Biomarkers Screening and Mechanisms Analysis of the Restraint Stress-Induced Myocardial Injury in Hyperlipidemia ApoE-/- Mice

doi:10.12116/j.issn.1004-5619.2023.430808

Abstract

Abstract:

Objective To explore the biomarkers and potential mechanisms of chronic restraint stress-induced myocardial injury in hyperlipidemia ApoE^-/- mice. Methods The hyperlipidemia combined with the chronic stress model was established by restraining the ApoE^-/- mice. Proteomics and bioinformatics techniques were used to describe the characteristic molecular changes and related regulatory mechanisms of chronic stress-induced myocardial injury in hyperlipidemia mice and to explore potential diagnostic biomarkers. Results Proteomic analysis showed that there were 43 significantly up-regulated and 58 significantly down-regulated differentially expressed proteins in hyperlipidemia combined with the restraint stress group compared with the hyperlipidemia group. Among them, GBP2, TAOK3, TFR1 and UCP1 were biomarkers with great diagnostic potential. KEGG pathway enrichment analysis indicated that ferroptosis was a significant pathway that accelerated the myocardial injury in hyperlipidemia combined with restraint stress-induced model. The mmu_circ_0001567/miR-7a/Tfr-1 and mmu_circ_0001042/miR-7a/Tfr-1 might be important circRNA-miRNA-mRNA regulatory networks related to ferroptosis in this model. Conclusion Chronic restraint stress may aggravate myocardial injury in hyperlipidemia mice via ferroptosis. Four potential biomarkers are selected for myocardial injury diagnosis, providing a new direction for sudden cardiac death （SCD） caused by hyperlipidemia combined with the restraint stress.

Key words: forensic pathology, bioinformatics, chronic restraint stress, hyperlipidemia, ferroptosis, biomarker, myocardial injury, mice

CLC Number:

Shang-heng CHEN, Sheng-zhong DONG, Zhi-min WANG, Guang-hui HONG, Xing YE, Zi-jie LIN, Jun-yi LIN, Jie-qing JIANG, Shou-yu WANG, Han-cheng LIN, Yi-wen SHEN. Biomarkers Screening and Mechanisms Analysis of the Restraint Stress-Induced Myocardial Injury in Hyperlipidemia ApoE^-/- Mice[J]. Journal of Forensic Medicine, 2024, 40(2): 172-178.

Add to citation manager EndNote|Ris|BibTeX

URL: http://www.fyxzz.cn/EN/10.12116/j.issn.1004-5619.2023.430808

http://www.fyxzz.cn/EN/Y2024/V40/I2/172

Figures/Tables 12

Tab. 1 Primer sequences of the target genesand reference gene

基因	引物序列（5′→3′）
Tfr-1	F：GTTTCTGCCAGCCCCTTATTAT
Tfr-1	R：GCAAGGAAAGGATATGCAGCA
Slc7a11	F：CCAAGAGGTGAGTGCTTCCC
Slc7a11	R：CTGTTGTTCAGACTCTCTCCCT
β-actin	F：GTGACGTTGACATCCGTAAAGA
β-actin	R：GCCGGACTCATCGTACTCC
mmu_circ_0001567	F：TCCTGTGGAAAAGGTGGATGTT
mmu_circ_0001567	R：CAGATGCGCTTTCACTGAGTC
mmu_circ_0001042	F：CAGGGTCAGAAGGATCGTCG
mmu_circ_0001042	R：GAGCGAGTCAGACAACTGCT
miR-7a	F：GCTGGAAGACTAGTGATTTTGTTGT
miR-7a	R：通用反向引物

Fig. 1 Electrocardiograms and M-mode echocardiographyimages of mice in the AP and APR groups

Tab. 2 The heart rate， EF and FS ofmice in the AP and APR groups

组别	心率/（次·min^-1）	FS/%	EF/%
AP	448.50±39.09	33.18±2.27	71.56±2.49
APR	373.00±22.77^1）	28.05±2.05^1）	62.25±3.73^1）

