基于转录组学探讨牛膝对大鼠脑缺血再灌注损伤的脑保护作用机制  

Mechanism of brain protection of Achyranthes on cerebral ischemia-reperfusion injury in rats based on transcriptomics

作　　者：谭丹 杨沛霖 张磊 王健英 袁颖[1] TAN Dan;YANG Peilin;ZHANG Lei;WANG Jianying;YUAN Ying(School of Pharmacy,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Innovation Center of TCM Health Service,Shanghai 201203,China)

机构地区：[1]上海中医药大学中药学院,上海201203 [2]上海中医健康服务协同创新中心,上海201203

出　　处：《中华中医药杂志》2024年第6期3078-3083,共6页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金面上项目(No.81973730,No.8177392,No.82374052);上海市自然科学基金(No.19ZR1452000)。

摘　　要：目的:通过转录组测序(RNA-seq)技术探讨牛膝对大鼠脑缺血再灌注损伤的脑保护作用机制。方法:将24只SD大鼠随机分为3组:假手术组、模型组、牛膝组,每组8只。牛膝组采用牛膝预给药(6 g·kg^(-1)·d^(-1))7 d后,进行大脑中动脉闭塞(MCAO)造模。2,3,5-三苯基氯化四氮唑(TTC)染色法观察各组大鼠的脑梗死面积;HE染色法观察大鼠脑组织病理学形态;生化试剂盒以及ELISA检测各组大鼠血清中的一氧化氮(NO)、还原型谷胱甘肽(GSH)、乳酸脱氢酶(LDH)以及内皮素-1(ET-1)表达水平;转录组测序分析假手术组vs模型组,模型组vs牛膝组的差异表达基因,再进行京都基因与基因组百科全书(KEGG)通路富集,最后通过qPCR验证相关通路。结果:TTC染色和组织病理分析显示,牛膝可显著降低脑梗死面积,改善脑组织病理学形态;与模型组比较,牛膝组可显著降低血清中的LDH以及ET-1水平(P<0.01),同时显著上升NO与GSH的水平(P<0.01)。RNA-seq分析结果显示,根据假手术组vs模型组,模型组vs牛膝组的差异基因KEGG通路富集分析结果,提示牛膝可能通过丝裂原活化蛋白激酶(MAPK)等信号通路发挥作用;qPCR结果显示,与模型组比较,牛膝组MEKK1、MKK4、JUN、JunD、Bax表达水平显著下降(P<0.01),Bcl-2表达水平有升高的趋势。结论:牛膝可能主要通过JNK/MAPK通路发挥抗脑缺血再灌注的作用。Objective:To investigate the cerebral protective mechanism of Achyranthes on cerebral ischemia-reperfusion injury in rats by RNA sequencing technology.Methods:Twenty-four rats were randomly divided into three groups:sham group,model group and Achyranthes group,8 in each group.Achyranthes(6 g·kg^(-1)·d^(-1))was pre-administered in Achyranthes group for 7 days,and then the middle cerebral artery occlusion(MCAO)model was established;TTC staining method was used to observe the cerebral infarction area of the rats in each group;hematoxylin-eosin(HE)staining method was used to observe the pathological morphology of the rat brain tissue;biochemical kits and enzyme-linked immunosorbent assay(ELISA)were used to detect the expression levels of NO,GSH,LDH and ET-1 in serum of rats in each group;Transcriptome sequencing was used to analyze the differentially expressed genes between the sham-operated group and the model group,and the Achyranthes and the model group,and then the KEGG pathway was enriched.Finally,qPCR was used to verify the relevant pathways.Results:TTC staining and histopathological analysis showed that Achyranthes can significantly reduce the area of cerebral infarction and improve the pathological morphology of brain tissue;compared with the model group,the Achyranthes group could significantly reduce the levels of LDH and ET-1 in serum(P<0.01),while the levels of NO and GSH were significantly increased(P<0.01).The results of transcriptome sequencing analysis showed that,according to the KEGG pathway enrichment analysis results of differential genes in the sham-operated group vs the model group,the model group vs the Achyranthes japonica group,it was suggested that Achyranthes may play a role through mitogen-activated protein kinase(MAPK)and other signaling pathways;qPCR results showed that compared with the model group,the expression levels of MEKK1,MKK4,JUN,JunD,and Bax in the Achyranthes radix group were significantly decreased(P<0.01),and the expression level of Bcl-2 had a tendency to increase.Conclus

关 键 词：牛膝 转录组学 脑缺血再灌注 JNK/MAPK 

分 类 号：R285.5[医药卫生—中药学]