【作者】 潘琳娜；

【导师】 郭莲军；

【作者基本信息】 华中科技大学 ， 药理学， 2013， 博士

【摘要】 本研究拟探讨TLR3(Toll-like receptor3)介导的TRIF (Toll/IL-1receptor domain containing adaptor inducing IFN-β)信号通路是否参与了脑缺血预适应后炎症抑制过程。在离体培养的星形胶质细胞上诱导IPC (ischemia preconditioning)保护模型,观察此过程中星形胶质细胞TLR3信号通路关键蛋白TLR3. TRIF及pIRF3(phospho-interferon regulatory factors)的表达规律及下游炎症抑制因子IFN-P (interferonβ)和IL-10(Interleukin-10)的释放量,同时检测TLR4(Toll-like receptor4)、pNF-KB (phospho-nuclear factor-KB)和其下游促炎因子TNF-a (Tumor necrosis factor-a)和IL-6(interleukin-6)的释放量。并进一步使用TLR3激动剂Poly I:C (Polyinosinic polycytidylic acid),观察其对离体培养的星形胶质细胞OGD (oxygen-glucose deprivation)损伤和整体小鼠脑缺血复灌损伤的保护作用及对缺血后炎症反应的影响,以阐明TLR3介导的脑缺血保护作用及作用机制。第一部分TLR3-TRIF信号通路及炎症反应在星形胶质细胞缺氧预适应保护作用中的研究目的：观察TLR3-TRIF信号通路关键蛋白在短暂缺氧缺糖预适应诱导的培养星形胶质细胞缺氧缺糖耐受中的表达规律及与星形胶质细胞炎症因子释放间的关系。方法：细胞分四组,短暂氧糖剥夺1h复灌24h为IPC组,致死性氧糖剥夺12h为OGD组,短暂氧糖剥夺1h复灌24h后再次氧糖剥夺12h为IPC+OGD组,细胞不进行任何处理为Control组。检测细胞活力和乳酸脱氢酶(lactatedehydrogenase, LDH)释放量评价细胞损伤程度,Hochest33258检测细胞凋亡和坏死,ELISA检测细胞培养液中炎性因子TNF-a, IL-6, IFN-β IL-10含量。免疫荧光检测细胞TLR3与TLR4蛋白表达；western-blot检测星形胶质细胞TLR3、TLR4、TRIF、pIRF3、pNF-KB蛋白质表达。结果：(1)与Control组相比,星形胶质细胞单纯IPC组细胞无明显损伤,除TLR3蛋白表达升高外其它蛋白表达无明显差异。(2)与Control组相比,星形胶质细胞OGD组细胞活力下降,LDH漏出量增多并且细胞凋亡增多,培养液中TNF-a、IL-6分泌增加,细胞TLR3、TLR4、pNF-KB蛋白表达升高。(3)与单纯OGD组比较,IPC+OGD组星形胶质细胞活力升高,LDH漏出量减少并且细胞凋亡减少,细胞培养液中炎性因子IL-6分泌和细胞中TLR4、pNF-KB表达下降,而IFN-β分泌及细胞中TRIF、pIRF3蛋白表达升高,细胞TLR3蛋白表达无明显变化。结论：短暂缺血缺氧预适应对随后的长时间氧糖剥夺所致的星形胶质细胞损伤有保护作用,其作用机制可能与预适应过程一方面激活TLR3信号通路使抗炎因子IFN-β表达上调,另一方面抑制TLR4及pNF-KB的表达使致炎因子IL-6的表达下调有关。第二部分Poly I:C对离体星形胶质细胞氧糖剥夺损伤及炎症反应的影响目的：观察Poly I:C特异性激活TLR3-TRIF信号通路对星形胶质细胞氧糖剥夺损伤是否有保护作用,并探讨保护作用是否与TLR3-TRIF信号通路介导的抑制炎性反应有关。方法：OGD12h诱导培养的星形胶质细胞缺血缺糖损伤,观察10μg/mL和20μg/mL Poly I:C预孵育对星形胶质细胞缺血缺糖损伤的保护作用。检测细胞活力和乳酸脱氢酶释放量评价细胞损伤程度。Hochest33258检测细胞凋亡和坏死。ELISA检测细胞培养液中炎性因子TNF-α、IL-6、IFN-β、IL-10含量。免疫荧光检测细胞TLR3与TLR4蛋白表达；western-blot检测星形胶质细胞TLR3、TRIF、pIRF3、TLR4、pNF-KB蛋白质表达。结果：(1)与Control组相比,星形胶质细胞氧糖剥夺12h后细胞活力下降,LDH漏出量增多,细胞凋亡增加。培养液中TNF-a、IL-6含量增加,细胞TLR3、 TLR4、pNF-KB蛋白表达升高。(2)与OGD组相比,Poly I:C预处理组能减轻细胞缺血缺氧损伤,抑制细胞培养液中炎性因子IL-6分泌减少,促进IFN-β分泌增加,促进细胞中TRIF、pIRF3表达,而对细胞TLR3、TLR4、pNF-KB蛋白表达无明显影响。结论：10μg/mL和20μg/mL Poly I:C预处理对星形胶质细胞的氧糖剥夺损伤有明显的保护作用,激动TLR3-TRIF信号通路后可以减轻炎症反应。第三部分Poly I:C对小鼠脑缺血再灌注损伤及炎症反应的影响目的：观察Poly I:C特异性激活TLR3-TRIF信号通路对小鼠的局灶性脑缺血损伤是否有保护作用,并探讨保护作用是否与TLR3-TRIF信号通路介导的抑制炎性反应有关。方法：0.3mg/kg Poly I:C一次性肌肉注射给药,24h后,左侧大脑中动脉栓塞2h复灌6h、12h、22h诱导小鼠的脑缺血再灌注损伤模型,脑组织切片TTC染色测定脑梗死体积：ELISA检测小鼠血清及脑组织匀浆炎性因子TNF-α、IL-6、IFN-β、IL-10的含量,western-blot检测缺血侧脑组织TRIF蛋白表达。结果：左侧大脑中动脉栓塞2h复灌6h、12h、22h能稳定地制作小鼠脑缺血再灌注损伤模型,小鼠出现明显的神经缺陷症状及脑组织梗死,同时外周血清TNF-α含量增加,缺血复灌6h、12h、22h缺血侧脑组织TNF-α、IL-6表达升高。0.3mg/kg Poly I:C肌肉注射给药能明显改善小鼠神经缺陷,减少脑梗死体积,降低缺血再灌12h和22h血清TNF-a含量,并提高血清中IFN-β含量。0.3mg/kg Poly I:C预处理亦降低缺血再灌6h和12h缺血侧脑组织TNF-a和IL-6的含量并升高IFN-B水平。0.3mg/kg Poly I:C预处理提高缺血侧脑组织TRIF蛋白的表达。结论：0.3mg/kg Poly I:C对小鼠脑缺血损伤有保护作用,激活TLR3-TRIF信号通路可以抑制小鼠脑缺血损伤过程中的炎症反应。更多还原

【Abstract】 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.更多还原