| Objective:Numerous studies have shown that the activation of microglia was involved in neonatal hypoxia-ischemia.Within the injured brain,classic activation(M1)and alternative activation(M2)of microglia exert proinflammatory and anti-inflammatory/repair functions,respectively.In the early stage of hypoxia,microglia rapidly activated,showing M1 phenotype,and switched to M2 phenotype after about 24h.The molecular mechanism of gliocyte activation in HIE could provide a novel target for the treatment of HIE children.The results showed that the expression of TSPO in resting microglia was very low.When the microglia was activated due to the damage of nervous system,the level of TSPO was significantly increased,indicating that the activation of microglia may be related to the expression of TSPO.The activation of PPAR?pathway could also participate in the clearance of hematoma and the protection of nerve function.In this study,we investigated whether TSPO mediating PPAR?pathway can regulate microglial cell polarization to M2 type through in vivo and in vitro experiments,and find a novel target for the treatment of neonatal ischemia and hypoxia.Methods:In vivo experiment:In this experiment,Rice-Vannucci method was used to establish hypoxia and hypoxic-ischemia to simulate hypoxia and hypoxic of human newborn.The rats were divided into four groups:group A(sham group,n=20),group B(model group,n=20),group C(model+PBS injection group,n=20),and group D(model+atriol injection group,n=20).Nissl staining was carried out on the third day after the model establishment in each group,and the water content and Evans blue(EB)content of brain tissue were detected.Western blot was used to detect the expression of PPAR?pathway protein and apoptosis related protein Bax,Bcl-2 and Caspase-3 in different groups.The costaining levels of caspase-3,Iba-1+CD16/32 and Iba-1+CD206were detected.In vitro experiment:The primary microglia of BALB/c mice were isolated,and the primary microglia in the logarithmic growth stage were inoculated into 48 well plates according to the cell number of 1×10~4.The microglia were treated with 20 ng/ml r IL-4 for0,6,12 and 24 hours respectively.The cells were divided into five groups:Control group,IL-4 group,IL-4+atriol group,IL-4+FGIN-1-27 group,and IL-4+HA-TSPO group.Five groups of cells were placed in a cell incubator at 37?for 12 hours.Western blot was used to detect the expression of TSPO and PPAR?protein in each group.The expression of TSPO,PPAR?m RNA and M2 polarized microglial markers CD206,Arg-1,YM-1 and FIZZ-1 were detected by real-time quantitative PCR,and the expression of BDNF,CNTF-1,IGF-1 and NGF-1 cytokines were detected by ELISA.Results:In vivo experiment:(1)On the third day after the model establishment,there were obvious edema on the injured side of rats in group B and C,while the edema on the injured side of rats in group D was significantly lower than that in group B and C.After statistical analysis,the water content of brain in group B,C and D were significantly higher than that in group A,while the water content of brain in group D was significantly lower than that in group B and C(P<0.05).At the same time,the content of EB in group B,C and D were significantly higher than that in group A,while the content of EB in group D was significantly lower than that in group B and C(P<0.05).Nissl staining showed that the cells in hippocampus and cerebral cortex of group A were arranged orderly and had normal structure,while the above cells in group B and C were arranged disorderly.The loss of cells in group B was significantly higher than that in group A(P<0.05).However,the degree of necrosis and degeneration of the right brain tissue in group D were significantly lower than that in group B and C,and the loss of cells in group D was significantly lower than that in group B and C(P<0.05).(2)Compared with group A,the expression level of Neu N+caspase-3 in brain tissues of group B,C and D increased significantly,while the expression level of Neu N+caspase-3 in brain tissues of group B and C was significantly higher than that of group D(P<0.05).There was no apoptosis in the neurons of group A,but the expression level of Caspase-3 and Bax protein in the brain tissue of group B,C and D were significantly increased,and the expression level of Bcl-2 protein was significantly decreased;at the same time,the expression level of Caspase-3 and Bax protein in the brain tissue of group B and C were significantly higher than that of group D,and the expression level of Bcl-2protein was significantly decreased compared with that of group D(P<0.05).