To investigate the effects of hydrogen inhalation on the expression of autophagy-related proteins and neural repair function in rats after spinal cord injury (SCI).

Forty-five female Sprague-Dawley rats were selected and divided into three groups using a random number table method, with 15 rats in each group: the control group (treated with only lamina resection), the SCI group (treated with a modified Allen method for modeling after laminectomy), and the hydrogen treatment group (SCI + H₂ group, given hydrogen inhalation after modeling). The Basso-Beattie-Bresnahan (BBB) score was used to evaluate the hind limb motor function of rats in each group at 1, 3, 7, 14, 21, and 28 days after SCI. Hematoxylin-eosin (HE) staining was used to observe the structural and morphological changes of the anterior horn of the spinal cord and the tissue at the injury site; Nissl staining was used to observe the morphological changes of spinal cord neurons. The protein expression levels of phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt), phosphorylated Akt (p-Akt), mammalian target of rapamycin (mTOR), and phosphorylated mTOR (p-mTOR) in the spinal cord tissue of rats in each group after SCI were detected by western-blotting. The levels of microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, glial fibrillary acidic protein (GFAP), microtubule-associated protein 2 (MAP2), myelin basic protein (MBP), and Tuj-1 of rats in each group after SCI were detected by immunofluorescence staining.

There were significant differences in the BBB scores of the three groups at 1, 3, 7, 14, 21, and 28 days after modeling (F = 4 805.000, 571.200, 323.400, 134.400, 62.740, 28.720, all P < 0.001), and at 21 and 28 days after modeling, the BBB scores in the SCI + H₂ group were significantly higher than those in the SCI group (both P < 0.05). HE staining revealed that the spinal cord structure of the control group was intact, while obvious tissue cavities and neuronal loss occurred in the SCI group. The injury degree of the SCI + H₂ group was significantly improved compared with the SCI group. The Nissl staining showed that the number of Nissl bodies in the SCI + H₂ group was significantly increased compared with the SCI group, but was less than that in the control group. Western-blotting showed that there were significant differences in the ratios of p-mTOR/mTOR (F = 17.030, P = 0.006) and p-AKT/Akt (F = 13.000, P = 0.004) among the three groups. Moreover, the ratios of p-mTOR/mTOR and p-AKT/Akt in the SCI + H₂ group and the control group were much higher than those in the SCI group (all P < 0.05). However, there was no statistically significant difference in the PI3K expression among the three groups (F = 0.267, P = 0.861). The immunofluorescence staining indicated that in the three groups, the average fluorescence intensities of GFAP (F = 181.100, P = 0.001), MAP2 (F = 264.800, P = 0.005), MBP (F = 239.800, P = 0.001), Tuj-1 (F = 105.400, P = 0.001), LC-3 (F = 105.500, P = 0.001), and Beclin-1 (F = 39.530, P = 0.003) all showed statistically significant differences. Moreover, compared with the SCI group, the average fluorescence intensities of GFAP, LC3, and Beclin-1 in the SCI + H₂ group were significantly decreased, while the average fluorescence intensities of MAP2, MBP, and Tuj-1 were significantly increased (all P < 0.05).

Hydrogen inhalation can improve the recovery of motor function in rats after SCI by inhibiting the excessive autophagy response and promoting the expression of proteins related to neural tissue repair.