| Objective To investigate the temporal changes of the circadian rhythm of spontaneous activity in Barb/c mice and the mechanism of acupuncture on phosphorylation of SCN nuclear protein at different time points.Method 108 male Barb/c mice were divided into six time points according to time aliquots,namely CT0(7:00)group,CT4(11:00)group,CT8(15:00)group,CT12(19:00)group,CT16(23:00)group,CT20(3:00)group;each group is divided into electroacupuncture group,false acupoint group,blank group.The screened Barb/c mice were placed in the isolation cells of the rhythm chamber for domestication.The temperature inside the isolation unit was constant at 23±1 °C,the relative humidity was kept at 50-60%,and the computer was used for automatic light-dark control LD12 :12,all animals are free to drink and drink.After 10 days of domestication,the spontaneous activity rhythm of the mice is synchronized with the light and dark cycle,turning the light into a constant darkness(dark-dark,DD)to make the spontaneous activity of the animal into free running(free running,FR).The spontaneous activity data of each mouse was continuously detected through the spontaneous activity automatic recording system.(1)After the mice entered the free-running for 10 days,the guiding experiment was carried out.The electroacupuncture group electroacupuncture the mice at the corresponding time,Baihui and Changqiang points,current frequency 2/15 Hz,sparse wave,current 0.5 mA Each time acupuncture is 15 min;the false acupoint group is stimulated by electroacupuncture at the non-acupoint points(at the intersection of the right midline and the iliac crest upper 10mm),the parameters are the same as the acupuncture group;And bundled processing.The spontaneous activity data of each mouse was continuously recorded by the MATLAB software,which was used to derive the activity rhythm map of the mouse runner,and the spontaneous rotation activity data of the animals were analyzed,and the peak phase,activity amount,start and stop activity time,and transfer amount of the activity amount of the daily mouse activity were calculated.The spontaneous activity of the mice after acupuncture was observed to change the phase characteristics of the circadian rhythm.(2)After the stimulation,the mice were further concealed,and the second experiment was performed after 10 days.The experimental method of each group of mice was the same as that of experiment one.After 2 hours of different treatment,the SCN brain tissue was taken out and TMT-labeled phosphorylation was applied.Group(IMAC enrichment)was used to analyze the mechanism of SCN nuclear protein phosphorylation by acupuncture at different times to adjust the circadian rhythm.Result(1)Electroacupuncture can adjust the initial activity time of spontaneous rhythm of mice,which is represented by CT4 and CT8 electroacupuncture.The initial activity time of meridian group is ahead of time.The initial activity time of meridian group is compared with that of false acupoint group P<0.05 The blank group was statistically significant compared with P<0.05.After the initial activity time of CT16 acupoint group,P<0.05 compared with the acupoint group and P<0.05,compared with the blank group,there was statistical significance.The change of initial activity time after electroacupuncture at other time points Not statistically significant.(2)The clustering analysis of the difference in protein phosphorylation at different time points showed a characteristic change in the clustering trend of CT8 and CT16.CT8 in Cluster1 and Cluster2 are the lowest points of the line graph,and CT16 is the highest point of the line graph.(3)Taking 1.2 times as the change threshold,take the difference between the common up-regulation and the common down-regulation in two replicate experiments.In this study,a total of 11232 phosphorylation sites on 3078 proteins were identified,of which 6631 were 2651 proteins.One site contains quantitative information.After filtering with a positioning probability of >0.75,a total of 7605 phosphorylation sites on 2829 proteins were identified,of which 6192 sites of 2488 proteins contained quantitative information.(4)According to the enrichment analysis of the signal pathway,there are 25 signal pathways corresponding to protein enrichment in the electroacupuncture group/blank group at the CT8 time point,and 5 significant enrichment points.CT8 time point electroacupuncture group/false There were 46 signal pathways corresponding to protein enrichment in the differentially modified sites of the acupoint group,and 11 pathways with significant enrichment.There were 41 signal pathways corresponding to protein enrichment in CT16 time point electroacupuncture group/blank group differential modification sites,10 were significantly enriched,CT16 time points,electroacupuncture group/false acupoint group differential modification site corresponding protein There are 17 enriched signal pathways and one significant enrichment.Conclusion(1)Electroacupuncture can adjust the beginning time of spontaneous behavior circadian rhythm in Barb/c mice,which has obvious time-dependent phase.it is characterized at CT4 and CT8,electroacupuncture induced phase advance at CT4?CT8,meanwhile at CT16,initial activity was delayed,there was no obvious transfer at the remaining time points(2)From the level of protein modification,CT8 acupuncture adjusted the phase of spontaneous activity,which may be due to the down-regulation of GABAB receptor and Gi/o protein phosphorylation in GABAergic synaptic signaling pathway,GABA involved in SCN to adjust circadian rhythm.The delayed spontaneous activity of CT16 acupuncture may be related to the up-regulation of AC protein and the inhibition of insulin secretion in the insulin signaling pathway. |
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