基于HIF-1α/NLRP3途径介导细胞焦亡探讨《古今录验》续命汤治疗缺血性卒中的机制  

Mechanism of Xumingtang in Gu Jin Lu Yan for Treatment of Ischemic Stroke Based on HIF-1α/NLRP3 Pathway-mediated Pyroptosis

作　　者：蔡昱哲 李定祥 罗政 刘艺璇 阳晶晶 吴琴[1,2] 张亚男 陈静[1,2] 邓奕辉 CAI Yuzhe;LI Dingxiang;LUO Zheng;LIU Yixuan;YANG Jingjing;WU Qin;ZHANG Yanan;CHEN Jing;DENG Yihui(School of Integrated Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;Hunan Province Key Laboratory of Cerebrovascular Disease Prevention and Treatment of Integrated Traditional Chinese and Western Medicine,Hunan University of Chinese Medicine,Changsha 410208,China;School of Traditional Chinese Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学中西医结合学院,长沙410208 [2]湖南中医药大学中西医结合心脑疾病防治湖南省重点实验室,长沙410208 [3]湖南中医药大学中医学院,长沙410208

出　　处：《中国实验方剂学杂志》2023年第17期9-17,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖南省科技创新团队项目(2020RC4050);湖南省教育厅科学研究项目(21A0228,20A365);湖南省中医药科研计划项目(E2022010);校级研究生创新课题(2022CX11)。

摘　　要：目的:研究《古今录验》续命汤通过缺氧诱导因子-1α(HIF-1α)/NOD样受体热蛋白结构域相关蛋白3(NLRP3)途径调控缺血性卒中(IS)细胞焦亡的作用机制。方法:将SD大鼠随机分为假手术组、模型组、续命汤低剂量组、续命汤高剂量组和二甲双胍组,每组20只,连续灌胃3 d取材。采用高通量测序筛选差异信使RNA(mRNA),对关键差异基因行基因本体(GO)功能及京都基因与基因组百科全书(KEGG)通路分析;改良神经功能评分(mNSS)法、2,3,5-氯化三苯四氮唑(TTC)染色评价脑梗死损伤效应;苏木素-伊红(HE)染色进行脑组织病理形态学观察;酶联免疫吸附测定法(ELISA)检测缺血侧脑皮质区白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)水平比较;荧光双标染色检测HIF-1α、NLRP3共定位情况;实时荧光定量聚合酶链式反应(Real-time PCR)检测NLRP3、HIF-1α、胱天蛋白酶-1(Caspase-1)、消皮素D(GSDMD)mRNA的表达情况;蛋白免疫印迹法检测HIF-1α、NLRP3、Caspase-1、GSDMD蛋白的表达情况。结果:mRNA测序共得到5 705个(下调2 733个,上调2 972个)差异基因,经同源基因转化后,与焦亡基因集取交集,获得95个关键差异焦亡基因。与假手术组比较,模型组的mNSS评分、脑梗死面积显著增大(P<0.01),神经元形态多样、排列错乱、细胞间隙增宽,缺血侧皮质区IL-1β、IL-18的含量显著增高(P<0.01),共定位荧光强度明显增强,HIF-1α、NLRP3、Caspase-1、GSDMD mRNA和蛋白的表达水平显著升高(P<0.01)。与模型组比较,续命汤高剂量组改善大鼠神经功能评分、脑梗死面积最显著(P<0.01);各药物组神经元较完整、排列较整齐、细胞间隙变窄,共定位荧光强度明显降低;续命汤能够降低IL-1β、IL-18的含量及HIF-1α、NLRP3、Caspase-1、GSDMD mRNA和蛋白的表达(P<0.05,P<0.01),尤以高剂量组最明显(P<0.01)。结论:《古今录验》续命汤能够抑制IS后细胞焦亡,促进神经功能恢复,可能与抑制HIObjective:To investigate the mechanism of Xumingtang in Gu Jin Lu Yan(《古今录验》)in regulating cell pyroptosis through the hypoxia-inducible factor-1α(HIF-1α)/NOD-like receptor pyrin domaincontaining protein 3(NLRP3)pathway in ischemic stroke(IS).Method:SD rats were randomly divided into a sham operation group,a model group,low-and high-dose Xumingtang groups,and a metformin group,with 20 rats in each group.Oral administration was performed for 3 days,and tissue samples were collected.Differential messenger RNA(mRNA)was screened using high-throughput sequencing,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses were performed on key differentially expressed genes.The modified neurological severity score(mNSS)and 2,3,5-triphenyltetrazolium chloride(TTC)staining were used to evaluate the effect of brain infarction.Hematoxylin-eosin(HE)staining was used for pathological morphological observation of brain tissue.Enzyme-linked immunosorbent assay(ELISA)was used to compare the levels of interleukin-1β(IL-1β)and interleukin-18(IL-18)in the ischemic cortical region.Double staining immunohistochemistry was used to detect the co-localization of HIF-1αand NLRP3.Real-time quantitative polymerase chain reaction(PCR)was performed to detect the mRNA expression of NLRP3,HIF-1α,Caspase-1(CASP-1),and gasdermin D(GSDMD).Western blot was used to detect the protein expression of HIF-1α,NLRP3,CASP-1,and GSDMD.Result:A total of 5705 differentially expressed genes(2733 downregulated and 2972 upregulated)were obtained by mRNA sequencing.After conversion to homologous genes and intersection with the pyroptosis gene set,95 key differentially expressed pyroptosis genes were obtained.Compared with the sham operation group,the model group showed significantly increased mNSS scores,larger brain infarction areas(P<0.01),diverse neuronal morphology,disordered arrangement,widened cell gaps,significantly increased levels of IL-1βand IL-18 in the ischemic cortical region(P<0.01),enhanced co-local

关 键 词：缺血性卒中 《古今录验》续命汤 细胞焦亡 缺氧诱导因子-1α(HIF-1α)/NOD样受体热蛋白结构域相关蛋白3(NLRP3) 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33