| Part ?Metabolics and oxidative stress validation of the anti-depressant effects of pioglitazoneObjective: To identify the pathophysiological changes of depression in mice challenged with LPS,and reveal the possible mechanisms underlying the anti-depressant effects of PPAR-? agonist PIO,by using a combination of behavioral tests,metabolic profiling,and oxidative stress-related parameters validation.Method: Forty-eight male C57BL/6 mice(aged 6–8 weeks and weighing 20–25 g)were housed singly in plastic cages at room temperature(22 ± 2°C)with a 12-hours light/dark cycle(lights on at 8 a.m.),with free access to food and water.After one-week acclimatization and the behavioral tests(OFT,FST and SPT)at baseline,mice were randomly divided into three groups:(1)LPS group was treated with LPS(1 ?L/mouse,i.c.v.)and NS(20 mL/kg,i.g.);(2)LPS+PIO group was treated with LPS(1 ?L/mouse,i.c.v.)and PIO(20 mL/kg,i.g.);(3)control group was treated with NS(1 ?L/mouse,i.c.v.)and NS(20 mL/kg,i.g.),which served as the vehicle control.LPS(serotype 0127:B8)and PIO were freshly dissolved in normal saline solution at a concentration of 10 ng/?L and 1 mg/mL,respectively.Intragastrical pretreatment with PIO or NS was conducted 2 hours before the LPS or NS i.c.v.administration.Body weight was recorded immediately after the surgery,and SPT within 24 hours post-operatively was performed closely followed by OFT and FST 24 hours later.As soon as the behavioral tests(n=14 per group)were finished,the mice were sacrificed,the entire brain was removed and the hippocampus was resected for further metabolic profiling(n=10 per group)and western blot(n=10 per group)analyses.The coordinate accuracy was confirmed by bromophenol blue dye injection of two mice per group.GC-MS was performed for metabolic profiling of mice hippocampus among the three groups.Multivariate statistical analyses,including PCA,PLS-DA and OPLS-DA were applied for the identification of the differential metabolites.Moreover,RPT with a 7-fold cross-validation and a 200 iteration permutation,was applied to validate the performance of the OPLS-DA model.The significance of variables in OPLS-DA was assessed using the variable influence on projection(VIP)method.The relative analysis of differential metabolites was performed using Ingenuity Pathway Analysis(IPA)to identify the most representative canonical pathways and biomolecular interaction networks,which were generated based on information stored in the Ingenuity Knowledge Base.According to the results of metabolic profiling,western blot analysis was conducted subsequently for further validation of oxidative stress-related parameters in mice hippocampus.Results: 1.Effects of PIO on behavioral changes in mice challenged with central inflammatory stress: central LPS administration precipitated depression-like behaviors in mice,including anhedonia,behavioral despair(manifested as the decrease of sucrose preference in SPT and increase of immobility duration in FST);accompanied with anxiety-like behavior(manifested as the decreased distance in center in OFT)as well as acute sickness responses,including reductions of exploratory and locomotor activities(measured as the decreased number of rears and total distance travelled in OFT),which were reversed by PIO pretreatment to different extents.However,as a consequence of negative energy balance in the acute sickness response,the loss in body weight was not altered by PIO pretreatment.Collectively,these results illustrate that PIO pretreatment is capable of reversing the depression-,anxiety-like behaviors,and certain acute sickness responses precipitated by i.c.v.LPS administration.2.Metabolic profiling of mice hippocampus based on GC-MS:(1)The chromatographic peaks in the TIC chromatograms represent corresponding metabolites,and their relative concentrations can be detected by peak area normalization method.In the present study,the representative TIC chromatograms of hippocampus samples from the three groups revealed strong signals for analysis,large peak capacity,and good R.T.reproducibility.A total of 229 metabolites were preliminarily detected in each group,and the corresponding concentrations of these metabolites were used in the subsequent multivariate statistical analyses.In the PCA score plot,the R~2 X values were > 0.5,indicating that it was able to discriminate among the three groups(R~2X = 0.609,Q~2 = 0.213).In PLS-DA score plots,the clear discriminations were observed in the three comparison sets,including LPS group vs.control group(R~2X = 0.421,R~2 Y = 0.997,Q~2 = 0.874),LPS+PIO group vs.LPS group(R~2X = 0.233,R~2 Y = 0.959,Q~2 = 0.564),and LPS+PIO group vs.control group(R~2X = 0.397,R~2 Y = 0.989,Q~2 = 0.938).Moreover,the OPLS-DA models used to maximize discriminations showed clear separations in the three comparison sets,including LPS group vs.control group(R~2X = 0.421,R~2 Y = 0.997,Q~2 = 0.864),LPS+PIO group vs.LPS group(R~2X = 0.233,R~2 Y = 0.959,Q~2 = 0.627),and LPS+PIO group vs.control group(R~2X = 0.397,R~2 Y = 0.989,Q~2 = 0.936).The PLS-DA and OPLS-DA models were robust and had good fitness and prediction,since the key parameter(Q~2)were all greater than 0.5.The RPT was used to validate the performance of the OPLS-DA model,in which the original R~2 and Q~2 values were higher than the corresponding permutated values,and all the Q~2 values <0,indicating that the OPLS-DA models were valid and not over-fitted(R~2 = 0.708,Q~2 =–0.492;R~2 = 0.786,Q~2 =–0.293;R~2 = 0.6,Q~2 =–0.471,respectively).