依达拉奉对大鼠星形胶质细胞水通透性的影响

目的 探讨依达拉奉对缺氧复氧大鼠星形胶质细胞模型水通透性和水通道蛋白4（AQP4）的影响.方法 原代培养大鼠星形胶质细胞,通过95％（体积分数）N2培养8h和95％（体积分数）的空气复氧培养6h,构建缺氧复氧水肿模型.设置空白组（正常细胞）、对照组（水肿细胞未经受药物刺激）和刺激组（接受0.1、1、10或100μg/mL的依达拉奉溶液刺激）.采用细胞水通透性系数（Pd）检测、荧光定量PCR和Western Blot定量空白组、对照组和刺激组细胞水肿、AQP4 mRNA AQP4蛋白表达.结果 缺氧8h和复氧6h后细胞水肿,且AQP4表达量上升.0.1μg/mL依达拉奉刺激后,模型细胞水通量、AQP4 mRNA水平和蛋白表达量与未刺激过的水肿星形胶质细胞比较,差异无统计学意义（P均＞0.05）,1、10或100μg/mL依达拉奉刺激后,水肿星形胶质细胞水通量、AQP4 mRNA水平和蛋白表达量显著低于未刺激过的水肿星形胶质细胞（P均＜0.05）,与正常的星形胶质细胞比较,差异无统计学意义（P均＞0.05）.结论 1、10或100 μg/mL依达拉奉可通过抑制AQP4 mRNA水平和蛋白表达,从而抑制细胞水肿.     

水通道蛋白4参与抑郁症发病机制研究进展

大脑具有高阶功能,由几种细胞组成,如神经元和神经胶质细胞。星形胶质细胞是一种神经胶质细胞。水通道蛋白4(AQP4)是一种调节水渗透性的膜结合蛋白,是水通道蛋白家族的成员,其在中枢神经系统(CNS)的星形胶质细胞的末端表达。最近,AQP4已被证明不仅可作为水通道蛋白,调节星形胶质细胞的各种生物学功能,例如水转运、钾空间缓冲、钙波传递、谷氨酸盐稳态和铁传递。而且还可作为通过长时程增强或长时程抑制参与神经兴奋和突触可塑性。尸检结果发现,抑郁症患者脑体积缩小,脑中AQP4表达减少;敲除AQP4会加重皮质酮诱发小鼠抑郁模型的症状,其机制与损伤星形胶质细胞功能和海马神经发生有关;AQP4与氟西汀治疗抑郁症过程中所表现的症状改善和神经发生增强有关;AQP4与部分难治性抑郁的发病机制相关。所以AQP4作为治疗抑郁症的新靶点值得进一步关注。     

体外缺氧／复氧对星形胶质细胞AQP4表达的影响及葛根素干预的实验研究

目的 观察缺氧,复氧条件下星形胶质细胞形态和AQP4 mRNA的表达变化以及葛根素对其表达变化的影响,探讨脑缺血再灌注损伤与AQP4的关系以及葛根索的干预作用.方法 原代培养星形胶质细胞,用5%CO2+95%N2混合气体造成缺氧,以LDH漏出率及MTT降解率作为细胞受损指标,应用RT-PCR技术检验星形胶质细胞缺氧/复氧各个时间点AQP4 mRNA的表达变化及葛根素的干预效果.结果 体外培养的星形胶质细胞在缺氧环境下损伤不明显,随着复氧时间的延长细胞损害加重.AQP4 mRNA在缺氧时表达与正常对照组无明显差异,复氧后表达升高并随时间延长呈增高趋势(P＜0.05).葛根素干预组AQP4 mRNA表达丰度与缺氧/复氧组无明显差异(P＞0.05).结论 星形胶质细胞AQP4 mRNA表达变化与细胞损伤有明显的相关性,葛根素对星形胶质细胞损伤的保护作用不是通过改变AQP4的表达来实现的.     