Tab. 3 The concentrations of corticosterone， BNP， CK and MDA in the serum of mice in the AP and APR groups

组别	皮质酮/（ng·mL^-1）	BNP/（pg·mL^-1）	CK/（U·mL^-1）	MDA/（ng·mL^-1）
AP	77.57±21.12	1 086.00±204.80	1.34±0.14	2.53±0.53
APR	158.90±40.45^1）	3 098.00±487.50^1）	1.70±0.15^1）	4.66±0.30^1）

Fig. 2 Volcano plot of differentially expressed proteins inthe myocardium of mice between the AP and APR groups

Tab. 4 Relative expression levels of differentiallyexpressed proteins in the myocardium of micebetween the AP and APR groups

组别	GBP2	TAOK3	UCP1	TFR1
AP	1.45±0.17	0.85±0.07	0.10±0.04	0.37±0.01
APR	0.49±0.08^1）	0.38±0.10^1）	0.92±0.29^1）	0.56±0.04^1）

Fig. 3 Differentially expressed proteins in the myocardium ofmice between the AP and APR groups （Western blotting）

Fig. 4 Bubble chart of the top 20 enriched KEGG pathways of differentially expressed proteinsin the myocardium of mice between the AP and APR groups

Fig. 5 Immunohistochemical staining results of TFR1 in the myocardium of mice in the AP and APR groups

Tab. 5 Relative expression levels of mRNA and proteinof ferroptosis-related genes in the myocardiumof mice in the AP and APR groups

组别	Tfr-1		Slc7a11
组别	mRNA	蛋白质	mRNA	蛋白质
AP	0.97±0.13	0.37±0.01	1.00±0.02	0.52±0.07
APR	6.67±0.30^1）	0.56±0.04^1）	0.25±0.08^1）	0.38±0.04^1）

Tab. 6 Relative expression levels of miR-7a and circRNAsthat interact with miR-7a in the myocardiumof mice in the AP and APR groups

组别	miR-7a	mmu_circ_0001567	mmu_circ_0001042
AP	1.01±0.16	1.03±0.31	1.01±0.18
APR	0.46±0.06^1）	6.04±1.38^1）	4.36±1.12^1）

Fig. 6 The predicted results of the interaction amongcircRNA， miRNA and Tfr-1 mRNA

