Sestrin2/AMPK信号通路调控新生鼠缺氧缺血脑损伤细胞自噬的新机制

Cellular autophagy plays an important role in neonatal rats with hypoxic-ischemic brain damage (HIBD). Recent studies found that the important autophagy regulator, adenosine monophosphate-activated protein kinase (AMPK), once activated by upstreams, could inhibit the mammalian target of rapamycin (mTOR) and then activate autophagy. Our previous study found that in neonatal rats after HIBD, activation of autophagy and apoptosis were associated with mTOR inhibition. However, whether AMPK/mTOR signaling pathway is involved in the activation of autophagy and apoptosis? What are the possible up and downstream target proteins in this signal transduction? Whether the recently identified signaling molecular Sestrin2, which can up-regulate AMPK in tumor cells, participates in the AMPK/mTOR pathway regulating? Whether Sestrin2 interacts with the AMPK upstream liver kinase B1 (LKB1) and mTOR key autophagic downstream Unc-51 like kinase 1 (ULK1)? The related signal mechanisms are largely unclear. This topic will establish HIBD model in neonatal rats and hypoxic-ischemic neuronal model in vitro, to detect the expression changes of each factor of Sestrin2/LKB1/AMPK/mTOR/ULK1 pathway. We also inhibit or enhance the main pathway factors, to reveal the mechanisms of modifing this Sestrin2/AMPK related signaling pathway in autophagy regulation, to promote certain neuroprotection. We believe that our research will supply new insights in clinical treatment for HIE.

细胞自噬在新生鼠缺氧缺血脑损伤(HIBD)中起重要作用。近年发现调控自噬的重要因子单磷酸腺苷活化蛋白激酶(AMPK)活化后，可抑制哺乳动物雷帕霉素靶蛋白(mTOR)而激活自噬。我们前期研究发现新生鼠HIBD后，细胞自噬及凋亡的激活与mTOR活性下降有关。但是，AMPK/mTOR通路是否参与调控自噬及凋亡激活？其可能的上下游信号分子是什么？新近发现的肿瘤中可调控AMPK的信号传导蛋白Sestrin2是否参与调节AMPK/mTOR？是否与AMPK上游肝酶B1(LKB1)及mTOR关键自噬下游Unc-51样激酶1(ULK1)相互联系？相关信号机制尚不清楚。本课题将建立新生鼠HIBD模型及体外神经元缺氧缺血模型，研究Sestrin2/LKB1/AMPK/mTOR/ULK1各因子表达，用信号通路抑制或增强方法，揭示Sestrin2/AMPK通路调控自噬促进神经保护的新机制，为HIE临床治疗提供新思路

目的：探讨新生大鼠缺氧缺血后海马脑组织中Sestrin2/AMPK信号通路在神经细胞自噬及存活中的可能机制。方法：将新生雄性10日龄(P10)SD大鼠随机分为缺氧缺血脑损伤组(HIBD)及假手术组，HIBD组行右侧颈总动脉结扎，低氧混合气(8%氧气，92%氮气)通气2.5小时。假手术组动物仅分离右侧颈总动脉，不结扎不缺氧。取假手术组及HI后4、8、12、24及72小时海马组织，Western Blot法检测Sestrin2，AMPK，p-AMPK，mTOR，p-mTOR，Beclin 1及LC3 (LC3Ⅱ/LC3Ⅰ比值)等蛋白表达变化。另将P10 SD乳鼠分为假手术组，HI组，HI+Compound C组(腹腔注射AMPK抑制剂Compound C)及HI+AICAR干预组(腹腔注射AMPK激动剂AICAR)。取各组HI后24 h海马检测。Western blot及免疫组化法检测p-AMPK，p-4EBP1，LC3，Beclin1及CC3表达情况，TUNEL法检测凋亡情况。另将P10 SD乳鼠分为HI+AICAR组及HI+AICAR+3-MA组。两组均腹腔注射AICAR，HI+AICAR+3-MA组侧脑室注射3-MA，TUNEL法检测神经细胞凋亡。结果：与假手术组相比，Sestrin2表达在HI后4小时开始上升，24小时达高峰；AMPK、mTOR总蛋白表达无明显变化；p-AMPK表达在HI后4小时上升，24小时后达高峰，持续到72小时；p-mTOR表达在HI后4 h开始增加，8 h达高峰，HI 24 h后逐渐下降；HI后Beclin 1、LC3蛋白表达在4小时上升，24小时后达高峰；HI后CC3蛋白表达在HI后4小时逐步上升，24小时达到表达高峰，72小时后开始下降。Compound C减少p-AMPK表达、增加p-4EBP1表达，减少Beclin1、LC3及CC3的表达。AICAR增加p-AMPK表达、减少p-4EBP1表达、增加Beclin1、LC3及CC3的表达。与HI组相比，HI+Compound C组中TUNEL染色阳性细胞数减少。与HI+AICAR相比，HI+AICAR+3-MA组TUNEL染色阳性细胞数减少。结论：缺氧缺血性脑损伤时，新生大鼠脑海马中Sestrin2可能通过AMPK/mTOR信号通路参与神经细胞自噬及凋亡的调节，从而提供神经保护作用。