水通道蛋白4在小鼠脑老化中的作用

水通道蛋白4(AQP4)主要分布在星形胶质细胞足突膜上,是脑内最重要的水通道亚型。已经发现,AQP4除参与调节脑内水平衡外,还参加学习记忆、脑外伤、脑缺血、脑肿瘤等一系列生理、病理过程。脑老化伴随着学习记忆下降,以及易发生脑缺血、脑肿瘤等老年相关性疾病,但AQP4在脑老化中的作用尚未见报道。目的观察AQP4在小鼠脑老化中的作用。方法对年轻(2～3个月)和老年(17～19个月)AQP4基因敲除小鼠(AQP4-/-)与野生型相应年龄的小鼠(AQP4+/+),观察AQP4基因敲除对老年小鼠自发活动、学习记忆、大体脑形态以及神经元、星形胶质细胞、小胶质细胞密度等的影响。结果老年小鼠自发活动减少,在穿梭箱被动避暗实验中,测试时进入暗箱的潜伏期明显短于年轻鼠,但AQP4+/+和AQP4-/-小鼠间无明显差异。甲苯胺蓝染色显示,老年小鼠皮层神经元密度(Ⅲ～Ⅳ层)显著低于年轻小鼠(P<0.05),海马CA1区厚度无明显差异,AQP4+/+和AQP4-/-小鼠间无明显差异。免疫组化显示,老年小鼠海马CA1的星形胶质细胞(GFAP阳性)数量明显高于年轻小鼠(P<0.01),而小胶质细胞数量在老年和年轻小鼠间无明显差异。免疫荧光显示,老年小鼠星形教胶质细胞的面积变小,而AQP4-/-小鼠的星形胶质细胞突起少而短、细胞面积明显小于AQP+/+小鼠(P<0.05),神经元和小胶质细胞形态无明显变化。结论 AQP4参与脑老化过程中的星形胶质细胞变化,而与运动、学习记忆相关的脑功能及神经元、小胶质细胞等脑结构变化无明显关系。     

谷氨酸引起星形胶质细胞核形态改变

谷氨酸是中枢神经系统中最主要的兴奋性神经递质,参与脑细胞问兴奋性信息的传递。星形胶质细胞表面丰富的谷氨酸转运蛋白可以有效清除细胞外谷氨酸,以终结谷氨酸的信号传递作用,但谷氨酸摄取的潜在生物学功能尚不明确。本研究首次证明谷氨酸进入星形胶质细胞后,直接引起细胞核发生形态改变。细胞核形态改变是谷氨酸的特异反应,与细胞外谷氨酸浓度密切相关。细胞膜表面的谷氨酸转运蛋白直接参与细胞核形态调节,而与离子型、代谢型谷氨酸受体,细胞内钙离子浓度改变无关。我们进一步证实水通道蛋白-1（AQP1）分布于星形胶质细胞核膜表面,并直接参与了谷氨酸引起的核形态调节。本项目证明细胞外谷氨酸通过转运蛋白和AQP1直接调节星形胶质细胞核形态,提示神经元兴奋性调节细胞核形态功能的潜在机制。     

体外培养新生大鼠星形胶质细胞在缺氧缺血损伤时β1整合素的表达变化

目的 了解缺氧缺血损伤时星形胶质细胞分泌β1整合素的变化.方法 采用免疫激光共聚集的方法检测体外培养的星形胶质细胞在缺氧缺血不同时段β1整合素的表达.结果 星形胶质细胞缺氧缺血12h、24h β1整合素表迭减少,细胞逐渐凋亡.结论 缺氧缺血损伤时星形胶质细胞合成β1整合素减少,可破坏细胞正常结构形态.     

吡拉格雷钠对氧糖剥夺/复氧后星形胶质细胞水肿及AQP4表达的影响北大核心CSCD

目的探讨氧糖剥夺并复氧培养后星形胶质细胞水肿及其水通道蛋白4(AQP4)表达的变化以及吡拉格雷(TSF)对其表达变化的影响。方法体外培养原代星形胶质细胞,建立氧糖剥夺/复氧细胞水肿模型,并随机分为正常组、氧糖剥夺/复氧损伤(OGD/Reox)组、奥扎格雷钠(Ozagrel)阳性对照组和TSF治疗组,在氧糖剥夺/复氧损伤后6、12、24和48 h 4个时间点测定细胞体积变化,乳酸脱氢酶(LDH)漏出率、MTT法检测细胞损伤及存活情况,Western blot法检测各组细胞膜AQP4蛋白的表达水平,RT-PCR法检测各组细胞AQP4 m RNA的转录水平。结果星形胶质细胞经缺氧再复氧处理,随着复氧时间的延长细胞损害加重(P<0.05,P<0.01);TSF治疗组的细胞体积明显低于OGD/Reox损伤组(P<0.05);给予TSF治疗后细胞在氧糖剥夺/复氧后的LDH漏出率较OGD/Reox损伤组明显降低(P<0.05);TSF治疗组与单纯氧糖剥夺/复氧相比较MTT检测细胞活力明显升高(P<0.05);给予TSF治疗组,AQP4表达明显降低(P<0.05);与Ozagrel相比较差异无显著性。Western blot及RT-PCR检测蛋白及m RNA的表达水平,给予TSF治疗组的AQP4蛋白及m RNA表达均明显降低(P<0.05)。结论TSF治疗能明显减轻体外氧糖剥夺/复氧损伤引起的星形胶质细胞水肿,可能是通过降低氧糖剥夺后AQP4的表达水平而实现。     

