TCM has a long history in the treatment of chronic diseases such as coronary artery disease and various complications. In the pharmacological research of active ingredients, due to the complex composition of TCM, conventional pharmacological approaches to identify the unique ingredients and mechanisms may not be feasible. However, the active ingredients and gene targets are important in uncovering the pharmacological mechanisms of TCM, which can provide a theoretical basis for drug development and design. Therefore, the network pharmacology provides a unique and innovative way to study the active ingredients and understand the mechanisms of multi-ingredient–multi-target–multi-pathway mode of TCM.
In this study, 111 active ingredients and 184 gene targets in TXL were determined by TCMSP database. Subsequently, 79 overlapping genes related to AMI were obtained. Herb-ingredient-overlapping target network suggested that the therapeutic effect of TXL on AMI was directly related to 66 active ingredients. Based on the degree value of each ingredient, beta-sitosterol was considered as the uppermost active ingredient of TXL against AMI. PPI analysis showed that the hub genes were IL6, AKT1, TNF, JUN, TP53, MAPK8, VEGF, MMP9, and NOS3. GO analysis results showed that the overlapping genes were mainly involved in oxidative stress process, the main cellular component was membrane, and the main molecular function was receptor ligand activity. The main pathways were pathways in cancer, AGE-RAGE signaling pathway in diabetic complications, and Fluid shear stress and atherosclerosis.
AMI is usually initiated by myocardial ischemia due to coronary artery obstruction. In the ischemic myocardium, reactive oxygen species (ROS) are generated, which can directly injure cell membrane and induce cell death. Meanwhile, in the ischemic and surrounding myocardium, inflammatory cytokines, e.g., tumor necrosis factor (TNF)-alpha can be produced via the generation of ROS in cardiac myocytes. TNF-alpha can regulate cell survival and cell death, and act as a trigger of another inflammatory reaction. Inversely, inflammatory cytokines can also stimulate ROS formation. In chronic stage, ROS and inflammatory cytokines activate the matrix metalloproteinase (MMP), which elicits the degradation of collagens and may cause a slippage in myofibrillar alignment and left ventricular dilatation. It is reported that in transgenic mice with cardiac overexpression of TNF-alpha, when MMP-2 and MMP-9 expressions were attenuated by inhibition of TNF-alpha, further collagen synthesis, deposition and denaturation were prevented and left ventricular diastolic function was improved. Furthermore, a clinical study also demonstrated a positive correlation between oxidative stress and relative level of MMP-2 and MMP-9 in patients with coronary artery disease.
In our analysis, beta-sitosterol, the uppermost active ingredient of TXL against AMI, is a natural ingredient widely found in many vegetable oils, nuts and plant medicines, and resembles cholesterol structurally, which is considered as a therapeutic agent to reduce serum cholesterol levels and suppress intestinal cholesterol absorption. Meanwhile, several studies also reported that beta-sitosterol functioned as important molecules in stabilizing the phospholipid bilayers of cell membranes and had the potential to suppress obesity-related chronic inflammations, revert immune abnormalities, and regulate the production of nitric oxide.  Beta-sitosterol was effective in cardiovascular protection by enhancing cellular glutathione redox cycling, which led to reduced oxidant injury in rat cardiomyocytes. Meanwhile, beta-sitosterol isolated from various plants can modulate antioxidant enzyme levels in pathogenesis and decrease free radical generation and act as scavengers of free radicals in vitro. In the recent experiment conducted by Lin et al, the protective effects of beta-sitosterol on myocardial ischemia/reperfusion (I/R) injury were confirmed in the in vivo animal model. The experiment suggested that I/R injury suppressed cell viability and induced cell apoptosis and ROS production, yet beta-sitosterol treatment concentration dependently increased the cell viability, reduced cell apoptotic rates and ROS production of I/R-stimulated H9c2 cells. Moreover, increased infarcted area and cell apoptosis in the heart tissues were also observed in the I/R mouse model, whereas beta-sitosterol treatment could alleviate infarction and cell apoptosis. Therefore, these results above implied that beta-sitosterol was effective in exerting protective actions against oxidative stress and myocardial I/R injury.
