Tianlong Huang1, Wangchun Wang2, Chao Tu3, Zhenyu Yang3, Donald Bramwell4, Xuejun Sun5. 1. Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China. 2. Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China. Electronic address: wwc1962@hotmail.com. 3. Department of Orthopaedics, Clinical Medicine for Eight-year-program, Xiangya School of Medicine, Central South University, Changsha, Hunan, China. 4. International Musculoskeletal Research Institute, Department of Orthopaedic Surgery, Flinders Medical Centre, Bedford Park, Australia. 5. Department of Naval Medicine, Second Military Medical University, Shanghai, China.
Abstract
BACKGROUND: To investigate the potential beneficial effect of hydrogen-rich saline (HRS) in ischemia-reperfusion (IR) injury of skeletal muscle. METHODS: 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. RESULTS: 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). CONCLUSIONS: HRS seems to be effective in attenuating IR injury in skeletal muscle via its antioxidant, anti-apoptosis, and anti-autophagy effect.
BACKGROUND: To investigate the potential beneficial effect of hydrogen-rich saline (HRS) in ischemia-reperfusion (IR) injury of skeletal muscle. METHODS: 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. RESULTS: 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). CONCLUSIONS:HRS seems to be effective in attenuating IR injury in skeletal muscle via its antioxidant, anti-apoptosis, and anti-autophagy effect.