| Background/ObjectiveMorphine is the first choice for the treatment of chronic,moderate and severe pain.After long-term treatment of morphine,its analgesia gradually weakened.More doses of morphine need to use to achieve the original effect,but it makes patients more prone to addiction.Compound 511 is specially developed for opioid addiction treatment by my tutor Lu Zhigang,based on the previous valuable experience combined with the latest pharmacological research results of TCM in preventing and controlling opioid addiction.The effect of Compound 511 on opioid addiction is still unclear,and its potential therapeutic mechanism needs further study.Electroacupuncture(EA)is also an effective means to fight against opioid addiction,but the mechanism is not completely clear.In this study,the effects of Compound 511,EA,511+EA on morphine addiction related behaviors in mice were observed.Then we used high-throughput sequencing technology to screen its possible mechanisms of action and further explore,in order to clarify the mechanisms of Compound 511,EA on morphine addiction in mice.Methods1.The effects and mechanisms of Compound 511 on morphine addictionExperiment 1:To observe the effect of different doses of Compound 511 on addictive behaviors of morphine in mice.Male C57BL/6 mice,20-25g,are divided into saline+vehicle(Con)group,morphine+vehicle(Mod)group,morphine+Compound 511(low,medium and high doses)(511-L,M,H)group and morphine+Jitai tablet(Jitai)group.(1)Conditioned place preference(CPP):The natural place preference of mice is determined before training.Mice are trained to regard non-natural preference box as morphine-paired box,natural preference box as saline-paired box.During the training,the mice are intervened with double distilled water(ddH2O)or Compound 511(3g/kg,6g/kg,12g/kg)or Jitai tablets(4.3g/kg),once a day.After training,place preference of mice is detected.(2)Locomotor activity:After a single dose of ddH2O or Compound 511(3g/kg,6g/kg,12g/kg),the movement distance of mice within 60 minutes is measured.ddH2O,Compound 511(3g/kg,6g/kg,12g/kg)or Jitai tablets(4.3g/kg)is given once a day,and morphine or saline is injected intraperitoneally 30 minutes later,on the 7th day,the movement distance of mice within 60 minutes is measured.(3)Naloxone precipitated withdrawal:The model of morphine dependence in mice is established by repeated subcutaneous injection of morphine,twice a day,with an interval of 4 hours for 5 consecutive days,and the mice are intervened by ddH2O,Compound 511(3g/kg,6g/kg,12g/kg)and Jitai tablets(4.3g/kg)once a day at the same time.3 hours after the last morphine injection,lmg/kg naloxone is injected to precipitate the withdrawal,and the withdrawal symptoms(jumping behavior,wet dog shaking,weight loss)are observed.(4)Self-administration(SA):Before the experiment,mice are trained to press the active bar to get food pellets for 5 days.During the experiment,mice are trained to press the active bar to get morphine injection for 15 days,and are intervened with ddH2O,Compound 511(3g/kg,6g/kg,12g/kg).The number of self-administration and inactive response in the last day of training are recorded.Experiment 2:Based on high-throughput sequencing technology,we study the effect of Compound 511 on gene changes in the mice VTA of morphine addiction.The VTA tissues of mice in Con group,Mod group and 511-M(511)group are used as the sequencing sample.The high-throughput sequencing technology is applied,combined with SeqPrep and Sickle software for raw data quality,HISAT2 and RSEM software for sequence alignment analysis and gene expression level analysis,and DESeq2 software for differential expressed genes(DEGs).GO and KEGG function enrichment were performed for part DEGs.Experiment 3:To study the effects of Compound 511 on BDNF signaling pathway and structural plasticity of dopaminergic neurons in the VTA of morphine addicted mice.The expression levels of BDNF and TrkB mRNA in the VTA of mice in Con,Mod and 511-M(511)group are detected by real-time quantitative RNA;the protein levels of BDNF and TrkB in each group are detected by immunoblotting and immunohistochemistry;the protein levels of BDNF signaling cascades(ERK pathway,PI-3-K/Akt pathway,PLCy pathway)are detected by Western blotting;the structural changes of dopaminergic neurons are detected by immunofluorescence imaging.2.The effects and mechanisms of EA on morphine addictionExperiment 1:To study the effect of EA on morphine addiction related behaviors in mice.Male C57BL/6 mice(20-25g)are divided into saline(Sal)group,morphine+sham EA(sham)group and morphine+EA(EA)group.(1)CPP experiment:the natural place preference of mice is measured before training,and the mice are trained with non-natural preference box as morphine-paired box and natural preference box as saline-paired box.During the training,EA at Zusanli and Sanyinjiao points are selected for intervention,once a day.