Male adult SD rats, provided by Xipu Bikai Laboratory Animal Co., Ltd. (Shanghai, China), were used in this experiment. Animals were housed under a 12 h light/12 h dark cycle and had free access to food and water. All experiments followed the Guidance on the Good Treatment of Laboratory Animals issued by the Ministry of Science and Technology of China.

All male adult SD rats (260–280 g) were adaptively bred for 1 week and then randomly divided into four groups (Fig. 1A): sham surgery (Sham), middle cerebral artery occlusion (MCAO), EA-preconditioned plus MCAO (EAM), and MCAO plus EA-treated (MEA) groups. In the MCAO, EAM, and MEA groups, cerebral I/R injury was induced by MCAO experiments in the rats. During the operation, rats were deeply anesthetized by inhalation of 5% isoflurane (R510-22-10; Reward, Shenzhen, China) and maintained under anesthesia with 2% isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. To maintain the rat’s body temperature at 37 °C, we used a heating pad during and after the operation. After 2 h of MCAO, the intraluminal suture (JiaLing, Guangzhou, China) was withdrawn to induce post-ischemic reperfusion. The rats without significant neurological deficits, intraoperative hemorrhage, subarachnoid hemorrhage, or thrombosis of the circle of Willis were excluded. For the Sham group, the external carotid artery was isolated and ligated, while the suture was not inserted.

Fig. 1.

Electroacupuncture Preconditioning and Treatment Both Reduce Cerebral Ischemia-Reperfusion Injury Stroke-Induced Rats. (A) Animal group and experimental protocol. All rats were randomly divided into four groups: sham surgery (Sham), middle cerebral artery occlusion (MCAO), EA-preconditioned plus MCAO (EAM), and MCAO plus EA-treated (MEA) groups. In brief, cerebral ischemia-reperfusion (I/R) injury was induced by MCAO experiments in rats in the MCAO, EAM, and MEA groups. (B) Representative image of TTC staining. (C) Quantitative results of TTC staining. The ischemic area was evaluated by calculating the hemispheric lesion area with ImageJ software, and the relative infarction volume percentage is presented as a bar graph. Data are presented as the mean $\pm$ standard deviation (SD), n = 6, *p 0.01 vs. the Sham group, #p 0.01 vs. the MCAO group. (D) EA preconditioning and treatment significantly decreased the blood–brain barrier (BBB) permeability induced by I/R injury. BBB permeability in each group was determined by EB extravasation. Data are presented as the mean $\pm$ SD, n = 5, *p 0.01 vs. the Sham group, #p 0.01 vs. the MCAO group. (E) EA preconditioning and treatment significantly decreased the neurologic function score in I/R injury rats. Neurologic deficit was scored on a five-point scale according to Zea Longa. Data are presented as the mean $\pm$ SD, n = 8, *p 0.01 vs. the Sham group, #p 0.01 vs. the MCAO group.

The Baihui acupoint (GV20), located at the midpoint of the connecting line between the auricular apices, was chosen for both EA preconditioning and treatment. In the MEA group, rats were subjected to EA treatment at the GV20 acupoint for 30 min per session, with a frequency of 2/15 Hz and an intensity level of 1 mA, which was stimulated with a stimulus isolation unit (WQ1002F; HanAcuten, Beijing, China). EA treatment at the GV20 acupoint was performed at 1 min after reperfusion and again at 2.5 h before sacrifice (all rats were sacrificed at 24 h after I/R or sham surgery), for a total of two times. In the EAM group, rats received EA preconditioning at the GV20 acupoint for 30 min per day for six consecutive days, with the same parameters as those in the MEA group. MCAO surgery was performed 2 h after the sixth EA pretreatment. Rats in the Sham and MCAO groups were immobilized in the same manner for 30 min without EA stimulation.

Neurologic function was assessed according to the Longa neurologic score, where a score of 0 indicates no neurologic deficit, 1 indicates a mild focal neurologic deficit (failure to extend left forepaw fully), 2 indicates a moderate focal neurologic deficit (circling to the left), 3 indicates a severe focal deficit (falling to the left), and 4 indicates rats that did not walk spontaneously and had a depressed level of consciousness. The first neurologic function assessment was performed at 2 h after ischemia, and the second evaluation was performed at 24 h after reperfusion.

