PI3K/Akt信号通路在HIV感染中的研究进展

[摘要]　磷脂酰肌醇3-激酶（phosphoinositide 3-kinase, PI3K）/蛋白激酶B（protein kinase B, PKB,普遍写作Akt）信号通路参与调控多种细胞功能，与多种疾病的发生密切相关。PI3K/Akt信号通路在HIV感染过程中也发挥重要作用，包括调节T细胞、线粒体功能，促进HIV复制，再激活潜伏HIV以及维持病毒储存库。因此，探索PI3K/Akt信号通路在HIV感染中的作用机制具有重要意义，针对PI3K/Akt的靶向治疗可能在抗HIV中发挥关键作用。本文对PI3K/Akt信号通路与HIV感染的相关研究进行综述，旨在为HIV的治疗提供新的思路与靶点。     

PI3K/Akt 信号通路与肿瘤细胞凋亡相关研究进展

PI3K/Akt信号通路作为细胞内重要信号转导通路之一,通过影响下游多种效应分子的活化状态,在细胞内发挥抑制凋亡、促进增殖的关键作用,它与人类多种肿瘤的发生发展密切相关.因此,通过对PI3K/Akt信号通路的研究有望寻求肿瘤药物治疗的新靶点.本文综述了PI3K/Akt信号通路的组成与功能、调节以及其抗肿瘤细胞凋亡作用机理等方面的研究进展,并就其抗细胞凋亡作用在肿瘤治疗中的应用作了评述,期待为以PI3K/Akt信号通路中关键分子为靶点的肿瘤治疗研究提供参考.     

PTEN在心肌缺血预适应和后适应中的调控作用

目的探讨人第10号染色体缺失的磷酸酶及张力蛋白同源的基因(PTEN)/Akt信号通路在心肌缺血预适应和后适应中的作用。方法雄性SD大鼠60只,随机分为4组:假手术组(sham组)、缺血再灌注组(IR组)、缺血预适应组(Ipre组)及缺血后适应组(Ipost组)。建立动物模型,实验结束后,计算心肌梗死面积,测定血清肌酸激酶(CK)、乳酸脱氢酶(LDH)活性,检测缺血心肌PTEN m RNA和蛋白含量及p-Akt蛋白表达水平。结果与IR组比较,Ipre组和Ipost组的心肌梗死面积明显缩小,CK、LDH的活性降低,PTEN m RNA和蛋白的含量降低,p-Akt蛋白的水平升高(P0.05)。结论 PTEN/Akt信号通路在心肌缺血再灌注损伤中发挥重要作用,心肌缺血预适应和后适应通过调节PTEN/Akt信号通路的表达在缺血再灌注损伤中发挥保护心肌的作用。     

磷脂酰肌醇3激酶信号通路在缺血后适应中对大鼠心肌保护机制的研究

目的研究缺血后适应(IPost)对大鼠心肌保护作用及磷脂酰肌醇3激酶(PI3K/Akt)信号通路机制。方法将32只雄性Wistar大鼠随机分为缺血再灌注组(A组),IPost组(B组),IPost+Wortmannin组(C组)和缺血再灌注+SB216763组(D组),每组8只。测定各组左心室收缩压(LVSP)和再灌注30 min冠状动脉流出液中乳酸脱氢酶和肌酸激酶含量。并测定心肌梗死面积,对心肌进行免疫组织化学染色,观察Akt磷酸化和GSK-3β磷酸化的表达。结果与B组比较,A组LVSP明显降低,乳酸脱氢酶和肌酸激酶含量明显升高(P0.05);同时IPost干预减小了心肌梗死面积(47.3% vs 29.5%),B组Akt磷酸化和GSK-3β磷酸化表达增加。结论 IPost对体外大鼠缺血再灌注损伤有明确的保护作用。Wortmannin可削弱IPost的保护作用,SB216763具有模拟IPost的心肌保护作用,Akt和GSK-3β的磷酸化水平在IPost的心肌保护作用信号通道传导机制中具有重要地位。     

Akt/FoxO传导通路调节细胞凋亡作用的研究进展

Foxo转录因子是PKB／Akt的下游靶点。Akt调节细胞生存和增殖。Akt磷酸化Foxo抑制Fox0的转录功能,促进细胞生存、生长和增殖。在癌症中FoxO在不同的细胞信号通路中发挥重要作用。FoxO通过两个途径抑制凋亡信号,促进细胞生长,其包括线粒体靶点蛋白Bcll2家族的多种前凋亡成员的表达、死亡受体配体如Fas配体和肿瘤坏死因子相关凋亡诱导配体（TRAIL）的表达,或是增加各种细胞周期蛋白依赖性激酶抑制蛋白的水平。本文主要概括了Akt／FoxO调节细胞生长和生存的机制,以期为抗癌治疗提供新的可能。     

