TLR4 and Cx43 signaling in dorsal spinal cord has been shown to be involved in the development of neuropathic pain. However, it is not clear whether TLR4 signaling is associated with the expression of MCP-1, CXCL1, and Cx43 in LPS (lipopolysaccharide)-treated rat dorsal spinal cord astrocytes under in vitro condition. In the present study, we found that TLR4 antagonist TAK-242 significantly inhibited LPS-induced MCP-1, CXCL1, and Cx43 expression, suggesting the role of TLR4 in response to LPS in cultured dorsal spinal cord astrocytes. Application of TAK-242 significantly blocked LPS-induced NF-κB and AP-1 activity and the expression of MCP-1, CXCL1 and Cx43. Furthermore, NF-κB inhibitor PDTC and AP-1 inhibitor SR11302 significantly blocked LPS-induced MCP-1, CXCL1, and Cx43 expression. DNA-binding activity of NF-κB, its effect on MCP-1 expression was suppressed by PDTC and SR11302. On the other hand, DNA-binding activity of AP-1, its effect on CXCL1 or Cx43 expression was also suppressed by PDTC and SR11302. In addition, PDTC was found to inhibit the nuclear translocation of AP-1 and the expression of c-Jun induced by LPS, which suggested that NF-κBp65 is essential for the AP-1 activity. Similarly, SR11302 significantly blocked LPS-induced the nuclear translocation of NF-κBp65 and the expression of NF-κBp65 induced by LPS. Pretreatment with CBX, Gap26, or Gap19 (Cx43 blockers) significantly inhibited abnormal astrocytic hemichannel opening and chemokines (MCP-1 and CXCL1) release in LPS-stimulated astrocytes. In summary, cell culture experiments revealed that LPS stimulation could evoke TLR4 signaling with the subsequent activation of NF-κB and AP-1, resulting in the expression of MCP-1, CXCL1, and Cx43. TLR4 activation increased Cx43 hemichannel, but not gap-junction activities and induced the release of the MCP-1 and CXCL1 from astrocytes via Cx43 hemichannel. These findings may help us to understand the role of astrocytic signaling in inflammatory response within dorsal spinal cord tissue.

Recent reports have suggested that abnormal miR-29c expression in hippocampus have been implicated in the pathophysiology of some neurodegenerative and neuropsychiatric diseases. However, the underlying effect of miR-29c in regulating hippocampal neuronal function is not clear. In this study, HT22 cells were infected with lentivirus containing miR-29c or miR-29c sponge. Cell counting kit-8 (CCK8) and lactate dehydrogenase (LDH) assay kit were applied to evaluate cell viability and toxicity before and after TNF-α administration. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Hoechst 33258 staining and TUNEL assay were used to evaluate cell apoptosis. The expression of key mRNA/proteins (TNFR1, Bcl-2, Bax, TRADD, FADD, caspase-3, -8 and -9) in the apoptosis pathway was detected by PCR or WB. In addition, the protein expression of microtubule-associated protein-2 (MAP-2), nerve growth-associated protein 43 (GAP-43) and synapsin-1 (SYN-1) was detected by WB. As a result, we found that miR-29c overexpression could improve cell viability, attenuate LDH release, reduce ROS production and inhibit MMP depolarization in TNF-α-treated HT22 cells. Furthermore, miR-29c overexpression was found to decrease apoptotic rate, along with decreased expression of Bax, cleaved caspase-3, cleaved caspase-9, and increased expression of Bcl-2 in TNF-α-treated HT22 cells. However, miR-29c sponge exhibited an opposite effects. In addition, in TNF-α-treated HT22 cells, miR-29c overexpression could decrease the expressions of TNFR1, TRADD, FADD and cleaved caspase-8. However, in HT22 cells transfected with miR-29c sponge, TNF-α-induced the expressions of TNFR1, TRADD, FADD and cleaved caspase-8 was significantly exacerbated. At last, TNF-α-induced the decreased expression of MAP-2, GAP-43 and SYN-1 was reversed by miR-29c but exacerbated by miR-29c sponge. Overall, our study demonstrated that miR-29c protects against TNF-α-induced HT22 cells injury through alleviating ROS production and reduce neuronal apoptosis. Therefore, miR-29c might be a potential therapeutic agent for TNF-α accumulation and toxicity-related brain diseases.