法舒地尔对大鼠局灶性脑缺血再灌注ICAM-1、MMP-9表达的影响
摘要
目的：探讨法舒地尔对大鼠局灶性脑缺血再灌注损伤后ICAM-1、MMP-9表达和神经功能缺损的影响。
方法：采用线栓法制备大鼠右侧大脑中动脉脑缺血2h再灌注模型。选用清洁级健康雄性SD大鼠126只,随机分为以下各组：正常对照组(NG)、假手术组(SG)、脑缺血再灌注模型组(MG)、生理盐水治疗组(PG)和法舒地尔治疗组(FG)。正常对照组6只大鼠,其他各组大鼠按照缺血再灌注后处死的时间随机分为3h、6h、12h、24h、48h和72h六个亚组,每个亚组5只动物。法舒地尔治疗剂量按15mg/kg,于再灌注同时经腹腔注射给药,生理盐水组则于再灌注同时予以与法舒地尔等体积的生理盐水,经腹腔注射给药。采用Longa五级别四分法,动态观察大鼠神经功能损伤变化；采用HE染色观察病理学变化；应用免疫组织化学检测ICAM-1及MMP-9免疫阳性细胞表达水平的动态变化。
结果：
1.法舒地尔对大鼠脑缺血再灌注损伤后神经行为学变化的影响：正常对照组和假手术组大鼠无明显神经功能缺损,脑缺血再灌注模型组、生理盐水治疗组及法舒地尔治疗组均有不同程度的神经功能缺损,但同时间点法舒地尔治疗组与模型组、生理盐水治疗组相比,神经功能缺损较轻,神经行为学评分差异有显著意义(P<0.05),提示法舒地尔能改善神经功能缺损症状。
2.法舒地尔对大鼠脑缺血再灌注损伤后ICAM-1、MMP-9免疫阳性细胞的影响：正常组未见明显ICAM-1、MMP-9免疫阳性细胞表达,假手术组偶见ICAM-1、MMP-9免疫阳性细胞表达,模型组缺血侧于再灌注24h ICAM-1、48hMMP-9免疫阳性细胞表达高峰,生理盐水治疗组、法舒地尔治疗组变化趋势与模型组相同,但同时间点法舒地尔治疗组与模型组、生理盐水治疗组相比,ICAM-1、MMP-9阳性细胞数目较少,且其差异有显著意义(P<0.05),提示法舒地尔能明显抑制ICAM-1、MMP-9表达。
结论：
1.大鼠脑缺血再灌注损伤能诱导ICAM-1、MMP-9表达上调。
2.脑缺血再灌注损伤后ICAM-1、MMP-9的活化及超量表达可能参与了缺血性神经损伤的病理生理过程。
3.法舒地尔可能通过抑制缺血再灌注过程中ICAM-1、MMP-9超量表达,而阻断炎症反应,从而减轻神经功能缺损。
Objective To study the effect of Fasudil on neuronal deficits, neuronal apoptosis,and the expression of ICAM-land MMP-9 after Focal Cerebral Ischemia/reperfusion in Rats.
Methods The middle cerebral artery occlusion (MCAO) models were established by using the intraluminal suture occlusion method in Sprague-Dawley rats.Adult male Sprague-Dawley rats weighing 250-300g were randomly divided into normal group, sham-operated group, ischemia/reperfusion model group, Fasudil treatment group (intraperitoneal injected Fasudil 15mg/kg immediately after ischemia/reperfusion) and saline treatment group (intraperitoneal injected the same dose physiological saline immediately after ischemia/reperfusion).Normal control group included six rats;Other rats were randomly divided into six sub-groups as follows:3h,6h,12h, 24h,48h and 72h treatment groups, according to the killing time after ischemia/reperfusion, each sub-group included five animals.The changement of neuronal deficits accepted dynamic observation. Pathologic changes were evaluated by Hematoxylin. The expressions of ICAM-land MMP-9 were detected by immunohistochemistry method.
Result
1.The effect of Fasudil on the changement of neuroethology after ischemia/reperfusion:The normal control group and sham-operated group had no significant neuronal deficits.The ischemia/reperfusion model group and saline treatment group respectively showed more severe nerronal deficits than Fasudil treatment group (P<0.05 for each), indicating Fasudil may improve the symptoms of neurological deficits.
2.The effect of Fasudil on the expressions of ICAM-1 and MMP-9 after ischemia/reperfusion:The expressions of ICAM-1、MMP-9 in the normal control group and sham-operated group were rare observed. The expressions of ICAM-land MMP-9 in ischemic part of model groups reached the peak at the time point of 24 hours and 48 hours after reperfusion (P<0.05),Fasudil significntly decreased the expressions of ICAM-1 and MMP-9 at the same time point(P<0.05),indicating Fasudil could inhibit the expressions of ICAM-1、MMP-9.
3.The effect of Fasudil on neuronal apoptosis after ischemia/ reperfusion:The apoptotic cells in the sham-operated group were rare observed. The amount of apoptotic cells in ischemic part of model groups reached the peak at the time point of 24 hours after reperfusion (P<0.05), Fasudil significntly decreased the amount of apoptotic cells at the same time point(P<0.05),indicating Fasudil could inhibit the amount of apoptotic cells.
Conclusion
1.The expressions of ICAM-land MMP-9 can be up-regulated by the focal cerebral ischemia/reperfusion in rats.
2.The increased expression of ICAM-1 and MMP-9 may participate in the pathophysiological injury process of neuronal deficits induced by ischemia/reperfusion.
