血管内皮生长因子对出血性脑水肿的保护与可能的机制：水通道蛋白4的作用
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摘要
脑出血(intracerebral hemorrhage, ICH)是一种非常严重的脑卒中类型,常导致患者严重的神经功能缺失,预后不良。产生这些后果与出血后脑水肿形成关系密切。血管内皮生长因子(vascular endothelial growth factor, VEGF)对多种神经系统疾病具有保护作用,包括ICH,但VEGF对ICH后脑水肿尚无研究。我们前期的研究证实,水通道蛋白4(aquaporin-4, AQP4)作为脑内含量最多的水通道蛋白,对ICH后脑水肿的清除起着重要作用。而且已有研究表明,两者可在血脑屏障(blood-brain barrier, BBB)破坏的星形胶质细胞终足上共表达,且脑内注入VEGF可诱导AQP4表达增加。既然两者之间有着密切联系,我们推测VEGF对ICH后脑水肿的影响可能与调节AQP4的表达有关。本课题首先探究侧脑室注入重组人VEGF165(recombinant human VEGF165, rhVEGF165)对野生小鼠ICH后神经功能缺损评分、脑组织含水量、BBB通透性和细胞凋亡的作用,以及对正常状态和ICH后脑组织AQP4表达的影响,然后通过与AQP4基因敲除小鼠比较,探讨rhVEGF165对ICH的上述作用是否与调节AQP4的表达相关。并进一步通过星形胶质细胞的离体培养,研究rhVEGF165影响AQP4表达可能的信号转导通路。本实验结果为VEGF下游信号通路、AQP4表达的调节及VEGF对ICH后脑水肿的影响机制提供理论依据,同时为治疗ICH后脑水肿提供双重作用靶点,可能为新药的研制提供新的思路。
第一部分：VEGF对ICH后脑水肿的作用
目的：研究侧脑室注入rhVEGF165对野生小鼠ICH后脑水肿的作用及脑组织AQP4蛋白表达的影响。
方法：将CD1小鼠随机分为对照组、对照+VEGF(3μg/kg)组、假手术组、ICH组、ICH+VEGF组和ICH+VEGFR抑制剂SU5416(60μg/kg)组,在干预后3d对前5组进行神经功能缺损评分、干湿重法测定脑组织含水量、伊文思蓝(Evans blue, EB)法测定BBB通透性、TUNEL染色法评价细胞凋亡。并对各组用Western blot法和免疫荧光法检测正常纹状体或血肿周围AQP4蛋白表达。
结果：1.侧脑室注入rhVEGF165在野生小鼠ICH后可显著减少神经功能缺损评分(p<0.05),减少脑组织含水量(p<0.01),减少血肿周围TUNEL阳性细胞数量(p<0.01),但不影响脑组织EB渗漏。2.Western blot结果显示,正常状态下侧脑室注入rhVEGF165可使纹状体AQP4蛋白表达明显增加(p<0.01)。ICH后侧脑室注入rhVEGF165也可使血肿周围AQP4蛋白表达增加(p<0.01),而给予SU5416可使血肿周围AQP4蛋白表达减少(p<0.01)。免疫荧光法显示AQP4蛋白的荧光表达数量及强度与Western blot结果相一致。
结论：1.侧脑室注入rhVEGF165可在野生小鼠ICH后减轻脑水肿,改善神经功能,减轻细胞凋亡,但不影响BBB通透性。2.侧脑室注入rhVEGF165可上调ICH后血肿周围AQP4蛋白表达。ICH后AQP4蛋白表达增加可能与内源性VEGF有关。
第二部分：AQP4在VEGF对ICH后脑水肿效应中的作用
目的：研究侧脑室注入rhVEGF165对AQP4基因敲除小鼠ICH后脑水肿的作用,并通过AQP4+/+和AQP4-/-小鼠的对比,探讨rhVEGF165对ICH后脑水肿作用是否与调节AQP4的表达相关。
方法：将AQP4-/-小鼠随机分为对照组、对照+VEGF组、假手术组、ICH组和ICH+VEGF组。在干预后3d对各组进行神经功能缺损评分、干湿重法测定脑组织含水量、EB法测定BBB通透性、TUNEL染色法评价细胞凋亡。
结果：1.ICH后AQP4-/-小鼠神经功能缺损较AQP4+/+小鼠严重(p<0.05),侧脑室注入rhVEGF165可减轻AQP4-/-小鼠神经功能缺损(p<0.05),但仍较AQP4+/+小鼠严重(p<0,05)。且rhVEGF165降低ICH后AQP4+/+小鼠神经功能缺损评分的百分比显著高于AQP4-/-小鼠(p<0.01)。2.正常状态下侧脑室注入rhVEGF165可增加AQP4-/-小鼠脑组织含水量(p<0.01)。ICH后AQP4-/-小鼠脑水肿较AQP4+/+小鼠严重(p<0.05),侧脑室注入rhVEGF165不影响AQP4-/-小鼠脑组织含水量。3.正常状态下侧脑室注入rhVEGF165可使AQP4-/-小鼠EB渗漏增加(p<0.01),且较AQP4+/+小鼠更为严重(p<0.01)。ICH后AQP4-/-小鼠脑组织EB含量较AQP4+/+小鼠增多(p<0.05),侧脑室注入rhVEGF165可使AQP4-/-小鼠EB渗漏显著增加(p<0.05)。4.ICH后AQP4-/-小鼠血肿周围TUNEL阳性细胞数高于AQP4+/+小鼠(p<0.01),侧脑室注入rhVEGF165可减少AQP4-/-小鼠TUNEL阳性细胞数(p<0.01),但仍高于AQP4+/+小鼠(p<0.01)。且rhVEGF165使ICH后AQP4+/+小鼠TUNEL阳性细胞数降低的百分比显著高于AQP4-/-小鼠(p<0.01)。
