Brain-derived neurotrophic factor prevents axotomized retinal ganglion cell death through MAPK and PI3K signaling pathways

PURPOSE: Brain-derived neurotrophic factor (BDNF) has a potential neuroprotective effect on axotomized retinal ganglion cells (RGCs); however, the mechanism, in regard to intracellular signaling, of BDNF-induced neuroprotection of RGCs is largely unknown. Intracellular signaling was investigated, by using axotomized RGCs and the relative contribution of the two major downstream signaling routes of TrkB determined--that is, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K)-Akt routes, mediated by BDNF. METHODS: Neuroprotective effects of BDNF were determined by quantifying the surviving RGCs after axotomy, by retrograde labeling. The MAPK and Akt levels were determined by Western blot analysis and activity assays. Quantification of the relative contribution of the two signaling pathways was performed by use of specific inhibitors for MAPK and PI3K (i.e., U0126 and LY294002, respectively). RESULTS: Intravitreous administration of BDNF had the most profound neuroprotective effects on axotomized RGCs among the neurotrophins. Burst phosphorylation of MAPK and Akt was induced by BDNF within 1 hour and was sustained over 2 weeks in the whole retina. Immunohistochemistry revealed that phosphorylated MAPK was detected in the RGCs and retinal Müller cells, and Akt was in the RGCs. BDNF-induced phosphorylation of MAPK and Akt was suppressed by their specific inhibitors. Moreover, administration of U0126 and LY294002 decreased significantly, but only partially, the neuroprotective effect of BDNF on the axotomized RGCs. CONCLUSIONS: BDNF-mediated signaling involves activation of both MAPK and Akt on the axotomized adult rat retina, and the collaboration of both MAPK and PI3K-Akt pathways seems to be necessary in neuroprotective signaling in axotomized RGCs.     

Ciliary neurotrophic factor protects rat retina cells in vitro and in vivo via PI3 kinase

PURPOSE: Neurotrophic factors and neurotrophins are well-known to have neuroprotective efficacy against retinal injury. The aim of this experiment is to investigate the signal transduction pathway of ciliary neurotrophic factor (CNTF) on the upregulation of viability of retinal primary culture and retinal protection against constant light damage in vivo. CNTF is known to enhance the viability of retinal culture and provide protection under constant light exposure conditions, but little is known about how the signal transduction pathways of CNTF affect retina function. METHODS: Primary retinal cultures were prepared from 7-day-old Wistar rats. Brain-derived neurotrophic factor (BDNF) (0.1, 1, 10 ng/ml), CNTF (0.1, 1, 10 ng/ml), PD98059 (10, 100, 1000 nM), or LY294002 (10, 100, 1000 nM) was added to these cultures at the time of cell preparation. After 3 days, the percentage of cells surviving was assessed using alamarBlue. For the in vivo experiment, inhibitors for the MAPKK (PD98059, 10 microg/eye) or PI3K (LY294002, 10 microg/eye) pathways were injected into the vitreous together with CNTF (1 microg/eye) 2 days before constant light exposure. Electroretinogram (ERG) analysis was performed to investigate which pathway was used by CNTF. RESULTS: CNTF at 1, 10, or 100 ng/ml enhanced cell viability in retinal cultures. The cell-survival activity of CNTF was blocked by 10 ng/ml LY294002 (Dunnet's test, p < 0.05). In vivo, the neuroprotective activity of CNTF in constant-light conditions was attenuated by 10 microg/eye LY294002 (Dunnet's test, p < 0.05). CONCLUSIONS: These data suggest that CNTF promotes cell survival via the PI3K signaling pathway in vitro and in vivo.     

