Study On The Anti-hypoxia Effect Of American Ginseng And Its Main Active Components Based On The Zebrafish Hypoxia Model

Objective: Establish a zebrafish hypoxia model,and evaluate the anti-hypoxia activity of the ethanol extract of American ginseng,explore the potential anti-hypoxia components of the ethanol extract of American ginseng,and verify the anti-hypoxia effect of the screened monomer Pseudoginsenoside F11.Methods: 1.The anoxic model of zebrafish was induced by anhydrous sodium sulfite.With Rhodiola capsule as the positive drug,the parameters such as the modeling treatment time and the drug pre-protection time were optimized,and the success of the anoxic model was evaluated according to the neural behavior of zebrafish after anoxia.2.Based on the established hypoxic model,the neurobehavior,mean heart rate,LDH release,HIF-1α,CS expression levels of zebrafish juveniles at different stages after hypoxia and The anti-hypoxic effects of the alcoholic extract of American ginseng were evaluated based on the established hypoxia model.3.The chemical characterization of the alcoholic extract of American ginseng was performed using UPLC-Q-Exactive Orbitrap-MS technology,and the potential targets of the anti-hypoxic effect of American ginseng were searched for with the help of a web-based pharmacology platform,and the identified components were molecularly docked with the potential targets to select the ones with lower binding energy to the target.4.Among the selected ingredients,Panax quinquefolium characteristic saponin Pseudoginsenoside F11 was selected for the next step of activity verification.The anti-hypoxia effect of Pseudoginsenoside F11 was verified by detecting the swimming speed,distance,swimming track,floating rate,average heart rate,anoxic marker index and cell apoptosis of young zebrafish.Results: 1.The experiment was conducted with the chemical reagent anhydrous sodium sulfate as the modeling agent,and zebrafish at 5 dpf were selected for the administration of pre-protection time of 48 h.Zebrafish larvae were established with the dissolved oxygen level of 0.6 mg/m L in water.2.The alcoholic extract of American ginseng could be safely administered in the range of 0～150 μg/m L,and the50,100 and 150 μg/m L American ginseng alcoholic extract administration groups significantly improved the zebrafish In the third stage of hypoxia,the swimming speed and distance of juvenile zebrafish in the model group decreased,and the pre-protection group of American ginseng extract significantly improved the reduction of vitality caused by hypoxia.In the third stage of hypoxia,the swimming speed and distance of juvenile fish in the model group decreased,while the pre-protection group of American ginseng alcohol extract significantly improved the reduced vigor caused by hypoxia.Compared with the model group,the zebrafish mean heart rate due to hypoxia was significantly enhanced in the ginseng alcoholic extract pre-protection group.All dose administration groups reduced LDH release,increased CS viability,reduced HIF-1α expression,and reduced apoptosis in brain tissue caused by hypoxia.3.A total of 42 components,mainly saponins,were identified by UPLC-Q-Exactive Orbitrap-MS.Using network pharmacology,a total of five potential anti-hypoxic targets were screened: EGFR,TNF,VEGFA,STAT3,and m TOR.dynamic molecular docking results showed that the key active ingredients of American ginseng had good binding ability to the targets,among which Pseudoginsenoside F11,Ginsenoside Rg5,Ginsenoside Rh1,and Adenosine stably bound to the key targets,which might be the key potential pharmacological substances of American ginseng against hypoxia.4.The highest administered mass concentration of Pseudoginsenoside F11 was 100 μg/m L;the survival time of juvenile fish under hypoxic conditions was significantly increased by pre-protection with 50μg/m L Pseudoginsenoside F11 administration.By comparing the locomotor trajectories of zebrafish larvae in the first stage of hypoxia,it was found that the drug-administered group could adapt to the hypoxic environment and swim more regularly than the model group;the Pseudoginsenoside F11-administered group could significantly enhance the vigor of zebrafish larvae,and the swimming speed and distance were significantly higher than those of the model group;all concentration-administered groups could effectively reduce the floating head rate and increase the mean heart rate;the medium and high dose administration groups could reduce The drug administration at medium and high doses reduced the release of LDH,increased CS viability,reduced HIF-1α expression,and reduced apoptosis in brain tissue caused by hypoxia.Conclusion: In this study,the anoxic model of juvenile zebrafish was successfully established,which is easy to operate and has good repeatability.The study found that the ethanol extract of American ginseng can improve the survival time of anoxic zebrafish by reducing the release of LDH,improving the activity of CS and reducing HIF-1 α The expression and reduction of apoptosis in brain tissue play an anti-hypoxic role.Four potential drug substances were screened by UPLC-Q-Empirical Orbitrap-MS combined with network pharmacology-dynamic molecular docking technology,and the characteristic saponin,Pseudoginsenoside F11,was selected to verify the anti-hypoxic activity.This study provides candidate drugs for the prevention and treatment of hypoxia,and provides a basis for the screening of anti-hypoxia components of traditional Chinese medicine.