| Purpose: 1.The zebrafish model was used to evaluate the safety of the water extracts from flowers,the ethanol and water extracts from stems,and the ethanol and water extracts from leaves of Three Gorges Yangju to determine the safe concentration of the extracts.2.A zebrafish model of nonalcoholic fatty liver injury was established.The liver protection experiments of the extracts from Three Gorges Yangju(chrysanthemum morifolium),stem and leaf were conducted,and the liver protection activities of the three Gorges Yangju’s flowers,stem and leaf were compared according to the experimental results.3 The network pharmacology was used to predict the key targets for liver protection of Three Gorges Yangju,and GO and KEGG enrichment analyses were performed on the key targets to predict the liver protection mechanism,and the zebrafish model was used to verify the liver protection mechanism of Three Gorges Yangju.4.According to the results of network pharmacology,the core components that play a role in liver protection of Three Gorges Yangju were determined,and the basic research on liver-protecting active substances of Three Gorges Yangju was completed.Methods: 1.Using wild-type AB zebrafish three days after fertilization,TG(L-FABP: EGFP)liver-labeled green fluorescent protein transgenic zebrafish as a model,we investigated the lethal rate and teratogenic rate of different extracts on zebrafish,as well as the effects on the heart,liver and kidney of zebrafish,and finally determined the safe concentration of different extracts.2.Three days after fertilization,Tg(LIPAN: ERFP)liver was used to mark the red fluorescent protein transgenic zebrafish,and the 4m M thioacetamide was used to construct the liver injury model of zebrafish.The liver protection experiments of zebrafish were conducted with the extracts of flowers,stems and leaves of Three Gorges Yangju,respectively.The liver protection activities of the extracts from different parts were compared by comparing the liver area,liver fluorescence intensity and yolk sac area of zebrafish protected by different extracts.3.Prediction of the active components and action targets of Three Gorges Yangju in the treatment of non-alcoholic fatty liver by network pharmacology was carried out.The liver protection effect of Three Gorges Yangju was verified by zebra fish model.The abnormal lipid accumulation in zebra fish was analyzed by zebra fish oil red staining.The liver injury was analyzed by using zebra fish liver sections.The expression levels of relevant genes in the signaling pathway predicted by network pharmacology were verified by real-time quantitative polymerase reaction(RT-q PCR).4.according to the main active components playing a role of liver protection found by network pharmacology,the active components of the three gorges yangju are screened out by measuring the content of each main active component in the three gorges yangju by using an HPLC method,and the liver protection effects of different compounds are compared by taking zebrafish as a model,the most main component of the three gorges yangju for treating non-alcoholic fatty liver is found out according to a liver protection result and the content of each compound,the zebrafish experiment is carried out,and T-CHO,TG and other biochemical indicators to explore the liver protection mechanism of the core component.Finally,RT-q PCR is used to verify the liver protection mechanism of the active component.The results were as follows: 1.The extracts from the leaves,stems and leaves of Three Gorges Chrysanthemum morifolium had no effect on zebrafish,but it could cause death and deformity of zebrafish only at high concentration.Among them,the safe concentration of flower water extract was 0.2 mg/m L.The safe concentration of the leaf extract was 2.8 mg/m L;The safe concentration of the leaf alcohol extract was1 mg/m L;The safe concentration of the aqueous extract from the stem was 2.8 mg/m L;The safety concentration of the stem alcohol extract was 2.8 mg/m L.2.The results of zebrafish experiments showed that the extracts from the stems,and leaves of Three Gorges Yangju had liver-protecting activity,and the water extract from the flowers had the best liver-protecting effect.3.Network pharmacology predicted that there were 20 active components of chrysanthemum in the treatment of non-alcoholic fatty liver,and51 common targets of chrysanthemum and non-alcoholic fatty liver.GO and KEGG predicted that the pathways of chrysanthemum in the treatment of non-alcoholic fatty liver involved multiple pathways.The results of zebrafish oil red staining showed that abnormal accumulation of liver appeared in the liver of zebrafish.Under the protection of the water extract of Three Gorges Yangju,the abnormal accumulation of lipid in the liver was improved.The results of zebrafish sections showed that in normal liver tissue,the cells were closely arranged,the polygonal outline was clear,and the nuclei were in the shape of normal spherical vacuoles.The morphology of the liver tissue in the model group was severely damaged,and there were a large number of gaps between the cells and a large number of vacuoles in the nucleus.Under the protection of different concentrations of water extract of Three Gorges Yangju,liver tissue morphology was improved and vacuoles were reduced.4.According to the prediction of network pharmacology,farnesoid,luteolin,kaempferol,cherry glycoside and quercetin are the core components of Three Gorges Yangju in the treatment of non-alcoholic fatty liver disease.Among them,luteolin content was 0.12 g/mg,cherry glycoside content was 9.515 g/mg,and quercetin content was 0.099 g/mg.No kaempferol and farnesol were detected.The zebrafish experiment showed that all five components had good liver-protecting activity and played a certain therapeutic effect on TAA-induced nonalcoholic fatty liver disease in zebrafish. |
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