| This study intends to explore whether the Toll receptor3(TLR3)-mediated TRIF signaling pathway is involved in the inflammation inhibition in the cerebral ischemic preconditioning. Ischemia preconditioning induced protection model in cultured astrocytes in vitro was induced and TLR3signaling pathway proteins (TLR3, TRIF and pIRF3) expression pattern and downstream anti-inflammatory cytokines IFN-beta and IL-10release were detected, at the same time TLR4, pNF-KB and its downstream pro-inflammatory cytokines TNF-a and IL-6release were measured. And further in order to clarify the role and mechanism of TLR3-mediated cerebral ischemia protection, the protective effect of TLR3agonist Poly I:C was observe against oxygen glucose deprivation (OGD) induced injury in cultured astrocytes in vitro and brain ischemia reperfusion injury in mice in vivo.First Part:The Effects of TLR3-TRIF Signaling Pathway in Preconditioning induced Protection against Simulated Ischemia in vitro in Cultured Rat AstrocytesAims:To observe TLR3signaling pathway proteins (TLR3, TRIF and pIRF3) expression pattern and the relationship with downstream inflammatory cytokines release.Methods:Sublethal1-h OGD or lethal12-h OGD were designed as IPC group or OGD group. Cultures subjected to OGD after IPC were IPC+OGD group. Cultures in normal medium were Control groups. Cell viability and lactate dehydrogenase (lactatedehydrogen-ase, LDH) release were measured to evaluate the degree of cell damage, cell apoptosis and necrosis was measured by Hochest33258detection, TNF-a, IL-6, IFN-β, IL-10levels in cultured medium were detected by ELISA. TLR3and TLR4protein expression were measured by Immunofluorescence and Western-blot. TRIF, pIRF3and pNF-KB protein expression were measured by Western-blot.Results:(1) Compared with Control group, cell viability%lactate dehydrogenase leakage、apoptosis、 inflammatory cytokines of IPC group have no significant difference except TLR3protein expression were up-regulated.(2) Compared with Control group, OGD12h caused decreased cell viability and increased LDH leakage, increased cell apoptosis. OGD12h caused also caused enhanced secretion of TNF-a、IL-6in cell supernatant and up-regulated expression of TLR3,TLR4,pNF-icB protein in cells.(3) Compared with OGD group, IPC+OGD reduced post-ischemia induced astrocytic damage, increased more IFN-β secretion but decreased IL-6secretion. TRIF and IRF3expression were up-regulated but TLR4was down-regulated in IPC+OGD group.Conclusions:IPC in cultured astrocytes reduced post-ischemia induced astrocytic damage activated TLR3-TRIF signaling, increased IFN-β secretion but decreased IL-6secretion. On the other hand the procession of preconditioning can inhibit the expression of TLR4and pNF-KB in post-ischemia period. Second Part:The Effect of Poly I:C against Oxygen-glucose Deprivation Injury and and Inflammatory Response on AstrocytesAims:To observe the effect of Poly I:C against oxygen-glucose deprivation injury on astrocytes in vitro and explore TLR3-TRIF signaling pathway on inhibition of inflammatory reaction.Methods: OGD12h induced ischemia injury in cultured astrocytes and the protective effect of lOμg/mL and20μg/mL Poly I:C pretreatment against ischemic injury was observed. Cell viability and lactate dehydrogenase (lactatedehydrogenase,LDH) release were measured to evaluate the degree of cell damage, cell apoptosis and necrosis was measured by Hochest33258detection, TNF-a, IL-6, IFN-β, IL-10levels in cultured medium were detect-ed by ELISA.TLR3and TLR4protein expression were measured by Immunofluorescence and Western-blot. TRIF, pIRF3and pNF-KB protein expression were measured by Western-blot.Results:(1)Compared with Control group, OGD12h caused decreased cell viability and increased LDH leakage, increased cell apoptosis. OGD12h caused also caused enhanced secretion of IL-6in cell supernatant and up-regulated expression of TLR3、TLR4、pNF-icB protein in cells.(2) Prereatment of Poly I:C (10and20μg/mL) attenuated OGD-induced astrocyte injury, upregulated TRIF and pIRF3expression accompanied by increased IFN-β production. Poly I:C(10μg/mL and20μg/mL) also suppressed the pro-inflammatory cytokine IL-6production.Conclusions:Pretreatment with Poly I:C has protective effects against oxygen-glucose deprivation injury on astrocytes in vitro. Activating TLR3-TRIF signaling pathways can reduce inflammation reaction. Third Part:The Effect of Poly I:C against Cerebral Ischemia/Reperufsion Injury and Brain Inflammatory Response in MiceAims:To observe the effect of Poly I:C against focal cerebral ischemia-reperfusion injury in mice subjected to ischmia/reperfusion injury and explore the effects of TLR3-TRIF signaling pathway on inhibition of inflammatory reaction.Methods:KM mice were treated with0.3mg/kg Poly I:C via one-time intramuscular injection,then subjected to2h of the left middle cerebral artery occlusion (MCAO) followed by6h,12h,22h of reperfusion. Brain infarction was indicated by TTC staining; TNF-a, IL-6, IFN-B, IL-10were measured by ELISA.TRIF protein in ischemic brain tissue expression were measured by western-blot.Results:In KM mice,2h of middle cerebral artery occlusion (MCAO) followed by6h,12h and22h of reperfusion can steadily make small mouse brain ischemia-reperfusion injury and severe nerve defects, increase cerebral infarction, lead to an enhanced secretion of TNF-a in I/R12h and22h in mice serum and increased production of TNF-a, IL-6and decreased expression of TRIF in ischemic brain tissue. Pretreatment of the mice with Poly I:C (0.3mg/kg) prior to MCAO reduced infarct volume. In addition, treatment of Poly I:C resulted in a reduction of the ischemia/reperfusion and improved the nerve defects, induced down-regulation of TNF-a and IL-6,enhanced secretion of IFN-β in mice serum and ischemic brain tissue, improved TRIF protein expression in the ischemic brain tissue.Conclusions:Pretreatment with Poly I:C have protective effects against cerebral ischemia-reperfusion injury. Activating TLR3-TRIF signaling pathways can reduce inflammation reaction. |
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