Antiepileptic and neuroprotective mechanism of ursolic acid based on full-length transcriptome analysis
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摘要:
基于转录组分析探讨熊果酸（ursolic acid, UA）抗癫痫和改善癫痫诱导的GABAergic神经元损伤的可能机制。选取对照组（NC组）、癫痫组（SE组）及癫痫UA给药组（UA组）大鼠的海马组织进行全长转录组测序；测序数据利用GO (gene ontology, GO)、KEGG (kyoto encyclopedia of genes and genomes, KEGG)及PPI (protein-protein interaction networks, PPI)对差异基因(differential genes, DEGs)进行分析；利用RT-qPCR验证海马组织中关键差异基因的表达量；最后在原代神经元上构建体外癫痫模型，采用RT-qPCR对差异基因的表达量进行验证，并利用免疫荧光、Western blot进一步检测神经元上GABAA受体γ2亚基（GABRG2）的表达量。两两样本表达量相关性热图及DEGs聚类分析结果显示：SE组距离NC组最远，UA治疗后，总体向正常组偏移。SE组与UA组对比，共筛选出220个差异基因，其中143个基因上调，77个基因下调。GO富集分析显示：在一级分类中涉及生物过程、细胞成分和分子功能3个过程。KEGG通路富集分析表明，DEGs涉及cAMP信号通路、钙信号通路等36条生物学通路。PPI分析表明DEGs与GABA及炎症关系密切。RT-qPCR结果表明UA处理增加了海马组织中GABA受体相关基因（Gng4）、GABA合成相关基因（Camk2a、Vgf和Npy）、炎症相关基因（Timp1和Spp1）的表达量，降低了GABA合成相关基因（Nptx2）、cAMP相关通路基因（Gnas）的表达量；并进一步证实UA处理增加了神经元上Gng4、Camk2a的表达量，降低了Gnas的表达量。免疫荧光与Western blot结果显示，与SE组相比，UA给药后原代神经元上GABRG2的表达量增加。本研究丰富了UA抗癫痫的转录组数据，也为深入研究UA抗癫痫和神经保护奠定理论基础。
Abstract:This study explores the potential antiepileptic mechanism of ursolic acid (UA) and its improvement of GABAergic interneuron damage induced by epilepsy based on transcriptome analysis. Hippocampal tissues from rats in the control group (NC group), epilepsy group (SE group), and epilepsy UA treatment group (UA group) were subjected to full-length transcriptome sequencing. The obtained sequencing data were analyzed, using gene ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) to perform the analysis of differential genes (DEGs). The expression levels of key differential genes were verified using RT-qPCR in hippocampal tissue. Finally, an epilepsy in vitro model was constructed on primary neurons, RT-qPCR was used to verify the expression levels of key differential genes, and the expression level of GABAA receptor γ2 subunit (GABRG2) on neurons was further examined using immunofluorescence and Western blot. The heatmap of pairwise sample expression correlation and the clustering analysis of differentially expressed genes showed that the SE group was farthest from the NC group, and that after UA treatment, the overall trend shifted towards the normal group. Compared with the SE group, a total of 220 differential genes were screened in the UA group, including 143 upregulated genes and 77 downregulated genes. GO enrichment analysis showed that it involved three processes in the primary classification: biological processes, cellular components, and molecular functions. KEGG pathway enrichment analysis showed that DEGs were involved in 36 biological pathways, including cAMP signaling pathway and calcium signaling pathway. PPI analysis showed that DEGs were closely related to GABA and inflammation. RT-qPCR results showed that UA treatment increased the expression levels of GABA receptor-related gene (Gng4), GABA synthesis-related gene (Camk2a,Vgf, and Npy) and inflammation-related gene (Timp1 and Spp1) in hippocampal tissue, and decreased the expression levels of GABA synthesis-related gene (Nptx2) and cAMP-related pathway gene (Gnas). It further confirmed that UA treatment increased the expression levels of Gng4 and Camk2a on neurons and decreased the expression level of Gnas. Immunofluorescence and Western blot results showed that, compared with the SE group, the expression level of GABRG2 on primary neurons increased after UA treatment. This study enriched the transcriptome data of UA's antiepileptic effect and laid a theoretical foundation for further research on UA's antiepileptic and neuroprotective effects.