Divergent NMDA signals leading to proapoptotic and antiapoptotic pathways in the rat retina

PURPOSE: To investigate the involvement of the p38 mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 (PI-3) kinase-Akt signaling pathways after pathologic stimulation by N-methyl-D-aspartate (NMDA) receptors of retinal neurons in vivo. METHODS: NMDA (2-200 nmol), SB203580 (0.2-10 nmol, an inhibitor of p38 MAP kinase), LY294002 (6 nmol, an inhibitor of PI-3 kinase), or control solution was injected into the vitreous of Long-Evans rats. To assess retinal ganglion cell (RGC) death quantitatively, we labeled RGCs retrogradely by injecting aminostilbamidine (FluoroGold) into the superior colliculus and subsequently counting fluorescently labeled RGCs in retinal wholemounts. Phosphorylation of p38 and Akt was assessed by immunoblot of whole retinal lysates, and activity was measured with in vitro kinase assays. To localize phospho-p38 and phospho-Akt, immunohistochemistry was performed. TUNEL staining coupled with morphologic assessment was performed to assess apoptotic cell death. RESULTS: Intravitreous injection of more than 10 nmol NMDA induced RGC death. Before death, NMDA-stimulated retinas manifested increased phospho-p38 and phospho-Akt in the ganglion cell and inner nuclear layers. Subsequently, pyknotic, TUNEL-positive cells were also localized to these regions. SB203580 partially rescued RGCs, whereas LY294002 enhanced death of RGCs due to 10 nmol NMDA. SB203580 and LY294002 specifically inhibited the activity of p38 MAP kinase and Akt, respectively. CONCLUSIONS: The p38 MAP kinase and PI-3 kinase-Akt pathways are involved in signal transduction after excessive stimulation of NMDA receptors in the retina. These inhibitor studies suggest that the p38 MAP kinase pathway is proapoptotic, whereas the PI-3 kinase-Akt pathway is antiapoptotic in RGC death induced by NMDA.     

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P<0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.     

Anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush are PI3K/AKT-dependent

The purpose of present study is to dissect the role of PI3K/AKT signaling in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on rat retinal ganglion cells (RGCs) after optic nerve (ON) crush. The ONs of seventy-two adult male Wistar rats were crushed by a standardized method. Control eyes received a sham operation. G-CSF or phosphate-buffered saline (PBS) was immediately administrated after the ON event for 5 days. Twelve rats were used to investigate the signaling pathways using western blot analysis. In other sixty rats, each eye also received intravitreal injections of PI3K/AKT inhibitor (LY294002) or PBS immediately after the experiments. Rats were euthanized at 1 or 2 weeks after the experiment. RGC density was counted by retrograde labeling with Fluorogold. Western blot analysis of p-AKT, TUNEL assays, and immunohistochemistry of the retinas were conducted. Two weeks after ON injury, RGC densities in the central and mid-peripheral retinas of ON-crushed, G-CSF treated rats were significantly higher than those of corresponding ON-crushed, G-CSF-treated and LY294002-injected rats (survival rates of 60% vs. 39% and 43% vs. 33%, respectively; p < 0.01). Decreased TUNEL staining and the up-regulations of p-AKT signaling in retinas of ON-crushed, G-CSF-treated rats were blocked by intravitreal injections of LY294002. The double staining showed that p-AKT expression co-localized with RGCs in the ON crushed, G-CSF treated retinas. In conclusion, the anti-apoptotic effects of G-CSF on RGCs are PI3K/AKT signaling dependent in the retinas to rescue RGCs after ON crush injury.     

Essential roles of the PI3 kinase/Akt pathway in regulating Nrf2-dependent antioxidant functions in the RPE

PURPOSE: To investigate functional interactions between the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant system in cultured human retinal pigment epithelium (RPE) cells. METHODS: Cultured ARPE-19 cells were treated with different concentrations of PI3K inhibitors, followed by exposure to sulforaphane, an Nrf2 inducer. Akt phosphorylation was detected by Western blot analysis. Intracellular glutathione (GSH) content was measured by HPLC. Expression of genes downstream of Nrf2, including glutamate-cysteine ligase (GCL) and glutathione S-transferase, was measured by quantitative RT-PCR. Nrf2 activity was measured by a dual luciferase assay after transfection of a reporter plasmid containing the antioxidant response element (ARE). The small interference RNA approach was used to knock down Nrf2 in the RPE. Nrf2 localization was determined by subcellular fractionation and Western blot analyses. RESULTS: PI3K inhibitors wortmannin and LY294002 caused dose-dependent cellular and mitochondrial GSH depletion and downregulation of the modulatory subunit of GCL in cultured RPE cells. Both the basal and the induced Nrf2 activities were inhibited by wortmannin and LY294002. Overexpression of a constitutively active form of Akt potentiated Nrf2 activation, and the effect of Akt was blocked by siRNA that knocked down Nrf2. LY294002 also inhibited sulforaphane-induced Nrf2 nuclear translocation. CONCLUSIONS: The PI3K/Akt pathway plays key roles in regulating Nrf2-ARE-dependent protection against oxidative stress in the RPE.     