5-氮杂胞苷通过PI3K/Akt/mTOR通路介导人脂肪间充质干细胞向心肌细胞定向分化的研究

目的:研究磷脂酰肌醇3激酶(PI3K)及其下游分子丝氨酸/苏氨酸激酶(Akt)和雷帕霉素靶蛋白(mTOR)所组成的信号通路在5-氮杂胞苷(5-aza)诱导人脂肪间充质干细胞(ADMSCs)向心肌细胞定向分化中的作用,探讨其信号转导机制。方法:利用胶原酶法分离、培养ADMSCs,并用10μmol/L的5-aza诱导其向心肌细胞定向分化,采用Western blot的方法分析5-aza诱导前后Akt通路相关蛋白的表达情况。结果:5-aza诱导前ADMSCs内Akt通路相关蛋白的表达水平较低,诱导后增强。PI3K抑制剂Ly294002处理后,Western blot结果显示,细胞内PI3K的磷酸化受到抑制和TnT的表达水平显著降低,具有统计学意义。结论:5-aza能诱导ADMSCs向心肌细胞分化,PI3K/Akt/mTOR信号通路在5-aza诱导ADMSCs向心肌细胞分化中发挥重要的调控作用。     

细胞自噬在心肌重构中的作用

自噬是普遍存在于真核细胞中高度保守的代谢过程。病理状态时,自噬被抑制或过度激活,可引起多种心脏疾病的发生。自噬参与心肌肥厚和心肌纤维化的病理生理过程,在心肌重构中发挥重要作用。     

肝部分切除术后PI3K/Akt信号途径对肝窦内皮细胞功能的影响

肝窦内皮细胞（HSEC）在一定条件下可以释放多种介质，如：肝细胞生长因子（HGF），转化生长因子β（TGFβ），白细胞介素-6（IL-6），一氧化氮（NO）及一氧化氮合酶（NOS）等，这些细胞因子和介质在肝再生中发挥重要的作用。PI3K/Akt信号途径在细胞增殖、细胞周期及生存中起到重要作用，Akt酶是PI3K下游直接靶蛋白，     

缺血后处理减轻大鼠肥厚心肌缺血再灌注损伤的观察

目的探讨缺血后处理对心肌肥厚大鼠离体心脏缺血再灌注损伤的影响及其信号机制。方法通过腹主动脉结扎建立大鼠心肌肥厚模型,用Landendorff装置建立心肌肥厚大鼠离体心脏缺血再灌注模型。观察缺血后处理对心肌肥厚大鼠离体缺血再灌注心脏左心室收缩压,冠状动脉流量,肌酸磷酸激酶和乳酸脱氢酶释放,心肌梗死范围,心肌组织中蛋白激酶B／Akt（Akt）、糖原合成酶激酶-3β（GSK-3β）磷酸化的影响。结果与缺血再灌注对照组相比,缺血后处理组心脏左心室收缩压、冠状动脉流量显著高,冠状动脉循环流出液中肌酸磷酸激酶、乳酸脱氢酶含量低,心肌梗死范围减小,心肌组织中磷酸化Akt（Ser473）、磷酸化GSK-3β（Set9）水平高,磷脂酰肌醇-3激酶（PI3K）抑制剂渥曼青霉素（wortmannin）能够抑制缺血后处理所致的磷酸化Akt（Ser473）、磷酸化GSK-3β（Set9）水平升高,但只能部分消除缺血后处理的心脏保护效应。结论缺血后处理能够减轻心肌肥厚大鼠离体心脏缺血再灌注损伤,PI3K／Akt／GSK-3信号途径参与介导缺血后处理对离体缺血再灌注肥厚心肌的保护作用。     

红花注射液改善大鼠心肌缺血再灌注氧化损伤的作用机制研究

目的:探讨红花注射液对大鼠离体心脏缺血/再灌注损伤的保护作用及其机制。方法:健康成年Wistar大鼠,通过Langendorff离体心脏灌流技术建立心肌缺血/再灌注模型并检测心肌血流动力学指标;检测心肌组织中超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性及丙二醛(MDA)含量;采用实时定量PCR及免疫印迹检测心肌组织PI3K、Akt、Sirt1表达变化。结果:红花注射液可明显恢复缺血/再灌注后大鼠离体心脏血流动力学病变;显著性升高机体心肌组织中SOD及GSH-Px活性,减少MDA含量;PI3K、Akt磷酸化水平及Sirt1蛋白表达明显增强。结论:红花注射液改善大鼠心肌缺血再灌注损伤的保护作用机制可能与其激活PI3K/Akt/Sirt1信号通路作用有关。     