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Keywords:
- ursolic acid /
- epilepsy /
- full-length transcriptome /
- sequencing analysis /
- GABA
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铜绿假单胞菌（Pseudomonas aeruginosa, PA）是一种常见的医院感染病原体[1]，可在患有慢性阻塞性肺疾病、囊性纤维化、癌症、创伤、烧伤及免疫缺陷个体中发生急性或慢性感染[2]。近年来，随着广谱抗生素的广泛使用，多重耐药（multidrug-resistant, MDR）的PA引起的感染变得越来越常见。多重耐药指的是对3种或3种以上类别的抗生素不敏感，因此临床上治疗手段非常有限，而多黏菌素类抗生素是为数不多的对MDR-PA仍然有效的药物[3]。
多黏菌素类抗生素是20世纪40年代开发的一类用于治疗革兰氏阴性菌感染的脂肽类抗生素[4]。由于其抗菌谱窄，并具有明显的肾毒性和神经毒性，逐渐退出了临床[5]。然而，随着MDR革兰氏阴性菌感染逐年增加，尤其是耐碳青霉烯类革兰氏阴性菌的出现，使得多黏菌素类抗生素又重新在临床上使用[6]。
临床上使用的多黏菌素类抗生素主要有多黏菌素B和多黏菌素E。其中，多黏菌素B作为带有正电荷的阳离子多肽，可与革兰氏阴性菌外膜上带负电荷的脂多糖（lipopolysaccharide, LPS）结合，使得外膜破裂进而胞内容物流失，最终导致细菌死亡[7]。然而，PA可通过修饰LPS的脂质A成分，从而降低多黏菌素B与LPS的亲和力，最终导致耐药[8]。由arnBCADTEF操纵子编码的蛋白质介导，在脂质A成分中添加带正电荷的4-氨基-L-阿拉伯糖（4-amino-4-deoxy-L-arabinose, L-Ara4N），是PA的LPS修饰的常见形式[9−10]。而上述操纵子基因的表达可由双组分系统PmrA-PmrB调控[11]。有研究表明，PA的pmrB上的碱基发生突变，可导致PA对多黏菌素B产生抗性。如Abraham等[12]从临床上发现的一株耐多黏菌素B的PA，其pmrB第292位碱基发生（T→C）突变；Barrow 等[13]发现pmrB在739位的（G→A）突变也可引起PA对多黏菌素B的耐药。
本研究通过阶梯式诱导的方式，获得了一株对多黏菌素B耐药的PA菌株。全基因组测序结果显示其pmrB基因第514−516位发生了缺失突变。通过同源重组、转录组测序（RNA sequencing，RNA-seq）、反转录荧光定量PCR（reverse transcription quantitative PCR, RT-qPCR）以及抗菌活性检测发现该缺失突变可导致pmrA、pmrB及arnBCADTEF操纵子基因的转录水平升高，细菌对多黏菌素B的敏感性下降。因此，本研究发现了一个新的可引起PmrA-PmrB功能改变的缺失突变形式，从而拓展了人们对多黏菌素B耐药机制的认识。
1. 材 料
1.1 菌株和质粒
PA标准菌株ATCC27853购自ATCC菌株保藏中心，pEX18Tc质粒购自上海禾午生物科技有限公司，大肠埃希菌DH5α感受态细胞、含有pUC57-pmrB⊿514-516质粒的大肠埃希菌Top10菌株购自上海生工生物工程有限公司。
1.2 培养基
LB液体培养基：10 g/L胰蛋白胨、5 g/L酵母粉、5 g/L NaCl。BHI（brain heart infusion）液体培养基：脑心浸粉17.5 g/L、葡萄糖2 g/L、胰蛋白胨10 g/L、NaCl 5 g/L、Na2HPO4 2.5 g/L。TYS10（tryptone yeast extract and sucrose 10）培养基：蛋白胨10 g/L，酵母提取物5 g/L，10%的过滤（0.22 µm）蔗糖。固体培养基：加琼脂粉于上述液体培养基至15 g/L。除特殊提及外，菌株在37 ℃的LB或BHI液体培养基中220 r/min振荡培养，37 ℃的LB或BHI固体培养基中静置培养。
1.3 试剂和仪器
MH（Mueller Hinton）培养基、胰蛋白胨、酵母粉、琼脂粉等（英国Oxoid公司）；NaCl、MgSO4、Na2HPO4（上海BBI生命科学有限公司）；质粒小提试剂盒、细菌基因组DNA提取试剂盒、细菌总RNA提取试剂盒（北京天根生化科技有限公司）；氨苄西林、四环素、RT Master Mix for qPCR Ⅱ试剂盒、SYBR Green qPCR Master Mix试剂盒（美国MedChem Express公司）。引物的合成由上海生工生物工程有限公司完成。微量分光光度计（美国Biochrom SimpliNano公司），PCR扩增仪、实时荧光定量PCR仪、Gene Pulser Xcell™ 电穿孔仪（美国Bio-Rad公司）。
2. 方 法
2.1 多黏菌素B自发耐药PA（PA-PBsr）的筛选
将单菌落的PA ATCC27853接种在含有多黏菌素B （1 mg/mL）2 µL的LB液体培养基2 mL中培养过夜。按1∶100转移到含有多黏菌素B（1 mg/mL）4 µL的LB液体培养基2 mL中继续培养过夜。重复上述操作，逐级增加多黏菌素B的浓度，直至细菌无法生长。将在多黏菌素B最大浓度下能够生长的细菌涂布在LB平板上，挑取单菌落后接种至LB液体培养基3 mL中培养至A600 为 0.6~0.8。采用微量肉汤稀释法测定出多黏菌素B对纯化菌株的最低抑菌浓度（ MIC），对MIC发生变化的菌株进行全基因组测序。
