Metabonomics Analysis of Brain Stem Tissue in Rats with Primary Brain Stem Injury Caused Death

doi:10.12116/j.issn.1004-5619.2022.420510

Abstract

Abstract:

Objective To explore the potential biomarkers for the diagnosis of primary brain stem injury （PBSI） by using metabonomics method to observe the changes of metabolites in rats with PBSI caused death. Methods PBSI, non-brain stem brain injury and decapitation rat models were established, and metabolic maps of brain stem were obtained by LC-MS metabonomics method and annotated to the HMDB database. Partial least square-discriminant analysis （PLS-DA） and random forest methods were used to screen potential biomarkers associated with PBSI diagnosis. Results Eighty-six potential metabolic markers associated with PBSI were screened by PLS-DA. They were modeled and predicted by random forest algorithm with an accuracy rate of 83.3%. The 818 metabolic markers annotated to HMDB database were used for random forest modeling and prediction, and the accuracy rate was 88.9%. According to the importance in the identification of cause of death, the most important metabolic markers that were significantly up-regulated in PBSI group were HMDB0038126 （genipinic acid, GA）, HMDB0013272 （N-lauroylglycine）, HMDB0005199 [（R）-salsolinol] and HMDB0013645 （N,N-dimethylsphingosine）. Conclusion GA, N-lauroylglycine, （R）-salsolinol and N,N-dimethylsphingosine are expected to be important metabolite indicators in the diagnosis of PBSI caused death, thus providing clues for forensic medicine practice.

Key words: forensic pathology, metabonomics, cause of death, primary brain stem injury, liquid chromato-graphy-mass spectroscopy （LC-MS）, partial least square-discriminant analysis （PLS-DA）, random forest algorithm, rats

CLC Number:

Qin SU, Qian-ling CHEN, Wei-bin WU, Qing-qing XIANG, Cheng-liang YANG, Dong-fang QIAO, Zhi-gang LI. Metabonomics Analysis of Brain Stem Tissue in Rats with Primary Brain Stem Injury Caused Death[J]. Journal of Forensic Medicine, 2023, 39(4): 373-381.

Figures/Tables 10

Fig. 1 Construction of rat models

Fig. 2 Pathological changes of rat brain tissues with different causes of death （HE staining）

Fig. 3 Pathological changes of rat brain stem tissues with different causes of death （Silver staining）

Fig. 4 PLS-DA score plots of rat brain stem tissues with different causes of death at different time points after death

Fig. 5 Permutation test results （200 times） of OPLS-DA of rat brain stem tissues with different causes of death at different time points after death

Fig. 6 Venn diagram of metabolic markers in rat brain stem tissues at 6 h， 1 d and 3 d after death

Fig. 7 Construction results of random forest model for 86 metabolic markers

Tab. 1 Prediction results of random forest modeling using 86 metabolic markers and 818 metabolic markers

组别	86种代谢标志物的预测值			818种代谢标志物的预测值
组别	对照组	非脑干脑损伤组	PBSI组	对照组	非脑干脑损伤组	PBSI组
对照	6	0	0	6	0	0
非脑干脑损伤	1	4	1	0	5	1
PBSI	0	1	5	0	1	5

Fig. 8 Relative expression levels of 15 metabolic markers in the rat brain stem tissues with different causes of death

