水平降低(P0.001)。结论 TRPV1可能通过PI3K/Akt通路抑制离体小鼠心脏缺血/再灌注所致的心肌细胞凋亡。     

米诺环素通过调控PI3K/Akt通路抑制硝普钠诱导的PC12细胞凋亡

 目的： 探讨PI3K/Akt信号通路在米诺环素（minocycline,MC）抑制硝普钠(sodium nitoprusside,SNP)诱导的PC12细胞凋亡中的作用。方法：将体外培养的PC12细胞分为4组：空白对照组、SNP组、MC+SNP组和PI3K抑制剂LY294002+ MC+SNP组。用四甲基偶氮唑盐（MTT）法检测细胞活力,流式细胞术检测细胞凋亡;Western blotting检测不同时点（0.5、1、2、3 h）各处理组PI3K/Akt通路蛋白p-Akt和Akt的表达。结果：SNP处理PC12细胞24 h能抑制细胞生长,加入10 μmol/L MC预处理30 min可明显提高细胞活力,降低细胞凋亡率（P<0.05）,抑制SNP诱导的PC12细胞凋亡。MC组的p-Akt表达高于其它组,而加入LY294002后可阻断MC的上述效应。结论：MC可通过调控PI3K/Akt通路抑制SNP诱导的PC12细胞凋亡。     

PI3K/Akt通路在滋养细胞增殖中的调控机制研究

旨在探讨PI3K/Akt信号转导通路在滋养细胞增殖中的作用及具体调控机制。体外培养滋养细胞系EVT。应用MTT法检测不同浓度表皮生长因子（EGF）刺激后EVT的增殖情况;应用流式细胞技术检测不同处理组EVT凋亡情况。使用PI3K抑制剂LY294002处理细胞后,检测以上各项结果的变化。结果发现：1.随EGF浓度增高,EVT增殖呈现增强趋势,EGF在10ng/ml及其以上时效应明显;2.EGF能够显著降低EVT的凋亡发生;3.使用PI3K抑制剂LY294002明显逆转EGF的促进EVT增殖的效应。提示表皮生长因子可以活化滋养细胞的PI3K/Akt信号通路,进而促进细胞增殖,并且抑制其凋亡,PI3K抑制剂可以明显抑制EGF的促EVT增殖作用。     

BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2

Spermatogonial stem cells (SSCs) capable of self‐renewal and differentiation are the foundation for spermatogenesis. Although several factors that govern these processes have been investigated, the underlying molecular mechanisms have not been fully elucidated. Here, we investigated the role of BMP4 in mouse SSC differentiation, and found that SSCs cultured in the presence of BMP4 underwent differentiation, characterized by downregulation of SSC self‐renewal markers, Plzf, and upregulation of SSC differentiation marker, c‐kit. Smad1/5/8 proteins were phosphorylated during BMP4‐induced differentiation. The effects of BMP4 on SSCs were blocked by BMP4 inhibitor (Dorsomorphin). The activation of BMP4/Smad signaling pathway in SSCs increased the expression of Sohlh2, which is involved in the early differentiation of spermatogonia. Knockdown sohlh2 expression by RNA interference abolished the effect of BMP4 on SSC differentiation and the upregulation of c‐kit expression. Overall, our results suggest that BMP4 plays an important role during the early differentiation of SSCs via upregulation of sohlh2. Anat Rec, 297:749–757, 2014. © 2014 Wiley Periodicals, Inc.     

淫羊藿甙腹腔注射对新生大鼠卵巢发育的影响

目的应用淫羊藿甙对新生大鼠进行腹腔注射,了解其对卵泡发育和卵母细胞凋亡的影响。方法用淫羊藿甙对出生后1～8d大鼠腹腔注射50mg/（kg·d）,分别取出生后2、4、8d龄卵巢,用苏木素-伊红（hematoxylin-eoxin,HE）染色观察不同发育阶段卵泡比例,末端脱氧核苷酰基转移酶介导性dUTP切口末端标记（TdT-mediated dUTP Nick-End Labeling,TUNEL）荧光染色检测卵巢内卵母细胞凋亡变化。结果在淫羊藿甙腹腔注射组的新生的鼠中,1、2d龄卵巢内未装配卵泡比例及4、8d龄卵巢内原始卵泡比例均高于对照组;各日龄组卵巢中卵母细胞TUNEL阳性率明显低于对照组。结论淫羊藿甙可能通过延缓卵母细胞巢破裂,抑制原始卵泡的发育启动,减少卵母细胞凋亡从而增加新生大鼠卵巢中卵母细胞的储备量。     

Expression and localization of Smad2 and Smad4 proteins in the porcine ovary

The objective of the present study was to investigate the temporal and spatial expression of Smad2 and Smad4 proteins, the downstream signaling molecules of the transforming growth factor beta (TGF-β) superfamily, in the porcine ovary. Cellular localization of Smad2 and Smad4 proteins was examined using immunohistochemistry. The specificity of the antibodies was examined using Western blot assay. Western blot analyses demonstrated that 52 kDa Smad2 and 60 kDa Smad4 proteins were expressed in the porcine ovary. Immunohistochemistry revealed that Smad2 and Smad4 were widely expressed in the porcine ovary, mainly localized in the oocyte, granulosa and thecal cells at different stages of folliculogenesis. Within the primordial and primary follicles, Smad2 and Smad4 showed strong staining in oocytes and follicular cells. In the antral follicle, strong staining was observed in oocytes, granulosa and theca cells. These findings suggest that Smad2 and Smad4 may be a key regulator of follicular development and growth of oocytes in the porcine ovary.     

