Effect of Electroacupuncture on the ceRNA Regulatory Network in the Ischemic Local Cerebral Cortex of Mice with Ischemic Stroke

Objective:This study aims to investigate the effects of electroacupuncture on the ceRNA regulatory network in the ischemic local cerebral cortex of mice with ischemic stroke. Methods: High-throughput sequencing technology was utilized to detect differential expression of long non-coding RNAs (lncRNAs) in middle cerebral artery occlusion (MCAO) mice. We analyzed the levels of lncRNA and miRNA transcripts involved in the ceRNA regulatory network targeting the gene TLR4, and verified the targeting interactions within the lncRNA/miRNA/TLR4 mRNA axis. We employed lentiviral transfection techniques to construct overexpression and knockdown vectors for multiple lncRNA/miRNA/TLR4 mRNA interactions. Changes in the protein levels of lncRNA, miRNA, and TLR4 mRNA were assessed to evaluate their effects on the proliferation and apoptotic regulation of vascular endothelial cells (VECs). Additionally, we constructed a TLR4-siRNA expression vector to assess the effects of electroacupuncture on the transcription levels of the lncRNA/miRNA/TLR4 mRNA axis in the cerebral cortex of MCAO mice. Results: (1) A dual-luciferase reporter assay confirmed the targeting relationship within the lncRNA/miRNA/TLR4 mRNA axis. (2) Bioinformatics analysis revealed that genes, such as WNT1, HIF1A, Netrin1, Malat1, and S1P were significantly upregulated, while TLR4, KLF4, and HEY1 were significantly downregulated. Upregulated genes mainly involved Wnt signaling pathways, while downregulated genes were primarily associated with Notch signaling pathways and neural cell apoptosis. (3) Overexpression of Malat1 promoted VEC proliferation and significantly increased TLR4 expression, along with the secretion of downstream cytokines IL-6, IL-8, and IL-1β. (4) lncRNA Malat1 promoted TMBIM1 expression, mediating TLR4 degradation and affecting the levels of downstream factors, with miRNA-182-5p identified as the interacting miRNA associated with TLR4. Conclusion: After ischemia-reperfusion, the expression of Malat1 was significantly positively correlated with TLR 4 levels, suggesting their synergistic role in neuroinflammatory