| Objective:To investigate whether the changes of 5-HT level,estrogen receptor beta and tryptophan,hydroxylase-2 in Ahil KO and stress model mice brains,and further explore the mechanisms in the lack of Ahil protein reducing 5-HT level and causing depressive behavior.Method:The study included three parts:First,preliminary study of Ahil regulation of 5-HT expression and depression-like behavior in mice;Second,the mechanisms of Ahil regulation of 5-HT expression and depression-like behavior in mice;Third,effects of ERβ agonist on depressive phenotype in Ahil KO mice;(1)Spatial restraint stress method was used to establish a mouse model of depression.The male ICR mice were randomly divided into two groups,control group and stress group.The stress group,mice were individually and gently placed into a modified,well-ventilated 50ml centrifuge tube daily for 2 hours(from 9 A.M)every day for 30 days,and mouse movements were restricted and were unable to move forward or backward.Control mice remained undisturbed in their original cages without water and food as stress mice.After stress,mice were returned to their original cages.The depressive phenotype in Spatial restraint stress model mice were further investigated by tail suspension test,forced swimming test and the basic state of mice.For Ahil knockout mice,we chose 8 weeks Ahil transgenic mice.The study found that Ahil KO mice have obvious depression,and in the classic experiments of depression-like behavior such as the tail suspension test(TST),forced swimming test(FST),mice showed significantly reduced activity.Therefore,the Ahil gene knockout(Ahil KO)animal model can be used to study the depression-like phenotype in mice.Determine the expression of ERβ,TPH2,ERα,SERT by Western blot.We also checked the expression levels of 5-HT in mice brains by ELISA.The expression of ERβ and TPH2 mRNA were detected by using Quantitative polymerase chain reaction(Q-PCR).Detect the expression of ERβ protein in mice brains by using immunofluorescence staining.The expression of ERβ in nucleus and cytoplasm was detected by Nuclear and Cytoplasmic Extraction Kit.Western blot and immunofluorescence staining test.(2)Cells were transfected with Ahil siRNA,and the cells were divided into control siRNA group and Ahil siRNA group.The expression of ERβ and GR in nucleus and cytoplasm was detected by Nuclear and Cytoplasmic Extraction Kit.Western blot.Quantitative polymerase chain reaction was used to determine the contents of 50HT in the two groups.PC 12 cells were treated with Dexamethasone(DEX),which were divided into control group and DEX group.Detect the protein expression of ERβ and GR in nucleus and cytoplasm by Nuclear and Cytoplasmic Extraction Kit,Western blot in control group and DEX group.Detect the expression of ERβ mRNA by quantitative polymerase chain reaction in control group and DEX group.The GR siRNA was transfected into PC 12 cells,and cells were divided into control group,GR siRNA group.Determine the protein expression of determination of GR,ERβ by Western blot in the two groups;detect the expression of ERβ mRNA by quantitative polymerase chain reaction(Q-PCR)in the two groups.PGL3-basic-ERβ plasmid was transfected into 293T cells which were treated DEX and GR siRNA.After treatment,the transfection and dual luciferase reporter assay were used to detect the expression of PGL3-basic-ERβ-promoter plasmid.(3)Ahil mice were randomly divided into AhilHet+Vehicle group,AhilHet+ ER beta agonist group,AhilKO+Vehicle group,and AhilKO+ ER beta agonist group.ER beta agonist(LY-500307)with a dose of 0.2mg/kg,subcutaneous injection every day,Vehicle group was given an equal amount of solvent.After continuous treatment 3 weeks,detect the behavior of each group mice.Western blot was used to determine the protein expression of TPH2;the contents of 5-HT was assay’ed by ELISA.Results:After being modeled,tail suspension test and forced swimming test showed that the immobility time of Stress group mice were significantly increased than the control group(P<0.05).The above behavioral tests showed that the Stress mice had significant depressive symptoms.Compared with the control group,the expression levels of ERβ and TPH2,and the ERβ and TPH2 mRNA levels significantly reduced in the Stress mice’s hippocampus,the differences were statistically significant.The expression levels of ERβ and TPH2,and the ERβ and TPH2 mRNA levels in Ahil KO mice’s hippocampus was significantly reduced than the control group(P<0.05).However,compared with the control mice,the expression levels of SERT and ERa had no significant difference,while the expression levels of 5-HT in Ahi1KO mice’s hippocampus was significantly lower than that in the control mice,the difference was statistically significant(P<0.05).Compared with the control group,the expression levels of ERβ in the nucleus was significantly reduced,the difference was statistically significant.and the expression level of ERβ was not significantly different in the cytoplasm.After being treated by ERβ agonist,the immobility time of Ahil KO mice in tail suspension test and forced swimming test was significantly reduced(P<0.05).(2)In PC12 cells transfected by Ahil-siRNA,the expression levels of ERβ protein and ERβmRNA levels were significantly decreased in Ahil-siRNA group than that in control-siRNA group.and the difference was statistically significant(P<0.05).Compared with the control group,the expression levels of Ahil protein in Ahil-siRNA significantly decreased than that in the control group,the difference was statistically significant(P<0.05).The expression levels of ERβ in Ahil-siRNA cells’ the nucleus was significantly reduced,while the expression levels of GR increased in Ahil-siRNA cells’ Ahil-siRNA cells’.the differences were statistically significant(P<0.05).Compared with the control group,the expression level of ERβ was not significantly different in Ahil-siRNA cells’ the cytoplasm,while the expression levels of GR in Ahil-siRNA cells’ the cytoplasm was significantly reduced,the differences were statistically significant(P<0.05),suggesting that Ahil may inhibit the GR protein into the nucleus to increase the expression levels of ERβ.In PC 12 cells with DEX treatment,the total protein levels of ERβ in the DEX group was significantly lower than that in the control group,and the difference was statistically significant(P<0.05);The results of nuclear and cytoplasmic extraction test showed that the expression level of ERβ was not significantly different in Dex group cells’ the cytoplasm,while the expression levels of ERβ in Dex group cells’ the nucleus was significantly reduced,the differences were statistically significant(P<0.05),In PGL3-basic-ERβ-promoter transfected 293T cells,the expression level of ERβ promoter significantly increased in 293T cells with GR siRNA and DEX co-treatment.It is suggested that GR can inhibit the expression levels of ERβ by binding to the promoter of ERβ,and down-regulate the signal pathway of ERβ,which made the decrease of the content of serotonin leads to the depression-like performance in mice.(3)The depression-like performance in Ahil KO mice had been significantly improved,the results suggesting that ERβ agonist injection can have therapeutic effects on depression like symptoms in mice.After giving ERβ agonist treatment,the expression levels of 5-HT in Ahil KO mice’s hippocampus was significantly increased than that in the control mice,the difference was statistically significant(P<0.05);the expression levels of TPH2 protein in Ahil KO mice’s hippocampus was significantly increased than that in the control mice,the difference was statistically significant(P<0.05).Conclusion:1.The decrease of Ahil protein resulted in the decrease of ERP and TPH2 protein expression and the decrease of 5-HT content,but ERa and SERT were not significantly changed in Stress model mice and Ahil knockout mice the brain.2.Decreased the content of Ahil leads to a large number of GR protein from cytoplasm into the nucleus,the nuclear GR binding to ERβ promoter on the inhibition expression of ERβ and down-regulation of ERβ-TH2 signaling pathway,resulting in serotonin content decreased and depression like performance of mice.3.ERβ agonist can improve the depressive like behavior and increase the expression levels of TPH2 protein in Ahil knockout mice. |
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