Fig. 9 Construction results of random forest model for 818 metabolic markers

References 23

1	左金华. 原发性脑干损伤患者的观察与刺激性护理[J].中国保健营养，2021，31（16）：178，182.
	ZUO J H. Observation and stimulating nursing care of patients with primary brain stem injury[J]. Zhongguo Baojian Yingyang，2021，31（16）：178，182.
2	SUN R， YANG S， ZHOU Z. Pathological and immunohistochemical study of lethal primary brain stem injuries[J]. Diagn Pathol，2012，7：54. doi：10.1186/1746-1596-7-54 .
3	向青青，李志刚. 原发性脑干损伤致死1例[J].广东公安科技，2022，30（2）：70-71.
	XIANG Q Q， LI Z G. Death due to primary brain stem injury： A case report[J]. Guangdong Gongan Keji，2022，30（2）：70-71.
4	姚青松，宋一璇，黄正光，等. 原发性脑干损伤组织学诊断要点[J].中国法医学杂志，2004，19（3）：134-137. doi：10.13618/j.issn.1001-5728.2004.03.002 .
	YAO Q S， SONG Y X， HUANG Z G， et al. Pathognostic findings of primary injuries on the brain stem[J]. Zhongguo Fayixue Zazhi，2004，19（3）：134-137.
5	SHUKLA D， MAHADEVAN A， SASTRY K V R， et al. Pathology of post traumatic brainstem and hypothalamic injuries[J]. Clin Neuropathol，2007，26（9）：197-209. doi：10.5414/npp26197 .
6	冯伟臣. 原发性脑干损伤死亡法医学鉴定中应注意的几个问题[C]//法医临床学专业理论与实践——中国法医学会·全国第二十届法医临床学学术研讨会论文集.乌鲁木齐，2017：26-27.
	FENG W C. Several problems should be paid attention to the forensic medical identification of death from primary brain stem injury[C]// Theory and Practice of Forensic Clinical Science — Chinese Forensic Medicine Association · Proceedings of the 20th Forensic Clinical Medicine Symposium. Urumqi，2017：26-27.
7	谷祯，牛佳佳，安国帅，等. 代谢组学在心脏性猝死鉴定中的应用前景及展望[J].法医学杂志，2021，37（3）：388-395. doi：10.12116/j.issn.1004-5619.2019.491116 .
	GU Z， NIU J J， AN G S， et al. Application prospects and prospect of metabolomics in the identification of sudden cardiac death[J]. Fayixue Zazhi，2021，37（3）：388-395.
8	SZEREMETA M， PIETROWSKA K， NIEMCUNO-WICZ-JANICA A， et al. Applications of metabolomics in forensic toxicology and forensic medicine[J]. Int J Mol Sci，2021，22（6）：3010. doi：10.3390/ijms22063010 .
9	DAWIDOWSKA J， KRZYŻANOWSKA M， MAR-KUSZEWSKI M J， et al. The application of metabolomics in forensic science with focus on forensic toxicology and time-of-death estimation[J]. Metabolites，2021，11（12）：801. doi：10.3390/metabo11120801 .
10	MYCHASIUK R， FARRAN A， ANGOA-PEREZ M， et al. A novel model of mild traumatic brain injury for juvenile rats[J]. J Vis Exp，2014（94）：51820. doi：10.3791/51820 .
11	TRIBA M N， LE MOYEC L， AMATHIEU R， et al. PLS/OPLS models in metabolomics： The impact of permutation of dataset rows on the K-fold cross-validation quality parameters[J]. Mol Biosyst，2015，11（1）：13-19. doi：10.1039/c4mb00414k .
12	马占君，李振国，王欢，等. 基于液相色谱-串联质谱的结肠癌血清氧固醇标志物筛选[J].色谱，2022，40（6）：541-546. doi：10.3724/SP.J.1123.2022.01001 .
	MA Z J， LI Z G， WANG H， et al. Screening of serum oxysterol biomarkers for colon cancer by liquid chromatography-tandem mass spectrometry[J]. Sepu，2022，40（6）：541-546.
