| Objective:Depression is a widespread mental disorder.At present,nearly 30%of patients have ineffective or ineffective clinical treatment.Although there has been some understanding of the pathogenesis of depression from the Pathophysiology perspective in recent years,it still can not satisfactorily explain the poor efficacy of depression.At present,most studies support the hypothesis of hippocampal neurogenesis disorder in depression.The amygdala is one of the key brain regions to regulate emotion.Studies have shown that the amygdala in patients with depression has reduced volume and abnormal function.Therefore,it suggests that amygdala neurogenesis disorder may be another important pathological mechanism of depression.Silent information regulator 1(SIRT1),as a NAD~+-dependent deacetylase,improves depressive mood disorders by promoting hippocampal neurogenesis.However,whether SIRT1 plays an antidepressant role by regulating amygdala neurogenesis has not been reported.So,our study intends to use the depression like rat model induced by chronic restraint stress(CRS).First,the changes of amygdala neurogenesis and depressive-like behavior in depressed rats were observed by immunofluorescence and behavioral experiments,and the effects of electroshock therapy(ECT)and fluoxetine(FLU)on them were also observed;On this basis,gene overexpression technique was used to observe the effects of SIRT1 up regulation on amygdala neurogenesis and depression like behavior in depressed rats.This study will provide theoretical basis for further elucidating the neurogenesis hypothesis of depressive affective disorder,and provide new ideas for the treatment of depression in the future.Methods:1.The first part of the experiment was randomly divided into six groups:(1)normal control group(Control group):Sprague Dawley male rats aged 8 weeks were selected and fed normally;(2)Chronic restraint stress group(CRS group):Sprague Dawley male rats of the same age were given chronic restraint stress for 3 weeks;(3)Chronic restraint stress+electroconvulsive therapy treatment group(CRS+ECT group):select the successful model of depression rats and treat them with electroconvulsive therapy once a day for 10 days;(4)Chronic restraint stress+pseudoelectric shock group(CRS+Sham ECT group):select the successful model of depression rats,and treat with pseudoelectric shock once a day,that is,clamp the ears with binaural clamps and do not give corresponding current for 10consecutive days;(5)Chronic restraint stress+fluoxetine treatment group(CRS+FLU group):select the successful model of depression rats and give fluoxetine(10mg/kg)by gavage once a day for 30 days;(6)Chronic restraint stress+normal saline group(CRS+Na Cl group):select the successful model of depression rats and give normal saline(10mg/kg)by gavage once a day for 30 days.The experiments of each group are as follows:(1)Behavioral experiment:A rat model of depression induced by chronic restraint stress was established.After fluoxetine and electric shock treatment,respectively,the depression behavioral indexes of rats in each group were detected by sucrose preference test(SPT),open field test(OFT)and forced swimming test(FST).(2)Immunohistochemical experiment:Using immunofluorescence staining cell proliferation marker Ki67~+,immature neuron marker DCX~+to detect the level of amygdala neurogenesis in each group.2.The second part of the experiment was randomly divided into four groups:(1)normal control group(Control group):Sprague Dawley male rats aged 8 weeks were selected and fed normally;(2)Chronic restraint stress group(CRS group):Sprague Dawley male rats of the same age were given chronic restraint stress for 3 weeks;.(3)Chronic restraint stress+overexpression SIRT1 group(CRS+AAV-SIRT1 group):select the successful model of depression rats,and inject AAV-SIRT1 virus into the amygdala of rats;(4)Chronic restraint stress+empty body group(CRS+AAV-EGFP group):select the successful model of depression rats,and inject AAV-EGFP virus into the amygdala of rats.The experiments of each group are as follows:(1)Western-blot and ELISA experiments:The level of SIRT1 protein was detected by Western-blot and the activity of SIRT1 enzyme was detected by enzyme-linked immunosorbent assay(ELISA).(2)Behavioral experiment:After overexpression of SIRT1 in the amygdala of depressed rats,the depression like behavior indexes of rats in each group were detected to evaluate the effect of SIRT1 on depression like behavior.