黄芩茎叶黄酮对慢性脑缺血大鼠脑内NMDA受体和VEGF表达的影响

目的: 探讨黄芩茎叶黄酮(SSF)对慢性永久性脑缺血大鼠脑内N-甲基-D-天门冬氨酸受体(NMDAR)和血管内皮生长因子(VEGF)表达的影响。方法: 雌性Sprague-Dawley(SD)大鼠双侧颈总动脉结扎2个月制备慢性脑缺血记忆障碍模型,通过RT-PCR测定海马细胞中NMDAR mRNA和皮层细胞中VEGF mRNA的含量。结果: 与假手术组相比,模型组大鼠海马细胞中NMDAR1 mRNA、NMDAR2A mRNA和NMDAR2B mRNA 的含量显著增加(P<0.01),皮层细胞中VEGF mRNA的含量明显增加(P<0.01);而 SSF 17.5 mg·kg^-1·d^-1、35 mg·kg^-1·d^-1和70 mg·kg^-1·d^-1灌胃给药38 d,能明显降低海马细胞中NMDAR1 mRNA、NMDAR2A mRNA和NMDAR2B mRNA 的含量,显著增加皮层细胞中VEGF mRNA的含量。结论: SSF 能够明显降低脑缺血大鼠海马细胞中NMDAR 的表达,明显增加皮层细胞中VEGF 的表达,提示SSF可能通过影响NMDAR和VEGF的生成发挥神经保护作用。     

CGRP和NGF对全脑缺血再灌注大鼠脑组织Erk表达的调节作用

目的 :检测大鼠脑缺血再灌注后细胞外调节蛋白激酶 (Erk)的表达 ,探讨降钙素基因相关肽 (CGRP)和神经生长因子 (NGF)对脑组织的保护作用及机制。方法 :采用颈动脉负压分流法制作大鼠脑缺血再灌注模型 ,采用免疫组织化学SABC法及显微图像分析检测海马及皮质内Erk的表达。结果 :大鼠缺血再灌注海马及皮质内Erk免疫反应阳性产物较正常组增多 (P <0 .0 5 ) ,而注射CGRP或NGF后阳性产物明显高于缺血再灌注组 (P <0 .0 1 ) ,二者联合应用效果更加显著 (P <0 .0 5 )。结论 :CGRP及NGF参与缺血神经元Erk的调节 ,二者对缺血神经元有协同修复作用     

NGF对局灶性脑缺血再灌注大鼠海马和顶叶皮质内CREB mRNA表达的影响

探讨外源性神经生长因子(nervegrowthfactor,NGF)对局灶性脑缺血再灌注大鼠海马和顶叶皮质内的cAMP反应元件结合蛋白(cyclicAMPresponseelementbindingprotein,CREB)mRNA表达的影响。用线栓法制作大鼠局灶性脑缺血再灌注模型,运用原位杂交和图像分析技术检测大鼠缺血侧海马CA1区和顶叶皮质内CREBmRNA的表达。结果显示:缺血再灌注组缺血侧海马CA1区和顶叶皮质CREBmRNA阳性反应产物平均光密度(OD)比假手术组减少(P<0.05),NGF组CA1区和顶叶皮质内的CREBmRNA阳性产物平均光密度高于缺血再灌注组(P<0.05)。本研究结果提示NGF可以上调局灶性脑缺血再灌注大鼠海马和顶叶皮质缺血神经元CREBmRNA的表达,NGF对缺血神经元的保护作用可能通过激活CREB的转录与翻译,从而启动一系列信号通路来实现。     

CGRP和NGF对全脑缺血再灌注大鼠脑组织Erk mRNA表达的调节作用

目的　检测大鼠脑缺血再灌注后细胞外信号调节蛋白激酶信使RNA(ErkmRNA)的表达,探讨降钙素 基因相关肽(CGRP)和神经生长因子(NGF)对脑组织的保护作用。　方法　采用颈动脉负压分流法制作大鼠脑缺 血再灌注模型,采用原位杂交及显微图像分析方法检测海马及皮质内Erk的表达。　结果　大鼠缺血再灌注海马 及皮质内ErkmRNA较正常组增多(P<0.05),而注射CGRP或NGF后ErkmRNA明显高于缺血再灌注组(P< 0.01),两者联合应用效果更加显著(P<0.05)。　结论　CGRP及NGF可能参与缺血神经元ErkmRNA的调节,两 者对缺血神经元有协同修复作用。     

