Animals and ethics statement

The animal experiments were reviewed and approved by the animal care and use committee, and husbandry was strictly performed in line with the recommendations of the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006) and the Nanjing Medical University Animal Care and Use Committee (NMJU-ACUC). During the experiments, pain and use of animals were minimized. Wild-type (WT, ICR/JCL, or C57BL/6) male mice (6–8 weeks) and MMP-9 knockout (C57BL/6) male mice (6–8 weeks) were approved by the Nanjing Medical University Animal Center. All animals were reared under pathogen-free conditions with controlled temperature (22 ± 2 °C) and a standardized light/dark cycle. They were given access to food and water ad libitum. All animals were allowed to acclimate to these conditions for approximately 1 week before being included in the experiments.

Surgery

Plantar surgery was performed according to a previous study [22]. All surgeries were performed under anesthesia induced by isoflurane (2% oxygen gas, 300 mL/min). The plantar aspect of the left hind paw was sterilized with a 10% povidone-iodine solution before and after surgery, and placed through a hole in a sterile drape. A 0.8-cm longitudinal and 2-mm plutonic incision extending from the proximal edge of the heel to the toes was made through the skin and fascia of the plantar aspect of the foot. Then the skin was elevated and longitudinally incised through leaving the origins and insertions of the muscle intact. The skin was then apposed using 5-0 nylon sutures. The incision was checked daily and any subject whose plantar incision had signs of wound infection or dehiscence was excluded from the study.

Drugs and reagents

NQDI-1 was purchased from Selleck Chemical Inc. (Houston, TX) (dissolved in 0.1% dimethyl sulfoxide [DMSO]). PX12 was purchased from MedChem Express (USA) (dissolved in DMSO). Antibodies for thioredoxin (#2429S), p-p38 (Thr180/Tyr182) (#9215S), p-JNK (Thr183/Tyr185) (#9255S), and p-ERK1/2 (Thr202/Tyr204) (#4377S) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies for p-ASK1 (Thr845) (sc-109911) and β-actin (sc-4778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for IBA-1 (ab178847) and MMP-9 (ab58803) were purchased from Abcam (Cambridge, MA). The antibody for IL-1β was purchased from R&D systems (USA). Elisa kit (IL-1β, IL-6, TNF-α) were purchased from MULTI SCIENCES. Lipopolysaccharide (LPS) and DMSO were purchased from Sigma (St. Louis, MO, USA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).

The procedures of saturated hydrogen-rich saline (HRS) were prepared as previously described [23]. They were prepared using H2 dissolved in normal saline for 12 h under high pressure (0.4 Mpa) and stored at 4 °C in an aluminum bag under normal pressure. To ensure that the hydrogen concentration was greater than 0.8 ppm, HRS was freshly prepared each time, and assessed using gas chromatography ENH-1000 (Trustiex Co, Japan) and Methylene Blue (Seo, Med Gas Res, Japan).

Grouping and treatment

Mice were randomly assigned to the following groups: sham, plantar incision (PI), PI + HRS (5 mL/kg, i.p.); HRS treatment alone, PI + NDQI-1 (5 μg/10 μl, i.t.); and NDQI-1 treatment alone groups, PI + HRS + PX12 (12 μg/10 μl, i.t.). In the HRS treatment groups, the mice received injections twice daily, and in the NDQI-1 treatment groups, the mice received injections once daily. HRS or NDQI-1 were injected 6 h before plantar incision surgery. PX12 was injected 0.5 h before HRS administration. Each group consisted of 12 mice. The experimental groups and the timing of analysis are presented in Additional file 1: Table S1. BV-2 cells were kept in humidified 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 U/ml). A total of 105 cells were seeded in 6-well plates overnight and divided into the following groups (n = 4/group): sham; LPS (1 μg/ml); LPS + H2 (5% CO2, 20% O2, 60% H2, PH-1-A, China); H2 alone; LPS + NQDI1 (10 μM); and LPS + H2 + PX12 (8 μM) groups. NQDI1 was added after LPS stimulation. PX12 was added 0.5 h before H2 treatment.

Behavioral analysis

Before baseline testing, all animals were placed in the testing environment for at least 2 days for acclimatization. The first researcher was responsible for numbering the animal groups and drugs, as well as for data analysis. The second researcher administered the drugs to the groups according to the assigned numbers, while the third researcher performed the behavioral tests. The staff involved were blinded. Mechanical sensitivity was detected using von Frey hairs (Woodland Hills, Los Angeles, CA, USA). The animals were placed in boxes with an elevated metal mesh floor for habituation 30 min before testing. A series of von Frey hairs (the pressure intensity of mice between 0.16 g and 0.008 g) with logarithmically incrementing stiffness were used to perpendicularly stimulate the plantar surface of each hind paw. Each mouse was tested three times and the average of the threshold was measured.