2.2 全基因组测序
挑取对MIC发生变化的菌株的单菌落，加入到LB培养液5 mL中培养过夜，按照细菌基因组DNA提取试剂盒提取DNA，取基因组DNA 1 µg（质量浓度≥10 ng/µL，A260/280=1.8~2.0）送至上海生工生物工程股份有限公司进行全基因组测序。使用Qubit 3.0对文库浓度进行初步定量，使用Agilent 2100检测文库片段的完整性。文库检测通过后，使用Illumina Hiseq 2000进行测序。测序得到的原始数据经过质控筛选、去除接头等后，利用SPAdes 3.12.0进行基因组组装，得到菌株PA-PBsr基因组序列。
2.3 PA的pmrB基因第514−516位缺失突变株（PA-pmrB⊿514-516）的构建
将含有pUC57-pmrB⊿514-516 质粒的大肠埃希菌TOP10单菌落接种于含有氨苄西林（50 µg/mL）的LB培养基5 mL中培养过夜；使用质粒小提试剂盒提取质粒。在冰上，将pUC57-pmrB⊿514-516 质粒5 µg与DH5α感受态细胞100 µL混合后孵育30 min，2.5 kV条件下于2 mm电转杯中电转, 添加LB培养基700 µL后转移至1.5 mL离心管培养1.5 h，
5000 r/min离心5 min，弃去部分上清液，留培养基100 µL重悬细菌并涂布于含氨苄西林（50 µg/mL）的LB平板继续培养；挑取单菌落接种至LB培养基3 mL培养过夜，提取质粒后使用引物pmrB-MUT-CXF1和pmrB-MUT-CXR1进行PCR验证。使用KpnⅠ和HindⅢ对pUC57-pmrB⊿514-516进行双酶切。酶切体系为：10×buffer（Cutsmart）10 µL，DNA模板4 µg，KpnⅠ（
20000 U/mL）2 µL，HindⅢ（20000 U/mL）2 µL，ddH2O补充至100 µL。使用KpnⅠ和HindⅢ对pEX18Tc质粒进行双酶切，酶切体系为：10 × buffer（Cutsmart）20 µL，DNA模板8 µg，KpnⅠ（20000 U/mL）4 µL，HindⅢ（20000 U/mL）4 µL，ddH2O补充至200 µL。37 ℃水浴酶切过夜进行胶回收。使用连接酶将回收后的目标片段和载体片段连接，酶连体系为：10 × T4 DNA 连接酶缓冲液 10 µL，T4 DNA 连接酶（40000 U/mL）2 µL，目标片段5 µg，载体片段5 µg，ddH2O补充至100 µL，16 ℃孵育过夜。将pEX18Tc-pmrB⊿514-516电转至（方法同上）DH5α感受态细胞100 µL，将菌液涂布于含四环素（20 µg/mL）的BHI平板继续培养。挑取单菌落培养后以pEX18-F和PA-pmrB-R引物进行PCR验证。将PCR阳性菌落接种至含有四环素（20 µg/mL）的LB培养基中，37 ℃振荡培养过夜后提取质粒（pEX18Tc-pmrB⊿514-516）。取PA ATCC27853单菌落，接种至LB培养基5 mL中，42 ℃静置培养过夜，
12000 r/min离心1 min后，使用MgSO4（1 mmol/L）1 mL洗涤3次，室温下重悬于MgSO4（1 mmol/L）50 µL中。冰上将pEX18Tc- pmrB⊿514-516 质粒5 µg与上述感受态50 µL混合孵育30 min，2.2 kV条件下于2 mm电转杯中电转，添加BHI培养基700 µL后转移至1.5 mL离心管后培养3 h，6000 r/min离心3 min后弃部分上清液，留培养基100 µL重悬细菌并涂布于含四环素（100 µg/mL）的BHI平板中培养64 h。挑取单菌落接种至含有四环素（20 µg/mL）的LB培养基3 mL中培养过夜，提取基因组DNA，以pEX18-F和PA-pmrB-R为引物进行PCR反应；将PCR结果为阳性的菌液涂布至含有四环素（100 µg/mL）的BHI平板培养过夜，挑取单菌落接种至TYS10平板上23 ℃培养64 h，挑取单菌落至LB培养基5 mL中培养过夜后提取基因组DNA，以pmrB-MUT-CXF1和pmrB-MUT-CXR1为引物进行PCR验证。扩增产物送上海生工生物工程有限公司进行测序验证。本研究所用引物参见表1。Table 1. Primers used in this studyPrimer Sequence(5′→3′) Sources pmrB-MUT-CXF1 CGCCTGCTGGTCAACCT This study pmrB-MUT-CXR1 CAGCAGGAGGTTGAGTTCGT This study pEX18-F GGCTCGTATGTTGTGTGGAATTGTG This study PA-pmrB-R GCAGGAGGTTGAGTTCGTCG This study rpsL-QF GTGGTGAAGGTCACAACCTG [14] rpsL-QR CCTGCTTACGGTCTTTGACA [15] pmrA-QF CACCAGGTGACCCTGTCC [14] pmrA-QR CGTAGAGGCTCTGCTCCAGT [15] pmrB-QF CCTCTCGCTGAAGCAGGTGA [15] pmrB-QR CTGGTCTTCGGTGGCAAGGT [16] arnB-QF CGCGATCAAGAACCTGACCT This study arnB-QR GGTCGGCCAGGTTGTATTTG This study arnC-QF AGTTGCGGTTGAGGATCACC