迷迭香酸通过AMPK/mTOR通路减轻新生大鼠缺血缺氧性脑损伤研究

目的 探讨迷迭香酸对新生大鼠缺血缺氧脑损伤（hypoxic-ischemic encephalopathy，HIE）的影响，及其对单磷酸腺苷活化蛋白激酶（adenosine monophosphate activated protein kinase，AMPK）/雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路的调控作用，初步探讨其脑保护机制。方法 取7 d龄SD新生大鼠，随机分为对照组、模型组、迷迭香酸（300 mg/kg）组、AMPK/mTOR激动剂MT6378（10 mg/kg）组、AMPK抑制剂GSK-690693（30 mg/kg）组和迷迭香酸（300 mg/kg）+MT6378（10 mg/kg）组，每组20只。建立HIE模型，给予相应药物进行干预，采用TTC染色法检测大鼠脑梗死情况；透射电镜（TEM）观察大鼠海马神经元结构损伤及自噬状况；免疫荧光法检测大鼠海马神经元自噬标记物微管相关蛋白1轻链3B（microtubule-associated protein 1 light chain 3B，LC3B）阳性表达；TUNEL法检测大鼠海马神经元凋亡率；免疫组化法检测大鼠海马神经元磷酸化AMPK（p-AMPK）阳性表达；Western blotting检测大鼠海马组织活化的半胱氨酸蛋白酶3（cleaved Caspase-3）、mTOR及其磷酸化蛋白（p-mTOR）、Unc-51样自噬激活激酶1（uncoordinated-51 like autophagy activating kinase 1，Ulk1）及其磷酸化蛋白（p-Ulk1）、LC3B表达。结果 与对照组相比，模型组大鼠脑梗死严重，海马神经元结构损伤及自噬空泡形成较多，细胞自噬及凋亡水平升高，AMPK/mTOR通路活化（P<0.05）。与模型组相比，迷迭香酸组及GSK-690693组大鼠脑梗死、海马神经元结构损伤、凋亡及自噬减弱，AMPK/mTOR通路被抑制（P<0.05）；MT6378组海马组织AMPK/mTOR通路进一步激活，大鼠脑梗死、海马神经元结构损伤、凋亡及自噬进一步加重（P<0.05）；MT6378可逆转迷迭香酸的上述作用（P<0.05）。结论 迷迭香酸可能通过抑制AMPK/mTOR通路激活，降低海马神经元自噬及凋亡进程，发挥抗HIE脑损伤作用。

Rosmarinic acid attenuates hypoxic-ischemic encephalopathy in neonatal rats through AMPK/mTOR pathway

Objective To investigate the effect of rosmarinic acid on neonatal rats with hypoxic ischemic encephalopathy (HIE) and regulation on adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, and preliminarily explore its brain protection mechanism. Methods Seven-day-old SD neonatal rats were randomly divided into control group, model group, rosmarinic acid (300 mg/kg) group, AMPK/mTOR agonist MT6378 (10 mg/kg) group, AMPK inhibitor GSK-690693 (30 mg/kg) group and rosmarinic acid (300 mg/kg) + MT6378 (10 mg/kg) group, with 20 rats in each group. HIE model was established, corresponding drugs were given for intervention, TTC staining method was used to detect cerebral infarction; Transmission electron microscope (TEM) was used to observe the structure damage and autophagy of hippocampal neurons; Immunofluorescence method was used to detect the positive expression level of autophagy marker protein microtubule-associated protein 1 light chain 3B (LC3B); TUNEL method was used to detect neuronal cell apoptosis rate; Immunohistochemistry was used to detect the positive expression level of phosphorylated AMPK (p-AMPK); Western blotting was used to detect the expressions of cleaved Caspase-3, mTOR and its phosphorylated protein (p-mTOR), uncoordinated-51 like autophagy activating kinase 1 (Ulk1) and its phosphorylated protein (p-Ulk1), LC3B in hippocampal tissue. Results Compared with control group, rats in model group had severe cerebral infarction, hippocampal neuron structure damage and autophagy vacuoles were more formed, autophagy and apoptosis levels were increased, AMPK/mTOR pathway was activated (P<0.05). Compared with model group, rats in rosmarinic acid group and GSK-690693 group had cerebral infarction, hippocampal neuron structural damage and apoptosis and autophagy were weakened, and AMPK/mTOR pathway was inhibited (P<0.05); AMPK/mTOR pathway was further activated in hippocampal tissue of rats in MT6378 group, cerebral infarction, hippocampal neuron structural damage, apoptosis and autophagy were further aggravated (P<0.05); MT6378 reversed the above effects of rosmarinic acid (P<0.05). Conclusion Rosmarinic acid may play an anti-HIE brain injury effect through inhibiting the activation of AMPK/mTOR pathway and reducing the autophagy and apoptosis of hippocampal neurons.