Study On The Mechanism Of Electroacupuncture On Depressive Disorder Based On Keap1-Nrf2-ARE Signaling Pathway

ObjectiveCurrently,the pathogenesis of depression has not been completely clarified,with certain limitations in the existing treatments.Besides,oxidative stress is closely related to the pathogenesis of depressive disorder Targeting such purpose,the electroacupuncture is a special therapy of TCM characteristic with positive anti-depression effect.Thus in this study,the clinical literature on acupuncture treatment of depressive disorder was firstly analyzed for the regularity of acupoint selection,based on which an animal depression model was constructed through chronic unpredictable mild stress(CUMS)combined with solitary parenting method,to explore the effects of electroacupuncture(EA)at Baihui(GV20)and Yintang(GV29)on oxidative stress and the Keap1-Nrf2-ARE signaling pathway in the amygdala of model depression mice.This study aimed to explore the mechanism of electroacupuncture in the treatment of depressive disorder,and to provide more available evidence for electroacupuncture clinical application.Methods1.Analysis of acupoint selection rulesDatabases such as CNKI,PubMed,and Cochrane Library were searched for clinical literature on depression,covering all related data till December 2020,and then screened according to the inclusion and exclusion criteria,in order to create a specific self-built database.By using SPSS 26.0 and SPSS Modeler 18.1 statistical analysis software,related processes including frequency analysis,systematic clustering and association rule analysis were performed on acupoints,and the summary was made on the common acupoints selection and compatibility rules of acupoint in treatment of depressive disorder.2.Experimental research(1)Animal groupingA total of 60 C57/BL6 male adult mice(aged 7-8 weeks,weighing 18.87±0.89 g)were randomly divided into four groups:control group(n=15),control+EA group(n=15),model group(n=15),and model+EA group(n=15).(2)ModelingCUMS combined with solitary parenting method was used to construct the models for mice in the model group and the model+EA group.The entire modeling cycle lasted for 28 days.There were 12 stimulation methods.During the modeling period,the mice were subjected to one short-term stimulation and one long-term stimulation each day.Short-term stimulation included restraint,icy water swimming,hot water swimming,tail clipping,foot shock,and noise interference.Long-term stimulation included food or water restriction,wet sawdust feeding,rat cage tilt,and reversing days and nights.Two groups of model mice were kept in single cages during the modeling period,and the rest were routinely rearing.(3)InterventionsMice of control+EA group and model+EA group received EA treatment after modeling.The mice were anesthetized with isoflurane and then put on a heating pad maintained at 32℃.Acupuncture needles were connected to the EA apparatus(continuous wave,2 Hz,1 mA,30 min)after piercing acupoints GV20 and GV29.Treatment was performed per day,lasting for 2 weeks with one day rest in between.The control group and the model group were only given anesthesia in the same way,without EA stimulation.(4)Evaluation Indicators①Macroscopic characterization observationDuring the modeling and electroacupuncture intervention,the mental state,hair,movement,behavior and defecation of the mice were observed.②Body weight and daily feed consumptionThe body weight and daily feed consumption of the mice in each group were measured one day before and after modeling,as well as one day after EA intervention.③Behavioral testAfter modeling,the behavioral changes of the depression model mice were evaluated by the Sucrose preference test(SPT);after the EA intervention,the SPT,Open field test(OFT)and Tail suspension test(TST)were applied to evaluate the depression level of mice in each group.The main observation indicators included:12h sugar water preference rate of SPT,the total distance travelled(mm)and the time spent in the center area(s)in OFT,and the immobility time(s)of TST.