Intrinsic activation of PI3K/Akt signaling pathway and its neuroprotective effect against retinal injury

PURPOSE: The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway can function as a neuroprotective pathway following induced retinal injury. METHODS: The activation of Akt was assessed by immunoblot analysis, and the role of PI3K/Akt pathway was evaluated by TUNEL staining and counting the number of retrogradely-labeled retinal ganglion cells (RGCs) in the whole retina at 168 h after injury with or without PI3K specific inhibitor, LY294002. RESULTS: Akt was induced within one hr and reached a maximum 6 hrs after optic nerve clamping. The activation was observed in the RGC layer including RGCs, the inner plexiform layer, inner nuclear layer, and in the photoreceptor outer segments. The number of surviving RGCs was decreased significantly 168 hrs after injury. LY294002 partially inhibited the activation of Akt, and significantly decreased the number of surviving RGCs as compared with that of injury alone. CONCLUSIONS: These results indicate that the PI3K/Akt signaling pathway is activated intrinsically and has a neuroprotective effect on injured RGCs.     

姜黄素通过抑制PI3K/Akt信号通路改善LPS诱导的视网膜炎症

目的:本研究旨在调查姜黄素是否能减轻动物模型和人视网膜色素上皮细胞(ARPE)-19细胞的视网膜炎症。方法:在体内,雄性C57/B6小鼠腹腔注射姜黄素3d,然后腹腔注射脂多糖(LPS;10mg/kg)诱导视网膜炎症。LPS作用24h后,通过RT-PCR检测促炎细胞因子的mRNA水平。伴刀豆球蛋白凝集素灌注标记技术评估了白细胞对视网膜脉管系统的粘附。用蛋白质定量试剂盒测量前房中的蛋白浓度。在体外,培养ARPE-19细胞。用细胞计数试剂盒8(CCK-8)法测量来选择姜黄素的最佳作用浓度。在用5μg/mL LPS刺激之前,将ARPE-19细胞在有或没有姜黄素的情况下孵育1h。通过RT-PCR和ELISA测量促炎细胞因子。通过蛋白质印迹分析PI3K/Akt表达。结果:姜黄素预处理可显著抑制EIU相关白细胞粘附于视网膜血管和前房蛋白渗漏。体内应用姜黄素后,炎症细胞因子(如IL-1β,IL-6和TNF-α)的mRNA表达水平也显著降低。同时,姜黄素在ARPE-19细胞的mRNA和蛋白质水平上均显著减弱IL-6,IL-8和MCP-1的表达。姜黄素抑制LPS激活的ARPE-19细胞中的PI3K/Akt磷酸化以及NF-κB激活。结论:姜黄素对LPS诱导的视网膜炎症具有预防作用,其作用似乎与PI3k/Akt信号传导途径的抑制有关。     

Curcumin alleviates LPS-induced retinal inflammation by inhibiting PI3k/Akt signaling pathway

AIM: To investigate whether the curcumin reduce retinal inflammation in animal model and human retinal pigment epithelium (ARPE)-19 cells.METHODS: In vivo, male C57/B6 mice received intraperitoneal injections of curcumin for 3d before intraperitoneal injection of lipopolysaccharide (LPS; 10 mg/kg) to induce retinal inflammation. 24h after LPS application, the mRNA levels of pro-inflammatory cytokines were detected by real-time polymerase chain reaction (RT-PCR). Concanavalin A lectin perfusion-labeling technique evaluated leukocyte adhesion to the retinal vasculature. The protein concentration in the anterior chamber was measured with a protein quantification kit. In vitro, ARPE-19 cells were cultured. The optimum concentration of curcumin was detected by cell counting kit-8 (CCK-8) assay. Before stimulated with 5 μg/mL LPS, ARPE-19 cells were incubated with or without curcumin for 1h. Pro-inflammatory cytokines were measured by RT-PCR and ELISA. PI3K/Akt expression was analyzed by Western blotting.RESULTS: Curcumin pre-treatment led to significant inhibition of EIU-associated leukocyte adhesion to retinal blood vessels and anterior-chamber protein leakage. The mRNA expression level of inflammatory cytokines was also significantly reduced with application of curcumin in vivo, such as IL-1β, IL-6 and TNF-α. Meanwhile, Curcumin significantly attenuated the expression of IL-6, IL-8 and MCP-1 at both mRNA and protein levels in ARPE-19 cells. Curcumin suppressed PI3K/Akt phosphorylation as well as NF-κB activation in LPS-activated ARPE-19 cells.CONCLUSION: Curcumin plays a preventive effect on LPS-induced retinal inflammation. The beneficial effect appears associated with inhibiting of the PI3k/Akt signaling pathway.     

