Exploring the anti hypoxia effect and potential targets of American ginseng based on zebrafish model and dynamic molecular docking technology
Hypoxia is a complex pathological process caused by multiple factors, which refers to varying degrees of breathing difficulties in the body. The inability to intake sufficient oxygen through the respiratory tract leads to pathological changes in the body, such as cyanosis of the skin, fingers, lips, and even symptoms such as wheezing, shortness of breath, and chest tightness. Hypoxia can induce irreversible damage to multiple organs such as the heart, brain, and lungs, which may develop into fatal diseases such as pulmonary edema or cerebral edema. Traditional Chinese medicine has obvious advantages in anti hypoxia research and clinical application, such as snow lotus and palm ginseng, which have been proven to have good anti hypoxia effects. Digging deeper into the unique medicinal plant resources of our country and discovering more efficient and low toxicity drugs is still of great significance for the prevention and treatment of hypoxia caused by high-altitude environments or pathology.

At present, the main models used for research on anti hypoxic diseases are rat and mouse models. The main methods include weight-bearing swimming experiments, normobaric hypoxia tolerance experiments, sodium nitrite poisoning survival experiments, and acute cerebral ischemic hypoxia experiments, which have limitations and high costs in large-scale drug screening. Research has found that zebrafish can establish hypoxia models through sodium sulfite, and zebrafish have high egg production, short experimental cycles, and low costs, which can achieve high-throughput screening. Therefore, we used the model animal zebrafish to investigate the anti hypoxia effect of American ginseng, and used bioinformatics techniques and methods to screen and predict the active ingredients and potential targets of American ginseng, in order to search for potential targets of American ginseng’s anti hypoxia effect and provide reference for the study of its anti hypoxia mechanism.

 

This article constructs a new model of zebrafish juvenile anti hypoxia, providing a new method for the study of traditional Chinese medicine anti hypoxia. This experiment used sodium sulfite to construct a low oxygen environment, replacing the common method of nitrogen filling to create an hypoxia model, and studied the anti hypoxia effect of American ginseng with a simpler and more direct operation. Using zebrafish as a model animal can reduce the experimental period, achieve high-throughput screening, and characterize hypoxia as a whole. After being deprived of oxygen, zebrafish will experience floating head phenomenon, and their swimming posture will be imbalanced. Their body will not be parallel to the horizontal, and they will swim irregularly in all directions, increasing their swimming speed within a certain period of time. This study evaluated the survival time of zebrafish in different groups under hypoxic conditions and their neurological behavior (floating head, swimming speed, and distance) after hypoxia. It was found that American ginseng extract could significantly improve the survival time of zebrafish under hypoxic conditions. With the increase of drug concentration, the floating head rate of each group of zebrafish also decreased significantly. The movement distance and speed after hypoxia were also significantly different from the model group. The neurological behavior of the high-dose drug group was close to that of the blank normoxic group, indicating that American ginseng extract has a significant anti hypoxia effect, laying the foundation for finding potential anti hypoxia targets in the next step.

American ginseng is a plant of the Panax genus in the Araliaceae family, native to Canada and widely cultivated in Jilin and Shandong provinces of China. As early as the Qing Dynasty Confucian physician Wang Ang compiled the book “Bu Tu Ben Cao Bei Yao”, which recorded that American ginseng has a cool nature, sweet taste, slight bitterness, and traditional effects such as tonifying qi and nourishing yin, clearing heat and generating fluids. Modern pharmacology shows that American ginseng has various pharmacological activities, including anti-tumor, cardiovascular protection, immune regulation, as well as anti myocardial ischemia, anti myocardial oxidation and other effects. However, so far, people’s understanding of the anti hypoxia effect and specific substance composition targets of American ginseng is still very limited. Previous studies have shown that ginsenoside Rg3 can inhibit the proliferation of Eca-109 and 786-0 cells under hypoxic conditions, and induce a significant decrease in the expression of vascular endothelial growth factor (VEGF) mRNA. It can also inhibit the expression of hypoxia inducible factor-1 α (HIF-1 α), cyclooxygenase-2 (COX-2), and nuclear factor kappa B (NF – κ B) induced by hypoxia under hypoxic conditions; Ginsenoside Rb3 can stabilize the cell membrane, inhibit the expression and activity of NOS, and has a significant protective effect on hypoxic-ischemic neuronal damage. Therefore, in-depth exploration of key anti hypoxia active substances in American ginseng is of great significance for quality evaluation and new drug development.