Hypothermia differentially increases extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun terminal kinase activation in the hippocampus during reperfusion after asphyxial cardiac arrest

Mitogen-activated protein kinases are signal transduction mediators that have been implicated in cell survival and cell death. This study characterized the activation of pathways in the hippocampus during reperfusion after global cerebral ischemia, as well as the influence of a regimen of hypothermia that reduces ischemic cell death in the hippocampus. Circulatory arrest was induced in rats by 8 min of asphyxia. Relative levels of phosphorylated and total extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were measured in the hippocampus after 6, 12 or 24h of reperfusion using immunoblotting. Asphyxia induced a progressive increase in phosphorylated extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase, but no change in phosphorylated p38 mitogen-activated protein kinase. Induction of mild hypothermia (33 degrees C) during reperfusion increased extracellular signal-regulated kinase phosphorylation and produced a smaller increase in stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation at 24h. Hypothermia did not alter extracellular signal-regulated kinase activation in rats not subjected to ischemia. Extracellular signal-regulated kinase activation was associated with an increase in phosphorylation of the mitogen-activated protein kinase kinase 1/2, and was inhibited by administration of the specific mitogen-activated protein kinase kinase 1/2 inhibitor SL327. Immunohistochemical staining showed an increase in active extracellular signal-regulated kinase in the CA1, CA2, CA3 and dentate gyrus regions of the hippocampus after ischemia and reperfusion. In contrast, active stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity was most intense in the CA3 and dentate gyrus regions.These data demonstrate that both extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase pathways are activated during the first 24h of reperfusion after global cerebral ischemia, and that hypothermia increases the activation of extracellular signal-regulated kinase relative to stress-activated protein kinase/c-Jun N-terminal kinase. Thus, an increase in extracellular signal-regulated kinase activation may be associated with improved neuronal survival after ischemic injury.     

Activation of the phosphatidylinositol 3'-kinase/AKT pathway in neuroblastoma and its regulation by thioredoxin 1

Neuroblastoma is a malignant pediatric tumor with poor survival. The phosphatidylinositol 3'-kinase/AKT pathway is a crucial regulator of cellular processes including apoptosis. Thioredoxin 1, an inhibitor of tumor-suppressor phosphatase and tensin homolog, is overexpressed in many tumors. The objective of this study was to explore phosphatidylinositol 3'-kinase/AKT pathway activation and regulation by thioredoxin 1 to identify potential therapeutic targets. Immunohistochemical analysis was done on tissue microarrays from tumor samples of 101 patients, using antibodies against phosphatidylinositol 3'-kinase, AKT, activated AKT, phosphatase and tensin homolog, phosphorylated phosphatase and tensin homolog, thioredoxin 1, epidermal growth factor receptor, vascular endothelial growth factor and receptors (vascular endothelial growth factor 1 and vascular endothelial growth receptor 2), platelet-derived growth factor receptors, insulin-like growth factor 1 receptor, neurotrophic tyrosine kinase receptor type 2, phosphorylated 70-kd S6 protein kinase, 4E-binding protein 1, and phosphorylated mammalian target of rapamycin. Using 3 neuroblastoma cell lines, we investigated cell viability with AKT-specific inhibitors (LY294002, RAD001) and thioredoxin 1 alone or in combination. We found activated AKT and AKT expressed in 97% and 98%, respectively, of neuroblastomas, despite a high expression of phosphatase and tensin homolog correlated with thioredoxin 1. AKT expression was greater in metastatic than primary tumors. Insulin-like growth factor 1 receptor, tyrosine kinase receptor type 2, vascular endothelial growth receptor 1, and downstream phosphorylated 70-kd S6 protein kinase were correlated with activated AKT. LY294002 and RAD001 significantly reduced AKT activity and cell viability and induced a G(1) cell cycle arrest. Thioredoxin 1 decreased cytotoxicity of AKT inhibitors and doxorubicin, up-regulated AKT activation, and induced cell growth. Thus, vascular endothelial growth receptor 1, tyrosine kinase receptor type 2, insulin-like growth factor 1 receptor, and thioredoxin 1 emerged as preferentially committed to phosphatidylinositol 3'-kinase/AKT pathway activation as observed in neuroblastoma. Thioredoxin 1 is a potential target for therapeutic intervention.     

