Heme Oxygenase-1 Activates AMPK Signaling Pathway To Regulate Microglia-Mediated Neuroinflammation After Spinal Cord Injury

Objectives1.To analyze the dynamics of inflammatory cells after spinal cord injury(SCI);2.To construct heme oxygenase-1(HO-1)overexpression recombinant lentivirus vector(LV-HO-1)and transfect microglia;3.To investigate the mechanism of adoptive transfer of LV-HO-1 transfected microglia to regulate inflammation after SCI;4.To explore the molecular mechanism of adoptive transfer of LV-HO-1 transfected microglia to enhance the anti-inflammatory response after SCI.Methods1.The rat-SCI-model was established by compression,and the monocyte enrichment and flow cytometry were used to analyze the proportion of microglia,macrophages and lymphocytes in spinal cord tissue,and q PCR were applied detect the level of HO-1 mRNA in these cells;2.Lentivirus were used to construct HO-1 overexpression vector(LV-HO-1),and transfected it into microglia to induce exogenous HO-1 overexpression.Real-time fluorescent quantitative PCR(q-RT-PCR)and Western blotting were used to detect the expression levels of HO-1 mRNA and protein in microglia;Annexin V-PI staining to detect the degree of microglia apoptosis;q-RT-PCR method were used to detect IL-1β,IL-18,IL-10 and TGF-β mRNA level;3.The adoptive transfer procedure of the microglia were applied after LV-HO-1 transfection.Flow cytometry and immunofluorescence staining were used to observe the changes of microglia;and q-RT-PCR was applied to detect HO-1 mRNA and a variety of pro-inflammatory and anti-inflammatory factors mRNA levels,and a kit were used to determine iron content in cells;flow cytometry to analyze the number of inflammatory cells;ELISA kit to detect IL-1β,TNF-α and IL-6 in spinal cord tissue expression;HE staining and immunofluorescence staining to observe the pathological morphology of spinal cord tissue;TUNEL method to label apoptotic cells;BBB score to evaluate changes in rat’s motor function;4.The LV-HO-1 transfected microglia was injected into the spinal cord,and the flow cytometry was used to screen out the adopted transfer microglia;q-RT-PCR method was used to screen the mRNA of transcription related to microglia activation factor(IRF5,IRF1,SP1,STAT2,IRF4,STAT6,PPARγ,STAT1,SP3,KLF4);Lipopolysaccharide was used to construct an inflammation model under in vitro conditions,and an inhibitor of AMPK signaling pathway was added for reverse verification.Results1.At 1,3,7,and 14 days after SCI,the expression of HO-1 mRNA in microglia first increased and then decreased,showing an opposite trend to the proportion;2.Successfully constructed LV-HO-1 and efficiently transfected microglia;3.LV-HO-1 transfected microglia still maintain a high expression of HO-1 in the spinal cord tissue after adoptive transfer,and can inhibit the secretion of a variety of pro-inflammatory factors,inhibit the infiltration of inflammation in the SCI site,and reduce the spinal cord pathological damage of tissues,inhibition of cell apoptosis and promotion of the recovery of rats’ motor function;4.HO-1 overexpression up-regulates AMPK signaling pathway in microglia and inhibits the secretion of pro-inflammatory factors IL-1β,TNF-α and IL-6 in microglia induced by lipopolysaccharide.Conclusions1.After SCI,the expression level of HO-1 in microglia is closely related to the proportion of microglia;2.LV-HO-1 can efficiently transfect microglia in vitro;3.The adoptive transfer of LV-HO-1 transfected microglia can inhibit the secretion of pro-inflammatory factors,reduce the infiltration of inflammatory cells,reduce the pathological damage of the spinal cord tissue and promote the recovery of rats’ motor function;4.Overexpression of HO-1 regulates spinal cord neuroinflammation by activating AMPK signaling pathway in microglia.