结论：1.ICH后AQP4表达增加可对神经功能、脑水肿、BBB完整性及细胞凋亡均起到保护作用。2.上调AQP4表达是rhVEGF165减轻ICH后神经功能缺损的机制之一。3.正常状态下AQP4有清除VEGF潜在引起脑水肿的作用。rhVEGF165减轻ICH后脑水肿的作用可能与增加AQP4表达,从而加强脑水肿的清除有关。4.正常状态下rhVEGF165有破坏BBB的效应,AQP4基因敲除可加重该效应。ICH后,rhVEGF165对BBB可能同时具有保护和破坏的作用,其保护作用可能与增加AQP4表达有关。5.上调AQP4表达是rhVEGF165减少ICH后血肿周围凋亡细胞数量的机制之一。
第三部分：VEGF影响AQP4表达的信号转导通路
目的：探讨rhVEGF165引起AQP4蛋白表达增高可能的信号转导通路。
方法：将传代并鉴定后的星形胶质细胞随机分为正常对照组、VEGF (50ng/ml)组、VEGF+JNK抑制剂SP600125(20μM)组、VEGF+ERK抑制剂U0126(10μM)组、VEGF+p38-MAPK抑制剂SB239063(20μM)组、VEGF+PI3K抑制剂Ly294002(20μM)组。分别在加药后2d用Western blot和免疫荧光法检测各组AQP4蛋白的表达。
结果：1.Western blot检测显示：rhVEGF165可使星形胶质细胞AQP4蛋白表达增加(p<0.01),该效应可被SP600125和U0126抑制(p<0.01),但不能被SB239063或Ly294002所抑制。2.免疫荧光法显示AQP4蛋白的荧光表达数量及强度与Western blot结果相一致。
结论：1.rhVEGF165可使离体培养的星形胶质细胞AQP4蛋白表达增加。2. rhVEGF165可能通过激活JNK和ERK通路,而非通过激活p38-MAPK或PI3K通路使AQP4蛋白表达增加。
Intracerebral hemorrhage (ICH) is a severe stroke type, which always leads to severe neurological deficits and poor prognosis. Brain edema formation after ICH is one of the major causes of these consequences. Vascular endothelial growth factor (VEGF) has protective effects on various neurological diseases, including ICH. But no related research on how VEGF acting on brain edema after ICH has been reported. Our previous study indicated that as the most abundant water channel in brain, aquaporin-4(AQP4) played an important role in edema resolution after ICH. In addition, it was reported that VEGF and AQP4colocalized on astrocyte processes after cerebral hypoxia and blood-brain barrier (BBB) disruption and intracerebral VEGF injection can highly up-regulate AQP4mRNA and protein. Since there is close connection between them, we speculated that the effect of VEGF on brain edema following ICH may result from regulating AQP4expression. We first studied the effects of recombinant human VEGF165(rhVEGF165) to wild type mice on neurological deficits, brain water contents, BBB permeability and cell apoptosis after ICH and the influence of rhVEGF165on AQP4expression in normal state and ICH. We also compared the parameters between AQP4+/+and AQP4-/-mice to indicate whether the above roles played by rhVEGF165resulted from regulating AQP4expression. We further explored the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression through astrocyte cultures. Our results can provide a theoretical basis to downstream signal transduction pathways of VEGF, regulation of AQP4expression and the mechanisms involved in the effects of VEGF on ICH. Double targets can be supplied to treatment of brain edema following ICH, which can present new insights for new drugs development.