This study arnC-QR TCTACAACGAGGAAGCCAGC This study arnA-QF CATCGGCATCCATTCGGAGT This study arnA-QR CGTTTGCCGTATTTCACGCA This study arnD-QF GCGACCTTCTTCTTCAGCGT This study arnD-QR AGCAGGATGTCCCAGCCATA This study arnT-QF CCGCAATTCACCTTCTGGGTC [16] arnT-QR CGAGGAAGCCCTTGGTCAGG [16] arnE-QF TCTGCTGGCTGCTGCTCCTG [16] arnE-QR CATCGAAGACGAAGCGTGCC [16] arnF-QF GTGCTTTCCTCGACGGATGA This study arnF-QR CAGTACCAGCAAGACCCTGG This study 2.4 MIC的测定
使用Ca2+、Mg2+调节过的MH培养基将对数生长期（A600 = 0.6~0.8）的PA野生株、PA-PBsr及PA-pmrB⊿514-516稀释至A600 为 0.001，96孔板第1孔加入待测菌液200 µL，第2孔至第10孔每孔加入菌液100 µL；第1孔加入多黏菌素B（1 mg/mL）6.4 µL，混匀后吸取菌液100 µL至第2孔，以此类推进行倍比稀释直至最后一孔混匀后弃掉100 µL。MH培养基、不含药物的菌液及DMSO 100 µL分别作阴性、阳性和空白对照。37 ℃静置培养18 h后，在每孔加入噻唑蓝（5 mg/mL）10 µL，37 ℃静置培养30 min后测量595 nm波长下的吸收度。
2.5 时间杀菌曲线
分别挑取PA野生株和PA-pmrB⊿514-516单菌落至LB培养基3 mL，培养至A600 为0.5。加入多黏菌素B至野生株与突变株菌液，使终浓度为0、16、32 µg/mL，在96孔板中按每孔总体积200 µL，37 ℃条件下培养。以2 h的间隔测量600 nm波长下的吸收度，平行试验3次，数据取均值。
2.6 抑菌圈
分别用棉拭子蘸取麦氏浊度0.5的野生株PA和突变株PA-pmrB⊿514-516的菌液，均匀涂布在MH平板上。滴加多黏菌素B（0.1 mg/mL）3 µL至MH平板，以生理盐水为阴性对照，37 ℃条件下培养过夜。游标卡尺测量抑菌圈直径，平行试验3次，数据取均值。
2.7 RNA-seq
分别培养野生株PA和突变株PA-pmrB⊿514-516 3 mL至对数生长期（A600 = 0.6~0.8），使用细菌总RNA提取试剂盒提取总RNA，取总RNA （质量浓度>50 ng/µL，A260/280=1.8~2.0）1 µg。使用RT Master Mix for qPCR Ⅱ 试剂盒进行反转录，使用NEB Next® Ultra™ Directional RNA Library Prep Kit for Illumina®试剂盒对样本进行建库。文库构建完成并质检后，使用Illumina Novaseq
6000 进行转录组测序。采用DESeq2进行基因差异表达分析。差异基因筛选条件以差异倍数（Fold change，FC）和P值作为参考指标。|log2FC| > 0.5和P < 0.05作为差异基因判断标准。测序工作主要由上海伟寰生物科技有限公司完成。2.8 RT-qPCR
取对数生长期（A600 = 0.6~0.8）的PA野生株及PA-pmrB⊿514-516 3 mL，使用细菌总RNA提取试剂盒提取总RNA。取总RNA 2 µg，使用RT Master Mix for qPCR Ⅱ 试剂盒进行反转录后，使用SYBR Green qPCR Master Mix试剂盒检测基因表达水平。以rpsL的表达作为内参对照，采用2−ΔΔCt法分析基因表达的相对变化。两组之间的比较采用非配对t检验。
2.9 数据处理及分析
采用GraphPad Prism 8.0.2对试验数据进行数据处理，P< 0.05作为显著性差异的标准。
3. 结 果
3.1 PA-PBsr的筛选及测序
通过阶梯式诱导的方式获得了一株对多黏菌素B耐药的PA菌株PA-PBsr（MIC=8 µg/mL），与野生型相比，其MIC增加到了原来的4倍。全基因组测序显示，与亲本株ATCC27853相比，PA-PBsr的pmrB基因第514−516位碱基发生了缺失突变，导致其编码蛋白同步发生了第172位亮氨酸缺失突变。
3.2 突变菌株的构建与验证
如图1所示，pmrB-MUT-CXF1和pmrB-MUT-CXR1扩增条带大小为657 bp，表明pUC57-pmrB⊿514-516成功电转至感受态细胞中；pEX18-F和PA-pmrB-R扩增条带大小为325 bp（图2），表明已成功将pmrB⊿514-516片段连接至pEX18Tc。引物pmrB-MUT-CXF1和pmrB-MUT-CXR1扩增产物测序结果见图3，测序结果表明pmrB的第514−516位碱基缺失突变株构建成功。
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Figure 1. pUC57-pmrB⊿514-516 positive bacteria were verified by PCRM: DL1200 DNA Maker; Lane 1: Negative control (empty plasmid); Lane 2: Blank control (without plasmid); Lane 3: Experimental hole![]()