4,4’-二异硫氰基芪-2,2’-二磺酸对大鼠缺血再灌注损伤的影响及机制

目的探讨广谱氯离子通道阻滞剂4,4’-二异硫氰基芪2,2’-二磺酸(DIDS)对大鼠缺血再灌注损伤心肌细胞凋亡的影响及其机制。方法雄性SD大鼠36只随机分为3组:缺血再灌注组(A组)、DIDS处理组(B组)和LY294002预处理组(C组)。伊文兰和TTC染色测定心肌梗死范围,TUNEL方法定性和定量检测心肌细胞凋亡指数,Western blot测定蛋白激酶B(Akt)的表达。结果与A组比较,B组心肌梗死范围和心肌细胞凋亡指数明显降低[(38.8±7.7)% vs (54.2±10.8)%,(8.9±1.8)% vs (17.6±3.5)%.P<0.01];磷酸化Akt表达水平明显增加(P<0.01)。与A组比较,C组梗死面积、凋亡指数无明显减小,磷酸化Akt水平无明显变化(P>0.05)。结论 DIDS能够抑制大鼠缺血再灌注所致的心肌细胞损伤,可能是通过信号分子磷脂酰肌酶三羟基激酶/Akt的调节。     

Estrogen replacement and cardiomyocyte protection

The effects of estrogen on vascular function have been studied extensively; however, much less is known regarding the effect of estrogen on myocardial function and cardiomyocyte biology. We recently examined the effects of estrogen on infarct size and left ventricular remodeling after induction of myocardial infarction by permanent coronary ligation in ovariectomized female mice. These studies demonstrated that physiologic replacement with 17beta-estradiol (E(2)) reduced infarct size and reduced cardiomyocyte apoptosis at 24 and 72 hours post-myocardial infarction. In vivo, physiologic E(2) replacement rapidly activated the Akt signaling pathway within the myocardium. In vitro, physiologic concentrations of E(2) rapidly activated Akt via estrogen receptor alpha (ERalpha) and phosphoinositide-3 (PI3)-kinase-dependent mechanisms. Moreover, estrogen mitigated anthracycline-induced apoptosis in vitro via ER- and PI3-kinase-Akt-dependent mechanisms. Our data therefore support that estrogen favorably influences cardiomyocyte survival both in a coronary occlusion model in vivo and in cultured cardiomyocytes in vitro. Further work is necessary to more completely understand the complex effects of sex hormones on cardiomyocyte biology.     

Aldehyde dehydrogenase 2 knockout accentuates ethanol-induced cardiac depression: Role of protein phosphatases

Alcohol consumption leads to myocardial contractile dysfunction possibly due to the toxicity of ethanol and its major metabolite acetaldehyde. This study was designed to examine the influence of mitochondrial aldehyde dehydrogenase-2 (ALDH2) knockout (KO) on acute ethanol exposure-induced cardiomyocyte dysfunction. Wild-type (WT) and ALDH2 KO mice were subjected to acute ethanol (3 g/kg, i.p.) challenge and cardiomyocyte contractile function was assessed 24 h later using an IonOptix edge detection system. Western blot analysis was performed to evaluate ALDH2, protein phosphatase 2A (PP2A), phosphorylation of Akt, and glycogen synthase kinase-3β (GSK-3β). ALDH2 KO accentuated ethanol-induced elevation in cardiac acetaldehyde levels. Ethanol exposure depressed cardiomyocyte contractile function including decreased cell shortening amplitude and maximal velocity of shortening/relengthening as well as prolonged relengthening duration and a greater decline in peak shortening in response to increasing stimulus frequency, the effect of which was significantly exaggerated by ALDH2 KO. ALDH2 KO also unmasked an ethanol-induced prolongation of shortening duration. In addition, short-term in vitro incubation of ethanol-induced cardiomyocyte mechanical defects was exacerbated by the ALDH inhibitor cyanamide. Ethanol treatment dampened phosphorylation of Akt and GSK-3β associated with upregulated PP2A, which was accentuated by ALDH2 KO. ALDH2 KO aggravated ethanol-induced decrease in mitochondrial membrane potential. These results suggested that ALDH2 deficiency led to worsened ethanol-induced cardiomyocyte function, possibly due to upregulated expression of protein phosphatase, depressed Akt activation, and subsequently impaired mitochondrial function. These findings depict a critical role of ALDH2 in the pathogenesis of alcoholic cardiomyopathy.     