Localization of Smad4 in the ovary of the European hedgehog (Erinaceus europaeus L.)

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor β (TGFβ) superfamily, TGFβ, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor (GDF) are present in the ovaries of many animals. In the present study, we investigated the immunolocalization of Smad4, a signaling molecule of the TGFβ superfamily, during folliculogenesis in the ovary of the European hedgehog (Erinaceus europaeus L., 1758). Immunolocalization studies revealed that Smad4 was widely seen in the ovary, mainly in the follicle, though its location and staining intensity varied with the different stages of the developing follicle. In the primordial follicles and early growing follicles, Smad4 protein was mainly localized in the cytoplasm of the oocyte with a half-moon staining pattern. In the pre-antral follicles, Smad4 protein was mainly located in the granulosa cells, theca cells and diffusely distributed in the interstitial cells surrounding the follicle. In the corpora lutea, the immunostaining for Smad4 was very intense. These results suggested that Smad signal transduction may play an important role in folliculogenesis and conceivably may participate in subsequent pregnancy.     

生育年龄妇女早期卵泡细胞凋亡和Bcl-2/BAX蛋白表达

目的:研究生育年龄妇女早期卵泡的细胞凋亡和Bcl-2/BAX蛋白表达。方法:12例卵巢组织标本来自进行妇科手术的生育年龄妇女(年龄23-38岁),并经过组织病理学检查证实无明显形态异常。利用末端标记法(TUNEL)和免疫组织化学方法研究早期卵泡(包括始基卵泡、间期和初级卵泡)细胞凋亡和Bcl-2/BAX蛋白表达。结果:早期卵泡内18.75%卵细胞表现TUNEL阳性,但是卵泡内未见TUNEL阳性颗粒细胞。BAX在早期卵泡的卵细胞内表达,阳性表达率76.07%;相反未见Bcl-2在卵细胞表达。另外,早期卵泡内颗粒细胞也没有表达Bcl-2和BAX。结论:生育年龄妇女早期卵泡的卵细胞发生凋亡,促凋亡蛋白BAX在卵细胞凋亡过程中发挥调节作用,由此提示BAX介导的卵细胞凋亡可能是生育年龄妇女早期卵泡闭锁的机制。     

Temporal and spatial regulation of bone morphogenetic protein signaling in late lung development.

Bone morphogenetic proteins (BMPs) play important roles in early lung development. No study to date has addressed a role for BMP signaling in late lung development. We describe changes in the expression and localization of BMP receptors (Bmpr1a, Bmpr1b, and Bmpr2) and Smad (Smad1, Smad4, Smad5, and Smad8) intracellular signaling proteins during the saccular and alveolarization stages of late lung development. BMP signaling, assessed by Smad1/5 phosphorylation, nuclear translocation, and induction of id1, id2, and id3 gene expression, was evident throughout late lung development. Our data indicate that BMP signaling is active during late lung development, and points to roles for the BMP system in septal and vascular development, and in the homeostasis of the epithelial layer of large conducting airways in the mature lung.     

卵丘颗粒细胞骨形发生蛋白-15、6信号通路分子表达对受精卵发育能力的影响

目的 探讨卵丘颗粒细胞骨形态发生蛋白（BMP）-15、BMP-6信号通路分子表达对受精卵发育能力的影响。 方法 收集2012年4月~2014年6月,因男性不育症行卵泡浆内单精子显微注射技术（ICSI）助孕的119例病例,456份单个卵子的卵丘颗粒细胞,采用免疫细胞化学,Western blotting方法检测颗粒细胞BMP-15、BMP-6信号通路分子蛋白的表达,采用Real-time PCR方法检测BMP-15、BMP-6信号分子基因表达。比较分析卵子ICSI后发育形成囊胚组与非囊胚组卵丘颗粒细胞BMP-15、BMP-6信号分子表达差异。 结果 对两组卵丘颗粒细胞,进行了免疫细胞化学,Real-time PCR,Western blotting分析,结果发现,BMP-15、BMP-6、AKL2、AKL6、BMPRⅡ、Smad1、Samd4、Smad5、Samd8及E盒结合锌指蛋白1（ZEB1）在两组颗粒细胞中均有表达,其中BMP-15、BMP-6、AKL2、AKL6、BMPRⅡ、Smad1 Samd4及ZEB1表达,组间比较差异具有显著性（经Bonferroni校正P＜0.005）。而Smad5、Samd8 表达,组间比较差异无显著性（P＞0.05）。 结论 卵丘颗粒细胞BMP-15、BMP-6、AKL2、AKL6、BMPRⅡ、Smad1、Samd4及ZEB1信号通路分子可能通过信号通路的调控作用,对受精卵发育能力产生影响。     

