| Objective:To investigate the glucose metabolism status and the expression of differentially expressed proteins in brain insulin / insulin resistance signaling pathways and the effects of Naoxintong on the above changes in rats with addiction to phenobarbital at different stages.Methods:150 SD rats were divided into 5 groups,30 rats in each group,male and female were divided into two groups.The model of addiction induced by phenobarbital was induced by dose escalation,and Naoxintong intervention:control group(0.5% CMC-Na),phenobarbital addicted group(phenobarbital dose-increment method),low-dose Naoxintong intervention group(phenobarbital;Naoxintong 250 mg / kg),middle-dose Naoxintong intervention group(phenobarbital;Naoxintong 350 mg / kg),high-dose Naoxintong intervention group(phenobarbital;Naoxintong 500 mg / kg).The natural withdrawal symptoms were scored and the body weight of the rats were withdrawn after the withdrawal to verify the model of addiction.The18F-FDG PET / CT technique was used to analyze the brain-related nuclei in the above groups after administration 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: the normal group did not have withdrawal symptoms after withdrawal;the addiction group and Naoxintong group began to show different degrees of withdrawal-related symptoms at 16 h after withdrawal,the most obvious was during 48-56 h,and then the symptoms were relieved.There was no withdrawal-related symptoms until 144 h.the addictive group and the Naoxintong low,middle dose group in the withdrawal period after 24-120 h abstinence score compared with the normal group had significant difference(P <0.05),and the Naoxintong high dose group after withdrawal of 24-96 h score than the normal group had significant difference(P <0.05).(2)weight change:the normal group weight gain after withdrawal.The weight of the intervention group was decreased in both the addiction group and the Naoxintong 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 the normal group,the glucose metabolism of brain related addicts(BLA,CA1-3,NAc,PFC,VTA)in phenobarbital addicted rats and Naoxintong low dose intervention groups were significantly increased;The brain tissue of high,Naoxintong medium dosage group had higher glucose metabolism(BLA,NAc,PFC and VTA),CA1-3 nucleus had no significant difference with normal group,while Naoxintong high dose intervention group CA1-3 and PFC nucleus and normal group no significant difference,only BLA,NAc,VTA nuclear glucose metabolism were significantly increased. 90d:Compared with the normal group,glucose metabolism was significantly increased in the relevant nuclei(BLA,CA1-3,NAc,PFC and VTA)of the rats with phenobarbital addiction and the Naoxintong low and medium dose intervention groups;However,only the BLA,CA1-3 and VTA nuclei in the Naoxintong high-dose intervention group showed a significant increase in glucose metabolism,while there was no significant difference in the NAc and PFC nuclei compared with the normal group.In the more addicted and Naoxintong low dose groups,the glucose metabolism of the CA1-3 nucleus significantly decreased,while the rest of the nuclei showed no significant difference.The glucose metabolism of each group in the Naoxintong medium and high dose groups was significantly decreased.Compared with the Naoxintong different dose groups,the glucose metabolism of BLA,NAc,PFC,VTA nucleus of the middle dose was significantly lower than the low dose intervention group,CA1-3 nucleus showed no significant difference,and the lower dose glucose metabolism of each nucleus of high dose group was significantly reduced;high dose group NAc,PFC nucleus than medium dose intervention Group glucose metabolism was significantly reduced,the remaining nuclei no significant difference.The results of 90 d and 60 d administration showed that glucose metabolism of BLA,CA1-3,NAc,PFC,VTA had no significant difference at 60 days and 90 days;90 days and 60 days after administration(BLA,CA1-3,NAc,PFC and VTA)in the addictive group were significantly higher than those in the control group.No significant difference(P <0.05)was observed in the nuclei.The BLA,CA1-3,PFC and VTA were significantly increased in the Naoxintong medium dose group,while there was no significant difference in the NAc nucleus.Only the BLA nucleus in the Naoxintong high dose group was significantly increased,nuclei no significant difference.(4)the proteomic analysis:60d:A total of 680 differentially expressed proteins were collected.Among them,444 proteins were up-regulated and 236 proteins were down-regulated.The 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.90d:A total of 501 differentially expressed proteins were collected.Among them,a total of 247 proteins were up-regulated and 254 proteins were down-regulated.The 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)Insulin / insulin resistance signaling pathways:60d:There were significant changes in the expression of differential proteins such as Cbl,Srebf1,Calm1,Araf and Prkacb in the insulin signaling pathway.Compared with the normal group,the differential expression of protein in the brain tissue of the addiction group mainly involved Calm1,Araf,Cbl three proteins,the three proteins were significantly down-regulated;Naoxintong intervention group involved more kinds of differential protein expression,mainly Calm1,Araf,Cbl,Srebf1 and Prkacb proteins.The expression of Calm1 was significantly different from those of addiction group in both low and high concentrations of Naoxintong,but Araf and Cbl were down-regulated in all Naoxintong groups The degree of addiction group was higher,Srebf1,Prkacb in Naoxintong dose groups were significantly reduced.After differential protein interaction analysis,we found that the differential protein enrichment in insulin resistance signaling pathway is not high.90d: The differential protein of insulin signaling pathway enrichment is not high.Only Naoxintong high-dose intervention group than the normal group has a protein differential expression,Nras(2.391266↑)was significantly up-regulated.Insulin resistance pathway mainly Slc27a3 and Nfkb1 differential protein expression changes significantly.Compared with the normal group,the difference expression of protein in the brain tissue of the addiction group mainly involved two kinds of Slc27a3 and Nfkb1,and the expression of the two differentially expressed proteins were significantly increased.Only the Nfkb1 expression in the Naoxintong intervention group was significantly up-regulated and the high dose was up-regulated More obvious.Conclusion :(1)Phenobarbital addiction can lead to increased glucose metabolism in the brain related to addiction in the rat brain(ventral tegmental area,nucleus accumbens,hippocampus,basolateral amygdala,ventral medial prefrontal cortex)With phenobarbital medication extended,the more obvious brain metabolism.(2)Naoxintong capsule to some extent can improve the metabolism of phenobarbital addicted to cerebral glucose metabolism.(3)Phenobarbital early addiction,will cause the insulin signaling pathway Cbl,Calm1,Araf and other differential protein expression down.Resulting in obstruction of insulin signaling pathway,resulting in increased glycogen synthesis,increased glucose uptake.With the intervention of Naoxintong,the expressions of Cbl and Araf in brain-derived insulin signal transduction pathway of phenobarbital-addicted rats are down-regulated and the expression of Calm1 is up-regulated,which may result in a relative decrease in glycogen synthesis and a relative decrease in glucose uptake.(4)With the prolongation of phenobarbital administration,the expression of Slc27a3 and Nfkb1 in the insulin resistance signaling pathway may be upregulated,which may lead to insulin resistance and prompt the body to excrete excess insulin to produce hyperinsulinemia,So that elevated blood sugar,which may increase glucose uptake,may lead to brain damage. |
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