Angiotensin-II type 1 receptor and NOX2 mediate TCF/LEF and CREB dependent WISP1 induction and cardiomyocyte hypertrophy

Angiotensin-II (Ang-II) plays a key role in myocardial hypertrophy, remodeling and failure. We investigated whether Ang-II-induced cardiomyocyte hypertrophy is dependent on WNT1 inducible signaling pathway protein 1 (WISP1), a pro growth factor. Ang-II induced hypertrophy and WISP1 expression in neonatal rat cardiomyocytes (NRCM), effects that were significantly inhibited by pre-treatment with the AT1 antagonist losartan and by WISP1 knockdown. Further, Ang-II induced WISP1 was superoxide-dependent, and inhibited by DPI, an inhibitor of NADPH oxidases, and by knockdown of NOX2. AT1 was physically associated with NOX2 both in vitro and in vivo, and Ang-II increased this interaction in vivo. Ang-II induced WISP1 expression via superoxide/Akt/GSK3β/β-catenin/TCF/LEF and by Akt-dependent CREB activation. Further, Ang-II also activated CREB via superoxide-mediated p38 MAPK and ERK activation. Continuous infusion of Ang-II for 7 days induced myocardial hypertrophy in rats, and was associated with increased Akt, p-Akt, p-p38 MAPK, p-ERK1/2, and WISP1 expression. These results demonstrate that Ang-II induced cardiomyocyte hypertrophy is mediated through AT1, NOX2 and the induction of WISP1, and may involve the direct interaction of AT1 with NOX2. Thus targeting both WISP1 and NOX2 may have a therapeutic potential in improving cardiomyocyte survival and growth following myocardial injury and remodeling. This article is part of a Special Issue entitled ‘Possible Editorial’.     

他汀类药物对心肌缺血再灌注损伤心肌细胞凋亡作用机制的研究进展

近年研究表明,细胞凋亡在心肌缺血再灌注损伤(MIRI)中占有重要地位,细胞凋亡与MIRI之间有着密切关系。实验证实他汀类药物具有除调脂以外的心肌保护效应。现主要从他汀类药物的抗氧化作用和激活磷脂酰肌醇-3-激酶丝氨酸-苏氨酸激酶/一氧化氮合酶(PI3K/Akt/eNOS)信号通路两个方面阐述在心肌缺血再灌注过程中他汀类药物抑制心肌细胞凋亡的主要机制。     

Dual actions of the Galpha(q) agonist Pasteurella multocida toxin to promote cardiomyocyte hypertrophy and enhance apoptosis susceptibility

Previous attempts to delineate the consequences of Galpha (q) activation in cardiomyocytes relied largely on molecular strategies in cultures or transgenic mice. Modest levels of wild-type Galpha(q) overexpression induce stable cardiac hypertrophy, whereas intense Galpha(q) stimulation induces cardiomyocyte apoptosis. The precise mechanism(s) whereby traditional targets of Galpha (q) subunits that induce hypertrophy also trigger cardiomyocyte apoptosis is not obvious and is explored with recombinant Pasteurella multocida toxin (rPMT, a Galpha(q) agonist). Cells cultured with rPMT display cardiomyocyte enlargement, sarcomeric organization, and increased atrial natriuretic factor expression in association with activation of phospholipase C, novel protein kinase C (PKC) isoforms, extracellular signal-regulated protein kinase (ERK), and (to a lesser extent) JNK/p38-MAPK. rPMT stimulates the ERK cascade via epidermal growth factor (EGF) receptor transactivation in cardiac fibroblasts, but EGF receptor transactivation plays no role in ERK activation in cardiomyocytes. Surprisingly, rPMT (or novel PKC isoform activation by PMA) decreases basal Akt phosphorylation; rPMT prevents Akt phosphorylation by EGF or IGF-1 and functionally augments cardiomyocyte apoptosis in response to H2O2. These results identify a Galpha(q)-PKC pathway that represses basal Akt phosphorylation and impairs Akt stimulation by survival factors. Because inhibition of Akt enhances cardiomyocyte susceptibility to apoptosis, this pathway is predicted to contribute to the transition from hypertrophy to cardiac decompensation and could be targeted for therapy in heart failure.     

