Exploring the mechanism of Fuzi Ganjiang in preventing myocardial ischemia-reperfusion injury based on network pharmacology and in vitro experiments
Ischemic heart disease (IHD) is the heart disease with the highest incidence rate, disability rate and mortality in China. With the rapid development of social economy and the aging of the population, IHD appears younger and the incidence rate is rising year by year. Myocardial ischemia-reperfusion injury (MI/RI) is an inevitable phenomenon during the treatment of ischemic heart disease, which leads to the expansion of myocardial infarction area and arrhythmia. MI/RI is an urgent problem that needs to be solved in modern medicine, but there is no specific drug yet.
MI/RI belongs to the category of chest obstruction in traditional Chinese medicine. Traditional Chinese medicine emphasizes a holistic approach and has unique experience and advantages in treating chest obstruction. Previous studies have shown that traditional Chinese medicine has good therapeutic effects in treating chest obstruction, such as Sini Tang, Yiqi Huoxue Tang, and Gualou Xiebai Tang. Aconiti Lateralis Radix Preparata Zingiberis Rhizama (AZ) is the core drug pair of Sini Tang. Previous studies by our research group have shown that the AZ group has the best therapeutic effect after the disassembly of Sini Tang, but its specific mechanism is still unclear. In addition, our research group has also demonstrated that the combination of AZ can reduce the apoptosis rate of myocardial cells during MI/RI, but its specific mechanism has not been studied yet. Due to the complex composition and unclear mechanism of action of AZ, it seriously hinders its clinical application and new drug development. Therefore, it is urgent to study the mechanism of AZ’s anti MI/RI effect.
Network pharmacology is an important tool for analyzing traditional Chinese medicine formulas. Network pharmacology has been widely used to analyze the biological processes and signaling pathways of traditional Chinese medicine compound treatments for diseases. To investigate the mechanism of action of AZ against MI/RI, this study intends to use network pharmacology to predict the mechanism of action of AZ against MI/RI, and verify the predicted results of network pharmacology through in vitro studies, laying a research foundation for the next development of AZ.

 

Myocardial ischemia-reperfusion injury belongs to the scope of chest obstruction and heartache in traditional Chinese medicine. The occurrence of chest obstruction is often related to factors such as invasion of cold pathogens, loss of emotional control, and old age and physical weakness. Fuzi and dried ginger have hot medicinal properties and are representative medicines for warming the body. Fuzi without dried ginger is not hot, and AZ combined with warming the body has better therapeutic effects. At the same time, AZ is used to treat chest obstruction because it has the effect of warming yang and promoting blood circulation. Warming yang refers to improving the energy metabolism of myocardial cells, while promoting blood circulation refers to relaxing blood vessels. AZ can improve energy metabolism and relax blood vessels to exert therapeutic effects. To verify whether AZ treats MI/RI by improving energy metabolism and relaxing blood vessels, we chose network pharmacology to predict the biological processes and specific mechanisms of AZ’s anti MI/RI effects.

Using network pharmacology to analyze the active ingredients in AZ and construct an active ingredient target network, the top key components in the network were identified as aconitine in Aconitum carmichaelii, β – sitosterol and 10 gingerol in dried ginger. Modern pharmacological studies have shown that aconitine has cardiotonic and antiarrhythmic effects; β – sitosterol has antioxidant and anti-tumor effects; 10 gingerol has anti-inflammatory properties and improves myocardial cell viability in H/R injury. These results indicate that the predicted components are likely to be the key active ingredients in AZ’s anti MI/RI activity.

Ten key biological processes were identified through GO-BP analysis of key targets. The positive regulation of nitric oxide biosynthesis process, negative regulation of apoptosis process, response to oxidative stress, impact of apoptosis signaling pathway on DNA damage, lipopolysaccharide mediated signaling pathway and other biological processes are interrelated with vasodilation, apoptosis, inflammation, and oxidative damage. Research has shown that reducing cell apoptosis, inflammatory response, oxidative damage, and promoting vasodilation can alleviate MI/RI. The GO-BP results predicted by network pharmacology are basically consistent with the known literature reports on MI/RI, indicating that their results have certain feasibility. Meanwhile, this study also found that AZ can increase the survival rate of RVEC cells with H/R injury, reduce apoptosis rate, and oxidative damage.

In order to further explore the mechanism of AZ anti MI/RI, KEGG signaling pathway analysis revealed that AZ anti MI/RI mainly exerts therapeutic effects through the HIF signaling pathway, PI3-AKT signaling pathway, and TNF signaling pathway, which are considered potential signaling pathways for AZ anti MI/RI. The HIF signaling pathway plays an important role during MI/RI. The hypoxia inducible factor HIF α is activated upon sensing hypoxia in the body, and the activated HIF α increases downstream expression of VEGF and eNOS. VEGF is an inducer of angiogenesis that can promote endothelial cell proliferation and improve vascular homeostasis. Nitric oxide synthase eNOS can promote the release of NO, leading to vasodilation and increased arterial blood flow. VEGF and eNOS activation during myocardial tissue hypoxia can promote vasodilation and angiogenesis, providing sufficient oxygen for myocardial tissue. The PI3K-AKT signaling pathway reduces cardiomyocyte apoptosis during MI/RI. The TNF signaling pathway reduces inflammatory response during MI/RI. Previous studies have also shown that promoting vasodilation through the HIF signaling pathway, improving myocardial cell energy metabolism through the PI3K-AKT signaling pathway, and reducing inflammatory response through the TNF signaling pathway are important signaling pathways for treating MI/RI. AZ has the effect of warming yang and promoting blood circulation, among which warming yang refers to improving myocardial energy metabolism and reducing myocardial cell apoptosis. It has been confirmed that it promotes vasodilation and exerts therapeutic effects, but it is still unclear. In this study, it was confirmed that AZ can stimulate the HIF α/VEGF/eNOS pathway, increase the expression of HIF α, VEGF, and NOS proteins in H/R damaged RVEC cells, and promote vasodilation/endothelial cell proliferation. It is worth noting that the therapeutic effect of the high-dose group is not significant in the WB index, which may be due to the unreasonable dose gradient we set and the toxic effects of the active ingredients in Aconitum. The research team will pay attention to this issue in future studies.

In summary, this study obtained 16 active ingredients and 171 therapeutic targets of AZ; Graph analysis revealed that AKT1, IL6, and TNF are potential targets; GO enrichment analysis revealed that AZ may exert therapeutic effects through apoptosis, inflammation, and vasodilation; KEGG analysis revealed that AZ may exert therapeutic effects through the PI3K-AKT signaling pathway, TNF signaling pathway, and HIF signaling pathway; In vitro studies have found that AZ can increase the survival rate, reduce apoptosis rate, and oxidative damage of RVEC cells damaged by H/R; Increase the expression of HIF – α, VEGF, and eNOS proteins. AZ activates the HIF/VEGF/eNOS signaling pathway to reduce oxidative damage and apoptosis of endothelial cells, promote vasodilation/endothelial cell proliferation, and exert anti myocardial ischemia-reperfusion injury effects, which is consistent with the results predicted by network pharmacology. AZ can promote vasodilation and endothelial cell proliferation through the HIF signaling pathway to exert therapeutic effects, providing a research basis for further development of AZ.