首乌益智胶囊对血管性认知障碍的促血管新生作用及机制研究
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摘要
目的：
1.观察首乌益智胶囊对人脐静脉血管内皮细胞增殖、迁移、管状结构形成的影响，及对VEGF、Ang-1、Tie-2、Notch4mRNA的影响，从而验证首乌益智胶囊是否能诱导血管内皮细胞血管新生，并探讨其可能机制。
2.通过观察首乌益智胶囊对血管性认知障碍模型大鼠缺血脑组织微血管密度的影响，探讨首乌益智胶囊促血管新生的疗效。
3.观察首乌益智胶囊对大鼠缺血脑组织VEGF、Ang-1、Tie-2、Notch4mRNA表达的影响，探讨首乌益智胶囊促进具有稳定功能的血管新生的信号调控机制。
方法：
1.培养人脐静脉内皮细胞，随机分为空白对照组、尼莫地平组（尼莫地平含药血清）、首乌益智胶囊组（首乌益智胶囊含药血清），分组用药，采用MTT法、划痕损伤实验、管样结构生成实验等检测内皮细胞增殖、迁移和管样结构生成；采用实时定量PCR检测HUVECs细胞VEGF、Ang-1、Tie-2、Notch4mRNA表达。
2.以Wistar大鼠为实验对象，采用大脑中动脉缺血再灌注法建立血管性认知障碍动物模型。将造模成功大鼠，随机分为假手术组、模型组、尼莫地平组（0.81mg/100g/d）、首乌益智胶囊组（54mg/100g/d），每组20只，造模后药物干预4周，Morris水迷宫试验检测各组大鼠行为学改变，免疫组化检测CD34表达观察缺血周围脑组织微血管密度的改变，电镜观察脑血管内皮细胞的超微结构变化，实时荧光定量PCR检测缺血脑组织VEGF、Ang-1、Tie-2、Notch4基因表达。
结果：
1.离体实验结果
（1）首乌益智胶囊对人脐静脉内皮细胞增殖、迁移、管状结构形成的影响：首乌益智胶囊组OD值为0.99±0.03,明显高于空白对照组的0.54±0.02(p<0.01)；首乌益智胶囊组迁移率为94.83%,明显高于空白对照组的70.94%(p<0.01)；高倍视野(200×)随机计算6个视野下形成的管腔个数，首乌益智胶囊为23.42±2.83,明显高于空白对照组的15.23±3.44(p<0.01)。
（2）首乌益智胶囊对人脐静脉内皮细胞VEGF、Ang-1、Tie-2、Notch4mRNA的影响：空白对照组、尼莫地平组、首乌益智胶囊组的VEGF、Ang-1、Tie-2、Notch4mRNA水平依次升高。与空白对照组比较，尼莫地平组和首乌益智胶囊组有显著性差异（p<0.01, p<0.05）；与尼莫地平组比较，首乌益智胶囊组Ang-1、Notch4mRNA表达有显著性差异（p<0.05），首乌益智胶囊组VEGF、Tie-2mRNA表达无显著性差异（p>0.05）。
2.在体实验结果
（1）首乌益智胶囊对VCI模型大鼠学习记忆能力的影响：水迷宫结果表明，首乌益智胶囊组大鼠的定位航行和空间探索实验测试成绩明显优于模型组和尼莫地平组(p <0.01, p <0.05)。
（2）首乌益智胶囊对缺血周围脑组织微血管密度的影响：模型组、尼莫地平组、首乌益智胶囊组较假手术组依次升高；各组与假手术组相比，有显著性差异（p <0.01,p<0.05）；与模型组比较，尼莫地平组、首乌益智胶囊组有显著性差异（p<0.01, p<0.05）；与尼莫地平组比较，首乌益智胶囊组差异无显著性（p>0.05）。
（3）首乌益智胶囊对VCI模型大鼠脑血管内皮细胞超微结构的影响：模型组缺血侧微血管内皮细胞胞质以及毛细血管周围高度水肿，血管内皮细胞内线粒体数目减少，线粒体的大部分嵴融合消失，部分核膜融合消失。首乌益智胶囊组和尼莫地平组：镜下表现较模型组均有明显改善，毛细血管周围水肿较模型组轻，基膜基本正常，血管内皮细胞可见较多的线粒体，已接近正常状态。以首乌益智胶囊组对内皮水肿改善最为显著。
（4）首乌益智胶囊对VCI模型大鼠缺血脑组织VEGF、Ang-1、Tie-2、Notch4mRNA的影响：模型组VEGF、Ang-1、Tie-2、Notch4、DLL4mRNA水平较假手术组大鼠升高，差异有显著性（p<0.05）。尼莫地平组和首乌益智胶囊组VEGF、Ang-1、Tie-2、Notch4mRNA水平与模型组有明显升高（p<0.01, p<0.05）；首乌益智胶囊组VEGF、Ang-1、Notch4mRNA表达升高，与尼莫地平组相比存在显著性差异（p<0.05）；首乌益智胶囊组Tie-2mRNA表达升高，与尼莫地平组相比无显著性差异（p>0.05）。
结论：
1.首乌益智胶囊能促进人脐静脉内皮细胞增殖、迁移、管状结构形成，从而具有促进血管新生的疗效。首乌益智胶囊可能通过上调VEGF、Ang-1、Tie-2、Notch4基因的表达发挥促血管新生的疗效。
2.首乌益智胶囊能明显提高VCI模型大鼠缺血脑组织微血管密度，促进血管新生。VEGF、Ang-1、Tie-2、Notch4基因表达的上调可能是首乌益智胶囊促血管新生的分子机制。
3.首乌益智胶囊通过促进血管新生，改善神经功能，从而提高VCI大鼠的学习和记忆能力，是治疗VCI的有效方药。
Objective:
