UPLC-QE-Orbitrap-MS/MS combined with network pharmacology and experimental verification to explore the mechanism of American ginseng in treating atherosclerosis
Atherosclerosis (AS) is the main cause of vascular diseases. Its lesions are characterized by partial lipid deposition in arteries, accompanied by proliferation of smooth muscle cells and fibrous matrix, and gradually develop into atherosclerotic plaques. The clinical manifestations of AS include ischemic heart disease, ischemic stroke, and peripheral vascular disease. The pathogenesis of AS is very complex, with multiple signaling pathways. Experimental and clinical studies have shown that it may be related to inflammation, oxidative stress, innate and adaptive immunity, infection, and other factors. At present, there is a tendency towards the “injury response inflammation theory”, which is similar in nature to inflammation, but there is no consensus yet.

Panax Quinquefolii Radix is a dried root of the Panax genus in the Araliaceae family, rich in ginsenosides. Research has shown that various ginsenosides have pharmacological activities such as anti-inflammatory, immune regulation, and protection of the cardiovascular system. The traditional drying method for American ginseng is drying, but the dried finished product has wrinkled skin, a burnt yellow color, a hard texture, and is difficult to slice or crush. Freeze drying (freeze-drying) can dehydrate and dry materials at low temperatures and under vacuum. Free drying Panacis Quinquefolii Radix (FDPQ) prepared by freeze-drying has a smooth skin, bright color, loose texture, good rehydration ability, easy to consume, thorough dehydration, and easy storage and transportation. In addition, the total content of ginsenosides Rg1, Re, and Rb1 in FDPQ was significantly higher than that in dried American ginseng. Overall, FDPQ has advantages over dried American ginseng in terms of appearance and internal composition, and has broad market prospects.
The previous research of the research group found that FDPQ can improve oxidative stress injury in atherosclerotic mice, reduce atherosclerotic plaque and lipid deposition in the aortic arch, but the mechanism is still unclear. Network pharmacology can reveal the regulatory effects of drugs on the body at a systemic level, providing new ideas for studying the interrelationships between traditional Chinese medicine components and diseases. This article intends to use UPLC-QE Orbitrap MS/MS technology combined with network pharmacology and molecular docking methods to explore the pharmacological substances, targets, and potential of FDPQ in the treatment of AS

 

