摘要
目的:探讨熟地黄对缺血性脑卒中大鼠神经功能损伤的影响,并探究其作用机制。方法:将雄性SD大鼠随机分为假手术组、模型组、熟地黄低剂量组(3.5 g·kg^(-1))、熟地黄高剂量组(7 g·kg^(-1))和尼莫地平组(0.01 g·kg^(-1))。参照改良线栓法制备大鼠脑中动脉闭塞(MCAO)模型,采用Zea-Longa5评分法进行神经功能评分,2,3,5-三苯基氯化四氮唑(TTC)染色检测大鼠脑梗死体积,苏木素-伊红(HE)染色和尼氏染色观察脑组织形态结构及损伤情况,同时检测血清乳酸脱氢酶(LDH)活力和氧化应激相关指标超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶4(GPX4)、丙二醛(MDA)及脑组织中铁含量变化情况;为进一步探究熟地黄干预缺血性脑卒中大鼠神经功能损伤的可能机制,采用蛋白免疫印迹法(Western blot)检测缺血侧脑组织中自噬相关蛋白:自噬效应蛋白(Beclin-1)、自噬微管相关蛋白轻链3(LC3B)、泛素结合蛋白p62(p62),铁死亡相关蛋白:转铁蛋白(TF)、转铁蛋白受体1(TFR1)、铁蛋白重链1(FTH1)、铁转运蛋白1(FPN1)和神经功能损伤相关蛋白:脑源性神经营养因子(BDNF)、酪氨酸激酶受体B(TrkB)的表达水平。结果:与假手术组比较,模型组大鼠神经功能评分升高,脑梗死体积显著升高,大脑皮层细胞出现细胞核固缩及空泡现象,熟地黄可降低神经功能评分,显著降低脑梗死体积,改善大脑皮层细胞病理现象;试剂盒检测结果显示,与假手术组比较,模型组LDH、MDA和铁含量显著升高(P<0.01),SOD、GPX4含量显著降低(P<0.01),与模型组比较,熟地黄干预后LDH、MDA和铁含量显著降低(P<0.05,P<0.01),SOD、GPX4含量明显升高(P<0.05,P<0.01)。缺血侧脑组织中自噬、铁死亡、神经功能损伤相关蛋白结果显示,与假手术组比较,模型组大鼠脑组织中自噬相关蛋白Beclin-1、LC3BⅡ/Ⅰ表达显著升高(P<0.01),p62表达显著降低(P<0.01);铁死亡相关蛋白TF、TFR1表达显著升高(P<0.01),FTH1、FPN1表达显著降低(P<0.01);同时神经功能损伤相关蛋白BDNF、TrkB表达降低(P<0.01),给予熟地黄后上述蛋白表达均得到改善。结论:熟地黄可能通过下调脑组织自噬水平,抑制铁死亡,改善缺血性脑卒中大鼠神经功能损伤。
Objective:To investigate the effect of Rehmanniae Radix Praeparata on neurological function injury in ischemic stroke rats and explore its mechanism.Method:Male SD rats were randomized into sham operation,model,low-and high-dose(3.5 g·kg^(-1) and 7 g·kg^(-1))Rehmannia Radix Praeparata,and nimodipine(0.01 g·kg^(-1))groups.The rat model of middle cerebral artery occlusion(MCAO)was established with the modified suture occlusion method.Zea-Longa 5-point scoring was employed to evaluate the neurological function of rats.The cerebral infarction volume was detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Hematoxylin-eosin staining and Nissl staining were employed to observe the morphology and damage of the brain tissue.Meanwhile,the serum levels of lactate dehydrogenase(LDH),oxidative stress-related indicators superoxide dismutase(SOD),glutathione peroxidase 4(GPX4),and malondialdehyde(MDA),and the iron(Fe)content in the brain tissue were determined.To explore the mechanism of Rehmanniae Radix Preparata in mitigating the neurological damage in ischemic stroke rats,Western blotting was employed to determine the expression levels of proteins in the ischemic brain tissue.The autophagy-associated proteins included autophagy effector(beclin-1),microtubule-associated protein light chain 3(LC3B),and ubiquitinbinding protein p62(p62).The ferroptosis-associated proteins included transferrin(TF),transferrin receptor 1(TFR1),ferritin heavy chain 1(FTH1),and ferropotin(FPN1).The neurological function injury-associated proteins included brain-derived neurotrophic factor(BDNF)and tyrosine kinase receptor B(TrkB).Result:Compared with the sham operation group,the model group showed increased neurological function score,cerebral infarction volume,and appearance of nuclear pyknosis and vacuole of cells in the cerebral cortex.In addition,the model group presented elevated levels of LDH,MDA,and Fe(P<0.01)and lowered levels of SOD and GPX4(P<0.01).Compared with the model group,Rehmanniae Radix Praeparata decreased the content of LDH,MDA,and Fe(P<0.05,P<0.01)and elevated the levels of SOD and GPX4(P<0.05,P<0.01).Compared with the sham operation group,the modeling promoted the expression of beclin-1,LC3BⅡ/Ⅰ,TF,and TFR1 and inhibited the expression of p62,FTH1,FPN1,BDNF,and TrkB(P<0.01).The expression levels of these proteins were recovered after the treatment with Rehmanniae Radix Praeparata.Conclusion:Rehmanniae Radix Praeparata may inhibit ferroptosis and improve the neurological function in ischemic stroke rats by down-regulating the autophagy level in the brain tissue.
作者
李赛菲
袁培培
魏亚新
高丽媛
李潘营
阮元
陈祎
傅阳
郑晓珂
冯卫生
LI Saifei;YUAN Peipei;WEI Yaxin;GAO Liyuan;LI Panying;RUAN Yuan;CHEN Yi;FU Yang;ZHENG Xiaoke;FENG Weisheng(School of Pharmacy,Henan University of Chinese Medicine,Zhengzhou 450046,China;Henan Engineering Technology Research Center for Traditional Chinese Medicine Development,Zhengzhou 450046,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2024年第8期26-33,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家重点研发计划项目(2017YFC1702800)
国家重点研发计划“中医药现代化研究”项目(2019YFC1708802)
河南省高层次人才特殊支持计划“中原千人计划”中原领军人才(ZYQR201810080)
河南中医药大学博士科研启动经费(RSBSJJ2018-04)。
关键词
熟地黄
缺血性脑卒中
铁死亡
自噬
Rehmanniae Radix Praeparata
ischemic stroke
ferroptosis
autophagy