Part Ⅰ Effect of VEGF on brain edema after ICH
Objective:To study the effect of intracerebroventricular rhVEGF165injection on brain edema after ICH and its influence on AQP4expression in normal state and ICH.
Methods:CD1mice were randomly divided into six groups:control, control plus VEGF (3μg/kg), sham operation, ICH, ICH plus VEGF, ICH plus SU5416(60μg/kg), a kind of VEGFR inhibitor. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of Evans blue (EB) and evaluated cell apoptosis through TUNEL staining in the first5groups3d after drug given. We also detected the expression of AQP4protein at striatum or perihemotoma using Western blot and immunofluorescence in each group at the same time point.
Results:1. Intracerebroventricular rhVEGF165injection significantly reduced neurological deficits (p<0.05), brain water contents (p<0.01) and TUNEL-positive cells (p<0.01) perihemotoma in AQP4+/+mice, but had no change of EB extravasation.2. Western blot showed an increase of AQP4protein at striatum (p<0.01) after rhVEGF165injected intracerebroventricularly. Furthermore, AQP4protein expression perihemotoma was increased by intracerebroventricular injection of rhVEGF165after ICH (p<0.01), but was decreased by SU5416(p<0.01). The intensity and number of fluorescence expression were in accord with Western blot.
Conclusion:1. RhVEGF165injected intracerebroventricularly can reduce brain edema, alleviate neurological deficits and reduce apoptotic cells in AQP4+/+mice after ICH, but has no influence on BBB permeability.2. Intracerebroventricular rhVEGF165injection can increase AQP4protein expression perihemotoma after ICH. Endogenous VEGF may be involved in AQP4protein up-regulation after ICH.
Part Ⅱ Roles played by AQP4in effects of VEGF to brain edema following ICH
Objective:To investigate the effects of rhVEGF165to AQP4-/-mice on brain edema after ICH. We also studied whether the above roles played by rhVEGF165resulted from regulating AQP4expression by comparison of AQP4+/+and AQP4-/-mice.
Methods:AQP4-/-mice were randomly divided into five groups:control, control plus VEGF, sham operation, ICH, ICH plus VEGF. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of EB and evaluated cell apoptosis through TUNEL staining.
