Experimental Study On The Therapeutic Effect Of Spinal Cord Injury Via Regulating The Nrf2-ARE Pathway In Mice

Spinal cord injury (SCI) is a frequent trauma type, and is a major cause of death and disability. The mechanisms underlying secondary spinal cord injury are known to be complex, and close interactions exist these pathological processes. It seems that the present therapeutic strategy aimed at single mechanism has been shown to be inefficacy in clinical trials, and excessive inhibition of one certain kind of secondary injury mechanisms, with ignoring its benefit to the body, will bring adverse reaction. Therefore, a new therapeutic strategy activating self-regulation mechanism and aimed at multiple targets may be desirable. Nrf2-ARE pathway is a kind of self-regulation mechanism in cells, which has been reported to be a pleiotropic regulator in cell survival mechanisms, consisted with several mechanisms underlying secondary spinal cord injury. The previous study has demonstrated the activation of Nrf2in the spinal cord tissue after spinal cord injury. Thus, we hypothesize that Nrf2-ARE pathway might be a new therapeutic target for protecting the neurological function after SCI. In this study, we used an SCI model induced by the application of vascular clips to the dura, and regulated the Nrf2-ARE pathway positively and negatively, in order to observe the neuroprotective effect of the Nrf2-ARE pathway and its mechanisms after SCI in mice.Part I:IntroductionIn this part, the background of the study is introduced, including reviews of the protective effect of Nrf2-ARE pathway in inflammation, the comparison of several models of experimental spinal cord injury, the research progress of post-spinal cord injury neuroinflammation, and the role of MMP-2、MMP-9in the central nervous system. Part Ⅱ:The therapeutic effect and anti-inflammation mechanism after spinal cord injury via positively regulating the Nrf2-ARE pathwayInflammation plays an important role in the pathogenesis of secondary damage after spinal cord injury (SCI). During the early-phase after acute SCI, palliating inflammation moderately is benefit for protecting neuron and recovery of neurological function. As an isothiocyanate present in cruciferous vegetables such as broccoli, sulforaphane (SFN) has been extensively investigated with regards to its ability to induce phase Ⅱ detoxification enzymes. It activates Nrf2and exerts anti-inflammatory effects. This study evaluated the neuroprotective role of activating Nrf2-ARE pathway via SFN in the inflammatory response after SCI in mice. Nrf2(-/-)ICR and Nrf2(+/+)ICR mice spinal cord compression injury was induced by the application of vascular clips (force of10g) to the dura. Sulforaphane (SFN) was used to activate Nrf2after SCI. Neurological function was assayed by the Basso open-field motor score. Pathological sections were used for histological injury score. Degenerating neuronal cells were stained with Fluoro Jade C, and observed by a confocal microscopy. Spinal cord percent water content was calculated as [(wet weight-dry weight)/wet weight]. NF-κB and Nrf2DNA binding activity was assessed by EMSA. The mRNA levels of MMP-9, AQP4, TNF-α, IL-6, IL-1β, NAD(P)H:quinone oxidoreductase1(NQO1) and glutathione S-transferase α1(GST-α1) were detected by RT-PCR ELISA was used to detect TNF-α, IL-6and IL-1β protein expression, and Western blot for MMP-9and AQP4protein expression. Colorimetric method was used to detect the enzyme activity of NQO1and GST-α1, and gelatin zymography for MMP-9activity. The results showed that SFN activated Nrf2and suppressed NF-κB in impaired spinal cord tissue, improved hindlimb locomotor function assessed by BMS, reduced inflammatory damage, histological injury, dying neurons count and spinal cord edema caused by SCI. Nrf2-/-mice demonstrated severer neurologic deficit and spinal cord edema after SCI and did not benefit from the protective effect of SFN. The results of this part suggest that regulating Nrf2-ARE pathway positively plays an important neurological protective role in limiting the spinal cord inflammatory response through modulating the proinflammatory NF-κB signaling pathway after SCI. Part Ⅲ:The influence on the neurological dysfunction and its mechanism after spinal cord injury via negatively regulating the Nrf2-ARE pathwayNrf2-ARE signaling is involved in attenuating inflammation-associated pathogenesis, such as autoimmune diseases, asthma, emphysema, colitis and atherosclerosis. Thus, disruption or loss of Nrf2signaling causes enhanced susceptibility to inflammatory tissue injuries. But the role of Nrf2-ARE pathway after SCI in mice is not very clear. Spinal cord compression injury of Nrf2(-/-)ICR and Nrf2(+/+)ICR mice was induced by the application of vascular clips (force of10g) to the dura. Neurological function was assayed by the Basso open-field motor score, footprint analysis and spinal motor-evoked potentials (MEPs). Pathological sections were used for histological injury score. Degenerating neuronal cells were stained with Fluoro Jade C, and observed by a confocal microscopy. Spinal cord percent water content was calculated as [(wet weight-dry weight)/wet weight]. NF-κB and Nrf2DN A binding activity was assessed by EMS A. The mRNA levels of MMP-9, TNF-a, IL-6, IL-1β, NAD(P)H:quinone oxidoreductase1(NQO1) and glutathione S-transferase al (GST-α1) were detected by RT-PCR ELISA was used to detect TNF-a, IL-6and IL-1β protein expression, and Western blot for MMP-9protein expression. Colorimetric method was used to detect the enzyme activity of NQO1and GST-al, and gelatin zymography for MMP-9activity. The results showed Nrf2KO mice developed severer hindlimb motor dysfunction, histological injury and neuronal death after SCI compared with WT mice. In correlation with neurological deficits, NF-κB DNA binding activity and the release of MMP-9, TNF-a, IL-6and IL-1β in the spinal cord of KO mice was higher than WT, whereas the Nrf2banding activity, the expression and activity of NQO1and GST-α1was all lower in KO mice24h after SCI compared with WT mice. The results of this part suggest genetic ablation of Nrf2exacerbated the neurological deficit and inflammation after SCI in mice. These findings raise the possibility that Nrf2-ARE pathway could be relevant in improving outcome after SCI. SummarySpinal cord injury (SCI) is a frequent trauma type, and aggravates the patients’quality of life. The emphasis and the nodus of treatment is to relieve and improve the neurological dysfunction after SCI. But the present therapeutic strategy in clinic has been shown to be inefficacy and unsure. Nrf2-ARE pathway is a kind of self-regulation mechanism in cells, which has been reported to be a pleiotropic regulator in cell survival mechanisms. But its neurological functional protective role and mechanisms after SCI are not very clear. In this study, we regulated the Nrf2-ARE pathway positively and negatively, and used an SCI model induced by the application of vascular clips to the dura. Then we counted degenerating neuronal cells, observed the extent of histological injury, spinal cord percent water content and hind limb neurological dysfunction. Moreover, we explored the anti-inflammation mechanism of Nrf2-ARE pathway after SCI in mice. According to the cross-check analysis, the study showed Nrf2-ARE pathway plays an important neurological protective role in limiting the spinal cord inflammatory response through modulating the proinflammatory NF-κB signaling pathway after SCI. Understanding the defense mechanism by which the Nrf2activation confers protection against SCI in mice can provide rationale to develop therapeutic and preventive strategies for the management of SCI in clinic.