(2)Naloxone precipitated morphine withdrawal:The model of morphine dependence in mice is established by repeated subcutaneous injection of morphine,twice a day,with an interval of 4 hours for 5 consecutive days.At the same time,EA is applied to Zusanli and Sanyinjiao points once a day.3 hours after the last morphine administration,lmg/kg naloxone is injected to precipitate the withdrawal,and the withdrawal symptoms(jumping behavior,wet dog shaking,weight loss)are observed.Experiment 2:To study the effect of EA on the expression profile of circRNA in NAc of morphine addiction in mice.The NAc tissues of mice in Sham group and EA group in morphine CPP experiment are used as sequencing samples.The high-throughput sequencing technology is used,combined with Cutadapt?FastQC software for raw data quality control,Tophat2?Bowtie2 software for sequence alignment,find circ software for identifying circRNA,and DESeq software for screening the differentially expressed circRNA between groups.The source genes of differentially expressed circRNA are enriched,and the miRNA targets of differentially expressed circRNA are predicted by miRanda software.Finally,the sequencing data are verified by fluorescent quantitative PCR.3.The effects of compound 511 combined with EA on morphine addictionTo study the effect of compound 511 combined with EA on morphine addiction related behaviors in mice.Male C57BL/6 mice were divided into control(Con)group model(Mod)group,compound 511(511)group,EA(EA)group,compound 511 +EA(511+ EA)group.(1)CPP experiment:the natural place preference of mice was determined before training.The mice were trained with non-natural preference box as morphine-paired box and natural preference box as saline-paired box.During the training,the mice were given double distilled water or compound 511 of 6g/kg,or EA at Zusanli and Sanyinjiao,or compound 511 of 6g/kg combined with EA at Zusanli and Sanyinjiao,After the training,place preference of mice was detected.(2)Naloxone precipitated morphine withdrawal:The model of morphine dependence in mice is established by repeated subcutaneous injection of morphine,twice a day,with an interval of 4 hours for 5 consecutive days.The mice were given double distilled water or 6g/kg compound 511 or EA at Zusanli and Sanyinjiao or 6g/kg compound 511 combined with EA at Zusanli and Sanyinjiao for intervention once a day.3 hours after the last morphine administration,lmg/kg naloxone is injected to precipitate the withdrawal,and the withdrawal symptoms(jumping behavior,wet dog shaking,weight loss)are observed.Results1.The effects and mechanisms of Compound 511 on morphine addictionExperiment 1:(1)In CPP experiment,Compound 511(3g/kg,6g/kg,12g/kg)could effectively inhibit the formation of CPP induced by morphine in mice,and the inhibition effect of 6g/kg Compound 511 is more significant.(2)In the locomotor activity,Compound 511(3g/kg,6g/kg,12g/kg)could effectively inhibit the formation of behavioral sensitization induced by morphine in mice,and does not affect the spontaneous activity of mice.The inhibition effect of 6g/kg Compound 511 is more obvious.(3)In the naloxone precipitated withdrawal,Compound 511(3g/kg,6g/kg,12g/kg)could significantly reduce the withdrawal symptoms,including the number of jumps,weight loss,wet-dog shaking,and 6g/kg Compound 511 has better inhibitory effect on jumping behavior.(4)In the self-administration experiment,Compound 511(3g/kg,6g/kg,12g/kg)could effectively alleviate the morphine self-administration in mice,and 6g/kg Compound 511 is more obvious.Experiment 2:A total of 712 DEGs are screened by RNA-seq and bioinformatics analysis,including 458(357 up-regulated and 101 down-regulated)DEGs between Con group and Mod group,and 469(84 up-regulated and 385 down-regulated)DEGs between Mod group and 511 group.BDNF,Nts,Penk,Slc18a3,Sfrp2,Tph2,Htrla,Plin4,Cartpt,Grin3b,Grip2,I13 1ra,Cdkn1a,Pdk4,Shox2,Trh,Tat and other DEGs are related to the mechanisms of morphine effect or addiction.In GO enrichment analysis,DEGs are mainly enriched in cellular component,molecular function,binding,biological regulation,regulation of biological process,regulation of cellular process,protein binding,membrane and other items closely related to morphine addiction.In KEGG enrichment analysis,DEGs are mainly enriched in morphine addiction,neuroactive ligand-receptor interaction,dopaminergic synapse,MAPK signaling pathway,cAMP signaling pathway,Ras signaling pathway and other pathways which play an important role in morphine addiction.Experiment 3:Combined with the transcriptome sequencing results of Experiment 2 and previous studies,it is found that Compound 511 may prevent and control morphine addiction by regulating BDNF in the VTA of mice.Firstly,the changes of BDNF are verified at gene and protein levels.Compared with the Con group,the mRNA and protein levels of BDNF in the Mod group are significantly decreased,Compound 511 intervention at the same time could significantly alleviate the decrease of BDNF mRNA and protein levels.Next,the changes of TrkB are observed,a high affinity receptor of BDNF.Compared with the Con group,TrkB mRNA and protein levels in the Mod group are significantly decreased,Compound 511 intervention at the same time could effectively reduce the decrease of TrkB mRNA and protein.In the downstream ERK pathway,compared with the Con group,morphine significantly increased the phosphorylation level of ERK1/2 protein,and Compound 511 could significantly inhibit the phosphorylation of ERK1/2 protein.In the downstream PI-3-K/Akt pathway,compared with the Con group,p-PI-3-K/PI-3-K,p-Akt/Akt are significantly down-regulated and p-S6/S6 are significantly up-regulated in the Mod group,Compound 511 intervention at the