| PART ? THE EFFECT OF 20-HETE INHIBITOR ON RAT MICROGLIA ACTIVATION AFTER TRAUMATIC BRAIN INJURYObjective: To investigate the characteristics of microglia activation after traumatic brain injury,as well as the effect of 20-HETE inhibitor(HET0016)on rat microglial cells.Methods: Male Sprague-Dawley rats at postnatal day 9 were divided into sham group,TBI+Vehicle group,TBI+HET0016 group randomly.Sham group was underwent craniotomy and stitching.The other two groups were underwent controlled cortical impact.to establish a TBI model The HET0016 group was treated with 1mg/kg HET0016 solution at 5min and 3h after injury through intraperitoneal injection.The TBI+Vehicle group was treated with 45% ?-cyclodextrin solution at equivalent volume.After 3 days,brains were removed after the transcardial perfusion with ice-cold PBS and 4% paraformaldehyde and then were dehydrated in 10%,20%,and 30% gradient sucrose solution.Brain tissue sections of different regions were collected to Iba-1 DAB staining to observe the regional distribution characteristics of the microglial cells after traumatic brain injury,and its morphological changes.We constructed CD68/Iba-1 immunofluorescence to observe the activation characters and the effect of HET0016 on microglia in those areas,including rat's cortex and hippocampus.Results: Iba-1 immunohistochemical analysis shows that the expression level of microglial cells are different in different areas.The results show that the microglial cells were widespread activation after TBI,the injured side was more serious than the other side and the morphology of microglia have changed.Immunofluorescence staining of CD68/Iba-1 indicated that with the treatment of HET0016,CD68/Iba-1 expression was attenuated in cortex and hippocampus areas.Conclusion: The overall results demonstrated that the distribution of microglial cells have regional character.Microglial cells were activated and experienced some morphological changes after injury stimulation.The number of microglia in the injuried side is more than the other side.it is probably that the activated microglia may happen proliferation or migration.The number of activated microglia were directly decreased by HET0016.PART ? THE EFFECT OF 20-HETE INHIBITOR ON PRIMARY MICROGLIA PROLIFERATION AND MIGRATIONObjective: To study the effect of HET0016 on primary microglial cells' including the proliferation and migration.Methods: Primary microglial cells were cultured in vitro,divided into Control group?HET0016 group?LPS group and LPS+HET0016 group.LPS group were treated with 50ng/m L LPS.HET0016 were treated with 1?M HET0016.Cell proliferation was measured by flow cytometry,CCK8 assay and MTT assay.Cell migration was detected by cell scratch and Transwell in 24 h.P50?P65 protein expressions were found out by Western blot.ELISA assay was used to detect the expression of 20-HETE.Results: The primary microglial cells have high purity with a clear and stable morphology.Cells were identified with CD11 b,which is the specific surface antigen of microglia.The flow cytometry results showed that the proportion of S phase in HET0016 group is lower than other groups(P<0.05).The result in LPS group is higher than control group(P<0.05).CCK-8 assay showed that the OD value of different times(12h,24 h,48h,72h)of HET0016 treated cells were lower than the control group(P<0.05).The MTT results had the same trend.The results of cell scratch showed that the change of migration area in LPS group is much higher than other groups(P<0.05).The area in HET00116 is lower than control group(P>0.05).According to the results of Transwell,the number of migration cells in LPS group are much higher than other groups(P<0.05).Western blot results indicated that HET0016 can decrease the expression of P50?P65 protein without significant difference.LPS group had a higher expression level(P<0.05).ELISA results showed that HET0016 can reduce the expression levels of 20-HETE(P<0.05).Conclusion: The results of this study showed that HET0016 can effectively inhibit the proliferation and migration of microglial cells,and its mechanism may be related to down-regulating the protein expression of P50,P65 through the NF-?B signaling pathway. |
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