糖尿病大鼠缺血再灌注脑组织MMP-9、RECK与VEGF蛋白表达的相关性研究
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摘要
目的建立糖尿病合并急性脑缺血再灌注大鼠模型;观察缺血脑组织中基质金属蛋白酶-9(MMP-9)、RECK及血管内皮生长因子(VEGF)的表达特点;研究MMP-9、RECK及VEGF的相关性;探讨它们在糖尿病缺血再灌注脑组织损伤中的作用。
方法
(1)采用高脂高糖饮食加腹腔注射小剂量链脲霉素法建立实验性大鼠糖尿病模型,选择成模者进一步采用线栓法建立大鼠局灶性脑缺血再灌注模型。120只雄性wistar大鼠被随机分为正常对照组(6只)、假手术组(6只)、单纯急性脑缺血再灌注组(CI组,54只)、糖尿病合并急性脑缺血再灌注组(DM+CI组,54只);其中CI组和DM+CI组各分成缺血再灌注后1h、3h、6h、12h、24h、3d、7d共7个亚组;
(2)采用Longa 5分制评分标准对各组脑缺血再灌注后大鼠进行神经功能评分;
(3)采用TTC染色法观察并标记梗死区域;利用HE染色和免疫组织化学技术在光镜下观察缺血脑组织病理变化特点;观察并分类计数脑组织缺血中心区和半暗带区MMP-9、RECK及VEGF蛋白表达阳性细胞数;
(4)各项计数指标均以均数±标准差((?)±s)表示,应用SAS12.0统计软件进行方差分析、q检验和直线相关分析。
结果
(1)神经功能评定结果CI组和DM+CI组大鼠麻醉清醒后均出现明显的右侧眼裂变小、眼球下陷等Horner征表现;CI组共有46只大鼠出现左侧前肢屈曲,向左侧转圈或向左侧倾倒,神经功能评分为1.54±0.71分;DM+CI组共有36只大鼠出现上述体征,神经功能评分为2.35±0.55分,两组相比有显著性差异(P＜0.05)。假手术组动物也出现明显的Horner征表现,但无其他神经功能缺损体征。
(2)TTC染色结果非梗死区脑组织被染成红色;梗死区呈现苍白色,与大脑中动脉供血分布区域一致;相同缺血再灌注时相点DM+CI组苍白区面积较CI组大。
(3)MMP-9表达结果假手术组和正常对照组大鼠脑组织中MMP-9阳性表达较少、散在分布;CI组和DM+CI组脑组织中MMP-9的阳性表达主要分布于梗死中心和半暗带区神经胶质细胞、神经元、血管内皮细胞及中性粒细胞等处。CI组和DM+CI组大鼠脑组织中MMP-9表达均较正常对照组明显增加(P＜0.05),其中DM+CI组较CI组明显增加(P＜0.05);DM+CI组MMP-9于再灌注3h开始升高,24h达到高峰,3d后逐渐下降。
(4)RECK蛋白表达结果假手术组和正常对照组大鼠脑组织中可见RECK蛋白阳性细胞主要表达在神经胶质细胞、神经元、血管内皮细胞等胞浆内;CI组和DM+CI组阳性表达散在分布于梗死周边区神经胶质细胞、神经元等处。CI组和DM+CI组大鼠脑组织中RECK蛋白表达均较正常对照组明显减少(P＜0.01),与CI组相比,DM+CI组阳性表达明显降低(P＜0.05)。DM+CI组RECK蛋白于再灌注3h表达开始降低,至24h达到最低,随后逐渐增加。
(5)VEGF蛋白表达结果假手术组和正常对照组鼠脑中仅见微量VEGF表达;DM+CI组和CI组脑组织VEGF阳性表达主要分布于梗死周边区神经元、神经胶质细胞、血管内皮、脉络丛等处。CI组和DM+CI组大鼠脑组织中VEGF表达均较正常对照组明显增加(P＜0.01),与CI组相比,DM+CI组阳性表达明显降低(P＜0.01)。DM+CI组VEGF表达于再灌注1h开始升高,至6h达到高峰,随后逐渐降低。
(6)直线相关分析表明,DM+CI组和CI组大鼠脑组织中MMP-9与RECK蛋白均呈直线负相关;DM+CI组和CI组大鼠脑组织中MMP-9与VEGF表达呈直线相关,其中DM+CI组于再灌注6～24h时段MMP-9与VEGF表达呈负相关。
结论
(1)高血糖条件下缺血再灌注后脑组织梗死面积较单纯脑缺血再灌注后显著增大,缺血后神经功能缺损症状较后者显著加重。
(2)糖尿病大鼠脑缺血后表达MMP-9、RECK和VEGF蛋白的细胞种类与单纯脑缺血相同,提示这几种细胞可能通过参与对MMP-9、RECK和VEGF蛋白的分泌调节而影响脑缺血后组织病理生理变化的过程。
(3)高血糖条件下,缺血再灌注后脑组织MMP-9表达较单纯脑缺血再灌注后表达明显升高,RECK和VEGF蛋白表达则明显减低,提示MMP-9参与调节RECK和VEGF蛋白表达变化过程。
(4)MMP-9、RECK及VEGF表达在缺血区空间分布上的一致性,和随时相变化的线性相关关系,提示:糖代谢异常引起MMP-9表达升高,RECK、VEGF蛋白表达较单纯脑缺血后降低,而高表达的MMP-9对VEGF具负性调节作用,进而影响缺血区血管新生和血管重构等病理生理过程,这可能是糖尿病合并脑梗死后组织损害较非糖尿病患者严重及功能恢复较差的重要原因。
OBJECTIVES: To establish a model of acute cerebral ischemia-reperfusion in diabetic rats; To observe the expression of MMP-9, RECK and VEGF protein in the cerebral ischemic tissues; To observe the relationships between the expression of MMP-9, RECK and VEGF in the cerebral ischemic tissues; To investigate the involvement of MMP-9, RECK and VEGF in the diabetic rat brain after focal cerebral ischemia.
METHODS:
