摘要
目的探讨痴呆健脑方调控NEK7/NLRP3炎症小体通路防治血管性痴呆的作用机制。方法按照改良双侧颈总动脉分次结扎法复制血管性痴呆大鼠模型。将100只SD大鼠随机分为假手术组,模型组,多奈哌齐组,痴呆健脑方高、中剂量组;采用Morris水迷宫法评价大鼠记忆功能,苏木精—伊红染色观察大鼠海马组织形态学变化,TUNEL法检测大鼠海马区神经元凋亡率,透射电子显微镜观察大鼠海马组织超微结构,分别采用Real-time PCR法和Western blot法检测大鼠海马NIMA相关激酶7(NIMA-related kinase 7,NEK7)、Nod样受体家族含pyrin结构域蛋白3(Nod-like receptor family,pyrin domain-containing protein 3,NLRP3)、含CARD结构域的凋亡相关斑点样蛋白(apoptosis-associated speck-like protein containing CARD,ASC)、半胱氨酸天冬氨酸特异蛋白酶(cysteinyl aspartate specific proteinase 1,Caspase-1)、消皮素D(gasdermin D,GSDMD)mRNA和蛋白表达水平,ELISA法检测各组大鼠海马区白细胞介素(interleukin,IL)-1β、IL-6、IL-18、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平。结果与模型组比较,多奈哌齐组,痴呆健脑方高、中剂量组大鼠逃避潜伏期明显缩短(P<0.05),穿越平台次数明显增加(P<0.05),目标象限时间比明显升高(P<0.05)。与模型组比较,多奈哌齐组,痴呆健脑方高、中剂量组大鼠海马区虽有部分核染色凋亡神经元,但神经元凋亡率明显低于模型组。与模型组超微结构不同,多奈哌齐组,痴呆健脑方高、中剂量组神经元整体形态规则,神经元间隙均匀致密,神经元结构层次分明、结构相似,核固缩、碎裂及坏死神经元极少。与模型组比较,多奈哌齐组,痴呆健脑方高、中剂量组海马区NEK7、NLRP3、ASC、Caspase-1、GSDMD mRNA和蛋白表达水平以及IL-1β、IL-6、IL-18、TNF-α水平均明显降低(P<0.05)。结论痴呆健脑方通过抑制NEK7/NLRP3炎症小体通路相关蛋白水平,降低神经元凋亡后下游炎症因子表达水平,达到改善血管性痴呆大鼠认知、记忆功能的目的。
Objective To investigate the mechanism of action of Chidai Jiannao prescription in the prevention and treatment of vascular dementia by regulating the NEK7/NLRP3 inflammasome pathway.Methods The modified bilateral carotid artery ligation method was used to establish an animal model of vascular dementia.A total of 100 Sprague-Dawley rats were randomly divided into sham-operation group,model group,donepezil group,and high-and middle-dose Chidai Jiannao prescription groups.The Morris water maze test was used to evaluate the memory function of rats;HE staining was used to observe the histomorphological changes of hippocampal tissue;the TUNEL method was used to measure the apoptosis rate of neurons in the hippocampus;electron microscopy was used to observe the ultrastructure of hippocampal tissue;real-time PCR and Western blot were used to measure the mRNA and protein expression levels of NEK7,NLRP3,ASC,Caspase-1,and GSDMD in the hippocampus,and ELISA was used to measure the levels of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-18(IL-18),and tumor necrosis factor-α(TNF-α).Results Compared with the model group,the donepezil group and the high-and middle-dose Chidai Jiannao prescription groups had a significant reduction in escape latency(P<0.05)and significant increases in the number of platform crossings(P<0.05)and the time ratio of the target quadrant(P<0.05).Compared with the model group,the donepezil group and the high-and middle-dose Chidai Jiannao prescription groups had a significantly lower apoptosis rate of neurons in spite of the presence of a certain number of apoptotic cells with nuclear staining.Compared with the model group in terms of ultrastructure,the donepezil group and the high-and middle-dose Chidai Jiannao prescription groups had regular morphology of neurons,uniform and dense space between neurons,and a clear and similar structure of neurons,with few neurons with karyopyknosis,fragmentation,or necrosis.Compared with the model group,the donepezil group and the high-and middle-dose Chidai Jiannao prescription groups had significant reductions in the mRNA and protein expression levels of NEK7,NLRP3,ASC,Caspase-1,and GSDMD and the levels of IL-1β,IL-6,IL-18,and TNF-αin the hippocampus(P<0.05).Conclusion Chidai Jiannao prescription can improve the cognitive and memory functions of rats with vascular dementia by inhibiting the levels of the NEK7/NLRP3 inflammasome pathway-related proteins and reducing the expression levels of downstream inflammatory factors after cell apoptosis.
作者
李伟峰
秦合伟
姬令山
董新刚
LI Weifeng;QIN Hewei;JI Lingshan;DONG Xingang(Henan University of Chinese Medicine,Henan Zhengzhou 450002,China;The Second Affiliated Hospital of Henan University of Chinese Medicine,Henan Zhengzhou 450002,China;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450008,China)
出处
《安徽中医药大学学报》
CAS
2024年第1期94-100,共7页
Journal of Anhui University of Chinese Medicine
基金
河南省中医药科学研究专项课题(2022ZYZD05)
国家中医药管理局第七批全国名老中医药专家学术经验继承工作项目(国中医药人教函〔2022〕76号)
中原英才计划中原青年拔尖人才项目(豫组通〔2021〕44号)。