(3)Immunohistochemical experiment:After overexpression of SIRT1 in the amygdaloid nucleus of depressed rats,the level of neurogenesis in the amygdaloid nucleus was detected by immunofluorescence staining to investigate the role of SIRT1 in the regulation of amygdala neurogenesis.Results:1.Behavioral experiment results:Compared to normal rats,the rats in CRS depression group showed obvious depression like behavior,including a significant decrease in the percentage of sucrose water preference(P<0.001),a significant decrease in the total distance of exercise(P<0.001)and the residence time in the central area(P<0.001)in the open field test,and a significant increase in the immobility time of forced swimming(P<0.001);Compared with CRS rats,the percentages of sucrose preference in electroshock group and fluoxetine group were significantly higher(P<0.001,P<0.01),the total distance of exercise(P<0.001,P<0.01)and central time(P<0.01,P<0.05)in open field test were prolonged,and the immobility time in forced swimming test was significantly shortened(P<0.001,P<0.05);There was no significant change in behavior in the electric shock group treated for 10 days compared with the fluoxetine group treated for 30 days;There were no significant changes in behavior in sham electric shock group and normal saline group compared with CRS depression group;Compared with CRS rats,the percentage of sucrose water preference in SIRT1 overexpression group was significantly higher(P<0.001),the total exercise distance(P<0.001)and central time(P<0.001)in open field test were prolonged,and the immobility time during forced swimming was significantly shortened(P<0.001);There was no significant difference in behavior between empty body group and CRS depression group.2.Western-blot results:Compared to normal rats,the expression of SIRT1 protein in amygdala of rats in CRS depression group decreased significantly(P<0.001);Compared with CRS rats,the expression of SIRT1 protein in amygdala of SIRT1 overexpression group was significantly increased(P<0.001);There was no significant difference in the expression of SIRT1 protein in amygdala between empty body group and CRS depression group.3.Results of enzyme linked immunosorbent assay(ELISA):Compared to normal rats,the activity of SIRT1 enzyme in amygdala of rats in CRS depression group decreased significantly(P<0.01);Compared with CRS rats,SIRT1 enzyme activity in amygdala of rats in SIRT1 overexpression group increased significantly(P<0.05);There was no significant difference in the activity of SIRT1 enzyme in amygdala between empty body group and CRS depression group.4.Immunofluorescence test results:Compared to normal rats,the number of cell proliferation marker Ki67~+cells and immature neuron marker DCX~+cells in amygdala of CRS depression group were significantly reduced(P<0.001);Compared with CRS rats,the number of Ki67~+cells(P<0.01,P<0.05)and DCX~+cells(P<0.001,P<0.01)in amygdala of electric shock group and fluoxetine group increased significantly;There was no significant difference in the number of Ki67~+cells and DCX~+cells in amygdala between the rats treated with electric shock for 10 days and the rats treated with fluoxetine for 30 days;There was no significant difference in the number of Ki67~+cells and DCX~+cells in amygdala between sham electric shock group and normal saline group and CRS depression group;Compared with CRS rats,the number of Ki67~+cells(P<0.01)and DCX~+cells(P<0.001)in amygdala of SIRT1 overexpression group increased significantly;There was no significant difference in the number of Ki67~+cells and DCX~+cells in amygdala between empty body group and CRS depression group.Conclusion:1.Promoting the regeneration of rat amygdala nerve can reverse the depression like behavior of rats.(1)Combining the results of three behavioral experiments:sucrose preference test,open field test and forced swimming test,CRS depressed rats showed significant depression like behavior compared with the normal control group.Electroshock and fluoxetine treatment were helpful to improve the depression like behavior of rats.(2)The number of Ki67~+cells and DCX~+cells in amygdala of CRS depressed rats decreased,suggesting that neurogenesis was impaired in CRS depressed rats;Electroshock and fluoxetine treatment can increase the number of Ki67~+cells and DCX~+cells in the amygdala of depressed rats and improve the damaged neurogenesis in CRS rats.(3)The antidepressant effect of electric shock treatment for ten days reached the therapeutic effect of fluoxetine for one month,indicating that the antidepressant effect of electric shock is faster than that of fluoxetine treatment.2.SIRT1 may play an antidepressant role by promoting amygdala neurogenesis.(1)SIRT1 protein and its enzyme activity in amygdala of CRS depressed rats decreased significantly.Overexpression of SIRT1 in amygdala significantly improved the depression like behavior of CRS rats.Firstly,the antidepressant effect of SIRT1 was clarified.(2)Overexpression of SIRT1 can partially restore the reduced number of Ki67~+cells and DCX~+cells in the amygdala of CRS depressed rats,suggesting that SIRT1 can promote neurogenesis. |
PDF Full Text Request