基于单细胞测序技术分析安宫牛黄丸减轻创伤性颅脑损伤的分子机制  

Analysis of Molecular Mechanism of Angong Niuhuangwan in Alleviating Traumatic Brain Injury Based on Single Cell Sequencing

作　　者：殷志如 田良良 曹光昭 张晶晶[1,4] 杨洪军 YIN Zhiru;TIAN Liangliang;CAO Guangzhao;ZHANG Jingjing;YANG Hongjun(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Jiangxi Province Key Laboratory of Traditional Chinese Medicine(TCM)Pharmacology,Institute of TCM Health Industry,China Academy of Chinese Medical Sciences,Nanchang 330115,China;Beijing Key Laboratory of TCM Basic Research on Prevention and Treatment for Major Diseases,Experimental Research Center,China Academy of Chinese Medical Sciences,Beijing 100700,China;Chinese Institute for Brain Research,Beijing 102206,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]中国中医科学院中医药健康产业研究所,中药药理江西省重点实验室,南昌330115 [3]中国中医科学院医学实验中心,中医药防治重大疾病基础研究北京市重点实验室,北京100700 [4]北京脑科学与类脑研究所,北京102206

出　　处：《中国实验方剂学杂志》2024年第23期35-45,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81974550);中国中医科学院科技创新工程项目(CI2021A04612,CI2021B017-03)。

摘　　要：目的:基于单细胞测序技术揭示安宫牛黄丸(AGNH)改善创伤性颅脑损伤(TBI)的分子作用机制。方法:将75只雄性SD大鼠随机分为假手术组、模型组、吡拉西坦组(3.6 g·kg^(-1))、AGNH低、高剂量组(0.09、0.27 g·kg^(-1)),每组15只。除假手术组外,其余4组采用改良Feeney自由落体撞击法制备TBI模型,造模后立刻灌胃给药,24 h后进行改良神经功能缺损评分(mNSS),并分离脑组织,测定脑水肿程度。苏木素-伊红(HE)染色观察脑组织皮层、CA1区、CA3区的损伤程度;免疫荧光(IF)染色观察损伤部位环氧合酶-2(COX-2)、干扰素调节因子1(IRF1)、Janus激酶2(JAK2)和细胞因子信号传导抑制因子3(SOCS3)的表达情况。酶联免疫吸附测定法(ELISA)测定脑组织白细胞介素(IL)-6、IL-18、IL-1β、IL-17A、肿瘤坏死因子-α(TNF-α)、胱天蛋白酶-1(Caspase-1)和核苷酸结合寡聚化结构域(NOD)样受体热蛋白结构域蛋白3(NLRP3)炎症小体含量。单细胞测序分析AGNH对各细胞群的调控情况,并对差异表达基因进行基因本体论(GO)与京都基因与基因组百科全书(KEGG)通路富集分析,进而构建小胶质细胞差异表达基因网络,寻找关键靶点,并通过ELISA与IF进行验证。结果:与假手术组比较,模型组mNSS与脑含水量均显著增加(P<0.01),与模型组比较,AGNH低、高剂量组均可降低mNSS与脑含水量(P<0.05);HE染色结果表明,与假手术组比较,模型组大鼠大脑皮层和海马部位的细胞丢失严重,细胞排列较为松散(P<0.01),与模型组比较,给药AGNH可明显提高大脑皮层及海马CA1和CA3区的神经元细胞密度,使排列更紧凑,同时改善细胞形态(P<0.05,P<0.01)。ELISA与IF染色结果表明,与假手术组比较,模型组大鼠脑组织Caspase-1、IL-17A、TNF-α、NLRP3及COX-2水平均显著升高(P<0.01);与模型组比较,AGNH组各炎症因子水平均明显降低(P<0.05,P<0.01)。单细胞测序共鉴定到13个细胞亚群,其中小胶质细胞在神经Objective:To reveal the molecular mechanism of Angong Niuhuangwan(AGNH)in improving traumatic brain injury(TBI)based on single cell sequencing.Method:Seventy-five male SD rats were randomly divided into the sham group,model group,piracetam group(3.6 g·kg^(-1)),AGNH low-and highdose groups(0.09,0.27 g·kg^(-1)),with 15 rats in each group.In addition to the sham group,the other 4 groups used the modified Feeney free-fall impact method to prepare TBI model,and the drugs were administered by gavage immediately after modeling,24 hours later,the modified neurological deficit score(mNSS)was performed,and brain tissue was isolated to determine the degree of cerebral edema.Hematoxylin-eosin(HE)staining was used to observe the injury degree in the cortex,CA1 region and CA3 region of brain tissue.The expression levels of cyclooxygenase-2(COX-2),interferon regulatory factor 1(IRF1),Janus kinase 2(JAK2)and suppressor of cytokine signaling 3(SOCS3)were observed by immunofluorescence(IF)staining.The levels of interleukin(IL)-6,IL-18,IL-1β,IL-17A,tumor necrosis factor-α(TNF-α),Caspase-1 and nucleotide binding oligomerization domain(NOD)-like receptor heat protein domain associated protein 3(NLRP3)inflammasome were determined by enzyme-linked immunosorbent assay(ELISA).The regulation of AGNH on each cell population was analyzed by single cell sequencing,and differentially expressed genes were analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG),which led to construct microglia differentially expressed gene network to search for the key targets,and validated by ELISA and IF.Result:Compared with the sham group,the mNSS and brain water content were significantly increased in the model group(P<0.01).Compared with the model group,mNSS and brain water content in the low and high dose AGNH groups were decreased(P<0.05,P<0.01).HE staining results showed that compared with the sham group,the cells in the cerebral cortex and hippocampus of rats in the model group were seriously lost,and the cells were arranged

关 键 词：安宫牛黄丸 创伤性颅脑损伤 单细胞测序 炎症反应 小胶质细胞 中医药 

分 类 号：R22[医药卫生—中医基础理论] R28[医药卫生—中医学] R651[生物学—遗传学] Q342