Hydrogen-rich saline attenuates ischemia–reperfusion injury in skeletal muscle

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Hydrogen-rich saline attenuates ischemiaereperfusion injury in skeletal muscle Tianlong Huang, MD,a Wangchun Wang, MD,a,* Chao Tu,b Zhenyu Yang,b Donald Bramwell,c and Xuejun Sun, PhDd a

Department of Orthopaedics, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China Department of Orthopaedics, Clinical Medicine for Eight-year-program, Xiangya School of Medicine, Central South University, Changsha, Hunan, China c International Musculoskeletal Research Institute, Department of Orthopaedic Surgery, Flinders Medical Centre, Bedford Park, Australia d Department of Naval Medicine, Second Military Medical University, Shanghai, China b

article info

abstract

Article history:

Background: To investigate the potential beneficial effect of hydrogen-rich saline (HRS) in

Received 20 May 2014

ischemiaereperfusion (IR) injury of skeletal muscle.

Received in revised form

Methods: Three experimental groups were established in male SpragueeDawley rats: (1)

25 November 2014

sham group, (2) IR with normal saline group, (3) and IR with HRS group. A rat model of skeletal

Accepted 8 December 2014

muscle IR injury was induced by 3-h tourniquet occlusion on its left hind limb and 4-h

Available online xxx

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

Keywords:

detecting the levels of myeloperoxidase (MPO), superoxide dismutase (SOD), malondialdehyde

Hydrogen

(MDA), and hydroxyl radical (
OH). Muscle samples were assessed by wet/dry rate, hema-

Ischemiaereperfusion injury

toxylin and eosin histologic assessment, Bcl2, Bax, cytochrome C, LC3B, terminal deoxy-

Skeletal muscle

nucleotidyl transferase-mediated dUTP-biotin nick end labeling, and electron microscopy.

Antioxidant

Results: The wet/dry ratio increased significantly in the IR group (P < 0.01 compared with

Apoptosis

that in the sham group) and decreased significantly in IR with HRS groups (4.12  0.14

Autophagy

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

* Corresponding author. Department of Orthopeadics, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China. Tel.: þ86 136 047 82026; fax: þ86 731 529 5828. E-mail address: [email protected] (W. Wang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.016

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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. ª 2015 Elsevier Inc. All rights reserved.

1.

Introduction

Skeletal muscle has high metabolic activity and therefore is acutely sensitive to reperfusion injury after ischemia. Many clinical events including trauma, primary thrombosis, arterial embolism, limb or flap replantation, arterial grafting, prolonged tourniquet application, and compartment syndrome can cause severe skeletal muscle ischemia and resulting reperfusion injury as blood flows back into the ischemic muscle. Ischemiaereperfusion (IR) injury of skeletal muscle can lead to severe damage to the extremities, including severe necrosis leading to amputation, or even multisystem organ dysfunction syndrome and threats to life. Given the range of events that can cause ischemia and the potential for severe damage from IR injury, it is important to develop some means to minimize limb IR injury and reduce the extent of damage to skeletal muscle to decrease amputation rates and improve the recovery of extremity function. Clinically, a therapeutic intervention, which changes the biochemical environment during the IR period and prevents subsequent damage, would be a significant benefit. Several pharmacologic agents with the potential to reduce IR injury have been evaluated, but none has yet been translated into clinical trials [1e4]. Ischemia can lead to energy depletion, accumulation of toxic metabolic products, activation of phospholipase and lysozymes, and cell damage. Reperfusion can then worsen the injury by inducing cellular infiltration and generating reactive oxygen species (ROS), leading to further cell damage. ROS and activated neutrophils are the most important factors responsible for local and systemic damage caused by IR [5]. Apoptosis is thought to be an inevitable phase in IR-induced cell death [6,7]. In addition, autophagy may play a critical role in the IR-induced cell death because regulation of the autophagic process can have a positive influence on IR injuries [8e13]. However, the evidence of IR-induced autophagy in skeletal muscle is sparse and divided although there is evidence of IR-induced autophagy in many other tissues and organs like the heart [8], the liver [9], the intestine [10], the kidney [11,12], and the brain [13]. It has been demonstrated that hydrogen could selectively reduce cytotoxic ROS and reactive nitrogen species, such as hydroxyl radical (
OH) and ONOO- in vitro and thus exert therapeutic antioxidant activity in a rat middle cerebral artery occlusion model [14]. However, to our knowledge, the protective effect of hydrogen on skeletal muscle IR injury has not been reported. Therefore, the present study investigated the possible therapeutic effects and underlying mechanisms of hydrogen-rich saline (HRS) on skeletal muscle IR injury in rats

and explored the possible mechanism of the therapeutic effects. Our study sought to determine whether HRS can attenuate IR injury in skeletal muscle through antioxidant, anti-apoptosis, and anti-autophagic mechanism.

