Effects Of IL-1β On Differentiation Of OPCs In The Corpus Callosum Of Septic Neonatal Rats And Mechanism Exploration

Neonatal sepsis was a common clinically serious disease, it had poorly prognosiss, it could cause nervous system dysplasia, even cerebral palsy. Recently studies had shown that infection-induced inflammation was one of the mechanisms of periventricular white matter white matter injury (periventriculars white matters damages, PWMD) which appears complete destruction of myelin, signaling abnormalities, may be one of the mechanisms of the nervous system dysplasia. Structural integrity of myelin was basis of nerve signals, which include the formations processs of differentiation of oligodendrocytes precursors cells (oligodendrocytes precursors cells, OPCs) mature, mature axons, then they are intertwined.oligodendrocyte precursors stages, immature oligodendrocytes, matured oligodendrocytes were the process.of differentiation. Studies have shown that oligodendrocytes was more wickness than the neuronal cell, so changes in internal environment may cause differentiations of OPCs obstacles. Studies had founds that hypoxia could cause inflammation, then caused the axon myelination disorder, it may be because of interaction of inflammatorys cytokine and theirs receptor causes, such as Interleukin-1β (Interleukin-1β, IL-1β) and IL-1R1 (IL-1 receptor 1), but the role of specific inflammatory factors is unclear. Studies have found that injection of IL-1βmay be cited oligodendrocytes developmental disorders in mice in vivo, leading to axon myelination, but the exact mechanism was unknown, and whether there were species differences were not clear.The impacts of experiment by intraperitonals injections of lipopolysacharides (Lipopolysacharides, LPS) sepsis newborn pups to study the effects of LPS on the expression of inflammatory cytokines and their receptors on oligodendrocyte apoptosis, proliferation, and differentiation in corpuss callosums in vivo, in vitro cultured OPCs and treated with IL-1β, in order to study the effect of IL-1βon OPCs.Chapter 1 Expression of IL-1βand IL-1R1 in the corpus callosum of septic neonatal ratsObject:To investigate whether sepsis model will affect inflammatory cytokines Interleukin -1β (Interleukin-lbeta, IL-1β) and its receptor IL-1R1 (IL-1 receptor 1) expressionMethod:Using the one-day SD rats, the experimental group were injected 1mg/kg of LPS, the controls groups was injected with equally volumes of salines. And female rats were raised to the same cage 7d, take 3h, 1d,3d,7d of time to experiment, to detect the expression of IL-1 and IL-1R1 by immunofluorescence and immunoblotting and PCR methods.First, confirmed the effectiveness of different concentrations of LPS and the stability of the model, selected 0.25mg/mg 0.5mg/kg, lmg/kg,2mg/kg,4mg/kg, 8mg/kg 6 concentration gradients, a rough estimate 7 days survival rate and body weight, the highest survival rate screened 0.25mg/kg,0.5mg/kg, 1mg/kg, and then based on the expression of changes in body weight and one day, three days IL-1β and IL-1R1, and determined to 1mg/kg was most appropriate.After determinings optimals doses of LPS, LPS whether increased expression of inflammatory cytokines IL-1βand its receptor IL-1R1, divided into salines controls groups and LPS groups, take 3h, Id,3ds,7d some point in time after appropriate treatment utilizing immunofluorescence, Western blot and PCR method to detect brain corpus callosum in vivo IL-1β and IL-1R1 expression and localization.Results:one-years-old SD rats, weighing about 5-7g, were injecteds with LPS and salines controls groups were observed after treatment known Id weighs about 7-9g about 12-13g,7d after about 18-20g,0.25mg/kg of body weight after LPS injection Id 3d after 8-9g,3 days for 12-13g,7d after for 18-20g, no differences