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.     

Attenuation of human lens epithelial cell spreading,migration and contraction via downregulation of the PI3K/Akt pathway

Background

Posterior capsule opacification (PCO) represents a major challenge in the postoperative management of cataract patients. Spreading, migration and contraction of residual human lens epithelial cells play a pivotal role in the pathogenesis of PCO. Therefore, we analyzed the effect of the alkylphosphocholine (APC) erufosine on these cellular features as well as on PI3K/Akt, a crucial pathway in PCO pathogenesis.

Methods

Human lens epithelial cells were cultured under standard cell culture conditions. Cell spreading was analyzed on fibronectin-coated wells and chemokinetic migration was assessed by time-lapse microscopy. For evaluation of cell-mediated collagen matrix contraction, the cells were seeded into collagen gels and incubated with an APC in different non-toxic concentrations before the surface area was measured on day 6. The activity of PI3K/Akt was assessed by an ELISA kit after incubation of the cells with different APC concentrations.

Results

Human lens epithelial cell spreading and migration were attenuated by APCs as follows: 7 % spreading, 48 % migration (0.1 μM APC), and 32 % spreading, 68 % migration (1.0 μM APC). APC concentrations of 0.1 μM reduced collagen gel diameter by 5 %, and 1.0 μM by less than 1 %, compared to untreated, cell-populated gels that resulted in a cell diameter contraction of 36 %. PI3K was downregulated in a concentration-dependent manner.

Conclusions

The crucial cellular features of PCO pathogenesis are attenuated by the APC erufosine via downregulation of the PI3K pathway. Thus, erufosine might become a valuable tool for pharmacologic PCO prophylaxis in the future.     

Vitreous levels of pigment epithelium-derived factor and vascular endothelial growth factor: implications for ocular angiogenesis

PURPOSE: Pigment epithelium-derived factor (PEDF) has been demonstrated to suppress ocular angiogenesis in several animal models. In this study, we sought to measure the levels of PEDF and vascular endothelial growth factor (VEGF) in the vitreous of patients with and without ocular neovascular disorders. DESIGN: Case-control study of patients undergoing intraocular surgery for a variety of neovascular and nonneovascular conditions. METHODS: Vitreous samples were collected from 65 eyes of 65 patients with no neovascular disorder (n = 24), choroidal neovascularization (n = 9), active proliferative diabetic retinopathy (n = 16), and inactive proliferative diabetic retinopathy (n = 16). The levels of VEGF and PEDF in these vitreous samples were determined by enzyme-linked immunosorbent assay. RESULTS: The VEGF levels were at or below the level of detectability in the reference and choroidal neovascularization groups. The VEGF levels were significantly elevated in both the active and inactive PDR groups, and significantly higher in the active PDR group as compared with the inactive PDR group. The PEDF levels, which were present at relatively high concentrations in all groups, were higher in patients with active PDR compared with the control and choroidal neovascularization groups. CONCLUSIONS: High levels of immunoreactive PEDF are present in the vitreous of individuals with or without ocular neovascularization, but PEDF levels are significantly higher in patients with active PDR compared with patients with choroidal neovascularization or nonneovascular retinal diseases. Although these results do not preclude the possibility that endogenous PEDF helps to modulate ocular neovascularization, they do not support ischemia-induced downregulation of PEDF as a mechanism for such modulation.     

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.     

Involvement of PI3K/Akt signaling pathway in hepatocyte growth factor-induced migration of uveal melanoma cells

