| Background:With the development of medicine,drug treatment has become the most important and most common means in medical activities,and tens of millions of children are receiving medication every year.Drug treatment brings good news to sick children,protects their health and improves their quality of life.They also bring some adverse reactions,especially in the Central Nervous System(CNS).Severe neurotoxicity often leave patients facing the dilemma of reducing the dosage of drugs or even stopping the drugs,and may damage the psychological,physical,and quality of life of patients.In the past few decades,scientists have done a lot of research on the toxicity mechanism of the CNS in order to explore the side effects of drugs,but the progress is very slow.Therefore,it is an urgent task to study the mechanism of drug neurotoxicity.Reserpine is widely used clinically in the treatment of hypertension and schizophrenia;The main pharmacological mechanism is to cause the consumption of dopamine at the synaptic terminal by restricting the access of neurotransmitters to vesicles by VMAT.Clinical observations and studies have found that reserpine has side effects that lead to depression-like and Parkinson-like symptoms.Studies have shown that high concentration of reserpine has an effect on the central nervous system of experimental animals,including slowing of behavioral movement and attenuation of related neurotransmitter levels;reserpine can inhibit the growth and development of dopaminergic neurons and reduce the number of tyrosine hydroxylase cells in the substantia nigra pars compacta of rats.in the central nervous system,dopaminergic neurons are distributed in the telencephalon,diencephalon,brainstem and spinal cord,forming four major projection pathways that perform different functions.As a rate-limiting enzyme that catalyzes the biosynthesis of catecholamines,TH is widely used as a marker for dopamine system expression.Studies have shown that the glycoprotein Wnt family,which regulates cell proliferation and differentiation,is differentially expressed in the development of dopaminergic neurons.The midbrain astrocyte-derived neurotrophic factor(MANF)plays an important role in protecting the dopaminergic neuron system against neurotoxic damage.In addition,Otpa and Otpb are associated with dopaminergic neurons development.MicroRNAs(miRs)are a class of highly conserved,non-coding RNAs that achieve their biological functions by base pairing with endogenous mRNAs to inhibit expression of target genes.Hundreds of miRs are expressed in mammalian brains and participate in a variety of functions.miRs are closely related to the etiology and pathophysiology of neurodegenerative and psychiatric diseases.miRs have important regulatory effects on the normal development and physiological activities of the CNS and the neurotoxicity of drugs.The intrinsic characteristics of miRs make them one of the key links to regulate neuronal stress.Different expression patterns of miRs in different diseases indicate that neuronal damage caused by different pathogenic mechanisms can be manifested in specific miRs.miRs play an active role in regulating almost all cellular processes.Exploring potential miRs target genes may lead to greater advances in clinical applications.miR-129 is present in mice,humans,rats,zebrafish,etc.miR-129 is produced by transcription of two genes,miR-129-1 and miR-129-2,and the miR-129 family is composed of miR-129-1 and miR-129-2;there is only one base difference between miR-129-1-3p and miR-129-2-3p,and it is almost impossible to distinguish functionally.They are collectively referred to as mir-129-3p.Using the miRBase database to look up the sequences of different species of miR-129,it was found that miR-129 is highly conserved among different species.Studies have shown that miR-129 regulates a series of genes closely related to the diagnosis and treatment of cancer and other physiological processes.This shows that miR-129 is involved in the regulation of various important physiological activities.Studies have confirmed that miR-129-3p is abundantly expressed in the brain of zebrafish embryos.Based on this,we speculate that miR-129-3p plays an important role in the zebrafish brain nervous system.However,the influence and regulation mechanism of miR-129-3p on cognitive function needs further study.The zebrafish is slender,has a strong reproductive capacity,and has low breeding costs.Importantly,the zebrafish genome is approximately 87%similar to the human genome,which means that the results of a drug experiment performed on it are also applicable to the human body in most cases.The zebrafish has a conserved nervous system structure and a rich behavioral pattern of vertebrate.The young zebrafish has a transparent brain and a simple structure,which is an ideal model for exploring the development of nervous system and the pathogenesis of drug neurotoxicity.Therefore,this study selected zebrafish as an experimental model to explore the effects of embryonic reserpine exposure on the development of embryonic nervous system and possible molecular mechanisms.Rcan2 is a thyroid hormone response gene and a regulator of calcineurin.Rcan2 is highly expressed in the mouse brain and is also distributed in the myocardium and skeletal muscle.The rcan2 protein is associated with pathology of diseases such as Down's syndrome,cardiac hypertrophy,and Alzheimer's disease.The rcan2 protein functions primarily by binding and transporting Ca2+ and serine-threonine phosphatase.Activated T cell nuclear factor is a substrate that mediates the regulation of a number of important signaling pathways,including neuronal development.Studies have shown that rcan2 knockdown reduces the amount of food consumed in mice,resulting in a reduction in body weight,a decrease in fat content,and a slight decrease in exercise.After comparison with the Targetscan database,it was found that miR-129-3p has a potential binding site with the rcan2 gene,suggesting that miR-129-3p may regulate the expression of rcan2.In order to clarify the neurotoxic effects of reserpine,the effect of miR-129-3p on the central nervous system of zebrafish embryos and its correlation