| BackgroundDepression is one of the most common mental illnesses in the world.There are 350million patients over the worldwide,and their lifetime prevalence is 4.4%to 20%.It has adverse effects on patients’physical and mental health,and also causes heavy burden to the family and society.Current methods for treating depression have limited efficacy with a cure rate of less than 50%.About 40%of patients with depression have no response to treatment,and also associated with greater side effects.Studies on the pathogenesis of depression have repeatedly proposed various hypotheses such as neurotransmitter and receptor dysfunction,neuroplastic changes,neuroendocrine dysfunction,signal transduction abnormalities,inflammation and neuroinflammation hypothesis,and neurotrophy,but the specific mechanism is still unclear.A lot of evidence shows that genetic and environmental factors play an important role in the development of depression,and stress is one of the important environmental risk factors leading to the development of depression.Inflammation is the basis of a variety of pathophysiology.In the face of noxious stimuli and various stresses,the inflammatory response protects the body,but this process must be strictly controlled and terminated to ensure steady home remodeling.When inflammatory cells are continuously over-activated,they cause a series of diseases,so the inflammatory process is considered to be a key factor in the occurrence and progression of the disease.Neuroinflammation is the immune response of the central nervous system（CNS）to stress and noxious stimuli,but persistent excessive neuroinflammation can also lead to neurodegenerative diseases.In short,inflammation is a normal immune response to stress,but persistent stress can impair the activation of surrounding macrophages,damage to central microglia and neural networks,and dysfunction.The study found that the levels of tumor necrosis factor alpha（TNF-α）and interleukin-1β（IL-1β）in the serum of depressed patients and depressed animals were significantly higher than those in the normal group,and the proinflammatory cytokines in the blood and brain of depressive animals were elevated.NF-κB is the most important regulator of the inflammatory pathway.It has been found that activation of the NF-κB pathway plays an important role in the prefrontal cortical inflammatory response induced by chronic unpredictable stress（CUMS）in rats.The expression of major inflammatory cytokines TNF-α,IL-1βand inducible nitric oxide synthase（iNOS）in the CNS of CUMS mice was significantly higher.In short,sustained stress can lead to an increase in inflammatory response,which in turn aggravates the occurrence of various nervous system-related diseases.G protein-coupled receptor 39（GPR39）is a rhodopsin-like G protein-coupled receptor.GPR39 is widely expressed in the body and is also widely expressed in the central nervous system such as amygdala and hippocampus.GPR39 is mediated by its ligand zinc to mediate different signal transduction pathways,resulting in cAMP response element（CRE）transcription,cAMP response element binding protein（CREB）and Brain derived neurotrophic factor（BDNF）expression.BDNF protects neurons and synapses and produces antidepressant effects.The study found that GPR39 knockout mice showed depression-like behavior and down-regulated CREB and BDNF expression in the hippocampus.The previous results of the research group also showed that stress can lead to a decrease in the expression of GPR39-CREB-BDNF.However,how stress causes down-regulation of GPR39 expression has not been reported.Previous research by the research team found that zinc can inhibit the NF-κB signaling pathway,reduce the level of neuroinflammation,and resist cell damage,such exerting neuroprotective effects.On the other hand,zinc can also activate the GPR39-CREB-BDNF signaling Neuroprotective function.The above findings suggest a possible link between neuroinflammation and GPR39.Later,we found a link between NF-κB activation and GPR39 down-regulation through pre-experimental experiments,but its regulatory mechanism has not reported.NF-κB and GPR39 do not have direct binding sites,so NF-κB does not directly regulate GPR39.What is the mechanism?Because NF-κB activation is inversely related to GPR39 down-regulation,and miRNA is an important negative regulatory molecule in vivo and has strong regulatory functions,we suspect that NF-κB may