Study Of The Neurotoxic Effects Of Lead And The Ameliorative Effects Of Chlorogenic Acid And It's Analogues Against Lead-induced Neurotoxicity

Objective:The widespread application of lead(Pb)has caused serious aquatic environment pollution.Other stressors such as thermal pollution also affect the aquatic environment.However,the effects of Pb alone and the combined effect of Pb on the aquatic environment are still largely unknown.In this study,zebrafish was used as a model to explore the effects and mechanism of repeated heat pulse(30,32,34?)on neurotoxicity induced by Pb(75 mg/L).In addition,the combined neurotoxic effects of dual environmental pressures were elaborated.Our study of the effects of low-dose(below environmentally relevant level)Pb(1,10,100 ?g/L)on adult zebrafish nervous system provided a reference for adjustment of aquatic toxic criteria.Based on above findings,this study used Pb(1000 ?g/L)-induced zebrafish model of nerve injury in vivo to study the neuroprotective effects of the natural products chlorogenic acid(CGA)and its analogues neochlogogenic acid(NCGA),cryptochlorogenic acid(CCGA)as well as the underlying mechanism,which lay a foundation for the study of the lead antagonists.Methods:1.The larval zebrafish was used as the model.Pb with repeated heat pulse at different temperatures(30,32,34?)were subject to zebrafish larvae.The wild type AB zebrafish were treated at 4 hours post fertilization(hpf),the mortality and development morphology of zebrafish at 24-144 hpf were observed and recorded.The hatching rate at 48-72 hpf,malformation rate at 96-144 hpf,and locomotion at 144 hpf were observed and recorded.The development of dopamine(DA)neurons and brain vessels were observed and recorded by using vmat2:GFP and fil1:GFP zebrafish at 72 hpf and 144 hpf,respectively.The real-time q PCR was used to test the expression of genes involved in nervous system to explore the effects of repeated heat pulse on Pb-induced neurotoxicity.Moreover,the apoptosis related mechanisms were studied by acridine orange(AO),TUNEL apoptosis staining,and immunofluorescence staining.2.The adult zebrafish were used as the model and treated with low-dose Pb for 14 days continuously.The neurotoxic effects of low-dose Pb on zebrafish was explored using the neurobehavior profiling assays including Black/White preference test,Novel tank diving test,and T-maze test.Real-time q PCR was used to analyze the expression of genes related to neurodevelopment,neurotoxicity,and stress responses,revealing the mechanism underlying low-dose Pb-induced neurotoxicity.In addition,brain pathological study was performed to investigate apoptosis associated underly mechanism.3.The nerve injury model of zebrafish was induced by Pb.Based On this model,CGA(100 ?mol/L),NCGA(100 ?mol/L),and CCGA(50 ?mol/L)were cotreated with Pb.The mortality rates at 24-120 hpf,hatching rates and development morphology at48-72 hpf were observed and recorded.The malformation rate and average toxicity score at 120 hpf,and locomotion at 120 hpf were observed and recorded.The development of DA neurons,brain vessels,and central nervous system(CNS)of 120 hpf zebrafish were observed and recorded using vmat2:GFP,fil1:GFP,and elavl3:EGFP zebrafish,respectively.The real-time q PCR was used to test the expression of genes related to neurodevelopment,oxidative stress,and autophagy,to explore the antagonistic effects of chlorogenic acid/analogues on Pb-induced neurodevelopmental toxicity in zebrafish and its underlying mechanism.Results:1.It was found that repeated heat pulse had no significant effect on the mortality of zebrafish induced by Pb.Repeated heat pulse increased the malformation and significantly affected disruption of locomotor activity,including the decrease in total swimming distance and average swimming speed.Repeated heat pulse worsen the loss of DA neurons and brain vasculature.The results showed that repeated heat pulse increased the number of apoptosis induced by Pb,increased the expression of Caspase3.The results of real-time q PCR showed that the expression of genes involved in nervous system was abnormal.Above all,repeated heat pulse worsens the neurodevelopmental toxicity induced by Pb.2.The behavior of adult zebrafish was affected by low-dose Pb treatment.Black/White preference test showed that low-dose Pb treatment reduced the swimming distance and time of zebrafish spent in the black area,suppressed the locomotion behaviors in both white and black areas,indicating that low-dose Pb led cognitive dysfunction in adult zebrafish.The novel tank diving test found that the swimming distance,time,and track of zebrafish exploration in the top zone of the tank were significantly reduced after low-dose Pb treatment.The frequency to top decreased significantly,suggesting that low-dose Pb treatment affected the exploration ability of zebrafish and caused anxiety behavior.The T-maze test is a behavioral detection method to test the learning and memory ability,low-dose Pb increased the swimming distance and time of zebrafish spent in the T-maze starting arm and wrong direction,prolonged the latency to the reservoir,suppressed the frequency of zebrafish to the reservoir,indicating that low-dose Pb affected the learning and memory ability of adult zebrafish.Low-dose Pb treatment did not affect the brain pathological structure of zebrafish,but the m RNA assays showed that low-dose Pb induced abnormal expression of genes related to neurodevelopment,neurotoxicity,and stress responses.The results showed that low-dose Pb could cause neurotoxicity of adult zebrafish.3.It was found that the co-treatment of chlorogenic acid/analogues with Pb did not affect the hatching and mortality rate of zebrafish larvae.It could alleviate Pb-induced malformation and reduce the malformation rate and average toxicity score,among which NCGA and CCGA have higher activity.CGA,NCGA,and CCGA ameliorated the development of DA neurons,CNS,and brain vascular induced by Pb.NCGA and CCGA significantly increased the length of DA neurons.CCGA ameliorated the development of blood vessels in brain and significantly increased the number of blood vessels in the brain of zebrafish,as well as protected the CNS development.CGA,NCGA,and CCGA co-treatment with Pb improved the locomotor impairment caused by Pb,increasing the total distance travelled and average swimming speed.The results of real-time q PCR showed that CGA,NCGA,and CCGA alleviated the abnormal expression of genes induced by Pb,including genes involved in neural development,oxidative stress,and autophagy.Our findings indicated that chlorogenic acid and its analogues could antagonize the Pb-induced neurodevelopmental toxicity and have neuroprotective effect,among which CCGA had the best effect.Conclusion:In this study,the model of larval zebrafish was used to reveal the combined toxic effects of repeated heat pulse and Pb,which are associated with apoptosis in the brain.Repeated heat pulse worsened the neurodevelopmental toxicity induced by Pb.The neurotoxicity of low-dose Pb on adult zebrafish was determined by neurobehavioral and biochemical studies.Based on our present and previous studies that Pb induces neurotoxicity through oxidative stress,apoptosis,and autophagy,it was found that the natural products chlorogenic acid and its analogues had activity against Pb-induced developmental neurotoxicity,among which CCGA showed the highest potential.