withs thes controls group,0.5mg/kg LPS injection 1d after weighing 7-9g,3 days for 11-13g, 7d after for 16-19g, littlely differences with thes controls groups, after lmg/kg LPS injection 1d weighing 6-7.5g (death 10%),3 days for 8-10g (mortality 40%),7d after for 12-13g, after 2mg/kg LPS injection 1d weighing 6-7.5g (mortality 20%),3 days for 8-10g (80% mortality),7d after for 12-13g, after 4mg/kg LPS injection 1d weighing 6-7.5g (40% mortality),3 days for 8-10g (death percentile of 100),7d free survival,,8mg/kg body weight after LPS injection 1d 6-7.5g (mortality 80%),3 days for 8-10g (100% mortality),7d-free survival, according to the model operability and stability select lmg/kgLPS intraperitoneal injection.Accordings to the screening resultsed, follow immunofluorescence, Western blot, PCR was found 1mg/kgLPS processing 3h, 1d,3d,7d after body of corpus callosum inflammatory cytokines IL-1β and IL-1R1 receptors expressions was significantlys increaseds compareds with the controls groups.Conclusion:LPS could activate the microglia in corpus callosum and release large amounts of inflammatory mediators IL-1β, presence of oligodendrocytes precursors cellss (OPCs) receptor on IL-1β and IL-1R1 receptor expression increased.Chapter 2 Impacts of sepsis on oligodendrocytes precursors cells apoptosis and proliferation and differentiation of OPCsObject:To investigate effects of lmg/kg LPS on apoptosis and proliferation and differentiation of OPCs after injection.Method:Using the double-labeled immunofluorescence method to explore the impact of sepsis model OPCs apoptosis and proliferation.1d SD rats aftered treatment, were divided into controls groups and LPS groups, and maternal same cage were raised to 28d, take Id,3d,7d,14d,28d of thes corpuss callosums were observeds. Detection of apoptosiss marker expressions Cleaved-Caspase-3 and the proliferations marker BrdUs. Using doubles-labeleds immunofluorescences, WB and PCR methods to achieve the goal. 1d of newborn SD rats aftered treatment, were divided into control groups and LPS groups, and maternal same cage were raised to 28d, take 7d,14d,28d in corpus callosum were observed. Detecting immature oligodendrocytes marker of PDGFRaand NG2, detection of matured oligodendrocytes, marker expression of MBP and CNPase. Detect the expression of ligodendrocytes specific transcription factors Oligl and Olig2.Results:Compared with the control groups, the nubers of apopttic OPCs incresed in 1d,3d,7d in LPS groups. Compared with controlls groups, LPSgroups reduced the numbers of proliferatings OPCs cells.Immunofluorescence assays and Westerns blots tests, PCR experiments showed PDGFRaand NG2 reduced compared with the control group at 7d, but increased at 14d and 28d when compareds withs the controls groups, MBP and CNPase expression decreased compareds withs the controls groups at 14d, 28d in the corpuss callosums in vivo Oligl and Olig2 expression levels lower than control group.Conclusion:LPS-induced sepsis model promotes early apoptosis of OPCs and inhibit the proliferation and of OPCs.Chapter 3 Impact of sepsis on the myelination of axons in corpus callosumObject:To investigate the effects of sepsis model on the myelination of axons in corpus callosum in vivoMethod:Using electron microscopy methods tos achieve goals. Id newborn SD ratss aftered treatment, were divideds into controls groups and LPS groups, and maternal same cage were raised to 28d, take the 28d corpuss callosums to electron microscopy. Detection of the numbers and thicknesss of the myelins sheaths myelinateds nervely fiberss in corpuss callosums.Results:Comparred with controlls groups, while the numbers of myelins sheaths were thicknesss in 28d LPS group and myelinated nervely fiberss were lower (P <0.05).Conclusion:LPS-induced sepsis model could lead to low