Figure 2. pEX18Tc- pmrB⊿514-516 positive bacteria were verified by PCRM: DL1200 DNA Maker; Lane 1: Negative control (empty plasmid); Lane 2: Blank control (without plasmid); Lane 3: Experimental hole![]()
Figure 3. Deletion mutation site sequencing verification resultsA: Wild-type strain; B: Strain after homologous recombination3.3 多黏菌素B对突变株的MIC、杀菌曲线和抑菌圈的测定
多黏菌素B对野生株PA及突变株PA-pmrB⊿514-516的MIC分别是2和8 µg/mL，表明pmrB第514−516位点缺失突变与PA对多黏菌素B的耐药性改变有关。
时间杀菌曲线结果表明，如图4所示，多黏菌素B对野生株PA及PA-pmrB⊿514-516的最低杀菌浓度分别为16和32 µg/mL，表明PA-pmrB⊿514-516对多黏菌素B表现出更强的耐受性。
当多黏菌素B质量浓度为0.1 mg/mL时，野生株PA和突变株PA-pmrB⊿514-516形成的抑菌圈如图5所示。野生株PA的抑菌圈平均值为11.4 mm，突变株PA-pmrB⊿514-516的抑菌圈平均直径为6.1 mm，表明突变株对多黏菌素B敏感性降低。
3.4 差异表达基因分析
如图6所示，与野生株PA相比，突变株PA-pmrB⊿514-516总共有787个基因发生显著变化，其中有418个基因表达上调，369个基因表达下调。其中，与多黏菌素B耐药性相关的pmrA、pmrB及arnBCADTEF都发生了上调（表2）。
Table 2. Changes in the related gene expression regulated by pmrBSymbol log2 Fold change P Stat Product pmrA 5.9 0 up PmrA: two-component regulator system response regulator PmrA pmrB 6.1 1.1 × 10−260 up PmrB: two-component regulator system signal sensor kinase PmrB arnB 7.5 7.3 × 10−5 up ArnB arnC 6.9 4.0 × 10−212 up ArnC arnA 7.2 0 up ArnA arnD 8.1 8.9 × 10−8 up ArnD arnT 7.0 0 up inner membrane L-Ara4N transferase ArnT arnE 7.4 1.6 × 10−26 up ArnE arnF 7.4 6.5 × 10−7 up ArnF 3.5 RT-qPCR结果分析
如图7所示，与野生型相比，在PA-pmrB⊿514-516中，受PmrB调控的基因pmrA、pmrB及arnBCADTEF的表达量均显著升高。其中pmrA和pmrB基因表达分别升高8.6倍和 3.4倍，而arnBCADTEF操纵子各基因组分的表达量分别升高31.3、56.9、43.8、5.1、45.2、5.3和19.9倍。结果与转录组测序结果相一致。
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Figure 7. Histogram of RT-qPCR to validate of microarray results**P < 0.01, ***P < 0.001, ****P < 0.000 1 vs control group4. 讨 论
随着抗生素的广泛使用，对抗生素产生耐药性的PA越来越常见。全国细菌耐药监测网显示，2021年我国PA对碳青霉烯类抗生素的耐药率为17.7%[17]。多黏菌素B是治疗对碳青霉烯耐药PA的重要手段。细胞外膜脂质A成分的修饰可使PA对多黏菌素B产生耐受性，其常见形式是在脂质A成分中添加正电荷的L-Ara4N，从而降低多黏菌素B与LPS的亲和力，而该过程是由arnBCADTEF操纵子编码的蛋白质介导的，该操纵子则受双组分系统PmrA-PmrB调控。目前已有文献表明，PA对多黏菌素B产生抗性，与PA的pmrB上的碱基发生突变存在一定关系[12−13]。
本研究使用多黏菌素B对PA进行亚抑菌浓度诱导，获得了一株PA-PBsr，该菌株在pmrB基因发生了第514−516位的缺失突变。我们通过同源重组，构建PA-pmrB⊿514-516突变株后发现，该突变可介导PA对多黏菌素B产生抗性。另外，RNA-seq和RT-qPCR结果显示该突变可引起pmrA、pmrB和操纵子arnBCADTEF表达量升高，提示PmrA-PmrB功能表达增强。因此，本研究明确了一个可以引起PA对多黏菌素B产生耐药性的新的pmrB突变形式，对于进一步认识和了解PA对多黏菌素B的耐药机制提供了新的数据。鉴于该突变形式目前还未在临床耐药株中发现，因此，在今后使用多黏菌素B治疗PA感染时应注意由该突变引起的耐药，从而体现了本研究对于临床用药的指导意义。然而，PA的pmrB⊿514-516突变是如何导致双组分系统PmrA-PmrB功能增强，其具体作用机制还需要进一步研究。