same time could effectively reduce the down-regulation of p-PI-3-K/PI-3-K,p-Akt/Akt,and inhibit the up-regulation of p-S6/S6.In the downstream PLCyl pathway,compared with the Con group,the expression level of PLCyl protein in the Mod group is significantly increased,and Compound 511 intervention at the same time could significantly inhibit the increased level of PLCyl protein.In addition,after morphine induced CPP in mice,the soma size of dopaminergic neurons in VTA are significantly reduced.At the same time,Compound 511 intervention at the same time could significantly reverse the change of soma size of dopaminergic neurons.2.The effects and mechanisms of EA on morphine addictionExperiment 1:In CPP experiment,EA can effectively inhibit CPP formation induced by morphine in mice,while in the naloxone precipitated morphine withdrawal,EA can significantly reduce morphine withdrawal symptoms,including jumping behavior,weight loss and wet dog shaking.Experiment 2:A total of 112 differentially expressed circRNAs are identified by RNA-seq and bioinformatics analysis,of which 51 are up-regulated and 61 are down regulated.Then,the source genes of differential circRNAs are enriched.In GO enrichment,the source genes of differential circRNAs are significantly enriched in 23 GO terms.Among them,cellular localization,cytoplasm,intracellular,enzyme binding,GTPase binding,protein binding and Golgi apparatus is related to morphine,while protein binding,intracellular part,intracellular,intracellular organelle,cellular localization is related to the effect of EA.In KEGG enrichment,the source genes of differential circRNAs were significantly enriched in 21 pathways.Among them,glutamatergic synapse,long-term potentiation,wnt signaling pathway,dopaminergic synapse,cGMP-PKG signaling pathway,axon guidance,oxytocin signaling pathway is related to morphine addiction,while renin secretion,VEGF signaling pathway,insulin secretion,glutamatergic synapse,wnt signaling pathway,dopaminergic synapse is related to acupuncture.In addition,we also predict the top five miRNA targets of differential circRNAs,and select 44 circRNAs and 162 interacting miRNAs to construct the circRNA-miRNA network.We find that mmu-miR-207,mmu-miR-669c-3p,mmu-miR-6946-3p and mmu-miR-6984-3p are associated with more circRNAs.3.The effects of compound 511 combined with EA on morphine addictionIn CPP experiment,compound 511,EA and compound 511 combined with EA can effectively inhibit CPP formation induced by morphine in mice,and compound 511 combined with EA has more significant inhibitory effect.In the naloxone precipitated morphine acute withdrawal,compound 511,EA and compound 511 combined with EA can significantly reduce the symptoms of morphine withdrawal,including jumping behavior,weight loss and wet dog shaking.Compound 511 combined with EA has more obvious effect.Conclusions1.Compound 511 can effectively inhibit the formation of CPP model,behavioral sensitization model and SA model induced by morphine in mice,which indicates that Compound 511 can effectively inhibit morphine induced mental dependence;The intervention of Compound 511 can effectively reduce the acute morphine withdrawal symptoms induced by naloxone,which suggests that Compound 511 can effectively reduce the physical dependence induced by morphine.These results suggest that compound 511 has a definite effect on morphine addiction.2.Compound 511 can prevent the down-regulation of BDNF and TrkB expression induced by morphine,inhibit the increased phosphorylation of ERK1/2 and S6 protein induced by morphine,avoid the decreased phosphorylation of PI-3-K and Akt protein,and reduce the increase of PLCyl protein;At the same time,it can reverse the reduced soma size of dopaminergic neurons induced by morphine,which suggests that Compound 511 may play an important role in anti-morphine addiction through regulating BDNF signaling cascades and structural plasticity of dopaminergic neurons.3.The intervention of EA can effectively inhibit the formation of CPP model induced by morphine in mice,which indicates that compound 511 can effectively inhibit morphine induced mental dependence;EA can effectively alleviate naloxone induced acute withdrawal in morphine dependent mice,which indicates that EA can effectively reduce morphine induced physical dependence.These results suggest that EA has a definite effect on morphine addiction.4.In this study,we screen the differentially expressed circRNAs in NAc of mice with morphine addiction after EA intervention,and predict the potential role of these circRNAs and the interaction between circRNAs and miRNAs.This provides a new target for the molecular mechanisms of EA intervention on morphine addiction.5.Compound 511 combined with EA can more effectively inhibit the formation of CPP induced by morphine in mice,which suggests that compound 511 combined with EA has more obvious inhibitory effect on morphine induced mental dependence;Compound 511 combined with EA can more effectively alleviate naloxone induced acute withdrawal symptoms in morphine dependent mice,which suggests that compound 511 combined with EA has more obvious effect on morphine induced physical dependence.It is concluded that compound 511 combined with EA has better effect on morphine addiction. |
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