13	TALLENT W H. Two new antibiotic cyclopentanoid monoterpenes of plant origin[J]. Tetrahedron，1964，20（7）：1781-1787. doi：10.1016/S0040-4020（01）99179-3 .
14	付庆，单淇，周钰通，等. 栀子中环烯醚萜类成分抗阿尔茨海默病的作用机制研究进展[J].中国药房，2022，33（1）：123-128. doi：10.6039/j.issn.1001-0408.2022.01.21 .
	FU Q， SHAN Q， ZHOU Y T， et al. Research progress on the mechanism of iridoids from Gardenia jasminoides against Alzheimer’s disease[J]. Zhongguo Yaofang，2022，33（1）：123-128.
15	NAM K N， CHOI Y S， JUNG H J， et al. Genipin inhibits the inflammatory response of rat brain microglial cells[J]. Int Immunopharmacol，2010，10（4）：493-499. doi：10.1016/j.intimp.2010.01.011 .
16	CHEN M K， PENG C C， MANER R S， et al. Geniposide ameliorated fluoxetine-suppressed neurite outgrowth in Neuro2a neuroblastoma cells[J]. Life Sci，2019，226：1-11. doi：10.1016/j.lfs.2019.04.003 .
17	张志华. 京尼平苷对APP/PS1小鼠行为学损伤和病理变化的保护作用及分子机制——抑制mTOR信号通路增强自噬的研究[D].太原：山西医科大学，2019.
	ZHANG Z H. Protective effects of geniposide on behavioral and pathologic changes in APP/PS1 mice and exploration of molecular mechanism： Down regulation of mTOR signal pathway and enhancement of autophagy[D]. Taiyuan： Shanxi Medical University，2019.
18	张青，李梦婷，李梅，等. 杜仲提取物多次给药后在正常大鼠和SHR模型体内的药动学比较研究[J].中国比较医学杂志，2022，32（1）：41-47. doi：10.3969/j.issn.1671-7856.2022.01.005 .
	ZHANG Q， LI M T， LI M， et al. Pharmacokinetic comparison of active components in an Eucommia ulmoides extract by multiple administrations between normal and spontaneously hypertensive rats[J]. Zhongguo Bijiao Yixue Zazhi，2022，32（1）：41-47.
19	BEYOĞLU D， IDLE J R. Metabolomic and lipidomic biomarkers for premalignant liver disease diagnosis and therapy[J]. Metabolites，2020，10（2）：50. doi：10.3390/metabo10020050 .
20	RYOU M， STYLOPOULOS N. Endoscopic ultra-sound-guided sampling and profiling of portal circulation in human patients for metabolic research studies and biomarker assessment[J]. Am J Physiol Gastrointest Liver Physiol，2020，319（5）：G584-G588. doi：10.1152/ajpgi.00135.2020 .
21	KURNIK-ŁUCKA M， PANULA P， BUGAJSKI A， et al. Salsolinol： An unintelligible and double-faced molecule — Lessons learned from in vivo and in vitro experiments[J]. Neurotox Res，2018，33（2）：485-514. doi：10.1007/s12640-017-9818-6 .
22	张芳芳，李梓晗，高怿宣，等. 基于转录组测序技术揭示salsolinol的神经毒性作用[J].生命科学仪器，2021，19（6）：29-34. doi：10.11967/2021191204 .
	ZHANG F F， LI Z H， GAO Y X， et al. Neurotoxic effects of salsolinol revealing by transcriptome analysis[J]. Shengming Kexue Yiqi，2021，19（6）：29-34.
23	MURAI Y， SEKIGUCHI A， HIRAKAWA T， et al. Evaluation of chiral N， N-dimethyl-sphingosine for the interaction between nerve growth factor and tropomyosin receptor kinase A[J]. Chirality，2022，34（5）：807-812. doi：10.1002/chir.23433 .