2.

Materials and methods

2.1.

Animals

Eighteen six-wk-old male SpragueeDawley rats (Animal Department of Xiangya School of Medicine, Central South University, China) weighing 250e300 g were housed in our research facility under standard conditions with a 12-h lighte dark cycle and free access to water and food. All animal care and experimental procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animals [15] and were approved by the Institutional Animal Care and Use Committee of Xiangya School of Medicine, Central South University.

2.2.

Chemicals

Phosphate-buffered saline (PBS) was purchased from Sigmae Aldrich (St. Louis, MO). Malondialdehyde (MDA), superoxide dismutase (SOD), and
OH assay kit were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit polyclonal antibody to LC3B (ab63817) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) in situ cell death detection kit (ab66108) were purchased from Abcam (Cambridge, MA). Rabbit polyclonal antibodies to Bcl-2 (#2870), Bax (#2772), and cytochrome C (#11940) were purchased from Cell Signaling Technology, Inc (Boston, MA). Rabbit polyclonal antibody to GAPDH and goat antierabbit IgG were purchased from Santa Cruz Biotechnology, Inc (Dallas, TX). A total Protein Extraction Kit (SJ-200501) was purchased from ProMab (Richmond, CA), 0.1% triton X-100 was purchased from MP Biomedicals (Solon, OH), and 0.1% citric acid was purchased from Mallinckrodt (Hazelwood, MO). All chemicals and reagents were of analytical grade.

2.3.

HRS production

Hydrogen was dissolved in normal saline (NS) for 6 h under high pressure (0.4 MPa) to a supersaturated level using apparatus produced by Beijing Hydrovita Biotechnology Co, Lt (Beijing, China). The saturated HRS was stored under

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atmospheric pressure at 4 C in an aluminum bag with no dead volume. HRS was sterilized by g radiation. Concentration is >0.6 mmol/L. Gas chromatography was applied to confirm the content of hydrogen in saline by the method described by Ohsawa et al. [14].

2.4.

Experimental design

Eighteen SpragueeDawley rats were randomly divided into three groups with six per group: i) sham group, in which rats were not operated with tourniquet occlusion on the left hind limb and given intraperitoneal injection with NS (1.0 mL/100g) 3 h later. ii) IR group, in which an orthodontic rubber band was applied to the left hind limb proximal to the greater trochanter to induce ischemia using a McGivney applicator [16]. Rats were exposed for 3 h to induce IR injury and the rubber band was removed to allow reperfusion for 4 h. These animals were given an intraperitoneal injection with NS (1.0 mL/100g) 10 min before the removal of tourniquet occlusion. iii) IR þ HRS group, in which ischemia was induced as in group ii, rats in this group were intraperitoneally injected with HRS (1.0 mL/100g) 10 min before the removal of tourniquet occlusion.

2.5.

IR injury model

The rats were fasted without water deprivation for 24 h before the experiments. After inducing anesthesia with an intraperitoneal administration of 10% chloral hydrate (0.3 mL/100 g), the rats were fixed on an operating table in a supine position with body temperature maintained between 36 and 37 C. After IR injury, all rats were sacrificed under anesthesia. A blood sample was collected from the abdominal inferior cava vein in each rat and centrifuged at 4000 rpm for 4 min. The harvesting of gastrocnemius in the left hind limb was made into a 10% tissue homogenate and centrifuged at 3000 rpm, 4 C for 10 min, after which the supernatant was collected. All samples were carefully reserved stored at 80 C for further examination.

2.6.

embedded with paraffin, sectioned into 5-mm slices, and stained with hematoxylin and eosin or 0.5% toluidine blue, respectively. Thirty visual fields (40) were randomly chosen in each slide and evaluated by two pathologists blinded to the treatment given, to assess the degree of pathologic injury. The muscle neutrophil and mast cell infiltration were expressed as cell number/high power field. The histologic damage score was determined as follows: disorganization and degeneration of the muscle fibers (0: normal, 1: mild, 2: moderate, and 3: severe); inflammatory cell infiltration (0: normal, 1: mild, 2: moderate, and 3: severe) [17].

