TLR4 Participates In The Expression And Release Of Chemokines From LPS-activated Cultured Dorsal Spinal Cord Astrocytes

Objective:To explore the possible mechanism that TLR4 participates in the LPS-evoked the expression or release of chemokines MCP-1 and CXCL1 from cultured rat dorsal spinal cord astrocytes.Methods:1.The cells were seeded on 12-well plates at a cell density of 2×105/well for 48 h.These cells were randomized into four groups(n=4): normal group(DMEM/F12 culture Medium);LPS(1 μg/m L,6 h)group;TAK-242(50 n M,30 min)+ LPS(1 μg/m L,6 h)group;TAK-242(50 n M,30 min).The effects of LPS on the expression and the release of MCP-1 and CXCL1 m RNA by using RT-q PCR.The expression of GFAP,TLR4,MCP-1,CXCL1,NF-κB p65,Cx-43 and p-Cx-43 were examined by western blot analysis.LPS-evoked MCP-1 and CXCL1 release from astrocytes were measured by ELISA assay.2.The cells were seeded on 12-well plates at a cell density of 2×105/well for 48 h.These cells were randomized into four groups(n=4): normal group(DMEM/F12 culture Medium);LPS(1 μg/m L,6 h)group;carbenoxolone(100 μM,1 h)+ LPS(1 μg/m L,6h)group;Gap26(100 μM,1 h)+ LPS(1 μg/m L,6 h)group;Gap27(100 μM,1 h)+LPS(1 μg/m L,6 h)group.The expression of GFAP/Cx-43 in cultured dorsal hom astrocytes were observed by using immunofluorescence.LPS-induced MCP-1 and CXCL1 release from astrocytes were measured by ELISA assay.Results:1.The RT-q PCR results show the LPS-evoked the increased expression of MCP-1 and CXCL1 m RNA from cultured dorsal spinal cord astrocytes(P<0.05).LPS-evoked the increased expression of MCP-1 and CXCL1 m RNA were obviously blocked after administration with TAK-242(P<0.05).2.The WB results show the LPS-evoked the increased expression of GFAP,IL-1β,MCP-1,CXCL1,TLR4,NF-κB p65,Cx-43 and p-Cx-43 from cultured dorsal spinal cord astrocytes(P<0.05).LPS-evoked the increased expression of GFAP,MCP-1,CXCL1,TLR4,NF-κB p65 were obviously blocked after administration with TAK-242(P<0.05).3.The ELISA results show the LPS-evoked the increased release of MCP-1 and CXCL1 from cultured dorsal spinal cord astrocytes(P<0.05).LPS-evoked the release of MCP-1 and CXCL1 were obviously blocked after administration with TAK-242(P<0.05).4.The ELISA results show the LPS-evoked the increased release of MCP-1 and CXCL1 from cultured dorsal spinal cord astrocytes(P<0.05).LPS-evoked the release of MCP-1 and CXCL1 were obviously blocked after administration with CBX,Gap26 and Gap27(P<0.05).Conclusion:TLR4 is involved in LPS-evoked the expression and the release of MCP-1 and CXCL1 from cultured dorsal spinal cord astrocytes.LPS-evoked the release of MCP-1 and CXCL1 from cultured dorsal spinal cord astrocytes may be mediated through the activation of the gap junction channel.