| Quercetin is one of important natural flavonoids,widely exists in the important source of food,such as fruits,vegetables and tea.Flavonoids are known as the medicinal properties of plant chemicals,and possess antioxidant,anti-inflammatory,antidepressant,antibacterial and anticancer effects.More and more studies have demonstrated the antioxidant,anti-inflammatory,anti-cancer and cardiac protective effects of quercetin,however,some negative effects of quercetin exposure have also been reported.Therefore,the complexity of the quercetin effects suggests that it is important for studies to assess its health effects.To investigate whether quercetin exposure exerts a beneficial or harmful impact on fish,the present study was designed to examine anxiety and shoaling behaviors in zebrafish(Danio rerio)after quercetin exposure at 0.01,0.1,1,10,100 and 1000 μg/L.After that,antioxidant enzymes,inflammatory cytokines and apoptosis in brains were analyzed.The results showed that(1)The quercetin has a tendency to increase shoal behavior at 0.1-10 μg/L,and it can reduce anxiety at 0.1-100 μg/L(shown as a decrease in latency into the upper half).However,quercetin at the concentration of 1000 μg/L might have adverse effects,manifested by the decrease of motor behavior and the increase of anxiety behavior.(2)The trends of brain antioxidant enzymes,such as superoxide dismutase and catalase,first decreased and then increased.The activities of superoxide dismutase was significantly different from that of the control group at 1-100 μg/L,while the catalase was significantly different from the control group at 10 μg/L.(3)The trends of inflammatory cytokines,such as interleukin(IL)-6,IL-1β,cyclooxygenase-2(COX-2)and tumor necrosis factor alpha,and anti-inflammatory cytokine IL-10 first increased and then decreased,a significant difference were found at 1 μg/L when compared with the control.(4)The expression of Caspase-3(the key performers of apoptosis)and Bax(proapoptotic factor)first decreased and then increased,a significant difference were found at 10 μg/L when compared with the control;while the expression of Bcl-2(anti-apoptotic factor)increased first and then decreased,a significant difference were found at 1 μg/L when compared with the control.(5)In addition,TUNEL assay showed that the glial cells in the subcutaneous subcutaneous regions of the 1 μg/L quercetin group were reduced;while no inhibition of apoptosis,and even more apoptosis,were observed at the 1000 μg/L quercetin group.From the above experimental results,it is known that the zebrafish has the most obvious improvement in anxiety and shoaling behaviors when the quercetin exposure concentration is 1 μg/L,which is beneficial for the fish;however,when the exposure concentration is 1000 μg/L,it has detrimental effects on zebrafish anxiety and shoaling.To further explore the mechanism by which quercetin affects neuro-behaviors,we chosed the representative concentrations of 1 μg/L,1000 μg/L and control for transcriptomic analysis.From the results of transcriptome analysis,we found that:(1)In the sequencing quality information,the Q20 of each group of samples reached 100%,and the proportion of each group of clean reads in the original sequence also reached 96%,indicating the quality of the sequencing data is good.(2)In the alignment of the sequencing sequence with the reference genome,the percentage of Reads in each group that can be compared to the reference genome is in the percentage of Clean reads,that is,the alignment efficiency is higher than 90%,indicating a better alignment ratio.(3)Bioinformatics analysis showed that a total of 1045 differentially expressed genes(DEGs)were screened in the Control VS 1 μg/L quercetin group,of which 874 DEGs were up-regulated and 171 DEGs were down-regulated;in the Control VS 1000 μg/L quercetin group,a total of 1278 DEGs were screened,of which 1149 DEGs were up-regulated and 129 DEGs were down-regulated;(4)Analysis of KEGG pathway revealed that up-regulation of Fanconi anemia pathway,downregulation of phagosome pathways might contribute to suppress brain apoptosis and the inflammatory cytokines in the lower concentrations of quercetin groups,which was consistent with the TUNEL and neuroinflammatory factor results.After exposure to 1000 μg/L quercetin,MAPK signaling and endocytosis pathways were up-regulated,Fanconi anemia pathways were down-regulated,which might be associated with the increase of stress.This would lead DNA damage,reduction of repair ability,andincrease of abnormal protein contents,which eventually result in aggravation of neuronal apoptosis.These results are also consistent with the results of TUNEL detection and neuroinflammatory factor detection.The present study demonstrated that an appropriate concentration of quercetin is beneficial to the health and can improve the shoaling and anxiety behaviors in fish.However,high concentrations of quercetin(eg,1000 μg/L)can adversely affect these behaviors.The beneficial or detrimental effects of quercetin may involve multiple mechanisms,such as induction of oxidative damage,pro-oxidation,regulation of neuroinflammatory cytokines,and neuronal apoptosis. |
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