电解水制备氢气对小鼠脊髓损伤的保护作用及机制研究

Objective: To explore the effects of hydrogen produced by water electrolysis on spinal cord injury in mice and the morphology, function of mitochondrial permeability transition pore(mPTP).Background: The incidence of spinal cord injury is increasing each year with no effective medications. Secondary spinal cord injury is the key and oxidative.stress plays an important role in it, which is closely related to the mPTP. However, the mechanism is still unclear. Electrolysis of water is a convenient way to obtain hydrogen(66.7%) whose effects on spinal cord injury remain unreported..Methods: spinal cord contusion model is established with C57BL/10 mice which are given hydrogen inhalation(hydrogen 66.7%, oxygen 33.3%). Spinal cord injury is evaluated through Basso mouse scale, nissl staining, Caspase-3, inflammatory cytokines and oxidative stress markers. Mitochondria are extracted and the functions and membrane potential changes are examined. mPTP and reactive oxygen species are detected..Hypothesis: Inhalation of hydrogen produced by electrolysis of water could protect spinal cord injury, promote functional recovery through inhibiting mPTP opening and reducing ROS generation.

目的：研究呼吸电解水制备氢气对小鼠脊髓损伤的保护作用及对线粒体形态、功能及内膜转运孔通道的影响。.背景：脊髓损伤发病率逐年升高，目前尚缺乏有效治疗药物。脊髓继发损伤是研究的重点，氧化应激是重要机制，其中线粒体内膜转位孔通道作用关键。电解水是获得氢气的简易途径，有望成为疾病治疗的新方法。目前呼吸电解水制备氢气（66.7%）对脊髓损伤的作用尚无报道。.方法：制备C57BL/10小鼠脊髓钳夹伤模型，给予呼吸电解水制备的氢氧混合气体（氢气66.7%，氧气33.3%），通过小鼠BMS评分、尼氏染色、凋亡酶、炎症因子、氧化应激标志物评估脊髓损伤程度，提取脊髓线粒体，测定线粒体功能及膜电位变化，测定通透性转位孔开放情况及活性氧自由基产生量。.预期结论：呼吸电解水制备的氢气能够抑制线粒体mPTP通道，保护小鼠脊髓损伤，促进脊髓功能恢复。

项目背景：. 脊髓损伤发病率逐年升高，目前尚缺乏有效治疗药物。脊髓继发损伤是研究的重点，氧化应激是重要机制，其中线粒体内膜转位孔通道作用关键。电解水是获得氢气的简易途径 ，有望成为疾病治疗的新方法。目前呼吸电解水制备氢气(66.7%)对脊髓损伤的作用尚无报道。我们希望通过研究呼吸电解水制备氢气对小鼠脊髓损伤的保护作用及对线粒体形态、功能及内膜转运孔通道的影响。探究脊髓损伤治疗的新方案。.研究内容：.1.在动物模型中确定氢气保护急性脊髓损伤的有效性及最优给药方案.2.在神经元机械损伤模型中确定氢气的保护效应.3.在细胞模型中探索氢保护脊髓急性损伤的分子机制.4.建立整套急性脊髓损伤动物模型的建立与评估体系.研究结果：. 通过制备C57BL/10小鼠脊髓钳夹伤模型，确定最佳给药方案，给予呼吸电解水制备的氢氧混合气体(氢气66 .7%，氧气33.3%)，脊髓损伤后所有小鼠均出现双侧后肢瘫痪。脊髓损伤后7天，脊髓损伤组有2只死亡，假手术组和氢气治疗组无小鼠死亡。BMS评分和足迹分析结果显示，呼吸高浓度氢气显著改善小鼠脊髓损伤后的恢复功能。H&E染色及尼氏染色显示，氢气治疗可减少小鼠前脚运动神经元的损伤和组织破坏。通过对脊髓神经元原代培养，我们发现氢气治疗组可明显改变神经元细胞及线粒体结构和形态。ROS、MDA、8-OHdG测定显示，高浓度氢气可降低神经元损伤后氧化应激水平。流式、Tunel染色、Western结果显示高浓度氢气可抑制神经元损伤后凋亡并增强胞质、线粒体中抗氧化酶表达。RT-PCR与RNA测序的结果显示，Cox8b, Cox6a2, Cox7a1, Hspb7, Atp2a1的表达在机械损伤后明显上升。同时，这种上调在氢气处理后得到了改善。.结论:. 呼吸电解水制备的氢气能够抑制线粒体mPTP通道，保护小鼠脊髓损伤，促进脊髓功能恢复，为临床脊髓继发损伤的治疗和防护提供了重要的科学依据。