Study On The Effect Of Electroacupuncture On CSDS Mice By Regulating P11

Research background and purpose In clinical practice,acupuncture has a long history of treating depression,but the mechanism of its antidepressant effect has not been well studied.In recent years,more and more studies have suggested that p11 protein plays an important role in mediating the pathogenesis of depression.The results suggest that the levels of p11 protein and mRNA of hippocampus,prefrontal lobe and nucleus accumbens in depression patients and depression model animals are significantly reduced.In our preliminary experiments,we verified the distribution of p11 in the prefrontal cortex and hippocampus and found that it has obvious expression in DRN.But there is no related research about p11 and depressive behavior in DRN.Therefore,in this experiment,we will focus on the role of p11 in DRN in the pathological process of depression and explore whether electroacupuncture will affect depression-like behavior by regulating p11 in DRN.Methods Experiment 1: The establishment of depression model and the effect of p11 protein on Depression Behavior 1.Samples were twenty male C57 mice aged 7-8 weeks.They were randomly divided into blank group and the Chronic Social Defeat Stress(CSDS)depression model.After the 10-day social frustration modeling,four behavioral examinations were carried out to evaluate the success of the model,which included the Sucrose Preference Test(SPT),the Open Field Test(OPT),the Social Interaction Test(SIT),and the Forced Swimming Test(FST).2.Using immunofluorescence to observe the changes of c-fos in DRN of normal and depression mice.3.Observing the changes in p11 mRNA and protein levels in normal and depressed mice by immunofluorescence.4.Brain stereotactic injection of the AAV-h Syn-sh-p11 virus would knock down the p11 protein in DRN.After 3 weeks of virus expression,sugar water experiments,open field experiments,social behavior tests,and forced swimming tests were used to observe relative behavioral changes.Experiment 2: Positioning and function of p11 1.Using colloidal gold particles to label p11,and observing the location of p11 in neurons by immunoelectron microscopy.2.Using the specificity of GammaAminobutyric Acid(GABA)neurons to label the protein GAD67,applying the specificity of 5-HT neurons to label the protein tph2,and employing glutamate neurons to specifically label the protein Camk?.These were co-standardized with p11 to observe which types of neurons were mainly enriched in p11.3.Practicing Coimmunoprecipitation(Co-IP)method to obtain p11 protein in DRN and its binding and interacting proteins.At the same time,this method was adopted to identify the protein that might interact with p11 using protein profiling technology after obtaining its binding interacting protein.Co-immunoprecipitation technology was used to verify the results of the protein profile to clarify the interacting protein of p11 as well.4.Exercising immunofluorescence to observe the changes of p11 and interacting proteins in depressed mice.Experiment 3: Effects of 5-HTDRN-specific Overexpression of p11 on Depressionlike Behaviors 1.Sixteen fev-cre mice 7 to 8 weeks old were randomly divided into two groups.One group of mice was injected with AAV-DIO-OE-p11 specifically over-expressed p11 in 5-HTDRN,and the other group was injected with AAV-DIO-OE-m Cherry as a control.Three weeks after the virus spread,mice were subjected to behavioral tests to see if purely specific overexpression of p11 could cause behavioral changes.2.After that,both groups of mice would undergo a full CSDS stimulation to perform another behavioral test,which would help us observe whether specific overexpression of p11 could reverse the depression-like behavior induced by CSDS modeling.Experiment 5: Effects of Electricity on Depressive Behavior and the Regulation of p11 in DRN 1.Thirty-two male C57 mice aged 7-8 weeks were randomly divided into a blank group,a model group,a sham-acupuncture group,and an electroacupuncture group. Among them,the sham-acupuncture group and the electroacupuncture group would be intervened for 3 weeks after modeling.2.After the intervention,a behavioral test will be performed,and the DRN of the mouse would be taken out to observe the changes of p11 mRNA levels in each group of mice.Results Experiment 1: The Establishment of the Depression Model and Effects of p11 Protein on Depressive Behavior 1.After social frustration modeling,the following scenes appeared: mice's sugar preference decreased;central time and central distance in the open field decreased;forced swimming immobility time increased;social behavior decreased.Compared with the