regulate GPR39expression through miRNA.Studies have found that miRNAs are widely involved in the regulation of many systems,including the nervous system,and play an important role in neurogenesis and neuronal development and plasticity.Studies have shown that some miRNAs are involved in the pathogenesis of depression by regulating multiple signaling pathways.Through bioinformatics prediction,we found that miR-182 has binding sites with both NF-κB and GPR39.In addition,clinical studies have also found that the expression of miR-182 in serum of patients with depression is elevated;inhibition of miR-182 expression in animal experiments has an antidepressant effect on CUMS-induced depression mice.The above findings suggest that miR-182 may be associated with depression.It is related to the occurrence and development,so we speculate that the neuroinflammatory signaling pathway may inhibit the expression of GPR39 through miR-182,and then participate in the development of depression.In summary,stress activation of the neuroinflammatory signaling pathway and inhibition of the GPR39 pathway are important factors in the pathogenesis of depression,so explore the relationship between neuroinflammation and GPR39 and whether neuroinflammation regulates the expression of GPR39 via miR-182.The role of chronic stress-induced depression is of great significance for the study of the pathogenesis of depression,and also provides a new experimental basis for the prevention and treatment of depression.ObjectiveBy establishing a chronic stress-induced depression animal model and a cell stress model,we observed the regulation and mechanism of stress-induced neuroinflammatory signaling pathway and GPR39,and further observed the role and mechanism of miR-182,in order to elucidate the mechanism of chronic stress-induced depression and provide experimental evidence.Experimental Methods1.Establishment of a mouse model of chronic stress-induced depressionMale C57 mice,4 weeks old,were randomly divided into 3 groups after 1 week of adaptive feeding,which were control group,CUMS group and chronic restraint stress group（CRS）.After the sugar water has been adapted,stress begins.（1）The CUMS group randomly used a method of stress every day at an unfixed time.There were 8 stress methods,namely,2h restraint,10min shaking,24h of diet deprivation,45°tilt of rat cage,12h,and 12h of litter.4°C ice water swimming for 5 min,day and night upside down and 45°C heat stress for 5 min.The behavioral index was measured after 6 weeks of stress.（2）The CRS group was placed in a plastic tube that could not move autonomously for 2 h every day from 16:00 to 18:00.The behavioral index was measured after 9 weeks of stress.2.Behavioral indicators testing（1）Sucrose preference testA sugar water preference experiment was conducted every 7 days.Each mouse was individually placed in a squirrel cage,and one bottle of pure water and one bottle of 1%syrup were placed in each cage to measure the amount of water consumed within 2 hours.Sugar water preference score（%）=sugar water consumption/total liquid consumption×100.（2）Tail suspension experimentThe tape was fixed to the gravity sensor of the test box at a distance of 1 cm from the tail end of the mouse,and the value of the immobile state of one mouse was set as a threshold.The immobility time of the mice within 6 min was recorded,and only the values of 5 min after the analysis were taken for analysis.3.Cell culture and interventionMouse hippocampal HT-22 cells were cultured in a 10%fetal bovine serum high glucose medium in a 37℃,5%CO2 incubator,and then treated with different concentrations（0M,500nM,1μM,10μM）CORT.HT-22 cells were treated with cells for6h or with NF-κB inhibitor SN50（final concentration of 18μM）for 24h.4.Cell transfectionThe HT-22 cells were seeded in the culture plate,and when the degree of fusion was80%,transfection was started.MiR-182 mimics or inhibitors and lipo2000 were diluted and mixed with Opti-MEM,respectively,and allowed to stand at room temperature for 5min,then the two liquids were gently mixed and allowed to stand at room temperature for 20 min.Finally,the mixture is added to the wells containing the cells and the culture solution.4-6h for liquid change,48h for the next experiment.5.Serum corticosterone concentration testThe corticosterone concentration in the serum of the mice was measured using a neuropeptide radioimmunoassay kit.6.Inflammatory