2.9. assay

Oxidant stress and antioxidant enzymatic activity

Activities of MDA and
OH were detected to assess the levels of oxidant stress, and activity of SOD was detected to assess the level of antioxidant ability. The supernatants from gastrocnemius homogenates and serum samples were used for MDA,
OH, and SOD measurement using a commercially available kit.

2.10.

TUNEL staining

TUNEL assay was performed according to the manufacturer’s instructions. Briefly, paraffin-embedded sections were deparaffinized with xylene followed by absolute ethanol and decreasing concentrations of ethanol (95% through to 70% ethanol). Sections were rinsed in PBS for 5 min and treated with protease K for 30 min at room temperature. After washing in PBS twice, these sections were incubated with 50 mL of TUNEL reaction solution in a humidified environment at 37 C for 60 min. After washing in PBS three times, these sections were treated with 50 mL of converter-POD in a humidified environment at 37 C for 30 min. After another washing step (three times in PBS), sections were mounted after washing in PBS. Cells with amethyst staining indicated apoptosis, and the integrated optical density (IOD) of positive

Muscle edema assessment

Fresh gastrocnemius samples were directly weighed (wet weight) after excision. Then the muscles were reweighed (dry weight) after drying at 60 C in a convection incubator for 72 h. Muscle edema index was calculated as wet/dry weight ratio (W/D ratio).

2.7.

Neutrophil infiltration degree assessment

Activity of myeloperoxidase (MPO) was detected to assess the neutrophil infiltration. The supernatants from gastrocnemius homogenates and serum samples were used for MPO measurement using a commercially available kit.

2.8.

Histologic assessment

To assess the degree of muscle damage, neutrophil accumulation, and mast cell infiltration, the excised gastrocnemius samples were sequentially fixed in 10% formaldehyde,

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Fig. 1 e The difference of W/D ratio in three groups. **indicates statistically significant differences between sham and IR groups (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01).

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1/3 volume protein denaturation solution, and boiled for 5 min to denature the protein. Samples were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. Polyvinylidene fluoride membranes were probed with polyclonal antibodies to LC3B, Bcl-2, Bax, and cytochrome C overnight at 4 C. The secondary antibody was conjugated to horseradish peroxidase with ECL. GAPDH was used to normalize for loading variations. Blots were quantified using Image-Pro Plus Software.

2.12.

Fig. 2 e The different MPO concentration of muscle and serum in three groups. ** indicates statistically significant differences between sham and IR groups (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01).

The gastrocnemius was cut into 2-mm slices, osmicated in 1% osmium tetroxide, dehydrated in acetone, and embedded in araldite epoxy resin in both transverse and longitudinal orientations sequentially. The ultrathin sections were contrasted with uranyl acetate and lead citrate and used for electron microscopy.

2.13. amethyst staining was analyzed by the software Images-Pro Plus 6.0 (Warrendale, PA).

2.11. Western blotting of LC3B, Bcl-2, Bax, and cytochrome C To clarify the influence of hydrogen on apoptosis in this IR model, the expression of Bcl2, Bax, and cytochrome C (the Bcl2 inhibit apoptosis and Bax, cytochrome C induce apoptosis) in skeletal muscle was assessed. Gastrocnemius protein was extracted according to the BCA method. The concentration of each sample was diluted to the same level, then mixed with

Electron microscopy

Statistical analysis

Histologic damage scores are expressed as median  interquartile range, other data were expressed a mean  standard error, and statistical analysis was performed by SPSS 17.0 (SPSS Inc, Chicago, IL) for Microsoft. Data plotting were performed using the Prism 5.0 software package (GraphPad Software, San Diego, CA). For histologic damage scores, statistical significance was calculated by KruskaleWallis H test, and P < 0.05 was considered statistically significant. For other data, statistical significance was calculated by one-way analysis of variance followed by an least significant difference test, and P < 0.05 was considered statistically significant.

Fig. 3 e Histologic evaluation and histologic damage scores of skeletal muscle. Representative photographs of hematoxylin and eosin pictures used to determine morphologic changes (3400) in skeletal muscle tissues were taken in sham (A), IR (B), and IR D HRS groups (C). Picture (D) shows the statistics of histologic damage scores in three groups. ** indicates statistically significant differences between sham and IR groups (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01). (Color version of the figure is available online.)