17beta-estradiol reduces cardiomyocyte apoptosis in vivo and in vitro via activation of phospho-inositide-3 kinase/Akt signaling

Female gender and estrogen-replacement therapy in postmenopausal women are associated with improved heart failure survival, and physiological replacement of 17beta-estradiol (E2) reduces infarct size and cardiomyocyte apoptosis in animal models of myocardial infarction (MI). Here, we characterize the molecular mechanisms of E2 effects on cardiomyocyte survival in vivo and in vitro. Ovariectomized female mice were treated with placebo or physiological E2 replacement, followed by coronary artery ligation (placebo-MI or E2-MI) or sham operation (sham) and hearts were harvested 6, 24, and 72 hours later. After MI, E2 replacement significantly increased activation of the prosurvival kinase, Akt, and decreased cardiomyocyte apoptosis assessed by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) staining and caspase 3 activation. In vitro, E2 at 1 or 10 nmol/L caused a rapid 2.7-fold increase in Akt phosphorylation and a decrease in apoptosis as measured by TUNEL staining, caspase 3 activation, and DNA laddering in cultured neonatal rat cardiomyocytes. The E2-mediated reduction in apoptosis was reversed by an estrogen receptor (ER) antagonist, ICI 182,780, and by phospho-inositide-3 kinase inhibitors, LY294002 and Wortmannin. Overexpression of a dominant negative-Akt construct also blocked E2-mediated reduction in cardiomyocyte apoptosis. These data show that E2 reduces cardiomyocyte apoptosis in vivo and in vitro by ER- and phospho-inositide-3 kinase-Akt-dependent pathways and support the relevance of these pathways in the observed estrogen-mediated reduction in myocardial injury.     

Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice

The serine-threonine kinase Akt seems to be central in mediating stimuli from different classes of receptors. In fact, both IGF-1 and IL6-like cytokines induce hypertrophic and antiapoptotic signals in cardiomyocytes through PI3K-dependent Akt activation. More recently, it was shown that Akt is involved also in the hypertrophic and antiapoptotic effects of beta-adrenergic stimulation. Thus, to determine the effects of Akt on cardiac function in vivo, we generated a model of cardiac-specific Akt overexpression in mice. Transgenic mice were generated by using the E40K, constitutively active mutant of Akt linked to the rat alpha-myosin heavy chain promoter. The effects of cardiac-selective Akt overexpression were studied by echocardiography, cardiac catheterization, histological and biochemical techniques. We found that Akt overexpression produced cardiac hypertrophy at the molecular and histological levels, with a significant increase in cardiomyocyte cell size and concentric LV hypertrophy. Akt-transgenic mice also showed a remarkable increase in cardiac contractility compared with wild-type controls as demonstrated by the analysis of left ventricular (dP/dt(max)) in an invasive hemodynamic study, although with graded dobutamine infusion, the maximum response was not different from that in controls. Diastolic function, evaluated by left ventricular dP/dt(min), was not affected at rest but was impaired during graded dobutamine infusion. Isoproterenol-induced cAMP levels, beta-adrenergic receptor (beta-AR) density, and beta-AR affinity were not altered compared with control mice. Moreover, studies on signaling pathway activation from myocardial extracts demonstrated that glycogen synthase kinase3-beta is phosphorylated, whereas p42/44 mitogen-activated protein kinases is not, indicating that Akt induces hypertrophy in vivo by activating the glycogen synthase kinase3-beta/GATA 4 pathway. In summary, our results not only demonstrate that Akt regulates cardiomyocyte cell size in vivo, but, importantly, show that Akt modulates cardiac contractility in vivo without directly affecting beta-AR signaling capacity.     

c-Jun N-terminal kinases mediate reactivation of Akt and cardiomyocyte survival after hypoxic injury in vitro and in vivo

Akt is a central regulator of cardiomyocyte survival after ischemic injury in vitro and in vivo, but the mechanisms regulating Akt activity in the postischemic cardiomyocyte are not known. Furthermore, although much is known about the detrimental role that the c-Jun N-terminal kinases (JNKs) play in promoting death of cells exposed to various stresses, little is known of the molecular mechanisms by which JNK activation can be protective. We report that JNKs are necessary for the reactivation of Akt after ischemic injury. We identified Thr450 of Akt as a residue that is phosphorylated by JNKs, and the phosphorylation status of Thr450 regulates reactivation of Akt after hypoxia, apparently by priming Akt for subsequent phosphorylation by 3-phosphoinositide-dependent protein kinase. The reduction in Akt activity that is induced by JNK inhibition may have significant biological consequences, as we find that JNKs, acting via Akt, are critical determinants of survival in posthypoxic cardiomyocytes in culture. Furthermore, in contrast to selective p38-mitogen-activated protein kinase inhibition, which was cardioprotective in vivo, concurrent inhibition of both JNKs and p38-mitogen-activated protein kinases increased ischemia/reperfusion injury in the heart of the intact rat. These studies demonstrate that reactivation of Akt after resolution of hypoxia and ischemia is regulated by JNKs and suggest that this is likely a central mechanism of the myocyte protective effect of JNKs.