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

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

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7d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(10mg/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 protein1light chain3B,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。

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 proteinmicrotubule-associated protein1light chain3B(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 inhibitingthe activation of AMPK/mTOR pathwayandreducingthe autophagy and apoptosis of hippocampal neurons.