西妥昔单抗对脑缺血再灌注大鼠神经元凋亡、胶质细胞活化及脑组织淀粉样前体蛋白表达的影响

Effects of cetuximab on neuronal apoptosis,glial cell activation and amyloid precursor protein expression in brain tissue in rats with cerebral ischemia/reperfusion

目的:探讨西妥昔单抗对大鼠脑缺血再灌注后神经元凋亡、胶质细胞活化及脑组织淀粉样前体蛋白(APP)表达的影响。方法:27只SD大鼠随机分为假手术组、缺血再灌注组、西妥昔单抗组,每组9只。结扎大脑中动脉制备缺血再灌注模型,立即经侧脑室注射给予西妥昔单抗(106μg/d),持续7 d。每组选取3只,在术后第3和7天进行神经功能评分。每组分别在缺血再灌注术后第3和7天处死3只,取脑组织制备冰冻切片,用免疫荧光染色观察APP及胶质细胞胶质纤维酸性蛋白(GFAP)表达,用TUNEL染色法检测神经元凋亡指数。提取新生大鼠大脑皮层组织制备胶质细胞悬液,培养的胶质细胞换成含有西妥昔单抗(10μg/mL)的无糖无血清培养基在缺氧培养箱中培养2 h,然后换成正常糖/血清的培养基在正常氧的培养箱中继续培养6 h,收集细胞,观察胶质细胞GFAP荧光强度的变化,对照组不进行缺氧/复氧处理,缺氧/复氧组不加西妥昔单抗。结果:与假手术组比较,缺血再灌注组大鼠神经功能评分升高,凋亡指数增加,胶质细胞GFAP免疫荧光强度增强,APP表达升高(P<0.05)。与缺血再灌注组相比,西妥昔单抗组神经功能评分下降,凋亡指数降低,GFAP免疫荧光强度减弱,APP表达下降(P<0.05)。体外实验结果显示,与对照组比较,缺氧/复氧组胶质细胞GFAP免疫荧光强度增强,而西妥昔单抗组较缺氧/复氧组降低(P<0.05)。结论:西妥昔单抗能促进脑缺血再灌注大鼠神经功能恢复,其机制可能与减少APP的表达、抑制神经元凋亡及胶质细胞活化有关。

Aim:To explore the effects of cetuximab on neuronal apoptosis,glial cell activation,and expression of amyloid precursor protein(APP)in brain tissue after cerebral ischemia/reperfusion in rats.Methods:A total of 27 SD rats were randomly allocated into sham surgery group,ischemia/reperfusion group,and cetuximab group,with 9 rats in each group.Animal model of the focal cerebral ischemia/reperfusion model was induced by ligating middle cerebral artery,then administering cetuximab(106μg/d)through lateral ventricular injection immediately after surgery,lasting for 7 days.Three animals were randomly selected from each group,and neurological function score was assessed on the 3rd and 7th day after surgery.The remaining 6 animals in each group were euthanized on the 3rd and 7th day after ischemia/reperfusion surgery,brain tissue was extracted,and the expression of APP and glial fibrillary acidic protein(GFAP)were observed using immunofluorescence staining.The neuronal apoptosis index was measured using TUNEL staining.Glial cell suspension was prepared by extracting cortical tissue from newborn rats.The glial cells were cultivated in a sugar free and serum free medium and cetuximab(10μg/mL)for 2 hours in a hypoxic incubator,and then the cells was replaced the medium with normal sugar/serum and continued to culture in a normal oxygen incubator for 6 hours.Then cells was collected and observed the changes of GFAP fluorescence intensity.The control group did not underwent hypoxia/reoxygenation treatment,and the hypoxia/reoxygenation group was not added with cetuximab.Results:Compared with the sham surgery group,the ischemia/reperfusion group showed an increase in neurological function score,apoptosis index,GFAP staining of glial cells and expression of APP(P<0.05).Compared with the ischemia/reperfusion group,the cetuximab group showed a decrease of neurological function score,apoptosis index,GFAP staining and APP expression(P<0.05).Compared with the control group,the GFAP immunofluorescence intensity of glial cells in the hypoxia/reoxygenation group increased,which decreased in the cetuximab group compared with the hypoxia/reoxygenation group(P<0.05).Conclusion:Cetuximab could promote the recovery of neurological function in rats with cerebral ischemia/reperfusion,and its mechanism may be related to reducing APP expression,inhibiting neuronal apoptosis and glial cell activation.