Intersection targets of clopidogrel and carotid artery stenosis

After calibration and deduplication in the GeneCards, SwissTargetPrediction, and ChEMBL databases, 250 targets related to clopidogrel were screened. From the GeneCards database, 2500 target genes related to carotid artery stenosis were screened. As shown in the Venn diagram, 127 intersecting genes between clopidogrel and carotid artery stenosis were identified (Fig. 2).

Fig. 2.

127 Potential Genes of Clopidogrel and Carotid Artery Stenosis. The blue section represents the targets associated with clopidogrel, while the yellow section represents the targets related to carotid artery stenosis. The overlapping area in the center indicates the intersecting targets shared between the two

Screening of PPI and core genes of potential targets

The initial PPI network was constructed using the STRING database (Fig. 3a). The downloaded PPI data were imported into Cytoscape 3.10.0 software, generating a new PPI network for visualization and analysis. Using the Centiscape 2.2 module in Cytoscape software, 25 core targets were screened and the corresponding PPI network was constructed. From light yellow to dark purple, the node colors indicate the increase in node degree within the PPI network (Fig. 3b). This network consists of 25 nodes and 208 edges, with node colors indicating the increase in node degree.

Fig. 3

PPI Network Diagram of Potential and Core Targets. Panel A shows the original PPI (protein–protein interaction) network. Panel B represents the core target network after screening. Each node corresponds to a gene, with darker colors indicating a higher number of interacting proteins. The five genes within the inner circle (TNF, MMP9, PTGS2, CCL2, TLR4, and IL-10) are identified as the most critical targets

The six core targets screened are TNF, MMP9, PTGS2, CCL2, TLR4, and IL-10. The TNF gene (tumor necrosis factor gene) can promote inflammatory responses, increase the expression of adhesion molecules in endothelial cells, and attract more monocytes into the vessel wall, leading to plaque formation and enlargement [15]. High levels of TNF-α are associated with vulnerable plaques (more likely to rupture, leading to thrombosis and embolic events). Studies show that serum TNF-α levels in patients with carotid artery stenosis may be higher than in those without stenosis, and TNF-α levels may correlate with the severity of carotid artery stenosis [16].

MMP9 is an enzyme that plays a role in extracellular matrix remodeling and can degrade various extracellular matrix components, including collagen and elastin. Studies show that MMP9 can degrade the extracellular matrix in plaques, making them more vulnerable and increasing the risk of rupture and thrombosis [17]. Increased MMP9 expression is associated with vascular inflammation, which is a key factor in the development of atherosclerosis [18]. MMP9 expression is positively correlated with the extent of neovascularization in carotid plaques and negatively correlated with plaque elasticity [19]. Therefore, MMP9 may play an important role in the occurrence, development, and complications of carotid artery stenosis and may serve as a potential therapeutic target or biomarker.

The PTGS2 gene encodes prostaglandin-endoperoxide synthase 2, also known as cyclooxygenase 2 (COX-2). PTGS2 is a key enzyme in the inflammatory process, promoting the synthesis of prostaglandins, which play a role in inflammation, pain, and fever responses [20]. In the development of atherosclerosis, upregulation of PTGS2 may be related to the inflammatory process in carotid artery stenosis [21]. In addition, PTGS2 may play a role in the formation and development of atherosclerotic plaques. PTGS2 is involved in the metabolism of lipids, collagen, and other extracellular matrix components in the vessel wall, which are related to plaque stability and vulnerability [21]. Therefore, inhibiting the activity of PTGS2 may help slow the progression of carotid artery stenosis.

CCL2, also known as Monocyte Chemoattractant Protein-1 (MCP-1), is a chemokine that plays a key role in inflammation and immune responses [22]. CCL2 can attract monocytes into the arterial wall, which may then differentiate into foam cells, a critical step in atherosclerotic plaque formation. Meanwhile, CCL2 exacerbates the inflammatory response in the vessel wall by promoting the migration and activation of inflammatory cells. Increased CCL2 expression is also associated with plaque instability and vulnerability, potentially increasing the risk of plaque rupture and thrombosis, thus worsening carotid artery stenosis [23]. Due to its role in the development of carotid artery stenosis, CCL2 may become a potential target for treating carotid artery stenosis. Inhibiting the activity of CCL2 or the signaling of its receptor CCR2 may help slow the progression of carotid artery stenosis.