(1) The rat models of diabetes mellitus were made by high sucrose, fat diet and streptozotion injection, in which the successful ones were selected to be made into focal cerebral ischemia-reperfusion rats. The models of the middle cerebral artery occlusion were made by improved zealong's method. 120 male wistar rats were assigned randomly into normal control group ([control I ],6 rats), sham-treated group ([control II],6 rats), cerebral ischemia-reperfusion and diabetic rats([DM+CI group] ,54 rats) and cerebral ischemia-reperfusion rats([CI group],54 rats). Middle cerebral artery occlusion models were made by DM+CI group and CI group, and the two groups were randomly assigned to 7 subgroups according to the reperfusion time (1h、3h、6h、12h、24h、3d、7d);
(2) The neural function of the rats after cerebral ischemia-reperfusion were assessed with longa method;
(3) TTC staining and HE staining were applied to observe and mark the clinical features of infarction zones; The pathologic characteristics of cerebral ischemic tissues were observed by HE staining and immunohistochemistry through microscope; Positive cells of MMP-9, RECK and VEGF protein in the infarction cores and penumbra zones of brain tissues were observed and counted;
(4) Statistical analyses were performed with the results by use of the SPSS 12.0 package. Mean values and standard error of mean were calculated for all studied areas, and statistical significance for intergroup was assessed by Student t test or ANOVA. The Spearman coefficient was used to study correlations between continuous variables. P<0.05 was considered statistically significant.
RESULTS:
(1) The assessment results of the rats neural functions The models of MCAO from CI group and DM+CI group appeared Homer sign; There were 46 rats with Horner sign, and the assessment score was 1. 54±0. 71; There were 36 rats with Horner sign from DM+CI group, and the score was2. 35±0. 55. A significant difference showed between the two groups (P<0.05). In sham-treated group, neural dysfunction sign wasn't found.
(2) TTC staining results of rat brain The non-infarcted areas of brain tissues were stained red. The infracted zones appeared pale in accordance with the distribution of the middle cerebral artery. Among samples from diabetic ischemic strokes, the infarct zones were larger and the damages were more severe than euglycemic ischemic strokes.
(3) Expression of MMP-9 In ischemic areas, MMP-9 was mainly located in the neuroglial, neurons and endothelial cells together with immunoreactive neutrophils. Positive cells are increased over time in diabetic and euglycemic ischemic rats but not in sham-treated or normal rats (P<0.05). However, at the same reperfusion time, the expression of MMP-9 was significantly higher in diabetic brains than in euglycemic ones (P<0.05). MMP-9 expression showed similar patterns in DM+CI and CI group: Increased at 3h, maximized at 24h, and gradually decreased after 3d.
