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

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免疫中发挥重要作用；对小胶质细胞差异表达基因的GO富集分析结果显示，AGNH改善TBI涉及细胞对炎症反应和TNF-α的反应等；KEGG富集到IL-17信号通路、TNF信号通路、Toll样受体信号通路等；网络分析结果显示，AGNH调控TBI的关键靶点可能为IL-6、IL-1β、JAK2、SOCS3、IRF1。IF与ELISA验证结果显示，与假手术组比较，模型组大鼠脑组织中IL-6、IL-1β含量升高，小胶质细胞中SOCS3表达减少，JAK2、IRF1的表达增加（P<0.01）；与模型组比较，AGNH组大鼠脑组织中IL-6、IL-1β含量降低，小胶质细胞中SOCS3表达增加，JAK2、IRF1的表达减少（P<0.05，P<0.01）。结论 AGNH可减轻TBI大鼠的脑水肿与脑损伤程度，减少炎症因子表达，抑制NLRP3及其下游Caspase-1的表达，可能通过作用于小胶质细胞中的IL-6、IL-1β、JAK2、IRF1、SOCS3等靶点发挥作用。

Abstract:

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 high-dose 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 loosely（P<0.01）. Compared with the model group， AGNH could significantly increase the density of neurons in the CA1 and CA3 regions of the cerebral cortex and hippocampus， making the arrangement more compact， as well as improved cell morphology（P<0.05，P<0.01）. ELISA and IF staining showed that AGNH could reduce the levels of Caspase-1， IL-17A， TNF-α， NLRP3 and COX-2 in brain tissue of TBI rats（P<0.05， P<0.01）. A total of 13 cell subsets were identified by single cell sequencing， among which microglia played an important role in neuroimmunity. The results of GO enrichment analysis of differentially expressed genes in microglia showed that AGNH improved TBI in response to inflammation and TNF-α. KEGG enriched IL-17 signaling pathway， TNF signaling pathway， Toll-like receptor signaling pathway， etc. The results of network analysis showed that the key targets of AGNH in regulating TBI might be IL-6， IL-1β， JAK2， SOCS3， IRF1. IF and ELISA verification results showed that compared with the sham group， SOCS3 expression in microglia was decreased in the model group， and the expressions of IL-6， IL-1β， JAK2 and IRF1 were increased（P<0.01）. Compared with the model group， AGNH could increase the expression of SOCS3， decrease the expression of IL-6， IL-1β， JAK2， IRF1 （P<0.05， P<0.01）.Conclusion AGNH can reduce the degree of brain edema and brain injury， decrease the expression of inflammatory factors， and inhibit the expression of NLRP3 and its downstream Caspase-1 in TBI rats， which may act on the targets of IL-6， IL-1β， JAK2， IRF1 and SOCS3 in microglia.