with rcan2.This study designed two parts of the experiment to:(1)the neurotoxicity of reserpine in zebrafish embryos;(2)miR-129-3p antagonized reserpine-induced zebrafish neurotoxicity by rcan2.Part I:The neurotoxicity of reserpine in zebrafish embryosObjective:Through the study of zebrafish embryos after reserpine treatment,the toxic effects on the early development of zebrafish nervous system were explored.Methods:The changes of embryonic locomotor activities of zebrafish after reserpine treatment were monitored by behaviour analysis,and the changes of sensitivity to light and sound in zebrafish embryos after reserpine treatment were also monitored by behavior analysis.The content of neurotransmitters level was measured by LCMS in zebrafish embryos after reserpine treatment.qRT-PCR was used to detect the effect of reserpine treatment on the expression of genes related to the development of dopamine system in zebrafish embryos.Changes in the number of dopaminergic neurons in the preoptic area of the embryonic brain of zebrafish after reserpine treatment were detected by in situ hybridization.Results:After treatment with different concentrations of reserpine,the average swimming distance of zebrafish embryos decreased significantly under light or dark conditions,and the average parade speed slowed down.The sensitivity of zebrafish embryos to external light or sound stimulation decreased after reserpine treatment.The average swimming distance decreased,the average parade speed slowed,and the response decreased when the stimulus appeared;and the levels of monoamine neurotransmitters in zebrafish embryos,including dopamine,serotonin and norepinephrine,were soaked in reserpine The zebrafish embryos decreased compared with the control group and the serotonin metabolite content did not change.At the same time,the number of dopaminergic neurons in the anterior preoptic area of the zebrafish embryo brain after reserpine treatment was lower than that of the control group.The genes involved in dopamine phylogeny,including otpa,otpb,wntl,wnt3,wnt5 and manf,were down-regulated in the reserpine-treated group.Conclusion:The use of reserpine drugs will have neurotoxic effects on the early nervous system of zebrafish,including developmental and functional effects,including impaired motor function,reduced sensitivity to the outside world,decreased monoamine neurotransmitter levels,and dopaminergic activity in the zebrafish brain.Neuronal damage and down-regulation of related gene expression.The parkinson-like model of zebrafish was successfully established in this experiment,which is a good attempt to study the central nervous system toxicity of the drug in the future and laid a good foundation.Part II:miR-129-3p antagonized reserpine-induced zebrafish neurotoxicity via rcan2Objective:By studying the function of miR-129-3p in zebrafish,the regulation of zebrafish embryonic body and early nervous system development,and the neurotoxicity of miR-129-3p-mediated rcan2 antagonizing reserpine were investigated.Methods:qRT-PCR was used to detect the expression profile of miR-129-3p in zebrafish embryos and adult fish;microinjection of miR-129-3p analogue and morpholino to express and knock down the expression of miR-129-3p in zebrafish embryos Level;immunofluorescence assay for high expression or knockdown of miR-129-3p on zebrafish embryonic head cell proliferation and apoptosis;in situ hybridization detection of knockdown miR-129-3p zebrafish embryo brain endoscopic Changes in the number of dopaminergic neurons in the pre-leaf area;behavioral detection of reserpine treatment of zebrafish embryonic motor function changes by microinjection of miR-129-3p analog;qRT-PCR detection of zebrafish embryo miR-129-3p expression after reserpine treatment;in situ hybridization technique was used to detect the change of the number of dopaminergic neurons in the anterior ocular area of zebrafish embryos after microinjection of miR-129-3p analog by reserpine;qRT-PCR detected high expression or The effect of knockdown of miR-129-3p on the expression of rcan2.Results:With the development of zebrafish embryos,the expression level of miR-129-3p increased;miR-129-3p was abundantly expressed in the brain of adult zebrafish;knockdown of high concentration of miR-129-3p caused zebrafish Embryonic developmental malformation,-while high expression of miR-129-3p leads to changes in embryo behavior of zebrafish;knockdown of miR-129-3p causes an increase in apoptosis of zebrafish embryo cells;knockdown miR-129-3p The number of dopaminergic neurons in the anterior optic lobes of the zebrafish embryonic brain was lower than that of the control group,while the high expression of miR-129-3p 45dopaminergic neurons in the anterior optic lobes in the brain.Reduction.After reserpine treatment,the level of miR-129-3p in zebrafish 'embryos was down-regulated.In addition,knockdown of miR-129-3p also led to an increase in the expression level of rcan2,while high expression of miR-129-3p caused a decrease in the expression level of rcan2.Conclusion:miR-129-3p is critical for the development of the embryonic body and nervous system of zebrafish.A decrease in miR-129-3p causes damage to dopaminergic neurons in the zebrafish brain.The level of miR-129-3p was decreased after reserpine treatment.The high expression of miR-129-3p can rescue the developmental damage of dopaminergic nervous system caused by reserpine exposure.Rcan2 is a potential target gene regulated by miR-129-3p.This experiment provides a new way to study the mechanism and prevention measures of drug neurotoxicity.Overall conclusions:The use of reserpine drugs will cause damage to dopaminergic neurons in the zebrafish brain;miR-129-3p is critical for the development of the zebrafish embryonic body and nervous system;the reduction of miR-129-3p causes the zebrafish brain Middle dopaminergic neurons are damaged;reserpine causes a decrease in miR-129-3p levels in zebrafish embryos.High expression of miR-129-3p has protective effects on retinal-induced zebrafish embryonic nervous system damage;rcan2 may be a potential target gene regulated by miR-129-3p. |
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