1. Introduction
Atherosclerosis (AS) is a chronic inflammatory disease characterized by intense immune activity. Inflammation plays an important role in the occurrence and development of atherosclerotic lesions [
1]. Atherosclerotic plaque formation is characterized by lipid accumulation, local inflammation, proliferation, apoptosis, necrosis and fibrosis of smooth muscle cells (SMCs) [
2]. The main cause of atherosclerotic stenosis is the activation of inflammatory cells and a series of chronic inflammatory reactions after initial endothelial cell injury [
3]. In the early stage of atherosclerosis, risk factors such as hyperlipidemia, diabetes, smoking and hypertension can induce an oxidative stress response, activate cytokines and increase LDL levels. Meanwhile, macrophages migrate to the vascular wall, and inflammatory stimulators promote endothelial dysfunction and initiate atherosclerotic lesions [
4]. At present, the most effective therapeutic drugs for AS are statins, which reduce the levels of atherogenic lipoproteins and prevent major cardiovascular events. However, treatment with statins is ineffective in reducing cholesterol levels in a small proportion of users, and prolonged use of statins may increase the risk of side effects [
5]. Therefore, it is urgent to find new anti-AS drugs of satisfactory therapeutic effect and suitable for long-term use.
Canagliflozin and dapagliflozin, known as sodium-glucose cotransporter-2 (SGLT2) inhibitors, are a class of anti-diabetic compounds that lower blood glucose by selectively blocking the reabsorption of glucose in the proximal convoluted tubule (PCT) [
6]. Recently, a growing number of studies have shown that canagliflozin and dapagliflozin are surprisingly effective in improving cardiovascular and kidney disease in both diabetic and non-diabetic patients. New clinical outcome trials have demonstrated that glucose-lowering drugs, especially SGLT2 inhibitors, overall reduce the risk of fatal and non-fatal atherosclerotic cardiovascular events and all-cause mortality [
7]. Similarly, in two trials involving patients with type 2 diabetes and an elevated risk of cardiovascular disease, canagliflozin reduced the risk for primary cardiovascular outcome compared with those who received placebos [
8]. In patients with heart failure and reduced ejection fraction, regardless of diabetes, dapagliflozin reduced the number of hospitalizations for heart failure or cardiovascular death compared to those who received placebos [
9]. Some evidence from animal models indicates that SGLT2 inhibitors could prevent AS. Canagliflozin attenuated the progression of AS in HFD-fed ApoE
−/− mice [
10]. In addition, dapagliflozin has been shown to inhibit AS in ApoE
−/− mice [
11].
Network pharmacology is based on the concept of a multilevel and multiangle interaction network of disease-gene-target-drug [
12]. It is associated with multiple disciplines in systems biology, pharmacology, computational biology, and network analysis [
13,
14]. Professional databases, resources, and software were used to visualize the relationship between drug–drug interaction and drug–disease interaction. Network pharmacology broke the traditional concept of single drugs, single target and single disease, and proposes that drugs act on multiple targets and multiple pathways [
15]. Thus, network pharmacology has been an effective way to search for new research drugs and discover their potential mechanisms.
In this article, we used network pharmacology to demonstrate the underlying mechanisms of canagliflozin and dapagliflozin in the management of atherosclerosis. Therefore, using a variety of databases, we systematically predicted and analyzed the complex relationship between the disease, drugs and targets. Meanwhile, we discussed the potential mechanisms of some important targets of canagliflozin and dapagliflozin in the treatment of atherosclerosis. This study provides a scientific basis for further research on SGLT2 inhibitors for cardiovascular diseases. The process for our study can be found in
Figure S1.
4. Discussion
In our study, we collected the relevant targets of drugs and diseases separately, took the intersection of the targets of drugs and diseases, and focused on analyzing the 191 targets that they had intersections with in the following study. The purpose of our study was to discover whether Akt1, MAPK1, MAPK14, RHOA, SRC and EGFR are central genes in different networks. These genes are both drug targets and disease-related targets. These genes, which are both drug- and disease-related targets, are six potential central targets for canagliflozin or dapagliflozin to improve AS. After verification of molecular docking, all five targets except RHOA were found to bind well to canagliflozin or dapagliflozin, so that Akt1, MAPK1, MAPK14, SRC and EGFR were determined as the final core targets.
Akt/PKB (protein kinase B) is very important for cell survival induced by growth factor. Active Akt can suppress apoptosis independently of transcription through phosphorylation and inactivation of apoptotic machine components. Akt1, Akt2 and Akt3 are three Akt isoforms in macrophages. In the mammalian genome, the major Akt isoform is encoded by Akt1, which modulates apoptosis [
26]. The PI3K/Akt pathway is a classic signaling pathway that plays a crucial role in cell survival and apoptosis. Akt activated m-TOR inhibits autophagy of macrophages in the inflammatory response [
27]. The change of Akt subtype or the regulation of Akt activity level significantly affects the polarized phenotype of macrophages, which may impact the progression of atherosclerosis [
28]. Canagliflozin can stimulate AMPK, Akt and eNOS and inhibits iNOS and NADPH oxidase isoform 4 (NOX4), all of which are associated with antioxidant and anti-inflammatory signaling pathways [
29,
30]. The combined treatment with dapagliflozin and rosuvastatin can synergistically inhibit apoptosis by activating the PI3K/AKt/mTOR signaling pathway in rats with myocardial ischemia [
31].