PI3K/Akt信号通路对高糖状态下视网膜Müller细胞的影响

目的探讨PI3K/Akt信号通路对高糖状态下视网膜Müller细胞的影响及机制。方法本实验分两部分,动物实验:雄性SD大鼠随机分成对照组、糖尿病组、胰岛素样生长因子-1(insulin-like growth factors-1,IGF-1)组、IGF-1+LY294002(PI3K/Akt信号通路抑制剂)组。后三组采用链脲佐菌素(streptozotocin,STZ)(50 mg·kg^-1)诱导成为糖尿病模型。模型诱导成功后,IGF-1组给予IGF-1 (80 g·L^-1) 5μL玻璃体内注射给药,IGF-1+LY294002组给予5μL(IGF-1+LY294002)(80μg·L^-1)玻璃体内注射给药。细胞实验:对培养的Müller细胞进行不同处理,分为对照组(空白对照)、高糖组(30 mmol·L^-1葡萄糖)、IGF-1组(30 mmol·L^-1葡萄糖+80μg·L^-1 IGF-1)、IGF-1+LY294002组[30 mmol·L^-1...     

Oxidant-mediated Akt activation in human RPE cells

PURPOSE: To determine whether a model oxidant, hydrogen peroxide (H2O2), influences Akt activation and, if so, whether Akt activation promotes retinal pigment epithelial (RPE) cell survival. METHODS: Cultured human RPE cells were pretreated with medium alone, with LY294002 (LY), an inhibitor of phosphatidylinositol-3 kinase (PI3K) and its downstream effector Akt, or with Akt/protein kinase B signaling inhibitor (API)-2, a specific Akt inhibitor, and then were stimulated with H2O2 at different doses for various times. Akt phosphorylation was evaluated by Western blot using antibody against phosphorylated Akt (Ser473). The effect of Akt blockade on RPE cell viability was assessed by tetrazolium salt (WST-1) assay and a lactate dehydrognease (LDH) release assay. Caspase-mediated cytokeratin cleavage, an early apoptosis marker, was assessed by M30 antibody staining. Caspase-independent apoptosis was determined by nuclear translocation of apoptosis-inducing factor (AIF). RPE cell morphology was evaluated by electron microscopy. The effect of H2O2 on downstream Akt targets was examined by Western blot using antibody against phosphorylated forkhead in rhabdomyosarcoma (FKHR) and phosphorylated glycogen synthase kinase (GSK)-3beta. RESULTS: H2O2 induced Akt phosphorylation in a dose-dependent manner and also induced the phosphorylation of downstream effectors FKHR and GSK-3beta. LY markedly inhibited H2O2-mediated Akt phosphorylation and significantly enhanced caspase-associated and caspase-independent RPE cell death. CONCLUSIONS: A model oxidant, H2O2, induces PI3K and thereby activates Akt. Akt activation enhances RPE cell survival and thus may protect RPE cells from oxidant-induced cell death under normal circumstances and in disease states such as age-related macular degeneration (AMD).     