References 16

1	中国血脂管理指南修订联合专家委员会. 中国血脂管理指南（2023年）[J].中国循环杂志，2023，38（3）：237-271. doi：10.3969/j.issn.1000-3614.2023.03.001 .
	Joint Committee on the Chinese Guidelines for Lipid Management. Chinese guidelines for lipid management （2023）[J]. Zhongguo Xunhuan Zazhi，2023，38（3）：237-271.
2	KIECOLT-GLASER J K， FAGUNDES C P， ANDRI-DGE R， et al. Depression， daily stressors and inflammatory responses to high-fat meals： When stress overrides healthier food choices[J]. Mol Psychiatry，2017，22（3）：476-482. doi：10.1038/mp.2016.149 .
3	KALYAN-MASIH P， VEGA-TORRES J D， MILES C， et al. Western high-fat diet consumption during adolescence increases susceptibility to traumatic stress while selectively disrupting hippocampal and ventricular volumes[J]. eNeuro，2016，3（5）：ENEURO.0125-16.2016. doi：10.1523/ENEURO.0125-16.2016 .
4	LI J H， LIU S， ZHOU H， et al. starBase v2.0： Decoding miRNA-ceRNA， miRNA-ncRNA and protein-RNA interaction networks from large-scale CLIP-Seq data[J]. Nucleic Acids Res，2014，42（Database issue）：D92-D97. doi：10.1093/nar/gkt1248 .
5	HINTERDOBLER J， SCHOTT S， JIN H， et al. Acute mental stress drives vascular inflammation and promotes plaque destabilization in mouse atherosclerosis[J]. Eur Heart J，2021，42（39）：4077-4088. doi：10.1093/eurheartj/ehab371 .
6	LIU B， ZHANG Y， WANG R， et al. Western diet feeding influences gut microbiota profiles in apoE knockout mice[J]. Lipids Health Dis，2018，17（1）：159. doi：10.1186/s12944-018-0811-8 .
7	CHIBA S， NUMAKAWA T， NINOMIYA M， et al. Chronic restraint stress causes anxiety- and depression-like behaviors， downregulates glucocorticoid receptor expression， and attenuates glutamate release induced by brain-derived neurotrophic factor in the prefrontal cortex[J]. Prog Neuropsychopharmacol Biol Psychiatry，2012，39（1）：112-119. doi：10.1016/j.pnpbp.2012.05.018 .
8	YE X， LIN J， LIN Z， et al. Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling[J]. Exp Cell Res，2017，359（2）：441-448. doi：10.1016/j.yexcr.2017.08.027 .
9	BIENENGRAEBER M， OZCAN C， TERZIC A. Stable transfection of UCP1 confers resistance to hypoxia/reoxygenation in a heart-derived cell line[J]. J Mol Cell Cardiol，2003，35（7）：861-865. doi：10.1016/ s0022-2828（03）00147-0 .
10	HOERTER J， GONZALEZ-BARROSO M D M， COUPLAN E， et al. Mitochondrial uncoupling protein 1 expressed in the heart of transgenic mice protects against ischemic-reperfusion damage[J]. Circulation，2004，110（5）：528-533. doi：10.1161/01.CIR.0000137824.30476.0E .
11	ZHANG J， ZHANG Y， WU W， et al. Guanylate-binding protein 2 regulates Drp1-mediated mitochondrial fission to suppress breast cancer cell invasion[J]. Cell Death Dis，2017，8（10）：e3151. doi：10.1038/cddis.2017.559 .
12	骆晓刚，周俊. 蛋白激酶TAOKs生物学功能及其与相关疾病关系的研究进展[J].中国细胞生物学学报，2022，44（6）：1129-1138. doi：10.11844/cjcb.2022.06.0018
	LUO X G， ZHOU J. Research progress on the biological functions and the relevant diseases of protein kinase TAOKs[J]. Zhongguo Xibao Shengwuxue Zazhi，2022，44（6）：1129-1138.
13	ZHANG H， ZHABYEYEV P， WANG S， et al. Role of iron metabolism in heart failure： From iron deficiency to iron overload[J]. Biochim Biophys Acta Mol Basis Dis，2019，1865（7）：1925-1937. doi：10.1016/
	j.bbadis.2018.08.030.
14	SUMNEANG N， SIRI-ANGKUL N， KUMFU S， et al. The effects of iron overload on mitochondrial function， mitochondrial dynamics， and ferroptosis in cardiomyocytes[J]. Arch Biochem Biophys，2020，680：108241. doi：10.1016/j.abb.2019.108241 .
15	HADIAN K， STOCKWELL B R. SnapShot： Ferroptosis[J]. Cell，2020，181（5）：1188-1188.e1. doi：10 .
	1016/j.cell.2020.04.039.
16	HU Z， ZHANG H， YANG S K， et al. Emerging role of ferroptosis in acute kidney injury[J]. Oxid Med Cell Longev，2019，2019：8010614. doi：10.1155/2019/8010614 .

