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 kinasemammalian 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。
[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 regulator2 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.
作者
程巨萍
戴奉德
郑娅
CHENG Juping;DAI Fengde;ZHENG Ya(LI Huili Hospital,Ningbo Medical Center,Ningbo(315040),China;Longsai Hospital of Ningbo Zhenhai;Ningbo Rehabilitation Hospital)
出处
《浙江中医药大学学报》
CAS
2022年第11期1189-1198,共10页
Journal of Zhejiang Chinese Medical University
基金
浙江省中医药管理局项目(2020ZB230)。
