The Molecular Mechanism Of SIRT1 Signaling Pathway In Depression And Cognitive Impairment Induced By Chronic Unpredictable Mild Stress And The Role Of Enriched Environment Intervention

Part Ⅰ The role of SIRT1 signaling pathway in hippocampus on cognitive impairment induced by chronic unpredictable mild stressObjective Chronic unpredictable mild stress(CUMS)leads to cognitive impairment.Resveratrol has been demonstrated to be a potent activator of Sirtuin 1(SIRT1).Previous studies reported that resveratrol treatment ameliorated CUMS-induced depressive-like behavior and cognitive deficits through upregulating cAMP response element-binding protein(CREB)and brain derived neurotrophic factor(BDNF)expression.However,the upstream signalling pathway mediating CREB/BDNF expression and then exerting a protective role on cognitive function remains unclear.The present study aims to investigate the possible mechanism of resveratrol on CUMS induced cognitive deficits.Methods Male Sprague Dawley rats(180–200g)were used in this study.All rats were randomly assigned to the following 4 groups: control group,stress group,stress+resveratrol(40mg/kg),stress+resveratrol(80mg/kg).Rats were adminstrated resveratrol(40 and 80mg/kg)every day for 4 consecutive weeks before exposure to CUMS procedure.Morris Water Maze test was used to appraise spatial learing and memory of rats.Sirt1/miR-134 signalling pathway and CREB/BDNF expression in hippocampus of rats were measured.We also explored Sirt1/miR-134 signalling pathway and CREB/BDNF expression in primary cultured hippocampus neurons with resveratrol(25,50 and 100μmol/L)treatment.Rusults The escape latency time on the 4th and 5th day of CUMS rats was significantly longer than control group(P < 0.05).Compared with rats in CUMS group,rats in resveratrol(80 mg / kg)treatment group displayed a shorter escape latency(P < 0.05).On the 6th day,the time of CUMS group was significantly shorter than that of resveratrol(40 mg / kg)treatment group(P < 0.01),resveratrol(80 mg / kg)treatment group(P < 0.001).Compared with control group,the expression of BDNF,p-CREB,CREB and SIRT1 in hippocampus of rats subjected to CUMS decreased significantly(P < 0.001),and the expression of miRNA134 increased(P < 0.001).Compared with CUMS group,resveratrol(40 mg / kg,80 mg / kg)treatment group can increase the expression level of BDNF,p-CREB,CREB and SIRT1 protein in hippocampus,and reduce the expression level of miRNA134.In vitro,resveratrol,a SIRT1 activator,increased the expression of BDNF,p-CREB,CREB and SIRT1,and decreased the expression of miRNA134.Conclusions Our study showed that the neuroprotective effect of resveratrol on CUMSinduced cognitive impairment may rely on activating Sirt1/ miR-134 pathway and then upregulating its downstream CREB/BDNF expression in hippocampus.Part Ⅱ Resveratrol exerts a protective effect in chronic unpredictable mild stress–induced depressive-like behaviorObjective Chronic unpredictable mild stress(CUMS)is an important contributing factor for depression with inflammatory response alteration,neuronapoptosis,and decreased neurogenesis.Previous study reported that the administration of resveratrol alleviated depression by normalizing the increased proinflammatory cytokine levels and inhibiting apoptosis in the hippocampus.However,the upstream signaling pathway that regulates cytokines and apoptosis in the antidepressant effect of resveratrol remains unclear.The objective of this study is to investigate the possible mechanism of the effect of resveratrol on depression.Methods Male Sprague Dawley rats(180–200g)were used in this study.Rats were randomly divided into the following eight groups: control,control+ LY294002,stress,stress+fluoxetine(10 mg/kg),stress+resveratrol(40 mg/kg),stress+resveratrol(80 mg/kg),stress+ resveratrol(40 mg/kg)+LY294002,and stress+resveratrol(80 mg/kg)+LY294002.SD rats were exposed to CUMS for four consecutive weeks to elicit depressive-like behavior.The rats in the drug treatment groups were injected with resveratrol(40 or 80 mg/kg/day)and fluoxetine(10 mg/kg/day)intraperitoneally for 4 weeks.Rats in two additional groups were administered LY294002 by bilateral stereotaxic microinjection into the lateral ventricle beforeresveratrol administration.Behavioral tests,including sucrose preference test,forcedswimtest,and open field test,were used after 4 weeks of a CUMS procedure to appraise depressive-like behavior.Then,the proinflammatory cytokines(TNF-α,IL-6,and