TLR4, or Toll-Like Receptor 4, is a pattern recognition receptor that plays an important role in the immune system. The activation of TLR4 can promote inflammatory responses, and inflammation is a key factor in atherosclerosis [24]. In atherosclerotic plaques, the activation of TLR4 can increase the expression of inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which further promote plaque development and instability [25]. TLR4 may also participate in the occurrence and development of atherosclerosis by affecting the autophagy process. Autophagy is an intracellular cleaning mechanism, and the activation of TLR4 may affect this process, thereby influencing plaque stability [26].

IL-10 is an anti-inflammatory cytokine produced by Th2 cells, macrophages, and other immune cells, regulating immune responses and inhibiting inflammatory processes [27]. IL-10 exerts its anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) and reducing immune cell activation [28, 29]. IL-10 exhibits anti-inflammatory properties that can attenuate endothelial cell damage and enhance the barrier function of endothelial cells. Endothelial cell damage is one of the critical factors in the development and progression of atherosclerosis. The protective effects of IL-10 on endothelial cells contribute to maintaining vascular integrity and reducing lipid infiltration and inflammatory cell adhesion. Changes in IL-10 levels can serve as a biomarker for assessing the inflammatory status and therapeutic efficacy in carotid artery stenosis. By monitoring serum IL-10 levels in patients with carotid artery stenosis, we can promptly evaluate the therapeutic response.

GO enrichment analysis of core targets

According to the GO functional enrichment analysis results of core targets for the effect of clopidogrel in treating carotid artery stenosis, there are 154 biological processes (BP), 17 cellular components (CC), and 23 molecular functions (MF). GO analysis was ranked based on false discovery rate (FDR) values and P-values, selecting the top 10 items with the lowest FDR values in BP, CC, and MF, and displayed through horizontal gradient bar charts and bubble charts (Fig. 4).

Fig. 4

GO Enrichment Analysis Results of Core Targets: Horizontal Gradient Bar Chart and Bubble Chart. a This histogram illustrated the top 10 enriched entries for each GO category (BP, CC, and MF) with smaller FDR values on the 127 potential targets. The FDR values reflected the statistical significance of the enrichment, with smaller values indicating higher significance. The height of each bar corresponds to the gene counts, reflecting the degree of enrichment within the respective category. These enriched entries highlighted key biological processes, cellular components, and molecular functions that are potentially influenced by clopidogrel exposure. b The size of each bubble corresponded gene expressions in a particular pathway. The enrichment significance was shown by the color saturation of the bubble

In the BP results, the main biological processes are closely related to the response to lipopolysaccharide (LPS). LPS can promote inflammatory responses by activating Toll-like receptors (such as TLR4), and inflammation is a key factor in atherosclerosis. Immune cells activated by LPS release various inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which further promote plaque development and instability [30]. LPS can also directly act on vascular endothelial cells, causing endothelial dysfunction, which is an important event in the early stages of atherosclerosis. LPS promotes the formation of atherosclerotic plaques by activating vascular endothelial cells and immune cells [31].

Additionally, platelet activation and angiogenesis are biological processes related to clopidogrel for treating carotid stenosis. Platelet activation is involved in multiple stages of atherosclerosis, including platelet adhesion, aggregation, and release reactions after endothelial injury, as well as the migration and proliferation of smooth muscle cells. Platelet parameters such as platelet count (PLT) and mean platelet volume (MPV) can reflect the quantity, size, and activation degree of platelets. These parameters are related to carotid artery stenosis and may serve as indicators for assessing the risk of carotid atherosclerosis [32]. Angiogenesis within atherosclerotic plaques may affect plaque stability. Newly formed vessels may be more prone to rupture, leading to the entry of plaque contents into the bloodstream, increasing the risk of thrombosis and embolic events. Angiogenesis-related factors, such as vascular endothelial growth factor (VEGF), may serve as biomarkers for the progression of carotid artery stenosis and patient prognosis [33].

