| Perinatal hypoxic-ischemic encephalopathy,HIE is the cause of childhood disability. So the mechanism of hypoxic-ischemic brain injury has become an important issue in today’s medical research.Its pathogenesis mainly include: excitatory amino acid poisoning, oxygen free radicals, calcium overload, and inflammatory response.Although some progresses have been made in the excitotoxicity, oxidative stress, and apoptosis, results of the clinical treatment of HIE is still not satisfied. In recent years, more and more attention were focused on the mechanisms of inflammatory. It is found that inflammation is one of the main causes of brain injury.Tim-3, a novel immunomodulatory factor, is expressed in a variety of immune cells and plays an important role in the regulation of the innate immunity and adaptive immunity.Tim-3 expressed on the Thl cell surface together with its ligand galectin-9, can suppress the immune response and induct Thl cells death. More immunomodulatory process,such as macrophages M1/M2 polarization,transplantation tolerance, and other related tumor immune responses can also be regulated by Tim-3. Tim-3 has shown its potential therapeutic effects on the inflammatory diseases. This study is the first time to investigate its role in the mechanism of HIBD.In this study, animal models and cell models were applied to investigate the role of Tim-3 in HIBD the role of Tim-3 in HIBD.We found:1、Hypoxic-ischemic promotes Tim-3 expression which focused on the activated microglia.Though histopathological analysis,apoptosis detection to determine the degree of neuronal damage; using RT-PCR, Western Blot, immunohistochemistry and confocal laser technology to detect brain Tim-3 expression and cellular localization. The results show that in the ischemia sensitive areas,Tim-3 expression was significantly up-regulated and the activation of microglia is the main source of Tim-3. 2、The mechanism of Tim-3 regulating microglia. In this study, we respectively, use Tim-3 overexpression and knock-down model to study how it regulate the microglial to secret inflammatory cytokines and NO. We found that, Tim-3 by NF-κB pathway, can enhance the secretion of microglial cells in TNF-α, IL-1β and NO, to mediate hypoxia ischemia and reperfusion brain inflammation.3, Specificly blocking Tim-3 can damage protect neurons in HIBD. We use Tim-3 block antibody and Tim-3 knock-out mouse to specificly blocking the effects of Tim-3.Though histopathological analysis and apoptosis detection method to determine the degree of neuronal damage; In water maze and open field experiment, mice were detected the ability of spatial learning and memory. In this study,we found that specificly blocking Tim-3 can significantly reduce inflammation after hypoxic-ischemic in mice brain, acting as a protective effect on neurons.In this study,it is the first time to reveal the role of Tim-3 in perinatal lhypoxic-ischemic brain injury and its mechanism.Our study show that Tim-3 is a critical factore in theprognostic evaluation, and provides a poteintial new treatment strategyResearch objective:In this study, we purpose is to use HIBD animal model and the activation of microglia in the process of hypoxic-ischemic reperfusion -to investigate the expression of Tim-3, and the regulation the microglia function by Tim-3. elucidate the role and mechanism of Tim-3 in hypoxia ischemia-induced brain inflammation and neurons death.Research methods:1, HIBD animal model and Tim-3 expression and localization:1) HIBD animal model:Artery ligation on the right side of day-7 newborn mice, hypoxia for 1.5 hrs under 8% oxygen atmosphere, kill the mice 3 days later.2) Using Western Blot, RT-PCR, immunofluorescence to detect Tim-3 expression in HIBD animal models3) Using RT-PCR to detect cytokines and iNOS expression in HIBD animal models.4) Using HE and tunel method to detect the degree of brain damage. 2, The mechanism of Tim-3 in regulation of microglia: 2.1, Stimulation of BV2 by OGD/R to detect the expression of Tim-3 and cytokine secretion:1) OGD/R Model:BV2 cells were cultured in the medium without glucose and serum,1% oxygen treatment for 3h, then replace to the normal medium to reoxygenation for 36 hrs.2) Using Western Blot, RT-PCR, immunofluorescence to detect Tim-3 expression.3) Using RT-PCR to detect cytokines and iNOS expression. 2.2, Effects of Tim-3 on the secretion of cytokines by BV2:1) Establish Tim-3 overexpression, knockdown and block model in BV2 cells, and OGD/R stimulation.2) Using RT-PCR to detect cytokines and iNOS expression.3) Using ELISA to detect cytokines and NO level in cell supernatant. 2.3, Tim-3 downstream signaling pathway: Construct Tim-3 overexpression and knockdown model of BV2 cell, giving OGD/R stimulation, reoxygenation for 30min, Werstern Blot were performed to detect Tim-3 downstream signaling pathway.3, Neuroprotection by blocking Tim-3 in HIBD:1) 5mg/kg of Tim-3 block antibody were injected intraperitoneally into Tim-3 knockout mice in HIBD model, the control group received the same type and amount of IgG antibody.2)Using tunel method to detect the degree of brain damage.3) Using TUNEL to detect the degree of brain damage.4) Tim-3-ko-mice and wt-mice are fed to four weeks old, Morris water maze and open-field test is used to detect the movement and spatial learning ability and memory.Research result:1, The expression and localization of Tim-3 in HIBD animal model:1) Tim-3 expression increased in HIBD.2) Tim-3 expression is positively correlated with CD11b expression. This results suggested that Tim-3 was mainly expressed on the activated microglia in HIBD animal model.2, The mechanism of Tim-3 in regulation of microglia:1) In Tim-3 overexpressed, knockdown and blocking cells model, the secretion of inflammatory cytokines and NO level were regulated by Tim-3 expression.2) The phosphorylation of NF-κB was regulated by Tim-3 expression.3, Blocking Tim-3 pathway in HIBD can protect neurons from damage:1) TUNEL positive cells are significantly reduced by blocking Tim-3 pathwy in HIBD model.2) The mRNA of inflammatory cytokines was reduced by blocking Tim-3 in HIBD model.3) Tim-3-knockout mice have better learning and memory capacity than the wild-type mice in HIBD model.Conclusion:1) The expression of Tim-3 increased in ischemic sensitive areas such as the cerebral cortex, hippocampus and striatum region, which may be one of the important causes of selective neuronal apoptosis in perinatal hypoxic-ischemic brain injury.2) Tim-3 regulates the secretion of inflammatory cytokines and NO level in microglia cell though NF-κB signaling pathway in the brain. Tim-3 plays an important role in perinatal hypoxic-ischemic encephalopathy.3) Specifically blocking the effect of Tim-3 can produce a protective effect on neurons in perinatal hypoxia-ischemia and reperfusion injury. |
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