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. 2024 Jul;14(7):e3608.
doi: 10.1002/brb3.3608.

RNA sequencing reveals the potential mechanism of exercise preconditioning for cerebral ischemia reperfusion injury in rats

Yan Wu  1 , Hui Yang  2 , Feifeng Chen  1 , Baohua Li  2 , Xiangbo Meng  3
Affiliations

RNA sequencing reveals the potential mechanism of exercise preconditioning for cerebral ischemia reperfusion injury in rats

Yan Wu et al. Brain Behav. 2024 Jul.

Abstract

Introduction: Cerebral ischemia reperfusion injury (CIRI) often leads to deleterious complications after stroke patients receive reperfusion therapy. Exercise preconditioning (EP) has been reported to facilitate brain function recovery. We aim to explore the specific mechanism of EP in CIRI.

Methods: Sprague-Dawley rats were randomized into Sham, middle cerebral artery occlusion (MCAO), and EP groups (n = 11). The rats in the EP group received adaptive training for 3 days (10 m/min, 20 min/day, with a 0° incline) and formal training for 3 weeks (6 days/week, 25 m/min, 30 min/day, with a 0° incline). Then, rats underwent MCAO surgery to establish CIRI models. After 48 h, neurological deficits and cerebral infarction of the rats were measured. Neuronal death and apoptosis in the cerebral cortices were detected. Furthermore, RNA sequencing was conducted to investigate the specific mechanism of EP on CIRI, and qPCR and Western blotting were further applied to confirm RNA sequencing results.

Results: EP improved neurological deficit scores and reduced cerebral infarction in MCAO rats. Additionally, pre-ischemic exercise also alleviated neuronal death and apoptosis of the cerebral cortices in MCAO rats. Importantly, 17 differentially expressed genes (DEGs) were identified through RNA sequencing, and these DEGs were mainly enriched in the HIF-1 pathway, cellular senescence, proteoglycans in cancer, and so on. qPCR and Western blotting further confirmed that EP could suppress TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions in MCAO rats.

Conclusion: EP can improve CIRI in vivo, the mechanism may relate to TIMP1 expression and HIF-1 pathway, which provided novel targets for CIRI treatment.

Keywords: RNA sequencing; TIMP1/HIF‐1 pathway; cerebral ischemia reperfusion injury; exercise preconditioning.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
EP alleviated the neurological deficit scores in MCAO rats. The neurological deficit scores in each group were assessed 48 h following CIRI. # p < .05, ## p < .01 versus Sham. *p < .05, **p < .01 versus EP. Results were presented as mean ± SD. n = 11. CIRI, cerebral ischemia reperfusion injury; EP, exercise preconditioning; MCAO, middle cerebral artery occlusion.
FIGURE 2
FIGURE 2
Exercise preconditioning (EP) alleviated neuronal death of the cerebral cortices in middle cerebral artery occlusion (MCAO) rats. At 48 h after cerebral ischemia reperfusion injury (CIRI), the neuronal death of the cerebral cortices in each group was measured by Nissl staining (magnification, 200×, scale bar = 100 µm; magnification, 400×; scale bar = 50 µm). # p < .05, ## p < .01 versus Sham. *p < .05, **p < .01 versus EP. Results were presented as mean ± SD. n = 3.
FIGURE 3
FIGURE 3
Exercise preconditioning (EP) reduced cerebral infarction for middle cerebral artery occlusion (MCAO) rats. At 48 h after cerebral ischemia reperfusion injury (CIRI), the cerebral infarct volume of the rats in each group were evaluated by TTC staining. # p < .05, ## p < .01 versus Sham. *p < .05, **p < .01 versus EP. Results were presented as mean ± SD. n = 3. TTC, 2,3,5‐triphenyl tetrazolium chloride.
FIGURE 4
FIGURE 4
Exercise preconditioning (EP) inhibited apoptosis of cerebral cortices in middle cerebral artery occlusion (MCAO) rats. At 48 h after cerebral ischemia reperfusion injury (CIRI), the apoptosis of cerebral cortices in each group were evaluated by TUNEL (magnification, 200×). # p < .05, ## p < .01versus Sham. *p < .05, **p < .01 versus EP. Results were presented as mean ± SD. n = 3. TUNEL, TdT‐mediated dUTP nick end labeling.
FIGURE 5
FIGURE 5
The differentially expressed genes (DEGs) identified by RNA sequencing among rats treated with or without exercise preconditioning (EP). (a) Principal component analysis. (b) Volcano plot showed significantly upregulated or downregulated DEGs among groups. (c) Venn diagram was applied to present the DEGs among group. (d) Hierarchical clustering analysis of DEGs among groups. n = 5. DEGs, differentially expressed genes.
FIGURE 6
FIGURE 6
Functional enrichment analysis of exercise preconditioning (EP) in middle cerebral artery occlusion (MCAO) rats. (a) Results of GO analysis. (b) Results of KEGG analysis. GO, Gene ontology; KEGG, kyoto encyclopedia of genes and genomes.
FIGURE 7
FIGURE 7
Exercise preconditioning (EP) suppressed TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions of the cerebral cortices in middle cerebral artery occlusion (MCAO) rats. At 48 h after cerebral ischemia reperfusion injury (CIRI), TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expression levels of cerebral cortices in each group were evaluated by qPCR (a) and Western blotting (b). # p < .05, ## p < .01 versus Sham. *p < .05, **p < .01 versus EP. Results were presented as mean ± SD. n = 3.
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