The Study Of APOE Polymorphism Affecting P38MAPK Signaling Mediated Post-traumatic Inflammatory Injury Of Astrocytes

Traumatic brain injury (TBI), ranking the second in systmetic trauma while the first in mortality and disability, causes direct and indirect economic loss amounting to 100 billion yuan every year in China, which has been a public nuisance, and is always the continuous exploration field for majority neuroscientists. Apolipoprotein E gene (APOE) has three alleles,ε2,ε3 andε4. Abundant foreign studies and previous works by our group showed that,ε4 carriers present lower tolerance to TBI, and are more susceptible to aggravation and bad outcome posttrauma. But the mechanism by which APOE influences the progression and outcome of TBI remains incompletely understood. In the end, apoE plays neurobiological roles via regulating lipid distribution and metabolism, and the close association between lipid metabolism and inflammation has been an increasing concern by foreigen scholars since the 1950s. Meanwhile, APOE itself can directly trigger multiple system organ acute or chronic inflammatory reaction. We speculated that, therefore, there is a difference in TBI triggered inflammation, and this inflammation induced neural injury due to the difference in APOE genotypes. APOEε4 carriers showed stronger traumatic inflammatory injury than the other two types, resulting difference in prognosis. This study takes the relationship between the APOE genotype and inflammatory injury posttrauma as a starting point, constructing the recombinant plasmid pEGFP-N1-APOE carrying each APOE genotype (ε2,ε3,ε4), transfecting the recombinant plasmid into astrocytes of APOE knock-out mice. Astrocytes stablely expressing APOE were purified by single cell cloning culture to establish an in vitro model of mechanical injury. Reverse transcription polymerase chain reaction (RT-PCR) and werstern blot were used to study the relationship between APOE subtype-specifcity and the early expression of p38 mitogen-activated protain kinase (MAPK)-neuclear factor kappa B (NF-κB) inflammatory signaling, aiming to uncover the possible mechanism by which APOE affects the second injury of neurons and glial cells.PartⅠConstruct and identification of eucaryotic expression vector carrying human APOE genotypeTotal RNA was abstracted from human embryo pallium of fetus, primer was designed to amplify the CDS region of APOEε3, then site-directed mutagenesis was used to obtain the cDNA encoding APOEε2 andε4 isoforms. Then restriction digested with EcoR I and BamH I and insert into pEGFP-N1. Recombinant plasmid was identified by enzyme digestion, PCR and sequence analysis. Recombinant plasmid was transfected into APOE knocked out astrocytes with Trans-EZ and an in vitro trauma model was produced by scratch injury. Results: Results of enzyme digestion, PCR and sequence analysis of recombinant plasmid demonstrated that APOE gene (ε2,ε3,ε4) were correctly inserted into eucaryotic expression vector pEGFP-N1. After transfected into astrocytes, recombinant plasmid could express successfully in cells. Conclusion: A green fluorescent protein reporter gene vector containing human APOE genotype was successfully constructed, thus providing an important and convenient tool to transfect NSCs of APOE knock-out mice, and the cell trauma model was created.PartⅡThe effect of APOE subtype specificity on the early intracellular Ca2+ concentration and expression of NF-κB posttrauma.Time point was setup at preinjury, 12h, 24h, 48h and 72h after injury, laser confocal scanning microscope (LCSM) was used to to observe the fluorescence intensity (FI) of intracellular Ca2+ of three genotype astrocytes, aiming to compare the mechanical force induced injury between three genotypes. At the same time point, expression of NF-κB of three genotype astrocytes was dectected by RT-PCR and western blot methods, aiming to compare the difference of traumatic inflammation between three genotypes. Results: Since 24h after injury, APOEε4 transfected astrocytes showed significantly higher Ca2+ FI thanε2 andε3 astrocytes, accordingly, the expression of NF-κB of APOEε4 astrocytes was significantly higher than the other two. Conclusions: There is a significant difference of NF-κB expression and intracellular Ca2+ between APOEε4 and the other two genotypes, which may contribute to bad outcome posttrauma.PartⅢThe effect of p38MAPK inflammatory signaling on differentiated expression of NF-κB posttrauma between three APOE genotype astrocytes.Time point was setup at preinjury, 12h, 24h, 48h and 72h after injury to detect expression of p38MAPK of three genotype astrocytes by RT-PCR and western blot methods. Meanwhile, SB203580, the specific inhibitor of p38MAPK was used to observe the difference of NF-κB expression before and after intervention. Results: Expression level of p38MAPK was significantly higher in APOEε4 as compared toε2 andε3 astrocytes. After inhibiting the expression of p38MAPK, expression of NF-κB of three genotype cells decreased, and there is no significant difference of expression of NF-κB betweenε4 and the other two types. Conclusions: In the early stage after injury, APOEε4 expressed higher level of p38MAPK, which may contribute to more intensive inflammation posttrauma.