Effects And Mechanisms Of Methionine Sulfoxide Reductase A On Microglial Activation And Microglia-mediated Inflammatory Responses

Part Ⅰ Effects of MsrA on microglial activation and microglia-mediated inflammatory responsesObjective:Methionine sulfoxide reductase A (MsrA) is a key member of methionine sulfoxide reductases (Msrs). In recent years, more and more attention has been paid to its role in antioxidant defense. MsrA is widely expressed in most tissues in mammal animals, especially in the heart and liver. Moreover, MsrA is also expressed in many brain regions including hippocampus, cortex and midbrain in the central nervous system (CNS). MsrA repairs oxidatively damaged proteins and protects against oxidative stress in many types of cells, including neuronal cells. Nonetheless, the expression and functions of MsrA in microglia remains virtually unknown. Considering the essential roles of oxidative stress in microglial over-activation and neuroinflammation, we asked whether MsrA was functionally expressed in microglia and acted as a molecular regulator to control microglia-mediated inflammatory responses.Methods:RT-PCR was performed to determine the expression of mRNA encoding MsrA in rat primary microglia. Western blotting was used to detect the protein expression of MsrA in rat primary microglia. Immunolabeling of MsrA and microglia in vitro and in vivo were performed to determine the existence and distribution of MsrA in rat microglia. Lentiviral expression of specific short hairpin RNAs (shRNAs) against MsrA was adopted to assess the roles of MsrA silencing in LPS-induced microglial activation and inflammatory responses. Supplement of MsrA by transduction of Tat-rMsrA was used to investigate the effects of up-regulation of MsrA on LPS-induced microglial activation and inflammatory responses. A rat model of acute neuroinflammation was used to evaluate the effects of Tat-rMsrA transduction on microglial activation and neuroinflammation in vivo. Results:(1) Both RT-PCR and western blotting analysis revealed that MsrA was expressed in rat microglia. A double-immunofluorescence-labeling study with an anti-MsrA/Ibal (a specific microglial marker) antibody revealed the existence of MsrA in rat microglia, which located in the cytoplasm and nucleus.(2) Microglial stimulation with LPS up-regulated the expression of MsrA.(3) MsrA silencing exacerbated LPS-induced activation of microglia and inflammatory responses.(4) Transduction of Tat-rMsrA fusion protein attenuated LPS-induced activation of microglia and inflammatory responses.(5) In vivo transduction of Tat-rMsrA fusion protein inhibited LPS-induced microglial activation and production of pro-inflammatory factors in a rat model of acute neuroinflammation.Conclusion:Our results suggest that MsrA is functional expressed in rat microglia and MsrA negatively controls micoglial activation and inflammatory responses. Part Ⅱ The mechanisms that MsrA negatively regulates microglial activation and inflammatory responsesObjective:Microglia-mediated inflammatory responses and reactive oxygen species (ROS) are interlinked. ROS can serve as signaling molecules and activate mitogen-activated protein kinases (MAPKs) and nuclear-factor kappa B (NF-κB) signaling cascades, which are thought to play a pivotal role in immune and inflammatory responses, thereby exacerbating the over-activation of microglia and neuroinflammation. Thus, endogenous antioxidants may put a "brake" on uncontrolled inflammatory responses. In proteins, the cyclic oxidation/reduction of methionine (Met) is believed to be an important process in scavenging ROS and preventing ROS from attack on key sites of proteins. Notably, MsrA is a key regulator of Met antioxidant defense. Therefore, we asked whether MsrA inhibits LPS-induced activation of p38, ERK and NF-κB through alleviating LPS-induced ROS production by catalyzing the reactions that ROS oxidize Met, thereby inhibiting LPS-induced microglial activation and inflammatory responses.Methods:Western blotting was used to detect the effect of Tat-rMsrA transduction on LPS-induced phosphorylation of p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and inhibitor of kappa Ba (IicBa). Immunocytochemical analysis was performed to evaluate the effect of Tat-rMsrA transduction on LPS-induced translocation of p65from the cytoplasm to the nucleus. The effect of Tat-rMsrA on LPS-induced production of ROS was assessed by intracellular ROS assay and extracellular superoxide measurement. Electron spin resonance (ESR), luminol-enhanced chemiluminescence measurement and methionine sulfoxide assay were adopted to determine the mechanisms that Tat-rMsrA decreased LPS-induced ROS production.Results:(1) Transduction of Tat-rMsrA fusion protein inhibited LPS-induced activation of p38and ERK MAPKs signaling pathways in microglia.(2) Transduction of Tat-rMsrA fusion protein inhibited LPS-induced activation of NF-κB signaling pathway in microglia.(3) Transduction of Tat-rMsrA alleviated oxidative stress during microglial activation.(4) MsrA displayed free radical-scavenging activity by catalyzing the reaction that ROS oxidized Met or Met residues of proteins to MetO.Conclusion:Our results indicate that MsrA decreases LPS-induced production of ROS by catalyzing the reactions that ROS oxidize Met, thereby inhibiting LPS-induced p38, ERK and NF-κB activation, which critically involved in the inhibitory effect of MsrA on LPS-induced microglial activation and inflammatory responses.