The Roles And Mechanisms Of Peroxiredoxin6 On Lipopolysaccharide-induced Acute Lung Injury In Mice Model

Part I : Roles of peroxiredoxin 6 in the regulation of oxidative stress to lipopolysaccharide-induced acute lung injuryObjective: Peroxiredoxin (Prdx) 6 is a novel non-seleno peroxidase that catalyze the reduction of H₂O₂, fatty acid hydroperoxides and phospholipid hydroperoxides. The present study aims at investigating roles of peroxiredoxin 6 in the regulation of oxidative stress to lipopolysaccharide-induced acute lung injury (ALI) in mice. Methods: From male Prdx6 (-/-) mice and C57BL/6J mice, primary macrophages were cultured for reactive oxygen species (ROS) test. ALI was induced by intratracheal administration of LPS (5mg/kg). Four hours after stimulation, morphology, wet/dry ratio, protein concentration in bronchial alveolar lavage fluid (BALF), myeloperoxidase (MPO) activity, hydrogen peroxide (H₂O₂), malondialdehyde (MDA), protein carbonylation, total superoxide dismutase (SOD) and total anti-oxidative capability (TAOC) were tested.Results: The level of ROS in macrophages from Prdx6 (-/-) mice was significantly higher than that from C57BL/6J mice, which was even higher after LPS administration. LPS-induced ALI was characterized by inflammation in morphology, increased wet/dry ratio and elevated protein concentration in BALF. Compared with vehicle treated mice, the levels of MPO activity, H₂O₂ and MDA were significantly increased, while SOD and TAOC were marked decreased in LPS treated wild type mice, which were more so in Prdx6 (-/-) mice accompanied with protein carbonylation.Conclusions: Prdx6 (-/-) mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that Prdx6 acts as an important scavenger of ROS under oxidative stress.Part II: Roles of peroxiredoxin 6 in the regulation of inflammation and fibrinolytic activity to lipopolysaccharide-induced acute lung injuryObjective: Matrix metalloproteinases (MMPs) are able to degrade the extracellular matrix so as to increase vascular permeability. Plasminogen activator inhibitor (PAI)-1 can inhibit plasminogen activation, fibrin degradation resulting in hyaline membrane production. The present study aims at investigating roles of peroxiredoxin 6 in the regulation of inflammation and fibrinolytic activity to LPS-induced ALI inmice.Methods: ALI was induced by intratracheal administration of LPS (5mg/kg). Four or 24hrs after LPS administration, histology, wet/dry weight ratio, protein concentration in bronchial alveolar lavage fluid (BALF), nuclear factor (NF)-κB activity, the expression of tumor necrosis factor (TNF)-α, interleukin (IL) -1β, MMP-9, MMP-2 and PAI-1 were measured.Results: After LPS administration, NF-κB was activated. The expressions of TNF-α, IL-1βand PAI-1 mRNA were significantly increased in a time-dependant manner and the concentration of TNF-α, IL-1βand PAI-1 in BALF were significantly increased at 4hrs and decreased nearly to baseline at 24hrs, which were more so in Prdx6 (-/-) mice than those in C57BL/6J mice. The concentration of PAI-1 in plasma was significantly increased in a time dependant manner in Prdx6 (-/-) mice compared to that in C57BL/6J mice. The expression of MMP9 and MMP2 mRNA were significantly increased in a time-dependant manner in Prdx6 (-/-) mice than those in C57BL/6J mice. MMP9 mRNA was only significantly increased at 24 hrs while MMP2 mRNA was not changed in C57BL/6J mice. The activity of MMP-9 was more significantly increased at 4hrs and 24hrs in Prdx6 (-/-) mice than that in C57BL/6J mice.Conclusions: NF-κB was more activated in Prdx6 (-/-) mice, consequently mediating higher expression of MMP9 and PAI-1, which resulted in LPS-induced lung injury more severely, and thus, suggested that Prdx6 possesses an important anti-inflammatory and anti-fibrinolytic activity under inflammation.Part III: Peroxiredoxin6 increases LPS-stimuIated MMP9 and cytokineexpression in macrophage by enhancing MAPKs pathwayObjective: MAPKs can be activated under LPS stimulation to mediate inflammatorysignal transduction. The present study aims at investigating roles of peroxiredoxin 6in the regulation of mitogen-activated protein kinase (MAPKs) activation and matrixmetalloproteinase (MMP)-9 secretion under LPS stimulation.Methods: Primary macrophages from male Prdx6 (-/-) mice and C57BL/6J mice werecultured and stimulated under LPS together with extracellular signal-regulated kinase (ERK) inhibitor PD98059, c-Jun N-terminal kinase (JNK) inhibitor SP600125 or p-38kinase inhibitor ML3403. The expression of reactive oxygen species (ROS), tumornecrosis factor (TNF)-α, interleukin (IL) -1βand MMP-9 were measured.Results: LPS was found to induce the production of reactive oxygen species (ROS)which was more significantly in Prdx6 (-/-) mice. LPS-induced higher MMP-9 andcytokines expression in Prdx6 (-/-) mice, were suppressed by ERK and JNK inhibitors,but not by p38 kinase inhibitor.Conclusions: ROS-ERK/JNK cascade mediating LPS signaling to MMP-9expression resulted in LPS-induced inflammation more severely in macrophages fromPrdx6 (-/-) mice. ERK and JNK were essential for the expression of MMP-9 in micemacrophages in response to LPS with Prdx6 deficiency.