2,3,5-triphenyl tetrazolium chloride (TTC; T8877; Sigma, St. Louis, MO, USA) was used to visualize and measure the ischemia infarct volume. First, rat brains were isolated and frozen at –20 °C for 30 min after 24 h of reperfusion. Then the brains were sliced into coronal sections that were approximately 2 mm thick. TTC staining was performed on brain slices using 2% TTC (1 g/50 mL) at 37 °C for 30 min, and the sections were fixed in 4% paraformaldehyde. The total infarcted area (IVA) and total area (TA) on the back side of five coronal slices per rat were evaluated using ImageJ software (Version 1.8.0; ImageJ, MD, United States). The relative infarction volume percentage (RIVP) was calculated as: RIVP = IVA/TA $\times$ 100%.

BBB permeability was evaluated by measuring the extravasation of Evans blue (EB). Five rats were randomly selected from each group and injected with 2% EB (E2129; Sigma) solution (3 mL/kg) via the femoral artery at 1 h prior to sacrifice. Then the rats were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital and immediately perfused with saline through the left ventricle until colorless fluid was obtained from the right atrium. The left and right brains were quickly removed and weighed. Then the appropriate amount of formamide (3 mL/600 mg) was added separately, and the samples were soaked in water at 60 °C overnight. After 24 h, the supernatant was extracted by centrifugation at 1000 rpm (radius 10 cm) for 5 min at room temperature. Subsequently, different concentrations of EB standard solution and supernatant from each group were added to a 96-well enzyme plate with three replicate wells for both standards and samples. The EB content was finally determined at 632 nm using a microplate reader (Synergy H1; BioTek, Winooski, VT, USA). A standard curve was constructed using gradient concentrations of EB to quantify the amount of EB retained in the hemisphere.

Total RNA was extracted from ischemic brain tissue with TRIzol reagent (15596018; Invitrogen). The RNA concentration was measured with the Qubit 1.2 fluorometric analyzer (Invitrogen; Carlsbad, CA, USA) using the Qubit RNA BR Assay Kit (Q10211, Invitrogen). RNA quality was assessed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Inc., Santa Clara, CA, USA) with an RNA integrity number of at least 8.0. Then RNA samples (3 replicates for each group, totaling 12 samples) were processed with the TruSeq RNA Kit (15025062; Illumina) to create DNA libraries, which were cluster-generated using cBOT recombinant hybridization plates and sequenced on the Illumina HiSeq 2000 System (Illumina, San Diego, CA, USA) using the TruSeq SBS kit v3 (15021668; Illumina). The resulting FASTQ files were extracted using Illumina’s CASAVA software (v.1.8.2; Illumina), and the sequencing data were assembled and quantified against the rat reference genome (UCSC Rn4) using the TopHat program and DESeq2 program, followed by analyses of the differentially expressed genes (DEGs) with the Cuffdiff program. Gene annotation, signaling pathway, functional analysis, and protein–protein interaction (PPI) network interaction analyses were performed with ClusterProfiler and Metascape. Only DEGs with p 0.05 after Benjamini-Hochberg correction (p values 0.05) were included in the bioinformatics analysis.

First, we examined the neuroprotective effects of EA pretreatment and EA treatment on I/R injury in SD rats. All rats were randomly divided into four groups: Sham, MCAO, EAM, and MEA groups. In the MEA group, rats were treated with EA at 1 min after reperfusion and 2.5 h before execution. In the EAM group, rats were pretreated with EA for six consecutive days, and MCAO surgery was performed 2 h after the sixth EA pretreatment. Cerebral infarction, BBB integrity, and neurological function of these four groups were evaluated at 24 h after reperfusion. The extent of I/R-induced cerebral injury was quantified by TTC staining. A marked escalation in infarct size was observed in the MCAO group relative to the Sham group, whereas both the MEA and EAM groups exhibited significantly diminished infarct volumes compared to the MCAO group (p 0.01; Fig. 1B,C). Compared with the Sham group, EB extravasation was significantly elevated in the MCAO, MEA, and EAM groups (p 0.01; Fig. 1D), with the MEA and EAM groups displaying significantly attenuated leakage compared to the MCAO group (p 0.05; Fig. 1D). Nevertheless, no significant disparity was discernible between the MEA and EAM groups (p $>$ 0.05; Fig. 1D). In alignment with EB extravasation outcomes, the neurological function score for the MCAO group was significantly elevated relative to the Sham group (p 0.01; Fig. 1E), whereas both the MEA and EAM groups had lower scores than the MCAO group (p 0.01; Fig. 1E). No significant differences were detected between the MEA and EAM groups (p $>$ 0.05, Fig. 1E).

