ZnR/GPR39 Protects Against CORT-induced Neuronal Injury And Its Mechanism In Hippocampal HT-22 Cells

BackgroundThe GPR39 receptor belongs to the class of rhodopsin-like receptor family and it is also classified as a member of the ghrelin family based on its amino acid sequence.It consists of 462 amino acid residues,with 7 transmembrane domains and expresses in all vertebrates.In 1997,Professor McKee first coloned GPR38 and GPR39 from the human fetal brain cDNA library.Initial reports suggest that these novel GPCRs are associated with ghrelin and neurotensin receptors.The GPR39 expresses in gastrointestinal tract,liver,pancreas,adipose tissue and,in the central nervous system it also has a wide range of expression,such as the amygdala,hippocampus,and these areas also are region enriched zinc,suggesting that the neurotransmitters role of zinc in central may be related to GPR39.Other studies have also shown that zinc release from glutamatergic terminals in the hippocampal CA3 region can activate postsynaptic GPR39 receptors and regulate mood and cognition.Recent studies confirm that zinc deficiency can lead to decrease the expression of GPR39,and the number of the GPR39 in prefrontal cortex reduced related to the immobility time in forced swimming test.More interestingly,such selective antidepressants,like escitalopram,reboxetine,bupropion can increase the expression of GPR39.More and more evidences suggest that GPR39 may be involved in antidepressant treatment,but the specific mechanism and its role in the occurrence and development of depression with zinc has not been elucidated.Depression is the affective disorder resulting from a multifactorial syndrome,the patients usually present with low self-esteem,immune system dysfunction,loss of appetite and accompanied by social barriers,work efficiency decline,psychological vulnerability and social life quality decreased.Depression is characterized by the change of mood and cognitive function and even generate the idea of death or suicide repeatedly.There are 200 thousand people with depression commit suicide in China every year,and have brought great pain not only to the patients themselves but also to their families.However,the etiology and pathogenesis of depression have not be fully elucidated yet.But it is certain that biological,psychological and social environment and many other factors involved in the pathogenesis of depression.Clinical studies find that depression is related to brain neurons and glial cells atrophy and apoptosis.Experimental studies have shown that stress break down the balance between neuronal damage and neuronal regeneration,which induced to the CA3 pyramidal neurons atrophy and hippocampal volume decrese and a series of hippocampal structural and functional changes which are closely related to the occurrence of depression.The hippocampus is the most abundant area of zinc in the central nervous system,and is closely related to learning and memory,behavior and emotion.Previous studies have shown that stress induce hippocampal zinc homeostasis disorder and can lead the animals to present the depressive behavior.And there is a significant decrease of GPR39 mRNA expression in hippocampus of rats with depression like behavior.Studies have shown that GPR39 gene knockout mice present the same depressive behavior in the forced swimming test and the tail suspension test and its hippocampal CREB and BDNF decrease.After treatment of the antidepressant,the expression of BDNF upregulate which may be related to the increase of the zinc receptor GPR39.Research shows that CREB has a variety of functions in the nervous system,in addition,CREB is the transcription factor of brain-derived neurotrophic factor(BDNF).CREB-BDNF plays an important role in the development of nerve growth,and closely relates to the nervous and emotional disorders such as anxiety and depression.BDNF distributs in the central nervous system widely,which plays an important role in the growth and development of neurons.We hypothesize that the signaling pathway of the CREB-BDNF induced by GPR39 may be one of the neuroprotective mechanisms of GPR39.In summary,we find that chronic stress can lead to the zinc homeostasis unbalanced that induce the increasing apoptosis of hippocampal neurons which plays an important role in the development of depression.And the expression of hippocampus GPR39 mRNA in mice decrease which present the depression-like behavior.Studies have further found that zinc deficiency diet can lead to a decrease expression of GPR39.So stress lead to hippocampus zinc decline is the material basis of he depression-like behavioral induced by stress,and stress cause brain hippocampal nerve cell