factor concentration detectionThe concentration of TNF-αand IL-6 in mouse serum and cell supernatant was measured using an ELISA kit.7.Western-BlottingThe total protein of hippocampus tissue and cells was extracted with RIPA lysate,mixed with 5×loading buffer,and denatured at 100°C for 10 min to become a protein sample.Protein samples were added,electrophoresed for 1.5h,transferred for 90 min,5%skim milk was blocked for 2h,primary antibody was incubated overnight,then washed 4 times with TBST for 5min each time,fluorescent secondary antibody was added,incubated for about 1h,and developed with ODYSSEY instrument perform gray value analysis.8.Real-time quantitative fluorescence PCRRNA was extracted from hippocampus tissues and cells using Trizol,and the concentration of RNA was measured using a microplate reader to obtain the volume of RNA to be added during reverse transcription,and cDNA was obtained after reverse transcription.The cDNA was mixed with primers,DEPC water,and SYBR green as a fluorescence quantitative system,amplified on a StepOne Plus instrument,and statistically analyzed based on the Ct value.9.Statistical methodsData analysis was performed using SPSS 16.0,graphs were drawn using graphpad,and data was collated using Excel 2007.Comparisons between groups were performed using t-test and one-way analysis of variance,and comparisons between groups were performed using Dunnett’s.When P<0.05,the difference was statistically significant.Results1.Multiple chronic stress leads to depression-like behavior and neuroinflammation in mice（1）Effects of CUMS and CRS on body weight of miceThe results showed that the body weight of the mice in the CUMS group and the CRS group was significantly lower than that in the control group（P<0.001;P<0.01）.（2）Effects of CUMS and CRS on serum corticosterone concentrations in miceThe results showed that the concentrations of corticosterone in the serum of the CUMS group and the CRS group were higher than those of the control group（P<0.01）.（3）Effects of CUMS and CRS on mouse behaviorThe results showed that compared with the control group,the sucrose preference scores of the CUMS group and the CRS group decreased（P<0.05;P<0.001）;the hangover time of the CUMS group and the CRS group prolonged（P<0.01;P<0.001）.（4）Effects of CUMS and CRS on the GPR39 pathway in mouse hippocampusAfter the end of stress in mice,hippocampus was taken and the expression of GPR39,CREB and BDNF was detected by Q-PCR and Western Blotting.The results showed that compared with the control group,the expression of GPR39（P<0.05;P<0.01）,CREB（P<0.05;P<0.01）and BDNF（P<0.01;P<0.05）decreased in hippocampus of CUMS and CRS mice..（5）Effects of CUMS and CRS on the concentration of NF-κB and serum TNF-αand IL-6 in hippocampus of miceAfter the end of stress in mice,hippocampus tissue and serum were taken,and the changes of NF-κB,TNF-αand IL-6 were detected by Western Blotting and ELISA.The results showed that compared with the control group,the expression of NF-κB in hippocampus of CUMS and CRS mice was increased（P<0.01;P<0.001）,and TNF-αin serum of CUMS group and CRS group（P<0.05;P<0.01）and IL-6（P<0.001;P<0.001）increased in concentration.2.The role of NF-κB activation in stress-induced decreased expression of GPR39 in hippocampal HT-22 cells（1）Effects of CORT on NF-κB,TNF-αand IL-6 in hippocampal HT-22 cellsHT-22 cells were treated with different concentrations of CORT（500nM,1μM,10μM）for 6h.The results showed that the expression of NF-κB was increased in the 500nM group,1μM and 10μM groups compared with the control group（P<0.05;P<0.001;P<0.001）;different concentrations of CORT intervention in HT-22 cells increased TNF-αand IL-6 concentrations（P<0.01;P<0.001）.（2）Effect of CORT on the expression of GPR39 in hippocampal HT-22 cellsThe results showed that there was no significant difference in the expression of GPR39 in the 500nM group compared with the control group,and the expression of GPR39 in the 1μM group and the 10μM group decreased（P<0.05;P<0.001）.（3）Effects of inhibition and overexpression of NF-κB on the expression of GPR39 in hippocampal HT-22 cellsThe results showed that compared with the control group,the expression of GPR39was increased after NF-κB inhibition（P<0.05）,while the expression of GPR39 was decreased after overexpression of NF-κB（P<0.001）.