Role And Mechanisms Of IL-6 Within Hippocampal CA1 Region In The Pathogenesis And Treatment Of Depression

Background:Depression is a chronic and recurrent neuropsychiatric disease,and its core symptoms are low mood,lack of pleasure and sluggishness.In severe cases,it is even accompanied by delusions and suicidal tendencies,but the treatment doesn't work well.The results of clinical and animal experiments show that the hippocampus,amygdala and other parts of depression patients are accompanied by neuroinflammatory reactions,but the mechanism is unclear.Recent studies had found that Interleukin-6(IL-6)was a pleiotropic cytokine that can regulate a variety of cellular functions.Its role in inflammation is two-way,and it has both inflammatory and anti-inflammatory effects.However,its specific role and mechanism in depression is still unclear.Therefore,this study constructs a rat depression model induced by chronic unpredictable mild stress(CUMS),we used behavioral,morphological,molecular biology and other experimental methods to investigate whether IL-6 in hippocampus CA1 region affects the occurrence of depression-like behavior in rats,and to study the role and mechanism of IL-6 in the pathogenesis and treatment of depression.Methods:(1)Male Wistar rats(5 to 6 weeks old,weighing 160 g to 180 g)were divided into a control group(non-stress stimulation group),a CUMS group,and an antidepressant fluoxetine-treated group(Fluoxetine+CUMS).The control group was the non-stress group;the CUMS group and the antidepressant fluoxetine treatment group(Fluoxetine+CUMS)were given chronic unpredictable mild stimulation for 6 weeks;The antidepressant fluoxetine-treated group was given intraperitoneal injection of fluoxetine(20mg/kg)30 minutes before the stress stimulus for six weeks.After six weeks,each group conducted behavioral tests,including sugar water preference test and forced swimming test,and the molecular biology and morphological examinationof the hippocampal CA1 region of each group of rats.(2)The expression levels of IL-6,Bcl-2,Bax,Caspase3,Caspase9 and other apoptotic factors were detected by RT-PCR.AAV-mRPP-GFP-LC3 were injected into the hippocampal CA1 region,then autophagy flow in hippocampal CA1 region of depression-like rats was detected by immunofluorescence.Besides,it also had detection of changes in expression levels of autophagy-related indicators such as LC3II/I,mTOR,p62 in the hippocampal CA1 region by Western Bolt.We observed autophagosome morphology by using electron microscopy.(3)The stereotactic injection of HBAAV2/9-r-IL-6 shRNAi-GFP(AAV-IL-6i)into the brain of the hippocampal CA1 region of normal rats to kockdown IL-6;Meanwhile the HBAAV2/9-r-IL-6-GFP(AAV-IL-6)were over-expressed by stereotactic injection,and the corresponding groups were injected with AAV-control.Two weeks after the virus infection,rats were tested for sugar water preference and forced swimming test.What's more,we detected the expression of IL-6 in hippocampal CA1 region,and related indexes such as oxidative stress,autophagy,neuronal apoptosis,inflammatory.Results:(1)After 6 weeks of CUMS exposure,the behavioral test results showed that compared with the control(non-stress stimulation group),the sugar water consumption of the CUMS group in the sugar water preference test was significantly reduced(P<0.05),suggesting that the rats had depression-like behavior with anhedonia.Resting time of the CUMS group increased significantly during the forced swimming experiment(p<0.05),suggesting that the rats had depressive behavior with behavioral despair.(2)RT-PCR results showed that compared with normal rats,the expression of IL-6 in hippocampal CA1 region of depression-like rats decreased,and the expression of IL-6 in hippocampal CA1 region of antidepressant drug fluoxetine-treated rats increased,and significantly improved depression-like behavior in rats.(3)Two weeks after injection of AAV-mRFP-GFP-LC3 into the hippocampal CA1 region of rats,immunofluorescence results showed that compared with control rats,autophagy in the hippocampal CA1 region of depression-like rats was expedite and significantly enhanced;The electron microscopyresults showed that compared with normal rats,autolysosomes in the hippocampal CA1 region of depression-like rats were clearly visible;Western Bolt results showed that compared with control rats,the autophagy pathway related molecules in the hippocampal CA1 region of depression-like rats significantly changed in expression.The results indicated that autophagy was increased in the hippocampal CA1 region of depression-like rats.RT-PCR results showed that the expression of anti-apoptotic factor Bcl-2 in the hippocampal CA1 region of depression-like rats decreased,but the expression of pro-apoptotic related factors increased significantly,indicating that neuronal apoptosis in depression-like rats increased.(4)After control rats were injected with HBAAV2/9-r-IL-6 shRNAi-GFP(AAV-IL-6i)in the hippocampal CA1 region,the expression level of IL-6 decreased,accompanied by anti-oxidative stress indicators decreased expression of Nrf2 and HO-1;the expression of LC3?/? increased,the expression of p-mTOR and p62 decreased;the expression of Bcl-2 decreased,the expression of Bax,Caspase3 and Caspase9 decreased;After injection of HBAAV2/9-r-IL-6-GFP(AAV-IL-6)in the hippocampal CA1 region of depression-like rats,the expression level of IL-6 increased,and the expression of Nrf2 and HO-1 increased;the expression of LC3?/? decreased,the expression of p-mTOR and p62 increased;the expression of Bcl-2 increased,but the expression of Bax,Caspase3 and Caspase9 increased;the expression of IL-1?,INF-? and TNF-? decreased,and significantly improved depression-like behavior in rats.Conclusions:(1)CUMS can induce depression-like behavior in rats effectively,and accompanied by a decrease in IL-6 expressive levels in hippocampal CA1 region,and fluoxetine can improve effectively;(2)CUMS can enhance oxidative stress response in hippocampal CA1 region,meanwhile the neuroinflammatory response,autophagy flow and neural apoptosis also increased significantly.(3)The knockdown of IL-6 in the normal rats hippocampal CA1 region leads to increased oxidation,autophagic flow,inflammation and apoptosis.(4)The overexpression of IL-6 in the depression-like rats hippocampal CA1 region can inhibit the oxidative stress enhancement caused by CUMS effectively,and reduce autophagy,inflammatory response and neuronal apoptosis,suggesting that exogenous administration of IL-6 can improve depression-like behavior by exerting a neuroprotective effect.