Cerebral heme oxygenase 1 and 2 spatial distribution is modulated following injury from hypoxia–ischemia and middle cerebral artery occlusion in rats

The regional and cellular distribution of heme oxygenase (HO)-1 and -2 following cerebral ischemia has not been ascertained. Employing the transient middle cerebral artery occlusion (MCAO) and hypoxia–ischemia (HI) models of unilateral brain injury, the aim was to elucidate immunolocalization of HO-1 and HO-2. Animals were sacrificed 3 days post-ischemia and immunohistochemistry and Western blotting were utilized to determine HO-1 and HO-2 expression. In the ipsilateral hemisphere following HI, HO-1 immunoreactivity was significantly upregulated in many neuronal and glial populations (including the cortex, hippocampus and thalamus). HO-1 was also detected in macrophages/microglia within the infarct. In addition to widespread neuronal HO-2 labelling, HO-2 was also expressed in vascular endothelial cells. Inflammatory cells within the infarct of MCAO and HI animals were surprisingly immunoreactive for HO-2, but only HI animals had significantly elevated HO-2 protein expression in the ipsilateral hemisphere. This may be due to the presence of global hypoxia in the HI model which can upregulate vascular endothelial growth factor and subsequent proliferation of endothelial cells. This report of HO-2 protein expression upregulation following HI coupled with an increase in HO-1 immunoreactivity suggests that this response may be implicated in reducing cell death or repairing damage induced by cerebral ischemia.     

Calcium signalling and control of cell proliferation by tyrosine kinase receptors (review)

Most peptidic growth factors (GFs) that act as mitogens for different cell types bind to tyrosine kinase receptors (RTKs) and trigger complex intracellular signal transduction pathways finally leading to cell proliferation. Among the early events induced by GFs, cytosolic calcium increase plays a key role, and in particular calcium entry from extracellular medium appears to be a widespread signal. Even if a great amount of data has been provided during the last decades, several issues remain to be fully clarified: the nature of the calcium-permeable channels involved, their regulation by intracellular messengers, and the mechanisms underlying calcium-dependent cell proliferation. This review focuses on the relationship between calcium signals and angiogenesis, a process in which endothelial cell proliferation, mainly triggered by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), is a critical step.     

Redox regulation of PI3K/Akt and p53 in bovine aortic endothelial cells exposed to hydrogen peroxide

To clarify the apoptotic and survival signal transduction pathways in activated vascular endothelial cells exposed to oxidative stress, the effects of inhibitors of signal transduction on hydrogen peroxide (H(2)O(2))-induced apoptosis in bovine aortic vascular endothelial cells (BAEC) were examined. Treatment of BAEC with 1 mM H(2)O(2) caused increases of DNA fragmentation, p53 expression, Bax/Bcl-2 ratio, and the activities of caspases 3 and 9. The increases of DNA fragmentation, Bax/Bcl-2 ratio, and caspase activities were abrogated by BAPTA-AM (an intracellular Ca(2+) chelator) and N-acetyl-L-cysteine (an antioxidant), and augmented by wortmannin [a phosphatidylinositol 3-kinase (PI3K) inhibitor]. The increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) observed in H(2)O(2)-stimulated cells was unaffected by wortmannin, suggesting that the potentiating effect of wortmannin on the apoptosis was not due to an alteration of [Ca(2+)](i). H(2)O(2) increased the levels of PI3K activity and Akt phosphorylation. Both were attenuated by wortmannin and, to a lesser extent, by genistein (a tyrosine kinase inhibitor) and suramin (a growth factor receptor inhibitor), but not affected by BAPTA-AM. These results suggest that H(2)O(2) induces Ca(2+)-dependent apoptosis and Ca(2+)-independent survival signals such as redox-regulated activation of PI3K/Akt, which is partly mediated by the activation of growth factor receptors in BAEC.     

A high n-6 polyunsaturated fatty acid diet reduces muscarinic M2/M4 receptor binding in the rat brain

The aim of this study was to examine the influence of different fat diets on muscarinic acetylcholine receptor binding. Nineteen male Sprague–Dawley rats were divided into four groups and fed a diet of either high saturated fat, n-6 polyunsaturated fatty acid (PUFA), n-3 PUFA or low fat (control) for 8 weeks. Using quantitative autoradiography, [³H]pirenzepine binding to muscarinic M1/M4 receptors and [³H]AF-DX384 binding to M2/M4 receptors were measured throughout the brain in all four groups. The main findings were that compared to the low fat control group, M2/M4 receptor binding was significantly reduced in the dorsolateral, dorsomedial and ventromedial parts of the caudate putamen (61–64%, p < 0.05), anterior cingulate cortex (59%, p < 0.01), dentate gyrus and CA1–3 fields of the hippocampus (32–43%, p < 0.01) of rats on a high n-6 PUFA diet; however, no differences in M1/M4 receptor binding densities between the four groups were observed. These results suggest that a diet high in n-6 PUFA, but not of n-3 PUFAs or saturated fat, may selectively alter M2/M4 receptor-mediated signal transduction in the rat brain.     