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3.

Results

3.1.

Muscle edema

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The results of W/D ratio in three groups and related statistics are shown in Figure 1. The W/D ratio of three groups are 3.76  0.15, 5.30  0.14, and 4.12  0.14 (n ¼ 6, means  standard error) individually. The results in both of sham group and IR þ HRS group showed a significant difference compared with those of IR group.

3.2.

Neutrophil infiltration degree

Figure 2 shows the comparison between the MPO activity in muscle and serum for the three groups. Compared with IR group, the IR þ HRS group has lower level of MPO activity both in muscle and serum (P < 0.01).

3.3.

Histologic changes

The hematoxylin and eosin pictures and histologic damage scores in three groups and related statistics are shown in Figure 3. In both IR þ HRS group and IR group, histologic damage scores were higher than those in the sham group (P < 0.01). Severely disorganized, degenerated muscle fibers and inflammatory cell infiltration could be observed in the IR group. Moderately disorganized, degenerated muscle fibers and mild inflammatory cell infiltration were observed in the IR þ HRS group. In the IR þ HRS group, histologic damage scores were significantly lower than those in the IR group (P < 0.01), showing a lower degree of muscle tissue degeneration and inflammatory cell infiltration.

3.4.

Oxidant stress and antioxidant enzymatic activity

The levels of MDA,
OH, and SOD in muscle and serum in three groups and related statistics are given in Figure 4. Compared with IR group, the IR þ HRS group has lower levels of MDA and
OH in both muscle and serum (P < 0.01) and higher activities of SOD in both muscle and serum (P < 0.01).

3.5.

Apoptosis

The TUNEL staining photographs and IOD in three groups and related statistics are shown in Figure 5. IOD in IR þ HRS group is significantly smaller compared with that in the IR group. The results of Bcl2, Bax, and cytochrome C in three groups and related statistics are given in Figure 6. Compared with the IR group, the IR þ HRS group has lower expression of Bax and cytochrome C and higher expression of Bcl2.

Fig. 4 e The different MDA,
OH, and SOD concentrations in muscle and serum. ** indicates statistically significant differences between sham and IR groups (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01). undigested cytoplasmic contents, organelles, proteins, and cytoplasm and are known as autolysosomes and autophagosomes. Contrasted to IR group, the IR þ HRS group showed a lower extent of autophagic phenomenon, which was in accordance with the levels of LC3B.

4. 3.6.

Discussion

Autophagy

The levels of LC3B in three groups and related statistics are showed in Figure 7. Contrasted to the IR group, the IR þ HRS group alleviated the levels of LC3B significantly (P < 0.01). Electron microscopy showed that, compared with sham group, the IR induced the accumulation of double-membrane structures within the cells (Fig. 8). These structures contain

The mechanisms of IR injury are still not fully understood. Multiple pathophysiological mechanisms are involved, including the oxidative and/or nitrosative stress, endoplasmic reticulum stress, neutrophils infiltration, no-reflow phenomenon, nitrogen monoxide, calcium overload, inflammation, necrosis, apoptosis, and autophagy [18,19]. Among them, ROS and activated neutrophils have been shown to be the

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Fig. 5 e TUNEL staining of skeletal muscle and IOD statistics. Representative photographs of TUNEL used to determine morphologic changes (3400) in skeletal muscle tissues were taken in sham (A), IR (B), and IR D HRS groups (C). Picture (D) shows the statistics of IOD in each group. ** indicates statistically significant differences between sham and IR group (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01). (Color version of the figure is available online.)

most important factors responsible for local and systemic damages [5]. Compared with other organs, the skeletal muscle can tolerate a comparatively longer time of ischemia without

reperfusion injury. With the rising impact of vehicle trauma and natural disasters nowadays, there is a risk of increased prevalence of skeletal muscle IR injury. For these types of injuries, we need treatments which are inexpensive, easily

Fig. 6 e The Western blot results (A) and the levels of Bcl2, Bax, and cytochrome C in muscle in three groups (BeD). ** indicates statistically significant differences between sham and IR groups (P < 0.01). && indicates statistically significant differences between IR and IR D HRS groups (P < 0.01). (Color version of the figure is available online.)

