CAJ | 학술논문

目的探讨单磷酸腺苷活化蛋白激酶(AMPK)信号通路参与蛛网膜下腔出血(SAH)后早期脑损伤中自噬激活的机制. 方法成年雄性SD大鼠60只按随机数字表法分为假手术组、SAH组、SAH+AICAR(AMPK激活剂)组、SAH+Compound C(AMPK抑制剂)组、SAH+生理盐水组,每组各12只.后4组采用血管内穿刺法建立SAH模型,后3组于造模前30 min分别经左侧侧脑室注射AICAR、Compound C或生理盐水.术后24 h采用免疫组化染色检测磷酸化雷帕霉素靶蛋白(p-mTOR)表达,采用Western blotting检测皮层自噬相关蛋白LC3、AMPK、磷酸化AMPK(p-AMPK)表达,采用Loeffler的5分制评分法进行神经行为学评分. 结果与假手术组相比,SAH组p-AMPK水平显著升高;p-mTOR组织学表达增高,主要表达于细胞浆,分布在出血周围的皮质、深部皮质与脑白质结合部;自噬相关蛋白LC3Ⅱ/LC3 Ⅰ比值显著增加;神经行为学评分显著降低,差异均有统计学意义(P＜0.05).与SAH组及SAH+生理盐水组相比,AICAR干预显著增高了SAH后p-AMPK水平,抑制了p-mTOR表达,显著增高了LC3Ⅱ/LC3 Ⅰ比值,降低了神经行为学评分,差异均有统计学意义(P＜0.05);而Compound C干预则有效降低了p-AMPK水平,抑制LC3Ⅱ/LC3Ⅰ比值的表达上调,有效改善了大鼠神经功能评分,差异均有统计学意义(P＜0.05),但对p-mTOR表达没有显著影响. 结论 AMPK信号通路参与了SAH后早期脑损伤中神经元自噬的激活,其机制可能与调控mTOR活性有关;调控AMPK可以发挥与自噬相关的神经保护作用.
목적 탐토단린산선감활화단백격매(AMPK)신호통로삼여주망막하강출혈(SAH)후조기뇌손상중자서격활적궤제. 방법 성년웅성SD대서60지안수궤수자표법분위가수술조、SAH조、SAH+AICAR(AMPK격활제)조、SAH+Compound C(AMPK억제제)조、SAH+생리염수조,매조각12지.후4조채용혈관내천자법건립SAH모형,후3조우조모전30 min분별경좌측측뇌실주사AICAR、Compound C혹생리염수.술후24 h채용면역조화염색검측린산화뢰파매소파단백(p-mTOR)표체,채용Western blotting검측피층자서상관단백LC3、AMPK、린산화AMPK(p-AMPK)표체,채용Loeffler적5분제평분법진행신경행위학평분. 결과 여가수술조상비,SAH조p-AMPK수평현저승고;p-mTOR조직학표체증고,주요표체우세포장,분포재출혈주위적피질、심부피질여뇌백질결합부;자서상관단백LC3Ⅱ/LC3 Ⅰ비치현저증가;신경행위학평분현저강저,차이균유통계학의의(P＜0.05).여SAH조급SAH+생리염수조상비,AICAR간예현저증고료SAH후p-AMPK수평,억제료p-mTOR표체,현저증고료LC3Ⅱ/LC3 Ⅰ비치,강저료신경행위학평분,차이균유통계학의의(P＜0.05);이Compound C간예칙유효강저료p-AMPK수평,억제LC3Ⅱ/LC3Ⅰ비치적표체상조,유효개선료대서신경공능평분,차이균유통계학의의(P＜0.05),단대p-mTOR표체몰유현저영향. 결론 AMPK신호통로삼여료SAH후조기뇌손상중신경원자서적격활,기궤제가능여조공mTOR활성유관;조공AMPK가이발휘여자서상관적신경보호작용.
Objective To investigate the role of AMP-activated protein kinase (AMPK) signal path in cell autophagy activation at early brain injury in rats after subarachnoid hemorrhage (SAH).Methods Adult male SD rats (weighting 300-350 g) were divided into five groups (n=12):sham-operated group,SAH group,and SAH+AICAR group,SAH+Compound C group and SAH+vehicle group.SAH models in the later four groups were established by endovascular perforation technique,and rats in the later three groups were performed left intracerebroventricular injection of AMPK agonist AICAR,AMPK inhibitor Compound C or normal saline 30 min before modeling;animals were subsequently sacrificed at 24 h after modeling.Immunohistochemical method was used to detect the phosphorylated mammalian target of rapamycin (p-mTOR) expression.Expressions of cortex autophagy related proteins LC3,AMPK and phosphorylated AMPK (p-AMPK) were observed by Western blotting.Loeffler's method was used to evaluate the neurologic behavior scores.Results As compared with those in the sham-operated group,the p-AMPK level,p-mTOR expression level and LC3Ⅱ/LC3Ⅰ ratio were significantly increased,while the behavioral deficit scores were significantly lower in the SAH group,with statistical differences (P＜0.05);the p-mTOR mainly expressed at cortex surrounding the hemorrhage areas,and integration areas of deep cortex and brain white matter.As compared with the sham-operated group and SAH+vehicle group,SAH+AICAR group had significantly increased p-AMPK level,decreased p-mTOR expression level,increased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05);as compared with the sham-operated group and SAH+vehicle group,SAH+Compound C group had significantly decreased p-AMPK level,decreased LC3Ⅱ/LC3Ⅰ ratio,and decreased behavioral deficit scores (P＜0.05).Conclusion AMPK is involved in the process ofautophagy activation after SAH through regulating mTOR,and the regulation of AMPK may contribute to neuroprotection related to autophagy.