| Objective: Based on the chemical biology theory of traditional Chinese medicine,using the main toxic-effective ingredients in Sini Decoction as a tool,and using the zebrafish model,combined with network pharmacology,to study the mechanism of reducing toxicity of Sini Decoction.Methods: Based on zebrafish model and traditional Chinese medicine theory,the toxicity of sini decoction was investigated.To study the developmental toxicity and mechanism of aconitine and isoliquiritin.The main toxic components in aconite were used as model drugs to select the toxic components in dried ginger and licorice.The mechanism of 6-Gingerol and glycyrrhizic acid antagonizing aconitine cardiotoxicity was studied by network pharmacology.Results: 1.AC,HC,and MC,which are the representative constituents of aconite,all have toxic effects on zebrafish,and AC has the most obvious cardiac toxicity.The representative ingredients of dried ginger,6-Gingerol,8-Gingerol,and 6-Shogaol,are all toxic to zebrafish.Glycyrrhizic acid,Liquiritin,and Liquiritigenin in licorice did not cause zebrafish death at a dose of 200 ?M.Isoliquiritin at 12 ?M had a significant toxic effect on zebrafish.2.When aconitine was administered to 72 hpf,the embryos with 7.27 ?M treatment group showed yolk retention.In the 8.23 ?M treated group,pericardial edema,yolk retention,and curved body shape were observed.At 96 hpf,in the 7.27 and 8.23 ?M treated groups continued to increase.Serious pericardial edema,swim bladder defects.Especially in the 8.23 ?M treated group,the malformation rate reached 100%?The pericardial area were increased,The ejection fraction ?stroke volume and fraction shortening were decreased in the 7.27 and 8.23 ?M treated groups at 96 hpf.The liver area and intensity were significantly decreased,the lengths of dopamine ganglion?total swimming distance and velocity of embryos were significantly decreased?The ROS generation was increased,the activities of T-SOD were significantly decreased,the MDA content was significantly increased?apoptotic cells were observed at heart and brain area,both in AO and TUNEL staining.qRT-PCR experiment results shows that the expression of JNK? Bax?Bad?Cyto c?Caspase-9?Caspase-3 were up-regulated,Erk1/2?Bcl-2 were down-regulated ? the expression of Apaf-1 and Keap-1 were not significantly changed?Autophagy related genes the expression of LC3 was down-regulated,Beclinl?Atg3 were not significantly changed.3.When ISL is administered to 96 hpf,the malformation rate reached 100% at 16.31 ?M,the body lengths were suppressed manner,pericardial edema,swim bladder deficiency,yolk retention,and curved body shape were both observed at 12.37 and 16.31 ?M ISL.ISL possessed severe toxicity on the developing heart,liver,and nervous system.The effect of ISL on Histopathology shows that,loose liver cells,irregular arrangement of intestinal wall cells,thinner heart walls,and myocardial fibrosis degeneration.The ROS generation was increased,the activities of T-SOD were significantly decreased,the MDA content was significantly increased?apoptotic cells were observed at heart both in AO and TUNEL staining.qRT-PCR experiment results shown the expression of JNK? Bad?Cyto c?apaf-1?Caspase-9?Caspase-3 ?Bax/Bcl-2 were up-regulated.The expression of Keap-1 was up-regulated,The genes that were related to oxidative stress were significantly down-regulated,such as Nrf2,HO-1,Sod-1,Sod-2? 4.Among the representative ingredients of dry ginger,6-Gingerol has the strongest antitachycardia effect on aconitine.It can significantly shorten SV-BA distance in zebrafish heart at 10 ?M 6-Gingerol;it can improve ejection fraction and increase stroke volume to antagonize aconitine-induced cardiac function impairment.6-Gingerol attenuates the increase of ROS content in the zebrafish heart area and reduces the apoptosis of zebrafish myocardial cells caused by aconitine. Glycyrrhizic acid has the strongest antagonistic effect on aconitine tachycardia among the representative components of licorice.Glycyrrhizic acid can shorten SV-BA distance in zebrafish heart;it can significantly improve ejection fraction and increase stroke volume to antagonize aconitine-induced cardiac function damage in zebrafish.Glycyrrhizic acid reduces the increase of ROS content in the heart areaand reduces the apoptosis of zebrafish myocardial cells caused by aconitine.5.Using network pharmacology analysis,the key targets of 6-Gingerol in antagonizing cardiac toxicity of aconitine were MAPK1,MAP2K1,AKT1,MAPK8,MAPK14 and SRC.The pathways with high enrichment degree include estrogen signaling pathway,pi3k-akt signaling pathway,Ras signaling pathway,etc.The key targets of glycyrrhizic acid in antagonizing cardiac toxicity of aconitine were MAPK,GRB2,CDC42,EGFR,GSK3 B and SCR.The pathways with high enrichment degree include PI3k-AKT signaling pathway,Ras signaling pathway and FoXo signaling pathway.Conclusion :1.Aconitine,hypaconitine,mesaconitine,6-Gingerol,8-Gingerol,6-Shogaol,Glycyrrhizic acid,Liquiritin,and Liquiritigenin in sini decoction have toxic effects on zebrafish,among which aconitine has obvious toxic effects and is the main toxic component in sini decoction.2.For the first time,aconitine has a developmental toxicity effect on zebrafish,causing serious abnormalities in zebrafish,resulting in developmental disorders of heart,liver and nervous system,oxidative stress,mitochondrial pathway apoptosis,and autophagy.3.For the first time,ISL caused developmental toxicity on zebrafish,indicated by morphological abnormalities.Moreover,ISL led to toxic effects on the developing heart,liver,and nervous system.The underlying mechanism might be associated with oxidative stress-induced apoptosis involving Nrf2-HO1/JNK-ERK/mitochondrion pathways.4.6-Gingerol and glycyrrhizic acid could significantly antagonize the cardiac toxicity of aconitine,and it was found for the first time that 6-Gingerol and glycyrrhizic acid could exert antagonistic effects mainly by regulating the pI3k-Akt signaling pathway and Ras signaling pathway through network pharmacology. |
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