| ObjectiveInsomnia is a common sleep disorder that affecting 10%-40%of the world’s population.Long-term insomnia can seriously damage people’s physical and mental health,and bring a heavy economic burden on society and families.Existing drugs for treating insomnia are limited by unavoidable adverse side effects.Therefore,it is necessary to find safer and more effective treatments.Traditional Chinese medicine is a promising complementary and alternative therapy.Suanzaoren decoction is a classic prescription for insomnia treatment in traditional Chinese medicine.This decoction is composed of 5 kinds of herbs including Suanzaoren,Chuanxiong,Zhimu,Fuling,and Gancao,and has the effect of nourishing the blood and tranquilization.Currently,the sedative and hypnotic effects of Suanzaoren decoction have been confirmed in clinical pharmacological studies and some animal models other than zebrafish.However,the substance basis and mechanisms of action underlying are still unclarified.The establishment of a zebrafish model biological behavioral platform provides new ideas and new methods for identifying the active ingredients of Suanzaoren Decoction and exploring their related pathways.This study was aim to evaluate the effects of Suanzaoren Decoction,Suanzaoren and Jujuboside A on sleep/wake behavior of zebrafish larvae based on the behavioral drug screening platform and then identify whether Jujuboside A is one of the active components that contributed to the sleep-promoting effect of Suanzaoren Decoction.Besides,by analyzing the behavioral phenotype induced by Jujuboside A and the well-characterized compounds,the candidate signaling pathway that associated with Jujuboside A were identified.Finally,the candidate target were further confirmed by conducting pathway-dependent behavioral assays as well as molecular biology experiments.This will provide data for better clarifying the pharmacodynamic basis of sleep-promoting effect and the involved mechanisms of Suanzaoren Decoction.MethodsAn automatic video tracking system was used to monitor the behavioural of zebrafsh larvae for 24 h after treatment.Six rest/wake parameters including rest total,rest bouts,rest bouts length,rest latency,activity total and waking activity were analyzed and then visualized by constructing a behavioral fingerprint.Total rest and waking activity were separately used for time series analysis by averaging in 10 min intervals and for quantitative analysis.To identify the candidate involved signaling pathway,correlation analysis were performed between the behavioral fingerprints induced by Jujuboside A and the well-characterized compounds.Pearson correlation coefficient were calculated and visualized with a heatmap.Hierarchical clustering were also performed for the behavioral phenotype of Jujuboside A and the well-characterized compounds.Besides,C.elegans mutant with phenotypic defects were used for pathway-dependent assay to confirm the effects of Jujuboside A on the candidate involved signaling pathway.Finally,quantitative real-time PCR were conducted to detect the effects of Jujuboside A on the mRNA levels of interesting genes associated with the signalling pathways for further confirmation.Results1.Suanzaoren Decoction at all tested concentrations generally increased total rest minutes,rest bout length,waking activity and the number of day-time rest bouts,and caused a general decrease in the number of night-time rest bouts,day-time total activity and night-time rest latency when compared with the untreated control.Besides,Suanzaoren Decoction caused an increase in day-time rest latency at low and medium concentrations while caused a decrease in it at high concentration.With regard to the night-time total activity,it was increased at medium and high concentrations and decreased at low concentrations.When compared with the untreated control,Suanzaoren at all tested concentrations generally increased total rest minutes,rest bout length,the number of day-time rest bouts and night-time waking activity,and caused a decrease in rest latency,day-time total activity and day-time waking activity.Besides,the number of night-time rest bouts was slightly increased at low concentration,and was slightly decreased at medium and high concentrations.The night-time total activity was slightly decreased at low concentration,and was opposite at medium and high concentrations.When compared with the untreated control,Jujuboside A at all tested concentrations generally increased total rest minutes,rest bout length and the number of day-time rest bouts,and caused a general decrease in the number of night-time rest bouts,total activity and day-time waking activity.In addition,at three different tested concentrations of Jujuboside A,rest latency and night-time waking activity in zebrafish larvae was decreased and increased with smaller amplitude separately.2.Time series analysis showed that normal zebrafish presented apparent day-night rhythms,with most sleep occurred at night and most waking activities occurred during the day time.Compared with the untreated control,zebrafish larvae treated with Suanzaoren Decoction at different concentrations presented a significant increase in total rest time and a varying degrees of increase in waking activity.Larvae treated with