Effect of Sakuranetin on Microglia-Mediated Neuroinflammation After Spinal Cord Injury

Abstract

Objective To investigate the effects of sakuranetin (SK) on motor functions in the mouse model of spinal cord injury (SCI) and decipher the mechanism. Methods Fifty-four C57BL/6J mice were randomized into sham,SCI,and SK groups.The mice in the sham group underwent only laminectomy at T9,while those in the SCI and SK groups were subjected to spinal cord contusion injury at T9.Behavioral tests were conducted at different time points after surgery to evaluate the motor functions of mice in each group.The pathological changes in the tissue were observed to assess the extent of SCI in each group.The role and mechanism of SK in SCI were predicted by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses.Reverse transcription real-time fluorescence quantitative PCR,ELISA,and immunofluorescence were employed to evaluate the inflammation and activation of microglia in SCI mice.BV2 cells in vitro were classified into control (Con),lipopolysaccharide (LPS),and LPS+SK groups.The effects of SK intervention on the release of inflammatory cytokines and the activation of BV2 cells were evaluated.Furthermore,the phosphatidylinositol-3-kinase(PI3K)/protein kinase B (AKT) signaling pathway activator insulin-like growth factor-1 (IGF-1) was used to treat the SK-induced BV2 cells in vitro (SK+IGF-1 group),and SK was used to treat the IGF-1-induced BV2 cells in vitro (IGF-1+SK group).Western blotting was conducted for molecular mechanism validation. Results Behavioral tests and histological staining results showed that compared with the SCI group,the SK group exhibited improved motor abilities and reduced area of damage in the spinal cord tissue (all P<0.001).The GO enrichment analysis predicted that SK may be involved in the inflammation following SCI.The KEGG enrichment analysis predicted that SK regulated the PI3K/Akt pathway to exert the neuroprotective effect.The results from in vitro and in vivo experiments showed that SK lowered the levels of tumor necrosis factor-α,interleukin-6,and interleukin-1β and inhibited the activation of microglia (all P<0.05).The results of Western blotting showed that SK down-regulated the phosphorylation levels of PI3K and Akt (all P<0.001) and inhibited the IGF-1-induced elevation of PI3K and Akt phosphorylation levels (all P<0.001).Conversely,IGF-1 had the opposite effects (P=0.001,P<0.001).The results of reverse transcription real-time fluorescence quantitative PCR,ELISA,and immunofluorescence showed that the SK+IGF-1 group had higher levels of inflammatory cytokines and more activated microglia than the SK group(all P<0.05). Conclusion SK may suppress the activation of the PI3K/Akt pathway to inhibit the inflammation mediated by SCI-induced activation of microglia,ameliorate the pathological damage of the spinal cord tissue,and promote the recovery of motor functions in SCI mice.

Keywords: inflammation; microglia; phosphatidylinositol-3-kinase/protein kinase B; sakuranetin; spinal cord injury.