[1]	Yong ZENG, Dong-hua ZOU, Ying FAN, Qing XU, Lu-yang TAO, Yi-jiu CHEN, Zheng-dong LI. Research Progress and Forensic Application of Human Vascular Finite Element Modeling and Biomechanics [J]. Journal of Forensic Medicine, 2023, 39(5): 471-477.
[2]	Yu-xin SUN, Xiao-juan GONG, Xiu-li HAO, Yu-xin TIAN, Yi-ming CHEN, Bao ZHANG, Chun-xia YAN. Screening of Genes Co-Associated with Sudden Infant Death Syndrome and Infectious Sudden Death in Infancy and Bioinformatics Analysis of Their Regulatory Networks [J]. Journal of Forensic Medicine, 2023, 39(5): 433-440.
[3]	Yu YANG, Fan-zhang LEI, Yu-you DONG, Jian-long MA, Qi-qiang SHI, Xue-song YE. Retrospective Analysis of Death Cases of Oral Diphenidol Hydrochloride Poisoning [J]. Journal of Forensic Medicine, 2023, 39(4): 393-398.
[4]	Qing-qing XIANG, Li-fang CHEN, Qin SU, Yu-kun DU, Pei-yan LIANG, Xiao-dong KANG, He SHI, Qu-yi XU, Jian ZHAO, Chao LIU, Xiao-hui CHEN. Research Progress on Microbial Community Succession in the Postmortem Interval Estimation [J]. Journal of Forensic Medicine, 2023, 39(4): 399-405.
[5]	Xu-dong ZHANG, Yao-ru JIANG, Xin-rui LIANG, Tian TIAN, Qian-qian JIN, Xiao-hong ZHANG, Jie CAO, Qiu-xiang DU, Jun-hong SUN. Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods [J]. Journal of Forensic Medicine, 2023, 39(2): 115-120.
[6]	Wu LONG, Peng-fei QU, Lin MA, Rui WANG, Yan-mei XI, Yu-hua LI, Sheng-jie NIE, Ting DUAN, Jin-liang DU, Xue TANG, Jing-feng ZHAO, Pu-ping LEI, Yue-bing WANG. Cytotoxicity of 4 Wild Mushrooms in a Case of Yunnan Sudden Unexplained Death [J]. Journal of Forensic Medicine, 2023, 39(2): 121-128.
[7]	Jun-wei GAO, Yang LU, Yan-jun LI, Dong-hua ZOU, Guang-long HE, Yan-bin WANG. Survey on the Construction Status of Forensic Virtual Autopsy Laboratory and the Applicability of Laboratory Accreditation [J]. Journal of Forensic Medicine, 2023, 39(2): 186-192.
[8]	Fang-Fang LIU, Hui WU, Wei WANG, Ying XIE. Changes of 1，5-AG in Vitreous Humor of Rabbit Cadavers with Hyperglycemic Metabolism [J]. Journal of Forensic Medicine, 2023, 39(1): 13-17.
[9]	Zhi-ling TIAN, Ruo-lin WANG, Jian-hua ZHANG, Ping HUANG, Zhi-qiang QIN, Zheng-dong LI, He-wen DONG, Dong-hua ZOU, Mao-wen WANG, Zhuo LI, Lei WAN, Xiao-tian YU, Ning-guo LIU. Comparison of CT Values between Thrombus and Postmortem Clot Based on Cadaveric Pulmonary Angiography [J]. Journal of Forensic Medicine, 2023, 39(1): 7-12.
[10]	Jia-yi WU, You-jia YU, Kai LI, Xin YIN, Han-ting FAN, Rong LI, Zhi-wei ZHANG, Wei TANG, Hui-jie HUANG, Feng CHEN. Report on Cardiac Gross Pathologic Measurements of Sudden Cardiac Death in Adults [J]. Journal of Forensic Medicine, 2023, 39(1): 1-6.
[11]	Hui WU, Fang-fang LIU, Jun-da WU, Ying XIE. Research Progress on Estimation of Postmortem Interval Based on Ocular Tissues Structure [J]. Journal of Forensic Medicine, 2023, 39(1): 50-56.
[12]	Tian-pu WU, Jian-long MA, Xin-biao LIAO, Dong-chuan ZHANG, Kai-jun MA, Yan-geng YU, Long CHEN. Research Progress on Molecular Changes in Pulmonary Hypoxia and Cause of Death Identification in Mechanical Asphyxia [J]. Journal of Forensic Medicine, 2023, 39(1): 57-65.
[13]	Yu-qing JIA, Tian-qi WANG, Rui ZHAO, Bao-li ZHU, Zhi-peng CAO. Effects of Postmortem Hemolysis and Ultrafiltration on Creatinine Detection Results [J]. Journal of Forensic Medicine, 2022, 38(6): 697-701.
[14]	Lan YANG, Xin WANG, Yong NIU. Research Progress of DNA-Based Technologies for Postmortem Interval Estimation [J]. Journal of Forensic Medicine, 2022, 38(6): 747-753.
[15]	Cheng-dong MA, Qiu-ping WU, Qian-hao ZHAO, Zhi-qiang ZHAO, Kun YIN, Nan ZHOU, Sai-qun WU, Jian-ding CHENG. Research Progress on Sport-Related Sudden Cardiac Death [J]. Journal of Forensic Medicine, 2022, 38(5): 618-624.