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目的探讨PI3K-AKT-m TOR信号通路在肺性脑病神经元自噬中的作用。方法对36只新生健康SD大鼠乳鼠建立离体培养大鼠皮层神经元,随机分为空白对照组、肺性脑病组、自噬抑制剂3-MA组后进一步建立了大鼠肺性脑病细胞模型,借助该模型运用CCK8法检测神经元活力、MDC染色观察神经元自噬泡数量、Western印迹法检测自噬相关蛋白LC3、Beclin-1及PI3K蛋白表达。结果与空白对照组比较,肺性脑病组神经元活力降低(P<0.05);神经元自噬泡数量增多;LC3-Ⅱ/Ⅰ的比值、Beclin-1及磷酸化PI3K表达水平增加(P<0.05)。使用自噬抑制剂3-MA预处理后,神经元活力降低更明显、自噬泡数量明显减少;缺氧诱导的自噬水平明显受到抑制,LC3-Ⅱ/Ⅰ的比值、Beclin-1及磷酸化PI3K表达水平明显降低。结论缺氧和二氧化碳潴留的信号使Beclin-1表达增加、LC3-Ⅰ向LC3-Ⅱ转化增加,加速PI3K磷酸化,使PI3K-AKT-m TOR信号通路失活,从而激活自噬信号通路,导致大脑神经元自噬增强。
Objective To investigate the role of PI3K-AKT-m TOR signaling pathway in autophagy of pulmonary encephalopathy. Methods Thirty-six neonatal SD rats were established into cortical neurons in vitro and were randomly divided into blank control group, pulmonary encephalopathy group and autophagy inhibitor 3-MA group to further establish rat pulmonary encephalopathy Cell model. CCK8 assay was used to detect the viability of neurons. MDC staining was used to detect the number of autophagic vacuoles. Western blotting was used to detect the expression of autophagy-related proteins LC3, Beclin-1 and PI3K. Results Compared with the control group, the viability of neurons in pulmonary encephalopathy group decreased (P <0.05), the number of autophagic vacuoles increased, the expression of Beclin-1 and phospho-PI3K increased in LC3-Ⅱ / Ⅰ group 0.05). After pretreatment with autophagy inhibitor 3-MA, the neuronal activity decreased more significantly and the number of autophagic vacuoles decreased significantly. The level of autophagy induced by hypoxia was significantly inhibited, and the ratio of LC3-Ⅱ / Ⅰ, Beclin-1 and phosphate The level of PI3K expression was significantly reduced. Conclusion The signal of hypoxia and carbon dioxide retention increases the expression of Beclin-1, and the conversion of LC3-Ⅰ to LC3-Ⅱ, accelerates the phosphorylation of PI3K, inactivates the PI3K-AKT-m TOR signaling pathway and activates autophagy signaling pathway, resulting in Autonomic enhancement of brain neurons.