吡拉格雷钠对氧糖剥夺/复氧后星形胶质细胞水肿及AQP4表达的影响

目的探讨氧糖剥夺并复氧培养后星形胶质细胞水肿及其水通道蛋白4(AQP4)表达的变化以及吡拉格雷(TSF)对其表达变化的影响。方法体外培养原代星形胶质细胞,建立氧糖剥夺/复氧细胞水肿模型,并随机分为正常组、氧糖剥夺/复氧损伤(OGD/Reox)组、奥扎格雷钠(Ozagrel)阳性对照组和TSF治疗组,在氧糖剥夺/复氧损伤后6、12、24和48 h 4个时间点测定细胞体积变化,乳酸脱氢酶(LDH)漏出率、MTT法检测细胞损伤及存活情况,Western blot法检测各组细胞膜AQP4蛋白的表达水平,RT-PCR法检测各组细胞AQP4 m RNA的转录水平。结果星形胶质细胞经缺氧再复氧处理,随着复氧时间的延长细胞损害加重(P<0.05,P<0.01);TSF治疗组的细胞体积明显低于OGD/Reox损伤组(P<0.05);给予TSF治疗后细胞在氧糖剥夺/复氧后的LDH漏出率较OGD/Reox损伤组明显降低(P<0.05);TSF治疗组与单纯氧糖剥夺/复氧相比较MTT检测细胞活力明显升高(P<0.05);给予TSF治疗组,AQP4表达明显降低(P<0.05);与Ozagrel相比较差异无显著性。Western blot及RT-PCR检测蛋白及m RNA的表达水平,给予TSF治疗组的AQP4蛋白及m RNA表达均明显降低(P<0.05)。结论TSF治疗能明显减轻体外氧糖剥夺/复氧损伤引起的星形胶质细胞水肿,可能是通过降低氧糖剥夺后AQP4的表达水平而实现。     

水通道蛋白-依赖性细胞迁移的研究进展

水通道蛋白(AQPs)是一类介导跨膜水运输的内在蛋白。近年来,一些研究成果证实,AQP具有介导细胞迁移的功能。该文综述了各种不同类型的AQPs-依赖性细胞迁移(AQPs-dependent cell migration):AQP1促进内皮细胞迁移;AQP4促进星形胶质细胞迁移;AQP3促进角膜上皮细胞、肠上皮细胞、皮肤角质形成细胞的迁移。此外,AQPs还能促进肿瘤细胞的迁移,增加肿瘤细胞的转移潜能。AQPs介导的细胞迁移与许多疾病有着内在联系,调控AQP的表达是一种尚待开发的治疗手段。     

水通道蛋白4缺失对丙泊酚催眠效应的影响

<正>水通道蛋白(aquaporins,AQPs)是一类广泛存在于真核生物细胞膜上、选择性高效转运水分子的特异孔道,对机体水液平衡和细胞微环境的稳定发挥重要调节作用[1]。其中,星形胶质细胞及广泛表达其上的AQP4还通过影响神经递质的释放,参与调节中枢神经传导[2]。本研究旨在通过对     

Aquaporin-4: orthogonal array assembly, CNS functions, and role in neuromyelitis optica

