| Purpose1:To study the expression of miRNA-182-5p and TLR4 in OGD-induced microglia and the regulatory relationship between the two;2:To study whether miRNA-182-5p regulates the inflammatory response in OGD-induced microglia and whether it is mediated through TLR4;3:To investigate whether miRNA-182-5p has a neuroprotective effect on cerebral ischemia-reperfusion rats;4:To investigate whether the neuroprotective effect of miRNA-182-5p on cerebral ischemia-reperfusion rats is through the regulation of TLR4-mediated inflammatory response;Methods1.In vitro:BV2 cells were transfected with miRNA-182-5p mimics and inhibitors and treated with OGD,RT-PCR and Western Blot were used to detect the expression of miRNA-182-5p and TLR4.BV2 cells were transfected with miRNA-182-5p mimics and treated with OGD,the levels of pro-inflammatory factors TNF-a,IL-6 and IL-1βwere detected by RT-PCR and ELISA.BV2 cells were transfected with siTLR4 and treated with OGD,the expression of TLR4 was detected by western blot and the levels of proinflammatory factors TNF-a,IL-6 and IL-1β were detected by RT-PCR.BV2 cells were co-transfected with miRNA-182-5p mimics and TLR4(ORF),Western blot was used to detect the expression of TLR4,and the levels of pro-inflammatory factors TNF-a,IL-6 and IL-1β were detected by RT-PCR.2.In vivo:MCAO model simulated cerebral ischemia-reperfusion and miRNA-182-5p mimics were administered once daily for 3 consecutive days prior to MCAO,ischemic area was detected by TTC staining and mNSS and foot-fault test were used to detect neurological functions.RNA and protein were extracted from brain tissue of ischemic penumbra and TNF-a,IL-6 and IL-1β were detected by RT-PCR and ELISA,the expression of TLR4 was detected by RT-PCR and western blot.Results1.OGD induced upregulation of TLR4 expression and downregulation of miR-182-5p expression in microglial cellsBV2 cells were treated with OGD and detected for microRNA-182-5p levels at 0,3,6 and 12 hours,the results showed that the expressions of TLR4 mRNA and miR-182-5p increased and significantly decreased,respectively.In addition,the protein expression level of TLR4 post-OGD also increased with time.2.miR-182-5p regulates TLR4 expression in microglial cellsWhen we overexpressed and knocked down miRNA-182-5p in BV2 cells,we found that the levels of TLR4 mRNA and protein decreased and elevated compared with the control group,respectively.Furthermore,we transfected BV2 cells with miR-182-5p mimics and inhibitors and then exposed to OGD,we found that the expressions of TLR4 mRNA and protein reduced and increased compared with the NC group,respectively.3.TLR4 is a direct target of miR-182-5pThe HEK293 cells or BV2 cells were co-transfected with the miR-182-5p mimics or NC and the reporter vectors containing the wt or mut TLR4 3’UTR,the results showed that the wt TLR4 3’UTR luciferase activity was weak compared with the NC group(p<0.05)and the mut TLR4 3’UTR luciferase activity was not significantly different compared with the NC group.4.miRNA-182-5p inhibits OGD-induced microglial inflammatory responseBV2 cell were transfected with the miR-182-5p mimics and then exposed to OGD,the results showed that the mRNA and protein relative values of TNF-a,IL-6 and IL-1βwere significantly decreased compared with the control group.5.TLR4 mediates the suppressed inflammatory response induced by miR-182-5p in post-OGD microglial cellsBV2 cell were transfected with siTLR4 and then exposed to OGD,the results showed that the mRNA relative values of TNF-α,IL-6 and IL-1β were significantly decreased compared with the NC group.Furthermore,BV2 cell were co-transfected with miR-182-5p mimics and TLR4(ORF)and then exposed to OGD,the results showed that the mRNA relative values of TNF-α,IL-6 and IL-1β were significantly increased compared with the NC group.6.miR-182-5p exerts neuroprotective effects on cerebral ischemia-reperfusion injury via the inhibition of TLR4 expression in microglial cellsThe area of cerebral ischemia was significantly reduced in the miRNA-182-5p treatment group compared with the NC group.mNSS and foot-fault test behavioral score display that the neurological function of the miRNA-182-5p-treated group improved from the 7th day and improved significantly on the 14th,21st and 28th days compared with the NC group.Compared with the NC group,Ibal and TLR4 positive cells were significantly reduced in the miRNA-182-5p treatment group.7.miRNA-182-5p inhibits TLR4 expression to attenuate inflammatory response in rats with cerebral ischemia-reperfusion Compared with the NC group,the mRNA and protein expression levels of TNF-a,IL-6 and IL-1β were significantly decreased and the relative value of mRNA and protein expression of TLR4 was decreased in miRNA-182-5p treatment group. |
