Huang Tianlong, Wang Wangchun, Tu Chao, Yang Zhenyu, Bramwell Donald, Sun Xuejun
Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China.
J Surg Res. 2015 Apr;194(2):471-480. doi: 10.1016/j.jss.2014.12.016. Epub 2014 Dec 15.
To investigate the potential beneficial effect of hydrogen-rich saline (HRS) in ischemia-reperfusion (IR) injury of skeletal muscle.
Three experimental groups were established in male Sprague-Dawley rats: (1) sham group, (2) IR with normal saline group, (3) and IR with HRS group. A rat model of skeletal muscle IR injury was induced by 3-h tourniquet occlusion on its left hind limb and 4-h reperfusion. Normal saline and HRS (1.0 mL/100 g) were administered intraperitoneally at 10 min before reperfusion, respectively. Muscle and serum samples were analyzed for detecting the levels of myeloperoxidase (MPO), superoxide dismutase (SOD), malondialdehyde (MDA), and hydroxyl radical (•OH). Muscle samples were assessed by wet/dry rate, hematoxylin and eosin histologic assessment, Bcl2, Bax, cytochrome C, LC3B, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, and electron microscopy.
The wet/dry ratio increased significantly in the IR group (P < 0.01 compared with that in the sham group) and decreased significantly in IR with HRS groups (4.12 ± 0.14 versus 4.12 ± 0.14, P < 0.01 compared with that in the IR group). Muscle tissues and serum of the IR group had significantly increased levels of MPO, MDA, •OH content, and decreased SOD activities compared with the sham group (P < 0.01). The activity of SOD in the IR with HRS group was greatly elevated compared with that in the IR group (295.028 ± 9.288 versus 249.190 ± 5.450 in muscle tissues; 91.627 ± 2.604 versus 73.4045 ± 6.487 in serum; P < 0.01), whereas the levels of MPO, MDA, and •OH content were clearly reduced (MPO: 0.5649 ± 0.0724 versus 1.0984 ± 0.0824 in muscle tissues; 0.7257 ± 0.1232 versus 1.3147 ± 0.0531 in serum. MDA: 4.457 ± 0.650 versus 7.107 ± 0.597 in muscle tissues; 2.531 ± 0.434 versus 4.626 ± 0.237 in serum. •OH: 16.451 ± 0.806 versus 19.871 ± 0.594 in muscle tissues; 500.212 ± 7.387 versus 621.352 ± 7.591 in serum, P < 0.01). The integrated optical density of positive amethyst staining increased significantly in the IR group (P < 0.01 compared with that in the sham group) and decreased significantly in IR with HRS group (928.79 ± 234.537 versus 3005.972 ± 83.567, P < 0.01 compared with that in the IR group). Muscle tissues of the IR group had significantly increased levels of Bax, cytochrome C, LC3B content, and decreased Bcl2 activities compared with those in the sham group (P < 0.01). The activity of Bcl2 in the IR with HRS group was greatly elevated compared with that in the IR group (0.2635 ± 0.0704 versus 0.1242 ± 0.0662; P < 0.01), whereas the levels of Bax, cytochrome C, and LC3B content were clearly reduced (Bax: 0.3103 ± 0.0506 versus 0.5122 ± 0.0148; cytochrome C: 0.4194 ± 0.1116 versus 0.8127 ± 0.0166; LC3B: 0.5884 ± 0.0604 versus 1.3758 ± 0.0319; respectively, P < 0.01).
HRS seems to be effective in attenuating IR injury in skeletal muscle via its antioxidant, anti-apoptosis, and anti-autophagy effect.
探讨富氢盐水（HRS）对骨骼肌缺血再灌注（IR）损伤的潜在有益作用。
将雄性Sprague-Dawley大鼠分为三个实验组：（1）假手术组，（2）生理盐水IR组，（3）HRS IR组。采用左后肢3小时止血带阻断和4小时再灌注诱导大鼠骨骼肌IR损伤模型。再灌注前10分钟分别腹腔注射生理盐水和HRS（1.0 mL/100 g）。分析肌肉和血清样本，检测髓过氧化物酶（MPO）、超氧化物歧化酶（SOD）、丙二醛（MDA）和羟自由基（•OH）水平。通过湿/干比、苏木精-伊红组织学评估、Bcl2、Bax、细胞色素C、LC3B、末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记和电子显微镜对肌肉样本进行评估。
IR组湿/干比显著升高（与假手术组相比，P < 0.01），HRS IR组显著降低（4.12 ± 0.14对4.12 ± 0.14，与IR组相比，P < 0.01）。与假手术组相比，IR组肌肉组织和血清中MPO、MDA、•OH含量显著升高，SOD活性降低（P < 0.01）。与IR组相比，HRS IR组肌肉组织和血清中SOD活性显著升高（肌肉组织中：295.028 ± 9.288对249.190 ± 5.450；血清中：91.627 ± 2.604对73.4045 ± 6.487；P < 0.01），而MPO、MDA和•OH含量明显降低（肌肉组织中MPO：0.5649 ± 0.0724对1.0984 ± 0.0824；血清中：0.7257 ± 0.1232对1.3147 ± 0.0531。MDA：肌肉组织中4.457 ± 0.650对7.107 ± 0.597；血清中：2.531 ± 0.434对4.626 ± 0.237。•OH：肌肉组织中16.451 ± 0.806对19.871 ± 0.594；血清中：500.212 ± 7.387对621.352 ± 7.591，P < 0.01）。IR组紫水晶染色阳性的积分光密度显著升高（与假手术组相比，P < 0.01），HRS IR组显著降低（928.79 ± 234.537对3005.972 ± 83.567，与IR组相比，P < 0.01）。与假手术组相比，IR组肌肉组织中Bax、细胞色素C、LC3B含量显著升高，Bcl2活性降低（P < 0.01）。与IR组相比，HRS IR组Bcl2活性显著升高（0.2635 ± 0.070对0.1242 ± 0.0662；P < 0.01），而Bax、细胞色素C和LC3B含量明显降低（Bax：0.3103 ± 0.0506对0.5122 ± 0.0148；细胞色素C：0.4194 ± 0.1116对0.8127 ± 0.0166；LC3B：0.5884 ± 0.0604对1.3758 ± 0.0319；P < 0.01）。
HRS似乎通过其抗氧化、抗凋亡和抗自噬作用有效减轻骨骼肌IR损伤。