damage may be the pathological basis of depression,we speculate that zinc can activate the GPR39,thereby activate the CREB-BDNF signaling pathway which are closely related to the nerve growth and development and inhibit apoptosis to perform neuroprotective effect.ObjectiveIn this study,we observe the protective effects of GPR39 on the injury of primary cultured hippocampal neuronal cell induced by corticosterone and at the same time,we test its possible mechanism.These findings suggest a potential neuroprotective action of GPR39 in prevention of neural damage and provide certain experiment basis for clinical treatment.Experimental Methods1.Cell cultureHippocampal neuronal cell line,HT22,a subline derive from parent HT4 cells that were originally immortalized from primary mouse hippocampal neuronal culture,were the generous gift of Dr.David Schubert(Salk Institute,SanDiego,CA,USA)Cells were bring up in DMEM media supplemented with 10% fetal bovine serum,and penicillin/streptomycin in monolayers in 100 mm Greiner tissue culture dishesat standard cell culture conditions(5% CO2,95% air).Media were changed three times weekly and then cultured at a confluence with every 3–5 days.Cells were observed with a phase-contrast microscope.2.Cell interventionFor cell growth condition is good,discard the above mentioned culture solution,and put the cells in batches to six orifices adding culture medium to continue culture 24 hours.Using 50?? concentration of Zinc sulfate for different time(0h? 3h?6h?12h? 24h)and using different concentrations of zinc sulfate(500nM?1???10???50?M)intervened HT-22 cells for 6h.Zinc sulfate was used to treat HT-22 cells for 30 min,and 10 ?M TPEN and DTPA were used to treat HT-22 cells for 6 h.The CORT and TCG-1008 intervened HT-22 cells as the same way.After the intervention,collect cells for later use.3.Cell transfecationHT-22 cells were inoculated with the platelets.When the degree of fusion was 80%,the GPR39 interfering RNA and the transfection reagent lipofctamine2000 were diluted with DMEM and then incubated at 37? and 5% CO2 20 min.Each well was preliminarily added with serum-free medium containing the antibiotic and the above mixed solution was added 6-8h after the liquid,cultured to 24 h after the follow-up experiments and collection of cell detection.4.Cell viability assayHT-22 cell activity was measured using CCK-8 kit.5.Mitochondrial membrane potential analysisThe changes of mitochondrial membrane potential in HT-22 cells were detected by JC-1 kit.6.Annexin V-FITC/PI double staining assayApoptosis of HT-22 cells was detected by Annexin V-FITC Apoptosis Detection Kit.7.Western Blotting analysisProtein heat denaturation was carried out for 10 min with the total protein of HT-22 cells extracted with RIPA lysate.SDS polyacrylamide gel electrophoresis 2h,transfer membrane 1.5h,5% milk closed 2h,incubation of primary antibody overnight,rinse 3 times each time 10 min,secondary antibody incubation 2h,rinse 3 times each time after 10 min,ImageJ image analysis software for gray value analysis.8.Real Time PCRThe total RNA of HT-22 cells was extracted with Trizol and quantified quantitatively.RNA was reverse transcribed into cDNA by reverse transcription kit.Using PBS as a template,it was mixed with primers,SYBRgreen and water and then amplified on a StepOne Plus real-time PCR instrument.According to the RQ value to 18 s as the reference.9.Statistical analysisStatistical analysis was recommended using SPSS16.0.Two independent samples were used t-test and one-way ANVOA.The SNK-q and Dunnett's test was used to compare the two groups.Results with P <0.05 as the significance level,P <0.01 was very significant level.Results1.The effect of zinc on the expression of GPR39 in HT-22 cellsThe experimental results showed that 50?? zinc sulfate treatment HT-22 cell at different time,the expression of GPR39 in 3h group and 6h group were significantly higher than the control group(P<0.05);different concentrations of zinc sulfate intervention HT-22 cell for 6h,compared with the control,the expression of GPR39 in 50?? group was significantly increase(P<0.05);give zinc chelating agent TPEN and DTPA to HT-22 cells,in TPEN group and DTPA group,the expression of GPR39 protein was decreased,(P<0.01),while the Zn2++TPEN and Zn2++DTPA groups GPR39 expression was significantly restored.2.The effect of CORT treated on the viability of HT-22 cellsThe results showed that compared with the control,different concentration of CORT treated in HT-22 cells with 6h,the 500 nM group cell viability shows no significant difference,1???10?? and 50?? groups cell viability was significantly decreased(P<0.05);500nM CORT treated HT-22 cells with different