The Role And Mechanisms Of Pregnane X Receptor (PXR) In Spinal Cord Injury

ObjectiveAs a severe neurological disease mainly caused by trauma,spinal cord injury（SCI）induces varying degrees of motor and sensory deficits and/or autonomic neuropathy resulting in declined quality of life and heavy medical burden to society and family.Pregnane X receptor（PXR）is a ligand-activated nuclear receptor,which regulates the expression of multiple metabolizing enzymes and transporters,and plays an important role in the metabolism of xenobiotic and endobiotic metabolism.PXR is mainly expressed in the liver and gut,and also expressed in brain,kidney and skin with moderate level.Recently,it has been implicated that PXR has various other functions including the regulation of apoptosis,proliferation,inflammatory response and oxidative stress,which are all involved in the pathological process of SCI.In the present study,we aimed to investigate the role and mechanisms of PXR in the development of SCI.MethodsThe clip-compressive SCI model was performed in male C57BL/6 background wild-type(PXR^+/+)and PXR-knockout(PXR^-/-)mice.H₂O₂-induced injury model in the mouse neuroblastoma N2a cells mimicked the pathological process of SCI in vitro.Pregnenolone16α?carbonitrile（PCN）,a mouse specific PXR agonist,was used to activate PXR in vivo and in vitro.The siRNA was applied to knockdown the PXR expression in vitro.The basso mouse scale（BMS）,inclined plane test,footprint analysis,Nissl staining,and hematoxylin-eosin staining were used to assess the recovery of motor function and pathological injury.The contents of malondialdehyde（MDA）,activities of superoxide dismutase（SOD）and glutathione peroxidase（GPx）were investigated by biochemical kits,the expression levels of 4-hydroxynonenal（4-HNE）and 8-hydroxydeoxyguanosine（8-OXO）were determined by immunofluorescence staining to evaluate the level of oxidative stress level comprehensively.Tunel staining and the expression of apoptosis-associated factors detected by Western blot were used to measure the level of apoptosis.Cell viability was determined by CCK-8 assay,and the inflammatory degree was evaluated by immunohistochemical staining of F4/80（as the marker of microglia）.Finally,transcriptome sequencing analysis was performed to discover the relevant mechanisms and further study the relevant mechanisms in vivo and in vitro.ResultsFor the first time in the spinal cord,PXR-specific expression was found in neurons,but not in astrocytes and microglia.We found that the protein level of PXR was decreased after SCI with the lowest expression on the third day after SCI,and then gradually returned to original level on day 7 after injury.In vivo,PXR knockout significantly improved motor function of mice after SCI,meanwhile,inhibited apoptosis,inflammation and oxidative stress induced by SCI.On the contrary,activation of PXR by PCN negatively influenced the recovery of SCI.We also found that PXR knockdown alleviated oxidative stress,increased cell viability and inhibited cell apoptosis in N2a H₂O₂-induced injury model.In contrast,activation of PXR aggravated H₂O₂-induced N2a cell damage.Mechanistically,transcriptome sequencing analysis revealed that PXR activation downregulated the m RNA level of heme oxygenase-1（HO-1）after SCI.We further verified that PXR inhibited the NRF2/HO-1 pathway in vivo and in vitro.ConclusionPXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway to control the oxidative stress,apoptosis and inflammation induced by SCI.Therefore,as a new therapeutic target,PXR has potential application value in the rehabilitation of SCI.