粪肠球菌感染小鼠的脑组织病理学观察及转录组学差异分析

doi:10.11843/j.issn.0366-6964.2022.09.030

摘要/Abstract

摘要： 旨在通过构建小鼠感染粪肠球菌后的脑部损伤模型，对不同感染时期脑组织的病理学观察及转录组学差异分析，探索粪肠球菌引起脑组织损伤的机制。本试验选择1株致脑膜炎粪肠球菌，以小鼠为感染动物，感染粪肠球菌后，分别在2、4、6、12、24、36、60和72 h采集小鼠脑组织，观察脑组织的损伤程度，挑选早期、明显期和转归期3个时间点，通过转录组学测序，对差异表达基因进行分析，使用荧光定量PCR对转录组学数据进行验证。结果显示：小鼠感染粪肠球菌12 h后，脑组织开始出现病变，通过病理组织学观察发现小鼠脑组织先后出现了脑膜及脑组织血管充血，血管周围间隙增宽，脑组织因水肿有网格状间隙，微血栓形成及炎性细胞浸润。对转录组学差异基因分析，GO富集结果显示主要富集到对β干扰素的反应、细胞对β干扰素的反应、对其他生物的防御反应、对γ干扰素的反应、对细菌的反应、外在凋亡信号通路、调节外在凋亡信号通路、神经元凋亡过程、内皮细胞迁移等生物进程中。KEGG富集结果显示差异信号通路主要富集在过氧物酶体、NOD样受体信号通路、氧化磷酸化、钙代谢信号传导途径、PI3K-Akt信号传导途径、Wnt信号通路、MAPK信号通路、GnRH分泌、VEGF信号通路、紧密连接等一些与脑组织损伤相关的通路上。粪肠球菌感染小鼠后，影响脑组织过氧物酶体、NOD样受体信号通路、氧化磷酸化、钙代谢信号传导途径、PI3K-Akt信号传导途径等通路改变，这些通路与蛋白磷酸化、炎症的发生及血脑屏障通透性的改变有关，为进一步研究粪肠球菌引起脑组织损伤机制提供研究方向和理论基础。

关键词: 粪肠球菌, 小鼠, 脑膜炎, 病理学, 差异基因

Abstract: The study aimed to explore the mechanism of brain tissue injury caused by Enterococcus faecalis, a brain injury model from the mice infected with E. faecalis was constructed. Pathological observation and transcriptomic difference analysis for different period of infection were conducted. In this experiment, a strain of E. meningitidis was selected. Mice were used as infected animals. After being infected with E. faecalis, the brain tissues of mice were collected in time periods:at 2, 4, 6, 12, 24, 36, 60 and 72 h, separately. Then, the degree of brain tissue damage were observed. Three time points:early stage, obvious stage and prognosis stage, were selected to analyze differentially expressed genes by transcriptomic sequencing, and fluorescent quantitative PCR was used to verify the transcriptomic data. After 12 h, lesions occurred in the brain tissues of the mice infected. Histopathological observation showed that the meninges presented and brain tissue blood vessels were congested successively in the mouse brain tissue. The perivascular space was widened, and the brain tissue presented grid gap due to edema. Microthrombosis and inflammatory cell occurred. For transcriptomic differential gene analysis, the GO enrichment results showed that the main enrichment was the response to interferon-β, the response of cells to interferon-β, the defense response to other organisms, the response to interferon-γ, the response to bacteria, response, extrinsic apoptosis signaling pathway, regulation of extrinsic apoptosis signaling pathway, neuronal apoptosis process, endothelial cell migration and other biological processes. KEGG enrichment results showed that differential signaling pathways were mainly enriched relative signal paths related to brain tissue damage:peroxisome, NOD-like receptor signaling pathway, oxidative phosphorylation, calcium metabolism signaling pathway, PI3K-Akt signaling pathway, Wnt signaling pathway, MAPK signaling pathway, GnRH secretion, VEGF signaling pathway, tight junction and some other related pathways. After the infection of mice, E. faecalis affected the brain tissue peroxisome, NOD-like receptor signaling pathway, oxidative phosphorylation, calcium metabolism signaling pathway, PI3K-Akt signaling pathway and other pathways. These are related to protein phosphorylation, the occurrence of inflammation and the change of the permeability of the blood-brain barrier. The study conclusion provides a research direction and theoretical basis for further research on the mechanism of brain tissue damage caused by E. faecalis.

Key words: Enterococcus faecalis, mice, meningitis, pathology, differential genes

中图分类号:

曹梦园, 陈明杰, 王晨豫, 李益涛, 闫雪琪, 陈杰, 齐亚银. 粪肠球菌感染小鼠的脑组织病理学观察及转录组学差异分析[J]. 畜牧兽医学报, 2022, 53(9): 3160-3171.