PURPOSE: Uveal melanoma is the most common primary intraocular malignancy in adult humans. Unlike cutaneous melanoma, uveal melanoma disseminates preferentially to the liver through the hematogenous system. To date, the mechanism underlying this metastatic homing is largely unknown. This study investigated the effect of hepatocyte growth factor (HGF)-triggered signaling pathways to identify the role of HGF and its downstream effectors in inducing the migration of uveal melanoma cells. METHODS: Migration of uveal melanoma cells was measured by in vitro wound healing and transwell migration assays. The expression and translocation of c-Met were detected using indirect immunofluorescence. The activation of extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathways was analyzed using specific antibodies against phospho-ERK1/2 and phospho-Akt. The impact of HGF treatment on the expression of cell adhesion molecules was measured using Western blotting. RESULTS: HGF was found to enhance cell migration, and that HGF-induced migration depends on PI3K/Akt pathway. The activation of PI3K/Akt pathway induced by the HGF/c-Met axis is involved in the downregulation of cell adhesion molecules E-cadherin and beta-catenin, contributing to the attenuation of cell-cell adhesion and promoting the enhanced motility and migration of uveal melanoma cells. On HGF stimulation, receptor c-Met is translocated to the nucleus in a ligand-dependent manner, suggesting that c-Met may modulate the expression of genes involved in melanoma cell migration. CONCLUSIONS: Data from this study directly linked the central PI3K/Akt pathway to uveal melanoma migration and pointed to new avenues for therapeutic intervention in hepatic metastasis.     

PI3K/Akt and mTOR/p70S6K pathways mediate neuroprotectin D1-induced retinal pigment epithelial cell survival during oxidative stress-induced apoptosis

The initiation and progression of several forms of retinal degenerations involve excessive, repetitive, and/or sustained oxidative stress that, in turn, mediate photoreceptor cell damage and death. Since phosphatidylinositol 3-kinase (PI3K)/Akt and mTOR/p70S6-kinase pathways are part of survival signaling in cells confronted with oxidative stress, we asked whether or not docosahexaenoic acid-derived neuroprotectin D1 (NPD1) mediates survival upon single-dose and/or repetitive oxidative stress through this pathway. For this purpose, we used human retinal pigment epithelial (ARPE-19) cells challenged by exposure to hydrogen peroxide (H₂O₂) plus tumor necrosis factor alpha (TNF-α). We found that in single-dose oxidative stress-induced apoptosis, phosphorylation of Akt, mTOR, and p70S6K was both time- and dose- dependent. Inhibition of PI3K or mTOR/p70S6K by wortmannin and rapamycin, respectively, increased apoptosis and inhibited phosphorylation of Akt and p70S6K induced by single-dose oxidative stress. While two exposures of a low dose, non-damaging oxidation induced apoptosis and upregulation of Akt, mTOR, and p70S6K, longer treatment of the cells with three exposures of low dose to low-dose stress showed no changes in the levels of Akt, mTOR, or p70S6K, and resulted in enhanced apoptosis compared to higher doses. Removing the oxidative stress-inducing agents following the single-dose or short term repetitive oxidative stress at the peak of Akt, mTOR, and p70S6K phosphorylation (i.e., 30 min after induction) led to recovery, with no apoptosis after 16 h of incubation. Cells that were induced with three low doses of stress did not show recovery when oxidative stress was removed 30 min after the last exposure. NPD1 protected the RPE cells against both single-dose and repetitive oxidative stress-induced apoptosis and promoted higher levels of phosphorylated Akt, mTOR, and p70S6K. Together, our results show that a) repetitive oxidative stress is dose dependent and may not be recovered by removing the oxidative stress-inducing agents, b) PI3K/Akt and mTOR/p70S6K pathways play a major role in the protection against oxidative stress-induced apoptosis in ARPE-19 cells, and c) NPD1 exerts protection under these conditions by inducing PI3K/Akt and mTOR/p70S6K pathways.     

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.     

Ghrelin inhibits autophagy mediated by AKT/mTOR pathway to ameliorate retinal angiogenesis induced by high glucose stress

AIM: To observe the effect of ghrelin, a growth hormone-releasing peptide, on retinal angiogenesis in vitro under high glucose (HG) stress and to explore the possible mechanism of autophagy. METHODS: Human retinal microvascular endothelial cells (HRMECs) were treated with high concentration of glucose alone or in combination with ghrelin. The cell migration, tube formation and the expression of the autophagy-related proteins LC3-II/I, Beclin-1, p62, phosphorylated AKT (p-AKT)/AKT and phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR were detected. Then, to clarify the correlation between ghrelin effect and autophagy, AKT inhibitor VIII was adopted to treat HRMECs, and cell migration, tube formation as well as the protein expressions of LC3-II/I, Beclin-1 and p62 were observed. RESULTS: Under HG stress, ghrelin inhibited migration and tube formation of HRMECs. Ghrelin inhibited the increases in the protein levels of LC3-II/I, Beclin-1 and the decreases in the protein levels of p62, p-AKT/AKT and p-mTOR/mTOR induced by HG stress. Moreover, under the action of AKT/mTOR pathway inhibitors, the effects of ghrelin on migration and tube formation were both reduced. In addition, the expression of LC3-II/I and Beclin-1 were significantly up-regulated and the expression of p62 was down-regulated. CONCLUSION: Retinal angiogenesis under in vitro HG stress can be inhibited by ghrelin through activating AKT/mTOR pathway to inhibit autophagy.     

