LPS对大鼠脊髓背角星形胶质细胞CXCL1和CCL2表达的影响

目的:观察脂多糖(LPS)诱发的大鼠脊髓背角星形胶质细胞趋化因子CXCL1和CCL2的表达及释放,分析Toll样受体2(TLR2)以及Toll样受体4 (TLR4)的作用。方法:培养新生SD大鼠(<3d)脊髓背角星形胶质细胞,免疫荧光鉴定纯度达95%之后分为空白对照组、LPS处理组、TAK-242+LPS处理组、LPS-RS+LPS处理组。在LPS(1μg/ml)作用下,采用real time RT-PCR法检测SD大鼠(<3d)脊髓背角星形胶质细胞CXCL1和CCL2 mRNA表达; Western Blot用于检测胶质纤维酸性蛋白(GFAP)、TLR2、TLR4、CXCL1以及CCL2表达;ELISA检测CXCL1和CCL2释放。结果:与正常对照组相比,LPS作用6 h,背角星形胶质细胞CXCL1和CCL2在mRNA和蛋白水平的表达均上调(P <0. 05),CXCL1和CCL2释放增加(P <0. 05)。LPS刺激CXCL1和CCL2表达和释放增加的效应可以被TLR4受体拮抗剂TAK-242(50 nmol)以及TLR2/TLR4受体拮抗剂LPS-RS(...

The effects of LPS on CXCL1 and CCL2 expressions in cultured dorsal horn astrocytes

Objective:The aim of this study is to investigate the expression and release of chemokines CXCL1 and CCL2 from LPS-activated cultured dorsal spinal cord astrocytes,and the roles of Toll like receptor 4(TLR4)and Toll like receptor 2(TLR2)were analyzed.Methods:Astrocytes obtained from SD rat spinal cord(<3d)were cultured and purified.Cultured neonatal SD rat astrocytes were divided into normal control group,LPS(1μg/ml)treated group、TAK-242(50 nmol)treated group(TAK-242+LPS)、LPS-RS(500 ng/ml)treated group(LPS-RS+LPS).Expression of CXCL1 and CCL2 mRNA in LPS-treated dorsal spinal cord astrocytes were assessed by using real time fluorescence quantitative PCR(RT-PCR).Western Blot analysis was used to detect the expression of GFAP,TLR2,TLR4,CXCL1 and CCL2.LPS-induced CXCL1 and CCL2 release were measured by using ELISA assay.Results:Compared with control group,LPS application induce the increased expression of CXCL1 and CCL2 at the mRNA and protein levels(P<0.05).LPS-induced CXCL1 and CCL2 release were also higher than that of control group(P<0.05).LPS-evoked the increased expression and release of CXCL1 and CCL2 were obviously blocked after administration of TAK-242(TLR4 receptor antagonist,50 nmol)and LPS-RS(TLR2/TLR4 receptor antagonist,500 ng/ml,P<0.05).Compared with control group,LPS-induced the increased expression of the GFAP,TLR2,TLR4 and nucleus NF-κBp65 was significantly increased(P<0.05).Compared with LPS group,the increased expression of GFAP,TLR2,TLR4 and nucleus NF-κBp65 were obviously inhibited after pretreatment with TAK-242 or LPS-RS(P<0.05).Conclusion:TLR4/NF-κB pathway may be involved in the LPS-induced the increased expression and release of chemokines CXCL1 and CCL2 from cultured rat dorsal spinal cord astrocytes.