The Impacts Of Rosiglitazone On Chronic Mild Stress-Induced Depressive Mice

Depression is one kind of mood disorder whose clinical symptoms included depressing mood,retardation of thought,cognition impairment,volitional activity drops and somatic symptom.The latest statistic data from World Health Organization(WHO)indicated that the ratio of patients with depression and dysthymic disorder raised to 12.8%,and predicted to become the second medical disorder all over the world.The clinical commonly used medicines on depression exerted limited therapeutical effects and significantly side effects.Thus,it is impressive to seek for drugs with better safety and curative effect.The low glucose metabolism of prefrontal cortex-limbic system in patients with depression provided evidence that energy metabolism may be the neurobiological mechanisms of depression.Besides,many common pathological characteristics were found between depression and metabolic disorders,such as glucose metabolism imbalance,chronic inflammation and neuroendocrine disorders.In conclusion,depression has a close relationship with metabolic disorders.As one kind of Thiazolidinedione(TZD),rosiglitazone always used as an antidiabetic to relieve Type 2 Diabetes Mellitus clinically.There is an increasing studies proved that rosiglitazone exerted important roles in multiple CNS diseases,included Parkinson’s disease(PD),Alzheimer’s disease(AD)and stroke,but the exact mechanism remains unclear.The present study was to evaluate the potential therapeutic action and mechanism of rosiglitazone in vivo(chronic mild unpredictable stress induced depressive mouse model)and in vitro(corticosterone induced cellular model)depressive models.AIM:To investigate and illustrate the therapeutic effect and the involved mechanisms of rosiglitazone on depressive model mice.METHODS:1.The depressive behaviors of mice were evaluated by behavior test including sucrose preference test(SPT),forced swimming test(FST)and open field test(OPT).2.Immunofluorescence(IF)assay was used to analyze the amount,cellular morphology of NeuN-labeled neuron,GFAP-labeled astrocyte,CDllb-labeled microglia and the expression of specific proteins in prefrontal and hippocampal paraffin sections.3.Enzyme linked immunosorbent assay(ELISA)was used to detect the concentration of IL-6,TNFα,IGF1,VEGF and CNTF in serum.4.Primary cortex neuron,astrocyte and N2a cell lines were cultured in vitro,and their cell positive rates were detected by immunocytochemistry combined with stereology technology.5.After exposing to corticosterone or rosiglitazone,cell activity was detected by methyl thiazolyl tetrazolium(MTT)and lactic dehydrogenase(LDH)assay.6.The formation and amount of autophagosome were observed by transmission electron microscope7.Flow cytometry(FCM)detected the apoptosis ratio of primary astrocyte by Annexin-V/PI double staining.8.Autophagy related proteins LC3,AMPK,P62,ULK1 and apoptosis related protein caspase3、Bax、Bcl-2、PARP of animal and cell protein samples were analyzed by western blotting assay(WB).9.The transfection efficiency of si-PPARγ and relative mRNA expression of cytokines,STRAD,M025 was detected by real-time quantification polymerase chain reaction(RT-PCR).RESULTS:1.Rosiglitazone improves CMS-induced depressive like behaviorsRosiglitazone(5 mg/ml)could raise the SPT of CMS modeling mice,and elevate the average weight for 3.7 g.Compared to CMS modeling,rosiglitazone administration increased the climbing durations of in FST and center remaining durations in OPT.2.Rosiglitazone ameliorates CMS-induced neuron/astrocyte injures in prefrontal cortex and neurogenesis decline in hippocampalRosiglitazone protected against CMS-induced apoptosis determined by TUNEL assay,and increased the amount of astrocyte and the density of neuron axon in CMS modeling mice prefrontal cortex.In addition,the amount of Ki67 labeled newborn neuron in CMS hippocampal DG region increased significantly,indicating a dramatic protective effect of rosiglitazone on depression.3.Rosiglitazone ameliorates inflammation respond and cytokines dysfunctionsCMS increased the concentration and mRNA level of IL-1β,IL-6 or TNFa,while rosiglitazone could reverse these decline tendencies in our qPCR and ELISA assay.Furthermore,rosiglitazone increased the concentration of CNTF and IGF1,and decreased the concentration of corticosterone in CMS mice to 366 ng/ml(compared to CMS group 713 ng/ml).4.Rosiglitazone up-regulates neuron autophagy and reduces astrocyte apoptosis in prefrontal cortexAutophagy related proteins were analyzed by western blotting assay,we found rosiglitazone could increase the expressions of LC3II/I,ULK1,Beclinl,p-AMPK/AMPK,and decreased P62 protein expression.Moreover,rosiglitazone elevated the expression of LC3B on NeuN labeled neuron compared to CMS modeling for 68%.We also found that rosiglitazone increased the levels of Bcl-2/Bax,p-mTOR/mTOR,p-IRS1/IRS1.5.Rosiglitazone promotes autophagy in corticosterone-treated primary neuron and N2a cell line via activating LKB1/AMPK pathwayCorticosterone at the concentration of 10μM decreased the cell vitality of primary cortex neuron and N2a cell,protecting against corticosterone-induced injury.Rosiglitazone increased the formation of autophagosomes observed by TEM,and reversed the corticosterone-induced decline of autophagy related protein such as ATG5、LC3、ULK1 and P62.All these results were reversed by T0070907,an antagonist of PPARγ.The neuroprotection role of rosiglitazone on N2a cell was influenced via increasing or decreasing the level of M025 and LKB1 by knocking down or overexpressing PPARγ.All these results indicated that rosiglitazone could activate LKB1/AMPK pathway by activating PPARy,and influenced autophagy,thus protected neuron and N2a cell.6.Rosiglitazone inhibits corticosterone induced astrocyte apoptosis via up-regulating IGF1R and activating Akt/CREB pathwayThe expression of Bcl-2/Bax,caspase 3,and PARPp85 were analyzed by AV/PI double staining and western blotting,the results show that rosiglitazone exerted an anti-apoptosis role on astrocyte exposing to corticosterone.IGF1R exerted a higher expression in astrocyte than neuron,and rosiglitazone increased the expression of IGF1R and the phosphorylation of its substrate IRS1(increased 175%and 86%,compared to corticosterone group),activated Akt/AMPK pathway,thus exerted protection roles on astrocyte.7.Rosiglitazone improve corticosterone-induced astrocytic dysfunction and thereby protects cortex neuronsPrimary cortex neuron cultured by option mediums from astrocyte exposing to rosiglitazone,exerted a higher cell vitality and less nuclear shrinking than the use of mediums exposing to corticosterone.Besides,rosiglitazone promoting the growth of axon in option mediums by immunocytochemistry observing MAP2 labeled-neuron.CONCLUSIONS:Rosiglitazone exerts anti-depressive role by enhancing neuron autophagy and inhibiting astrocyte apoptosis.INNOVATION POINTS OF PAPER1.Rosiglitazone alleviates CMS induced neuron/astrocyte injuries,providing evidence for developing new antidepressant.2.Rosiglitazone enhances autophagy in neuron by activating LKB1/AMPK pathway,and inhibits apoptosis in astrocyte by activating Akt/CREB pathway,which is responsible for rosiglitazone-mediated anti-depression effects.