The Role Of Non-muscle Myosin Regulatory Light Chain In Cerebral Ischemia/Reperfusion Injury And The Underlying Mechanisms

Background and purposeRestoration of blood flow following ischemic stroke plays a critical role in tissue repair and functional recovery. However, after a period of ischemia, reperfusion may exacerbate the injury initially caused by ischemia, producing a so-called "cerebral ischemia/reperfusion (CI/R) injury". Multiple pathological processes are involved in ischemic neuronal damage, including energy metabolism disturbance, excitotoxicity, oxidative stress, inflammation, necrotic and apoptotic cell death. Among them, oxidative stress is attracted special attention.Oxidative stress refers to a steady-state level of oxidative damage in a cell, tissue, or organ, caused by over production of reactive oxygen species (ROS). Potential sources of ROS in the body are produced by a variety of enzymes. Among them, NADPH oxidases (NOX) are considered as a major source of ROS production in the cardiovascular system.It has been reported that NOX activity is increased following cerebral ischemia/reperfusion (CI/R), accompanied by the increased ROS level and celebral oxidative injury. Although the role of NOX-derived ROS in CI/R injury is well recognized, the role of NOX isoforms in this process is not fully investigated and it still remains poorly understood on the mechanisms underlying the changes of NOX expression.Non-muscle myosin regulatory light chain (MLC2o) is a component of non-muscle myosin. The phosphorylation of its regulatory light chain (p-MLC2o) is controlled by myosin light chian kinase (MLCK). Through alteration in phosphorylation level, MLC20was reported to be involved in the development of many diseases (including stroke). There were reports to show that the elevation of MLC20phosphorylation level was closely correlated with CI/R injury, but the underlying mechanisms remain unknown. As mentioned before, NOX plays a key role in CI/R oxidative injury and our preliminary data also showed that NOX expression was up-regulated following CI/R. It is not known, however, whether this phenomena was related to the changes in MLC2o phosphorylation level.Based on the literatures as well as our preliminary data, we hypothesized that MLC2o participates in regulation of NOX expression through the alteration in phosphorylation level follwing CI/R.MethodsIschemia/reperfusion model was established by occlusion of the middle cerebral artery (MCAO) in rats for2h followed by reperfusion for24h. The NADPH oxidase inhibitor DPI and apocynin, MLCK inhibitor ML-7were used in this study.70male SD rats (250-300g) were randomly divided into7groups (n=10): the control group, the sham group, the CI/R group, the CI/R+ML-7group, the CI/R+DPI, the CI/R+apocynin and the CI/R+vehicle group. The infarct volume, neurological behavior, histopathological change and cellular apoptosis were assessed by TTC staining, Bederson neuroscore, HE staining and TUNEL assay, respectively. Expression of NOX1-4and phosphorylation level of MLC20were measured by Real-Time PCR or Western Blotting. The activites of NOX and caspase-3and the level of H2O2were determined by spectrophotometry or ELIS A kit.Results(1) Compared with control group, rats in the CI/R group significantly displayed defect in neurological behavior and cerebral infarct accompanied by the increase of H2O2level, NOX activity, apoptosis ratio and caspase-3activity, these effects were attenuated by ML-7, DPI or apocynin treatment.(2) Compared with control group, rats in the CI/R group showed edema and vacuole in brain tissue, pyknosis and lysis in cell, nucleolus vanishment, increase in cell gap and disarray in cell alinement. These phenomenon were significant decreased by ML-7, DPI or apocynin treatment.(3) Compared with control group, rats in the CI/R group showed higher apoptosis ratio and caspase-3activity in brain tissue. Inhibition of MLCK or NOX significantly blocked the effects of I/R on apoptosis and caspase-3activity.(4) Compared with control group, the mRNA and protein expression of NOX2and NOX4was upregulated in the CI/R group accompanied by increase in NOX activity and H2O2production, which was inhibited by ML-7, DPI or apocynin. There was no significant change in NOX1and NOX3expression.(5) Compared with control group, MLCK activity and MLC2o protein phosphorylation level were markedly elevated in the CI/R group, which was attenuated by MLCK but not NOX inhibitor.Conclusions(1) The up-regulation of NOX2and NOX4expression played an important role in brain oxidative stress injury following ischemia/reperfusion.(2) MLC2O is involved in regulation of NOX2and NOX4gene expression through the change in its phosphorylation level following cerebral ischemia/reperfusion.