Mitogen-activated protein kinase (MAPK), a kind of serine-threonine protein kinase, plays a more important role in many important physiological and pathological processes such as cell proliferation, differentiation and apoptosis [
32]. There are four main subfamilies of the MAPK pathway: ERK1/2, c-Jun N-terminal kinase (JNK), P38/MAPK and ERK5 [
33]. MAPK1 (MAP kinase ERK2) is a subfamily of MAPK, form ERK1/2 [
34]. MAPK14, also named as p38α, is an isoform of the p38 MAPK family, and it is ubiquitously expressed in the family [
35]. The MAPK/ERK pathway can be activated with proatherogenic stimuli in vitro and in vivo [
36]. p38 MAPK is an important component of inflammatory signaling that can be activated by various stimuli such as oxidative stress, cytokines, and growth factors, all of which are involved in the formation of atherosclerosis [
35,
37,
38].
A recent study suggests that canagliflozin has atheroprotective effects against atherosclerosis by promoting the Akt-eNOS pathway and inhibiting the activation of p38 MAPK [
39]. Dapagliflozin shows a protective effect in complicated T2DM with CVD via the MAPK signaling pathway [
40]. Similarly, dapagliflozin alleviates diabetic cardiomyopathy by upregulating the AKT/JAK/MAPK pathway via erythropoietin in diabetic rats [
41].
Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in the regulation of cellular metabolism, survival, and proliferation [
42,
43]. Many studies have shown that SRC plays an important role in the functional activation of macrophages and the regulation of cholesterol levels, which are involved in atherosclerosis [
44,
45]. A study demonstrated that canagliflozin and dapagliflozin protect endothelial cells from glucose-induced oxidative stress by blocking ROS-activated SRC, EGF receptor, protein kinase C and Rho kinase [
46].
Epidermal growth factor receptor (EGFR) is a member of the ERBB family of tyrosine kinase receptors. EGFR plays a very important role in cell survival, proliferation, migration, differentiation and division [
47,
48]. Recently, several studies have indicated that EGFR is involved in the regulation of inflammation and oxidative stress in macrophages [
49]. As we all know, inflammation and oxidative stress are significant manifestations of atherosclerosis development. Moreover, blocking EGFR induced anergia of T cells in vitro and in vivo and reduced atherosclerosis development [
48]. These results suggest that EGFR plays an important role in the pathogenesis of atherosclerosis. There is currently little research on SGLT2 inhibitors and EGFR. Only one of the studies we mentioned above showed that SGLT2 inhibitors can block EGFR-related signaling pathways and play a role in protecting the vascular endothelium [
46]. In our research, we found a very high degree of binding of EGFR to both canagliflozin and dapagliflozin, so we believe that EGFR may be a key target for canagliflozin and dapagliflozin in the treatment of AS.
Arterial bifurcations and intra-arterial curves are the most common sites of atherosclerotic plaque formation [
50]. The same related experimental studies have found that the endothelium of vessels in these two locations is often affected by disorder or low blood flow rate. Contrarily, vascular areas exposed to high-speed blood flow within the same vessel are less prone to form plaques [
51]. Numerous experimental studies have shown that low shear stress (LSS) on the surface of vascular endothelial cells is an important factor for the occurrence and development of atherosclerosis [
52]. However, there is no research on SGLT2 inhibitors to improve atherosclerosis by adjusting the shear stress. In our study, KEGG pathway analyses showed that the fluid shear stress was closely related to atherosclerosis; our research found that canagliflozin and dapagliflozin may alter fluid shear force-related pathways to improve atherosclerosis. In detail, we found that the genes
AKT1,
MAPK14 and
SRC are involved in fluid shear stress and the atherosclerosis pathway. In the results of the analysis of biological processes, the five core targets were mainly involved in biological processes, including cellular response to hormone stimulus, cellular response to lipid, muscle cell proliferation, a response to lipopolysaccharide, wound healing and positive regulation of cell migration. These biological processes are critical to the development of atherosclerosis. In addition, the five targets also affected molecular functions (phosphotransferase activity, protein kinase binding, protein serine/threonine kinase activity and phosphatase binding) and cellular composition (membrane raft, focal adhesion, postsynapse and postsynapse) related to atherosclerosis. Therefore, through our analysis, we speculate that canagliflozin and dapagliflozin may affect the fluid shear stress and then regulate these target genes, leading to further improvement of AS.
However, this study also has some limitations. Drug and disease targets were collected through databases with limited numbers, so some data may be biased. At the same time, since our current research results were only based on the analysis of the collected data, we need to further confirm our conclusions in future experiments.