| Part One Dopamine transmitter transfer and Percoll density gradient centrifugation for preparation of synaptosomeObjective:(1)To comprehend the complete process of dopamine transmitter synaptic transmission and clarify the significance of each transfer link,providing theoretical support for experimental research in the later period.(2)To understand the preparation methods and development history of synaptosomes in China and abroad and summarize the key technical steps in each period.To compare the advantages and disadvantages of various methods,master the core technology of synaptosome preparation,and provide technical support for experimental research.Methods:Through the literature collection and analysis and the technology exploration,the complete process of dopamine neurotransmitter synaptic transmission was mastered,and the development of synaptosome preparation technology was summarized.The synaptosome was prepared by Percoll density gradient centrifugation,and its morphology and structure were observed by transmission electron microscopy.Results:The dopamine system is the focus of the exploration of the pathogenesis of ADHD.The dopamine transmitter synaptic transmission is a complex process that includes the synthesis,storage,transfer,release,and inactivation of transmitters.Abnormalities at all levels can affect dopamine in the brain,s level.The synaptosome preparation technology has undergone four key technical stages:the classic sucrose density gradient method?classical improvement method?Ficoll/sucrose method?discontinuous Percoll density gradient centrifugation,and Percoll density gradient centrifugation is the latest international technology at present,domestically No professional technical report has been made yet.The presynaptic membrane,synaptic cleft and postsynaptic membrane of the synaptosome prepared by the research group in this study are preserved well,and the density of the synapse is high.It has a typical synaptosome morphological structure.Conclusions:The generation,release,transport,and clearance of dopamine synapse in the synaptic transmission are all performed under the mediation of corresponding mediators in the synaptophysin.From the level of synapses,the dopamine synaptic transmission is studied for the level of dopamine in the brain.The impact is very promising.Synaptosomes are the basis for neurophysiological,biochemical,pharmacological,and toxicological studies.Understanding and mastering different stages of synaptosome preparation technology is the basis for in-depth study of brain diseases and neurotransmitter molecular water products in the brain.It is worth learning in depth.And master.The technology of synaptosome preparation mastered by the research group has the advantages of less time consuming and high output rate.The prepared synapse body has a complete structure and typical morphology,which makes up for the deficiencies of traditional techniques and provides technical support for the study of diseases of the nervous system.Part Two Effect of Anshen Dingzhiling on signal pathways which regulates dopamine production,release and clearanceObjective:To investigate the effect of Anshen Dingzhiling on the synaptosome dopamine production,release and clearance-related factors and signaling pathway in ADHD model SHR rats,and to explore the efficacy of Anshen Dingzhiling in the treatment of ADHD.Methods:Forty SHR rats were divided into model group,western medicine group,Anshin Dingzhiling low-,middle-,and high-dose groups according to random numbers,eight rats in each group.Eight WKY rats were set as normal groups.All the rats were separately lavage for 4 consequent weeks separately.Percoll density gradient centrifugation was used to prepare brain synaptosomes as experimental specimens.The morphology of synaptosomes was observed by electron microscope.The activities of ATPase and lactate dehydrogenase in synaptosomes were detected by colorimetry.Real-time PCR,Western blot,and enzyme-linked immunosorbent assay,immunohistochemical method and fluorescence method were selectivity used to detect the expression of TH,DDC,AC/cAMP/PKA,VMAT2,SNAP25,Synataxinla,ERK1/2,CAMKII and other related markers in synaptophysin of SHR rats.Results:The synaptosome extracted from the experiment was structurally complete and had enzyme activity,which met the relevant requirements.The Anshen Dingzhiling middle dose group can significantly increase the activity of ATPase in synaptosomes and significantly reduce the activity of LDH enzyme(P<0.01);the concentration of AC,cAMP and PKA in the brain synapses of the middle dose of Anshen Dingling Elevated(P<0.05).The AC and cAMP contents in brain synaptosomes of high-dose group of Anshen Dingzhiling were increased(P<0.05);the expression levels of TH and DDC mRNA in the low dose group of Anshen Dingling were increased.