This study assessed the impact of EA on the DEG profile in the ischemic ipsilateral brain of stroke-induced rats. In the MCAO group, relative to the Sham group, 4798 DEGs were discerned, with 67.84% exhibiting an expression fold change (FC) of at least 1.5 (1.5 FC, $|$log${}_2$ (FC)$|$ $\ge$0.585), and 34.16% demonstrating a FC of 2 or more (2.0 FC, $|$log${}_2$ (FC)$|$ $\ge$1). A total of 1785 and 4020 were identified in the MEA and EAM groups, respectively, relative to the MCAO group, and with equivalent proportions of DEGs with $|$log${}_2$ (FC)$|$ $\ge$0.585 and $|$log${}_2$ (FC)$|$ $\ge$1. Notably, the downregulated genes outnumbered the upregulated genes in both the MEA and EAM groups, particularly the 2.0 FC DEGs. By contrast, in the MCAO group, the number of upregulated genes was significantly more than that of the downregulated genes (Table 1).

Upon considering all DEGs across the three groups, 1395 genes were found to be co-regulated by the MEA and MCAO groups, with 771 MCAO-upregulated genes downregulated by MEA, and 616 MCAO-downregulated genes upregulated by MEA. These MEA reverse-regulated genes accounted for 28.91% of the total DEGs in the MCAO group, and accounted for 77.7% of the total DEGs in the MEA group. In the context of the EAM and MCAO groups, 1550 coregulated DEGs were identified, with 428 genes upregulated post-MCAO surgery but downregulated by EA, and 470 genes downregulated post-MCAO surgery but upregulated by EA. Additionally, 299 co-downregulated genes and 353 co-upregulated genes were observed in these two groups. EAM reverse-regulated genes accounted for 18.72% of the total DEGs of the MCAO group, and accounted for 22.34% of the total DEGs in the EAM group (Table 2). These results suggest that post-stroke, EA mainly specifically attenuates aberrant gene expression induced by I/R injury, whereas pre-stroke EA preconditioning not only exerts specific regulatory effects on the abnormal genes induced by I/R injury but also modulates other genes, potentially contributing to the neuroprotective effects of acupuncture preconditioning on ischemic stroke.

Upon examination of the distribution of DEG expression magnitudes, we found that approximately 60% of the total DEGs in each group exhibited an expression FC exceeding 1.5. This provides a comprehensive reflection of transcriptome changes of the brain tissue following I/R injury under different conditions. To explore functional changes at the cellular levels, we performed Gene Ontology (GO) annotation enrichment and functional analyses of these DEGs, employing the Metascape bioinformatics platform. Furthermore, we conducted cluster comparative analysis of the three subgroups based on different gene expression levels to gain a better understanding of the relatively significant and unique characteristics of each intervention.

GO enrichment analysis outcomes revealed that the upregulated DEGs in the MCAO group were significantly enriched in biological processes related to both internal and external stimuli, with the top 20 processes encompassing a response to wounding, response to lipopolysaccharide, oxygen levels, and cellular response to growth factor stimulus. Response to these stimuli also manifested in immune and inflammatory reactions, encompassing cytokine production, innate immune response, regulation of cell adhesion, leukocyte migration, and myeloid leukocyte activation. Additionally, processes related to cell death, such as positive regulation of cell death and negative regulation of cell population proliferation were discerned (Fig. 2A). Among the top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways, the pathways related to these functions included the tumor necrosis factor (TNF) signaling pathway, cytokine–cytokine receptor interaction, PI3K/AKT signaling pathway, nuclear factor kappa B (NF-$\kappa$B) signaling pathway, chemokine signaling pathway, human T-cell lymphotropic virus type 1 infection, and apoptosis (Fig. 2B). By contrast, the downregulated DEGs in the MCAO group were predominantly linked to biological processes pertinent to the regulation of neuronal excitability. The top 20 processes included synapse organization, modulation of chemical synaptic transmission, neuron projection morphogenesis, regulation of neurotransmitter levels, and the GABAergic synapse (Fig. 2C). The top 20 KEGG pathways enriched by these downregulated DEGs were associated with neurotransmitter transduction such as the calcium signaling pathway, axon guidance, glutamatergic synapse, serotonergic synapse, oxytocin signaling pathway, and the synaptic vesicle cycle. Furthermore, amino acid metabolic pathways related to neurotransmitter synthesis such as propanoate metabolism, histidine metabolism, alanine, aspartate and glutamate metabolism were also enriched (Fig. 2D).