(1) To observe the effects on proliferation,migration, capillary-like structures ofHUVECs and expresstion of VEGF,Ang-1,Tie-2,Notch4treatment with ShouWuYiZhicapsule,then to study the effect of ShouWuYiZhi capsule on angiogenesis and investigatethe mechanisms of ShouWuYiZhi capsule.
(2)To investigate the effect ShouWuYiZhi capsule on angiogenesis through observingthe expression of CD34of VCI model rats.
(3) To investigate the potential mechanisms of ShouWuYiZhi capsule on angiogenesisthrough observing the expression of the genes of VEGF,Ang-1,Tie-2,Notch4of VCI modelrats.
Methods:
(1) At the cellular level，HUVECs were cultured,we observed the catalpol effects onHUVECs prolifersion,migration,capillary-like structures using cell counting,MTT,respectively.In addition,the expression of the genes of VEGF,Ang-1,Tie-2,Notch4onHUVECs induced by ShouWuYiZhi capsule were observed with real time PCR.
(2)Rats were used and the VCI model was established with MCAO.80rats werescreened after Morris water maze test and randomly divided into4groups: sham operationgroup, model group, Nimodipine group and Shouwuyizhi Capsule group, and each grouphas20rats. After4-week administration, behavioral change of rat was recorded by Morriswater maze. Ultrastructure alternation of vascular endothelial cells was observed viatransmission electric microscope.CD34expression was measured by immunohistochemistrymethod and the expression of the genes of VEGF,Ang-1,Tie-2,Notch4were measured using real time PCR.
Results:
1. Results in vitro
(1) ShouWuYiZhi capsule caused a significant increase in MTT test of HUVECs(OD(0.99±0.03) vs (0.54±0.02) in control group (p <0.01)). The incubation withShouWuYiZhi capsule led to a significantly augmented migratory rate(94.83%vs70.94%in cont rol group (p <0.01)).ShouWuYiZhi capsule induced the differentiation ofendothelial cells into capillary-like structures ((23.42±2.83) mm vs (15.23±3.44)mm incontrol group (p <0.01)).
(2) The content of cerebral tissue of angiogenesis factors (VEGF,Ang-1,Tie-2,Notch4)of rats model with vascular congnitive impairment were higher than the rats of sham group（p <0.01）;After the treatment,the content of theses factors were further highter thanmodel group（p <0.01, p <0.05）.Except VEGF,Tie-2,the content of Ang-1,Notch4ofShouwuyizhi group were highter than Nimodipine group（p <0.05）.