Aquaporin-4 (AQP4) is a water-selective transporter expressed in astrocytes throughout the central nervous system, as well as in kidney, lung, stomach and skeletal muscle. The two AQP4 isoforms produced by alternative spicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular structures called orthogonal arrays of particles (OAPs). Phenotype analysis of AQP4-null mice indicates the involvement of AQP4 in brain and spinal cord water balance, astrocyte migration, neural signal transduction and neuroinflammation. AQP4-null mice manifest reduced brain swelling in cytotoxic cerebral edema, but increased brain swelling in vasogenic edema and hydrocephalus. AQP4 deficiency also increases seizure duration, impairs glial scarring, and reduces the severity of autoimmune neuroinflammation. Each of these phenotypes is likely explicable on the basis of reduced astrocyte water permeability in AQP4 deficiency. AQP4 is also involved in the neuroinflammatory demyelinating disease neuromyelitis optica (NMO), where autoantibodies (NMO-IgG) targeting AQP4 produce astrocyte damage and inflammation. Mice administered NMO-IgG and human complement by intracerebral injection develop characteristic NMO lesions with neuroinflammation, demyelination, perivascular complement deposition and loss of glial fibrillary acidic protein and AQP4 immunoreactivity. Our findings suggest the potential utility of AQP4-based therapeutics, including small-molecule modulators of AQP4 water transport function for therapy of brain swelling, injury and epilepsy, as well as small-molecule or monoclonal antibody blockers of NMO-IgG binding to AQP4 for therapy of NMO.     

Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression

Brain edema is a common and serious complication of ischemic stroke with limited effective treatment.We previously reported that methylene blue(MB)attenuated ischemic brain edema in rats,but the underlying mechanisms remained unknown.Aquaporin 4(AQP4)in astrocytes plays a key role in brain edema.We also found that extracellular signal-regulated kinase 1/2(ERK1/2)activation was involved in the regulation of AQP4 expression in astrocytes.In the present study,we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema.Rats were subjected to transient middle cerebral artery occlusion(tMCAO),MB(3 mg/kg,for 30 min)was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia(1.5 mg/kg,for 15 min).Brain edema was determined by MRI at 0.5,2.5,and 48 h after tMCAO.The decreases of apparent diffusion coefficient(ADC)values on diffusion-weighted MRI indicated cytotoxic brain edema,whereas the increase of T2 MRI values reflected vasogenic brain edema.We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO.In addition,MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO.In a cell swelling model established in cultured rat astrocyte exposed to glutamate(1 mM),we consistently found that MB(10μM)attenuated cell swelling,AQP4 increases and ERK1/2 activation.Moreover,the ERK1/2 inhibitor U0126(10μM)had the similar effects as MB.These results demonstrate that MB improves brain edema and astrocyte swelling,which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway,suggesting that MB may be a potential choice for the treatment of brain edema.     

Down-regulation of aquaporin 4 in human placenta throughout pregnancy

BACKGROUND: The family of mammalian aquaporins (AQP) consists of 12 known members, each with a specific tissue distribution and membrane localization pattern. AQP4 is the first member of this family identified in biological membranes. This water channel protein is primarily expressed in astrocytes but is also localized in ependymocytes and endothelial cells, suggesting its involvement in the movement of water between the blood and brain, and between the brain and cerebrospinal fluid (CSF). To date, the regulation of AQP4 expression in the human placenta has not been studied. The purpose of this work was to investigate AQP4 localization and expression in the human placenta during gestation. MATERIALS AND METHODS: A total of 30 samples, 15 full-term placentae and 15 chorionic villous samples from first trimester, for the immunohistochemical analysis of AQP4 expression were used. The gestation period ranged from 5 to 40 weeks. RESULTS: A decrease of AQP4 expression in the syncytiotiophoblast from the first to the third trimester of gestation, in contrast with an increased expression shown by endothelial cells and stroma of placental villi was found. CONCLUSION: Our results may suggest that AQP4-mediated maternal-fetal fluid exchange could play an important role in the control of ion homeostasis and water balance in the human placenta throughout pregnancy.     

Aquaporin water channels and brain edema

Brain edema accounts for much of the morbidity and mortality associated with common neurological conditions such as head trauma, brain tumors, stroke and liver failure. Treatment options are limited to osmotic agents such as mannitol, surgical decompression, and other maneuvers, none of which correct the molecular-level mechanisms responsible for brain swelling. Recent data suggest that aquaporin (AQP) water-transporting proteins may provide a key route for water movement in the brain. AQP1 is expressed in choroid plexus and probably facilitates cerebrospinal fluid secretion. AQP4 is expressed in astrocyte foot processes near capillaries and in ependymal cells lining the ventricles -- key sites for water movement between the cellular, vascular, and ventricular compartments. AQP4 expression is markedly altered in experimental models of brain injury and swelling, and transgenic mice lacking AQP4 are partially protected from brain swelling in response to acute hyponatremia and ischemic stroke. Aquaporins and regulators of brain aquaporin expression are thus potential targets for discovery of compounds for treatment of brain swelling.     

