| Objectives:1.Investigate the effect of glutamate excitotoxicity on the expression of PPARγ and its possible regulatory mechanism.2.Explore the changes of PPARγ activity after glutamate injury and its possible mechanism through observing the change of PPARγ phosphorylation 3.Observe the effects of PPARγ agonists rosiglitazone,pioglitazone and MAPK/ERK1/2 inhibitor U0126 on glutamate excitotoxicity,and further explore the protection of activation of PPARγ or protection of its activity to against toxic damage.Method:Adult healthy male Sprague Dawley rats were used as experimental animals for testing.1.SD rats were randomly divided into normal group,control group,glutamate(Glu)group and NMDA group.The normal group was given no treatment.The control group was injected with vehicle in the cortex.Glutamate group and NMDA group were treated with 50,100,and 200 mmol/L(in 1μL)Glu/NMDA for 24 h,or with 200 mmol/L Glu/NMDA for 3,6,12,and 24 hours,respectively..(1)Western blot was used to detect the expression of inflammation and apoptosis-related proteins,i.e.COX-2,iNOS and cleaved-3,.(2)Immunofluorescence staining was carried out for immunoreactivity of COX-2,iNOS,or cleaved aspase-3 and their co-localiztion with neuron marker NeuN(3)Histological changes were detected by HE and TTC staining.2.SD rats were randomly divided into control group,MK-801 alone group,Glu group and Glu plus MK-801 group.The control group was givenvehicle.MK-801 alone group was given 0.15 mg/kg MK-801 intraperitoneally.Glu group was with 200 mmol/L glutamate(1 μL)cortex injection.Glu plus MK-801 group was treated with 0.375,0.75,0.15mg/kg MK-801 intraperitoneally30 min before the 24-hour Glu cortical injection(1)Western blot was used to detect the expression of inflammation and apoptosis-related proteins(COX-2,iNOS and cleaved caspase-3).(2)HE staining was used to detect the histological changes.3.SD rats were randomly divided into normal group,control group,Glu or NMDA group,and the treatment was the sameas described above.(1)Western blot was for the expression of PPARγ,pPPARγ,ERK1/2 and pERK1/2.(2)Immunofluorescence staining was for the immunoreactivity of PPARγ and co-localization with NeuN.4.SD rats were randomly divided into control group,intervention alone group,Glu/NMDA group,and Glu/NMDA plus intervention group.The intervention agents were MK-801(intraperitoneal injection),GSH(tail vein injection),microinjection of BAPTA-AM(cortex injection),calpeptin(cortex injection)and Ac-DEVD-CHO(cortex injection).The control group,Glu/NMDA group were treated as described above,intervention alone group was treated with only the intervention agent,and Glu/NMDA plusintervention group was treated with individual intervation agent 30 min before the 24-hour NMDA(200 mmol/L)cortical injection glutamate Western blot was used to detect the expression of PPARγ in each group.5.SD rats were randomly divided into control group,U0126 alonegroup,Glu/NMDA group,Glu/NMDA plus U0126 group.The control group,Glu/NMDA group were treated asdescribed above.U0126 alone group is treated with U0126.The Glu/NMDA plus U0126 group was treated with U0126 30 minutes before the 24-exposure of NMDA(200 mmol/L).Western blot was used to detect the effect of U0126,inhibitor of ERK1/2 activation,on the expression of p-PPARγ,ERK1/2 and p-ERK1/2.6.SD rats were randomly divided into normal group,control group,protectant alone group,NMDA group and NMDA plus protectant group.The protectants were rosiglitazone(Ros),Piolitazone(Pio)(intraperitoneal injection),and U0126(cortical injection).The normal group,the control group and the NMDA group were treated as above.The NMDA plus protectant group was treated with protectant 30 minutes before the 24-hour treatment earlier of glutamat NMDA(200 mmol/L).(1)HE and TTC staining were applied for histological changes.(2)Western blot was used to detect the expression changes of inflammation and apoptosis related proteins in each group.(3)Immunofluorescence staining was for the immunoreactivity of COX-2,iNOS,cleaved caspase-3 and their co-localization with NeuN.staining with NeuN.Result:1.Assessment of glutamate excitotoxic damage model:(1)Western blot analysis showed that glutamate or NMDA caused increased expression of COX-2,iNOS,and cleaved caspase-3 proteins,while MK-801 inhibited the above protein expression induced by glutamate(P<0.05,the difference was statistically significant).