(P<0.05),the TH and PKA mRNA expression levels in the middle dose group of Anshen Dingzhiling were significantly higher(P<0.01),but there was no statistical difference in the high dose group.The middle-dose group significantly increased the number of TH and DDC positive cells;the middle dose of Anshen-Ding-ling had significant up-regulation of SNAP25,VMAT2,and Synataxinla expression(P<0.01);Anshen Dingzhiling could significantly affect ERK1/2 and CaMKII signaling Pathways regulate the expression of signaling pathways and are most effective at medium and high doses.Conclusions:Anshen Dingzhiling can increase the activity of ATPase in synaptosomes,significantly reduce the activity of LDH enzyme,and have a protective effect on synaptosomes.Anshen Dingzhiling can upregulate the expression level of AC/cAMP/PKA,increase the expression of TH and DDC,increase the number of positive cells,accelerate the synthesis of dopamine,and increase the up-regulation of VMAT2,SNAP25 and Synataxinla involved in dopamine transport and release.,And through the regulation of CAMKII and ERK1/2 protein function,accelerate the dopamine vesicle efflux process,reduce the removal efficiency,to increase the concentration of dopamine in the synaptic cleft,thereby increasing the dopamine content in the synaptic cleft and improving the dopamine defect state.Anshen Dingzhiling has a certain degree of regulation on the generation,release,transport and clearance of dopamine transmitters in synaptic transmission,especially with moderate doses.Part Three Baicalin modulates the core symptoms of ADHD and its preliminary mechanismObjective:To study the therapeutic effects of Baicalin,which was the main effective ingredient of Anshen Dingzhiling,on the regulation of ADHD core symptoms and explore its preliminary efficacy mechanism.Methods:A total of 40 SHR rats were randomly divided into model group,methylphenidate hydrochloride(MPH)group,and low-,medium-,and high-dose baicalin groups,with 8 rats in each group.8 WKY rats were selected as normal control group.The MPH group(0.07 mg/mL)and the baicalin in low(3.33 mg/mL),medium(6.67 mg/mL),high(10 mg/mL)dozes groups were given the drug by gavage,the WKY and the model group were given normal saline by gavage at the same dose.The frequency of gavage was twice a day for 4 weeks.Rats' total moving distance and average moving speed were observed at 0 days and 7,14,21 and 28 days after intragastric administration with open field test to evaluate the effects of drugs on control the rats' hyperactivity and impulsive behavior.Morris water maze was used to observe the latency,target quadrant activity and the number of wearing in each group to evaluate the influence of the drug on the attention of rats.Brain synaptosomes were prepared by Percoll density gradient centrifugation,and the expression of ATP,LDH,AC,cAMP,PKA,SNAP25,VMAT2,and Synataxinla in synaptosomes were detected by Western blot,PCR,ELISA and colorimetry.High performance liquid chromatography(HPLC)was used to detect the content of DA in rat prefrontal cortex and striatum.Results:Baicalin did not affect the body weight and food intake of rats.The open-field test data showed that the total exercise distance and average movement speed of the methylphenidate hydrochloride group and the high dose group of baicalin were significantly lower than those of the model group(P<0.05).The water maze space exploration experiment results showed that the residence time ratio of the target quadrant of the methylphenidate hydrochloride group and the baicalin high dose group was significantly higher than that of the model group(P<0.05).The target quadrants of the methylphenidate hydrochloride group and the high dose group of baicalin were observed.The proportion of exercise distance was also significantly higher than that of the model group(P<0.05);the high dose group of baicalin traversed the platform more frequently than the other groups(P<0.05),showing a great advantage.Baicalin can regulate the ATPase and LDH activity of synaptosomes,and the levels of AC,cAMP,and PKA in the low,middle,and high dose groups can increase(P<0.01),which is significantly higher than that in the western medicine group.High doses of methylphenidate hydrochloride and baicalin significantly increased the expression of SNAP25,VMAT2 and Synataxin la protein(P<0.05 or P<0.01).Baicalin high dose group can increase striatal dopamine content.Conclusions:Baicalin does not affect the normal growth of rats.Baicalin can modulate the exercise ability and learning and memory ability of ADHD model SHR rats,and thus control the ADHD hyperactivity,impulsivity and inattention core symptoms;this mechanism may be With baicalin,it can regulate ATPase and LDH activity of synaptosomes in model rats,increase the content of AC,cAMP,PKA in synaptosomes,up-regulate the expression of SNAP25,VMAT2 and Synataxin la protein and mRNA,and thus affect the content of dopamine in striatum..The efficacy of baicalin was dose-dependent,and the efficacy was most significant in the high-dose group,but the efficacy of baicalin was later than that of methylphenidate hydrochloride. |
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