Study On The Effect Of Acupuncture At Different Time On The Phosphorylation Modification Of Glu Signaling Pathway Protein In SCN Of Mice

Objective: To explore the effect mechanism of electroacupuncture at different time points on the phosphorylation level of Glu signaling pathway protein in SCN of suprachiasmatic nucleus of mice.Method: 108 male Barb/c mice were placed in separate laboratory compartments for acclimatization,Clocklab system software was set to simulate light-dark alternating environment LD 12:12,acclimatization was conducted for 10 days,and behavioral data was recorded through the Clock Lab system To synchronize the spontaneous activity rhythm of the mouse with the light-dark cycle.After the cycle is synchronized,the light is set to constant dark(dark-dark,DD),and the phase shift value generated by the freerunning mouse day and night wheel is recorded and randomly divided into CT0 group,CT4 group,CT8 group,CT12 group,CT16 group,CT20 group was divided into electroacupuncture group,sham point group and control group at each time point.According to the initial activity time of mice during acclimation,the CT12 of the treatment day is predicted to infer the time of each electroacupuncture.The electroacupuncture group electroacupuncture the mouse at Baihui,Changqiang,and two meridian points at the corresponding time.Use the acupuncture needle to percutaneously prick a needle as an auxiliary needle.The electroacupuncture parameters were set as: density wave,electroacupuncture frequency 2/15 Hz,current 0.5 m A,acupuncture for 15 minutes in total;mice in the sham acupuncture group at the same time under the right side of the electroacupuncture non-menstrual and nonacupoint points,assist The stimulation parameters of acupuncture and electroacupuncture are the same as above;the control group does not do any other treatment.After 2 hours of acupuncture treatment of all animals,10% chloral hydrate intraperitoneal injection was used for anesthesia.The brain tissue including the SCN area was surgically removed and immediately placed into-80° liquid nitrogen.Each group collected 108 samples at the corresponding time points in the same way,TMT labeling,peptide enrichment and mass spectrometry-based protein phosphorylation modification quantitative omics were used to analyze the phosphorylation modification level of SCN protein.Results:(1)A total of 11,232 phosphorylation sites on 3,078 proteins were identified in this experiment,of which 7,633 sites on 2,651 proteins had quantitative information,and 1.2 times were used as the difference threshold for screening.It appears as up-and down-regulated differential protein sites.The results showed that at the CT0 time point,the electroacupuncture group and the control group were mainly overexpressed for phosphorylation modification,and the electroacupuncture group and the sham point group were mainly overexpressed;at the CT4 time point,the electroacupuncture group and the control group Compared with the expression above,the electroacupuncture group and the sham point group mainly expressed the following adjustment;CT8 time point,the electroacupuncture group was compared with the control group,and the electroacupuncture group was compared with the sham point group;CT12,CT16,CT20 time points,compared with the electroacupuncture group and the control group,the electroacupuncture group and the sham point group,the modification level was mainly up-regulated.(2)After analyzing the KEGG pathway with significantly enriched proteins corresponding to the differential modification sites at 6 time points,it was found that the Glutamatergic synapse pathway has a high difference value,and its significance is different at different time points,manifested as CT0,CT4,At the time point of CT8,the pathway enrichment was significant,but the enrichment of CT12,CT16,and CT20 pathways was not obvious.(3)At the time points of CT4,CT8,and CT0,there are co-upregulated protein calcium channel proteins VGCC and SHANK proteins in the phosphorylation of Glutamatergic synapse pathway proteins.By analyzing the amino acid sequence of the differentially modified protein sites,it was found that the gene corresponding to the calcium channel protein VGCC is Cacna1 a,and the gene of the SHANK protein is Shank3.After electroacupuncture at different time points,the VGCC protein was at the corresponding site of 394 and the SHANK protein 2200,2202,2303 The upstream and downstream amino acid sequences of the corresponding sites showed a differential distribution.conclusion:(1)The effect of electro-acupuncture at different time points on the phosphorylation modification of Glutamatergic synapse pathway protein in SCN of mice is different,which may be the internal mechanism of acupuncture signal regulating circadian rhythm.(2)The up-regulation of the protein phosphorylation modifications produced by CT4 and CT8 may be related to the amino acid sequences of the upstream and downstream positions 2200,2202,2303 of the calcium channel protein VGCC corresponding to the acupuncture signal regulation Glutamatergic synapse pathway and the corresponding position 394 of the SHANK protein.