人宫颈癌细胞端粒酶活性与PI3K/AKT信号转导通路及细胞生物学行为

目的 观察磷脂酰肌醇-3激酶(P13K)/AKT信号转导通路特异性抑制剂LY294002对人官颈癌细胞株(HeLa细胞)端粒酶活性的影响,同时观察其对细胞生物学行为的改变,探讨肿瘤细胞内端粒酶活性可能的调节机制.方法 ccK.8法测定LY294002作用于HeLa细胞的Ic50值;Western blot检测总AKT以及磷酸化AKT(P-AKT);端粒重复序列扩增-酶联免疫吸附测定法(TRAP-ELISA)测定细胞的端粒酶活性;生长曲线、流式细胞术和Hoechst33258染色分别检测细胞增殖、细胞周期和细胞的凋亡;最后用划痕实验检测细胞的迁移能力.结果 LY294002作用于HeLa细胞的Ic50值为1.73 mg/L;Western blot结果显示LY294002作用使细胞在总AKT蛋白表达相同的情况下明显抑制了P-AKT,此时细胞内端粒酶活性由对照组的98.61%显著降低至36.72%.LY294002降低了细胞内端粒酶活性同时,使细胞的生长增殖能力明显降低,处于Go/G.期(静止期)的细胞由对照组的47.36%增加到实验组的66.88%,凋亡细胞的比例则由2.4%增加至14.9%,同时细胞的运动迁移明显降低,迁移距离由对照组62.57%明显降低为24.6%.结论 LY294002抑制P13K/AKT信号转导通路能明显改变HeLa细胞内端粒酶活性,同时引起细胞生物学行为的改变,其细胞内端粒酶活性的调节可能与P13K/AKT信号转导通路密切相关.     

Nerve Growth Factor Protects the Ischemic Heart via Attenuation of the Endoplasmic Reticulum Stress Induced Apoptosis by Activation of Phosphatidylinositol 3-Kinase

Background: Increased expression of nerve growth factor (NGF) has been found in the myocardium suffered from ischemia and reperfusion (I/R). The pro-survival activity of NGF on ischemic heart has been supposed to be mediated by phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway. Endoplasmic reticulum (ER) stress, which is activated initially as a defensive response to eliminate the accumulated unfolded proteins, has shown a critical involvement in the ischemia induced myocardial apoptosis. This study was aimed to investigate whether NGF induced heart protection against I/R injury includes a mechanism of attenuation of ER stress-induced myocardial apoptosis by activation of PI3K/Akt pathway.Methods: Isolated adult rat hearts were perfused with a Langendörff perfusion system. Hearts in the Sham group were subjected to 225 min of continuous Krebs-Henseleit buffer (KHB) perfusion without ischemia. Hearts in I/R group were perfused with KHB for a 75-min of equilibration period followed by 30 min of global ischemia and 120 min of KHB reperfusion. Hearts in the NGF group accepted 45 min of euilibration perfusion and 30 min of NGF pretreatment (with a final concentration of 100 ng/ml in the KHB) before 30 min of global ischemia and 120 min of reperfusion. Hearts in K252a and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 groups were pretreated with either a TrkA inhibitor, K252a or a phosphatidyl inositol 3-kinase inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 for 30 min before NGF (100 ng/ml) administration. Cardiac hemodynamics were measured from the beginning of the perfusion. Cardiac enzymes and cardiac troponin I (cTnI) were assayed before ischemia and at the end of reperfusion. Myocardial apoptosis rate was measured by TUNEL staining, and expression of glucose-related protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, total- and phospho-(Ser473)-Akt were assessed by Western blot analyses.Results: NGF pretreatment significantly improved the recovery of post-ischemia cardiac hemodynamics. Reduced creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) activity and cTnI levels, as well as decreased myocardial apoptosis ratio were observed in the NGF group. The improvement of NGF on recovery of cardiac function and alleviation of myocardial injury were completely abolished by K252a or {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. GRP78, caspase-12 and CHOP were highly expressed in ischemic myocardium, while NGF significantly inhibited the overexpression of these proteins which were involved in ER stress-induced myocardial apoptosis. NGF pretreatment also induced phosphorylation of Akt. When the activation of PI3K/Akt pathway is blocked by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, the NGF induced suppression of the apoptosis-related proteins expression was reversed.Conclusions: NGF pretreatment may protect the ischemic heart via inhibition of the ER stress-induced apoptosis; this pro-survival effect is mediated by PI3K/Akt pathway.