2. Results in vivo
(1)The results of Morris water maze showed that assessment of orientation test andspatial probe test from ShouWuYiZhi capsule group were significantly higher than modelgroup and Nimodipine group (p <0.01, p <0.05).
(2) About the microvessel dencity of ischemia cerebral tissue,all of the other threegroups were higher than sham group with the significant difference (p <0.01, p <0.05)；Compared with model group, Nimodipine group and Shouwuyizhi group had thesignificant difference (p <0.01, p <0.05)；Shouwuyizhi group had no difference withNimodipine group（p>0.05）.
(3) The changes of BCECs microstructure were observed by transmission electronmicroscopy.Compared with sham group and model group, ShouWuYiZhi capsule reducedthe BCECs edema significantly.The number of chondriosome in ShouWuYiZhi capsulegroup is more than that of in model group, and near normal levels.
(4)The content of cerebral tissue of angiogenesis factors (VEGF,Ang-1,Tie-2,Notch4)of rats model with vascular congnitive impairment were higher than the rats of sham group（p <0.01）; After the treatment,the content of theses factors were further highter thanmodel group（p <0.01, p <0.05）.Except Tie-2,the content of VEGF,Ang-1,Notch4of Shouwuyizhi group were highter than Nimodipine group（p <0.05）.
Conclusion:
(1) ShouWuYiZhi capsule enhences endothelial cell prolifersion,stimulatesendothelial cell migration,capillary-like structures accompanied by upregulating theexpression of the genes of VEGF, Ang-1, Tie-2,Notch4.
(2) ShouWuYiZhi capsule given after pMCAO in adult rats provides significantneuroprotection and angiogenesis,mediated partly by activation of the the expression ofthe genes of VEGF, Ang-1, Tie-2,Notch4.
(3) ShouWuYiZhi capsule could improve the learning and memory ability of VCImodel rats, and ShouWuYiZhi capsule is a valid therapy to VCI,which might be moreeffective than Nimodipine.
1.观察首乌益智胶囊对人脐静脉血管内皮细胞增殖、迁移、管状结构形成的影响，及对VEGF、Ang-1、Tie-2、Notch4mRNA的影响，从而验证首乌益智胶囊是否能诱导血管内皮细胞血管新生，并探讨其可能机制。
2.通过观察首乌益智胶囊对血管性认知障碍模型大鼠缺血脑组织微血管密度的影响，探讨首乌益智胶囊促血管新生的疗效。
3.观察首乌益智胶囊对大鼠缺血脑组织VEGF、Ang-1、Tie-2、Notch4mRNA表达的影响，探讨首乌益智胶囊促进具有稳定功能的血管新生的信号调控机制。
