The roles and the kinetics of gp91 phox after traumatic brain injury in mice

摘要：我们假设gp91phox （NOX2）是NADPH氧化酶的一个亚基，产生超氧阴离子（O2-），并在创伤性脑损伤（TBI）中起主要的致病作用。为了评估gp91phox和活性氧（ROS）在TBI中的功能作用，我们对gp91phox基因敲除小鼠（gp91phox-/-）进行了控制性皮质影响。我们还使用小胶质细胞系来确定促进gp91phox产生的活化细胞表型。方法：在gp91phox-/-和野生型（Wt）小鼠（C57/B6J） （25-30 g）中诱导单侧TBI。采用免疫印迹和染色技术研究gp91phox在脑外伤后的表达及其作用。原位测定小鼠脑内氧和过氧亚硝酸盐的水平。在IFNgamma或IL-4存在的情况下，在小胶质细胞系BV-2中测定了表达gp91phox的小胶质细胞的活化表型。结果：脑外伤后，Gp91phox主要在Wt小鼠同侧半球阿米巴样小胶质细胞中表达增加。gp91phox-/-小鼠的挫伤面积、tunel阳性细胞数量以及产生的O2-和过氧亚硝酸盐代谢物的数量比Wt少。在IFNgamma存在下，BV-2细胞的诱导型一氧化氮合酶和一氧化氮水平增加，符合经典的活化表型，并急剧增加gp91phox的表达。结论：经典激活的小胶质细胞通过gp91phox促进ROS的形成，在脑外伤后脑损伤中起重要作用。调节gp91phox和gp91phox衍生的ROS可能为治疗创伤后脑损伤提供新的治疗策略。

INTRODUCTION :We hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.METHODS :Unilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNgamma or IL-4.RESULTS :Gp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNgamma, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.CONCLUSIONS :Classical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.