IL-1β)in the hippocampus and prefrontal cortex(PFC)tissues of rats were measured.Apoptosis-related molecules such as Bax and Bcl-2 m RNA levels in the hippocampus were analyzed.Furthermore,p-Akt/Akt and pGSK3β/GSK3β protein expression in the hippocampus were also measured.Rusults The results show that ratswere subjected to CUMS procedure exhibited depressivelikebehavior,increased TNF-α,IL6,and IL-1β levels in hippocampus and PFC,alteration of Bax and Bcl-2 m RNA levels in hippocampus,decreased p-Akt/Akt and p-GSK3β/GSK3β protein expression in hippocampus,and an increased apoptotic cell percentage in the hippocampal CA1 region.However,resveratrol(40 or 80 mg/kg)treatment reversed these behavioral and molecular changes in CUMS rats.The positive control drug fluoxetine showed a similar effect as the resveratrol treatment.When rats were injected with LY294002 before resveratrol treatment,the antidepressant effect of resveratrol was significantly attenuated,TNF-α,IL-6and IL-1βlevelsin hippocampus and PFC increased again,Bax m RNA levels increased and Bcl-2 m RNA levels decreased in hippocampus,and Akt/GSK3β protein expression in hippocampus decreased.Conclusions The findings in the present study suggest that the antidepressant effect of resveratrol treatment may act through activation of the Akt/GSK3β signaling pathway and then regulation of proinflammatory cytokine expression and alteration of apoptosis.Part Ⅲ The enriched environment ameliorates chronic unpredictable mild stressinduced depressive-like behaviors and cognitive impairment by activating the SIRT1/miR-134 signaling pathway in hippocampusObjective Chronic unpredictable mild stress(CUMS)is an important risk factor for depression and cognitive deficits in humans.Enriched environment(EE)showed a beneficial effect on depression and cognition by enhancing brain derived neurotrophic factor(BDNF)expression and synaptic plasticity.However,it is still not clearly understood whether an epigenetic mechanism is involved in the BDNF modulation and synaptic plasticity that occurs after EE treatment for the depressive-like behaviors and cognitive deficits elicited by CUMS.In this study,we investigated the possible mechanism of the neuroprotective effect of EE.Methods Male Sprague Dawley rats(180–200g)were used in this study.All rats were randomly assigned to the following 5 groups: Control,CUMS,CUMS+EE,CUMS+EX527+EE and CUMS+sh-SIRT1+EE.All rats were exposed to the 5-week CUMS procedure except the control group.After CUMS procedure,some rats were stereotaxically injected with SIRT1 pharmacologic inhibitor EX527 or SIRT1 knocking down lentivirus(shSIRT1)in the hippocampus followed by EE treatment for 3 weeks.Other rats were directly subjected to EE treatment without stereotaxic injection.Behavioral tests were used to appraise depression and cognition after EE treatment.Then epigenetic molecules,synaptic proteins,dendritic spine density and branches,and synaptic morphology of the dorsal hippocampus were determined.Rusults We found that CUMS induced depressive-like behaviors including decreased sucrose preference ratio,prolonged immobility and reduced locomotor and exploratory activity;cognitive deficits including spatial learning and memory impairment;reduced dendritic spine density and number of branches;thinned postsynaptic density;downregulated SIRT1/micro RNA-134 pathway,decreased BDNF and synaptic proteins including synaptophysin(SYN)and postsynaptic density protein 95(PSD95)expression in the hippocampus.However,the CUMS-induced depressive-like behaviors,cognitive deficits,dendritic spine density and branch number reduction,postsynaptic density thinning,SIRT1/micro RNA-134 pathway downregulation,BDNF and synaptic proteins reduction,including synaptophysin(SYN)and postsynaptic density protein 95(PSD95),were reversed by EE treatment.However,depressive-like behaviors and cognitive deficits were observed again in rats subjected to stereotaxic injection with EX527 or sh-SIRT1.Furthermore,this study also found that SIRT1/ micro RNA-134 regulates the downstream molecules BDNF,and the synaptic proteins SYN and PSD95 in primary cultured hippocampal neurons.Conclusions This study provides evidence for the neuroprotective role of EE on depression and cognitive deficits by activating the SIRT1/micro RNA-134 pathway,which accounts for the regulation of synaptic proteins,including BDNF,PSD95 and SYN,dendritic remodeling and ultrastructure changes of synapses in the hippocampus.