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Figure 1. Analyses of ORF, transcription factor, and transcript expression after epilepsy treated by ursolic acid
A:Amino acid sequence length distribution in ORF coding region;B:Distribution of transcription factors;C:Box plots of expression levels of three groups of transcripts NC：Normal control；SE：Status epilepticus；UA：Ursolic acid
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Figure 2. Heat map of expression correlation between every two samples after epilepsy following the ursolic acid treatment
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Figure 3. Differentially expressed genes overall situation after ursolic acid treatment with epilepsy
A:Venn analysis of differentially expressed genes between every two groups; B:Volcano plot of DEGs between SE group and UA group
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Figure 4. GO enrichment analysis of DEGs between the SE group and the UA group The numbers on the right of the figure have the following meanings:the black font represents the number of transcripts, and the blue is the number of differential transcripts
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Figure 5. KEGG annotation results of the DEGs between the SE group and the UA group were classified according to the pathway types in KEGG
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Figure 6. PPI network of the DEGs after UA treatment of epilepsy
A:Protein interaction between the DEGs and GABA receptor genes; B:Protein interaction between the DEGs and GABA synthesis gene; C:Protein interaction between the DEGs and some inflammatory-related genes (Red: up-regulated genes, green: down-regulated genes)
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Figure 7. mRNA expression level of representative differential genes associated with ursolic acid treatment after epilepsy on the hippocampus and primary neurons
A:Representative genes associated with GABA receptors and GABA synthesizes;B: Representative genes associated with inflammation;C:Expression of differential genes Gng4, Gnas, and Camk2a was verified by the epilepsy model constructed from primary neurons cultured with low magnesium in vitro. The relative mRNA levels were determined by normalizing to the level of GAPDH. The relative gene expression data were analyzed using the ΔΔCT method ($ \bar{x} $±s, n=3) ****P<0.001, ***P<0.001, **P<0.01, *P<0.05 vs SE group
