CD36 Inhibition Mitigates White Matter Injury After Traumatic Brain Injury By Regulating Microglia Polarization

Part Ⅰ.Effects of CD36 inhibition on microglial polarization and white matter function in mice after TBIObjective: To establish a traumatic white matter injury model and explore the effects of CD36 receptor inhibition on white matter injury,neurological recovery and the polarization of microglia around the injury in mice.Methods: C57 mice were randomly divided into Sham group(n=36),TBI group(n=36)and SSO group(n=36).Neurobehavioral experiments were performed on mice at different time after TBI to observe the recovery of nerve function.7 days after TBI,HE staining,LFB staining,immunofluorescence staining,transmission electron microscopy and real-time quantitative PCR(q PCR)were performed on the brain tissues of mice in each group to observe the polarization of microglia and the recovery after WMI.LFB staining,immunofluorescence staining and transmission electron microscopy were performed on the brain tissues of mice in each group 35 days after injury to observe the recovery of white matter injury in the long term.Results: HE staining showed that the cortex of the brain tissue of the mice in the TBI and SSO groups had defects,and the corpus callosum and part of the striatum were broken,but the degree of brain tissue damage in the SSO group was less than that in the TBI group.LFB staining showed that both the TBI and SSO groups showed demyelination,while the demyelination degree of the SSO group was lower than that of the TBI group.Immunofluorescence showed that the fluorescence intensity of MBP in the SSO group was stronger than that in the TBI group,while the fluorescence intensity of SMI-32 in the SSO group was weaker than that in the TBI group.Electron microscope observation revealed that the axons in both TBI and SSO groups were demyelinated,and the demyelination in the SSO group was milder than that in the TBI group.Neurobehavioral experiments showed that the recovery of neurological function in the SSO group was better than that in the TBI group.Immunofluorescence cell counts showed that there were no co-stained cells in the Sham group.Besides,i NOS+ and Arg1+ cells were found in both the TBI group and the SSO group.The number of Arg1+ cells in the SSO group was significantly more than that in the TBI group,while the number of i NOS+ cells was significantly less than that in the TBI group.q PCR test showed that the m RNA expressions of i NOS and CD16 in the SSO group were significantly lower than those in the TBI group but significantly higher than those in the Sham group.The m RNA expressions of TGF-β and CD206 in the TBI group were significantly lower than those in the SSO group but significantly higher than those in the Sham group.Conclusions: Traumatic white matter injury was successfully established using the CCI devi,and significant myelin damage,oligodendrocyte reduction,and microglial aggregation were found.In addition,after traumatic white matter injury,CD36 inhibitors can significantly promote the repair of white matter structure and function.Finally,CD36 inhibitors can inhibit the formation of M1-type microglia and promote the increase of M2-type microglia in the brain tissue surrounding the injury in micePart Ⅱ.The mechanism of CD36 regulating microglial polarizationObjective: Cell experiments were used to further clarify the mechanism of CD36 receptor regulation of M1 and M2 microglia formation.Methods: BV2 cells and CD36-KD BV2 cells were divided into BV2 group or CD36-KD BV2 group,LPS BV2 group and LPS CD36-KD BV2 group according to whether LPS(100ng/ml)intervention was performed.The cells were incubated in the incubator for 24 h and collected for immunofluorescence staining,q PCR detection,NO detection by Griess method and Western blot detection.Results: Compared with the BV2 group,the relative expressions of CD36 m RNA and protein in the CD36-KD BV2 group were significantly decreased,while there was no other significant difference between the two groups.The relative expressions of i NOS,CD16,TGF-β and CD206,the density of CD16/32+ cells and CD206+ cells,and the NO content in the LPS BV2 and LPS CD36-KD groups were significantly higher than those in the BV2 group.Compared with LPS BV2 group,the m RNA expressions of i NOS and CD16,the density of CD16/32+ cells and the content of NO were significantly decreased in the LPS CD36-KD BV2 group,while the m RNA expressions of TGF-β and CD206,and the density of CD206+ cells were significantly increased in the LPS CD36-KD BV2 group.WB results showed that,compared with the BV2 group,the phosphorylation levels of ERK1/2,JNK,and p38 in the LPS BV2 group and LPS CD36-KD BV2 were all increased,while the p38 phosphorylation level in the LPS CD36-KD BV2 group was lower than that in the LPS BV2 group.Conclusions: The expression of CD36 exists on the surface of microglia,and lentiviral RNA interference with microglia can significantly inhibit the expression level of CD36 in microglia.In addition,microglia can be polarized into M1-type microglia after LPS stimulation.Inhibition of CD36 can promote the transformation of LPS-induced M1-type microglia to M2-type.Finally,when microglia were stimulated by LPS,the phosphorylation level of MAPK pathway was up-regulated,and inhibition of CD36 receptor can make LPSinduced M1 microglia transform to M2 type by inhibiting the phosphorylation of p38.Part Ⅲ.Inhibition of CD36 relieves white matter injury by regulating microglial polarizationObjective: Microglia-conditioned medium was used to further clarify the mechanism of how inhibition of CD36 reduces white matter injury.Methods: According to whether CD36 was knocked down and whether LPS was used for intervention,microglia were divided into BV2 group,CD36-KD BV2 group,LPS BV2 group and LPS CD36-KD BV2 group.Meanwhile,oligodendrocytes were subjected to oxygen-glucose deprivation(OGD)treatment for 2 h,and then normal culture for 24 h.The culture medium of the microglial cells collected from each group was added to the oligodendrocyte culture medium,and the oligodendrocytes were also divided into BV2 group,CD36-KD BV2 group,LPS BV2 group and LPS CD36-KD BV2 group.After 24 hours of incubation,the OGD oligodendrocytes in each group were subjected to cellular immunofluorescence staining,CCK-8 detection and LDH release detection.Results: The results of immunofluorescence staining,CCK-8 and LDH release showed that there was no significant difference in MBP expression,cell viability,LDH release and branch structure complexity of oligodendrocytes between BV2 group and CD36-KD BV2 group.Compared with the BV2 group,the MBP expression,branch structure complexity and cell viability of the oligodendrocytes in the LPS BV2 group were significantly decreased,while the LDH release was significantly increased.Compared with the LPS BV2 group,the MBP expression,branch structure complexity and cell viability of the oligodendrocytes in the LPS CD36-KD BV2 group were significantly increased,while the LDH release was significantly decreased.Conclusions: Oxygen-glucose deprivation experiment can lead to the decrease of oligodendrocyte cell viability and function,and the increase of cell membrane permeability.In addition,cytokines in BV2 cells after LPS induction can aggravate the damage of OGD oligodendrocytes,while inhibition of CD36 can significantly alleviate the effects above.Taken together,CD36 inhibition mitigates white matter injury after traumatic brain injury by regulating microglia polarization via MAPK signal pathway.