The topological analysis of PPI network suggests that AKT1, STAT3, HSP90AA1, JUN, and TNF may be the core target genes of American ginseng for anti hypoxia. AKT1, as a serine/threonine protein kinase, can be activated by PI3K binding to growth factors and phosphorylated to activate or inhibit downstream substrates such as apoptosis related proteins Bad, Caspase9, mTOR activity, thereby participating in regulating biological processes such as cell proliferation, differentiation, apoptosis, and migration. Research has found that recombinant lentivirus mediated Akt1 gene transfection can significantly enhance the hypoxia tolerance of rat BMSCs by inhibiting apoptosis. STAT3 is a member of the STAT family and plays a crucial role in regulating cell proliferation, differentiation, apoptosis, and other processes. STAT3 plays an important role in hypoxia induced pulmonary artery smooth muscle cells, and blocking the STAT3 signaling pathway can effectively inhibit the proliferation of vascular smooth muscle cells. HSP90AA1 is a member of the heat shock protein family and one of the most abundant cytoplasmic proteins in non stress cells. It plays a critical role in maintaining cellular homeostasis and can enhance cell resistance to external stress under hypoxic stress conditions. JUN belongs to the oncogene family and is a member of the MAPKs superfamily. It can participate in processes such as cell proliferation, inflammation, migration, and invasion. Under hypoxic conditions, an increase in the expression level of phosphorylated c-jun N-terminal kinase can promote the proliferation of rat pulmonary artery smooth muscle cells. TNF is a pro-inflammatory cytokine mainly secreted by monocytes and macrophages, which plays a crucial role in regulating inflammatory effects and host defense against microbial pathogens. During hypoxia stress, TNF regulates the transcription level of HIF-1 α (hypoxia inducible factor-1 α) through the NF – κ B pathway.

This study found through dynamic molecular docking that key compounds of American ginseng (such as ginsenoside F11, papaverine, ginsenoside RO, ginsenoside Rb3, ginsenoside Rg1, ginsenoside Rg3) can bind well with hypoxia core target genes (AKT1, HSP90AA1, JUN, STAT3, TNF). This molecular docking study determined that the original ligand position in the crystal structure is the active docking pocket, ensuring that the binding site between key compounds of American ginseng and hypoxic target proteins is the key active region. The lower the binding energy, the stronger the binding ability between the ligand and the target protein. Through the docking of various compounds with the target, it was found that the pro-inflammatory factor TNF and the heat shock protein HSP90AA1 have a strong binding ability with key active ingredients, which may be a potential key target of American ginseng for anti hypoxia. Through further verification by molecular dynamics, it was found that the dynamic simulation of HSP90AA1 and ginsenoside Rg1, TNF, and ginsenoside Ro can form stable conformations, revealing and verifying the binding tightness and stability of the core target and compound complex. This provides a reliable theoretical basis for the study of potential hypoxia targets of American ginseng for anti hypoxia effects.

In summary, this study evaluated the anti hypoxia effect of American ginseng using a zebrafish model, and explored the active ingredients and core targets involved in anti hypoxia using network pharmacology techniques. Molecular docking technology and molecular dynamics were used to further verify the binding of active ingredients and core targets, revealing potential targets of American ginseng for anti hypoxia. Due to the complexity of traditional Chinese medicine ingredients and the molecular structure of saponins in American ginseng, preliminary exploration based on network pharmacology is still insufficient. Regarding the predicted targets of the anti hypoxia effects of Chinese and Western ginseng, further experimental verification will be conducted in the later stage, in order to provide theoretical basis and reference for the study of the anti hypoxia pharmacological components of American ginseng, and to provide a basis for related mechanism research in the later stage.