(4) Expression of RECK protein RECK protein expressed mainly in the neuroglial, neurons in the normal rat brains and peri-infarct zones, barely in the ischemic cores. RECK protein expression significantly decreased compared to normal and sham-treated rat brain (P<0.01). At the same reperfusion time, there were less RECK positive cells in DM+CI group than CI group (P<0.05). RECK protein expressed similarly in DM+CI and CI group: Decreased at 3h, minor at 24h, and gradually increased after 3d
(5) Expression of VEGF VEGF mainly localized in the neurons, neuroglial and endothelial cells in the ischemic penumbra. It significantly increased in diabetic and euglycemic ischemic rats (P<0.01). At the same reperfusion time, VEGF expression in euglycemic ischemic rats is more than diabetic ischemic rats(P<0.01). In diabetic ischemic rats, VEGF expression peaked at 6h; in euglycemic ischemic rats, it peaked at 24h.
(6) A correlation study in diabetic ischemic rats between MMP-9 and RECK, MMP and VEGF demonstrated a strong relation between MMP-9 and RECK protein(r=-0.880, P <0.01), MMP-9 and VEGF (r=0.758, P <0.05).
CONCLUSIONS:
(1) There were significantly larger infarction areas in DM+CI group after ischemia-reperfusion compared with CI group. The signs of neural dysfunction after ischemia in diabetic rats were more severe than euglycemic rats.
(2) The categories of cells in which MMP-9, RECK and VEGF protein were expressed plentifully in the brain after cerebral ischemia in diabetic rats were the same with those in euglycemic rats. It indicated that the several cells mentioned above were likely to played roles in the courses after ischemia-reperfusion.
(3) There were significantly higher expression of MMP-9 in the ischemic tissues after ischemia-reperfusion in diabetic rats paralleled with lower expressions of RECK and VEGF. It suggested MMP-9 might be involved in the regulation of RECK and VEGF expression.
(4) The coherence between MMP-9 and RECK, MMP-9 and VEGF in the space distribution and the correlation between them along with the reperfusion time indicated that hyperglycemia caused increased MMP-9 and decreased VEGF and RECK protein ,while increased MMP-9 was involved in VEGF regulation. This mechanism probably was one of the reasons for the larger infarction areas and the worse outcome in cerebral ischemia of diabetic patients.
