Protective Effect And Mechanism Of Butylphthalide On Rats With Chronic Alcoholism

Background:Drinking has great harm physically and socially,and also has great damage on the cognitive function of the patients.drinking heavily for a long time will seriously affect the patient's work,life,learning,the overall health and other social functions.Alcoholism is a chronic and recurrent disease.Negative emotional states and negative emotional events more easily lead to alcohol abuse.Due to long-term drinking,most patients is combined with physical damage especially in heart,liver,nervous system.Long-term drinking can lead to the central and peripheral nervous system damage,which leads to the symptoms of alcohol poisoning.As a neurotransmitter,physiological dose of H₂S can relax blood vessels,increase microcirculation,directly antagonize the injury caused by oxidative stress,induce long-term potentiation?LTP?in hippocampus CAl region,strengthen the excitatory postsynaptic potential?EPSP?and improve the ability of learning and memory.The high concentration of H₂S has toxic effects on living organ:on the one hand,it reflects in the inhibitory effects on the central nervous system,which specifically inhibits excitatory postsynaptic potential?the effect is reversible?,and then synaptic transmission is blocked and the function of neurons is inhibited.On the other hand,it can increase the calcium overload mediated by NMDA receptor,which cause the swelling of mitochondria,the disturbance of energy metabolism and cell necrosis progressively.However,chronic alcoholism can activate CBS,which catalyzes the formation of H₂S.In China,the prevalence rate of chronic alcohol poisoning is increasing year by year,Which has become one of the social problems of public health concern.There is a pressing need to develop antialcoholismic drug.The main purpose of this experiment is to explore the influence of butylphthalide on chronic alcoholism rats hippocampus H₂S/CBS system,learning and memory,memory related proteins and biology.Part I:Effects of butylphthalide on H₂S and F-actin in hippocampus of rats with chronic alcoholismObjective:To investigate the effects of NBP on the learning and memory ability,the activity of cystathionine-?-synthase?CBS?,the content of hydrogen sulfide?H₂S?and fibrous actin?F-actin?in hippocampus of rats with chronic alcoholism.Methods:100 SD male rats were randomly divided into five groups.Except the control group,each group were fed with 6%alcohol for 28 days.The alcohol solution was prepared and replaced daily at 9:00.After 14 d,the low,medium and high dose NBP group rats were fed different doses of NBP?around 14 d?.The learning and memory abilities were tested by Y-shaped maze detection after drinking.Subsequently,Hydrogen sulfide content in hippocampus and the expression of F-actin in hippocampus of chronic alcoholism rats were measured.Results:Compared with the control group rats,the training times for requiring?38.3750±3.9306?,the activity of CBS?55.4640±3.5170?,H₂S?13.9589±2.1119?content of hippocampus and the expression of F-actin in positive cells.The training times for requiring?83.0625±4.6686?,the activity of CBS?76.7890±4.2084?and H₂S content?25.6796±2.4960?of rats in the model group were increased,and theexpression of F-actin was decreased,?P<0.01?.Compared with the rats in the model group,the training times for requiring?77.8750±4.3340??65.1875±4.2461??62.1250±4.9514?,the activity of CBS?70.9180±3.0542??64.7020±2.4332??62.4570±2.0700?and H₂S content in the hippocampus?20.8504±1.5303??17.4829±1.4241??16.2821±0.8353?in low,medium and high dose NBP group rats were decreased,and the expression of F-actin were increased?P<0.01?.Conclusion:NBP could reduce the symptom and improve the ability of learning and memory in chronic alcoholism rats,which may be related to the effect of NBP on the activity of CBS,the content of H₂S and F-actin.PartII: Effects of butylphthalide on hydrogen sulfide and myelin basic protein in hippocampus of rats with chronic alcoholismObjective:This study was carried out to explore the protective effect of butylphthalide?NBP?on learning and memory of chronic alcoholism rats.In this study,learning and memory abilities of rats in each group,hydrogen sulfide?H₂S?content in the hippocampus,the activity of cystathionine-beta-synthase?CBS?,myelin basic protein?MBP?content and mitochondria and demyelination in the electron microscope were discussed.Methods:60 SD male rats were randomly divided into five groups.Except the control group,each group were fed with 6% alcohol for 28 days.The alcohol solution was prepared and replaced daily at 9:00.After 14 d,the treatment group rats were fed medium doses of NBP?around 14 d?.The learning and memory abilities were tested by Y-shaped maze detection after drinking.Subsequently,Hydrogen sulfide content in hippocampus and the expression of MBP in hippocampus of chronic alcoholism rats were measured.mitochondria and demyelination in the electron microscope were observed.Results:The results show that,compared with the normal group,learning and memory ability and MBP content were decreased significantly in model group;H₂S content and CBS activity were significantly increased?P <0.01?;The number of mitochondria was reduced,and the mitochondria were seriously swelling;Myelin was loose and fell off.Compared with the model group,learning and memory abilities and MBPwere significantly increased in the NBP treatment groups?P <0.01?;H₂S content in the hippocampus and CBS activity were significantly decreased?P <0.05?;The number of mitochondria was increased;The swelling of mitochondria were reduced.;The degree of loose myelin was reduced.Conclusion:The appropriate concentration of NBP can reduce chronic alcoholism damage on learning and memory ability,which may be related to the effect of NBP on H₂S/CBS system expression and the protective effect of NBP on mitochondria and MBP.Part III: Bioinformatic analysis of differentially expressed genes in rats with chronic alcoholism after being treated by butylphthalideObjective:To analyze the differentially expressed genes in the hippocampus of SD rats after chronic alcoholism and the rats being treated with NBP for two weeks,and to explore its biological significance.Methods:Illumina sequencing chip was used to obtain the raw data of rat hippocampus.According to the reserch on digital differential gene detection method published by Audic and Claverie Genome in 1997,We preprocess the data and screen out the differentially expressed genes.Gene Ontology function enrichment analysis and Pathway enrichment analysis were also carried out.At the same time,we relied on STRING online database to carry out DEGs protein-protein-interaction?protein-protein-interaction,PPI?network analysis.Results:208 DEGs was found in chronic model group,compared with normal group,51 of them were up-regulated,and the expression of 157 genes was down regulated.Pathway analysis shows that the Oxidative phosphorylation,Huntington,Parkinson and Alzheimer pathways associated with neurodegenerative diseases were more significant pathways.Compared with the model group,there were 585 DEGs in the treatment group,of which the expression of the 320 genes was up-regulated and the expression of the 265 genes was down regulated.Pathway analysis shows that Oxidative phosphorylation pathway,Adhesion junction pathway,Proteasome pathway,Adipocyte Factor Pathway and MAPK signaling pathway are highly enriched.352 DEGs was found in treatment group,compared with normal group,124 of them were up-regulated,and the expression of 228 genes was down regulated.Pathway enrichment analysis showed that the Wnt signaling pathway,protein digestion and absorption pathway,Other glycan degradation pathway,valine,leucine and isoleucine degradation pathway,and Adherens junction pathway are highly enriched.Conclusion:NBP may up-regulate/down-regulate Gls2,Tars2,Slc25a18,Ckmt1,Cox6 c,Ndufb8,Ndufa4,Ndufa2,Ndufb6 and LOC679739 genes which expressed reversed after alcoholism,protecting mitochondria and myelin and improving the ability of learning and memory in rats.