（4）Inhibition of NF-κB on the down-regulation of GPR39 expression in HT-22cells by CORTThe results showed that compared with the control group,the expression of NF-κB was increased in the 10μMCORT group（P<0.001）,and the expression of GPR39 was decreased（P<0.05）.The expression of NF-κB was decreased in the SN50 group（P<0.05）,and the expression of GPR39 was increased（P<0.05）.;Compared with the10μM CORT group,NF-κB expression was decreased in the SN50+CORT group（P<0.001）,and GPR39 expression was increased（P<0.001）.3.The role of miR-182 in the negative regulation of NF-κB in the expression of GPR39（1）Bioinformatics predicts that miR-182 has binding sites with NF-κB and GPR39.All miRNAs downstream of NF-κB and upstream of GPR39 were predicted by Target Scan software.After the intersection of the two,we found that miR-182 has a binding site with both.（2）Changes in miR-182 expression in animal models and cell modelsThe results showed that the expression of miR-182 in hippocampus of CUMS group and CRS group was higher than that of the control group（P<0.01;P<0.001）.The results of cell experiments showed that the expression of miR-182 was increased in the10μM CORT group（P<0.05）.（3）Effects of inhibition and overexpression of NF-κB on the expression of miR-182 in HT-22 cellsAfter inhibition of NF-κB,Q-PCR results showed that the expression of miR-182was increased in the CORT group compared with the control group（P<0.01）;the expression of miR-182 was decreased in the SN50 group（P<0.01）;compared with the CORT group The expression of miR-182 was decreased in the SN50+CORT group（P<0.001）.After overexpression of NF-κB,the results showed that the expression of miR-182 was elevated in the OE group compared with the control group（P<0.01）.（4）Effect of inhibition and overexpression of miR-182 on the expression of GPR39 in HT-22 cellsHT-22 cells were treated with different concentrations of miR-182 inhibitors（50nM,100nM）for 24h.The results showed that inhibition of miR-182 expression promoted the expression of GPR39 protein（P<0.001;P<0.001）.Treatment of HT-22 cells with different concentrations of miR-182 mimics（10nM,50nM,100nM,200nM）for 24h showed that promotion of miR-182 expression inhibited the expression of GPR39 protein（P<0.001）.（5）Effect of miR-182 on the expression of GPR39 after overexpression of NF-κB in HT-22 cellsThe cells were divided into 4 groups:（1）control group（CON）;（2）NF-κB overexpression plasmid group（OE）;（3）miR-182 inhibitors group;（4）NF-κB overexpression plasmid+miR-182 inhibitors group（OE+inhibitor）,observed changes in NF-κB,miR-182 and GPR39 expression.The results of Q-PCR and Western Blotting showed that there was no significant change in NF-κB in miR-182 inhibitor group,miR-182 expression was decreased（P<0.05）,and GPR39 protein expression was increased（P<0.01）.Compared with miR-182 inhibitors group,NF-κB expression was increased in OE+miR-182 inhibitors group（P<0.05）,and there was no significant change in miR-182 and GPR39 expression.（6）Effect of overexpression of miR-182 on the expression of GPR39 after NF-κB inhibition by HT-22 cellsThe cells were divided into 4 groups:（1）control group（CON）;（2）NF-κB inhibitor group（SN50）;（3）miR-182 mimics group;（4）SN50+miR-182 mimics group（S+M）,changes in the expression of NF-κB,miR-182 and GPR39 were observed.The results of Q-PCR and Western Blotting showed that compared with the control group,the expression of NF-κB and miR-182 was decreased in SN50 group（P<0.001;P<0.05）,and the expression of GPR39 protein was increased（P<0.05）;miR-The expression of miR-182 was increased in the 182 mimics group（P<0.001）,and the expression of GPR39 protein was decreased（P<0.001）.Compared with miR-182 mimics group,there was no significant difference in NF-κB,miR-182 and GPR39 expression between SN50+miR-182 mimics group.ConclusionIn this study,two chronic stress-induced depression models and CORT-treated cells were used to observe changes in neuroinflammation,GPR39 and miR-182;the relationship between GPR39 and GPR39 was observed by inhibiting and over-expressing NF-κB,and on this basis Further explore the role of miR-182 in down-regulating GPR39in inflammation.The main conclusions are as follows:First,depression-like behavioral changes caused by chronic stress may be related to the occurrence of neuroinflammation caused by chronic stress and inhibition of GPR39-CREB-BDNF signaling pathway.Second,stress-induced neuroinflammation may up-regulate miR-182 by activating NF-κB signaling pathway,thereby inhibiting GPR39-CREB-BDNF signaling pathway. |