Results:1. Neurological deficits were more severe in AQP4-/-mice than AQP4+/+mice after ICH (p<0.05). Injection of rhVEGF165intracerebroventricularly alleviated neurological deficits of AQP4-/-mice (p<0.05), but the difference between them was the same as that mentioned above (p<0.05). Moreover, the percentage of neurological deficits decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice (p<0.01).2. An increase of brain water contents in normal AQP4-/-mice after intracerebroventricular injection of rhVEGF165was observed (p<0.01). AQP4-/-mice had more severe brain edema after ICH than AQP4+/+mice (p<0.05). Injection of VEGF intracerebroventricularly had no effect on AQP4-/-mice.3. The amount of EB extravasation was markedly increased by intracerebroventricular rhVEGF165injection in AQP4-/-mice (p<0.01), which was much more than AQP4+/+mice (p<0.01). Injection of VEGF intracerebroventricularly after ICH developd an increased effect in AQP4-/-mice (p<0.05).4. There were more TUNEL-positive cells found perihemotoma after ICH in AQP4-/-mice than AQP4+/+mice (p<0.01). RhVEGF165injected intracerebroventricularly reduced TUNEL-positive cells in AQP4-/-mice (p<0.01), but the difference between them was the same as that mentioned above (p<0.01). In addition, the percentage of TUNEL-positive cells decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice(p<0.01).
Conclusion:1. Increase of AQP4expression after ICH may have protective effects on neurological functions, brain edema, BBB integrity and cell apoptosis.2. Up-regulation of AQP4expression is one of the possible mechanisms by which rhVEGF165significantly alleviates neurological deficits after ICH.3. AQP4has an effect in normal state on eliminating brain edema potentially induced by VEGF. However, rhVEGF165can reduce brain edema following ICH, which has close relationship with enhancement of edema clearance by AQP4up-regulation induced by rhVEGF165.4. RhVEGF165can damage BBB in normal mice, which can be worsened by AQP4knock-out. The effect of rhVEGF165on BBB after ICH can be both protective and destructive and increase of AQP4expression may participate in the positive aspect.5. Apoptotic cells perihemotoma after ICH can be markedly reduced by exogenous rhVEGF165. Up-regulating AQP4expression is one of the possible mechanisms.
Part Ⅲ The signal transduction pathways through which VEGF regulates AQP4expression
Objective:To explore the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression.
Methods:The subcultured and determined astrocytes were randomly divided into six groups:control; VEGF (50ng/ml); VEGF plus SP600125(20μM), a kind of JNK inhibitor; VEGF plus U0126(10μM), a kind of ERK inhibitor; VEGF plus SB239063(20μM), a kind of p38-MAPK inhibitor; VEGF plus Ly294002(20μM), a kind of PI3K inhibitor. We detected the expression of AQP4protein using Western blot and immunofluorescence2d after drug given.
Results:1. Western blot showed that rhVEGF165increased AQP4protein expression of astrocytes (p<0.01), which was inhibited by SP600125and U0126(p<0.01), but not SB239063or Ly294002.2. The intensity and number of fluorescence expression were in accord with Western blot.
Conclusion:1. RhVEGF165can make cultured astrocytes increase expression of AQP4.2. RhVEGF165up-regulates AQP4expression via activating JNK and ERK pathways, but not p38-MAPK or PI3K pathways.