Fig. 2.

Bioinformatics Analyses of Differentially Expressed Genes in Sprague Dawley Rat Brains After Ischemic Stroke. (A) The top 20 Gene Ontology (GO) categories enriched by the upregulated differentially expressed genes (DEGs) with $|$log${}_2$ (fold change [FC])$|$ $>$0.585 of the middle cerebral artery occlusion (MCAO) group (MCAO_up_ 1.5 FC), n = 3. (B) The top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by MCAO_up_ 1.5 FC, n = 3. (C) The top 20 GO downregulated DEGs with $|$log${}_2$ (FC)$|$ $>$0.585 of the MCAO group (MCAO_down_ 1.5 FC), n = 3. (D) The top 20 KEGG pathways enriched by MCAO_down_ 1.5 FC, n = 3.

In the MEA group, the upregulated DEGs were primarily implicated in ion and neurotransmitter transport, regulated by G protein-coupled receptor (GPCR) signal transduction. These processes encompassed regulation of ion transport, potassium ion transmembrane transporter activity, regulation of neurotransmitter levels, active ion transmembrane transporter activity, and presynaptic membrane. Additionally, a variety of metabolic processes were noted such as positive regulation of organic acid transport, catecholamine metabolic process, regulation of catecholamine secretion, fatty acid binding, and positive regulation of alkaline phosphatase activity (Fig. 3A). KEGG analyses identified eight signaling pathways enriched by the upregulated DEGs including the cell cycle, neuroactive ligand–receptor interaction, adrenergic signaling in cardiomyocytes, calcium signaling pathway, and gap junction (Fig. 3A). On the other hand, the downregulated DEGs in the MEA group were significantly enriched in biological processes related to the response of the organism to physical and chemical stimuli including cellular response to growth factor stimulus, inflammatory response, wound healing, response to mechanical stimuli, response to lipopolysaccharides, and cellular response to interleukin 1 (IL-1). The top 20 enriched KEGG signaling pathways were also closely associated with the aforementioned functions such as the inflammatory responses mediated by the TNF, PI3K/AKT, NF-$\kappa$B, and hypoxia inducible factor 1 (HIF-1) signaling pathways; exogenous apoptosis mediated by the mitogen-activated protein kinase (MAPK), transforming growth factor beta (TGF-b), and p53 signaling pathways; and transcriptional misregulation in cancer and the hemodynamics pathway (Fig. 3B).

Fig. 3.

Bioinformatics Analyses of the Differentially Expressed Genes in Rat Brains in the Middle Cerebral Artery Occlusion Plus Electroacupuncture-Treated (MEA) Group. (A) The top 20 Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in the middle cerebral artery occlusion (MCAO) plus electroacupuncture (EA)-treated (MEA)-upregulated DEGs with $|$log${}_2$ (fold change [FC])$|$ $>$0.585. (B) The top 20 GO categories and KEGG pathways enriched by the MEA-downregulated DEGs with $|$log${}_2$ (FC)$|$ $>$0.585. (C) The top 20 functional clusters enriched by the MCAO downregulated but MEA-upregulated DEGs at different expression levels. (D) The significant KEGG pathways enriched by the MCAO-downregulated but MEA-upregulated DEGs at different expression levels. (E) Protein–protein interaction (PPI) network analyses of the MCAO-downregulated but MEA-upregulated DEGs. (F) The top 20 functional clusters enriched by the MCAO-upregulated but MEA-downregulated DEGs at different expression levels. (G) The KEGG pathways enriched the MCAO-upregulated but MEA-downregulated DEGs at different expression levels. (H) PPI network analyses of the MCAO-upregulated but MEA-downregulated DEGs.