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Fig. 7 e The Western blot results (A) and the levels of LC3B (B) in three groups. *indicates statistically significant differences between sham and IR groups (P < 0.01). **indicates statistically significant differences between IR and IR D HRS groups (P < 0.01). (Color version of the figure is available online.)

stored, and administered to the patient immediately so we can safely decompress the injured extremities in time. Numerous treatments or drugs are available for IR injury in different tissues and organs, and some of those have proved to be effective. IR injury of skeletal muscle has its unique properties compared with that of other organs. First, the ischemia of skeletal muscle often results from sudden unpredictable events such as trauma. For those patients, prolonged tourniquet application might be an indispensable part of rescue before transported to hospital though it might be longer than clinical allowed time. Although pretreated with ischemic preconditioning [20] or other methods [21e23] have been shown to be useful in ameliorating the IR injury, these measures cannot be used into clinic treatment because of various limitations. Second, skeletal muscle have longer endurance to IR injury than other susceptible organs. Drugs such as silibinin [24], iloprost [25], and dextran sulfate [26], which can attenuate the heart, renal, and brain IR injury have little effect in skeletal muscle injury. Finally, some drugs like hydrogen sulfide [2] and carbon monoxide [21], which are effective in physiologic dosage might be poisonous in moderate or high doses. Hydrogen is the lightest and most abundant chemical element in nature. Since Ohsawa et al. [14] discovered that hydrogen gas has antioxidant and anti-apoptotic properties, which can protect the brain IR injury and stroke by selectively neutralizing
OHs, hydrogen has received increasing focus in

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therapeutic medical research. Accumulated evidences have shown that hydrogen, administered either through gas inhalation or by consumption of hydrogen-containing solution, can act as a scavenger, which is able to reach all cellular compartments because of its small size. Hydrogen is thus able to selectively alleviate ROS and exert potent cellular protective effects [10,27e32]. Hydrogen is inexpensive, safe, and easily produced, stored, and transported for clinical application [33,34]. Therefore, HRS could be a much better choice if it can attenuate the skeletal muscle IR injury. MPO is a peroxidase enzyme abundantly expressed in neutrophil granulocytes. The activated neutrophils are responsible for local and systemic damage caused by IR [5]. In our study, compared to IR group, the W/D ratio and MPO were significantly decreased after the treatment of HRS. Furthermore, from the microscopic view, inflammatory cell infiltration, muscle edema, muscle fiber injury, and necroses were also markedly relieved by the HRS treatment. We could therefore conclude that the HRS attenuates the damages caused by IR injury both macroscopically and microscopically. The oxidation family mainly includes H2O2, O 2 ,
OH, and ONOO. Previous studies have focused on finding substances strong enough to cause sufficient change. However, antioxidant drugs demonstrated in former studies are too strong that they interfere with the normal physiological functions of H2O2 and O 2 . Recently, a consensus has been reached that the most suitable antioxidant species are those that aim at the pathologic oxidation species but seldom influence the physiological oxidation species [35]. It has been reported that hydrogen is such a type of antioxidant substance, which will only focus on antioxidizing$
OH and ONOO [14]. The main medium of mediating cell oxidative damage, which should be neutralized at the early stage of IR injury. MDA production during free radical attack on membrane lipoproteins and polyunsaturated fatty acids is an indicator of lipid peroxidation. SOD, an antioxidant enzymes and super oxygen free radical scavenger, is critical to clean the superoxide anion into hydrogen peroxide and water [36,37]. Our study has demonstrated that the notable increase in MDA in the IR group confirmed the oxidative damage in the skeletal muscle, and the marked decrease in SOD levels showed the exhaustion of antioxidant enzymatic activity. In contrast, HRS treatments markedly reduced MDA and
OH levels and increased the SOD level. By measuring changes in some type of important oxidative stress biomarkers in both plasma and tissue, we found that the decrease of oxidative damage and the increase of endogenous antioxidant enzymatic activities in serum and skeletal muscle might contribute to the protective effect of HRS treatment, which is similar to the results in previous studies for others organs [32]. Collective evidence from various IR models has indicated apoptosis might be the initial mode of cell death in the progress to ultimate cell death [38]. Increasing researches have confirmed that apoptosis plays an important role in the pathophysiology of skeletal muscle IR injury [6,7,39,40]. Cytochrome C is a vital protein located in the mitochondrial intermembrane space, which could initiate apoptosis, in a process called the cytochrome C-initiated pathway [41,42]. Anti-apototic protein Bcl-2 and proapoptotic protein Bax are widely regarded apoptotic regulators, and their relative levels