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Figure 8. Ursolic acid promoted the GABRG2 expression after epilepsy induction on primary neurons
A-B:Western blot of GABRG2; C-D:Immunofluorescence staining of GABRG2, Cell nuclei were stained by DAPI (blue). The GABRG2 marked by anti-GABRG2 was visualized with Alexa Fluor®488 secondary antibodies (green) ($ \bar{x} $±s, n=3) ****P<0.0001, *P<0.05 vs SE group
Table 1 Primer list used to verify differential genes
Gene F-sequence (5' to 3') R-sequence (5' to 3') Gng4 CTGAAGGAAGCCTGCATGGAC AGGCACAGGGATGATGAGAGGATC Nptx2 GCATTCAAAGTGTCCCTCCCTCTC CAGGCAGATGAAGGCATACAG Camk2a CTGAGAGCACCAACACCACCATC GTCATTCCAGGGTCGCACATCTTC Vgf GAACTGTCCACCAAACTCCACCTG CTTCTTCCGCTTCCGTTTCTCCTC Npy TGTGTTTGGGCATTCTGGCTGAG TGAGATTGATGTAGTGTCGCAGAGC Timp1 CTTCCTGGTTCCCTGGCATAATCG TCCACAAGCAATGACTGTCACTCTC Spp1 GACGATGATGACGACGACGATGAC GTGTGCTGGCAGTGAAGGACTC Gapdh TGATTCTACCCACGGCAAGTT TGATGGGTTTCCCATTGATGA 
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Table 2 New isoform annotation of all samples
Annotated database New isoform number COG 3230 GO 17480 KEGG 19042 KOG 11771 Pfam 12323 Swiss-Prot 18006 TrEMBL 23761 eggNOG 15071 NR 25207 All 25427
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Table 3 Annotation of DETs between every two groups
DET set DET Number Upregulated Down-regulated COG GO KEGG KOG NR Pfam Swiss-Prot egg NOG NC1_NC2_NC3_vs_SE1_SE2_SE3 1683 1270 413 225 966 1214 625 1475 763 1035 840 NC1_NC2_NC3_vs_UA1_UA2_UA3 3334 2478 856 407 1799 2544 1059 3075 1397 2 035 1 941 SE1_SE2_SE3_vs_UA1_UA2_UA3 143 76 67 24 116 100 71 129 102 114 104
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Table 4 Biological pathways of the DEGs
Biological pathway Gene number Gene Neuroactive ligand-receptor interaction 9 Tac1, Penk, Npy, Calcb, Calca, Gal, Mchr1, Pyy, Ntsr MAPK signaling pathway 6 Flnc, Grasp, Fos, Sbno2, Gadd45b, Flna PI3K/Akt/mToR signaling 6 Lamc2, Col4a1, Spp1, Gng4, Tnc, AC114343.1 Calcium signaling 5 Itpka, Sphk1, Camk2a, Gnas, Asphd2 cAMP signaling 4 Fos, Npy, Camk2a, Gnas Dopaminergic synapse 4 Fos, Camk2a, Gnas, Gng4 Glutamatergic synapse 3 Gnas, Homer1, Gng4 Cholinergic synapse 3 Fos, Camk2a, Gng4 Toll-like receptor signaling pathway 3 Fos, Irf7, Spp1
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