LY294002对人晶状体上皮细胞蛋白激酶B活化的影响

背景蛋白激酶B（Akt）与许多细胞信号转导通路密切相关,从而参与多种细胞功能活动的调控,包括细胞的增生、迁移和代谢等,其中磷脂酰肌醇3-激酶（P13K）可催化Akt活性并启动各种相关的细胞信号通路。P13K抑制剂能够抑制Akt活性,但是否影响后发性白内障发生过程中残留的晶状体上皮细胞（LECs）的生物学行为尚不清楚。目的观察P13K抑制剂LY294002对人LECs中Akt活化的影响。方法对人LECs系HLEC-B3细胞进行常规培养和传代,然后接种于96孔板中,培养24h后在培养板中加入LY294002,终浓度分别为0、10、20、30、40、50、60、70、80μmol／L,继续培养24h后采用细胞计数试剂盒-8（CCK-8）法检测各组细胞的增生抑制率。在同一批接种于6孔板内无菌盖玻片上的传代细胞中加入10μg／L转化生长因子-β2（TGF—β2）为诱导组,用20μmol／LLY294002预培养1h后再加入10Ixg／LTGF—β2进行共培养为TGF—B,与LY294005共培养组,以未加入TGF,p：和LY294002的细胞作为对照组,应用激光扫描共焦显微镜观察各组细胞中磷酸化Akt（p-Akt）的荧光表达情况,并用Westernblot法检测各组细胞中p-Akt表达量的差异。结果CCK-8法检测结果显示,随着LY294002浓度的增加,HLEC—B3的吸光度（A）值逐渐减小,即LY294002对HLEC—B3细胞增生的抑制作用随浓度的增加而逐渐增强,不同浓度LY294002组间HLEC—B3的A值比较差异有统计学意义（F分组=9．72,P=0．00）;随着检测时间点的延长,HLEC—B3的4值逐渐增大,不同检测时间点A值比较差异有统计学意义（F时间=1737．54,P=0．00）。激光扫描共焦显微镜观察显示,对照组细胞仅有微量的p-Akt红色荧光,诱导组p-Akt表达明显增多,并向细胞膜聚集,细胞轮廓清晰,而TGF—β2与LY294005共培养组细胞中p-Akt表达的红色荧光明显减弱,细胞形态不清。Westernblot法检测结果显示,对照组、诱导组和TGF—β2与LY294005共培养组细胞中p-Akt的表达量分别为0．91±0．08、1．48±0．13和0．95±0．19,3个组间差异有统计学意义（F=15．04,P=0．00）。结论LY294002能够拈抗TGF—β2诱导的Akt磷酸化激活过程,有望成为新的有效防治后发性白内障的药物。     

磷脂酰肌醇-3-激酶/丝氨酸-苏氨酸激酶抑制剂LY294002对小鼠视网膜新生血管形成的抑制作用

目的　探讨磷脂酰肌醇-3-激酶（phosphatidylinositol-3-kinase,PI3K）/丝氨酸-苏氨酸激酶（Serine/threonine kinase,AKT）信号转导通路抑制剂LY294002对小鼠氧诱导视网膜病变（oxygen-induced retinopathy,OIR）的视网膜新生血管（retinal neovascularization,RNV）形成的影响。方法　取C57BL/6J小鼠60只,随机分为实验组和对照组,每组各30只,均制备OIR模型。小鼠出氧箱前1 d即鼠龄11 d时实验组玻璃体内注射0.5 μL的LY294002,对照组玻璃体内注射等体积的PBS。病理切片计数突破视网膜内界膜的新生血管内皮细胞核数,免疫组织化学和RT-PCR法检测pAKT、VEGF蛋白及mRNA的表达情况。结果　实验组小鼠新生血管内皮细胞核数为（12.53±1.71）个,较对照组（25.31±1.42）个明显减少（P<0.05）;实验组小鼠pAKT、VEGF的蛋白表达呈弱阳性,阳性细胞的吸光度值（9.12±1.35、13.91±1.49）均较对照组（15.11±2.17、19.72±2.61）明显下降（均为P<0.05）;实验组小鼠AKT、VEGF mRNA相对表达量均较对照组明显下降（均为P<0.05）。结论　LY294002通过抑制PI3K/AKT信号转导通路,可有效抑制小鼠OIR的RNV形成,LY294002有望成为防治血管增生性视网膜病变的一种有效方法。     

