Animals and ethics statement
Adult male wild type and MMP-9 knockout mice (20 ± 2 g) were approved by Nanjing Medical University animal center. All animals were bred under pathogen-free conditions with controlled temperature (22 ± 2 °C) and a standardized light/dark cycle. They were given ad libitum access to food and water. All animals were allowed to acclimate to these conditions for about one week before inclusion in the experiments.
Surgery
The plantar surgery was performed according to previous study [30]. All surgeries were done 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 surgeries, and was placed through a hole in a sterile drape. A 1 cm longitudinal incision extending toward the toes from the proximal edge of the heel was made through skin and fascia of the plantar aspect of the foot. Then elevated and longitudinally incised through, leaving muscle origin and insertion intact, then the skin was apposed with sutures of 5-0 nylon. The incision was checked daily, and any plantar incision with signs of wound infection or dehiscence was excluded from the study.
Drugs and reagents
NQDI-1 was purchased from Selleck Chemical Inc. (Houston, TX). PX12 was purchased from MedChem Express (USA). 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). Antibody for IL-1β was purchased from R&D systems (USA). Lipopolysaccharide (LPS) and Dimethyl Sulfoxide (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 [31]. They were prepared by hydrogen gas which was 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. In order to make sure the concentration of hydrogen was more than 0.8 ppm, it was prepared freshly every time and detected with gas chromatography ENH-1000 (Trustiex Co, Japan) and Methyene Blue (Seo , Med Gas Res, Japan).
Grouping and treatment
Mice were randomly assigned to the following groups: sham, plantar incision (PI), PI plus HRS (5 ml/kg, i.p.), HRS treatment alone, PI plus NDQI-1(5 μg/10 μl, i.t.) and NDQI-1 treatment alone. Each HRS treatment group, the mice received injections twice daily, and NDQI-1 treatment groups were administered once daily. HRS and NDQI-1 were injected 6 h before plantar incision surgery. Each group consisted of 12 mice.
BV-2 cells were maintained 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). 105 cells were seeded onto 6-well plates overnight and divided into groups(n = 4/group) as sham, LPS (1μg/ml), LPS plus H2(5% CO2, 20% O2, 60% H2, PH-1-A, China), H2 alone, LPS plus PX12 (8 μM), and LPS plus H2 and PX12. In LPS plus PX12 treatment groups, PX12 was added 2 h before LPS.
Behavioral analysis
All animals were placed in the testing environment daily for at least 2 days before baseline testing for acclimatization. The first researcher was responsible for numbering the animal groups and drugs as well as data analysis. The second researcher administrated the drugs to the groups according to the numbers, and the third researcher performed the behavioral tests. All staffs involved were blinded. Mechanical sensitivity was detected using von Frey hairs (Woodland Hills, Los Angeles, CA, USA). Animals were placed into boxes with an elevated metal mesh floor for habituation 30 min before testing. A series of Von Frey hairs with logarithmically incrementing stiffness were used to stimulate the plantar surface of each hind paw perpendicularly. Each mouse was tested three times, and the average of the threshold was measured.
Gelatin zymography
Mice were anesthetized and spinal cord segments were rapidly dissected and homogenized in 1% Nonidet P-40 lysis buffer. Then the solubilized proteins were resolved on gels (8% polyacrylamide gels 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 blotting analysis
The protein concentrations were determined by BCA protein assay (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of protein per lane were separated by 8-15% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA, USA). After 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 the 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), IBA-1 (1:1000). For loading control, the blots were probed with 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, the data were acquired with the Molecular Imager (Gel DocTM XR, 170-8170) and analyzed by the associated software Quantity One 4.6.5 (Bio-Rad Laboratories, Hercules, CA).
Immunofluorescence staining
After deep anesthesia, the animal was perfused transcardially with normal saline followed by 4% paraformaldehyde. L4 and/or L5 lumbar segment was dissected out and post-fixed in the same fixative. The embedded blocks were sectioned into 20 μm thickness. Then, sections from each group (six animals in each group) were incubated with rabbit antibodies for IBA-1 (1:100), mouse antibodies for MMP-9 (1:100), the 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 washing out three times with PBS, all slides were carried out blindly and then studied under a confocal microscope (Olympus FV1000 confocal system, Olympus, Japan) for morphological details of the staining. Images were randomly coded and the fluorescence intensities were analyzed by 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 specimens were sectioned into 5 μm thickness, then incubated with first antibodies for Thioredoxin (1:100) and mouse antibodies for MMP-9 (1:100) in 10% donkey serum and 0.3% Triton-X100. After quenching endogenous peroxidase activity, slides were washed in PBS and incubated with the HRP conjugated secondary antibody for 2 h. Diaminobenzidine was used as a chromogen and counterstaining was done with hematoxylin. All immunohistochemistry staining sections were evaluated by 2 independent pathologists. The score for each slide was measured as the cross-product of the value of immunostaining intensity and the value of proportion of positive-staining cells. The immunostaining intensity 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%; 4, >75%. The score was calculated using the following formula: Total score = Intensity score × Proportion score.
Statistical analysis
SPSS Rel 15 (SPSS Inc., Chicago, IL) 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 as statistically significant.