方法
(1)采用高脂高糖饮食加腹腔注射小剂量链脲霉素法建立实验性大鼠糖尿病模型,选择成模者进一步采用线栓法建立大鼠局灶性脑缺血再灌注模型。120只雄性wistar大鼠被随机分为正常对照组(6只)、假手术组(6只)、单纯急性脑缺血再灌注组(CI组,54只)、糖尿病合并急性脑缺血再灌注组(DM+CI组,54只);其中CI组和DM+CI组各分成缺血再灌注后1h、3h、6h、12h、24h、3d、7d共7个亚组;
(2)采用Longa 5分制评分标准对各组脑缺血再灌注后大鼠进行神经功能评分;
(3)采用TTC染色法观察并标记梗死区域;利用HE染色和免疫组织化学技术在光镜下观察缺血脑组织病理变化特点;观察并分类计数脑组织缺血中心区和半暗带区MMP-9、RECK及VEGF蛋白表达阳性细胞数;
(4)各项计数指标均以均数±标准差((?)±s)表示,应用SAS12.0统计软件进行方差分析、q检验和直线相关分析。
结果
(1)神经功能评定结果CI组和DM+CI组大鼠麻醉清醒后均出现明显的右侧眼裂变小、眼球下陷等Horner征表现;CI组共有46只大鼠出现左侧前肢屈曲,向左侧转圈或向左侧倾倒,神经功能评分为1.54±0.71分;DM+CI组共有36只大鼠出现上述体征,神经功能评分为2.35±0.55分,两组相比有显著性差异(P＜0.05)。假手术组动物也出现明显的Horner征表现,但无其他神经功能缺损体征。
(2)TTC染色结果非梗死区脑组织被染成红色;梗死区呈现苍白色,与大脑中动脉供血分布区域一致;相同缺血再灌注时相点DM+CI组苍白区面积较CI组大。
(3)MMP-9表达结果假手术组和正常对照组大鼠脑组织中MMP-9阳性表达较少、散在分布;CI组和DM+CI组脑组织中MMP-9的阳性表达主要分布于梗死中心和半暗带区神经胶质细胞、神经元、血管内皮细胞及中性粒细胞等处。CI组和DM+CI组大鼠脑组织中MMP-9表达均较正常对照组明显增加(P＜0.05),其中DM+CI组较CI组明显增加(P＜0.05);DM+CI组MMP-9于再灌注3h开始升高,24h达到高峰,3d后逐渐下降。
(4)RECK蛋白表达结果假手术组和正常对照组大鼠脑组织中可见RECK蛋白阳性细胞主要表达在神经胶质细胞、神经元、血管内皮细胞等胞浆内;CI组和DM+CI组阳性表达散在分布于梗死周边区神经胶质细胞、神经元等处。CI组和DM+CI组大鼠脑组织中RECK蛋白表达均较正常对照组明显减少(P＜0.01),与CI组相比,DM+CI组阳性表达明显降低(P＜0.05)。DM+CI组RECK蛋白于再灌注3h表达开始降低,至24h达到最低,随后逐渐增加。
(5)VEGF蛋白表达结果假手术组和正常对照组鼠脑中仅见微量VEGF表达;DM+CI组和CI组脑组织VEGF阳性表达主要分布于梗死周边区神经元、神经胶质细胞、血管内皮、脉络丛等处。CI组和DM+CI组大鼠脑组织中VEGF表达均较正常对照组明显增加(P＜0.01),与CI组相比,DM+CI组阳性表达明显降低(P＜0.01)。DM+CI组VEGF表达于再灌注1h开始升高,至6h达到高峰,随后逐渐降低。
(6)直线相关分析表明,DM+CI组和CI组大鼠脑组织中MMP-9与RECK蛋白均呈直线负相关;DM+CI组和CI组大鼠脑组织中MMP-9与VEGF表达呈直线相关,其中DM+CI组于再灌注6～24h时段MMP-9与VEGF表达呈负相关。
结论
(1)高血糖条件下缺血再灌注后脑组织梗死面积较单纯脑缺血再灌注后显著增大,缺血后神经功能缺损症状较后者显著加重。
(2)糖尿病大鼠脑缺血后表达MMP-9、RECK和VEGF蛋白的细胞种类与单纯脑缺血相同,提示这几种细胞可能通过参与对MMP-9、RECK和VEGF蛋白的分泌调节而影响脑缺血后组织病理生理变化的过程。
(3)高血糖条件下,缺血再灌注后脑组织MMP-9表达较单纯脑缺血再灌注后表达明显升高,RECK和VEGF蛋白表达则明显减低,提示MMP-9参与调节RECK和VEGF蛋白表达变化过程。
(4)MMP-9、RECK及VEGF表达在缺血区空间分布上的一致性,和随时相变化的线性相关关系,提示:糖代谢异常引起MMP-9表达升高,RECK、VEGF蛋白表达较单纯脑缺血后降低,而高表达的MMP-9对VEGF具负性调节作用,进而影响缺血区血管新生和血管重构等病理生理过程,这可能是糖尿病合并脑梗死后组织损害较非糖尿病患者严重及功能恢复较差的重要原因。
OBJECTIVES: To establish a model of acute cerebral ischemia-reperfusion in diabetic rats; To observe the expression of MMP-9, RECK and VEGF protein in the cerebral ischemic tissues; To observe the relationships between the expression of MMP-9, RECK and VEGF in the cerebral ischemic tissues; To investigate the involvement of MMP-9, RECK and VEGF in the diabetic rat brain after focal cerebral ischemia.