方法：
1.培养人脐静脉内皮细胞，随机分为空白对照组、尼莫地平组（尼莫地平含药血清）、首乌益智胶囊组（首乌益智胶囊含药血清），分组用药，采用MTT法、划痕损伤实验、管样结构生成实验等检测内皮细胞增殖、迁移和管样结构生成；采用实时定量PCR检测HUVECs细胞VEGF、Ang-1、Tie-2、Notch4mRNA表达。
2.以Wistar大鼠为实验对象，采用大脑中动脉缺血再灌注法建立血管性认知障碍动物模型。将造模成功大鼠，随机分为假手术组、模型组、尼莫地平组（0.81mg/100g/d）、首乌益智胶囊组（54mg/100g/d），每组20只，造模后药物干预4周，Morris水迷宫试验检测各组大鼠行为学改变，免疫组化检测CD34表达观察缺血周围脑组织微血管密度的改变，电镜观察脑血管内皮细胞的超微结构变化，实时荧光定量PCR检测缺血脑组织VEGF、Ang-1、Tie-2、Notch4基因表达。
结果：
1.离体实验结果
（1）首乌益智胶囊对人脐静脉内皮细胞增殖、迁移、管状结构形成的影响：首乌益智胶囊组OD值为0.99±0.03,明显高于空白对照组的0.54±0.02(p<0.01)；首乌益智胶囊组迁移率为94.83%,明显高于空白对照组的70.94%(p<0.01)；高倍视野(200×)随机计算6个视野下形成的管腔个数，首乌益智胶囊为23.42±2.83,明显高于空白对照组的15.23±3.44(p<0.01)。
（2）首乌益智胶囊对人脐静脉内皮细胞VEGF、Ang-1、Tie-2、Notch4mRNA的影响：空白对照组、尼莫地平组、首乌益智胶囊组的VEGF、Ang-1、Tie-2、Notch4mRNA水平依次升高。与空白对照组比较，尼莫地平组和首乌益智胶囊组有显著性差异（p<0.01, p<0.05）；与尼莫地平组比较，首乌益智胶囊组Ang-1、Notch4mRNA表达有显著性差异（p<0.05），首乌益智胶囊组VEGF、Tie-2mRNA表达无显著性差异（p>0.05）。
2.在体实验结果
（1）首乌益智胶囊对VCI模型大鼠学习记忆能力的影响：水迷宫结果表明，首乌益智胶囊组大鼠的定位航行和空间探索实验测试成绩明显优于模型组和尼莫地平组(p <0.01, p <0.05)。
（2）首乌益智胶囊对缺血周围脑组织微血管密度的影响：模型组、尼莫地平组、首乌益智胶囊组较假手术组依次升高；各组与假手术组相比，有显著性差异（p <0.01,p<0.05）；与模型组比较，尼莫地平组、首乌益智胶囊组有显著性差异（p<0.01, p<0.05）；与尼莫地平组比较，首乌益智胶囊组差异无显著性（p>0.05）。
（3）首乌益智胶囊对VCI模型大鼠脑血管内皮细胞超微结构的影响：模型组缺血侧微血管内皮细胞胞质以及毛细血管周围高度水肿，血管内皮细胞内线粒体数目减少，线粒体的大部分嵴融合消失，部分核膜融合消失。首乌益智胶囊组和尼莫地平组：镜下表现较模型组均有明显改善，毛细血管周围水肿较模型组轻，基膜基本正常，血管内皮细胞可见较多的线粒体，已接近正常状态。以首乌益智胶囊组对内皮水肿改善最为显著。
（4）首乌益智胶囊对VCI模型大鼠缺血脑组织VEGF、Ang-1、Tie-2、Notch4mRNA的影响：模型组VEGF、Ang-1、Tie-2、Notch4、DLL4mRNA水平较假手术组大鼠升高，差异有显著性（p<0.05）。尼莫地平组和首乌益智胶囊组VEGF、Ang-1、Tie-2、Notch4mRNA水平与模型组有明显升高（p<0.01, p<0.05）；首乌益智胶囊组VEGF、Ang-1、Notch4mRNA表达升高，与尼莫地平组相比存在显著性差异（p<0.05）；首乌益智胶囊组Tie-2mRNA表达升高，与尼莫地平组相比无显著性差异（p>0.05）。
结论：
1.首乌益智胶囊能促进人脐静脉内皮细胞增殖、迁移、管状结构形成，从而具有促进血管新生的疗效。首乌益智胶囊可能通过上调VEGF、Ang-1、Tie-2、Notch4基因的表达发挥促血管新生的疗效。
2.首乌益智胶囊能明显提高VCI模型大鼠缺血脑组织微血管密度，促进血管新生。VEGF、Ang-1、Tie-2、Notch4基因表达的上调可能是首乌益智胶囊促血管新生的分子机制。
3.首乌益智胶囊通过促进血管新生，改善神经功能，从而提高VCI大鼠的学习和记忆能力，是治疗VCI的有效方药。
Objective:
(1) To observe the effects on proliferation,migration, capillary-like structures ofHUVECs and expresstion of VEGF,Ang-1,Tie-2,Notch4treatment with ShouWuYiZhicapsule,then to study the effect of ShouWuYiZhi capsule on angiogenesis and investigatethe mechanisms of ShouWuYiZhi capsule.