补肾活血中药血清对加压纯化培养视网膜神经节细胞PI3K/AKT信号转导通路的影响

目的 研究补肾活血中药血清对加压纯化培养视网膜神经节细胞(retinal ganglion cells,RGCs)凋亡模型PI3K/Akt信号转导通路主要成员PDK及Akt表达的影响,探索补肾活血法保护RGCs的机制.方法 制备补肾活血中药含药血清,体外纯化SD大鼠RGCs,采取开放式压力控制培养系统建立体外加压培养RGCs凋亡模型,以50g·L-1、100g· L-1、200g· L-1血清浓度梯度补肾活血中药血清分别处理.将RGCs分为5组,分别为正常培养组(N组)、对照组(C组)、50 g· L-1补肾活血中药血清组(50 g·L-1BSHX组)、100g· L-1补肾活血中药血清组(100 g·L-1BSHX组)、200g·L-1补肾活血中药血清组(200 g·L-1BSHX组),Annexin V-FITC/PI双染检测细胞凋亡率,实时荧光定量PCR(qRT-PCR)检测补肾活血中药血清对RGCs PDK及AktmRNA表达水平的影响,Western blot检测各组PDK、Akt蛋白表达量.结果 Q-PCR检测各组mRNA结果:C组(0.04±0.01)与N组(1.00±0.04)相比,RGCs中PI3K、Akt的mRNA表达水平下降,差异均有统计学意义(均为P ＜0.05),而50 g·L-1、100 g·L-1、200 g·L-BSHX组(0.18±0.01、0.21±0.02,0.22±0.01、0.36±0.01,0.84±0.10、1.07±0.17)与C组相比,PI3K、Akt mRNA含量逐渐升高,差异均有统计学意义(均为P＜0.05).Western blot检测各组蛋白表达,C组与N组相比,细胞PI3K、Akt的蛋白表达水平下降,差异有统计学意义(P＜0.05),而50 g·L-1、100 g·L-1、200 g·L-BSHX组与C组相比,PI3K、Akt蛋白表达量逐渐升高,差异均有统计学意义(均为P＜0.05).结论 补肾活血中药血清抑制加压诱导的RGCs凋亡,其机制可能与激活PI3K/Akt信号转导通路有关.     

Retinal ischemic injury rescued by sodium 4-phenylbutyrate in a rat model

Retinal ischemia is a common cause of visual impairment for humans and animals. Herein, the neuroprotective effects of phenylbutyrate (PBA) upon retinal ischemic injury were investigated using a rat model. Retinal ganglion cells (RGCs) were retrograde labeled with the fluorescent tracer fluorogold (FG) applied to the superior collicoli of test Sprague-Dawley rats. High intraocular pressure and retinal ischemia were induced seven days subsequent to such FG labeling. A dose of either 100 or 400 mg/kg PBA was administered intraperitoneally to test rats at two time points, namely 30 min prior to the induction of retinal ischemia and 1 h subsequent to the cessation of the procedure inducing retinal ischemia. The test-rat retinas were collected seven days subsequent to the induction of retinal ischemia, and densities of surviving RGCs were estimated by counting FG-labeled RGCs within the retina. Histological analysis revealed that ischemic injury caused the loss of retinal RGCs and a net decrease in retinal thickness. For PBA-treated groups, almost 100% of the RGCs were preserved by a pre-ischemia treatment with PBA (at a dose of either 100 or 400 mg/kg), while post-ischemia treatment of RGCs with PBA did not lead to the preservation of RGCs from ischemic injury by PBA as determined by the counting of whole-mount retinas. Pre-ischemia treatment of RGCs with PBA (at a dose of either 100 or 400 mg/kg) significantly reduced the level of ischemia-associated loss of thickness of the total retina, especially the inner retina, and the inner plexiform layer of retina. Besides, PBA treatment significantly reduced the ischemia-induced loss of cells in the ganglion-cell layer of the retina. Taken together, these results suggest that PBA demonstrates a marked neuroprotective effect upon high intraocular pressure-induced retinal ischemia when the PBA is administered prior to ischemia induction.     

Inhibition of LY294002 in retinal neovascularization via down-regulation the PI3K/AKT-VEGF pathway in vivo and in vitro