(2)Immunofluorescence staining showed that the signal intensity of COX-2,iNOS and cleaved caspase-3 protein in glutamate or NMDA group was increased when compared with that of control group(P<0.05),and most of them could overlap with NeuN signal,suggesting they were mainly expressed in neurons.(3)HE staining showed cortical damage caused by glutamate or NMDA,which could be inhibited by MK-801,suggesting that excitotoxic damage of glutamate was mainly mediated by the NMDA receptor.2.The effectof glutamate on the protein expression of PPARγ:(1)glutamate treatment: 1)Western blot analysis showed that PPARγ protein expression increased in both time-and dose-dependent manner(P<0.05,the difference has statistically significant).2)Immunofluorescence staining showed that the PPARγ protein signal density were enhanced by Glu,and most of them could be overlaid with NeuN signal,suggesting that it was predominantly expressed in neurons.(2)NMDA treatment: Western blot analysis showed that PPARγ expression increased in NMDA group in both time-and dose-dependent manner when compared with the control group(P<0.05,the difference was statistically significant),consistent with the alterations induced by glutamate.(3)MK-801 treatment: Western blot analysis showed that MK-801 significantly inhibited the increase of PPARγ expression induced by glutamate in a dose-dependent manner,indicating that the effect of glutamate on PPARγ protein is mainly mediated by NMDA receptors.(4)Other treatments: Western blot analysis showed that compared with NMDA group,with the administration of GSH,BAPTA-AM,Calpeptin and Ac-DEVD-CHO,the increase of PPARγ expression induced by NMDA was inhibited(P<0.05,the difference was Statistically significant),demonstrating that the effect of glutamate on PPARγ is related to oxidative stress and calcium-calpain-caspase 3 signaling pathway.3.The effect of glutamate on PPARγ activity:(1)p-PPARγ expression: Western blot analysis showed that NMDA treatment caused a time-dependent increase in p-PPARγ expression from 3-24 h.(2)PPARγ expression: Western blot analysis showed that there was no significant change in PPARγ at 3 and 6 h after NMDA treatment,and PPARγ expression was significantly increased at 12 and 24 h.(3)p-PPARγ/ PPARγ ratio: NMDA treatment caused a biphasic change in the ratio,which increased 3-6 h and decreased at 12-24 h,suggesting that NMDA can cause transient inhibition of PPARγ activity by inducing PPARγ phosphorylation.(2)p-ERK1/2 expression: Western blot analysis showed that NMDA caused an increase in p-ERK1/2 expression in a time-and dose-dependent manner(P<0.05,the difference is statistically significant),and the total ERK1/2 wasunchanged.(3)The role of ERK1/2 inhibitor U0126: U0126 inhibited the expression of p-ERK1/2 and p-PPARγ induced by NMDA,suggesting that NMDA can induce PPARγ phosphorylation by activating ERK1/2.4.Protective effects of protectant(Ros,Pio and U0126)on glutamate excitotoxicity:(1)HE staining and TTC staining showed that the treatment with individual protectant significantly reduced NMDA-induced cortical injury when compared with NMDA group.(2)Western blot analysis showed that the protectants Ros,Pio and U0126 significantly inhibited the expression of COX-2,iNOS and cleaved caspase-3 induced by NMDA(P<0.05,the difference has statistically significant).These suggest that activating PPARγ or protecting its activity has a protective effect against glutamate excitotoxicity.Conclusion:1.Glutamate produces excitotoxic damage mainly through the NMDA receptor.2.Glutamate damage caused increased expression of PPARγ in neurons,which effect may be NMDA receptor-dependent,calcium ion-calpain-caspase-3 mediated,and oxidative stress-related and is possibly in association with theself-defense response of neuron to oxidative stress.3.Glutamate excitotoxic damage caused transient PPARγ activity inhibition by increasing PPARγ phosphorylation modification in early phase,which may be related to glutamate activation of MAPK/ERK1/2 pathway.4.Rosiglitazone,piolitazone,and U0126 have protective effects on the excitotoxicity caused by NMDA.Its mechanism may be related to the activation of PPARγ or protection its activity,resulting in direct or indirect anti-inflammatory,anti-oxidative,anti-apoptotic effects. |
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