3.Fasudil could be a potent neutoprotector by blocking the inflammation process mediated by ICAM-1 and MMP-9 to reduce the nerronal deficits.
方法：采用线栓法制备大鼠右侧大脑中动脉脑缺血2h再灌注模型。选用清洁级健康雄性SD大鼠126只,随机分为以下各组：正常对照组(NG)、假手术组(SG)、脑缺血再灌注模型组(MG)、生理盐水治疗组(PG)和法舒地尔治疗组(FG)。正常对照组6只大鼠,其他各组大鼠按照缺血再灌注后处死的时间随机分为3h、6h、12h、24h、48h和72h六个亚组,每个亚组5只动物。法舒地尔治疗剂量按15mg/kg,于再灌注同时经腹腔注射给药,生理盐水组则于再灌注同时予以与法舒地尔等体积的生理盐水,经腹腔注射给药。采用Longa五级别四分法,动态观察大鼠神经功能损伤变化；采用HE染色观察病理学变化；应用免疫组织化学检测ICAM-1及MMP-9免疫阳性细胞表达水平的动态变化。
结果：
1.法舒地尔对大鼠脑缺血再灌注损伤后神经行为学变化的影响：正常对照组和假手术组大鼠无明显神经功能缺损,脑缺血再灌注模型组、生理盐水治疗组及法舒地尔治疗组均有不同程度的神经功能缺损,但同时间点法舒地尔治疗组与模型组、生理盐水治疗组相比,神经功能缺损较轻,神经行为学评分差异有显著意义(P<0.05),提示法舒地尔能改善神经功能缺损症状。
2.法舒地尔对大鼠脑缺血再灌注损伤后ICAM-1、MMP-9免疫阳性细胞的影响：正常组未见明显ICAM-1、MMP-9免疫阳性细胞表达,假手术组偶见ICAM-1、MMP-9免疫阳性细胞表达,模型组缺血侧于再灌注24h ICAM-1、48hMMP-9免疫阳性细胞表达高峰,生理盐水治疗组、法舒地尔治疗组变化趋势与模型组相同,但同时间点法舒地尔治疗组与模型组、生理盐水治疗组相比,ICAM-1、MMP-9阳性细胞数目较少,且其差异有显著意义(P<0.05),提示法舒地尔能明显抑制ICAM-1、MMP-9表达。
结论：
1.大鼠脑缺血再灌注损伤能诱导ICAM-1、MMP-9表达上调。
2.脑缺血再灌注损伤后ICAM-1、MMP-9的活化及超量表达可能参与了缺血性神经损伤的病理生理过程。
3.法舒地尔可能通过抑制缺血再灌注过程中ICAM-1、MMP-9超量表达,而阻断炎症反应,从而减轻神经功能缺损。
Objective To study the effect of Fasudil on neuronal deficits, neuronal apoptosis,and the expression of ICAM-land MMP-9 after Focal Cerebral Ischemia/reperfusion in Rats.
Methods The middle cerebral artery occlusion (MCAO) models were established by using the intraluminal suture occlusion method in Sprague-Dawley rats.Adult male Sprague-Dawley rats weighing 250-300g were randomly divided into normal group, sham-operated group, ischemia/reperfusion model group, Fasudil treatment group (intraperitoneal injected Fasudil 15mg/kg immediately after ischemia/reperfusion) and saline treatment group (intraperitoneal injected the same dose physiological saline immediately after ischemia/reperfusion).Normal control group included six rats;Other rats were randomly divided into six sub-groups as follows:3h,6h,12h, 24h,48h and 72h treatment groups, according to the killing time after ischemia/reperfusion, each sub-group included five animals.The changement of neuronal deficits accepted dynamic observation. Pathologic changes were evaluated by Hematoxylin. The expressions of ICAM-land MMP-9 were detected by immunohistochemistry method.
Result
1.The effect of Fasudil on the changement of neuroethology after ischemia/reperfusion:The normal control group and sham-operated group had no significant neuronal deficits.The ischemia/reperfusion model group and saline treatment group respectively showed more severe nerronal deficits than Fasudil treatment group (P<0.05 for each), indicating Fasudil may improve the symptoms of neurological deficits.
2.The effect of Fasudil on the expressions of ICAM-1 and MMP-9 after ischemia/reperfusion:The expressions of ICAM-1、MMP-9 in the normal control group and sham-operated group were rare observed. The expressions of ICAM-land MMP-9 in ischemic part of model groups reached the peak at the time point of 24 hours and 48 hours after reperfusion (P<0.05),Fasudil significntly decreased the expressions of ICAM-1 and MMP-9 at the same time point(P<0.05),indicating Fasudil could inhibit the expressions of ICAM-1、MMP-9.
3.The effect of Fasudil on neuronal apoptosis after ischemia/ reperfusion:The apoptotic cells in the sham-operated group were rare observed. The amount of apoptotic cells in ischemic part of model groups reached the peak at the time point of 24 hours after reperfusion (P<0.05), Fasudil significntly decreased the amount of apoptotic cells at the same time point(P<0.05),indicating Fasudil could inhibit the amount of apoptotic cells.
Conclusion
1.The expressions of ICAM-land MMP-9 can be up-regulated by the focal cerebral ischemia/reperfusion in rats.
2.The increased expression of ICAM-1 and MMP-9 may participate in the pathophysiological injury process of neuronal deficits induced by ischemia/reperfusion.
3.Fasudil could be a potent neutoprotector by blocking the inflammation process mediated by ICAM-1 and MMP-9 to reduce the nerronal deficits.
引文
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