Inhibitor of vascular endothelial growth factor receptor tyrosine kinase attenuates cellular proliferation and differentiation to mature neurons in the hippocampal dentate gyrus after transient forebrain ischemia in the adult rat

Neurogenesis in the adult hippocampal dentate gyrus is promoted by transient forebrain ischemia. The mechanism responsible for this ischemia-induced neurogenesis, however, remains to be determined. It has been suggested that there may be a close relationship between neurogenesis and the expression of vascular endothelial growth factor, an angiogenic factor. The purpose of the present study was to examine the relationship between vascular endothelial growth factor and cell proliferation in the dentate gyrus after transient forebrain ischemia. The mRNA expression of vascular endothelial growth factor was increased in the dentate gyrus on day 1 after ischemia. Immunohistochemical analysis on day 9 after ischemia, when a significant increase in cell proliferation was seen, showed that the cerebral vessel space in the subgranular zone of the dentate gyrus had not been affected by the ischemia. Neither were the vascular densities on days 1 and 3 after ischemia altered compared with those of non-operated naïve control rats. Furthermore, the distance from the center of the proliferative cells to the nearest cerebral vessel of ischemic rats was comparable to that of the sham-operated rats. We demonstrated that transient forebrain ischemia-induced cell proliferation and differentiation to mature neurons in the hippocampal dentate gyrus was attenuated by the i.c.v. administration of a vascular endothelial growth factor receptor tyrosine kinase inhibitor. These results suggest that vascular endothelial growth factor receptor at the early period of reperfusion may contribute to neurogenesis rather than to angiogenesis in the hippocampal dentate gyrus.     

降钙素基因相关肽对局灶性脑缺血再灌注大鼠脑cAMP反应元件结合蛋白mRNA表达的影响

目的 探讨外源性降钙素基因相关肽(CGRP)对局灶性脑缺血再灌注大鼠海马和顶叶皮质cAMP反应元件结合蛋白(CREB)mRNA表达的影响.方法 用线栓法制作大鼠局灶性脑缺血再灌注模型,应用原位杂交和图像分析技术检测大鼠缺血侧海马CA1区和顶叶皮质CREB mRNA表达.结果 假手术组右侧海马CAl区和顶叶皮质CREB mRNA有明显表达,缺血再灌注组右侧海马CAl区和顶叶皮质CREB mRNA阳性产物吸光度值减少,CGRP组缺血侧海马CAl区和顶叶皮质CREB mRNA表达的吸光度值比缺血再灌注组增高(P＜0.05).结论 CGRP上调局灶性脑缺血再灌注大鼠海马和顶叶皮质CREB mRNA的表达,CGRP对缺血神经元的保护作用可能通过激活CREB的转录与翻译,从而启动一系列信号通路来实现.     

脑缺血诱导大鼠皮层和海马二价金属离子转运体1表达增加的研究

目的:探讨脑缺血对大鼠皮层、海马二价金属离子转运体1(DMT1)表达的影响。方法:雄性Wistar大鼠随机分为脑缺血1、3、7、28 d和假手术组。结扎双侧颈总动脉建立脑缺血模型组,假手术组仅分离双侧颈总动脉但不结扎。采用RT-PCR测定DMT1+/-IRE mRNA的表达;采用免疫组化染色测定大鼠皮层及海马组织DMT1的表达。结果:大鼠皮层和海马DMT1+/-IRE mRNA的表达随缺血时间的延长逐渐增加。与假手术组比较,皮层DMT1+/-IRE mRNA的表达在缺血1、3 d时无差异(P>0.05);缺血7 d时表达增加(P<0.01),缺血28d时增加更明显(P<0.01)。海马DMT1-IRE mRNA表达除在缺血1 d时与假手术组无差异外(P>0.05),其余时间点DMT1+/-IRE mRNA表达均高于假手术组(P<0.01)。随缺血时间的延长,大鼠皮层、海马的锥体细胞、颗粒细胞及血管内皮细胞DMT1的表达逐渐增加。DMT1的表达除缺血1 d组与假手术组无差别外(P>0.05),其余各组均高于假手术组(P<0.05)。结论:脑缺血可诱导大鼠皮层及海马DMT1表达升高,DMT1表达的改变可能参与了脑缺血引起大鼠脑铁含量升高及神经元铁沉积过程。     