(3)There was no significant difference in the levels of Iba-1+CD206 and Iba-1+CD16/32 fluorescent protein Co located positive cells between group B and group C(P>0.05),but the levels of Iba-1+CD206 and Iba-1+CD16/32 fluorescent protein Co located positive cells in group B and group C were significantly higher than those in group A(P<0.05).In group B and C,the level of Iba-1+CD16/32 cells was significantly higher than that of Iba-1+CD206 cells.But in group D,the level of Iba-1+CD16/32 cells was significantly lower than that of Iba-1+CD206 cells(P<0.05).(4)The expression level of PPAR?protein in group B,C and D was significantly lower than that in group A(P<0.05),while the expression level of TSPO protein in group D was significantly higher than that in group B and C(P<0.05),while the expression level of TSPO protein in group D was significantly lower than that in group B and C(P<0.05).In vitro experiments:(1)The microglia mainly showed long fusiform,round and Amoeba like,and the cells grew close to the wall under the microscope.The purity of microglia was more than 96%by double immunofluorescence.(2)To investigate the expression and function of TSPO in M2-polarized microglia,primary microglia of mice were cultured in vitro.After purification,microglia were exposed to 20ng/ml r IL-4 for 0,6,12,and 24 h,and the protein and m RNA levels of TSPO and PPAR?in microglia were evaluated.Results of Western blot showed that protein expression of TSPO was gradually decreased by r IL-4 at 6 and 12 h,and slightly recovered at 24 h after treatment.The PPAR?protein level was significantly increased after r IL-4treatment;the highest expression was achieved at 12 h.As expected,similar trends of m RNA levels of TSPO and PPAR?were found using quantitative PCR.(3)To further examine the role of TSPO in M2-polarized microglia,microglia were treated with different TSPO ligands for 12h and the expression level of PPAR?was determined.It is well established that activated PPAR?is localized in the nucleus and causes activation or repression of target genes.Nuclear protein and cytoplasmic protein of microglia were extracted before immunoblot to analyze the expression level of PPAR?in the cytoplasm and nucleus.Interestingly,TSPO antagonist Atriol enhanced PPAR?expression in M2-polarized microglia,while TSPO agonist FGIN-1-27 inhibited PPAR?expression both in the cytoplasm and nucleus.TSPO overexpression in microglia also significantly suppressed PPAR?expression induced by IL-4 in the cytoplasm and nucleus.There was no change of PPAR?expression in the group treated with Atriol,FGIN-1-27,or TSPO overexpression alone.These results indicated that PPAR?expression and activation in M2-polarized microglia were regulated by TSPO.(4)To determine whether the M2 polarization of microglia induced by r IL-4 was affected by TSPO,expression of M2 markers in microglia was assessed using real-time PCR after TSPO ligand treatment.Compared with the control group,r IL-4 clearly increased the expression of CD206,Arg-1,YM-1,and FIZZ-1 in microglia,which indicated that microglia were M2 polarized.The results showed that TSPO antagonist Atriol enhanced the expression of CD206,Arg-1,YM-1,and FIZZ-1 induced by r IL-4,while FGIN-1-27 and TSPO overexpression attenuated the expression of these genes,comparing with the r IL-4 group.These results suggest that TSPO is an important regulator that participates in M2 polarization of microglia.(5)To determine the involvement of TSPO in the pro-trophic capacity of microglia,we exposed microglia in culture to TSPO ligands and detected levels of brain-derived neurotrophic factor(BDNF),ciliary neurotrophic factor 1(CNTF-1),insulin-like growth factor 1(IGF-1),and nerve growth factor 1(NGF-1)in the medium.Exposure to r IL-4markedly induced the release of BDNF,CNTF-1,IGF-1,and NGF-1 from microglia,comparing with the control group.Levels of BDNF,CNTF-1,IGF-1,and NGF-1 were enhanced by Atriol and suppressed by FGIN-1-27 and TSPO overexpression,thereby suggesting that the TSPO pathway is involved in pro-trophic capacity of microglia.Conclusions:In this study,TSPO mediating PPAR?pathway can promote polarization of M2 microglia and alleviate secondary neurological injury in nenonatal rats with brain ischemic and hypoxic injuries. |
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