Study On The Protection Effects And Mechanisms Of Necrostatin-1 On Spinal Cord Post The Injury

Objective:Spinal cord injury (SCI) is a serious central nervous system (CNS) trauma, which leads to irreversible damage of sensory and motor function below the wound area. Given that the pathological and physiological mechanisms of SCI are complex and variable, there are no satisfactory drugs or surgical treatments for SCI so far, and it remains as a serious public health problem. This study mainly focuses on the new pathological mechanism of injury on nerve cells, the possible therapeutic targets, new effective therapeutic compounds and methods after injury, for the better protection after injury of spinal cord and function.Methods:In this study, rats were used to generate SCI model using dorsal spinal cord impact method. Necrostatin-1 (Nec-1) is an inhibitor of the receptor interacting protein (RIP)1 kinase, thus acts as an inhibitor of necroptosis, a special programmed necrosis. To investigate the role of Nec-1 in the recovery of SCI, Nec-1 was used to treat the SCI rats 20 minutes before injury in this study. Spinal cord viability and tissue damage 12 h and 24 h post-injury were detected by TTC staining to detect the protection effects and optimum dose. As a specific inhibitor of RIP1, Necrostatin-1 (Nec-1) can effectively inhibit necroptosis. In 20 minutes before the spinal cord injury are treated with different concentrations of Nec-1 to be protected, to observe the efficacy of. HE staining was used for pathological study on the protective effects of Nec-1 on tissues and cells. Recovery of motor function was detected using BBB score test after spinal cord injury. The effects of Nec-1 on inflammatory cytokines 24 h after spinal cord injury were detected using ELISA kits. TUNEL detection for cell apoptosis and the effect inhibition of Nec-1 on spinal cord 12 h and 24 h after injury was performed. Further Western blot for quantitative analysis and IHC detection for localization analysis on Caspase-3, Bax, Bcl-2 and p53 expression were also performed. The expressions of RIP 1, RIP3 and MLKL were also detected using Western blot for quantitative analysis, not only to determine the necroptosis and the inhibition effect of Nec-1. For the detection of protective effect on mitochondrial of Nec-1 Study on the protection effects and mechanisms of Necrostatin-1 on spinal cord post the injury ABSTRACT after SCI, transmission electron microscopy was used to analyze the mitochondrial ultra-structure 24 h post-injury. The concentrations of mitochondrial calcium 12 h and 24 h post-injury were detected using kit, while the mitochondrial membrane potential (MMP) using JC-1 assay. HPLC was used to detect the cytosolic cytochrome c and total cytochrome c (including mitochondrial and cytosolic cytochrome c) and calculate the ratio, together with the ATP generation 12 h and 24 h after spinal cord injury. The effects of Nec-1 on mitochondrial respiratory chain complex Ⅰ, Ⅱ, Ⅲ, Ⅳ and the products of oxidative stress were detected 24 h after spinal cord injury using the kits. Realtime PCR was performed to detect the mitochondrial genes cytochrome b, PGC-1, NRF-1 and TFAM 12 h post-injury and mitochondrial fusion genes Mfn1 and Mfn2, mitochondrial fission genes Drpl and Fisl 6 hand 12 h post-injury.Results:TTC staining indicated that 1 μl Nec-1 with concentration more than 1μg/μl significantly decreased the lesions and this viability protection effect was dose-dependent, which saturated at concentration more than 25μg/μl. Thus this optimum dose (1 μl 25μg/μl) of Nec-1 was used throughout the entire study. Nec-1 was found to reduce the lesions, improve the pathological conditions and blood supply in the spinal cord trauma area. After the treatment of Nec-1, the cell structure was much better while decreased vacuolization was found. Mild necrosis was detected in the focal zone, but not as obvious nor as serious as in the SCI group. The performance and function of spinal cord were much better after the treatment of Nec-1. Ethological performance of SCI rats confirmed the improvement and protection of physiological function by Nec-1. No obvious toxicity was found for Nec-1. All these indicated the high safety of Nec-1. In the primary and secondary stagesof spinal cord injury, secondary injury is the more destructive phase. Inhibition or decrease of the damages to the spinal cord after neural cell death, relieving pathological damage and promotion on the recovery of neurological function are much more important. Our study indicated that Nec-1 inhibited the apoptosis by doen-regulation of Caspase 3 and Bax while up-regulation of Bcl-2. P53 is not likely involved in this process. Not only apoptosis, necroptosis was also found after SCI and Nec-1 could also inhibit the necroptosis by inhibiting RIP1/3-MLKL recruitment. Besides supplying cellular energy (adenosine triphosphate, ATP), mitochondria are also involved in cell signaling, cellular differentiation, cell death, cell cycle and cell growth. Mitochondrial dysfunction following SCI may be critical for the development of secondary pathophysiology and neuronal cell death. After the spinal cord injury, intramitochondrial concentration of Ca2+ increase sharply. With the inflow of large amounts of Ca2+ and thus calcium overload in mitochondria obviously. A further decline in mitochondrial membrane stability and reduced membrane potential (MMP) are also detected, which are important for the maintenance of the function of the mitochondrial respiratory electron chain capacity (ATP). With the rapid increased concentration of Ca2+, the mitochondrial respiratory chain is also damaged, and the ATP formation decrease, which results in the accumulation of ROS. These ROS can destroy the stability of mitochondria. After spinal cord injury, with the inflow of large amounts of Ca2+ mitochondrial swelling and resulting in rupture of the outer membrane, cytochrome c release into the cytoplasm, activation of apoptosis related factors and inflammatory cytokines. All these would aggravate the neuron cell damage. These injuries also result in a marked reduction of cytochrome b. The expression of transcription activator is significantly decreased. The expressions of mitochondrial fusion and fission factors were abnormal. Nec-1 reduces the increased Ca2+ concentration in mitochondria due to the injury and preserves the remarkable decreased MMP level after SCI. It also increases the generation of ATP and the activity of mitochondrial respiratory chain complex I which are decreased due to the injury. Nec-1 inhibits the generation of ROS and the release of cytochrome c in mitochondria and protects the spinal cord from mitochondrial swelling. Nec-1 promotes mitochondrial biogenesis by the activation of PGC-1, NRF-land Tfam especially up-regulating mitochondrial transcription factor A (Tfam), in accordance with the mtDNA content. The expressions of mitochondrial fusion factors Mfnl, Mfn2 and fission factors Drp1 and Fis1 are tend to be normal.Conclusions:Not only the apoptosis, but also necroptosis were found after spinal cord injury. As an inhibitor of necroptosis, Nec-1 can effectively improve the spinal cord function post-injury, playing the protective effect on the injuryed spinal cord. It is likely be used as a potential drug for the treatment of spinal cord injury, which deserves the further study.