blank group,the depressive symptoms and behavioral statistics of the model mice were statistically different.The protein and mRNA levels of p11 in the dorsal raphe nucleus of the depressed mice were significantly lower than those of the blank group.2.Three weeks after the virus injection,we performed a behavioral test on the samples.Compared with the AAV-h Syn-sh-e GFP sample group,the sample group injected with AAV-h Syn-sh-p11 showed the following situations: social time was significantly reduced;forced swimming immobility time was significantly increased;sugar preference was reduced,all of which are all statistically different.However,there is no difference in central time and distance in open field experiments.Results showed that the total distance was less,and the speed of exercise was slower.Experiment 2: Positioning and Function of p11 1.The results of immunoelectron microscopy showed that p11 protein is widely present in the soma before and after synapses and intramyelinic.Especially before and after synapses,p11 protein mostly existed in the form of aggregates.2.Immunofluorescence indicated that p11 was co-standard with Tph2,which mainly presented in dorsal raphe serotonergic neurons.3.Protein profiling results revealed that 178 proteins might bind to p11.In this experiment,the co-immunoprecipitation method was used to verify the interaction between Annexin A? and m Glu R5 and p11.Confocal observation of normal mice p11 and m Glu R5 accumulated on the cell membrane,while depressed mice p11 and m Glu R5 mostly fell into the cytoplasm.Experiment 3: Effects of 5-HTDRN-specific Overexpression of p11 on Depressionlike Behaviors 1.After overexpression of p11 by 5-HTergic neurons in fev-cre mice,social index increased in basic behavior which were statistically different.There were no significant differences in other behavioral tests.2.After CSDS modeling,behaviors suggested that the comparison group had shorter social time,lower social index,lower sugar preference,longer forced immobility time,and reduced central time and central distance,all of which were statistically different.In the p11 overexpression group,there was no significant difference in social time,social index,and sugar preference before and after modeling.While situations that forced swimming immobility time was prolonged,and central time and central distance happened in the p11 overexpression group,all of which were statistically different.After modeling,the social time,social index,and sugar preference of the p11 overexpression group were significantly higher than those of the control group.These data were statistically different.No difference in other behavioral tests was found.Experiment 4: Effects of Electricity on Depressive Behavior and the Regulation of p11 in DRN.1.Compared with the blank control group,the social index of the mice in the model group and the sham-acupuncture group was reduced,the percentage of sugar and water preference was reduced,the duration of forced swimming immobility was extended,and the number of uprights,central time and central distance were less.All the data showed statistical differences.Compared with the blank control group,the electroacupuncture group had no difference in various behavioral tests.Compared with the model group,the electroacupuncture group had less time for forced swimming,and the percentage of sugar and water preference increased with central time.These data were all statistically different.Compared with the sham-acupuncture group,the electroacupuncture group showed less time for forced swimming,and the percentage of sugar preference increased,both of which had statistical differences.Besides,the number of uprights,central time,and central distance increased but indicated a negative statistical difference.2.The p11 mRNA level of the electroacupuncture group was not different from that of the normal group but was higher than that of the model group with statistical differences.There was an upward trend after comparison with the shamacupuncture group,but there was no statistical difference.Conclusion1.The level of p11 mRNA and protein were denmonstrated to decrease in the dorsal raphe nucleus of depression model.p11-KO mice showed depression behavior rather than anxiety behavior.2.P11 is mainly located in the postsynaptic membrane of 5-HTDRN,and is located on the cell membrane by binding to m Glu R5.m Glu R5 membrane binding decreased with the decrease of p11 and resulted in anhedonia of interest and other depression-like behaviors.3.Electroacupuncture can improve depressive behavior by reversing the decrease of p11 in the dorsal raphe nucleus of depressive mice and exciting the dorsal raphe nucleus.