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TLR4介导小鼠小胶质细胞自噬在脑出血后炎症反应的作用机制研究 预览

Mechanism of TLR4-mediated Autophagy of Microglia in the Inflammation after Cerebral Hemorrhage in Mice
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摘要 目的研究TLR4介导小胶质细胞自噬在脑出血后炎症反应的作用机制。方法从C57BL/6J小鼠中提取分离原代小胶质细胞,分组1中,A组:小胶质细胞;B组:小胶质细胞+阴性siRNA转染;应用C组:小胶质细胞+TLR4-siRNA转染。应用Q-PCR和Western Blot检测分组1中TLR4的表达。分组2中,D组:小胶质细胞+等量的对照溶剂(0.9%NaCl);E组:小胶质细胞+自噬抑制剂(3-MA);F组:小胶质细胞+control-siRNA+3-MA;G组:小胶质细胞+TLR4-siRNA+等量的对照溶剂(0.9%NaCl);H组:小胶质细胞+TLR4-siRNA+3-MA。Western Blot检测LC3-Ⅱ/Ⅰ比值、TLR4、MyD88和核转录因子(NF-κB)蛋白表达。ELISA检测细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)水平。结果 (1)A组和B组TLR4 mRNA和蛋白表达水平无明显变化(P>0.05);与B组相比,C组TLR4 mRNA和蛋白表达水平下调(P <0.05)。(2)D组、E组和F组TLR4、MyD88和NF-κB p65蛋白水平无明显变化(P>0.05);与D组相比,G组TLR4、MyD88和NF-κB p65蛋白水平下调(P <0.05);与F组相比,H组TLR4、MyD88和NF-κB p65蛋白水平下调(P <0.05)。E组和F组LC3-Ⅱ/Ⅰ比值、TNF-α、IL-1β、IL-6水平无明显变化(P>0.05);与F组相比,H组LC3-Ⅱ/Ⅰ比值、TNF-α、IL-1β、IL-6水平下降(P <0.05);与G组相比,H组LC3-Ⅱ/Ⅰ比值、TNF-α、IL-1β、IL-6水平下降(P <0.05)。结论 TLR4-siRNA可抑制TLR4介导的小胶质细胞自噬,从而减轻自噬引起的脑出血炎症损伤。 Objective To investigate the effects of toll-like receptor 4(TLR4)-mediated autophagy of microglia in inflammation after cerebral hemorrhage.Methods The original microglia cells from C57 BL/6 J mice isolated from C57 BL/6 J mice were divided into group A(microglia cells),group B(microglia cells+control siRNA),group C(microglia cells+TLR4 siRNA)and The quantitative polymerase chain reaction(q-PCR)and western blot were used to detect the expression level of toll-like receptor-4(TLR4).The microglia cells were divided into group D(microglia cells+0.9%NaCl),group E(microglia cells+3-MA),group F(microglia cells+control-siRNA+3-MA),group G(microglia cells+TLR4-siRNA+0.9%NaCl),group H(microglia cells+TLR4-siRNA+3-MA).Q-PCR and western blot were used to detect the expression level of TLR4.Western blot was used to detect the expression level of LC3-Ⅱ/Ⅰrate,TLR4,MyD88 and nuclear factor-kappa B(NF-κB)p65.Results There was no difference in the TLR4 mRNA and protein levels between group A and group B(P>0.05).Compared with B group,the TLR4 mRNA and protein levels were lower in group C(P<0.05).There was no difference in the TLR4,MyD88 and NF-κB p65 protein levels in group D,group E,and group F(P>0.05).Compared with group D,the TLR4,MyD88 and NF-κB p65 protein levels were lower in group G(P<0.05).Compared with group F,the TLR4,MyD88 and NF-κB p65 protein levels were lower in group H(P<0.05).There was no difference in the LC3-Ⅱ/Ⅰrate,tumor necrosis factor(TNF)-α,interleukin(IL)-1β,and IL-6 levels between group E and group F(P>0.05).Compared with group F,the LC3-Ⅱ/Ⅰrate,TNF-α,IL-1β,and IL-6 levels were lower in group H(P<0.05).Compared with group G,the LC3-Ⅱ/Ⅰrate,TNF-α,IL-1β,and IL-6 levels were lower in group H(P<0.05).Conclusion TLR4-siRNA can Inhibit TLR4 mediated-autophagy of microglia in inflammation after cerebral hemorrhage.
作者 陆梦茹 朱祖福 张慧萍 孔玉 高志强 杨江胜 陆强彬 柏燕燕 周国庆 沈丽萍 LU Mengru;ZHU Zufu;ZHANG Huiping;KONG Yu;GAO Zhiqiang;YANG Jiangsheng;LU Qiangbin;BAI Yanyan;ZHOU Guoqing;SHEN Liping(Jiangyin People′s Hospital,Jiangyin 214400,Jiangsu,China)
机构地区 江阴市人民医院
出处 《中西医结合心脑血管病杂志》 2019年第5期689-693,共5页 Chinese Journal of Integrative Medicine on Cardio-/Cerebrovascular Disease
基金 2016年江阴科技局指令性计划项目(No.JYKJ3033).
关键词 脑出血 TLR4 小胶质细胞自噬 核转录因子 肿瘤坏死因子-α 白细胞介素-1Β 白细胞介素-6 cerebral hemorrhage toll-like receptor 4 autophagy of microglia cells nuclear factor-kappa tumor necrosis factor-α interleukin-1β interleukin-6
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