The Role And Mechanism Of Cx36 In HMGB1 Mediating Depressive-like Behaviors Induced By Chronic Stress

Depressive disorder is a common mental illness characterized by significant low mood and loss of interest.It is often accompanied by several non-specific symptoms,such as insomnia,pain,and fatigue.In severe cases,self-injury or even suicidal behavior may occur.In China,the latest nationwide mental illness epidemiological survey shows that the incidence of mental illness is getting higher and higher,and the lifetime prevalence of mood disorders ranks second,as high as 7.4%,of which more than 90% are depressive disorders.Although most patients can get remission within one year,there’s a high probability of recurrence,and a considerable number of patients will progress to the chronic stage.Depressive symptoms may last a lifetime,thus seriously impairing the psychosocial function of patients and lowering their quality of life.With the advent of new antidepressants,the clinical treatment has been significantly improved,and the side effects have been also reduced.However,the current antidepressant drugs still have many limitations,such as slow and limited effects.The clinical diagnosis is largely dependent on patients’ complaints and clinicians’ experiences without objective and effective indicators.The diagnosis and treatment of depressive disorder are still faced with great challenges.Therefore,it is of great value and importance to clarify the mechanism of depressive disorder and search for standard biomarkers for diagnosis,as well as provide rapid and effective targeted treatment.So far,there are many hypotheses about the pathogenesis of depressive disorder,such as monoamine,inflammation,HPA-axis change,neuroplasticity and so on.However,these hypotheses still cannot clarify the specific molecular mechanisms underlying the development of depressive disorder.In recent years,the hypothesis that connexins(Cxs)dysfunction leads to depressive disorder has attracted much attention,which is likely to be the molecular basis of the above hypotheses,and plays a vital role in the development of depressive disorder.Cxs are key structures of intercellular communications,which are abundantly expressed in the central nervous system(CNS)and exists in almost all types of cells,such as neurons,microglia,astrocytes,oligodendrocytes and so on.However,the phenotypes of Cxs differ in different cells.Cxs can also form two functional forms,namely gap junctions(GJ)and hemichannls(HC).Under normal circumstances,Cxs participate in both embryonic development and neural functioning processes.The abnormal expression or function of Cxs may lead to many neurological diseases,such as epilepsy,Alzheimer’s disease(AD),Parkinson’s disease(PD),CNS inflammation and tumors.Studies have reported that knockdown of Cxs in the hippocampus of mice could play antidepressant and anti-anxious roles in the tail suspension test(TST)and the novelty suppression feeding test(NSFT)in a model of acute stress.Injection of CBX,a nonspecific inhibitor of connexins and pannexins,into the hippocampus of mice also showed similar effects.Therefore,these results suggest that Cxs may play an important role in depressive disorder.Our lab found in previous studies that the high mobility group protein B1(HMGB1)released by hippocampal neurons and microglia was involved in depressive-like behaviors induced by chronic stress.Studies also demonstrated that the release of HMGB1 can be regulated by Cxs.Furthermore,Cx36 is co-expressed in hippocampal neurons and microglia.Therefore,we focused mainly on the role and mechanism of Cx36 in HMGB1-mediated depressive-like behaviors.In our study,we first established a chronic unpredictable mild stress(CUMS)model,and four weeks later,behavioral tests like sucrose preference test(SPT),tail suspension test(TST),forced-swimming test(FST)and open field test(OFT)were performed to assess depressive-like behaviors in order to see whether the model was successful.In addition,mice were also injected with HMGB1 inhibitor-glycyrrhizic acid(GZA,30 mg/kg),Cx36 specific inhibitors-mefloquine(MQ,15 mg/kg)and quinine(Q,50 mg/kg)respectively to see their effects on depressive-like behaviors and study the downstream mechanisms.Western-Blot analysis was used to detect the expression of Cx36 protein in the hippocampus and prefrontal cortex(PFC)of mice in different groups.Then,immunofluorescence staining was used to label Cx36 with neurons,astrocytes and microglia in the hippocampus to determine the cellular location and expression of Cx36.In order to further study the release of inflammatory mediators in CUMS mice treated with different drugs,we adopted enzymelinked immunosorbent assay(ELISA)to determine the levels of HMGB1,TNF-α and IL-1βin the hippocampus and serum.In order to further observe the change of neuronal excitability,we used whole-cell patch-clamp to record the action potentials and inward currents of hippocampal neurons.During electrophysiological experiments,LPS(10ug/ml)was applied to model stressful in-vivo environment.Mefloquine(50μM),quinine(200μM)and GZA(50μM)were used to observe their effects on the changes of neuronal excitability after stress.The results showed that CUMS model was successfully established after four weeks.CUMS procedures could induce obvious depressive-like behaviors,which were manifested by decreased sucrose preference percentage,prolonged immobility duration in tail suspension test and forced swimming test.After treatment with GZA,mefloquine or quinine,the depressive-like behaviors of mice were all significantly improved.Thereafter,WB results showed that the expression of Cx36 in the hippocampus of CUMS mice was significantly increased,while administration of GZA could significantly reduce Cx36 expression.The immunofluorescence staining further confirmed that Cx36 was mainly expressed in hippocampal neurons and microglia,while it was hardly expressed in astrocytes.Under chronic stress,neurons expressing Cx36 increased significantly,while GZA could reduce them.The above results suggested that GZA might reduce the expression of Cx36 through inhibition of HMGB1.At the same time,ELISA results also showed the levels of HMGB1,TNF-α and IL-1β in hippocampus and serum were significantly increased in the CUMS group.Mefloquine and quinine could reduce the them,which suggested Cx36 might be involved in the regulation of inflammatory cytokines after exposure to chronic stress.Electrophysiological results showed that LPS could increase the amplitude and frequency of action potentials in hippocampal neurons,decrease the threshold potenial and rheobase current,as well as increase the inward current significantly,suggesting that the excitability of hippocampal neurons was increased.After application of GZA,mefloquine or quinine,the neuronal excitability was reduced.In summary,Cx36 in hippocampal neurons might play a key role in HMGB1-mediated depressive-like behaviors induced by chronic stress,which will provide a new target for clinical intervention in depression.