Functional domains of aquaporin-1: keys to physiology, and targets for drug discovery

Aquaporins (AQPs) are expressed in physiologically essential tissues and organs in which edema and fluid imbalances are of major concern. Potential roles in brain water homeostasis and edema, angiogenesis, cell migration, development, neuropathological diseases, and cancer suggest that this family of membrane proteins is an attractive set of novel drug targets. A problem in pursuing therapeutic and basic research strategies for dissecting contributions of AQPs to cell and tissue functions is that little is known regarding the pharmacology of AQP channels; currently defined agents such as tetraethylammonium and phloretin as blockers for aquaporins suffer from a lack of specificity and potency. Subtypes of AQPs modulated by signaling pathways could enable discrete localized control of fluid homeostasis, volume and morphology in cells and intracellular organelles, and might be found to participate in many different aspects of physiology, such as the control of paracellular permeability, process extension, growth, migration, and other responses involving changes in cell shape or surface to volume ratios. Recognizing that AQP1 is a water channel and, under permissive conditions, also a cGMP-gated cation channel, evidence in various tissues for a coupling of the cGMP signaling cascade to a physiological outcome that might involve AQP1 dual ion-and-water channel functions is of interest. Groundbreaking advances in defining aquaporin gating mechanisms suggest conformational changes are important elements in regulation and gating across classes of aquaporins. With a rapidly expanding knowledge of aquaporin structure and functional regulation, new avenues for manipulation of aquaporin channels are likely to be discovered. In parallel, a discovery for novel compounds with specificity and potency for aquaporins is a compelling goal. The need for pharmacological agents to dissect the roles of aquaporins in physiological and pathological processes is a clear call for further research in the field.     

Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.     

Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy

Autophagy is a highly regulated cellular mechanism that leads to degradation of long-lived proteins and dysfunctional organelles. The process has been implicated in a variety of physiological and pathological conditions relevant to neurological diseases. Recent studies show the existence of autophagy in cerebral ischemia, but no consensus has yet been reached regarding the functions of autophagy in this condition. This article highlights the activation of autophagy during cerebral ischemia and/or reperfusion, especially in neurons and astrocytes, as well as the role of autophagy in neuronal or astrocytic cell death and survival. We propose that physiological levels of autophagy, presumably caused by mild to modest hypoxia or ischemia, appear to be protective. However, high levels of autophagy caused by severe hypoxia or ischemia and/or reperfusion may cause self-digestion and eventual neuronal and astrocytic cell death. We also discuss that oxidative and endoplasmic reticulum (ER) stresses in cerebral hypoxia or ischemia and/or reperfusion are potent stimuli of autophagy in neurons and astrocytes. In addition, we review the evidence suggesting a considerable overlap between autophagy on one hand, and apoptosis, necrosis and necroptosis on the other hand, in determining the outcomes and final morphology of damaged neurons and astrocytes.     

Aquaporins as potential drug targets

The aquaporins (AQP) are a family of integral membrane proteins that selectively transport water and, in some cases, small neutral solutes such as glycerol and urea. Thirteen mammalian AQP have been molecularly identified and localized to various epithelial, endothelial and other tissues. Phenotype studies of transgenic mouse models of AQP knockout, mutation, and in some cases humans with AQP mutations have demonstrated essential roles for AQP in mammalian physiology and pathophysiology, including urinary concentrating function, exocrine glandular fluid secretion, brain edema formation, regulation of intracranial and intraocular pressure, skin hydration, fat metabolism, tumor angiogenesis and cell migration. These studies suggest that AQP may be potential drug targets for not only new diuretic reagents for various forms of pathological water retention, but also targets for novel therapy of brain edema, inflammatory disease, glaucoma, obesity, and cancer. However, potent AQP modulators for in vivo application remain to be discovered.     

脑水肿分子机制的研究进展

脑水肿是指脑内水分增加导致脑容积增大的一种病理现象,是脑组织对各种致病因素的反应。颅内损伤、缺血、缺氧、炎症、脑代谢障碍、肿瘤以及中毒都会引起脑水肿。脑水肿可导致颅内压的升高,当颅内压升高到一定程度时,脑组织就会发生功能和结构的损害,严重者导致脑死亡。先前对脑水肿发病机制的研究包括血脑屏障学说、钙离子学说、脑微循环障碍学说、脑细胞代谢障碍等。但是近年的研究表明脑水肿的发生与水通道蛋白4、基质金属蛋白酶、紧密连接蛋白、炎性细胞因子等密切相关。本文就脑水肿发生的分子机制进行综述。