| Objective: Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is the core metabolite of compound Tanshin formula in the organism,which has anti-hypoxic,anti-inflammatory,antioxidant,vasodilatory and anti-myocardial ischemic and cardiomyocyte protective effects on in vitro cellular models.In order to verify whether it has the same effects on the overall animal model,we used the model organism zebrafish for the evaluation of the antihypoxic activity and the mechanism of action of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate.Previously,when we conducted literature research,we did not find any reports on the safety evaluation of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate in zebrafish model.In order to determine the safe concentration of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate in zebrafish model and to better investigate the anti-hypoxic activity and mechanism of action,we conducted a systematic experimental study on the safety evaluation of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate.Methods: 1.The safety evaluation of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was performed using a zebrafish model to determine the safe concentration of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate and to systematically evaluate the effects of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on the heart,liver,kidney and development of zebrafish.2.The anti-hypoxic activity of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was investigated using a zebrafish model,and the effects of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on mortality,overall morphology,dopamine neurons,brain,blood vessels,locomotor behavior and oxidative stress of zebrafish damaged by hypoxia were used as evaluation indicators.The anti-hypoxic activity of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was evaluated in zebrafish model,and the effects of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on mortality,overall morphology,dopamine neurons,brain vascularization,motor behavior and oxidative stress were used as evaluation indexes.3.The binding mode and activity of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate were analyzed by molecular docking.The anti-inflammatory and antioxidant abilities of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate were evaluated by investigating the effects of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on inflammatory cells,apoptotic cells and reactive oxygen species in zebrafish damaged by hypoxia,and the expression levels of genes related to inflammation and oxidative stress were examined by Real time qPCR.The mechanism of anti-hypoxic injury of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate was verified by molecular docking technique.Results: 1.The experimental results showed that low concentrations of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate(1.0 mM and below)had little or no effect on zebrafish larvae.High concentrations of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate were toxic to juvenile zebrafish,causing morphological and functional damage to zebrafish tissues and organs.Based on the results,we selected 0.5,1.0 and 2.0 mM as the working concentrations for the subsequent safety evaluation experiments.The experimental results revealed that at the concentration ofisopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate of 2 mM,the heart rate of juvenile zebrafish was significantly reduced,the liver showed atrophy,blurred boundary,deepened color and other malformations,and the relative fluorescence area and relative fluorescence intensity of the liver were significantly reduced.Compared with the blank group,there was no edema on the body side and eye of zebrafish in each dosing group of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate,indicating that the kidney of zebrafish was not affected by each concentration of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate.In the study of the effect of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on zebrafish development,it was found that zebrafish in the isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 1.0 mM and 2.0 mM groups had delayed development,incomplete embryos,and slight pericardial edema in hatched juveniles.As the exposure time increased,zebrafish showed deformities such as curved body shape,missing swim bladder and shrunken liver.The degree of deformation and mortality of zebrafish in the isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 2.0 mM group was severe.2.Compared with the blank group,there was no difference in the overall morphology,tissues and organs of juvenile zebrafish in the anoxic group and the isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate group,and no zebrafish death occurred in each group.Compared with blank group,the relative length of dopamine neurons of zebrafish in hypoxia group was significantly reduced.Compared with anoxic group,the relative length of zebrafish dopamine neurons in isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 200 μM group was significantly increased.Compared with the blank group,the cerebral blood vessels of zebrafish in the hypoxia group were damaged and the number of blood vessels decreased,and the number of blood vessels in the brain increased significantly after the administration of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate.Compared with blank group,swimming behavior of zebrafish in anoxic group decreased,and the total swimming distance and average speed decreased.Isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoateimproved the decreased swimming ability of zebrafish induced by hypoxia.Compared with anoxic group,the swimming distance and average speed of zebrafish in isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate treatment group increased.Compared with blank group,the glutathione content of zebrafish in hypoxia group was decreased,while the glutathione content in isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 100 and 200 μM groups was significantly increased.Compared with blank group,malondialdehyde content of zebrafish in anoxic group had an increasing trend,and decreased after administration of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate,but there was no significant difference.3.Transcriptome sequencing results revealed that the GO entries with higher enrichment of biological processes in the isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 50 μM group compared with the hypoxic group were protein hydrolysis,chemokine-mediated signaling pathways,inflammatory response to trauma,and neutrophil chemotaxis.The entries with higher enrichment of biological processes in theisopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 100 μM group were neutrophil chemotaxis,chemokine-mediated signaling pathways,immune response and inflammatory response.The biological processes that were more enriched in the isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate 200 μM group were neutrophil chemotaxis,chemotaxis,response to bacteria,chemokine-mediated signaling pathways,and immune response.The KEGG pathway enrichment analysis revealed that the four comparison groups were jointly enriched to the neuroactive ligand-receptor interaction signaling pathway.We selected key genes in the neuroactive ligand-receptor interaction signaling pathway for Real time qPCR validation,and found that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate could regulate the abnormal expression of genes in this pathway.We also selected some target proteins such as F2RL1,NPY1 R,PTGER2 andisopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate for molecular docking,and found that isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate has good binding activity with these target proteins.By analyzing the transcriptome sequencing results,we speculated that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate might be involved in the regulation of inflammation and oxidative stress in vivo through the neuroactive ligand-receptor interaction signaling pathway,so we examined the effects of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate on the migration number of inflammatory cells,the number of apoptotic cells and the reactive oxygen species content in zebrafish with hypoxic injury.The results showed that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate reduced the inflammatory cells,apoptotic cells and reactive oxygen species in zebrafish damaged by hypoxia.We selected genes related to inflammation and oxidative stress and performed Real time qPCR to detect their expression changes,and found that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate could regulate the abnormal expression of related genes.Also the molecular docking results showed that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate had good binding activity with related target proteins such as CXCL8,HSP70.1 and PRKCEB.Conclusions: 1.Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate is less toxic to zebrafish.Low concentration of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate(1.0 mM and below)had little or no effect on the overall morphology and organs of zebrafish,while high concentration of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate(>2.0 mM)was toxic to zebrafish,causing damage to the morphology and function of organs of zebrafish.It is suggested that we should pay attention to control the dosage of isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate in clinical application.2.Isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate of different concentrations was given to zebrafish with hypoxia injury,and it was found that isopropyl3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate could reverse the damage caused by hypoxia,such as increasing the relative length of dopamine neurons,increasing the number of blood vessels in the brain,and alleviating the injury of zebrafish motor behavior by increasing the swimming distance and average speed of zebrafish.3.Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate may be involved in the regulation of inflammation,oxidative stress and blood vessels in vivo through the neuroactive ligand-receptor interaction pathway,so as to play an anti-hypoxia effect on zebrafish. |
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