Changes of mitochondrial autophagy in rats after spinal cord injury and efficacy of targeted therapy
Objective To investigate the changes of mitochondrial autophagy after spinal cord inju-ry in rats and the effect of targeted Nix on spinal cord injury.Methods Totally,30 SD rats were randomly divided into control group,spinal cord injury group and Nix down-regulated group according to the digital table method.Mice in the Nix down-regulated group were injected with Nix short hairpin RNA(shRNA)adeno-associated virus in the spinal cord at 24th day before surgery,and the control group and the spinal cord injury group were injected with adeno-associated virus at the same location.Spinal cord injury model was prepared by aneurysm clamp compression injury method in spinal cord injury group and Nix down-regu-lated group,and no treatment was given in control group.The number of denatured neurons in the spinal cord was analyzed by staining after 14 days.Basso-Beattie-Bresnahan(BBB)motor function rating scale was used to analyze the changes of motor function after injury in the three groups.Mitochondrial outer membrane protein TOM20,mitochondrial inner membrane protein Tim23 and phosphatase and tensin homo-logue deleted on chromosome ten-induced putative kinase(PINK1)and Parkin of the three groups were an-alyzed by Western blotting.The number of autophagosomes in spinal cord cells of three groups of rats was analyzed by transmission electron microscopy.The mitochondrial membrane potential of spinal cord cells in three groups of mice was analyzed by flow cytometry.Univariate analysis of variance was used for measure-ment comparison between groups.Results The expression level of Nix protein in bone marrow of rats in the down-regulated Nix group(0.89±0.31)was significantly lower than that in the spinal cord injury group(1.79±0.11,t=15.320,P＜0.05).The expression levels of TOM20 and Tim23 protein in bone marrow tissue of rats in Nix down-regulated group(0.80±0.07,0.69±0.06)was significantly higher than that in spinal cord injury group(0.53±0.12,0.39±0.09,t=6.029,8.270,P＜0.05).The ex-pression levels of PINK1 and Parkin protein in bone marrow tissue of rats in Nix down-regulated group(1.04±0.11,0.83±0.07)were significantly lower than those in control group(1.48±0.15,1.24±0.13,t=7.565,9.019,P＜0.05).The number of dead neurons in bone marrow tissue of rats in Nix down-regulated group(6.70±1.34)was significantly less than that in spinal cord injury group(17.9±5.26,t=6.527,P＜0.05).The number of autophagy in bone marrow cells in the down-regulated Nix group(14.00±2.58)was significantly greater than that in the spinal cord injury group(29.70±3.56,t=11.290,P＜0.05).The fluorescence intensity of mitochondrial membrane potential of bone marrow cells in the down-regulated Nix group(179.54±12.02)was significantly higher than that in the spinal cord injury group(108.14±12.89,t=12.810,P＜0.05).The BBB score of rats in Nix down-regulated group[(12.50±2.80)points]was significantly higher than that in spinal cord injury group[(5.80±1.48)points,t=6.960,P＜0.05].Conclusion The level of mitochondrial autophagy increases signifi-cantly after spinal cord injury.Down-regulating Nix can inhibit the inhibition of mitochondrial autophagy,reduce mitochondrial damage,protect spinal cord neurons,and improve neural function in rats.
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