(2)A total of 52 differential metabolites were identified among the three groups by VIP > 1.0 in the OPLS-DA models and Bonferroni-or Dunnett-corrected P < 0.05.They were primarily involved in amino acid,energy and nucleotide(purine and pyrimidine)metabolisms.In the 52 differential metabolites,there were 41,15 and 35 metabolites found in the three comparisons(LPS group vs.control group,LPS+PIO group vs.LPS group,and LPS+PIO group vs.control group),which were deemed to be potentially related to the development of inflammation-associated depression,the effects of PIO,and the interaction between the two factors above,respectively.The top five influenced canonical pathways and the most significantly altered networks in the three comparisons were revealed by IPA.Moreover,based on the biochemical relationships among these metabolites,a summary figure was developed by integrating the pathways involving the most affected metabolites,which suggests that processes of neurotransmissions,energy metabolisms and oxidative stress containing most differential metabolites may participate in the pathogenesis of inflammation-associated depression,as well as the anti-depressant effects of PIO.3.Validation of oxidative stress-related parameters in mice hippocampus: Aside from neurotransmissions,the GSH metabolic signalling was the most perturbed pathway in the comparison between the LPS+PIO and LPS groups;moreover,the IPA network analysis predicted that SOD and MPO associated with the anti-oxidant process,may be involved in the effects of PIO.In our study,the activation of oxidative stress was found in the LPS group,with an up-regulation of pro-oxidative NOX subunits gp91 phox,p47phox(pro-oxidant activity),and a down-regulation of anti-oxidative SOD1 and GPX1,compared with the control group.However,the imbalance was ameliorated to a certain extent in the LPS+PIO group compared with the LPS group,consistent with the metabolic findings.These results indicate that PIO pretreatment effectively attenuates the imbalance between pro-oxidant and anti-oxidant status in a mouse model of depression challenged with central inflammatory stress.Conclusion: The present study demonstrated the beneficial effects of PIO on depression-,anxiety-like behaviors and certain acute sickness responses elicited by central inflammatory stress.Perturbations in amino acid,energy and nucleotide metabolisms were identified among the three groups using GC-MS-based metabolic profiling,suggesting that the perturbations of neurotransmissions,energy metabolisms and oxidative stress may,at least partially,contribute to the pathogenesis of inflammation-associated depression,through which it may yield the anti-depressant effects of PPAR-? agonist.Moreover,the alterations of oxidative stress were further confirmed by western blot analysis.The work provides novel insights into the pathogenesis of inflammation-associated depression as well as the molecular mechanisms of anti-depressant effects of PPAR-? agonist on it,which may lead to a new therapeutic target for inflammation-associated depression.Part ? Protein detection of related signaling pathway involved in the anti-depressant effects of pioglitazoneObjective: To investigate the beneficial effects of PPAR-? activation on depression-like behaviors in mice challenged with central inflammatory stress by applying the PPAR-? agonist PIO and antagonist GW9662,and to further explore the underlying mechanisms by protein detection of related signaling pathway.Method: Forty-five male C57BL/6 mice,aged 6–8 weeks and weighing 18–22 g,were housed individually at room temperature(22 ± 2°C)with a 12/12 hours light/dark cycle(lights on at 8 a.m.)and food and water provided ad libitum.At baseline,OFT and FST were performed followed by a 5-day acclimatization in which mice were trained to adapt to the presence of two calibrated drinking bottles containing water,and SPT was conducted subsequently.Of note,OFT and FST were conducted in advance compared with Experiment I,for avoiding the potential influence due to the short time interval between the pre-and post-operative tests.After the baseline behavioral assessments,mice were randomly divided into four groups:(1)control group(NS,i.c.v.;NS,i.g.;NS,i.p.);(2)LPS group(LPS,i.c.v.;NS,i.g.;NS,i.p.);(3)LPS+PIO group(LPS,i.c.v.;PIO,i.g.;NS,i.p.);(4)LPS+PIO+GW9662 group(LPS,i.c.v.;PIO,i.g.;GW9662,i.p.).LPS(serotype 0127:B8),PIO and GW9662 were diluted in normal saline at 10 ng/?L,1 mg/mL,and 2 mg/mL;and their dose were 1 ?L/mouse,20 mL/kg and 1 mL/kg,respectively.PIO(or NS)and GW9662(or NS)were administered 2 hours and 30 min prior to the cerebral stereotaxic surgery,respectively.Subsequently,all mice were received a central LPS(or NS)injection.All