Interleukin (IL)-6 is a cytokine with both proinflammatory and anti-inflammatory effects on many cell types, affecting B-cell immunoglobulin production, T-cell cytotoxic activity, platelet production and reactivity, and endothelial function.  Meta-analysis performed by the Emerging Risk Factors Collaboration demonstrated that for each SD increase in log IL-6, there was a 25% increase in risk of future vascular events. Moreover, in a randomized trial including 3,489 patients, circulating IL-6 was a strong independent marker of increased risk for mortality in patients with unstable coronary artery disease. TNF is one of the most important cytokines produced by macrophages and released very rapidly after all types of injuries and stimuli, which plays a very important role in host defense. However, prolonged TNF production is also associated with pathology. Therefore, removal of a upregulated cytokine can make a clinical difference. A retrospective cohort study including 8,845 patients suggested that patients treated with TNF inhibitors had approximately half the risk of developing MI (hazard ratio [HR], 0.50; 95% CI, 0.32–0.81) compared with psoriasis patients treated with topical agents. Another protein, MMP-9 is a collagenase, which upregulated in the diabetic heart, and ablation of MMP-9 decreased infarct size in the non-diabetic MI heart, which provided a novel intracellular role of MMP-9 in mediating cell death via apoptosis and pyroptosis. Consistently, the results based on mouse model fed with high fat diet suggested that TXL treatment could lower the expressions of inflammatory cytokines including IL-6, TNF-alpha and MMP-2 in a dose-dependent manner when compared with the control group. AKT1 is the predominant isoform in vascular endothelial cells and plays a crucial role in physiological and pathological angiogenesis. Many of the angiogenic functions attributed to vascular endothelial growth factor (VEGF) are mediated by intracellular activation of the phosphoinositide 3-kinase–Akt signaling pathway. Similarly, VEGF is important in vasculogenesis, which defined as the formation of blood vessels from de novo generation of endothelial cells and angiogenesis. Again, the effect of TXL on VEGF had been explained by previous research, which indicated that the VEGF expression in TXL treated bone marrow mesenchymal stem cells was increased compared to the control group.
Most importantly, activation of endothelial nitric oxide synthase (eNOS) in cardiac microvascular endothelial cells, whose gene name is NOS3, plays a critical role in the protection against myocardial I/R injury. In our recent experiment where ex vivo, in vivo, and in vitro settings of I/R were used, we identified the signals produced by cardiomyocytes that can regulate cardiac microvascular endothelial cells biology during myocardial I/R injury. We found that cardiac microvascular endothelial cells-derived eNOS activity was required for the cardioprotection of TXL, which again confirmed the mechanism of TXL in the treatment of AMI.
KEGG enrichment analysis suggested that the main pathway was pathways in cancer. Similar with cancer cells, ischemic myocardium are also characterized by high levels of oxidative stress. At low levels, ROS increase cell proliferation and survival through the post-translational modification of kinases and phosphatases, which is required for homeostatic signaling events. At moderate levels, ROS induce the expression of stress-responsive genes, which in turn trigger the expression of proteins providing prosurvival signals, such as VEGF. At high levels, however, ROS can result in damage to macromolecules, including DNA, and cause permeabilization of the mitochondria, leading to the release of cytochrome c and apoptosis. The abovementioned process was similar to that of I/R injury in patients with myocardial infarction. Consistent with our KEGG pathway analysis, in a study investigating the anti-oxidation properties of TXL, the authors found that the remarkably up-regulated expression of NADPH oxidase subunits p22phox, p47phox and inflammatory factors TNF-alpha, IL-1β and NF-κB induced by C16 could be obviously decreased following pretreatment with TXL, which again highlighted the important role of TXL in the process of oxidative stress. Meanwhile, the main pathway of TXL against AMI was also involved in AGE-RAGE signaling pathway in diabetic complications, which indicated that TXL may be effective in decreasing diabetic complication. The Fluid shear stress and atherosclerosis pathway has been confirmed.
In our China Tongxinluo Study for myocardial protection in patients with Acute Myocardial Infarction (CTS-AMI) trial, we are going to recruit 3,796 eligible patients with ST-segment elevation myocardial infarction (STEMI) from 120 centers and randomize them in a 1:1 ratio to TXL or placebo groups. The primary endpoint is 30-day major adverse cardiovascular and cerebrovascular events composed of cardiac death, myocardial reinfarction, emergency coronary revascularization, and stroke. Based on the results of CTS-AMI trial, we sought to determine the clinical efficacy, safety, and mechanisms of TXL in the treatment of STEMI patients in the reperfusion era.
There are several limitations in our study. First, our results need to be further verified by experiments. However, findings from our study provided insight into the research of TXL. Meanwhile, in our CTS-AMI trial, we are going to systematically elucidate the efficacy and mechanisms of TXL in the treatment of AMI, which is helpful in explaining the role of TXL. Second, the TCMSP database had not been updated since 2014, therefore, we still could not completely understand the accurate therapeutic mechanism of TXL on AMI despite the results of network pharmacology. More comprehensive and timely updated TCM databases are needed to make the results of network pharmacology analysis more reliable. A comprehensive understanding of the effect of TXL on AMI depends on the common development of multi-disciplines and further invalidation both in experimental and clinical situation.