文章摘要
程巨萍,戴奉德,郑娅.地黄饮子通过SIRT1-AMPK-mTOR/eNOS通路减轻老龄大鼠脑缺血再灌注损伤[J].浙江中医药大学学报,2022,46(11):1189-1198.
地黄饮子通过SIRT1-AMPK-mTOR/eNOS通路减轻老龄大鼠脑缺血再灌注损伤
Dihuang Yinzi Alleviates Cerebral Ischemia-reperfusion Injury in Aged Rats through SIRT1-AMPK-MTOR/eNOS Pathway
DOI：10.16466/j.issn1005-5509.2022.11.003
中文关键词: 地黄饮子  SIRT1-AMPK-mTOR/eNOS  脑缺血再灌注损伤  调控  老龄大鼠  脑梗死率  神经功能  尼莫地平
英文关键词: Dihuang Yinzi  SIRT1-AMPK-mTOR/eNOS  cerebral ischemia-reperfusion injury  regulation  aged rats  cerebral infarction rate  neurological function  Nimodipine
基金项目:浙江省中医药管理局项目(2020ZB230)
作者单位
程巨萍 宁波市医疗中心李惠利医院　浙江，宁波　315040 
戴奉德 宁波市镇海区龙赛医院 
郑娅 宁波市康复医院 
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中文摘要:
      [目的] 探究地黄饮子(Dihuang Yinzi，DY)减轻老龄大鼠脑缺血再灌注损伤(cerebral ischemia-reperfusion injury，CIRI)的作用机制。[方法] 30只SD大鼠随机分为假手术组、模型组、DY低剂量组(6 g·kg-1)、DY高剂量组(12 g·kg-1)、尼莫地平组(10 mg·kg-1)。建立CIRI模型，进行神经功能评分，血小板聚集实验，采用2，3，5-氯化三苯基四氮唑(2，3，5-triphenyltetrazole chloride，TTC)、苏木素-伊红(hematoxylin-eosin，HE)和尼氏染色检测脑梗死率及病理改变，流式细胞术测定血小板α颗粒膜糖蛋白140(CD62P)表达，实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction，Real-time qPCR)检测沉默信息调节因子2同系物1-腺苷酸活化蛋白激酶-雷帕霉素靶蛋白/内皮型-氧化氮合酶(silent information regulator 2 homolog 1-adenylate activated protein kinase-mammalian target of rapamycin/endothelial nitric oxide synthase，SIRT1-AMPK-mTOR/eNOS)通路相关mRNA表达，免疫印迹法检测各相关蛋白表达情况。[结果] 与假手术组比较，模型组神经功能评分、脑梗死率、血小板聚集率、CD62P表达、腺苷酸活化蛋白激酶(adenylate activated protein kinase，AMPK)mRNA，磷酸化腺苷酸活化蛋白激酶(phosphorylation-adenylate activated protein kinase，p-AMPK)和磷酸化一氧化氮合酶(phosphorylation-nitric oxide synthase，p-eNOS)蛋白表达显著上升(P＜0.01)，SIRT1、mTOR的mRNA及SIRT1和磷酸化-雷帕霉素靶蛋白(phosphorylation-mammalian target of rapamycin，p-mTOR)蛋白表达显著降低(P＜0.05，P＜0.01)，病理染色显示脑组织损伤严重，细胞核固缩。与模型组比较，DY高剂量组神经功能评分、脑梗死率、CD62P表达量、AMPK的mRNA及p-AMPK和p-eNOS蛋白表达显著降低(P＜0.05，P＜0.01)，SIRT1、mTOR的mRNA及SIRT1、p-mTOR蛋白表达显著升高(P＜0.05，P＜0.01)，病理染色结果显示脑组织损伤情况减轻；尼莫地平组与DY高剂量组相似。[结论] DY能够减轻CIRI引起的脑组织损伤，并起到神经保护作用，其作用机制可能与影响SIRT1-AMPK-mTOR/eNOS通路相关蛋白的表达有关。
英文摘要:
      [Objective] To investigate the mechanism of Dihuang Yinzi(DY) in alleviating cerebral ischemia-reperfusion injury(CIRI) in aged rats. [Methods] Thirty SD rats were randomly divided into sham operation group， model group， DY low-dose group(6 g·kg-1)， DY high-dose group(12 g·kg-1) and Nimodipine group(10 mg·kg-1). CIRI model was established in aged rats， then the neurological function score， platelet aggregation test， 2，3，5-triphenyltetrazole chloride(TTC)， hematoxylin-eosin(HE) and Nissl staining were performed to detect cerebral infarction rate and pathological changes， and CD62P expression was determined by flow cytometry. The mRNA expression of silent information regulator 2 homolog 1-adenylate activated protein kinase-mammalian target of rapamycin/endothelial nitric oxide synthase(SIRT1-AMPK-mTOR/eNOS) pathway was detected by Real-time quantitative polymerase chain reaction(Real-time qPCR)， and the expression of related proteins was detected by Western blot. [Results] Compared with sham operation group， neurological function score， cerebral infarction rate， platelet aggregation rate， CD62P expression， adenylate activated protein kinase(AMPK) mRNA and phosphorylation-adenylate activated protein kinase(p-AMPK) and phosphorylation-nitric oxide synthase(p-eNOS) protein expression were significantly increased(P＜0.01). The mRNA expression of SIRT1 and mTOR， protein expression of SIRT1 and phosphorylation-mammalian target of rapamycin(p-mTOR) were significantly decreased(P＜0.05， P＜0.01). Pathological staining showed severe brain tissue injury and nuclear pyrosis. Compared with model group， neurological function score， cerebral infarction rate， CD62P expression， mRNA expression of AMPK， protein expression of p-AMPK and p-eNOS in DY high-dose group were significantly decreased(P＜0.05， P＜0.01). The mRNA expression of SIRT1 and mTOR， protein expression of SIRT1 and p-mTOR were significantly increased(P＜0.05， P＜0.01). Pathological staining results showed that brain tissue damage was relieved， and the results of Nimodipine group were similar to those of DY high-dose group. [Conclusion] DY can alleviate CIRI-induced brain tissue injury and play a neuroprotective role， which may be related to regulating the expression level of SIRT1-AMPK-MTOR/eNOS pathway related proteins.
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