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目的探讨排斥性导向分子a(RGMa)特异性抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注引发的自噬及对神经功能恢复的影响。方法雄性成年SD大鼠立体定位侧脑室注射不同浓度6FNⅢ后建立大脑中动脉闭塞(MCAO)/再灌注模型,Western blotting检测RGMa下游分子脑衰反应调节蛋白-2(CRMP-2)蛋白表达水平,确定最佳6FNⅢ干预浓度。将72只雄性成年SD大鼠随机分成4组:假手术组(sham)、脑缺血再灌注组(I/R)、生理盐水干预组(I/R+NS)、6FNⅢ干预组(I/R+6FNⅢ),每组18只。缺血2h再灌注后24h,采用神经功能缺损评分评估各组大鼠神经功能恢复情况;Western blotting检测自噬相关蛋白LC3Ⅱ/Ⅰ水平;免疫荧光双标检测LC3在神经元内的表达情况;透射电子显微镜观察自噬体的形成情况。结果根据各浓度6FNⅢ干预组CRMP-2表达,确定中浓度(0.5 g/L)为本实验的最适浓度。缺血再灌注后24h,I/R组较sham组神经功能评分降低(P<0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平升高(P<0.05)、自噬体形成增多;I/R+6FNⅢ组较I/R组神经功能评分增高(P<0.05)、自噬相关蛋白LC3Ⅱ/Ⅰ水平降低(P<0.05)、自噬体形成减少;而I/R+NS组与I/R组以上各项均无统计学意义(P>0.05)。结论 RGMa特异性抑制剂6FNⅡ可在缺血再灌注急性期抑制自噬发生,促进神经功能恢复。
Objective To investigate the effect of exclusive 6RNⅢ, a repulsive targeting molecule a (RGMa) inhibitor, on the autophagy induced by ischemia / reperfusion in rat cortical neurons and the recovery of neurological function. Methods Adult male Sprague - Dawley rats were injected intracerebroventricular injection of 6FN Ⅲ with different concentrations to establish middle cerebral artery occlusion (MCAO) / reperfusion model. Western blotting was used to detect the expression of CRMP - 2 protein downstream of RGMa. Determine the best 6FNⅢ intervention concentration. Seventy-two male adult SD rats were randomly divided into 4 groups: sham, I / R, I / R + NS, 6FNⅢ intervention group, R + 6FNⅢ), 18 in each group. Neurological deficit scores were used to evaluate the neurological function recovery of rats in each group at 2 h after ischemia, and the level of LC3 Ⅱ / Ⅰ was detected by Western blotting. The expression of LC3 in neurons was detected by double immunofluorescence staining. Electron microscopy was used to observe the formation of autophagosomes. Results According to the expression of CRMP-2 in each concentration of 6FNⅢ intervention group, the optimal concentration of 0.5 g / L was determined. The neurological function score of I / R group was lower than that of sham group (P <0.05), and the level of autophagy-related protein LC3Ⅱ / Ⅰ was increased (P <0.05) The neurological score of 6FNⅢgroup was higher than that of I / R group (P <0.05), and the level of LC3Ⅱ / Ⅰwas decreased (P <0.05) None of the above was statistically significant (P> 0.05). Conclusion RGMa-specific inhibitor 6FNⅡ can inhibit autophagy in the acute phase of ischemia-reperfusion and promote the recovery of neurological function.