第一部分：VEGF对ICH后脑水肿的作用
目的：研究侧脑室注入rhVEGF165对野生小鼠ICH后脑水肿的作用及脑组织AQP4蛋白表达的影响。
方法：将CD1小鼠随机分为对照组、对照+VEGF(3μg/kg)组、假手术组、ICH组、ICH+VEGF组和ICH+VEGFR抑制剂SU5416(60μg/kg)组,在干预后3d对前5组进行神经功能缺损评分、干湿重法测定脑组织含水量、伊文思蓝(Evans blue, EB)法测定BBB通透性、TUNEL染色法评价细胞凋亡。并对各组用Western blot法和免疫荧光法检测正常纹状体或血肿周围AQP4蛋白表达。
结果：1.侧脑室注入rhVEGF165在野生小鼠ICH后可显著减少神经功能缺损评分(p<0.05),减少脑组织含水量(p<0.01),减少血肿周围TUNEL阳性细胞数量(p<0.01),但不影响脑组织EB渗漏。2.Western blot结果显示,正常状态下侧脑室注入rhVEGF165可使纹状体AQP4蛋白表达明显增加(p<0.01)。ICH后侧脑室注入rhVEGF165也可使血肿周围AQP4蛋白表达增加(p<0.01),而给予SU5416可使血肿周围AQP4蛋白表达减少(p<0.01)。免疫荧光法显示AQP4蛋白的荧光表达数量及强度与Western blot结果相一致。
结论：1.侧脑室注入rhVEGF165可在野生小鼠ICH后减轻脑水肿,改善神经功能,减轻细胞凋亡,但不影响BBB通透性。2.侧脑室注入rhVEGF165可上调ICH后血肿周围AQP4蛋白表达。ICH后AQP4蛋白表达增加可能与内源性VEGF有关。
第二部分：AQP4在VEGF对ICH后脑水肿效应中的作用
目的：研究侧脑室注入rhVEGF165对AQP4基因敲除小鼠ICH后脑水肿的作用,并通过AQP4+/+和AQP4-/-小鼠的对比,探讨rhVEGF165对ICH后脑水肿作用是否与调节AQP4的表达相关。
方法：将AQP4-/-小鼠随机分为对照组、对照+VEGF组、假手术组、ICH组和ICH+VEGF组。在干预后3d对各组进行神经功能缺损评分、干湿重法测定脑组织含水量、EB法测定BBB通透性、TUNEL染色法评价细胞凋亡。
结果：1.ICH后AQP4-/-小鼠神经功能缺损较AQP4+/+小鼠严重(p<0.05),侧脑室注入rhVEGF165可减轻AQP4-/-小鼠神经功能缺损(p<0.05),但仍较AQP4+/+小鼠严重(p<0,05)。且rhVEGF165降低ICH后AQP4+/+小鼠神经功能缺损评分的百分比显著高于AQP4-/-小鼠(p<0.01)。2.正常状态下侧脑室注入rhVEGF165可增加AQP4-/-小鼠脑组织含水量(p<0.01)。ICH后AQP4-/-小鼠脑水肿较AQP4+/+小鼠严重(p<0.05),侧脑室注入rhVEGF165不影响AQP4-/-小鼠脑组织含水量。3.正常状态下侧脑室注入rhVEGF165可使AQP4-/-小鼠EB渗漏增加(p<0.01),且较AQP4+/+小鼠更为严重(p<0.01)。ICH后AQP4-/-小鼠脑组织EB含量较AQP4+/+小鼠增多(p<0.05),侧脑室注入rhVEGF165可使AQP4-/-小鼠EB渗漏显著增加(p<0.05)。4.ICH后AQP4-/-小鼠血肿周围TUNEL阳性细胞数高于AQP4+/+小鼠(p<0.01),侧脑室注入rhVEGF165可减少AQP4-/-小鼠TUNEL阳性细胞数(p<0.01),但仍高于AQP4+/+小鼠(p<0.01)。且rhVEGF165使ICH后AQP4+/+小鼠TUNEL阳性细胞数降低的百分比显著高于AQP4-/-小鼠(p<0.01)。
结论：1.ICH后AQP4表达增加可对神经功能、脑水肿、BBB完整性及细胞凋亡均起到保护作用。2.上调AQP4表达是rhVEGF165减轻ICH后神经功能缺损的机制之一。3.正常状态下AQP4有清除VEGF潜在引起脑水肿的作用。rhVEGF165减轻ICH后脑水肿的作用可能与增加AQP4表达,从而加强脑水肿的清除有关。4.正常状态下rhVEGF165有破坏BBB的效应,AQP4基因敲除可加重该效应。ICH后,rhVEGF165对BBB可能同时具有保护和破坏的作用,其保护作用可能与增加AQP4表达有关。5.上调AQP4表达是rhVEGF165减少ICH后血肿周围凋亡细胞数量的机制之一。
第三部分：VEGF影响AQP4表达的信号转导通路
目的：探讨rhVEGF165引起AQP4蛋白表达增高可能的信号转导通路。
方法：将传代并鉴定后的星形胶质细胞随机分为正常对照组、VEGF (50ng/ml)组、VEGF+JNK抑制剂SP600125(20μM)组、VEGF+ERK抑制剂U0126(10μM)组、VEGF+p38-MAPK抑制剂SB239063(20μM)组、VEGF+PI3K抑制剂Ly294002(20μM)组。分别在加药后2d用Western blot和免疫荧光法检测各组AQP4蛋白的表达。
结果：1.Western blot检测显示：rhVEGF165可使星形胶质细胞AQP4蛋白表达增加(p<0.01),该效应可被SP600125和U0126抑制(p<0.01),但不能被SB239063或Ly294002所抑制。2.免疫荧光法显示AQP4蛋白的荧光表达数量及强度与Western blot结果相一致。
结论：1.rhVEGF165可使离体培养的星形胶质细胞AQP4蛋白表达增加。2. rhVEGF165可能通过激活JNK和ERK通路,而非通过激活p38-MAPK或PI3K通路使AQP4蛋白表达增加。
Intracerebral hemorrhage (ICH) is a severe stroke type, which always leads to severe neurological deficits and poor prognosis. Brain edema formation after ICH is one of the major causes of these consequences. Vascular endothelial growth factor (VEGF) has protective effects on various neurological diseases, including ICH. But no related research on how VEGF acting on brain edema after ICH has been reported. Our previous study indicated that as the most abundant water channel in brain, aquaporin-4(AQP4) played an important role in edema resolution after ICH. In