the post-operative procedures,including body weight measurement,behavioral tests and sample collection were the same as Experiment 1(n = 9-10).The coordinate accuracy was confirmed by bromophenol blue dye injection of two mice per group.Western blot analysis was performed for the identification of related signaling pathways and proteins in mice hippocampus,including the JAK2/STAT3,CREB/BDNF pathways and their downstream effectors,as well as 5-HT1AR(n = 6).Results: 1.Effects of PPAR-? activation on behavioral changes in mice challenged with central inflammatory stress: As Experiment I,i.c.v.LPS administration elicited anhedonia and behavioral despair,the core symptoms of depression;accompanied with anxiety-like behavior,and the acute sickness responses,including loss in body weigh and reductions of exploratory and locomotor activities(LPS group vs.control group).PIO pretreatment ameliorated the alterations to different extents,except for body weight change(LPS+PIO group vs.LPS group),and the ameliorative effects were abolished by GW9662(LPS+PIO+GW9662 group vs.LPS+PIO group,LPS+PIO+GW9662 group vs.control group).Pretreatment of PIO or GW9662 did not affect central LPS-induced loss in body weight.Collectively,these results suggest the PPAR-? dependent beneficial effects of PIO on depression-,anxiety-like behaviors,and certain acute sickness responses precipitated by central inflammatory stress.2.Identification of related signaling pathways and proteins in mice hippocampus:(1)To examine whether PIO exerts antidepressant-like effects through specific signaling pathways by activating PPAR-?,we measured the expression of PPAR-? and protein kinases which have been identified to be related to depression-like behaviors induced by inflammation,including JAK2 and STAT3 in mice hippocampus.A significant decrease in PPAR-? was observed following central LPS administration(LPS group vs.control group),which was reversed by PIO pretreatment(LPS+PIO group vs.LPS group),and the effect of PIO was abolished by GW9662(LPS+PIO+GW9662 group vs.LPS+PIO,LPS+PIO+GW9662 group vs.control group).(2)Increased expression of IL-6,and activation of the JAK2/STAT3 signaling pathway downstream,manifested as the increased ratios of phosphorylated to total JAK2 and STAT3(p-JAK2/t-JAK2,p-STAT3/t-STAT3)were observed following central LPS administration.Intriguingly,PIO pretreatment inhibited the alterations above,which was significantly blocked by GW9662.(3)As an important downstream effector of the JAK2/STAT3 pathway under inflammatory conditions,the increased expression of IDO1 following central LPS administration was significantly reversed by PIO pretreatment,and this effect was inhibited by GW9662.(4)5-HT1 AR was significantly lower in mice that received LPS than in control mice.PIO pretreatment reversed the decrease of 5-HT1AR(LPS+PIO group vs.LPS group)to a certain extent,even though it failed to be back to normal(LPS+PIO group vs.control group),and the effect of PIO was blocked by GW9662.As a rate-limiting enzyme for the degradation of serotonin precursor tryptophan along the kynurenine pathway which is highly inducible by inflammation,the activation of IDO1 resulted in the enhanced tryptophan catabolism which could well explain the decrease in serotonin synthesis as well as deficiency of central serotonergic neurotransmission in depression.Moreover,5-HT1 AR also participates in the central serotonergic neurotransmission by binding to 5-HT.Our results of IDO1 and 5-HT1 AR may provide a possibility that PPAR-? activation may have potential impact on central serotonergic neurotransmission,which could also contribute to the beneficial effects of PIO.(5)The neurotrophic CREB/BDNF pathway accompanied with synaptic plasticity-related proteins downstream were also assessed in the present study.A significant decrease in the ratio of p-CREB/t-CREB and the expression of BDNF was observed following central LPS administration,which was significantly reversed in response to PIO pretreatment;moreover,synaptic plasticity-related proteins,including SYN1,GAP-43 and PSD-95,which have been proven to mediate the neurotrophic effects of BDNF were also evaluated as the downstream effectors of CREB/BDNF pathway in our study.Central LPS-treated mice displayed a significant reduction in expression of SYN1,GAP-43 and PSD-95 compared with the control group which was ameliorated by PIO as well.The improving effects of PIO on the down-regulation of the CREB/BDNF pathway as well as the effectors downstream were all inhibited by GW9662.Conclusion: Our data confirms the PPAR-?-denpendent beneficial effects of PIO on depression-like behaviors,coupled with certain acute sickness responses and anxiety-like behavior in mice precipitated by central inflammatory stress.Western blot analysis implies that the modulation of the JAK2/STAT3 and CREB/BDNF pathways,as well as the potential impact on central serotonergic neurotransmission,may be involved in the PPAR-?-dependent effects of PIO on depression-like behaviors challenged with central inflammation. |
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