Low-Dose Lipopolysaccharide Alleviates Neuronal Apoptosis and Oxidative Stress in Rats with Spinal Cord Injury by Inducing Nrf2 m6A Methylation Modification via Suppressing ALKBH5.

This work reported the neuronal protection of low-dose lipopolysaccharide (LD-LPS) after spinal cord injury (SCI). SCI rat model was constructed, after adenovirus-mediated ALKBH5 vectors and shRNA transfection and LD-LPS pre-treatment. Hematoxylin and eosin, Nissl, TUNEL staining of spinal cord tissues were adopted to monitor pathological changes, neuronal survival and apoptosis. PC12 cells transfected with ALKBH5 vectors and ALKBH5/Nrf2 siRNAs were treated by LD-LPS, followed by oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability and apoptosis were assessed by cell counting kit-8 and TUNEL assays. Neuronal oxidative stress was evaluated by appraising MDA and SOD levels. ALKBH5 and Nrf2 expression was monitored through immunohistochemistry, Western blot and qRT-PCR. Methylated RNA immunoprecipitation assay and Dot-blot experiment were for Nrf2 m6A modification detection, while RNA pull-down assay was for the binding validation between ALKBH5 and Nrf2. In rats with SCI, LD-LPS relieved spinal cord tissue damage and neuronal apoptosis; enhanced neuronal survival; decreased MDA content; elevated SOD activity; down-regulated ALKBH5; up-regulated Nrf2; and facilitated Nrf2 m6A methylation. These above influences by LD-LPS were eliminated by ALKBH5. Similar results were found in the OGD/R-induced PC12 cells after LD-LPS treatment. ALKBH5 significantly blocked Nrf2 m6A methylation, and pulled down Nrf2 protein. In the OGD/R-induced PC12 cells, the repressed oxidative stress and apoptosis by ALKBH5 silencing was abrogated by Nrf2 knockdown. LD-LPS might alleviate neuronal apoptosis and oxidative stress after SCI by facilitating Nrf2 m6A methylation via reducing ALKBH5. It was proposed to be a novel strategy for SCI treatment.