Suanzaoren at different concentrations presented a varying degrees of increase in total rest time and decrease in waking activity.Similar to the effects of Suanzaoren,total rest was increased and waking activity was decreased in larvae treated with Jujuboside A.Suanzaoren Decoction-,Suanzaoren-and Jujuboside A-treated larvae remained the day-night rhythms,with a longer duration of rest at night and higher waking activity levels during the day time.While the day-night rhythms tend to flatten in larvae treated with melatonin.3.Quantitative analysis showed that compared with the untreated control,total rest minutes was significantly increased during both the day-time and night-time in zebrafish larvae treated with Suanzaoren Decoction at low and medium concentrations(P<0.05).While Suanzaoren Decoction treatment at high concentration significantly increased total rest time of larvae only during the day-time(P<0.05),without significantly affecting the night-time total rest(P>0.05).Besides,Suanzaoren Decoction treatment at medium concentration significantly increased both the day-time and night-time waking activity(P<0.05).Suanzaoren Decoction treatment at high concentrations significantly increased the night-time waking activity(P<0.05).There was no significant changes in waking activity of larvae after treatment of Suanzaoren Decoction at low concentration(P>0.05).When compared with the untreated control,Suanzaoren at medium and high concentrations significantly increased total rest time during the night-time and the day-time,separately(P<0.05).Suanzaoren at low concentration did not significantly affect total rest time(P>0.05).In addition,Suanzaoren at high concentration significantly decreased the day-time waking activity(P<0.05).While no significant changes in waking activity was found in larvae treated with other tested concentrations(P>0.05).When compared with the untreated control,total rest time in zebrafish larvae was significantly increased after treatment of Jujuboside A at medium concentrations(P<0.05).Besides,total rest time was significantly increased during the night-time and the day-time after treatment of Jujuboside A at low and high concentrations,separately(P<0.05).Jujuboside A at medium concentrations significantly decreased the day-time waking activity(P<0.05).But all tested concentrations did not significantly affect the night-time waking activity(P>0.05).4.Analysis of the stability of drug fingerprints over multiple concentrations showed that there was a high degree of correlation between the behavioral phenotypes of Suanzaoren Decoction across 3 different concentrations(all Pearson correlation coefficients>0.7).Besides,there was a high degree of correlation between the behavioral phenotypes of Suanzaoren across 3 different concentrations(all Pearson correlation coefficients>0.65).A high degree of correlation was also found between the behavioral phenotypes of Jujuboside A across 3 different concentrations(all Pearson correlation coefficients>0.8).5.Correlation analysis of behavioral phenotypes induced by Jujuboside A and well-characterized compounds found that the behavioral effects of Jujuboside A on larvae were highly correlated with the behavioural phenotype induced by compounds targeting serotonergic and dopaminergic transmitter systems.Among them,serotonergic compounds accounted for a larger proportion.Hierarchical clustering analysis found that all drugs/compounds were clustered into 4 categories.Jujuboside A at all tessted concentrations and compounds regulating the serotonin,DA,GABA,histamine,adenosine,adenosine and melatonin signaling were classified into one category.Among them,serotonergic compounds accounted for a larger proportion.Further clustering found that low concentrations of Jujuboside A,D2 dopamine agonists,5-HT reuptake inhibitors,D2/3 dopamine agonists,5-HT reuptake inhibitors and mGluR5 glutamate antagonists are grouped together.In addition,medium and high concentrations of Jujuboside A,D2/3 dopamine agonists,A3 adenosine antagonists,5-HT1A receptor agonists,and α1 adrenaline antagonists are grouped together.6.The serotonin-dependent pharyngeal pumping assay in C.elegans showed that Jujuboside A at tested concentrations(30 and 100 μM)significantly increased the pumping rate of the N2 strain(P<0.05).However,Jujuboside A did not induce any apparent effect on the pumping rate of tph-1 mutants at any tested concentration(P>0.05).7.Results of quantitative real-time PCR showed that the mRNA levels of sert were significantly decreased in larvae exposed to Jujuboside A compared to the untreated control(P<0.05),whereas 5-HT1A mRNA expression did not significantly changed(P>0.05).Conclusion1.Suanzaoren Decoction,Suanzaoren and Jujuboside A all induced sleep-promoting effects in larvae zebrafish,and Jujuboside A is one of the active components that contributed to the sleep-promoting effects of Suanzaoren Decoction.2.Suanzaoren Decoction affected the sleep/wake behavior of larvae zebrafish through a bidirectional regulation of both inhibition and excitement.While Suanzaoren and Jujuboside A exerted only the inhibitory effects.3.Serotonin signaling may be involved in the sleep regulation of Jujuboside A in larvae zebrafish. |
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