Identification Of Anti-heart Failure And Anti-inflammatory Substances Of Panacis Quinquefolii Radix Based On Zebrafish Model

Objectives:As a traditional Qi-tonifying herb,the efficacy of American Ginseng（Panacis Quinquefolii Radix）in tonifying qi and clearing heat is closely related to anti-heart failure and anti-inflammatory effects,but the knowledge of its pharmacodynamic substances is still not comprehensive.In this study,we established a new method to identify the potent substances of Chinese medicine with the help of network pharmacology technology,zebrafish and metabolomics technology of Chinese medicine,and explored the potential of anti-heart failure and anti-inflammatory effects of American ginseng.Methods:1.Network pharmacology was used as a tool to characterize the anti-heart failure and anti-inflammatory network regulation of Panacis Quinquefolii Radix from a holistic perspective;According to the key targets of molecular docking,the potential anti heart failure and anti-inflammatory active ingredients were found,which provided reference for the follow-up experiments.2.Wild AB line zebrafish developing until 2 dpf（days post fertilization,dpf）and Neutrophils transgenic zebrafish Tg（lyz:Ds RED2）labled by red fluorescence until 3 dpf were used to establish heart failure and inflammation models of zebrafish,and to evaluate the activity differences of American ginseng from different habitats.3.According to the samples of Panacis Quinquefolii Radix from different areas with different activities,the metabolomics of traditional Chinese medicine was analyzed by UPLC-MS/TOF technology,and the mathematical models of PCA and OPLS-DA were established to screen the components that may be related to the anti-heart failure and anti-inflammatory activities of Panacis Quinquefolii Radix.4.Zebrafish model was used to verify the activity of different components identified by metabolomics of traditional Chinese medicine,and then the anti-heart failure and anti-inflammatory substances of American ginseng were determined.Finally,based on the"drug target"view of network pharmacology,the possible network action mechanism of identified pharmacodynamic substances is predicted again.Results:1.The network pharmacology was used to predict the anti-heart failure and anti-inflammatory activities of Panacis Quinquefolii Radix.By constructing the multi-dimensional network of traditional Chinese medicine,it was predicted that the components of Panacis Quinquefolii Radix might act on 8 signaling pathway（JAK-STAT,TNF,HIF1,Estrogen,c AMP,TNF,c GMP-PKG,regulation of actin cytoskeleton and m TOR）play an important role in anti-heart failure.It can play an anti-inflammatory role by acting on multiple targets in PI3K-Akt signaling pathway.Network prediction analysis showed that Ginsenoside Rg3,3-epioleanolic acid,16-oxoseratonediol and Gypenoside IX in Panacis Quinquefolii Radix might be the important components of anti-heart failure and anti-inflammatory.2.The results of zebrafish activity test showed that the WE,AE and SMEW of American ginseng had significant improvement on zebrafish heart failure caused by verapamil hydrochloride.The anti-heart failure activity of Panacis Quinquefolii Radix from different habitats fluctuated significantly,and the anti-heart failure activity index of Panacis Quinquefolii Radix from Shandong Province was significantly better than that of Liaoning Province.The treatment of 3 dpf zebrafish with Cu SO₄significantly induced the migration of neutrophils to the lateral colliculus,which proved that the zebrafish inflammation model was successfully constructed.Using this model,it was found that the anti-inflammatory activities of Panacis Quinquefolii Radix collected from Shandong and Shaanxi were significantly different,and the anti-inflammatory activities of Panacis Quinquefolii Radix collected from Shandong were better than those from Shaanxi.3.Using metabolomics of traditional Chinese medicine,we detected and analyzed Panacis Quinquefolii Radix from Shandong,Shaanxi and Liaoning provinces,and made pattern discrimination.It was found that Ginsenoside Rg3,Ginsenoside Rg5,Ginsenoside Rg6,DL-malic acid,Quinic acid,L-arginine succinic acid,3-methyl-3-butenyl-apinyl-（1→6）glucoside,（-）-Annonaine and so on were the components with different anti-heart failure activities.The different components related to anti-inflammatory activity may be Ginsenoside Rs3,Ginsenoside Rg1,Ginsenoside Rg4,3-o-methylgallic acid,α-Linolenic acid,Oleanolic acid,Falcarindiol and so on.4.The results showed that Ginsenoside Rg3,Gginsenoside Rg5,Ginsenoside Rg6,DL-malic acid and Quinic acid were the main effective substances anti-heart failure;Ginsenoside Rg1,Ginsenoside Rg4,3-o-methylgallic acid,Oleanolic acid and Falcarindiol are the main effective substances for anti-inflammatory effect.Conclusion:In this study,a novel strategy of"network prediction-omics discrimination-activity evaluation"was constructed,which could realize the rapid identification of major pharmacodynamic substances.We discovered five major anti-heart failure components,five major anti-inflammatory components in American ginseng.It could provide new evidences for the basic research of pharmacodynamic substances in American ginseng.