Protective Effect And Mechanism Of Activated PPAR-? On White Matter After Traumatic Brain Injury

Objective To investigate the protective effect of PPAR-?agonist,rosiglitazone?RGZ?on the white matter of traumatic brain injury,and examine whether epigenetic regulation of microglia polarization is involved in the mechanism of promoting remyelination.Methods In vivo,TBI was induced in C57/BL6 mice by a controlled cortical impact?CCI?mouse model.Different doses of RGZ were intraperitoneally administrated at 2 h,12 h,24 h and 48 h after TBI.Effect of RGZ on the neurological function of mice was examined at 1,3,5,7,14,and 28 days after brain injury by rotarod and beam walking test.Cognitive performance was detected in the Morris water maze test at 30-35 days post-TBI.The changes of brain lesion volume and myelin of corpus callosum were observed at 35 days post injury by nissl and fast blue staining.Immunohistochemistryfluorescencelabelingandreal-time quantitative PCR were adopted to detect the polarization of M1 and M2microglia.Remyelinationwasobservedby immunohistochemistry and western blotting at days 7 and 35 after injury.Double labeling of NG-2/Brdu and Olig-2/Brdu were used to observe the proliferation and differentiation of oligodendrocyte precursor cells?OPCs?.The protein changes of histone deacetylase and acetylase histone H3 and H4 were examined by western blotting.PPAR-?was knockdown in the corpus callosum of mouse by lentivirus interfered at 3 days after TBI,and the changes of myelin and microglia polarization in brain tissue were further observed.In vitro,PMA induced the suspension of THP-1 cells adherent,then treated with LPS+IFN-?,LPS+IFN-?+RGZ,IL-4+IL-13,RGZ,and PBS Control group respectively.After cultured for 48 hours,cell immunofluorescence,Nile red staining,western blotting and real-time PCR were detected the effect of RGZ on macrophage polarization.In vitro experiments using a neuron and OPCs co-cultured system was used to further elucidate the effect of RGZ on the remyelination.Results 1.Compared with the control group,RGZ?2 mg/kg and 4 mg/kg?significantly increased the time spent on the roller wheel and reduced the number of hindlimb staggered times when crossing the balance beam after TBI.However,the dose of RGZ?1mg/kg?treatment had no significant effect.In addition,the latency that mice found the platform in morris water maze test was also significantly shortened by RGZ treated.In the open-field experiment,the total numbers that RGZ-treated group explored two objects were significantly more than that of the Control group,even close to the sham group.2.At 35 days after TBI,it was showed that RGZ?2 mg/kg and4 mg/kg?significantly reduced the volume of brain tissue defects and increased the area of injury corpus callosum?p<0.05?.There was no difference between these two treatment groups.3.RGZ treatment elicited robust white matter protection at both 7 and 35 days after TBI,as demonstrated by reductions in abnormally dephosphorylated neurofilament proteins SMI-32,increases in MBP staining.The similar results of MBP expression was confirmed by Western blotting?p<0.05?.4.Compared with the brain injury group at 3 and 7 day,RGZ?2 mg/kg?significantly reduced the number of M1(CD16^+/Iba-1⁺co-staining)microglia,while M2?CD206⁺/Iba-1⁺?microglia significantly increased.Real-time quantitative PCR also showed that CD16 mRNA was down-regulated and CD206 mRNA up-regulated?p<0.05?by RGZ treated.5.In vitro,RGZ significantly down-regulated the mRMA levels of TNF-?and IL-1?and the fluorescence intensity of CD86?p<0.01?in macrophages,and upregulated the expression of CD206mRNA and fluorescence intensity.Morever,RGZ significantly increased the relative numbers of phagocytosed Nile red granules in macrophages and up-regulated the gene expression of PPAR-?mRNA?p<0.05?.6.RGZ treatment produced a significant increase in co-localization of NG-2/Olig-2 and BrdU cells at 7 and 21 days post injury.Furthermore,RGZ promoted the differentiation of OPCs toward mature oligodendrocyte,as revealed by increased expression of MBP?oligodendrocyte marker?by neuron-OPC co-culture system.7.RGZ treatment group had no obvious effect on HDAC1 and3 protein expression,but increased the expression of acetylated histones H3,H4 and PPAR-??p<0.05?at 3 days after TBI.PPAR-?and acetylated-H3,H4 expression were significantly down-regulated following lentivirus interference with PPAR-??p<0.01?,and increased HDAC3 expression.Moreover,it was also found that M1type microglia was decreased while the number of M2 type microglia increased in RGZ-treated group by fluorescent labeling.Conclusions RGZ improved the sensory,motor and cognitive function of mice through potent protection of white matter after TBI.Inaddition,RGZpromotedthepolarizationof microglia/macrophages from harmful M1 type to beneficial M2 type,which reduced neuroinflammation response,and might provide a favorable micro-environment for the differentiation and maturation of oligodendrocyte precursor cells and remyelination of neurons.Furthermore,RGZ activated PPAR-?can increase the level of acetylated-H3,H4 and may reduce the activity of HDAC3,then help to promote the normal transcription of genes and protein translation.The present study provide a new therapeutic directions and theoretical basis for the application of PPAR-?Agonists in the treatment of white matter injury after TBI.