myelination of axons.Chapter 4 The effect of IL-1βon apoptosis and proliferation and differentiation of OPCsObject:To investigate the effect of IL-1β on apoptosis and proliferation and differentiation of OPCs.Method:Remove the raw 1-2d SD neonatal rat cerebral cortex, and after trypsin digestion mixed glials, replacement of oligodendrcyte precuror cells proliferations medium, the proliferation of cultured for 7 days, and then one day training group, divided into a control group (PBS group),30ng/ml IL-1β group,30ng/ml IL-1β +30ng/ml IL-1Ra group (IL-1Ra, IL-1R antagonists specificity). Apoptsis and prolifration were detected by imunofluorecence.The same method as chapter five, and then the same number of species after digestion packet cultured cells were divideds into controls groups (PBS group),30ng/ml IL-1β group,30ng/ml IL-1β+ 30ng/ ml IL-1Ra group (IL-1Ra, IL-1R antagonist specificity),30ng/ml IL-1Ra group. Replacing oligodendrcyte differentation medium for 3d, immunofluorescence and Western blot 3d after treatment in each group to detect cell differentiation.Results:Immunofluorescence had shown that compared with controls groups, after treament 3d, IL-1β group naive cells marker by NG2 increased. Compared with IL-1β, IL-1β+IL-1Ra group naive cells decreased. Compred with control groups, IL-1β group mature cells marker by MBP decreased. Compared with IL-1β, IL-1β+ IL-1Ra group mature cells increased. Westerns blot (Westerns blotting, WB) showed that compared with the control group, IL-1β group NG2, PDGFRa expression increased, while MBP, CNPase, Oligl, Olig2 expression decreased, compared with IL-1β group, IL-1β+IL-1Ra group NG2, PDGFRa expression decreased, MBP, CNPase, Oligl, Olig2 expression rebounded. The expression of IL-1Ra indicators had no difference compared with the control group.Conclusion:IL-1β inhibits the proliferation and differentiation of OPCs, had no effects on apoptosiss of OPCs.Chapter 5 The effect of IL-1βon FYN-MEK-ERK pathway and the relationshipbetween IL-1β and ERK pathway on differentiation of OPCsObjective:To investigate the effect of IL-1β on FYN-MEK-ERK pathway the relationship between IL-1β and ERK pathway on differentiation of OPCsMethod:The same method as chapter four, and then the same number of species after digestion packet cultured cells were divideds into controls groups (PBS groups),30ng /ml IL-1βgroup,30ng/ml IL-1β+30ng/ml IL-1Ra group (IL-1Ra, IL-1R antagonist specificity),30ng/ml IL-1Ragroup. Replacing oligodendrocyte sdifferentiations culture differentiations mediums 6h, perform Western blot detection 6h after treatment in each group (Western blotting, WB) cells were observed phosphorylation-FYN, phosphorylation-MEK, phosphorylation -ERK expression. cultured cells were divideds into controls groups (PBS groups),30ng/ml IL-1β group, ERK target gene group,30ng/ml IL-1β+ERK object gene, the empty vector pEGFP group. Replacing, oligodendrocyte differentiation culture differentiation medium 3d, each group after processing 3d Western blot detection (Western blotting, WB) cell observation NG2, PDGFRa, Oligl,Olig2, MBP, CNPase expression.Results:Compared with control group, the IL-1βtreatment after 6h, phosphorylation-FYN, phosphorylation-MEK, phosphorylation-ERK expression decreased. Compared with IL-1β, IL-1β+IL-1Ra group phosphorylation-FYN, phosphorylation-MEK, phosphorylation-ERK expression rebounded. The expression of IL-lRa indicators had no difference compared with the control group.Compared with the control group, NG2, PDGFRα expression increased, MBP, CNPase, Oligl, Olig2 expression decreased in IL-1βgroup,compared with IL-1βgroup, NG2, PDGFRa expression has decline, MBP, CNPase, Oligl, Olig2 expression increased in IL-1β+ERK purpose gene group.Conclusion:IL-1βmay regulated differentiation of OPCs by inhibiting ERK channel.