The CC results indicate that clopidogrel for treating carotid stenosis may act on the extracellular region, plasma membrane external side, extracellular space, and extracellular exosomes. The MF results suggest that the molecular processes of clopidogrel for treating carotid stenosis may include protease binding, peroxidase activity, heme binding, and lipopolysaccharide binding.

KEGG pathway enrichment analysis of core targets

To explore the potential signaling pathways of clopidogrel in the treatment of carotid artery stenosis, core target genes were input into the DAVID database for KEGG pathway analysis. A total of 86 typical signaling pathways were enriched, and the top 20 pathways with the lowest false discovery rate (FDR) values were selected, with biological pathways, horizontal gradient bar charts, and bubble charts drawn (Fig. 5).

Fig. 5

KEGG Enrichment Analysis Results of Core Targets: Biological Pathways, Horizontal Gradient Bar Chart, and Bubble Chart. a Biological schematic diagram of atherosclerosis progression. b The bubble diagram visualized the top 20 enriched KEGG signal pathways in reverse order of FDR values. Each bubble represented a specific pathway, where the bubble area indicating the number of enriched genes in the pathway. The intensity of the bubble’s color indicated the significance of the enrichment, with darker shades of red representing higher significance. The 127 intersection genes were mainly involved in KEGG pathways, with a notable emphasis on neuroactive ligand–receptor interactions and signaling pathways. c The histogram illustrated the frequency and significance of enrichment for each pathway. The length of each bar corresponded to the gene counts, indicating enrichment score and the level of significance, with taller bars representing larger counts and higher enrichment. It presented a concise and visually informative depiction of the top 20 enriched KEGG signal pathways, emphasizing the pathways that were particularly relevant to clopidogrel for treating carotid stenosis

KEGG analysis results show that the signaling pathways of clopidogrel for treating carotid stenosis are most likely closely related to lipid and atherosclerosis. This result is consistent with previous studies. Abnormal lipid metabolism is a major risk factor for atherosclerosis. Atherosclerosis is characterized by intimal lesions of the affected arteries, accumulation of lipids and complex carbohydrates, fibrous tissue proliferation, and calcium deposition forming plaques [34]. Accumulated low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) are oxidized or chemically modified to form oxidized low-density lipoprotein (ox-LDL). These modified lipoproteins induce inflammatory responses, recruit macrophages to engulf lipids, form foam cells, and promote the development of atherosclerosis [35, 36].

The main cause of carotid artery stenosis is atherosclerosis, accounting for more than 90% [37]. Atherosclerotic plaques involve the carotid artery, leading to arterial stenosis or even occlusion, causing cerebral ischemia and stroke symptoms. Studies show that pericarotid fat density is positively correlated with the degree of carotid artery stenosis; patients with symptomatic carotid stenosis and recurrent stenosis-related ischemic cerebrovascular events have higher pericarotid fat density [38].

In summary, abnormal lipid metabolism plays a central role in the occurrence and development of atherosclerosis, which is the main cause of carotid artery stenosis. Controlling blood lipid levels and improving lipid metabolism are crucial for preventing and treating atherosclerosis and carotid artery stenosis.

Molecular docking results

The interactions between clopidogrel and six core target genes (TNF, MMP9, PTGS2, CCL2, TLR4, IL-10) were studied through molecular docking analysis. Molecular docking was completed using AutoDock software, and the results showed that the binding energies were all less than − 4.20 kcal/mol (see Table 1), indicating that clopidogrel can tightly bind to the core target genes. This result demonstrates the strong affinity between clopidogrel and these target genes, further confirming the important role of clopidogrel in the molecular mechanism of treating carotid artery stenosis [39]. Finally, PyMOL software was used to visually display the binding details of clopidogrel with the core targets (Fig. 6).

Table 1 Binding energy of clopidogrel with six core target genesFig. 6

Molecular docking results of clopidogrel with core targets