Thermal hyperalgesia was detected using an analgesia meter (UGO Basile, Gemonio, Varese, Italy). Briefly, each mouse was placed on a 55 °C hot-plate apparatus. The heat source was focused on a portion of the hind paw, which was flush against the glass, and a radiant thermal stimulus was delivered to that site. The stimulus was then shut off when the hind paw moved (or after 20 s to prevent tissue damage). The intensity of the heat stimulus was kept constant throughout all the experiments. The elicited paw movement occurred at a latency of between 9 and 14 s in control animals. Thermal stimuli were delivered three times to each hind paw at 5- to 6-min intervals. Behavioral tests were performed blindly.

Gelatin zymography

The lumbar spines (L4–L5) of mice were collected and analyzed at 24 h or 5 days after surgery. Mice were anesthetized, and spinal cord segments were rapidly dissected and homogenized in 1% Nonidet P-40 lysis buffer. The solubilized proteins were then resolved on gels (8% polyacrylamide gel containing 0.1% gelatin). After electrophoresis, each gel was incubated with 50 mL of developing buffer for 48 h (37.5 °C) in a shaking bath. Finally, the gels were stained with Coomassie Brilliant Blue (1%, with 10% acetic acid and 10% isopropyl alcohol, diluted with double-distilled H2O).

Western blot analysis

Lumbar spine samples (L4–L5) were collected and analyzed at 24 h or 5 days after surgery. Cell samples were collected and analyzed 24 h after H2 treatment. Protein concentrations were determined by the BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of protein per lane were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel, and transferred to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). After being blocked with 5% bovine serum albumin and 5% skim milk for 2 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. The primary antibodies used included thioredoxin (1:500), p-p38 (1:1000), p-JNK (1:1000), p-ERK1/2 (1:1000), p-ASK1(1:1000), IL-1b (1:500), and IBA-1 (1:1000). For loading control, the blots were probed with an antibody for β-actin (1:5000). The filters were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) with secondary antibodies, Sigma (St. Louis, MO, USA). Finally, data were acquired using the Molecular Imager (Gel DocTM XR, 170–8170) and analyzed using the associated software Quantity One 4.6.5 (Bio-Rad Laboratories, Hercules, CA).

Immunofluorescence staining

The lumbar spines (L4–L5) of the mice were collected and analyzed 5 days postoperatively. After deep anesthesia, the animal was perfused transcardially with normal saline followed by 4% paraformaldehyde. The lumbar segment L4 and/or L5 were dissected and postfixed in the same fixative. The embedded blocks were sectioned to a thickness of 20 μm. The sections of each group (six animals in each group) were then incubated with rabbit antibody for IBA-1 (1:100, #ab178846, abcam, Cambridge, MA, USA), mouse antibody for MMP-9 (1:100, #10375-2-AP, Proteintech, Rosemont, IL USA), secondary antibody (1:300, Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit/Mouse IgG, #711–545-152/#715–545-150, Jackson ImmunoResearch Laboratories, USA) at room temperature. After being washed three times with PBS, all slides were processed blindly and then studied under a confocal microscope (Olympus FV1000 confocal system, Olympus, Japan) to observe morphological details after staining. Images were randomly coded and fluorescence intensities were analyzed using Image Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD, USA). The average green and red fluorescence intensity of each pixel was normalized to the background intensity in the same image.

Immunohistochemistry staining

The lumbar spines (L4–L5) of the mice were collected and analyzed at 24 h or 5 days after surgery. The samples were sectioned to 5 μm thickness, then incubated with the first antibodies for thioredoxin (1:100), mouse antibodies MMP-9 (1:100) and rabbit antibodies for IBA-1(1:300) in 10% donkey serum and 0.3% Triton-X100. After quenching endogenous peroxidase activity, the slides were washed in PBS and incubated with HRP conjugated secondary antibody for 2 h. Diaminobenzidine was used as a chromogen and counterstaining was performed with hematoxylin. Two independent pathologists evaluated all immunohistochemistry staining sections. The score for each slide was measured as the cross-product of the value of immunostaining intensity and the value of the proportion of positive-staining cells. The intensity of immunostaining was divided into four grades: 0, negative; 1, weak; 2, medium; 3, strong. The proportion of positive-staining cells was also divided into four grades: 1, 0 − 25%; 2, 26 − 50%; 3, 51 − 75%; and 4, > 75%. The score was calculated using the following formula: total score = intensity score × proportion score.

Statistical analysis

GraphPad Prism 6 software (GraphPad Software, San Diego, CA, USA) was used to conduct all statistical analysis. Continuous variables were presented as means ± SEM. Normally distributed alteration of protein detected expression and changes in behavioral responses were tested with Student’s t tests and one-way ANOVA. The differences in latency over time among groups were tested with two-way ANOVA. Bonferroni post hoc comparisons was performed between multiple groups. A criterion value of P < 0.05 was considered statistically significant.