Effects of inhaled hydrogen on autophagy and neurological function after spinal cord injury in rats

doi:10.3877/cma.j.issn.1674-6880.2024.05.002

Abstract

Abstract:

Objective

To explore the effect of hydrogen inhalation therapy on the expression of autophagy-related proteins and motor function recovery after spinal cord injury in rats.

Methods

Forty-five female rats were randomly divided into a control group (Sham group)，a spinal cord injury group (SCI group) and a hydrogen-treated group (SCI+H₂ group)， with 15 rats in each group.The Sham group only performed laminectomy without injury to the spinal cord.The SCI group and the SCI + H₂ group used the modified Allen method to injury the spinal cord in the T10 segment and established a spinal cord injury model.The SCI group did not undergo any treatment after injury， whereas rats in the SCI+H₂ group were given inhalation of hydrogen for 30 minutes twice a day.The hindlimb motor function of rats in each group was evaluated by the Basso-Beattie-Bresnahan (BBB) score at 1， 3， 7 and 14 days after injury.At 3 d after injury， the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I and the expression of polyubiquitin-binding protein P62 and Beclin-1 were detected by western-blotting.Quantitative real-time PCR was used to detect the expression of Beclin-1 and LC3 messenger RNA (mRNA) in the injured area of rats in each group.The number of positive cells of LC3 and Beclin-1 in the injured area of each group was detected by immunofluorescence staining.

Results

At 1， 3， 7， and 14 days after injury， the BBB scores of the rats in the three groups were compared， and the differences were statistically significant (F=1 055.216， 771.909， 446.000，215.438； all P ＜0.001).Further two-by-two comparisons showed that the BBB score of the SCI group and SCI+H₂ group was lower than that of the Sham group at all time points after injury，and the BBB score was significantly higher in the SCI + H₂ group than in the SCI group at 7 and 14 days after injury (all P ＜0.05).At 3 d after injury， the LC3-II/LC3-I ratio， P62 protein，Beclin-1 protein， LC3 mRNA and Beclin-1 mRNA were compared among the three groups， and the differences were statistically significant (F=384.212， 49.279， 28.249， 17.904， 50.674； all P ＜0.001).Further two-by-two comparisons revealed that compared with the Sham group， the LC3-II/LC3-I ratio， Beclin-1 protein and the expression of LC3 and Beclin-1 mRNA were significantly higher in the SCI group and the SCI + H₂ group， and the expression of P62 protein was significantly lower (all P ＜0.05).Compared with the SCI group， the LC3-II/LC3-I ratio， Beclin-1 protein and the expression LC3 and Beclin-1 mRNA were significantly decreased， while the P62 protein expression was significantly increased in the SCI+H₂ group (all P ＜0.05).At 3 d after injury， immunofluorescence staining showed that LC3 and Beclin-1 positive cells in the Sham group were less than those in spinal cord injury groups， and they were significantly higher in the SCI group than in the SCI+H₂ group.

Conclusions

Autophagy is activated in the injured area after spinal cord injury with nerve damage in rats.Hydrogen inhalation can regulate the overactivation of autophagy and promote it to a homeostatic environment， which is beneficial to the recovery of spinal cord injury.

Key words: Spinal cord injury, Hydrogen, Autophagy, Microtubule-associated protein 1 light chain 3, Beclin-1, P62

Jie Wu, Zhiqiang Zhou, Jing Fu, Xigong Li, Qin Zhang. Effects of inhaled hydrogen on autophagy and neurological function after spinal cord injury in rats[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2024, 17(05): 363-371.

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Figures/Tables 6

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组别	只数	1 d	3 d	7 d	14 d
Sham组	5	21.0±0.0	21.0±0.0	21.0±0.0	21.0±0.0
SCI组	5	0.9±0.7^a	2.0±0.7^a	3.7±0.9^a	7.2±1.1^a
SCI+H₂组	5	1.6±1.1^a	3.2±1.3^a	5.5±1.5^ab	10.3±1.6^ab
F值		1 055.216	771.909	446.000	215.438
P值		＜0.001	＜0.001	＜0.001	＜0.001

组别	只数	1 d	3 d	7 d	14 d
Sham组	5	21.0±0.0	21.0±0.0	21.0±0.0	21.0±0.0
SCI组	5	0.9±0.7^a	2.0±0.7^a	3.7±0.9^a	7.2±1.1^a
SCI+H₂组	5	1.6±1.1^a	3.2±1.3^a	5.5±1.5^ab	10.3±1.6^ab
F值		1 055.216	771.909	446.000	215.438
P值		＜0.001	＜0.001	＜0.001	＜0.001

组别	只数	LC3-Ⅱ/LC3-Ⅰ比值	P62	Beclin-1
Sham组	3	0.413±0.015	0.85±0.06	0.51±0.09
SCI组	3	0.823±0.026^a	0.43±0.04^a	0.88±0.04^a
SCI+H₂组	3	0.496±0.015^ab	0.67±0.06^ab	0.74±0.05^ab
F值		384.212	49.279	28.249
P值		＜0.001	＜0.001	＜0.001

组别	只数	LC3-Ⅱ/LC3-Ⅰ比值	P62	Beclin-1
Sham组	3	0.413±0.015	0.85±0.06	0.51±0.09
SCI组	3	0.823±0.026^a	0.43±0.04^a	0.88±0.04^a
SCI+H₂组	3	0.496±0.015^ab	0.67±0.06^ab	0.74±0.05^ab
F值		384.212	49.279	28.249
P值		＜0.001	＜0.001	＜0.001

组别	只数	LC3	Beclin-1 mRNA
Sham组	3	1.01±0.18	1.00±0.12
SCI组	3	1.66±0.07^a	1.72±0.04^a
SCI+H₂组	3	1.31±0.12^ab	1.51±0.10^ab
F值		17.904	50.674
P值		＜0.001	＜0.001

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