AIM: To investigate the effects of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 on retinal neovascularization (RNV) in the oxygen-induced retinopathy (OIR) mouse model and human umbilical vein endothelial cells (HUVECs). METHODS: C57BL/6J mice were randomly divided into normoxia-control, OIR-control and LY294002 treatment groups. LY294002 or phosphate-buffered solution was intraperitoneally injected daily into mouse pups from P6 to P9 in LY294002 treatment group or OIR-control group. Morphological and pathological changes in RNV, as well as expression levels of PI3K, serine-threonine kinase (AKT) and vascular endothelial growth factor (VEGF) were observed. HUVECs treating with LY294002 were exposed to hypoxia; the expression of PI3K, AKT and VEGF were examined by Western blot and RT-PCR analyses. RESULTS: Compared with the OIR-control group, LY294002 significantly inhibit RNV. Adenosine diphosphatase (ADPase) staining and hematoxylin and eosin staining indicated that the clock hour scores of neovascularization and the nuclei of pre-retinal neovascular cells in the LY294002 treatment group were clearly less than those in the OIR-control group (1.41±0.52 vs 6.20±1.21; 10.50±1.58 vs 22.25±1.82, both P<0.05). Intravitreal injection of LY294002 (in the LY294002 treatment group) markedly decreased PI3K/AKT-VEGF expression compared with the OIR-control group by immunohistochemistry, Western blotting and RT-PCR (all P<0.05). In HUVECs treated with hypoxia, expression of PI3K, AKT and VEGF were downregulated in the hypoxia-LY294002 group (all P<0.05). CONCLUSION: The PI3K inhibitor LY294002 can inhibit RNV by downregulating PI3K, AKT, and VEGF expression in vivo and in vitro. LY294002 may provide an effective method for preventing retinopathy of prematurity (ROP).     

Retinal ganglion cell survival in development: mechanisms of retinal growth hormone action

Several variants of growth hormone (GH) are found in the retina and vitreous of the chick embryo, where they appear to act as cell survival factors, having neuroprotective effects on retinal ganglion cells (RGCs). Here, we investigate the molecular mechanisms of the anti-apoptotic effect of GH in cultured RGCs. GH treatment increased Akt phosphorylation in these cells, which is an anti-apoptotic event. Whereas unphosphorylated Akt was detected in both nucleus and cytoplasm of RGCs by immunocytochemistry, the phosphorylated form of Akt (Akt-phos) was located primarily in the cytoplasm of both normal and apoptotic cells, although levels were markedly lower in the latter. It was found that GH treatment of RGCs reduced Akt levels, while concomitantly raising Akt-phos levels, consistent with a role for Akt signaling pathways in GH neuroprotective action. This was substantiated using Wortmannin, which, like GH antiserum, inhibited Akt phosphorylation and initiated apoptosis. The addition of Wortmannin to RGC cultures simultaneously with GH significantly reduced the anti-apoptotic effect of GH. The induction of apoptosis by GH antiserum was clearly accompanied by an increase in caspase-3 activation and PARP-1 cleavage, both of which were significantly reduced in the presence of the broad spectrum caspase inhibitor, Q-VD-OPh, which itself had a dramatic neuroprotective effect on cultured RGCs. Calpain activation appeared to be a major caspase-independent pathway to PARP-1 cleavage and apoptosis in these cells. Calpain inhibitor III (MDL 28170) was able to reduce PARP-1 cleavage and abrogate the apoptogenic effect of GH antiserum. The results support the view that caspase and calpain inhibitors are major neuroprotective agents for RGCs, and that pathways that activate both caspases and calpains are important for the anti-apoptotic actions of GH in these cells.     