④Molecular biological detectionIn order to evaluate the effect of EA on the function of GABAergic neurons in the amygdala of depression model mice,real-time quantitative polymerase chain reaction(qRTPCR)and immunofluorescence were used to detect the expression of GABAergic neuron markers,GABA synthase,GABA transporter and synaptic protein,enzyme-linked immunosorbent assay was to detect serum GABA concentration;DHE staining,Western blotting,MDA content and antioxidant enzyme activity detection were used to measure the expression level oxidative stress and NOX2 protein,and antioxidant capacity in amygdala region of mice;western blotting and immunofluorescence experiments were used to evaluate the effect of EA intervention on the Keap1-Nrf2-ARE signaling pathway in the amygdala of depression model mice.Results1.Analysis of acupoint selectionIn this study,the acupoint prescriptions of 809 literatures were analyzed,and it was concluded that the most frequently used acupoints and meridians were Baihui point and Du meridians.The results of systematic cluster analysis showed that high-frequency acupoints could be classified into six categories,which were mainly composed of acupoints of Du meridians on the head,acupoints of Ren meridians in the abdomen and Beishu acupoints,with the functions of dredging Du Meridian and regulating spirit,soothing the liver and regulating qi,invigorating the yang qi,and harmonizing the internal organs.The results of association rule analysis indicated that,the Tiaoshen acupoint pair of Baihui-Yintang points had the highest confidence.2.Experimental research(1)Behavioral changes of depression model mice of CUMS combined with solitary parenting methodAfter modeling,the mice changed from being full of energy,irritable,and violently resisting when stimulated to mental fatigue,to the state of curling up with less movement,increased grooming behavior,and weakened resistance;compared with the control group,the body weight and daily feed consumption of model group decreased significantly(P<0.01),and the 12h sugar water preference rate was dramatically decreased(P<0.01).(2)The effect of EA treatment on the behavior of depression model miceAfter EA treatment,the macroscopic features of model+EA group were improved,the specific manifestations including mental state,fur gloss,eye canthal cleanliness,autonomous activities,grooming behavior,stool texture,etc.,returned to normal,with the body weight and daily feed consumption increased compared with the model group(P<0.01).In conclusion,EA treatment could improve the depression-like behavior of model mice,mainly manifested in the following ways:The preference rate was significantly higher than that of the model group(P<0.01);the total distance travelled and the time spent in the center area in OFT of model+EA group were significantly higher than those in the model group(P<0.01);compared to the model group,the immobility time in TST of model+EA group was significantly reduced(P<0.01).(3)The effect of EA intervention on the function of GABAergic neurons in the amygdala of depression model miceEA treatment significantly improved the function of GABAergic neurons in the amygdala of depression model mice.The specific manifestations were as follows:in contrast to the model group,the CCK,NPY,GAD67,GAD65,VGAT mRNA in the amygdala of mice in the model+EA group were markedly enhanced(P<0.05),and PV,SST,GAT1,Gephyrin mRNA were showed an increasing trend(P>0.05);the number of GAD67+ cells in amygdala and the serum GABA concentration was effectively increased(P<0.01).(4)The effect of EA treatment on the oxidative stress level of amygdala in depression model miceEA treatment effectively reduced the oxidative stress level in the amygdala of depression model mice and increased the activity of antioxidant enzymes.In contrast to the model group,the average fluorescence density of DHE in the amygdala of depression model mice in the model+EA group obviously decreased(P<0.01).The content of MDA and the expression of NOX2 protein were down-regulated(P<0.01),while the activities of SOD,CAT,GSH-PX and GSH content were up-regulated(P<0.01).(5)The effect of EA treatment on the Keap 1-Nrf2-ARE signaling pathway in the amygdala of depression model miceEA treatment activated the Keap 1-Nrf2-ARE signaling pathway in the amygdala of depression model mice.In relative to model group,the protein expression of Keap l in the model+EA group were obviously decreased(P<0.01).EA treatment remarkably enhanced the expressions of p-Nrf2,HO-1,NQO-1,and SOD-1(P<0.01),and increased the number of Nrf2 nuclear translocated cells(P<0.01).Conclusion1.Depression-like behavioral changes in model mice induced by CUMS combined with solitary parenting.2.Electroacupuncture ameliorated the behavioral disorder of depression model mice.3.Electroacupuncture intervention improved the dysfunction of GABAergic neurons in the amygdala of depression model mice.4.Electroacupuncture reduced the level of oxidative stress in the amygdala of depression model mice and increased the activity of antioxidant enzymes.5.Electroacupuncture activated the Keap1-Nrf2-ARE signaling pathway of the amygdala of depression model mice and regulated the expression levels of related proteins.