DHA玻璃体注射对ARMD大鼠模型光感受器细胞凋亡和PI3K/Akt通路的影响

目的：探讨二十二碳六烯酸(DHA)玻璃体注射对年龄相关性黄斑变性(ARMD)大鼠光感受器细胞凋亡和磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(PI3K/Akt)通路的影响。

方法：大鼠随机分为空白对照组、模型组、低剂量DHA组(L-DHA组)、中剂量DHA组(M-DHA组)和高剂量DHA组(H-DHA组)。采用光损伤法建立干性ARMD大鼠模型。HE染色观察视网膜病理变化,TUNEL法检测视网膜细胞凋亡情况,透射电镜观察视网膜神经节细胞超微结构,酶联免疫吸附法测定视网膜组织TNF-α和IL-6水平,Western Blot检测视网膜组织p-PI3K、p-Akt、Bax、Bcl-2、p-NF-κBp65和cleved-caspase-3蛋白表达。

结果：与空白对照组比较,模型组大鼠视网膜总厚度、外核层和内核层厚度、视网膜组织p-PI3K、p-Akt和Bcl-2蛋白表达均降低(P<0.05),神经节细胞层和外核层细胞凋亡指数、视网膜组织TNF-α和IL-6水平、Bax、p-NF-κBp65和cleved-caspase-3蛋白表达均升高(P<0.05)。与模型组比较,M-DHA组和H-DHA组大鼠视网膜总厚度、外核层和内核层厚度、视网膜组织p-PI3K、p-Akt和Bcl-2蛋白表达均升高(P<0.05),神经节细胞层和外核层细胞凋亡指数、视网膜组织TNF-α和IL-6水平、Bax、p-NF-κBp65和cleved-caspase-3蛋白表达均降低(P<0.05)。

结论：DHA可能通过激活PI3K/Akt通路抑制ARMD大鼠光感受器细胞凋亡。     

miR-519d-3p靶向HIF-1α抑制高糖诱导的人视网膜微血管内皮细胞功能障碍及血管生成

目的:明确miR-519d-3p对高糖诱导的人视网膜微血管内皮细胞(HRMEC)功能障碍与血管生成的影响,并阐明其对低氧诱导因子-1α(HIF-1α)的调控机制。方法:通过5、30mmol/L葡萄糖分别诱导HRMEC建立正常(NG)和高糖(HG)细胞模型。将HRMEC分为对照组(HG细胞模型转染阴性对照模拟物)、甘露醇组(对照组加入25mmol/L甘露醇)、miR-519d-3p过表达组(HG细胞模型转染miR-519d-3p模拟物)、miR-519d-3p联合HIF-1α过表达组(HG细胞模型共转染miR-519d-3p模拟物和HIF-1α过表达载体)。实时荧光定量PCR法检测各组miR-519d-3p的表达情况。Western blotting法检测各组HIF-1α蛋白的表达情况。荧光素酶报告基因实验检测miR-519d-3p和HIF-1α的结合位点情况。CCK-8法检测各组细胞增殖情况。Hoechst 33342染色法检测各组细胞凋亡情况。ELISA法检测各组细胞外液炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6蛋白的表达情况。小管形成实验检测各组新生...     