At present, the number of people who die from ischemic cardiovascular and cerebrovascular diseases every year ranks first in the world, and myocardial infarction and cerebral infarction caused by AS are the most deadly vascular diseases. The previous research of the research group found that FDPQ may have the effect of intervention or treatment of atherosclerosis. Compared with traditional dried American ginseng, FDPQ has increased the content of some saponin components, but it is still uncertain whether the types of saponin components have changed due to different drying methods and mechanisms; At the same time, the information sources of active ingredients in traditional Chinese medicine databases are single and even outdated, which cannot fully and timely reflect the ingredients contained in FDPQ. Therefore, this article first identified the saponin components in FDPQ through UPLC-QE Orbitrap MS/MS, and then explored its therapeutic substances and mechanisms for treating AS through network pharmacology combined with molecular docking, supplemented by cell experiments for preliminary verification.
UPLC-Q Exactive Orbitrap MS/MS analysis identified 28 types of ginsenosides, including protopanaxadiol types such as Rb1, Rc, and Rd, protopanaxatriol types such as Re, Rh1, and Rg4, and anthropomorphic ginsenoside F11 of the Oktyrone type. Based on database screening, 117 candidate targets for FDPQ treatment of AS were obtained, including 21 core targets such as STAT3, EGFR, MAPK1, AKT1, PIK3CA, VEGFA, etc. Indicating that FDPQ treatment for AS has the characteristics of multi-component and multi-target. The analysis of drug ingredient target disease pathway network shows that there are many targets associated with components such as ginsenoside Rk3, ginsenoside Rh4, ginsenoside Rg4, anthropomorphic ginsenoside F11, and 20 (R/S) – ginsenoside Rh1, which may be important components for FDPQ treatment of AS. Existing studies have shown that ginsenoside Rk3 has antioxidant, anti apoptotic, and anti-inflammatory effects, significantly reducing the expression of inflammatory factors such as NF – κ B, TNF – α, IL-6, and IL-1 β, significantly lowering serum levels of AST and ALT, and reducing the occurrence of oxidative stress. Ginsenoside Rg4 can significantly clear ROS and inhibit ROS induced activation of p38 mitogen activated protein kinase (MAPK), which helps maintain the integrity of endothelial cells. Personification ginsenoside RF11 is a unique component of American ginseng. Studies have shown that it can exert neuroprotective effects on stroke by reducing autophagy/lysosome defects and inhibiting calcium overload. It can also improve long-term neurological function damage after stroke by activating the BDNF/TrkB pathway, promote neurogenesis after stroke, and have great potential in the chronic rehabilitation of ischemic stroke. The enrichment results of GO and KEGG suggest that the mechanism of FDPQ in treating AS is mainly related to PI3K Akt signaling pathway, endocrine resistance, lipid and atherosclerosis related pathways, MAPK signaling pathway, VEGF signaling pathway, etc., which shows that FDPQ has the characteristics of multiple targets and pathways in treating AS. The molecular docking results indicate that the active ingredient has good binding activity with potential targets, and the analysis results of network pharmacology have a certain degree of reliability.
More and more research supports that AS is a chronic inflammatory disease that begins with endothelial injury, and the associated inflammation is mediated by pro-inflammatory cytokines, inflammatory signaling pathways, bioactive lipids, and adhesion molecules. Recent studies have confirmed that the PI3K/Akt signaling pathway plays an important role in inflammatory responses. PI3K is a type of protein kinase that specifically catalyzes phosphatidylinositol lipid substances, while Akt is a serine/threonine protein kinase, also known as protein kinase B. PI3K specifically catalyzes the production of PIP3 by PI, which can fully activate Akt and trigger a cascade reaction in the PI3K/Akt signaling pathway, such as regulating the release of inflammatory factors such as NF κ B, TNF – α, IL-6, and IL-1 β. Regulating these signaling pathways can achieve the effects of inhibiting inflammatory responses, improving oxidative stress damage, and reducing lipid deposition and endothelial damage. The activation of NF – κ B pathway plays an important role in inflammatory response. Protein kinase MAPK is an important substance that transduces signals and triggers cellular responses. After activation of the MAPK family subfamily p38MAPK stress sensitive kinase, NF – κ B can be activated through phosphorylation or pro-inflammatory cytokines (such as TNF α). NF – κ B also activates p38MAPK in reverse through the pro-inflammatory cytokines it produces. The bidirectional effect between the two accelerates the development of atherosclerosis. Vascular endothelial growth factor (VEGF) is secreted by various cells and binds to the homologous tyrosine kinase VEGF receptor (VEGFR-1, VEGFR-2, VEGFR-3) in endothelial cells, producing various downstream effects, promoting the formation of new blood vessels and the growth of vascular endothelial cells, thereby promoting the functional recovery of heart or brain tissue after ischemia-reperfusion. VEGF Akt is a classic signaling pathway that promotes endothelial cell proliferation, inhibits cell apoptosis, and promotes angiogenesis. Research has shown that VEGF inhibits cell apoptosis through the downstream PI3KAkt signaling cascade via the BAD pathway, and promotes endothelial cell proliferation and angiogenesis through mTORC2 and FOXO1. In this study, the screened possible mechanism was verified by cell experiments in vitro. The results showed that FDPQ could increase the mitochondrial membrane potential of PC12 cells induced by H2O2, increase the activities of antioxidant enzymes such as SOD and CAT, reduce the level of MDA in cells, and increase the expression of PI3K/Akt, suggesting that FDPQ may be related to improving oxidative damage, inhibiting apoptosis and inflammation in treating atherosclerosis. This study provides a preliminary explanation of the mechanism of action of FDPQ in treating AS through multiple components, targets, and pathways, but further experimental verification is still needed. However, another limitation of this study is that it did not include large molecular compounds such as polysaccharides and proteins, and further research is needed.