(2)To investigate the effect ShouWuYiZhi capsule on angiogenesis through observingthe expression of CD34of VCI model rats.
(3) To investigate the potential mechanisms of ShouWuYiZhi capsule on angiogenesisthrough observing the expression of the genes of VEGF,Ang-1,Tie-2,Notch4of VCI modelrats.
Methods:
(1) At the cellular level，HUVECs were cultured,we observed the catalpol effects onHUVECs prolifersion,migration,capillary-like structures using cell counting,MTT,respectively.In addition,the expression of the genes of VEGF,Ang-1,Tie-2,Notch4onHUVECs induced by ShouWuYiZhi capsule were observed with real time PCR.
(2)Rats were used and the VCI model was established with MCAO.80rats werescreened after Morris water maze test and randomly divided into4groups: sham operationgroup, model group, Nimodipine group and Shouwuyizhi Capsule group, and each grouphas20rats. After4-week administration, behavioral change of rat was recorded by Morriswater maze. Ultrastructure alternation of vascular endothelial cells was observed viatransmission electric microscope.CD34expression was measured by immunohistochemistrymethod and the expression of the genes of VEGF,Ang-1,Tie-2,Notch4were measured using real time PCR.
Results:
1. Results in vitro
(1) ShouWuYiZhi capsule caused a significant increase in MTT test of HUVECs(OD(0.99±0.03) vs (0.54±0.02) in control group (p <0.01)). The incubation withShouWuYiZhi capsule led to a significantly augmented migratory rate(94.83%vs70.94%in cont rol group (p <0.01)).ShouWuYiZhi capsule induced the differentiation ofendothelial cells into capillary-like structures ((23.42±2.83) mm vs (15.23±3.44)mm incontrol group (p <0.01)).
(2) The content of cerebral tissue of angiogenesis factors (VEGF,Ang-1,Tie-2,Notch4)of rats model with vascular congnitive impairment were higher than the rats of sham group（p <0.01）;After the treatment,the content of theses factors were further highter thanmodel group（p <0.01, p <0.05）.Except VEGF,Tie-2,the content of Ang-1,Notch4ofShouwuyizhi group were highter than Nimodipine group（p <0.05）.
2. Results in vivo
(1)The results of Morris water maze showed that assessment of orientation test andspatial probe test from ShouWuYiZhi capsule group were significantly higher than modelgroup and Nimodipine group (p <0.01, p <0.05).
(2) About the microvessel dencity of ischemia cerebral tissue,all of the other threegroups were higher than sham group with the significant difference (p <0.01, p <0.05)；Compared with model group, Nimodipine group and Shouwuyizhi group had thesignificant difference (p <0.01, p <0.05)；Shouwuyizhi group had no difference withNimodipine group（p>0.05）.
(3) The changes of BCECs microstructure were observed by transmission electronmicroscopy.Compared with sham group and model group, ShouWuYiZhi capsule reducedthe BCECs edema significantly.The number of chondriosome in ShouWuYiZhi capsulegroup is more than that of in model group, and near normal levels.
(4)The content of cerebral tissue of angiogenesis factors (VEGF,Ang-1,Tie-2,Notch4)of rats model with vascular congnitive impairment were higher than the rats of sham group（p <0.01）; After the treatment,the content of theses factors were further highter thanmodel group（p <0.01, p <0.05）.Except Tie-2,the content of VEGF,Ang-1,Notch4of Shouwuyizhi group were highter than Nimodipine group（p <0.05）.
Conclusion:
(1) ShouWuYiZhi capsule enhences endothelial cell prolifersion,stimulatesendothelial cell migration,capillary-like structures accompanied by upregulating theexpression of the genes of VEGF, Ang-1, Tie-2,Notch4.
(2) ShouWuYiZhi capsule given after pMCAO in adult rats provides significantneuroprotection and angiogenesis,mediated partly by activation of the the expression ofthe genes of VEGF, Ang-1, Tie-2,Notch4.
(3) ShouWuYiZhi capsule could improve the learning and memory ability of VCImodel rats, and ShouWuYiZhi capsule is a valid therapy to VCI,which might be moreeffective than Nimodipine.
引文
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