CAO Mengyuan, CHEN Mingjie, WANG Chenyu, LI Yitao, YAN Xueqi, CHEN Jie, QI Yayin. Pathological Observation and Transcriptomic Difference Analysis from Mice Infected with Enterococcus faecalis[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3160-3171.

参考文献 30

[1]	STUCKI K, HARBARTH S, NENDAZ M. Enterococcal infections:from simple to most complex[J]. Rev Med Suisse, 2014, 10(446):1918, 1920-1923.
[2]	VAN TYNE D, GILMORE M S. Friend turned foe:evolution of enterococcal virulence and antibiotic resistance[J]. Annu Rev Microbiol, 2014, 68:337-356.
[3]	ARIAS C A, MURRAY B E. The rise of the Enterococcus:beyond vancomycin resistance[J]. Nat Rev Microbiol, 2012, 10(4):266-278.
[4]	CH'NG J H, CHONG K K L, LAM L N, et al. Biofilm-associated infection by enterococci[J]. Nat Rev Microbiol, 2019, 17(2):82-94.
[5]	FUKUOKA K, FURUICHI M, ITO K, et al. Longer duration of urinary catheterization increases catheter-associated urinary tract infection in PICU[J]. Pediatr Crit Care Med, 2018, 19(10):e547-e550.
[6]	DAHL A, MIRO J M, BRUUN N E. Enterococcus faecalis bacteremia:please do the echo[J]. Aging (Albany NY), 2019, 11(23):10786-10787.
[7]	BONAZZETTI C, MORENA V, GIACOMELLI A, et al. Unexpectedly high frequency of Enterococcal bloodstream infections in coronavirus disease 2019 patients admitted to an Italian ICU:an observational study[J]. Crit Care Med, 2021, 49(1):e31-e40.
[8]	PERICàS J M, AMBROSIONI J, MUÑOZ P, et al. Prevalence of colorectal neoplasms among patients with Enterococcus faecalis endocarditis in the GAMES cohort (2008-2017)[J]. Mayo Clin Proc, 2021, 96(1):132-146.
[9]	SUN J L, SONG X B, KRISTIANSEN B E, et al. Occurrence, population structure, and antimicrobial resistance of enterococci in marginal and apical periodontitis[J]. J Clin Microbiol, 2009, 47(7):2218-2225.
[10]	邹晓旭, 张敏, 赵露, 等. 我国抗菌药物临床应用专项整治情况调查与分析[J]. 中国医院管理, 2014, 34(2):10-12.ZOU X X, ZHANG M, ZHAO L, et al. Investigation and analysis of antibiotic stewardship program in China[J]. Chinese Hospital Management, 2014, 34(2):10-12. (in Chinese)
[11]	KHODABANDEH M, MOHAMMADI M, ABDOLSALEHI M R, et al. High-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium;as a serious threat in hospitals[J]. Infect Disord Drug Targets, 2020, 20(2):223-228.
[12]	狄婷婷, 高原. 粪肠球菌主要毒力因子研究进展[J]. 中国病原生物学杂志, 2012, 7(3):231-234.DI T T, GAO Y. Advances in research on pathogenicity islands of Enterococcus faecalis[J]. Journal of Pathogen Biology, 2012, 7(3):231-234. (in Chinese)
[13]	李慧, 王蒙蒙, 李傲寒, 等. 致动物脑膜炎粪肠球菌人工感染小鼠脑部模型的建立[J]. 动物医学进展, 2019, 40(8):34-39.LI H, WANG M M, LI A H, et al. Establishment of brain model of artificially infected mice with Enterococcus faecalis causing animal encephalitis[J]. Progress in Veterinary Medicine, 2019, 40(8):34-39. (in Chinese)
[14]	李益涛, 曹梦园, 陈明杰, 等. 体外血脑屏障模型的构建及致脑膜炎粪肠球菌对其的影响[J]. 中国畜牧兽医, 2021, 48(7):2549-2558.LI Y T, CAO M Y, CHEN M J, et al. Establish of a blood-brain barrier model in vitro and the influence of meningitis-causing Enterococcus faecalis on the model[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(7):2549-2558. (in Chinese)
[15]	周霞, 剡根强, 马勋, 等. 致羔羊脑炎粪肠球菌人工感染小鼠模型的建立[J]. 西北农业学报, 2007, 16(5):15-17, 21.ZHOU X, YAN G Q, MA X, et al. An animal model for encephalitis in lamb caused by Enterococcus faecalis[J]. Acta Agriculturae Boreali-Occidentalis Sinica, 2007, 16(5):15-17, 21. (in Chinese)
