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.

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

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