水蛭提取液通过VEGF/PI3K/AKT通路抑制WERI-RB-1细胞表达VEGF

目的：研究水蛭提取液对视网膜母细胞瘤细胞(WERI-RB-1细胞)表达血管内皮生长因子(VEGF)的影响及相关分子机制。

方法：将体外培养的WERI-RB-1细胞分为对照组、0.04U/mL水蛭提取液组、0.08U/mL水蛭提取液组,对照组采用完全培养基培养48h,水蛭提取液组分别采用含0.04、0.08U/mL含药培养基培养48h。采用ELISA法检测各组细胞培养基上清液中VEGF表达水平; 采用RT-PCR法检测各组细胞缺氧诱导因子-1a(HIF-1a)和基质金属蛋白酶-9(MMP-9)mRNA相对表达水平; 采用Western Blot法检测各组细胞HIF-1a、MMP-9、磷脂酰肌醇3-激酶(PI3K)、人磷酸化蛋白激酶(p-AKT)相对表达水平。

结果：与对照组比较,水蛭提取液组细胞培养基上清液中VEGF表达均降低(P<0.05),0.04、0.08U/mL水蛭提取液对VEGF表达抑制率分别为32.43%、38.92%。与对照组比较,水蛭提取液组细胞HIF-1a和MMP-9 mRNA相对表达水平均明显降低(P<0.05),0.04、0.08U/mL水蛭提取液对HIF-1a mRNA表达抑制率分别为27.64%、24.75%,对MMP-9 mRNA表达抑制率分别为43.97%、51.48%。与对照组比较,水蛭提取液组细胞HIF-1a、MMP-9、PI3K、p-AKT蛋白相对表达水平均明显降低(P<0.05),0.04、0.08U/mL水蛭提取液对HIF-1a蛋白表达抑制率分别为55.81%、43.85%,对MMP-9蛋白表达抑制率分别为39.49%、47.23%,对PI3K蛋白表达抑制率分别为33.27%、29.83%,对p-AKT蛋白表达抑制率分别为52.07%、30.21%。

结论：水蛭提取液可能通过VEGF/PI3K/AKT通路及HIF-1a、MMP-9因子抑制WERI-RB-1细胞VEGF的表达。     

PI3K/Akt信号通路在眼科疾病发生发展过程中的调控作用研究进展

眼科疾病的发生发展与眼组织的异常发育和功能障碍有关,其中相关信号通路的活化在眼科疾病的发生和发展过程中发挥重要作用.PI3 K/Akt信号通路(磷脂酰肌醇3激酶/蛋白激酶B信号通路)广泛存在于各类细胞中,是参与细胞生长、增殖、分化调节的重要信号转导通路.研究发现,PI3 K/Akt信号通路在白内障、糖尿病视网膜病变等多...     

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).     

丁香酚通过PI3K/AKT信号通路减轻镰刀菌诱导的角膜炎症

目的：探讨丁香酚对镰刀菌(F.solani)诱导的小鼠真菌性角膜炎(FK)的保护作用,并初步探讨可能的潜在机制。

方法：采用改良的表层镜法制备FK小鼠模型。二甲基亚砜(DMSO)组大鼠右眼结膜囊涂抹等量DMSO(0.05%)。丁香酚组将丁香酚(160 μg/mL)涂抹至小鼠右眼结膜囊。类胰岛素生长因子-1(IGF-1)组除施用丁香酚外,还于右眼结膜囊涂抹PI3K/AKT通路激活剂IGF-1(10 nmol/mL)。分别在接种镰刀菌悬浮液的第1、3、5 d,于裂隙灯显微镜下观察角膜形态。采用苏木精伊红(HE)染色评估角膜组织病理损伤。测定角膜组织载菌量。酶联免疫吸附试验和蛋白免疫印迹用于分析炎症介质白介素-6(IL-6)和白介素-1β(IL-1β)的水平和PI3K/AKT通路蛋白的表达。

结果：丁香酚治疗可改善FK小鼠的角膜炎形态症状和炎症反应,减轻角膜病理组织损伤和真菌负荷。在镰刀菌感染3 d,与DMSO组相比,丁香酚组角膜组织IL-6水平明显升高,而IL-1β水平明显降低(均P<0.05); 且丁香酚组角膜组织IL-6水平明显高于IGF-1组、IL-1β水平明显低于IGF-1组(均P<0.05)。在感染5 d,丁香酚组角膜组织中IL-6和IL-1β水平均明显低于DMSO组和IGF-1组(P<0.05)。与DMSO组相比,丁香酚组角膜组织中p-PI3K和p-Akt表达均明显降低(P<0.05); 且丁香酚组角膜组织p-PI3K和p-Akt表达明显低于IGF-1组(均P<0.05)。

结论：丁香酚可能通过抑制PI3K/AKT通路来减轻镰刀菌引起的角膜炎症,对小鼠镰刀菌角膜炎具有保护作用。