Heart failure (HF) is a major complication of diabetes mellitus (DM). It is necessary to prevent HF in patients with DM. In clinical studies, SGLT2 inhibitors were effective in treating HF among patients with DM, which aroused great interest and attention. However, the mechanism is not clear. Based on the network pharmacological analysis, this study explored the mechanism of SGLT2 inhibitors in the therapy of HF and DM. In the present study, four SGLT2 inhibitors (canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin) were obtained to analyze the effects on HF and DM based on the network pharmacology analysis. According to the drugs-diseases target network and PPI network, the 33 core genes of the SGLT2 inhibitors acting on HF and DM were obtained from 125 common genes. Among the 33 core genes, the top 5 hub targets SRC, MAPK1, NRAS, MAPK3 and EGFR were screened according to the degree. The module analysis confirmed that SGLT2 inhibitors have the potential to influence varieties of biological pathways that play an important role in the pathogenesis of HF and DM. The key pathways were screened by KEGG analysis, mainly involved Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance and AGE-RAGE signaling pathway in diabetic complications, etc.
1. Further analysis of the core genes
Among the top 5 core genes, the first one, SRC, is a non-receptor tyrosine kinase that serves roles in numerous biological process including cell adhesion, cell cycle and cell migration. Haendeler et al. demonstrated that SRC kinases could be activated by Ang II, which plays an important role in Ang II-mediated processes, including the pathophysiology of cardiac hypertrophy and remodeling, vascular thickening and heart failure[24]. Meanwhile, Pandey et al. found that SRC activation would contribute to the alteration of non-myofibrillar tension, which will impact the baseline tension in fibrotic hearts after MI or in dilated cardiomyopathy[25]. And inhibition of SRC(c-Src) activation can decrease endogenous ROS production and increase ATP production in diabetic GK rat islets [26]. Safari-Alighiarloo et al. also identified that SRC is one of the key genes for type 1 diabetes through analysis of gene expression profiles[27]. SRC may be a potential target for HF and DM disease.
And the next two hub genes, MAPK1(mitogen-activated protein kinase 1) and MAPK3(mitogen-activated protein kinase 3), also named ERK1 and ERK2 respectively, are the members of the MAP kinase (MAPK) family. MAPK family act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, transcription regulation and development. Bueno et al. indicated that the activation of ERK1/2 seems to be related to a beneficial form of myocardial hypertrophy, which may be advantageous to a failing or dilated myocardium[28]. Besides, stimulation by glucose can activate the ERK1/2 signaling pathway in rat islets, and blocking the activation of the ERK1/2 signaling pathway can reduce glucose-stimulated insulin secretion (GSIS)[29]. This suggests that MAPK1 and MAPK3 may be the potential targets in HF and DM.
Next, the following gene is, NRAS, an N-ras oncogene encoding a membrane protein that shuttles between the Golgi apparatus and the plasma membrane, works in various differentiation processes and signal transduction, involving the regulation of cell survival and growth, T cell activation and apoptosis. Katoh et al. has reported that NRAS was one of the representative targets on cardio-miR-214 that were upregulated in human heart failure, showing that NRAS might be associated with the progression of heart failure[30]. And it’s observed that NRAS expression elevated with tumor advancement in diabetic rats owing to increased levels of IRS-1, leading to the activation of MAP kinase cascade[31]. Thus, NRAS has the potential to be the one target of HF and DM.
And the last one of top 5 hub gene is EGFR, epidermal growth factor receptor, a cell surface protein binding to epidermal growth factor, can regulate cell growth, proliferation, and survival, which involved in blood pressure regulation, neointimal hyperplasia, atherogenesis and reactive oxygen. Xu et al. revealed that the enhanced myogenic constriction of the mesenteric artery in heart failure might be related to the loss of plasmalemmal caveolae in mesenteric VSMCs, while the increased activity of EGFR and AT1 – receptor was considered to be one of the mechanisms leading to this result[32]. Furthermore, EGFR transactivation also could mediate the Ang II to affect the pathophysiology of left ventricular (LV) hypertrophy[33]. And Belmadani et al. found that elevated EGFR phosphorylation contributed to resistance artery dysfunction in type 2 diabetes[34]. Zhang et al. concluded that inhibiting EGFR could slow the progression of diabetic nephropathy by decreasing endoplasmic reticulum stress and increasing autophagy[35]. The findings demonstrate that EGFR plays an irreplaceable role in HF and DM. Above all, the top 5 core genes of the present study based on network pharmacology are supported by previous studies.