addition, it was reported that VEGF and AQP4colocalized on astrocyte processes after cerebral hypoxia and blood-brain barrier (BBB) disruption and intracerebral VEGF injection can highly up-regulate AQP4mRNA and protein. Since there is close connection between them, we speculated that the effect of VEGF on brain edema following ICH may result from regulating AQP4expression. We first studied the effects of recombinant human VEGF165(rhVEGF165) to wild type mice on neurological deficits, brain water contents, BBB permeability and cell apoptosis after ICH and the influence of rhVEGF165on AQP4expression in normal state and ICH. We also compared the parameters between AQP4+/+and AQP4-/-mice to indicate whether the above roles played by rhVEGF165resulted from regulating AQP4expression. We further explored the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression through astrocyte cultures. Our results can provide a theoretical basis to downstream signal transduction pathways of VEGF, regulation of AQP4expression and the mechanisms involved in the effects of VEGF on ICH. Double targets can be supplied to treatment of brain edema following ICH, which can present new insights for new drugs development.
Part Ⅰ Effect of VEGF on brain edema after ICH
Objective:To study the effect of intracerebroventricular rhVEGF165injection on brain edema after ICH and its influence on AQP4expression in normal state and ICH.
Methods:CD1mice were randomly divided into six groups:control, control plus VEGF (3μg/kg), sham operation, ICH, ICH plus VEGF, ICH plus SU5416(60μg/kg), a kind of VEGFR inhibitor. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of Evans blue (EB) and evaluated cell apoptosis through TUNEL staining in the first5groups3d after drug given. We also detected the expression of AQP4protein at striatum or perihemotoma using Western blot and immunofluorescence in each group at the same time point.
Results:1. Intracerebroventricular rhVEGF165injection significantly reduced neurological deficits (p<0.05), brain water contents (p<0.01) and TUNEL-positive cells (p<0.01) perihemotoma in AQP4+/+mice, but had no change of EB extravasation.2. Western blot showed an increase of AQP4protein at striatum (p<0.01) after rhVEGF165injected intracerebroventricularly. Furthermore, AQP4protein expression perihemotoma was increased by intracerebroventricular injection of rhVEGF165after ICH (p<0.01), but was decreased by SU5416(p<0.01). The intensity and number of fluorescence expression were in accord with Western blot.
Conclusion:1. RhVEGF165injected intracerebroventricularly can reduce brain edema, alleviate neurological deficits and reduce apoptotic cells in AQP4+/+mice after ICH, but has no influence on BBB permeability.2. Intracerebroventricular rhVEGF165injection can increase AQP4protein expression perihemotoma after ICH. Endogenous VEGF may be involved in AQP4protein up-regulation after ICH.
