Effects Of Interferon Alpha On P11、5-HTR1b And 5-HTR4 Expression In Neurons

BackgroundDepression is the major side effect of interferon alpha(IFN-α)treatment,but the molecular mechanism underlying IFN-α induced depression remains unclear.Several studies have shown that the serotonin receptors 5-HTR1b and 5-HTR4 play key roles in the anti-depression effects associated with p11(S100 calcium binding protein A10,S100A10),and IFN-α can decrease p11 level.Thus,we hypothesized that the depression originating from IFN-α treatment could be mediated by p11 and the serotonin receptors.To understand the role of such molecules in the onset of IFN-α-induced depression,we investigated the effects of IFN-α on the regulation of 5-HTR1b and 5-HTR4 in human neuroblastoma cells(SH-sy5y).We found that intraperitoneal injection withIFN-α in Balb/c mice resulted in an increased immobility in FST and TST.Treatment withIFN-αremarkably reduced the 5-HTR1b and 5-HTR4 protein levels in a time and dose-dependent manner in SH-sy5 y cells.Our study revealed 5-HTR1b and 5-HTR4 as down-stream effectors of p11 by over-expression and knockdown experiments.Over-expression of p11 is sufficient to prevent the IFN-α-induced inhibition on 5-HTR1b and 5-HTR4.The results indicated that IFN-α treatment resulted in p11 down-regulation which subsequently decreased 5-HTR1b and 5-HTR4.p11 was identified as a potent regulator on 5-HTR1b and 5-HTR4 and therefore it could be a potential target in the risk evaluation of side effects and therapies for IFN-α induced depression.PurposeThrough the behavior experiment and cell experiment in mice,study the effect of IFN-αon 5-HTR1b 、5-HTR4 and p11,research the mechanism of 5-HTR1b 、5-HTR4 and p11 involved in IFN-α-induced depression.Methods1.Animals and drug treatments.Adult male wild-type Balb/c mice were used in the animal tests.For drug treatments,three or four mice were housed per cage and hIFN-α-2b was injected into mice for 10 uninterrupted days(1000 IU/g/day,intraperitonealinjection,total volume = 0.2 mL).2.Mice behavior tests.For FST,mice were individually and gently placed in a glass cylinder(30cm high,10 cm in diameter)filled with water at 24-26℃ to a depth of 18 cm,and allowed to swim for 6 min.The data of immobility within 2-6 min were collected to measure the behaviors..For TST,mice were suspended in the testing equipment(Bioseb,USA)by taping the distal part of the tail(1-1.5 cm)to a flat metallic surface 40 cm above the floor.Escape movements were recorded for 6 min.The time spent in an immobile posture within 2-6 min was measured as an index of depression-like behavior.3.Cell culture.SH-sy5 y cell was cultured in Dulbecco’s modified Eagle medium(DMEM)containing 10% fetal bovine serum,50 U/mL penicillin,and 50 mg/mL streptomycin at 37°C,with 5% CO2.All of the experiments were performed when the cells were 80%-90% confluent.4.hIFN-a-2b treatment.Dose-dependent test Cells were treated with a series of hIFN-α-2b(0,50,500,1000,2000,and 3000 IU/mL).Time-dependent test Cells were treated with a single dose of 1000 IU/mL hIFN-α-2b and harvested at 0,6,12,24,36,and48 h for protein detection.5.Plasmid construction.To construct p11 over-expression plasmid,a 319-bp product of p11 cDNA were cut using enzymes HindIII and EcoRI,followed by cloning into the pcDNA3.0 vector.The p11-SiRNA plasmid were constructed using pcDNA6.2-GW/EmGFP-miR vector.6.Plasmid transfection.The plasmids were transfected into cells with Liposome Transfast2000(Invitrogen,USA).The transfected plasmids contained p11-pcDNA3.0,pcDNA3.0(control),P11-SiRNA,and Si RNA-control(control).At 5 h after transfection,the cells were washed with PBS and supplemented with complete DMEM.7.Cell membrane proteins extraction.Cytomembrane proteins were extracted with the Mem-PER Eukaryotic Membrane Protein Extraction kit(Thermo Fisher Scientific,USA).Anti-Pan-cadherin(Abcam,USA)and anti-tublin(Abcam,USA)antibodies were applied to test the levels and the purity of membranal proteins.8.Western blots.Tricine-SDS-PAGE gel electrophoresis was uesed to detect the p11,5-HTR1b,5-HTR4 protein level.9.RNA extraction.Cells were collected at 16 h and the total RNA was extracted usingan RNA extraction kit(Minico,USA)..The real-time PCR primers were obtained from Biosail10.real-time PCR The cDNA was assayed using real-time PCR.The real-time PCR primers were obtained from Biosail11.Statistical analysis.The P values in all experiments were determined with Prism software(GraphPad Software Inc.)for nonparametric unpaired T tests.F tests were used to compare variances.We considered P-values <0.05 to be significant and the degree of significance is indicated as follows: *,P < 0.05;**,P < 0.01.Results1.IFN-α treated mice showed increased immobility in the FST and TST.2.IFN-α remarkably decreased the 5-HT1Rb/4 protein levels in a time and dose-dependent manner.3.The protein levels of p11,5-HTR1b and 5-HTR4 in the cytomembrane of SH-sy5 y cells were significantly reduced after IFN-α treatment.4.p11 controlled protein levles of 5-HTR1b and 5-HTR4,and over-expression of p11 prevented the IFN-α-induced down-regulation of 5-HTR1b and 5-HTR4.Conclusion p11 involved in IFN-α-induced depression by influencing the 5-HTR1b/4 levels.