| Objectives: 1.Set up a model of ischemic preconditioning(IPC)to study the mechanism of PPARγ in IPC.2.Observe the expression of PPARγ and LPL,CD36,CAT,Bcl-2 in brain and peripheral blood mononuclear cells(PBMCs)over time.Observe the location of PPARγ in brain.Discuss the influence and meanings of these changes.3.Observe the expression and activity of PPARγ and NF-κB in brain and PBMCs after IPC-ischemia/reperfusion(IPC-I/R).Observe the expression of COX-2 and 3-NT in brain and CD36 in PBMCs after IPC-I/R over time.Evaluate whether IPC exert a protective effect on subsequent brain ischemia/reperfusion injury and discuss the mechanism at the same time.Methods: 1.Use the methods of bilateral common carotid artery occlusion(BCCAO)to set up a model of IPC,and evaluate the feasibility of the model via mNSS,TTC staining,Hoechst staining and expression of HSP70.2.Adult male SD mice were randomized into two groups,IPC group and Sham group.Each group was divided into 4 subgroups according to different times.Use western blot to detect the expression of PPARγ,LPL,CD36,CAT,Bcl-2 in brain and PBMCs.Use double immunofluorescence to detect the location of PPARγ in different kinds of cells.3.Adult male SD mice were randomized into two groups,IPC-I/R group,I/R group and Contrl group.The first two groups were divided into 4 subgroups according to different times.(1)Compare mNSS and infarct volume between IPC-I/R group and I/R group.(2)Western blot detect the expression of PPARγ in brain and immunofluorescence detect the location.Compare the expression and activity of PPARγ between IPC-I/R group and CI group.(3)Western blot detect the expression of NF-κB and COX-2 in brain.Immunofluorescence detect the location of NF-κB and the expression 3-NT in brain.Evaluate the inflammation in brain after IPC-I/R.(4)Western blot detect the expression of PPARγ and CD36 in PBMCs.Compare the expression and activity of PPARγ between IPC-I/R group and I/R group over time.(5)Western blot detect the expression of NF-κB in PBMCs.Evaluate the inflammation in PBMCs after IPC-I/R.Result: 1.Evaluation of IPC model: The model had no neurological deficits,infarct volume,and apoptotic cells,with HSP70 expression significantly increasing(P<0.001).2.The expression of PPARγ in brain and PBMCs after IPC: WB detected that the expression of PPARγ in brain and PBMCs increased first and then decreased,with the third day highest.The target genes of PPARγ,such as LPL,CAT,Bcl-2,CD36,presented the same trend.Immunofluorescence detected that expression of PPARγ was mainly located in neurons and little in microglia,astrocytes and endothelial.3.Comparision of IPC-I/R group and I/R group.(1)Evaluation of mNSS and infarction volumes: IPC-I/R group has less mNSS and infarction volume(all P<0.05).(2)Western blot analysis: 1)PPARγ in brain: the expression of PPARγ in both IPC-I/R group and I/R group increased first and then decreased,with the third day highest.Compared to I/R group at the same time,IPC-I/R-1d,IPC-I/R-7d and IPC-I/R-12 d subgroups were siginificantly higher(all P<0.05),while IPC-I/R-3d subgroup was lower(P<0.05).2)NF-κB in brain: In IPC-I/R group,the expression of NF-κB decreased gradually with “IPC-I/R-1d subgroup” highest.In I/R group,the expression of NF-κB increased first and then decreased,with the third day highest.Compared to I/R group at the same time,all IPC-I/R subgoups were significantly lower(all P<0.05).3)COX-2 in brain: the expression of COX-2 in both IPC-I/R group and I/R group increased first and then decreased,with the third day highest.Compared to I/R group at the same time,all IPC-I/R subgoups were significantly lower(all P<0.05).4)PPARγ in PBMCs: the expression of PPARγ in both IPC-I/R group and I/R group increased first and then decreased,with the third day highest.Compared to I/R group at the same time,IPC-I/R-1d subgroup was similar to I/R subgroup(P>0.05),while IPC-I/R-3d,IPC-I/R-7d,IPC-I/R-12 d subgoups were significantly higher(all P<0.05).5)CD36 in PBMCs: the expression of CD36 in both IPC-I/R group and I/R group increased first and then decreased,with the third day highest.Compared to I/R group at the same time,all IPC-I/R subgoups were significantly higher(all P<0.05).6)NF-κB in PBMCs: NF-κB expression in IPC-I/R group had no significant trend over time.NF-κB expression in I/R group increased first and then decreased,with the third day highest.Compared to I/R group at the same time,all IPC-I/R subgoups were significantly lower(all P<0.05).(2)Immunofluorescence analysis: 1)PPARγ in brain: PPARγ in IPC-I/R group was mainly located in nucleus of neurons,while PPARγ in I/R group mainly located in cytoplasm of neurons.2)NF-κB in brain: NF-κB in I/R group was mainly located in nucleus of neurons,while NF-κB in IPC-I/R group mainly located in cytoplasm of neurons.3)3-NT in brain: Compared to I/R group at the same time,all IPC-I/R subgoups were significantly lower(all P<0.05).Conclusion: 1.The expression and activity of PPARγ in brain and PBMCs are enhanced after IPC.IPC improves the anti-inflammatory,anti-oxidant and anti-apotptic capacity of neurons via increasing the expression and activity of PPARγ in neurons.IPC can also provide system protection via regulating PPARγ expression and activity in PBMCs.2.The expression and activity of PPARγ after IPC in brain and PBMCs are highly concordant,therefore,PPARγ can be a biomarker for IPC.3.IPC can protect subsequent brain ischemia/reperfusion injury.IPC can attenuate inflammation both in central nervous system and peripheral immune system.The mechanism may be related to the increasing expression and activity of PPARγ. |
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