Part Ⅱ Roles played by AQP4in effects of VEGF to brain edema following ICH
Objective:To investigate the effects of rhVEGF165to AQP4-/-mice on brain edema after ICH. We also studied whether the above roles played by rhVEGF165resulted from regulating AQP4expression by comparison of AQP4+/+and AQP4-/-mice.
Methods:AQP4-/-mice were randomly divided into five groups:control, control plus VEGF, sham operation, ICH, ICH plus VEGF. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of EB and evaluated cell apoptosis through TUNEL staining.
Results:1. Neurological deficits were more severe in AQP4-/-mice than AQP4+/+mice after ICH (p<0.05). Injection of rhVEGF165intracerebroventricularly alleviated neurological deficits of AQP4-/-mice (p<0.05), but the difference between them was the same as that mentioned above (p<0.05). Moreover, the percentage of neurological deficits decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice (p<0.01).2. An increase of brain water contents in normal AQP4-/-mice after intracerebroventricular injection of rhVEGF165was observed (p<0.01). AQP4-/-mice had more severe brain edema after ICH than AQP4+/+mice (p<0.05). Injection of VEGF intracerebroventricularly had no effect on AQP4-/-mice.3. The amount of EB extravasation was markedly increased by intracerebroventricular rhVEGF165injection in AQP4-/-mice (p<0.01), which was much more than AQP4+/+mice (p<0.01). Injection of VEGF intracerebroventricularly after ICH developd an increased effect in AQP4-/-mice (p<0.05).4. There were more TUNEL-positive cells found perihemotoma after ICH in AQP4-/-mice than AQP4+/+mice (p<0.01). RhVEGF165injected intracerebroventricularly reduced TUNEL-positive cells in AQP4-/-mice (p<0.01), but the difference between them was the same as that mentioned above (p<0.01). In addition, the percentage of TUNEL-positive cells decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice(p<0.01).
Conclusion:1. Increase of AQP4expression after ICH may have protective effects on neurological functions, brain edema, BBB integrity and cell apoptosis.2. Up-regulation of AQP4expression is one of the possible mechanisms by which rhVEGF165significantly alleviates neurological deficits after ICH.3. AQP4has an effect in normal state on eliminating brain edema potentially induced by VEGF. However, rhVEGF165can reduce brain edema following ICH, which has close relationship with enhancement of edema clearance by AQP4up-regulation induced by rhVEGF165.4. RhVEGF165can damage BBB in normal mice, which can be worsened by AQP4knock-out. The effect of rhVEGF165on BBB after ICH can be both protective and destructive and increase of AQP4expression may participate in the positive aspect.5. Apoptotic cells perihemotoma after ICH can be markedly reduced by exogenous rhVEGF165. Up-regulating AQP4expression is one of the possible mechanisms.
Part Ⅲ The signal transduction pathways through which VEGF regulates AQP4expression
Objective:To explore the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression.
Methods:The subcultured and determined astrocytes were randomly divided into six groups:control; VEGF (50ng/ml); VEGF plus SP600125(20μM), a kind of JNK inhibitor; VEGF plus U0126(10μM), a kind of ERK inhibitor; VEGF plus SB239063(20μM), a kind of p38-MAPK inhibitor; VEGF plus Ly294002(20μM), a kind of PI3K inhibitor. We detected the expression of AQP4protein using Western blot and immunofluorescence2d after drug given.
Results:1. Western blot showed that rhVEGF165increased AQP4protein expression of astrocytes (p<0.01), which was inhibited by SP600125and U0126(p<0.01), but not SB239063or Ly294002.2. The intensity and number of fluorescence expression were in accord with Western blot.
Conclusion:1. RhVEGF165can make cultured astrocytes increase expression of AQP4.2. RhVEGF165up-regulates AQP4expression via activating JNK and ERK pathways, but not p38-MAPK or PI3K pathways.
引文
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