To gain a comprehensive understanding of the protective effects of EA treatment on brain injury in stroke-induced rats, we further focused on the DEGs that were induced by stroke but reversed by EA treatment. Among the 277 DEGs that were downregulated by MCAO but upregulated by MEA with $|$log${}_2$(FC)$|$ $>$0.585, the majority were responsible for neurotransmitter transmission and neural excitability regulation including axon guidance, GPCR ligand binding, calcium signaling pathway, establishment or maintenance of the transmembrane electrochemical gradient, neuroactive ligand-receptor interaction, and the GABAergic synapse (Fig. 3C,D). Moreover, when all 616 MCAO-downregulated but MEA-upregulated DEGs were analyzed together, they were also involved in fatty acid metabolism, carbon metabolism, and mannose-type O-glycan biosynthesis, which are associated with brain substance metabolism and energy supply (Fig. 3C,D). PPI analyses revealed nine enriched clusters consistent with biological functions (Fig. 3E). On the other hand, the MCAO-upregulated but MEA-downregulated DEGs were primarily involved in immune inflammatory pathways (e.g., TNF, NF-$\kappa$B, and IL-17 signaling pathways), hemodynamic phase pathways (e.g., hematopoietic cell lineage, fluid shear stress, and atherosclerosis), and signaling pathways related to cell apoptosis, survival, and differentiation (e.g., MAPK, TGF-b, p53, and HIF-1 signaling pathways) (Fig. 3F,G). PPI analyses also revealed that the functional clusters of proteins encoded by these genes were primarily involved in a range of GPCR-mediated stress responses to internal and external stimuli, along with innate or adaptive immune responses in various organs of the body (Fig. 3H). These responses are the most significant pathological and pathogenic processes that occur in the brain after stroke.

In the EAM group, GO analyses of the upregulated DEGs indicated that the top 20 significant biological processes were primarily related to three main categories: energy metabolism, protein synthesis, and stress response. Examples of these processes include the mitochondrial protein-containing complex, cytoplasmic side of the rough endoplasmic reticulum membrane, and cellular response to toxic substances. In addition, KEGG analyses revealed that energy supply and substance metabolism accounted for the largest proportion, involving oxidative phosphorylation, proteasome, and glycolysis, among others. Furthermore, these DEGs were also found to participate in the synaptic vesicle cycle, which is associated with neural transmission signaling and RNA degradation (Fig. 4A). For the downregulated DEGs, the top 20 significant biological functions clusters were mainly related to the composition and function of synapses and neurons such as integral component of the synaptic membrane/presynaptic membrane, positive regulation of cell projection organization, forebrain development, and negative regulation of neurotransmitter secretion. Furthermore, these DEGs were involved in the regulation of gene expression such as mRNA metabolic processes, ATP-dependent chromatin remodeler activity, histone modification, and mRNA processing. The results of KEGG analyses revealed eight enriched signaling pathways including lysine degradation, axon guidance, and the extracellular matrix (ECM)–receptor interaction (Fig. 4B).

Fig. 4.

Bioinformatics Analyses of Differentially Expressed Genes in Stroke-Induced Rats with Electroacupuncture Preconditioning. (A) The top 20 Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by EA-preconditioned plus middle cerebral artery occlusion (MCAO) (EAM)-upregulated differentially expressed genes (DEGs) with $|$log${}_2$ (fold change [FC])$|$ $>$0.585. (B) The top 20 GO categories and KEGG pathways enriched by the EAM-downregulated DEGs with $|$log${}_2$ (FC)$|$ $>$0.585. (C) The top 20 functional clusters enriched by the MCAO-downregulated but EAM-upregulated DEGs at different expression levels. (D) The significant KEGG pathways enriched by the MCAO-downregulated but EAM-upregulated DEGs at different expression levels. (E) Protein-protein interaction (PPI) network analyses of the MCAO-downregulated but MEA-upregulated DEGs. (F) The top 20 functional clusters enriched by the MCAO-upregulated but EAM-downregulated DEGs at different expression levels. (G) The KEGG pathways enriched the MCAO-upregulated but EAM-downregulated DEGs at different expression levels. (H) PPI network analyses of the MCAO-upregulated but EAM-downregulated DEGs.

