All animal procedures were performed with the approval and guidance of the Animal Care and Use Committee of our institution. The experiments were conducted in accordance with the ARRIVE guidelines.
In vitro experiments: cell preparation
Achilles tendons were excised from 15 healthy male Sprague-Dawley (SD) rats of eight weeks of age. Tendons were washed twice with phosphate-buffered saline (PBS) and cut into small pieces measuring approximately 1.5 to 2.0 mm3. Several pieces were placed on a culture plate and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum (FBS), 100 μg/mL streptomycin, and 100 U/mL penicillin. The explants were incubated at 37 °C in a humidified atmosphere of 5 % CO2/95 % air. After the tenocytes from explants had attained a subconfluent state, the cells were subcultured after trypsin digestion. The culture medium was changed every five days. Cells from passages 2 to 3 were used in this study.
In vitro experiments: cell proliferation assays
Cell proliferation was measured by a water-soluble tetrazolium salt (WST) assay using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). A total of 5000 cells were seeded in 100 μL of DMEM in each well of a 96-well plate. Cells were cultured for 24 hours in a CO2 incubator at 37 °C before the WST assay evaluation and then incubated for another 48 hours in DMEM containing four different DHEA (Tokyo Chemical Industry, Tokyo, Japan) concentrations (0 as a control, 1, 10, 20, and 50 µM). DHEA was dissolved in 0.1 % dimethyl sulfoxide (DMSO). For the WST assay, each well was supplemented with 10 μL of WST for four hours at 37 °C in a CO2 incubator before spectrophotometric evaluation at 450 nm (n=15 per group).
In vitro experiments:experimental protocol
Tenocytes were seeded onto 12-well culture plates at 105 cells per well and incubated in DMEM with two different glucose concentrations: 6 mM in the low-glucose (LG) group, and 33 mM in the high-glucose (HG) group according to a previous study24. DHEA was dissolved in DMSO to obtain a 2 mM stock solution and diluted to a final concentration of 10 µM19. DHEA was added at cell seeding and the tenocytes were divided into four groups: the control group (LG DHEA-), LG with DHEA (LG DHEA+), HG without DHEA (HG DHEA-), and HG with DHEA (HG DHEA+) (n = 12 per group). The same amount of DMSO was added to all groups (n=15 per group).
In vitro experiments: Quantitative real-time polymerase chain reaction (PCR) analysis
At 48 hours, total RNA from tenocytes was extracted using a RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA), total RNA was reverse-transcribed into single-strand complementary DNA (cDNA). PCR in triplicate was performed on the cDNA with 7900HT Fast Real-Time PCR System and SYBR Green reagents (Applied Biosystems). The messenger RNA (mRNA) levels of NOX1, NOX4, and IL-6 were analyzed. The list of primer sequences are shown in Table 1. Results were normalized to the mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed relative to their levels in the control culture using the 2-ΔΔCt method25 as previous studies16,17 (n=15 per group).
Table 1
Primer sequences used for polymerase chain reaction.
Gene
|
Oligonucleotide sequence
|
NOX1
|
Forward 5' GTGGCTTTGGTTCTCATGGT 3' Reverse 5' TGAGGACTCCTGCAACTCCT 3'
|
NOX4
|
Forward 5' GGGCCTAGGATTGTGTTTGA 3' Reverse 5' CTGAGAAGTTCAGGGCGTTC 3'
|
Type Ⅰ collagen
|
Forward 5' TGGAGACAGGTCAGACCTG 3' Reverse 5' TATTCGATGACTGTCTTGCC 3'
|
Type Ⅲ collagen
|
Forward 5' TAAAGGGTGAACGGGGCAGT 3' Reverse 5' ACGTTCCCCATTATGGCCAC 3'
|
MMP-2
|
Forward 5' GGAAGCATCAAATCGGACTG 3' Reverse 5' GGGCGGGAGAAAGTAGCA 3'
|
TIMP-1
|
Forward 5' ATAGTGCTGGCTGTGGGGTGTG 3' Reverse 5' TGATCGCTCTGGTAGCCCTTCTC 3'
|
TIMP-2
|
Forward 5' GGACACGCTTAGCATCACCCAGA 3' Reverse 5' GTCCATCCAGAGGCACTCATCC 3'
|
IL-6
|
Forward 5' GGTCTTCTGGAGTTCCGTTTC 3' Reverse 5' GGTCTTGGTCCTTAGCCATCT 3'
|
GAPDH
|
Forward 5' GGTGGTCTCCTCTGACTTCAACA 3' Reverse 5' GTTGCTGTAGCCAAATTCGTTGT 3'
|
In vitro experiments: detection of ROS accumulation
According to previous reports16,17, intracellular ROS levels in tenocytes of each group were detected by the oxidation-sensitive fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA) using the Total ROS/Superoxide Detection Kit (Enzo Life Science, Farmingdale, NY, USA) in accordance with the manufacturer’s protocol. Tenocytes (5 × 104) were incubated with DCFH-DA at a final concentration of 10 µM for 60 minutes at 37 °C in the dark, washed three times with PBS, trypsinized, and resuspended. For quantification, the number of ROS-positive cells and DAPI-positive cells in four rectangular areas (0.75 mm × 1.0 mm) in each slide were counted and the mean values were calculated. The percentage of ROS-positive cells was calculated the formula (number of ROS-positive nuclei/number of DAPI-positive nuclei) × 100 and expressed as the mean of the four areas (n=15 per group).
