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中华危重症医学杂志(电子版) ›› 2024, Vol. 17 ›› Issue (05) : 363 -371. doi: 10.3877/cma.j.issn.1674-6880.2024.05.002

论著

吸入性氢气对大鼠脊髓损伤后自噬及神经功能的影响
吴杰1, 周志强2, 符菁3, 李喜功4, 张钦1,()   
  1. 1.044000 山西运城,山西医科大学附属运城市中心医院脊柱外科
    2.330000 南昌,南昌大学第一附属医院赣江新区医院疼痛科
    3.310023 杭州,杭州市西溪医院口腔科
    4.310003 杭州,浙江大学医学院附属第一医院脊柱外科
  • 收稿日期:2024-03-25 出版日期:2024-10-31
  • 通信作者: 张钦
  • 基金资助:
    山西省卫生健康委科研课题项目(2020168)北京医学奖励基金会项目(HX2021004)浙江省医药卫生科技计划项目(2024KY211)

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

Jie Wu1, Zhiqiang Zhou2, Jing Fu3, Xigong Li4, Qin Zhang1,()   

  1. 1.Department of Spine Surgery,Yuncheng Central Hospital Affiliated to Shanxi Medical University,Yuncheng 044000,China
    2.Pain Department,Ganjiang New Area Hospital of the First Affiliated Hospital of Nanchang University,Nanchang 330000,China
    3.Department of Stomatology,Xixi Hospital of Hangzhou,Hangzhou 310023,China
    4.Department of Spine Surgery,the First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310003,China
  • Received:2024-03-25 Published:2024-10-31
  • Corresponding author: Qin Zhang
引用本文:

吴杰, 周志强, 符菁, 李喜功, 张钦. 吸入性氢气对大鼠脊髓损伤后自噬及神经功能的影响[J]. 中华危重症医学杂志(电子版), 2024, 17(05): 363-371.

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.

目的

探讨氢气吸入对大鼠脊髓损伤后自噬相关蛋白的表达和运动神经功能恢复的影响。

方法

将45 只雌性大鼠分为对照组(Sham 组)、脊髓损伤组(SCI 组)和氢气组(SCI+H2 组),每组各15 只。Sham 组仅进行椎板切除,而不损伤脊髓;SCI 组和SCI+H2 组均采用改良Allen 法在T10 节段损伤脊髓,建立脊髓损伤模型;SCI 组模型建立完成后不进行任何治疗,而SCI+H2 组大鼠在脊髓损伤后给予吸入氢气治疗,每次30 min,每天2 次。采用Basso-Beattie-Bresnahan(BBB)评分量表评估各组大鼠脊髓损伤后1、3、7、14 d 的后肢运动功能。脊髓损伤后3 d,采用western-blotting 法检测各组大鼠损伤组织区域微管相关蛋白1 轻链3(LC3)-Ⅱ和LC3-Ⅰ的比值、多泛素结合蛋白P62 和Beclin-1 的表达量;实时荧光定量PCR 法检测各组大鼠损伤区域LC3 和Beclin-1 信使RNA(mRNA)的表达量;同时采用免疫荧光染色法检测各组大鼠损伤区域LC3 和Beclin-1 的阳性细胞情况。

结果

脊髓损伤后1、3、7、14 d,3 组大鼠BBB 评分比较,差异均有统计学意义(F=1 055.216、771.909、446.000、215.438,P 均<0.001)。进一步两两比较发现,脊髓损伤后各时间点SCI 组和SCI+H2 组大鼠BBB 评分均低于Sham 组;且脊髓损伤后7、14 d,SCI+H2 组大鼠BBB 评分均显著高于SCI 组(P 均<0.05)。脊髓损伤后3 d,3 组大鼠LC3-Ⅱ/LC3-Ⅰ比值、P62、Beclin-1 蛋白及LC3、Beclin-1 mRNA 的表达量比较,差异均有统计学意义(F=384.212、49.279、28.249、17.904、50.674,P 均<0.001)。进一步两两比较发现,与Sham 组相比,SCI组和SCI+H2 组LC3-Ⅱ/LC3-Ⅰ比值、Beclin-1 蛋白及LC3、Beclin-1 mRNA 的表达量均明显升高,P62 蛋白表达量明显下降(P 均<0.05);与SCI 组相比,SCI + H2 组LC3-Ⅱ/LC3-Ⅰ比值、Beclin-1 蛋白及LC3、Beclin-1 mRNA 的表达量均明显下降,P62 蛋白表达量则明显升高(P 均<0.05)。免疫荧光染色检测结果显示,脊髓损伤后3 d,Sham 组大鼠自噬蛋白LC3 和Beclin-1 的阳性细胞表达较少,SCI 组自噬蛋白LC3 和Beclin-1 的阳性细胞表达数明显多于Sham 组;而与SCI组相比,SCI+H2 组LC3 和Beclin-1 的阳性细胞则呈低表达。

结论

脊髓损伤后神经受损,自噬被激活,氢气吸入能够调控自噬的过度激活,使自噬恢复到稳态环境,有利于脊髓损伤的恢复。

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.

表1 脊髓损伤后不同时间点各组大鼠BBB 评分比较(分, ±s)
表2 脊髓损伤后3 d 各组大鼠LC3-Ⅱ/LC3-Ⅰ比值、P62 和Beclin-1 蛋白的表达量比较( ±s)
图1 脊髓损伤后3 d 各组大鼠自噬相关蛋白的表达情况 注:LC3.微管相关蛋白1 轻链3;Sham 组、SCI 组、SCI+H2 组分别为对照组、脊髓损伤组和氢气组
图2 脊髓损伤后3 d 各组大鼠自噬蛋白LC3 的表达情况 注:LC3.微管相关蛋白1 轻链3;DAPI.4',6-二脒基-2-苯基吲哚;Sham 组、SCI 组、SCI+H2 组分别为对照组、脊髓损伤组和氢气组;DAPI 定位细胞核,为蓝色荧光;LC3 定位细胞膜,为红色荧光,其标记的细胞为神经元细胞;Sham 组显示表达自噬蛋白LC3 的阳性细胞较少;SCI 组和SCI+H2 组表达LC3 的阳性细胞明显增多;SCI+H2 组表达LC3 的阳性细胞较SCI 组有所减少(免疫荧光染色法 ×400)
图3 脊髓损伤后3 d 各组大鼠自噬蛋白Beclin-1 的表达情况 注:DAPI.4',6-二脒基-2-苯基吲哚;Sham 组、SCI 组、SCI+H2 组分别为对照组、脊髓损伤组和氢气组;DAPI 定位于细胞核,为蓝色荧光;Beclin-1 和NeuN 定位于细胞膜,分别为红色荧光和绿色荧光,均标记神经元细胞;Sham 组显示表达自噬蛋白Beclin-1 的阳性细胞较少;SCI 组和SCI+H2 组表达Beclin-1 的阳性细胞明显增多;且SCI+H2 组表达Beclin-1 的阳性细胞较SCI 组有所减少(免疫荧光染色法 ×400)
表3 脊髓损伤后3 d 各组大鼠LC3 和Beclin-1 mRNA 的表达量比较( ±s)
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