[1]	Jie YANG, Yi-lei HU, Jin-ming WANG, Zheng-dong LI. Analysis of the Injury Mechanism of Traumatic Craniocerebral Injury from Falls Based on Finite Element Simulation [J]. Journal of Forensic Medicine, 2026, 42(1): 1-7.
[2]	Qiong JIA, Kai-jun JIN, Shi-qi LIU, Yi-meng ZHANG, Yun-le MENG, Hao NIE, Sheng HU, Fan YANG, Xing-chun ZHAO, Jian YE. Research Progress on the Methodological System for Decomposition Stage Classification in Forensic Medicine [J]. Journal of Forensic Medicine, 2026, 42(1): 34-42.
[3]	Xin NIU, Zhe CAO. Forensic Significance of Mesenteric Injury in Child Abuse Cases [J]. Journal of Forensic Medicine, 2026, 42(1): 43-48.
[4]	Zhen WANG, Tai-qi ZANG. Comprehensive Identification of the Vulnerant by Examining Marks on Clothing and Bodies Combining with Trace Comparison： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(1): 69-72.
[5]	Jia MENG, Ling-yu LI, Wu-lin HUANG, Tong ZHOU, Meng-qin ZOU, Jian-wei PENG, Bo LI, Xin HUANG, Qing-bo ZHANG. Multidisciplinary Identification in a Hit-and-Run Traffic Accident： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(1): 73-76.
[6]	Cong-da SHEN, Shi-fan WU, Yi WU, Wen-dong MAO, Xiang-tao MA. Fatal Rupture of the Abdominal Aorta Caused by Trampling： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(1): 77-78.
[7]	Jun-jiang CHEN, Lan-jiang LI, Pu-ping LEI, Shang-wen WANG, Xin-biao LIAO, Li-fang CHEN, Huan ZHANG. Forensic Identification of a Fatality from Confined Container Deflagration Complicated by Tertiary Blast Injury： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(1): 79-83.
[8]	Jian YE, Yong-liang HU, Ya-ling XIE, Yu CAI, Wen-hao LI. Medical Damage Identification of CT Guided Pulmonary Nodule Localization Needle Escapes to the Left Ventricle： A Case Report [J]. Journal of Forensic Medicine, 2026, 42(1): 83-86.
[9]	Ning-guo LIU. Innovative Development and Future Prospects in Forensic Pathology [J]. Journal of Forensic Medicine, 2025, 41(6): 517-530.
[10]	Yu-mei WANG. Postmortem Submersion Interval Estimation of Bodies Recovered from the Jingzhou Reach of the Yangtze River by Aquatic Decomposition Score [J]. Journal of Forensic Medicine, 2025, 41(6): 552-558.
[11]	Jin-xuan YANG, Dong-an TIAN, Xin-yu TANG, Zhan-ying ZHAO, Xi-yue JING, Dong-mei WANG, Xiao-fei TIAN. The Role of Iron Dysregulation in White Matter Injury Induced by Carbon Mono- xide Poisoning [J]. Journal of Forensic Medicine, 2025, 41(6): 545-551.
[12]	Li-liang LI, Jian-hua ZHANG, Feng CHEN, Rui ZHAO, Ya-dong GUO, Zhen-yuan WANG, Yi-wu ZHOU, Da-wei GUAN, Bin CONG. Expert Consensus on Cause of Death Determinations in Antipsychotics-Related Deaths with Negative Autopsy Findings [J]. Journal of Forensic Medicine, 2025, 41(6): 537-544.
[13]	Ji CHEN, Yu-rong ZHAO, Xin HUANG, Yi-ling QU, Yan-fang LU, Yu XING, Han ZHANG, Jian-ye ZENG, Shi-lin LI, Su-hua ZHANG. Effect of Temperature on Microbial Succession in Different Tissues of Cadavers and Estimation of Postmortem Interval [J]. Journal of Forensic Medicine, 2025, 41(5): 456-467.
[14]	Xiao-feng ZHANG, Lin CHEN, Xiao-hui CHEN, Jian ZHAO, Tian-chun LIN, Dang-en GU, Feng WANG, Zhi-yong LIU, Shi-yun MENG, Xing-yi YANG, Qu-yi XU. Diagnosis of Drowning by qPCR Detection of Plankton DNA in Cardiac Blood of Cadavers [J]. Journal of Forensic Medicine, 2025, 41(5): 477-481.
[15]	Hao-jie QIN, Chen MO, Hong-wei LI, Zhi-jiang LIU, Zhe ZHENG. Role of Ferroptosis in Paraquat-Induced Acute Lung Injury in Mice [J]. Journal of Forensic Medicine, 2025, 41(5): 494-501.