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Fig. 8 e The representative photographs of transmission electron microscopy used to determine morphologic changes in skeleton muscle (A) Normal view from sham group (magnification 1: 5000). (B)The overview of IR-induced autophagy (magnification 1: 15,000). (C) The typical autophagosome from IR group (magnification 1: 25,000). (D) The typical autolysosome from IR group (magnification 1: 25,000).

determine the fate of cells [43]. Besides, it has also been reported that an anti-apoptotic protein via its inhibitory interaction with Beclin 1 [44]. In the present study, we disclosed that compared to the IR group, the apoptosis ratio, cytochrome C, and Bax were notably decreased, whereas Bcl-2 was significantly increased by the treatment of HRS. From these results, we have shown that HRS can attenuate the apoptosis of skeletal muscle by regulating cytochrome C, Bax, and Bcl-2. The function of autophagy in IR injury is still controversial [8,45e48]. From one perspective, autophagy provides cells with a survival mechanism to withstand stressful conditions [18,49]. However, it has also been shown that uncontrolled autophagy can lead to the irreversible demise of the cell [48]. Compared with other metabolic tissues such as liver and pancreas, skeletal muscle showed a persistent generation of autophagosomes that continued for days during fasting [50], which probably is one of the mechanisms by which skeletal muscle has a better endurance ischemia of IR injury than other organs. Our study explored whether autophagy could regulate the process of skeletal muscle in IR injury for the first time. From transmission electron microscopy [51], we find that the focal degradation of cytoplasmic areas sequestered by the phagophore, which matures into the autophagosome, is the morphologic hallmark of autophagy. LC3 is a core component of autophagosome and functions as an adaptor for delivering the cargoes to autophagosomes [52]. Both results of transmission electron microscopy and LC3B revealed that autophagy in the skeletal muscle was significantly enhanced by the IR injury, but could be relieved by the HRS treatment.

These findings showed that the autophagy can aggravate the skeletal muscle IR damages. Further investigations are still needed before it could pave the way for a new target treatment for skeletal muscle IR injury. Many stress pathways sequentially elicit autophagy and apoptosis within the same cell. It has already confirmed that autophagy and apoptosis could interact with each other [53,54]. A more precise understanding of the relationship between autophagy and apoptosis in skeletal muscle IR injury is still required as the interplay between autophagy and apoptosis might be variable depending on different IR periods. We find that autophagy can increase apoptosis during 3-h ischemia and then 4-h reperfusion from our results. Further research is needed to clarify the complex relationship between autophagy and apoptosis in skeletal muscle IR injury. Our results have demonstrated a significant effect of HRS in IR injury of skeletal muscles. First, we evaluated the pathologic changes after 3-h ischemia and 4-h reperfusion in a small sample size. IR injury is a progressing pathologic injury, which will endanger the survivorship of limbs. It might be better to explore the progress of IR injury in multiple time points with bigger sample sizes for more details. Second, there might be differences in effect depending on the way HRS is administered, with HRS given by oral or intravenous injection. In this study, intraperitoneal injection was chosen for convenience, and later we will explore oral administration in further study. Finally, rat models have some limitations in mimicking human biological and physiological processes. Future research will focus on larger animal models.

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5.

Conclusions

This study demonstrates that HRS is able to attenuate the skeletal muscle IR injury, possibly by the reduction of oxidative stress, anti-apoptosis, and anti-autophagy. We have shown that HRS may be a potential candidate for therapeutic manipulation of skeletal muscle IR injury in the clinical setting because of its safety, and ease of production and transportation. It should be noted, however, that the exact mechanism and signaling pathway involved in the protection role of hydrogen in the skeletal muscle IR injury needs to be studied in more detail in the future.

Acknowledgment Authors’ contributions: T.H. performed all the experiments, data collection, data analysis, and wrote the article. W.W. proposed the conception, designed the experiment, guided all the experiments and data analysis, and then decided which journal to submit. C.T. assisted with the experiments and data interpretation. Z.Y. attended to the experiments and the data analysis. D.B. made the critical revision to the article. X.S. helped to revise the design of the experiment, especially with regard to the production and detection of hydrogen-rich saline.

Disclosure All authors declare no conflicts of interest.

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