In vitro experiments: Immunofluorescence staining for analysis of apoptotic cells
According to previous studies16,17, nuclear fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining with an APO-DIRECT Kit (Phoenix Flow Systems, San Diego, CA, USA) according to the manufacturer’s protocol, using fixed cells (4% paraformaldehyde/PBS) with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI). For quantification, the number of apoptosis-positive and DAPI-positive cells in four rectangular areas (0.75 mm × 1.0 mm) in each slide were counted and the mean values were calculated. The percentage of apoptosis-positive cells was calculated using the formula (number of apoptosis-positive nuclei/number of DAPI-positive nuclei) × 100 and expressed as a mean of the four areas (n=15 per group).
In vivo animal experiments: type I diabetic rat model
To induce DM, a single dose of streptozotocin (STZ; 65 mg/kg; Sigma-Aldrich) dissolved in sodium citrate buffer (pH 4.5) was intravenously injected into 18 eight-week-old healthy male SD26. Following the injections, all animals were housed in standard cages with unrestricted food, water, and activity. All STZ-injected rats became diabetic 10 days after STZ injection. Their mean blood glucose level was 405.8 ± 62.5 mg/ml (mean ± standard deviation), although that of healthy control rats was <150mg/dl26.
In vivo animal experiments: experimental protocol
DM rats were randomly divided into 2 groups: a control group and a DHEA-injected group (DHEA group) (n = 9 in each group). In the DHEA group, two weeks after STZ injection, 50 mg/kg DHEA27,28 and vehicle (10 % DMSO) were injected intraperitoneally every other day for 4 weeks. In the control group, vehicle alone was injected in the same manner. The animals were sacrificed 4 weeks after the first DHEA or vehicle injection (6weeks after STZ injection) according to a previous study16. The right Achilles tendon was used for immunohistological evaluation, and the left was used for quantitative real-time PCR.
In vivo animal experiments: quantitative real-time PCR analysis
Nine left tendons in each of DHEA group and control group were used for quantitative real-time PCR. The Achilles tendons were cut into small pieces and minced. Isolated Achilles tendons were enzymatically dissociated with type II collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) and prepared for RNA isolation25. Total RNA was extracted using a RNeasy Mini Kit. Reverse transcription into single-stranded cDNA and real-time PCR was performed as previously described. Expression of NOX1, NOX4, IL-6, MMP-2, TIMP-2, and type I and III collagen (col1 and col3) was evaluated as previously described.
In vivo animal experiments: Achilles tendon histology and immunohistochemistry for NOX analysis
For immunohistological analysis, hematoxylin and eosin (H&E) staining, and staining for NOX1 and NOX4 were performed using the nine right tendons of diabetic rats from each group according to previous study16. Frozen, long-axis sections of Achilles tendons were sectioned into 7 μm thick specimens and fixed by using 10 % phosphate-buffered paraformaldehyde at room temperature. Histological evaluation of fiber structure and arrangement, nuclear morphology, and zonal variations in tendon cellularity was performed using H&E staining29. Each variable was scored between 0 and 3; 0 being normal, 1 slightly abnormal, 2 abnormal, and 3 markedly abnormal29. The grading of H&E-stained sections was performed in five randomly selected optical fields in each section and evaluated by two blinded investigators.
The immunohistochemical evaluation of NOX expression was performed by using anti-NOX1 and anti-NOX4 antibodies (Abcam, Cambridge, UK). Sections were incubated with proteinase for 10 minutes, treated with 3 % hydrogen peroxide (Wako Pure Chemical Industries, Osaka, Japan) to block endogenous peroxidase activity, and incubated with anti-NOX1 or anti-NOX4 antibodies (1:100 for both) at 4 °C overnight16. Then, sections were incubated with a peroxidase-labeled immunoglobulin antibody (Nichirei Bioscience, Tokyo, Japan) at room temperature for 30 minutes16. The signal (NOX1 and NOX4) was detected by the formation of a brown color following incubation with the peroxidase substrate 3,3’-diaminobenzidine (Nichirei Bioscience). Sections were counterstained with hematoxylin and examined microscopically. For semi-quantitative analysis, the ratio of NOX-positive tendon cells per field was determined in five randomly selected fields for each tissue section16.
Statistical analysis
All data are expressed as means and standard deviations. All statistical analyses of recorded data were performed using the Excel statistical software package (Ekuseru-Toukei 2015; Social Survey Research Information Co., Ltd., Tokyo, Japan). Comparisons between more than two groups were performed by analysis of variance and Tukey’s post hoc test. Comparisons between two groups were performed using analysis of variance and the Mann-Whitney U test. A p-value < 0.05 was considered statistically significant.