[16]	李慧. 粪肠球菌对小鼠血脑屏障通透性及脑组织损伤相关因素的研究[D]. 石河子:石河子大学, 2019.LI H. Effect of Enterococcus faecalis on blood-brain barrier permeability and brain tissue injury in mice[D]. Shihezi:Shihezi University, 2019. (in Chinese)
[17]	GOLDSTEIN L D, CAO Y, PAU G, et al. Prediction and quantification of splice events from RNA-Seq data[J]. PLoS One, 2016, 11(5):e0156132.
[18]	MOHAMED J A, HUANG D B. Biofilm formation by enterococci[J]. J Med Microbiol, 2007, 56(Pt 12):1581-1588.
[19]	周霞. 肠球菌性羔羊脑炎的发现及其病原特性和诊断方法研究[D]. 雅安:四川农业大学, 2007.ZHOU X. The finding of lamb encephalitis induced by enterococcus and studies on its pathogenic characteristics and diagnosis method[D]. Yaan:Sichuan Agricultural University, 2007. (in Chinese)
[20]	黎满香. 感染猪的粪肠球菌的分离鉴定及部分特性和诊断方法研究[D]. 长沙:湖南农业大学, 2010.LI M X. Isolation and Identification of E. faecalis from infected pigs and studies on its partial characteristics and diagnosis method[D]. Changsha:Hunan Agricultural University, 2010. (in Chinese)
[21]	王玉峰, 于宁, 刘晓军, 等. 熊源粪肠球菌小鼠感染模型的建立及其毒力基因的检测[J]. 畜牧与兽医, 2019, 51(9):95-99.WANG Y F, YU N, LIU X J, et al. Establishment of mice pathological model of Enterococcus faecalis and detection of its virulence genes[J]. Animal Husbandry & Veterinary Medicine, 2019, 51(9):95-99. (in Chinese)
[22]	RASOULI J, CASELLA G, ISHIKAWA L L W, et al. IFN-β acts on monocytes to ameliorate CNS autoimmunity by inhibiting proinflammatory cross-talk between monocytes and Th cells[J]. Front Immunol, 2021, 12:679498.
[23]	权瑜, 王举波, 程格庆, 等. lncRNA MALAT1靶向miR-532-3p对脑出血继发脑损伤大鼠脑微血管内皮细胞模型凋亡和氧化损伤的影响[J]. 山西医科大学学报, 2021, 52(7):874-882.QUAN Y, WANG J B, CHENG G Q, et al. Effect of lncRNA MALAT1 targeting miR-532-3p on apoptosis and oxidative damage of cerebral microvascular endothelial cell model in rats with secondary brain injury after intracerebral hemorrhage[J]. Journal of Shanxi Medical University, 2021, 52(7):874-882. (in Chinese)
[24]	嵇祝星, 王晓泉, 刘晓文, 等. 干扰素刺激基因15(ISG15)在天然免疫中的抗病毒作用研究进展[J]. 中国免疫学杂志, 2022, 38(2):253-258.JI Z X, WANG X Q, LIU X W, et al. Research progress of antiviral effect of ISG15 in natural immune response[J]. Chinese Journal of Immunology, 2022, 38(2):253-258. (in Chinese)
[25]	YIN K J, DENG Z, HAMBLIN M, et al. Peroxisome proliferator-activated receptor δ regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury[J]. J Neurosci, 2010, 30(18):6398-6408.
[26]	杨理. 长链非编码RNA在慢性脊髓损伤大鼠的差异表达研究[D]. 天津:天津医科大学, 2019.YANG L. Differentially expression of long non-coding RNA in chronic spinal cord injury rat[D]. Tianjin:Tianjin Medical University, 2019. (in Chinese)
[27]	HAORAH J, RAMIREZ S H, SCHALL K, et al. Oxidative stress activates protein tyrosine kinase and matrix metalloproteinases leading to blood-brain barrier dysfunction[J]. J Neurochem, 2007, 101(2):566-576.
[28]	WANG Z G, CHENG Y, YU X C, et al. bFGF protects against blood-brain barrier damage through junction protein regulation via PI3K-Akt-Rac1 pathway following traumatic brain injury[J]. Mol Neurobiol, 2016, 53(10):7298-7311.
[29]	SALVADOR E, BUREK M, FÖRSTER C Y. Stretch and/or oxygen glucose deprivation (OGD) in an in vitro traumatic brain injury (TBI) model induces calcium alteration and inflammatory cascade[J]. Front Cell Neurosci, 2015, 9:323.
[30]	OAK C H, WILSON D, LEE H J, et al. Potential molecular approaches for the early diagnosis of lung cancer (review)[J]. Mol Med Rep, 2012, 6(5):931-936.