We subsequently conducted functional enrichment and protein interaction analyses on the DEGs that were co-regulated by MCAO and EAM. GO enrichment analyses revealed that the protein products, translated by these DEGs that were downregulated by MCAO but upregulated by EAM, particularly those with expression FC exceeding 1.5, were predominantly implicated in two clusters of biological processes. The first cluster pertained to regulating neurotransmitter transmission and the maintenance of neural excitability, encompassing modulation of chemical synaptic transmission, vesicle-mediated transport in synapse, cognition, regulation of neuronal synaptic plasticity, and catecholamine secretion (Fig. 4C). The corresponding enriched KEGG signaling pathways included the synaptic vesicle cycle, GABA biosynthesis, endocytosis, gap junction, and the glutamatergic synapse (Fig. 4D). The second cluster was associated with the brain’s energy supply and the regulation of substance metabolism including the ethanol catabolic process, energy derivation by oxidation of organic compounds, and cellular oxidant detoxification. The corresponding enriched KEGG signaling pathways included valine, leucine, and isoleucine degradation; glutathione metabolism; the tricarboxylic acid cycle; oxidative phosphorylation; and insulin secretion (Fig. 4C,D). PPI analyses confirmed that these DEGs were primarily involved in the biological function of neural excitability (e.g., serotonin neurotransmitter release cycle, activation of N-methyl-D-aspartate receptors, and synaptic vesicle exocytosis), energetic metabolism (e.g., metabolism of xenobiotics by cytochrome P450, oxidative phosphorylation, Rab geranylgeranylation), and protein modification (e.g., neddylation and histone modification) (Fig. 4E). The DEGs that were upregulated in the MCAO group but downregulated in the EAM group were largely associated with immune inflammatory responses and gene transcriptional activity including the response to hypoxia, regulation of cell adhesion, leukocyte cell-cell adhesion, and inflammatory responses (Fig. 4F). The corresponding enriched signaling pathways were the IL-17 signaling pathway, T helper 17 (Th17) cell differentiation, focal adhesion, and others (Fig. 4G). The functional clusters enriched by PPI analyses were in alignment with the GO and KEGG results including ribonucleoprotein complex biogenesis, antigen processing: ubiquitination and proteasome degradation, the innate immune system, negative regulation of myeloid leukocyte differentiation, and positive regulation of Th1 cell cytokine production (Fig. 4H). Additionally, these DEGs also regulated the advanced glycation end product (AGE)-receptor for AGE (RAGE) signaling pathway in diabetic complications, which was associated with vascular injury (Fig. 4F,G).

Excluding the EAM-reversed co-regulated DEGs, we observed that approximately 40% (652/1550) of the co-regulated DEGs in the MCAO and EAM groups exhibited the same expression pattern. To thoroughly decipher the potential mechanisms through which acupuncture pretreatment mitigates brain injury in stroke rats, we subjected these genes to biological analyses. The co-upregulated DEGs in both the MCAO and EAM groups were primarily linked to the augmentation of immune capacity (e.g., immunoglobulin-mediated immune response, innate immune response, regulation of the defense response), the promotion of non-neurotic cell death (e.g., apoptotic signaling pathway, ferroptosis, positive regulation of programmed cell death), and the regulation of ribosomal protein synthesis (Fig. 5A,C). PPI analyses demonstrated that these co-upregulated DEGs were predominantly enriched in functions such as the ribosome, p53-dependent G1 DNA damage response, neutrophil degranulation, defense response to bacterium, and cellular response to oxidative stress (Fig. 5E). Conversely, the co-downregulated DEGs in these two groups were chiefly tasked with regulating neuronal excitability including modulation of the neuronal membrane potential, glutamate receptor complex, and potassium ion transmembrane. Additionally, these DEGs were also implicated in the regulation of myocardial contractile function (Fig. 5B,D). PPI analyses indicated that these downregulated DEGs were primarily involved in neuron recognition, chloride transmembrane transport, neuronal action potential, and protein ubiquitination (Fig. 5F). In summary, the co-regulated DEGs in the MCAO and EAM groups appeared to bolster the organism’s defense ability and maintain neural excitability and function.

Fig. 5.

