| Objective:This experiment aims to investigate the effect of hydrogen inhalation therapy on the regulation of autophagy after spinal cord injury in rats,and to provide a relevant experimental basis for the clinical application of hydrogen in the treatment of spinal cord injury.Methods:120 female SD rats weighing 200-220 g were selected and randomly divided into sham surgery/Sham group(n=40),spinal cord injury/SCI group(n=40),and hydrogen treatment/SCI+H2 group(n=40).the Sham group only underwent laminectomy without injury to the spinal cord;spinal cord injury models were constructed at the T10 level for the SCI and SCI+H2 groups using the vascular forceps clamping method.The SCI+H2group was treated with inhaled hydrogen gas for 30 minutes each time,twice a day after successful modeling.The motor function of the hind limbs of each group was assessed by BBB scoring method;9 rats were randomly executed in each group at 1,3,7 and 14 days after surgery,respectively,of which 3 were used for WB(Western blot)to detect the expression of autophagy proteins Beclin-1,LC3 and P62,3 rats were used for immunohistochemical fluorescence staining co-localization to detect the level of autophagy in each group,and 3 for reverse transcription-polymerase chain reaction(RT-PCR)to detect the expression of autophagy protein m RNA.Results:On postoperative days 1 and 3,the rat BBB motor function scores were not statistically significant between the SCI and SCI+H2 groups(P > 0.05),and on postoperative days 7 and 14,the motor function scores of rats in the H2 intervention group were significantly higher than those in the SCI group(P < 0.05).Western Blot results showed that compared with the Sham group,the expression of Beclin-1 protein and LC3-II/LC3-Ⅰ ratio increased on day 1 after spinal cord injury,peaked on day 3,and then gradually recovered,and the levels remained stable on day 14 after SCI.Similarly,compared with the Sham group,the expression levels of Beclin-1and LC-3 m RNA increased on day 1 after spinal cord injury,reached the highest level on day 3,and then gradually recovered,and basically stabilized on day 14 after SCI.The expression of P62 protein,an autophagy-related marker,was also measured.Compared with the Sham group,the expression of P62 protein showed an opposite trend,started to decrease on day 1 after spinal cord injury,reached its lowest level on day 3 after SCI,and then gradually recovered and stabilized on day 14.To further confirm the expression of Beclin-1 and LC3 in spinal cord tissues after spinal cord injury,paraffin-embedded sections and immunofluorescence staining were performed on spinal cord tissues,and the fluorescence expression of LC3 and Beclin-1 was observed under the microscope.The results showed that the number of positive cells expressing LC3 and Beclin-1 in spinal cord tissues after spinal cord injury was significantly increased compared with the Sham group.After hydrogen inhalation treatment,both Beclin-1 protein expression and m RNA expression were reduced in spinal cord tissue on day 3 compared with the SCI group,and the ratio of LC3-II/LC3-Ⅰ was reduced,and the m RNA expression level of LC3 was also reduced,all with statistically significant differences.Similarly,the expression level of P62 protein in spinal cord tissues was significantly higher in the hydrogen intervention group on day 3.At 14 days after SCI,paraffin sections of spinal cord tissue were processed using Nissl staining,and the number of motor neurons in the anterior horn of the spinal cord was observed and counted under the microscope,and the number of motor neurons in the anterior horn of the spinal cord was significantly increased in the hydrogen-treated group compared with the SCI group.Conclusion:Autophagy was activated after spinal cord injury,and hydrogen inhalation treatment improved the recovery of hind limb motor function after SCI in rats,attenuated the activity of cellular autophagy,promoted the recovery of neurological function and played a neuroprotective role. |
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