2. Enrichment analysis based on core targets
GO functional enrichment and KEGG pathway analysis have illustrated the role of the SGLT2 inhibitors in the gene function and signaling pathway. According to the biological processes that mainly reflects the GO functional enrichment, these core targets are focused on kinase activity, primarily in positive regulation of MAP kinase activity, positive regulation of protein serine/threonine kinase activity, regulation of MAP kinase activity, activation of protein kinase activity and so on. In the enrichment of the KEGG pathway, Rap1 signaling pathway, MAPK signaling pathway, EGFR tyrosine kinase inhibitor resistance and AGE-RAGE signaling pathway in diabetic complications are the significant pathways.
Rap1 signaling pathway is implicated in a wide range of biological processes from cell proliferation and differentiation to cell adhesion[36].Rap1 has been proved to play a part in the regulation of integrin affinity, adhesion, and migration in postnatal neovascularization[37], mainly mediating in the angiogenesis pathway served an important role in cardiac hypertrophy. Furthermore, Rap1B can prevent excessive vascular leakage in early diabetes mellitus by inhibiting VEGF signal transduction. Through controlling telomere length, Rap1 can decrease the occurrence and development of diabetes-related cardiovascular disease [38]. Downregulation of Rap1B to reduce VEGF signal transduction can impede excessive vascular leakage in early diabetes mellitus [39]. At the same time, Rap1 also regulates MAP kinase (MAPK) activity in a manner highly dependent on the context of cell types[40].
Consistent with the core target results above, the MAPK pathway is also predicted to play a role in HF and DM. It’s demonstrated that the MAPK signaling pathway cascade initiated in cardiomyocytes through activation of g protein-coupled receptors, receptor tyrosine kinases, and stress stimulation [41]. Zhang et al. informed that the MAPK signaling pathway could regulate cardiomyocyte apoptosis in mice with heart failure after MI, indicating that this pathway has a potential role in heart failure disease [42]. During the process of insulin resistance, there exists a chronic inflammatory response, which makes the MAPK pathway serve on the inflammatory response of type 2 diabetes, such as diabetic nephropathy and liver disease[43, 44].
EGFR tyrosine kinase inhibitor resistance, acts on EGFR tyrosine kinase, also identical with the core genes predicted based on the PPI. Previous studies had indicated that inhibition of EGFR activity protected against progressive DN in T1 DM and T2 DM [45]. And Zeng et al. inferred that EGFR inhibition reduced ROS production in the left ventricle and blunted hypertensive myocardial hypertrophy in spontaneously hypertensive rats [46]. Therefore, the EGFR tyrosine kinase inhibitor resistance may have a common effect to accelerate the progression of HF and DM.
AGE-RAGE signaling pathway, is a well-studied cascade in DM. It's found that the AGE-RAGE signaling pathway can directly mediate vascular calcification in diabetes[47]. Additionally, the pathway can also impact diabetic complications, as it leads to oxidative stress, increased inflammation, and enhanced extracellular matrix accumulation resulting in diastolic and systolic dysfunction[48]. Fukami et al. proved that the activation of the AGE-RAGE signaling pathway in diabetic complications could cause excessive production of advanced glycation end products to hurt cardiomyocyte, leading to HF[49], which pointed that the AGE-RAGE signaling pathway in diabetic complications predicted in the study may mediate the progression of heart failure, as the same as the results of pathway analysis in another study[50]. Therefore, our results suggest that the 4 signaling pathways might be involved in the mechanisms of SGLT2 inhibitors affecting HF and DM.
Network pharmacology is indeed considered to be a new method to study the relationship between drugs and diseases. In our study, the network pharmacology revealed the potential targets of SGLT2 inhibitors, as well as HF and DM related targets, and bioinformatics was used to study the main enrichment pathways. Based on the network pharmacology, our study predicted the potential therapeutic targets of SGLT2 inhibitors on HF and DM, revealed its action on the main pathways through core genes, which explained the mechanisms of SGLT2-inhibitors on HF and DM and provided scientific evidence for SGLT2-inhibitors to HF and DM. However, the main limitation of this study is the lack of experimental verification. Consequently, it is of great significance to undertake pharmacological studies to research the relationship between SGLT2 inhibitors in HF and DM. Moreover, the validation of molecular levels of our findings is necessary for the future.