Signaling pathways induced by vascular endothelial growth factor (review)

Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. Angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, while vasculogenesis is the development of new blood vessels from the differentiation of endothelial precursors (angioblasts) in situ. Vascular endothelial growth factor (VEGF) family members are major mediators of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to promote endothelial cell viability, mitogenesis, chemotaxis, and vascular permeability. It mediates its activity mainly via two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), although other receptors, such as neuropilin-1 and -2, can also bind VEGF. Another tyrosine kinase receptor, VEGFR-3 (flt-4) binds VEGF-C and VEGF-D and is more important in the development of lymphatic vessels. While the functional effects of VEGF on endothelial cells has been well studied, not as much is known about VEGF signaling. This review summarizes the different pathways known to be involved in VEGF signal transduction and the biological responses triggered by the VEGF signaling cascade.     

Functions and mechanisms of receptor tyrosine kinase Torso signaling: lessons from Drosophila embryonic terminal development.

The Torso receptor tyrosine kinase (RTK) is required for cell fate specification in the terminal regions (head and tail) of the early Drosophila embryo. Torso contains a split tyrosine kinase domain and belongs to the type III subgroup of the RTK superfamily that also includes the platelet-derived growth factor receptors, stem cell or steel factor receptor c-Kit proto-oncoprotein, colony-stimulating factor-1 receptor, and vascular endothelial growth factor receptor. The Torso pathway has been a model system for studying RTK signal transduction. Genetic and biochemical studies of Torso signaling have provided valuable insights into the biological functions and mechanisms of RTK signaling during early Drosophila embryogenesis.     

血管内皮生长因子及其受体家族在角膜组织及角膜病变中的作用和研究进展

BACKGROUND: Vascular endothelial growth factors are a family of multifunctional cytokines that can enhance vascular permeability, induce angiogenesis, promote endothelial cell growth and migration, and inhibit cell apoptosis. OBJECTIVE: To elaborate the latest progress in the role of vascular endothelial growth factor and its receptors in the corneal tissue. METHODS: A computer-based search of PubMed databases was performed for relevant articles published from 2005 to 2015. The key words were “vascular endothelial growth factor, cornea”. According to the inclusion and exclusion criteria, 43 articles were included in result analysis. RESULTS AND CONCLUSION: Vascular endothelial growth factor and its receptors are involved in the regulation of corneal neovascularization by causing Tip cell activation that affects the Notch signaling pathways. Corneal lymphatic regeneration mainly relies on macrophages to secrete vascular endothelial growth factor-C or vascular endothelial growth factor-D that further activate vascular endothelial growth factor receptor-3 in the lymphatic endothelial cells to cause cell proliferation and migration, and eventually lead to the formation of new lymphatic vessels. But herpes simplex keratitis HSK induces the corneal lymphatic regeneration by vascular endothelial growth factor-A/vascular endothelial growth factor receptor-2 pathway. Vascular endothelial growth factor family can significantly improve the damaged corneal nerve endings, epithelium and corneal sensitivity, has the function of nerve nutrition and promote restoration of the corneal epithelium.       

CGRP和NGF对全脑缺血再灌注大鼠脑组织PKC表达的调节作用

目的　研究大鼠脑缺血再灌注后蛋白激酶C(proteinkinaseC ,PKC)在海马和顶叶皮质的表达 ,探讨降钙素基因相关肽 (CGRP)和神经生长因子 (NGF)对脑组织缺血再灌注的保护作用及机制。方法　采用颈动脉负压分流法制作大鼠脑缺血再灌注模型 ,采用免疫组织化学SABC法及显微图像分析检测海马及皮质内PKC的表达。结果　大鼠缺血再灌注海马CA1区及顶叶皮质内PKC阳性产物平均灰度值较正常组低 (P <0 .0 5 ) ,注射CGRP或NGF后PKC阳性产物平均灰度值明显高于缺血再灌注组 (P <0 .0 1) ,二者联合应用时平均灰度值比单独应用高 (P <0 .0 1)。结论　CGRP及NGF参与缺血神经元PKC的调节 ,二者对缺血神经元有协同保护作用