X. Wu1, P. Chang1, R. Zeng1, Y. Deng1, Y. Zhao1, W. Chong1 1The First Affiliated Hospital Of China Medical University,Emergency,Shenyang, LIAONING, China
Introduction: Secondary hit is regarded as a lethal complication of traumatic brain injury (TBI), due to severe inflammation response induced by ischemia and hypoxia. Recently, it has been demonstrated that macrophages are mainly involved in inflammation with two different functional phenotype (or polarization): ones that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation are called M2 macrophages. In TBI, microglia have the similar phenotype conversion as macrophages. Researches show that valproic acid (VPA) and molecular hydrogen (H2) attenuate cerebral swelling and manifest nervous protection in animal model of TBI in uncertain ways. The hypothesis in this study was that hypoxia enhances microglia polarization toward the pro-inflammatory phenotype. Both VPA and H2 inhibit hypoxia-induced inflammatory effects on microglia.
Methods: Mouse BV2 microglia were divided into 4 groups: Sham (no hypoxia, no VPA), hypoxia group, VPA group and H2 group. Hypoxia and VPA groups were exposed to hypoxic conditions (0.5% O2, 10% CO2, and 89.5% N2) at 37°C in the absence or presence of VPA (3mM) for 2 hours. H2 group were treated with H2 (0.5%O2, 2%H2, 10%CO2, 87.5%N2) for 1 hour under same hypoxic condition. Enzyme-Linked Immunosorbent Assay (ELISA), flow cytometry and Real-Time PCR were performed to assess protein and mRNA levels of phenotypic markers in microglia respectively.
Results: Hypoxia increased mRNA levels of M1 markers and reduced mRNA levels of M2 markers in microglia (P<0.05). In addition, the ratio between the mRNA levels of M1 markers and M2 markers were also increased by hypoxia (P<0.05). VPA reduced protein and mRNA levels of M1 markers (P<0.05), decreased the ratio between the mRNA levels of M1 markers and M2 markers of microglia (P<0.05) compared with hypoxia group. H2 not only decreased protein and mRNA levels of M1 markers (P<0.05) but also increased cytokine secretion and mRNA levels of M2 markers in microglia (P<0.05), and lowered the ratio between the mRNA levels of M1 markers and M2 markers of microglia as well in comparison with hypoxia group (P<0.05).
Conclusion: Hypoxia enhances microglia polarization toward the pro-inflammatory phenotype. Both VPA and H2 attenuate hypoxia-induced pro-inflammatory effects on microglia.