右归丸对糖尿病视网膜病变大鼠PI3K/Akt信号通路的影响

目的：探讨“阴中求阳”立法之右归丸对糖尿病视网膜病变大鼠PI3 K/Akt信号通路的影响。方法：SD大鼠80只,随机分组,10只为正常组,余大鼠通过一次性腹腔注射链脲佐菌素溶液(60 mg/kg )联合大鼠玻璃体腔注射VEGF(0.05μg)的方式建立糖尿病视网膜病变增殖期大鼠模型。模型建立成功后,将最终成模大鼠随机分为4组：右归丸高中低剂量组、模型组,右归丸高中低剂量组每天给予相应右归丸浓缩液浓度进行灌胃,模型组、正常组每天给予等剂量蒸馏水灌胃。连续灌胃3 mo后,采取SP免疫组织化学、Western-blot法分别观察检测大鼠视网膜组织PI3 K和Akt的表达情况。结果：免疫组织化学观察显示PI3 K和Akt免疫组化染色的阳性表达,在视网膜上均为棕黄色颗粒;正常组PI3 K和Akt表达部分分布于神经节细胞层,内核也有少量表达,呈弱阳性免疫反应;模型组、右归丸各治疗组大鼠视网膜神经节细胞层、内丛状层及内外颗粒层都有阳性表达;与模型组比较,右归丸中、高剂量组PI3 K和Akt表达均减弱,右归丸低剂量组表达减弱不明显;与右归丸低剂量组比较,右归丸中、高剂量组PI3 K和Akt表达减弱;右归丸中、高剂量组表达强弱比较无差异。 Western-blot检测结果显示与正常组比较,模型组、治疗组表达差异有显著统计学意义( P<0.01),模型组、治疗组PI3 K和Akt蛋白表达水平显著升高;与模型组比较,右归丸中、高剂量组表达差异有统计学意义(P<0.05),右归丸中、高剂量组PI3 K和Akt蛋白表达水平均降低,右归丸低剂量组表达差异无统计学意义(P>0.05);与右归丸低剂量组比较,右归丸中、高剂量组表达差异有统计学意义(P<0.05),右归丸中、高剂量组PI3 K和Akt表达水平下降;右归丸中、高剂量组表达比较,差异无统计学意义(P>0.05)。结论：基于“阴中求阳”立法之右归丸可以通过影响PI3 K和Akt蛋白表达水平,抑制PI3 K/Akt 信号通路活化,延缓糖尿病视网膜病变的病变进程,为糖尿病视网膜病变防盲治疗提出新的治疗思路与科学依据。     

人参皂苷Rg3对糖尿病视网膜病变大鼠PI3K-Akt/PKB通路和血管内皮生长因子、细胞间黏附分子-1表达的影响

目的　研究人参皂苷Rg3对糖尿病视网膜病变大鼠PI3K-Akt/PKB通路和血管内皮生长因子（vascular endothelial growth factor,VEGF）、细胞间黏附分子-1（intercellular adhesion molecule-1,ICAM-1）表达的影响。方法　将60只SD大鼠随机分为对照组、模型组、人参皂苷Rg3治疗高剂量组（H-Rg3组,5.0 mg·kg^-1 Rg3）、低剂量组（L-Rg3组,0.5 mg·kg^-1 Rg3）,每组各15只,构建糖尿病视网膜病变大鼠模型,共治疗28 d。检测各组大鼠视网膜组织中丙二醛（malondialdehyde,MDA）、超氧化物歧化酶（superoxide dismutase,SOD）和乳酸脱氢酶（lactate dehydrogenase,LDH）表达;HE染色和TUNEL染色分析病理变化;免疫组织化学检查ICAM-1和VEGF蛋白的表达;Western blot检测视网膜组织中PI3K、Akt、p-Akt蛋白的表达;采用LY294002抑制剂（静脉注射,0.5 mg·kg^-1）验证PI3K-Akt/PKB通路。结果　与对照组相比,模型组凋亡细胞比例、视网膜组织中MDA和LDH表达均升高（均为P<0.05）,SOD、PI3K和p-Akt蛋白表达均降低（均为P<0.05）,VEGF、ICAM-1、Bad、cleaved-Caspase-3蛋白表达均升高（均为P<0.05）。与模型组相比,L-Rg3组和H-Rg3组凋亡细胞比例、视网膜组织中MDA和LDH表达均降低（均为P<0.05）,SOD、PI3K和p-Akt蛋白表达均升高（均为P<0.05）,VEGF、ICAM-1、Bad、cleaved-Caspase-3蛋白表达均降低（均为P<0.05）。与L-Rg3组相比,H-Rg3组凋亡细胞比例、视网膜组织中MDA和LDH表达均降低（均为P<0.05）,SOD、PI3K和p-Akt蛋白表达均升高（均为P<0.05）,VEGF、ICAM-1、Bad、cleaved-Caspase-3蛋白表达均降低（均为P<0.05）。与LY-模型组相比,LY294002组VEGF、ICAM-1、Bad和cleaved-Caspase-3蛋白表达均升高（均为P<0.05）,Bcl-2蛋白表达降低（P<0.05）。与LY-Rg3组相比,LY-Rg3+LY294002组VEGF、ICAM-1、Bad和cleaved-Caspase-3蛋白表达均升高（均为P<0.05）,Bcl-2蛋白表达降低（P<0.05）。结论　人参皂苷Rg3能够通过激活PI3K-Akt/PKB信号通路,下调VEGF和ICAM-1蛋白的表达,保护糖尿病视网膜病变大鼠的视网膜组织。     

Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells

PURPOSE: To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS: Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS: P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. CONCLUSIONS: Bacterial infection of HCECs induces EGFR transactivation through HB-EGF ectodomain shedding. EGFR and its downstream ERK and PI3K signaling pathways play a role in preventing epithelial apoptosis in the early stage of bacterial infection.     

Blocking LINGO-1 function promotes retinal ganglion cell survival following ocular hypertension and optic nerve transection

PURPOSE: LINGO-1 is a functional member of the Nogo66 receptor (NgR1)/p75 and NgR1/TROY signaling complexes that prevent axonal regeneration through RhoA in the central nervous system. LINGO-1 also promotes cell death after neuronal injury and spinal cord injury. The authors sought to examine whether blocking LINGO-1 function with LINGO-1 antagonists promotes retinal ganglion cell (RGC) survival after ocular hypertension and optic nerve transection. METHODS: An experimental ocular hypertension model was induced in adult rats using an argon laser to photocoagulate the episcleral and limbal veins. LINGO-1 expression in the retinas was investigated using immunohistochemistry and Western blotting. Soluble LINGO-1 protein (LINGO-1-Fc) and anti-LINGO-1 mAb 1A7 were injected into the vitreous body to examine their effects on RGC survival after ocular hypertension and optic nerve transection. Signal transduction pathways mediating neuroprotective LINGO-1-Fc effects were characterized using Western blotting and specific kinase inhibitors. RESULTS: LINGO-1 was expressed in RGCs and up-regulated after intraocular pressure elevation. Blocking LINGO-1 function with LINGO-1 antagonists, LINGO-1-Fc and 1A7 significantly reduced RGC loss 2 and 4 weeks after ocular hypertension and also promoted RGC survival after optic nerve transection. LINGO-1-Fc treatment blocked the RhoA, JNK pathway and promoted Akt activation. LINGO-1-Fc induced Akt phosphorylation, and the survival effect of LINGO-1 antagonists was abolished by Akt phosphorylation inhibitor. CONCLUSIONS: The authors demonstrated that blocking LINGO-1 function with LINGO-1 antagonists rescues RGCs from cell death after ocular hypertension and optic nerve transection. They also delineated the RhoA and PI-3K/Akt pathways as the predominant mediator of LINGO-1-Fc neuroprotection in this paradigm of RGC death.     

Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells

PURPOSE: FGF-2 is a potent mitogen of rabbit corneal endothelial cells (CECs). This study was undertaken to investigate whether PI 3-kinase participates in cell cycle regulation in response to stimulation with FGF-2 in CECs. METHODS: Cell proliferation was assayed by counting the cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of cyclin-dependent kinase 4 (Cdk4), p27(Kip1) (p27), phosphatidylinositol 3 (PI 3)-kinase, protein kinase B/Akt (Akt), and beta-actin was analyzed by immunoblot. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. LY294002 was used to inhibit PI 3-kinase. RESULTS: CEC required prolonged and continuous exposure to FGF-2. FGF-2 at 10 ng/mL markedly stimulated PI 3-kinase enzyme activity, and stimulation with FGF-2 also caused activation of Akt. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase in cell cycle stimulation was determined: FGF-2 markedly upregulated expression of Cdk4 and stimulated translocation of Cdk4 into nuclei, whereas LY294002 markedly blocked upregulation of Cdk4 expression, and the inhibitor facilitated nuclear export of Cdk4. In contrast, FGF-2 significantly downregulated expression of p27 and facilitated phosphorylation of p27. LY294002 completely blocked the action of FGF-2 on the expression and phosphorylation of p27. CONCLUSIONS: These data indicate that PI 3-kinase ultimately leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating expression of Cdk4, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei as it simultaneously downregulates expression of p27 and facilitates the proteolysis of the molecule by phosphorylation.