time,compared with the control group,cell viability shows no significant difference between the 6h group,the 12 h and 24 h groups' cell viability decreased significantly(P<0.01).3.Effects of interfering and activating GPR39 on the activity of corticosterone-treated HT-22 cellsThe experimental results showed that compared with the group of control,the SiRNA group cell viability decreased significantly(P<0.01);compared with 500 nM CORT group,SiRNA+500nM CORT group,cell viability decreased significantly(P<0.05).Compared with control,the cell viability of SiRNA group and 1?? CORT group decreased significantly(P<0.01);compared with the group of SiRNA,the cell viability of SiRNA+TCG-1008 group increased(P<0.05);compared with the 1?? CORT group,the cell viability of the 1?? CORT+TCG-1008 group was significantly increased(P<0.01)4.Effects of silencing and activating GPR39 on the apoptosis of corticosterone-treated HT-22 cellsThe experimental results showed that compared with the control,the 500 nM CORT group and TCG-1008 group cell apoptosis rate had no significant change,and cell apoptosis rate of the SiRNA group and 1?? CORT group increased significantly(P<0.001);compared with 500 nM CORT group,apoptotic cells in SiRNA+500nM CORT group were significantly incresed(P<0.001);compared with 1?? CORT,the group of 1??CORT +TCG-1008 apoptosis rate was improved(P<0.001).5.Effects of silencing and activating GPR39 on the mitochondrial membrane potential of corticosterone-treated HT-22 cellsThe experimental results showed that compared with the control group,the 500 nM CORT and TCG-1008 groups mitochondrial membrane potential had no obvious change,mitochondrial membrane potential of the SiRNA group and the 1?? CORT group was significantly decreased(P<0.001);compared with 500 nM CORT group,mitochondria membrane potential of SiRNA+500nM CORT group decreased significantly(P<0.001);compared with the 1?? CORT group,the mitochondrial membrane potential of the 1?? CORT +TCG-1008 group was improved significantly(P<0.001).6.Effects of zinc sulfate on expression of CREB and BDNF in HT-22 CellsCompared with the control,50?M zinc sulfate could promote the expression of CREB and BDNF mRNA and protein(P <0.05),and the expression of CREB and BDNF mRNA and protein were significantly decreased in 200?M zinc sulfate group(P <0.05).7.The effect of corticosterone on GPR39 and downstream CREB-BDNF signaling pathway in HT-22 cellsThe results showed that the different concentration of CORT treated with HT-22 cells 6 h,the expression of GPR39 in the 500 nM CORT group significantly increased,and in the 1??,25?? and 50?? CORT groups,GPR39 expression decreased significantly;500nM CORT intervented 6h can promote the expression of GPR39 better,while CREB and BDNF expression changes consistent with GPR39.8.Effects of silencing and activating GPR39 on expression of CREB-BDNF signaling pathwayThe experimental results showed that the expression of CREB and BDNF in the SiRNA group were significantly reduced,and the CREB,BDNF protein and mRNA levels were significantly increased by 0.5?? TCG-1008 treated with HT-22 cells.9.Effects of silencing and activating GPR39 on apoptosis signal pathwayThe results show that compared with the control,in the 500 nM CORT group,Caspase3,Caspase9,AIF and BAX mRNA had no obvious change,and Bcl-2 mRNA increased significantly(P<0.01);compared with 500 nM CORT group,in the Si+500nM CORT group,the expression of Caspase3,Caspase9 AIF and BAX mRNA was significantly increased,but Bcl-2 mRNA expression was significantly decreased.Compared with 1?? CORT group,the expression of Caspase3,Caspase9,AIF and BAX in the group of 1?? CORT +TCG-1008 was significantly lower,Bcl-2 mRNA expression was significantly increased(P <0.01).ConclusionThis study is the first time to systematic research about the neuroprotection effect of the znic sensing receptor-GPR39 in the hippocampal neural HT-22 cells injuried by corticostercone and its possible mechanism.Through the stimulation of CORT on HT-22 cells,observe the effects of stress on cell viability and mitochondrial function etc.;and the role of interfering and activating GPR39 in cellular stress,in order to study the protection effect of GPR39 on HT-22 cell in condition of cell stress and further explore its mechanism.The main conclusions are as follows:1.As a ligand of GPR39,zinc can activate GPR39 and promote its expression.2.GPR39 has protective effect on HT-22 cell injury induced by corticosterone.3.GPR39 may play a neuroprotective role by promoting CREB-BDNF expression and inhibiting the expression of pro-apoptotic proteins BAX,Caspase3,Caspase9 and AIF,up-regulating the expression of anti-apoptotic protein Bcl-2.