| Objective:Depression is a common mental illness.Depression brings infinite pain to the patient,makes patients lose pleasure of life,and it is accompanied by severe physical symptoms.However,the molecular mechanism of depression remains to be elucidated.Mitotic arrest deficient protein(MAD2B)is a highly conserved protein,also known as hRev7 or MAD2L2.Previous studies have shown that MAD2 B is widely expressed in the nervous system,mainly in neurons.MAD2 B inhibits the active of the anaphase promoting complex(APC)in the late mitotic process,and the Cdh1/APC complex plays an important role in regulating neuronal development,synaptic plasticity,learning and memory and other physiological processes.However,the role of MAD2 B in depression and its mechanism are not yet known.Therefore,this study first explored the expression of MAD2 B in the hippocampus of depressive mice.Then we assessed the effects of MAD2 B on depression-like phenotype and its molecular mechanism by using the adeno-associated virus microinjection method to interfere with the expression of MAD2 B in the hippocampus and transfecting lentivirus into the primary cultured hippocampal neurons to interfere with the expression of MAD2 B.Methods1.Three weeks of intraperitoneal injection of corticosterone(CORT)and consecutive five weeks of chronic unpredictable stress(CUS)were taken to establish depression models with 7-8 weeks male C57BL/6J mice.The sucrose preference test,forced swimming test,and tail suspension test were used to detect depression-like behaviors in mice;the fatigue rotating rod test was used to detect the locomotivity of mice.Through the above behaviors,we can judge whether these chronic depression modelling methods are successful.2.We used Western blot to detect the expression of MAD2 B in the hippocampus of the control and chronic depression model mice.3.The C57BL/6J mice prepared for CUS model were randomly divided into the following four groups: Ctrl+AAV8-eGFP,Ctrl+AAV8-MAD2B-OE-eGFP,CUS+AAV8-eGFP,CUS+AAV8-MAD2B-OE-eGFP.Similarly,C57BL/6J mice prepared for CORT model were randomly divided into the following four groups:Ctrl+AAV8-eGFP,Ctrl+AAV8-MAD2B-OE-eGFP,CORT +AAV8-eGFP,CORT+AAV8-MAD2B-OE-eGFP.The control and MAD2 B overexpression adeno-associated virus(AAV8)were injected into the hippocampus by stereotactic injection.After the virus expressed for three weeks,the mice were injected with corticosterone for three weeks or carried out CUS for five weeks.Before and after modelling,depressive behaviors such as "heditation" and "despair" were evaluated by sucrose preference test,forced swimming test and tail suspension test.And we used the fatigue rotating rod test to detect the locomotivity.The above results can be used to evaluate the effect of MAD2 B on depression-like behavior in mouse.4.Primary cultured hippocampal neurons were treated with CORT(100 μM)for 24 h.MAD2 B,glucocorticoid receptor(GR),metabolic glutamate receptor 4(GRM4),miR-29b-3p,etc were detected by Western blot and Q-RT-PCR.On this basis,MAD2 B was intervened using lentivirus to investigate the mechanisms.Results1.Mice treated with CORT and CUS treatment exhibited depression-like behaviors as i decreased sucrose preference in sucrose preference test and increased immobility in forced swimming test and tail suspension test,which suggested that the depression models were successfully established and could be used for follow-up studies.2.The expression of MAD2 B in the hippocampus were detected by Western Blot.The results showed that the levels of MAD2 B protein was lower in the hippocampus of mice treated with CORT and CUS compared with control mice.3.We injected control and MAD2 B overexpression adeno-associated virus into hippocampus of mice.After three weeks,sucrose preference test,forced swimming test and tail suspension test were performed to detect depression-like behaviors in mice.The results revealed that there was no difference in sucrose preference percent in each group,and there was no difference in the immobility time of forced swimming test and tail suspension test.It suggested that under normal conditions,MAD2 B did not affect the depressive behavior of mice.However,after 3 weeks,we constructed chronic depression models and the tested above behaviors.The results showed that the modeled mice had depression-like behaviors,but the overexpression of MAD2 B in the hippocampus could reverse the depression-like behaviors induced by CUS and CORT.These mice behaved as the increased preference for sucrose,deceased immobility time in the forced swimming test and tail suspension test.It reveals that overexpression of MAD2 B in mouse hippocampus can reverse the depression-like behavior caused by chronic depression modelling.4.It was showed that,there was no significant difference in GR expression after MAD2 B overexpression in depressive models,which suggested that GR did not play a key role in the antidepressant effect of MAD2 B.5.After chronic depression modelling,the expression of GRM4 in the hippocampus was up-regulated.Overexpression of MAD2 B in hippocampus could decrease the expression of GRM4.These results indicate that MAD2 B exerts antidepressant effect by down-regulating GRM4.6.In the primary cultured hippocampus neurons,the expression of MAD2 B was significantly down-regulated and the expression of GRM4 was up-regulated after treatment with CORT drugs compared with the control group.The expression of miR-29b-3p was up-regulated,GRM4 expression was down-regulated in hippocampus neurons transfected with MAD2 B overexpression lentivirus,which suggested that overexpression of MAD2 B reversed effects of CORT.Conclusion:Through the following methods such as animal modelling,behavioral test,brain stereotactic injection,viral transfection,and molecular biology experiments,we found that MAD2 B exerts its antidepressant effect by regulating miR-29b-3p/GRM4. |
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