The Effect Of The Tetrandrine On The Brain Glucose Metabolism Rate And The Differential Protein Expression Of The Insulin/insulin Resistance Signaling Pathway In Rats Which Were Phenobarbital Addiction

Objective:to study different dosing phase,the rat which had phenobarbital addiction brain tissue changes in glucose metabolism and brain insulin signaling pathway differences in protein expression,and the influence of tetrandrine.Methods:150 SD rats were divided into 5 groups,each group of 30,male and female half,adopts the method of increasing doses of phenobarbital addictive rats model induced,for the tetrandrine intervention:normal group,phenobarbital addiction group（dose escalation to phenobarbital）,low dose group of tetrandrine(phenobarbital;tetrandrine6.25mg·kg^-1·d^-1),the middle dosage group(phenobarbital;tetrandrine 18.75mg·kg^-1·d^-1),the high dosage group(phenobarbital;tetrandrine 32.25mg·kg^-1·d^-1).The addiction model was verified by natural withdrawal symptom score and rat weight change after withdrawal.Using ¹⁸F-fludeoxyglucose positron emission tomography/computerized tomography(¹⁸F-FDG PET/CT)technical analysis each group rats brain tissue differences in glucose metabolism for 60 days and 90 days:the prefrontal cortex（PFC）,ventral tegmental area（VTA）,the field CA1-3 of hippocampus（CA1-3）,the basolateral amygdala（BLA）,the nucleus accumbens（NAc）.Analysis of each group rats tissue homogenate differences in protein expression with Application of Tandem Mass Tag（TMT）and Liquid chromatography-Tandem Mass spectrometry（LC-MS/MS）.Results:（1）score of natural withdrawal symptoms:normal group did not have withdrawal symptoms after withdrawal;The addiction group and the tetrandrine intervention group began to have different degrees of withdrawal symptoms at 16h after withdrawal,the most obvious during the 48-56h period,and then the symptoms were reduced until 144h no longer withdrawal symptoms.Addiction group and the tetrandrine intervention low-does and middle-dose group of 24-120h after withdrawal,the withdrawal score results compared with normal group,there was statistically significant of the difference（P<0.05）,and after 24-96h the tetrandrine intervention high-dose group during withdrawal,score results compared with normal group,there was statistically significant of the difference（P<0.05）.（2）weight change:normal group weight gain after withdrawal.The weight of the intervention group was decreased in both the addiction group and the tetrandrine control group,among which the reduction in the group was the most obvious,while the high-dose intervention group was relatively small.On the 6th day after abstinence,the weight stopped and began to recover on the 7th day after withdrawal.（3）glucose metabolism rate:60d,compared with normal group,addiction group and the tetrandrine low-dose group,middle-dose group of the brain tissue which related with addiction （BLA,CA1-3,NAc,PFC,VTA）a significant rise in glucose metabolism,there was statistically significant of the difference（P<0.05）;And then,compared with the addiction group,the glucose metabolism rate in the low,middle and high-dosage groups of the powder was decreased,and the high dose group was the most significant,and there was statistically significant of the difference（P<0.05）.The comparison between the intervention groups of the tetrandrine intervention groups showed that the glucose metabolism rate of the high-dosage group was significantly lower than that in the other two tetrandrine control groups,and there was difference which is statistically significant（P<0.05）.90d,To comparing with the normal group,the brain tissue glucose metabolism rate of the addiction group and tetrandrine control groups was significantly higher,and there has difference which was statistically significant（P<0.05）.Compared with the addiction group,the glucose metabolism rate in each group was decreased,with the difference between the medium and high dose group（P<0.05）.There was a significant decrease in the glucose metabolism rate in the high-dose group and the medium dose group,and there was difference which is statistically significant（P<0.05）.For 60d and 90d comparison results show that addiction group and the tetrandrine intervention dose groups of cerebral glucose metabolism rate,when dosing 90d is 60d further increase,but low-dose group of the VTA, middle-dose group of the VTA,CA1-3,NAc and high-dose group of VTA,CA1-3 glucose metabolism rate increase is not significant,no statistically significant difference.（4）the proteomic analysis:use 60d after GO and KEGG analysis shows that differentially expressed proteins are mainly located in vacuoles,cell membrane,organelle,the place such as synapses,mainly with enzyme,cytoskeleton,the g protein coupled glutamate receptor combination,such as participate in the regulation of enzyme activity,extracellular stimulation reaction adjustment,ion balance biological processes in the cell.The main enrichment is in the insulin signaling pathway,phosphatidyl inositol signaling system,calcium signaling pathway and other metabolic pathways.Use 90d differentially expressed proteins mainly locate in cytoplasm and organelles,the place such as neuronal dendrites,adjusting and enzyme activity,calcium ion regulation,cell apoptosis and other associated program,to participate in the regulation of cell metabolism,intracellular p H adjustment, biological processes such as transmission of nerve impulses.It mainly concentrates on metabolic pathways such as fatty acid metabolism pathway,insulin resistance signaling pathway and calcium signaling pathway.（5）the effects of insulin signaling pathway for 60d,there are differences in insulin signal transduction protein concentration significantly,mainly Calm1 and Araf,Cbl,Srebf1,Prkacb etc.Among them,Araf and Cbl were down-regulated in the intervention groups of the addiction group and the tetrandrine groups.Srebf1 and Prkacb were only down-regulated in the low,medium and high dose groups of the tetrandrine.The difference protein Calm1 was down-regulated in the addiction group,and the expression was up-regulated in the low-dose and high-dose group.For the drug 90d,after the analysis of the interaction between different proteins,it was found that the difference protein enrichment in the insulin signaling pathway was not high,but was significantly enriched in the insulin resistance signaling pathway.There are mainly Slc27a3and Nfkb1.Among them,the difference protein Slc27a3 was only expressed significantly in the addiction group,and the difference protein Nfkb1 was significantly increased in the intervention groups of the addiction group and the tetrandrine control groups.Conclusion:（1）phenobarbital addiction can lead to the increase of glucose metabolism in rats with brain addiction,and the increase of phenobarbital duration is more obvious.（2）The tetrandrine can reduce the cerebral glucose metabolism rate of phenobarbital addiction.（3）in the early stage of phenobarbital addiction,the expression of differentially expressed proteins such as Cbl,Calm1 and Araf in the brain insulin signaling pathway was down-regulated.The expression of Cbl and Araf was down-regulated in the brain insulin signaling pathway of tetrandrine intervention,and the expression of Calm1 was up-regulated.（4）with the extension of phenobarbate,the expression of differentially expressed proteins such as Slc27a3 and Nfkb1 in the insulin resistance signaling pathway was increased,leading to insulin resistance.