METHODS:
(1) The rat models of diabetes mellitus were made by high sucrose, fat diet and streptozotion injection, in which the successful ones were selected to be made into focal cerebral ischemia-reperfusion rats. The models of the middle cerebral artery occlusion were made by improved zealong's method. 120 male wistar rats were assigned randomly into normal control group ([control I ],6 rats), sham-treated group ([control II],6 rats), cerebral ischemia-reperfusion and diabetic rats([DM+CI group] ,54 rats) and cerebral ischemia-reperfusion rats([CI group],54 rats). Middle cerebral artery occlusion models were made by DM+CI group and CI group, and the two groups were randomly assigned to 7 subgroups according to the reperfusion time (1h、3h、6h、12h、24h、3d、7d);
(2) The neural function of the rats after cerebral ischemia-reperfusion were assessed with longa method;
(3) TTC staining and HE staining were applied to observe and mark the clinical features of infarction zones; The pathologic characteristics of cerebral ischemic tissues were observed by HE staining and immunohistochemistry through microscope; Positive cells of MMP-9, RECK and VEGF protein in the infarction cores and penumbra zones of brain tissues were observed and counted;
(4) Statistical analyses were performed with the results by use of the SPSS 12.0 package. Mean values and standard error of mean were calculated for all studied areas, and statistical significance for intergroup was assessed by Student t test or ANOVA. The Spearman coefficient was used to study correlations between continuous variables. P<0.05 was considered statistically significant.
RESULTS:
(1) The assessment results of the rats neural functions The models of MCAO from CI group and DM+CI group appeared Homer sign; There were 46 rats with Horner sign, and the assessment score was 1. 54±0. 71; There were 36 rats with Horner sign from DM+CI group, and the score was2. 35±0. 55. A significant difference showed between the two groups (P<0.05). In sham-treated group, neural dysfunction sign wasn't found.
(2) TTC staining results of rat brain The non-infarcted areas of brain tissues were stained red. The infracted zones appeared pale in accordance with the distribution of the middle cerebral artery. Among samples from diabetic ischemic strokes, the infarct zones were larger and the damages were more severe than euglycemic ischemic strokes.
(3) Expression of MMP-9 In ischemic areas, MMP-9 was mainly located in the neuroglial, neurons and endothelial cells together with immunoreactive neutrophils. Positive cells are increased over time in diabetic and euglycemic ischemic rats but not in sham-treated or normal rats (P<0.05). However, at the same reperfusion time, the expression of MMP-9 was significantly higher in diabetic brains than in euglycemic ones (P<0.05). MMP-9 expression showed similar patterns in DM+CI and CI group: Increased at 3h, maximized at 24h, and gradually decreased after 3d.
(4) Expression of RECK protein RECK protein expressed mainly in the neuroglial, neurons in the normal rat brains and peri-infarct zones, barely in the ischemic cores. RECK protein expression significantly decreased compared to normal and sham-treated rat brain (P<0.01). At the same reperfusion time, there were less RECK positive cells in DM+CI group than CI group (P<0.05). RECK protein expressed similarly in DM+CI and CI group: Decreased at 3h, minor at 24h, and gradually increased after 3d
(5) Expression of VEGF VEGF mainly localized in the neurons, neuroglial and endothelial cells in the ischemic penumbra. It significantly increased in diabetic and euglycemic ischemic rats (P<0.01). At the same reperfusion time, VEGF expression in euglycemic ischemic rats is more than diabetic ischemic rats(P<0.01). In diabetic ischemic rats, VEGF expression peaked at 6h; in euglycemic ischemic rats, it peaked at 24h.
(6) A correlation study in diabetic ischemic rats between MMP-9 and RECK, MMP and VEGF demonstrated a strong relation between MMP-9 and RECK protein(r=-0.880, P <0.01), MMP-9 and VEGF (r=0.758, P <0.05).
CONCLUSIONS:
(1) There were significantly larger infarction areas in DM+CI group after ischemia-reperfusion compared with CI group. The signs of neural dysfunction after ischemia in diabetic rats were more severe than euglycemic rats.
(2) The categories of cells in which MMP-9, RECK and VEGF protein were expressed plentifully in the brain after cerebral ischemia in diabetic rats were the same with those in euglycemic rats. It indicated that the several cells mentioned above were likely to played roles in the courses after ischemia-reperfusion.
(3) There were significantly higher expression of MMP-9 in the ischemic tissues after ischemia-reperfusion in diabetic rats paralleled with lower expressions of RECK and VEGF. It suggested MMP-9 might be involved in the regulation of RECK and VEGF expression.
(4) The coherence between MMP-9 and RECK, MMP-9 and VEGF in the space distribution and the correlation between them along with the reperfusion time indicated that hyperglycemia caused increased MMP-9 and decreased VEGF and RECK protein ,while increased MMP-9 was involved in VEGF regulation. This mechanism probably was one of the reasons for the larger infarction areas and the worse outcome in cerebral ischemia of diabetic patients.
引文
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