Bioinformatics Analyses of the Co-upregulated or Co-downregulated Differentially Expressed Genes in the Electroacupuncture-Preconditioned Plus Middle Cerebral Artery Occlusion (MCAO) (EAM) and MCAO groups. (A,B) The top 20 functional clusters enriched by co-upregulated or co-downregulated differentially expressed genes (DEGs) between the middle cerebral artery occlusion (MCAO) and electroacupuncture-preconditioned plus MCAO (EAM) groups. (C,D) The Kyoto Encyclopedia of Genes and Genomes pathways enriched by co-upregulated or co-downregulated regulated DEGs between the MCAO and EAM groups. (E,F) Protein-protein interaction network analyses of the co-upregulated or co-downregulated DEGs between the MCAO and EAM groups.

Based on the analyses, we determined that the biological processes implicated in the DEGs coregulated by either the MEA or EAM group and the MCAO group individually bore resemblance to each other, such as oxidative stress, immunoinflammatory responses, cell death, excitatory neurotoxic responses, and neurological dysfunction, which are the primary pathological processes of stroke. This observation prompted us to determine whether the mechanisms of EA preconditioning and treatment were identical. To this end, we compared the DEGs between the MEA and EAM groups and discovered that there were 532 identical genes in these two groups, constituting only 29.8% and 13.23% of the total DEGs in the MEA and EAM groups, respectively. Among these identical DEGs, 185 genes were upregulated and 201 genes were downregulated in both groups. Interestingly, 80 genes were upregulated in the EAM group but downregulated in the MEA group, while 66 genes exhibited the opposite pattern (Fig. 6A). To delve deeper into the shared mechanisms of EA preconditioning and treatment, we compared the co-regulatory genes of the MEA and EAM groups with the DEGs in the MCAO group. We found that a significant majority (81.08% or 150/185) of the co-upregulated genes in these two groups were the downregulated DEGs in the MCAO group. These genes were primarily involved in substance and energy metabolism and glial cell activation such as the citric acid cycle and respiratory electron transport, cellular biogenic metabolic process, and mitochondrial ATP synthesis-coupled proton transport (Fig. 6B). On the other hand, of the 201 co-downregulated genes in the MEA and EAM groups, 159 were upregulated DEGs in the MCAO group. These genes were primarily involved in the stress and immune response to hypoxic environments such as the response to hypoxia, ECM–receptor interaction, regulation of cell adhesion, and cellular response to organic cyclic compounds (Fig. 6C).

Fig. 6.

Bioinformatics Analyses of the Differentially Expressed Genes in the Middle Cerebral Artery Occlusion (MCAO) Plus Electroacupuncture (EA)-Treated (MEA) Group Versus EA-Preconditioned Plus MCAO (EAM) group. (A) Venn diagrams show the numbers of differentially expressed genes (DEGs) overlapping in the middle cerebral artery occlusion (MCAO) plus electroacupuncture (EA)-treated (MEA)-upregulated, MEA-downregulated, EA-preconditioned plus MCAO (EAM)-upregulated, and EAM-downregulated DEGs at different expression levels. (B,C) The top 20 functional clusters enriched by co-upregulated or co-downregulated DEGs between the MEA and EAM groups. (D,E) Comparative enrichment analyses of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways enriched by the upregulated and downregulated DEGs in the MCAO, MEA, and EAM groups.

Subsequently, we conducted comprehensive analyses of all upregulated and downregulated DEGs in the MEA, EAM, and MCAO groups using GO and KEGG pathways, with the aim of identifying the key characteristics and pathways of EA treatment and preconditioning in the regulation of ischemic stroke pathophysiological processes. Our findings revealed clear directionality in the biological processes and signaling pathways enriched by I/R-induced DEGs. Specifically, These I/R-induced upregulated DEGs were primarily involved in the activation, migration, and differentiation of immune inflammatory cells; and the downregulated DEGs were highly enriched in neural functions that primarily regulate synaptic excitability (Fig. 6D,E). The biological functions involved in the DEGs of the MEA group were mainly specifically targeted at the abnormal pathological processes caused by the MCAO group. Interestingly, for the EAM group, although most of the GO clusters enriched by the DEGs overlapped with those in the MCAO and MEA groups, the three top significant GO and KEGG clusters were related to mitochondrial energy metabolism (Fig. 6D,E), which were not the main regulatory targets of DEGs in the MCAO and MEA groups.