HQWD preparation
HQWD was provided by the Department of Pharmacy in the Affiliated Hospital of Nanjing University of Chinese Medicine. HQWD is composed of crude herbs, including Phellodendron chinense Schneid, Chinemys reevesii, Anemarrhena asphodeloides Bge, Rehmannia glutinosa Libosch, Citrus reticulata Blanco, Paeonia lactiflora Pall, Cynomorium songaricum Rupr, Canis familiaris L, and Zingiber oj-jicinale Rosc (dosages shown in Table 1). The medicinal herb mixture was extracted in boiling water, and the aqueous extracts were vacuum-dried at 60 °C to obtain a powder. The powder was then stored at -20 °C. The content of crude herbs was determined and used for experiments by dissolution in pure water at desired concentrations (0.5 mg/ml). The fingerprint of HQWD was determined by high-performance liquid chromatography (Fig. S1, Supporting Information).
Animal experiments
All the experimental procedures were performed with the approval of the Experimental Research Institute of Nanjing University of Chinese Medicine and followed the guidelines of the Institutional Animal Care and Use Committee. A total of 72 rats (four-week-old Sprague-Dawley (SD) rats, male, 70–100 g, purchased from Bejing Charles River Company) were used for in vivo experiments. Rats were housed in an airconditioned room at 25°C with a 12 h light/dark cycle. Nine rats were used in BMSCs extraction and 63 rats were used in rotator cuff reconstruction model building. Rotator cuff reconstruction model included two groups: control group and HQWD group, and each group included 18 rats. Eighteen rats were used in tissue section analysis (Control group, 9; HQWD group, 9). Eighteen rats were used in biomechanical test analysis (Control group, 9; HQWD group, 9). Twenty-seven rats were used in interleukin-1β (IL-1β) and IL-1RA experiments (Control group, 9; IL-1β group, 9; IL-1RA group, 9). The rats were sacrificed after anesthesia of isoflurane, and tissues were obtained.
RCT and reconstruction model building: After successful anesthesia with isoflurane, we incised the shoulder joint laterally and pulled the trapezius muscle away. The insertion of the supraspinatus to the humeral head was exposed. The supraspinatus insertion was resected. Approximately 2 mm of the distal tendon of the supraspinatus was cut off. Then, the skin was sutured. Four weeks later, a 3-0 nonabsorbable Propathene line was used to fix the supraspinatus tendon to the humeral head through bone tunnels. The subjects were randomly divided into two groups: equal amounts of either normal saline (NS) or HQWD. The intragastric administration of the treatments to the rats was performed after rotator cuff reconstruction.
The interleukin-1β (IL-1β) and IL-1RA drugs were administered intravenously into the rat tail at 1 and 10 μg/kg, respectively, once a day after rotator cuff reconstruction.
Extraction and culture of rat BMSCs
BMSCs inside their femurs and tibiae were obtained after anesthesia, according to previous literature [22]. Isolated BMSCs were incubated with standard media comprising DMEM (Gibco) supplemented with 10% FBS and 1% double-antibiotics (streptomycin + penicillin; Gibco). After 3–5 days of incubation (at 80% confluence), cells were re-plated in 10 cm Petri dishes and maintained at 37 ℃ in a humidified atmosphere of 5% CO2 and 95% air.
Extraction of exosomes
Blood was collected from rat hearts after administering anesthesia, and the serum was extracted after centrifugation. Supercentrifugal extraction was adopted, and the cell debris was removed from the serum with 300× g centrifugation for 10 min, followed by 29,500× g centrifugation for 20 min. The supernatant was filtered through a 0.22 µm disposable sterile filter and centrifuged at 120,000× g for 90 min. The resulting supernatant was discarded. The precipitate was dissolved in 100 μL of PBS and stored at −80 °C after packaging.
After BMSCs had grown to about 80% fusion, the culture medium was replaced by exosome-depleted FBS-containing medium (EXO-FBS-250A-1; System Biosciences, Mountain View, CA, USA). The cells were then cultured for a further 48 h. The medium was collected and centrifuged at 4 °C at 300 × g for 10 min and at 2000 × g for 10 min. Supercentrifugal extraction was adopted, and the cell debris was removed from the serum with 300 × g centrifugation for 10 min, followed by 29,500 × g centrifugation for 20 min. The supernatant was filtered through a 0.22× µm disposable sterile filter and centrifuged at 120,000 × g for 90 min. The resulting supernatant was discarded. The precipitate was dissolved in 100 μL of PBS and stored at −80 °C after packaging. The protein content of the BMSC exosomes was measured by the bicinchoninic acid assay (BCA; Thermo Fisher, Waltham, MA, USA). A microplate reader (ELx800, BioTek, USA) was used to measure the absorbance at a wavelength of 562 nm.
Grouping of drug and experimental animals
HQWD was provided by the Department of Pharmacy in the Affiliated Hospital of Nanjing University of Chinese Medicine. The drug composition was as follows: Phellodendron chinense Schneid, Chinemys reevesii, Anemarrhena asphodeloides Bge, Rehmannia glutinosa Libosch, Citrus reticulata Blanco, Paeonia lactiflora Pall, Cynomorium songaricum Rupr, Canis familiaris L, and Zingiber oj-jicinale Rosc (Table 1). The water decoction of HQWD contained 0.5 mg/ml of crude drug. Rats were randomly assigned to the control group or the HQWD group after reconstructing the supraspinatus muscle. Normal saline was administered to the control group intragastrically. The HQWD group received HQWD decoction 5 g/kg/day intragastrically.
Immunohistochemistry
For the immunohistochemistry assays, the paraffin sections were de-waxed, dehydrated, and incubated overnight at 4 °C with anti-ColⅠ, anti-NLRP3, and anti-IL-1RA (diluted 1:100; Abcam). After the primary antibody was removed, the secondary antibody (diluted 1:100, Thermo Fisher) was added, and the sections were incubated for 1 h at room temperature. The stained cells were developed with diaminobenzidine and counterstained with hematoxylin.
Safranin and fast green staining
After the shoulder joint was fixed and decalcified, the shoulder was embedded in paraffin, sliced at 5 μm in the coronal position, and hydrated after dewaxing. For fast green staining, the slices were placed in the fast green dye liquid for 5–10 min. Excess dye was washed with water until the cartilage became colorless. The slices were then slightly soaked in the differentiation fluid and then washed with tap water. For staining with safranin, the slices were placed in safranin dye liquid for 15–30 s and then quickly dehydrated with three cylinders of anhydrous ethanol. For the transparent seal, sections were cleared with xylene for 5 min and then sealed with a neutral gum seal. The cartilage stained red or orange with a green background.
Western blot analysis
Cells and tissues were placed on ice immediately following treatment and washed with ice-cold Hank’s balanced salt solution. All the wash buffers and final resuspension buffer included a 1× protease inhibitor cocktail (Pierce, Rockford, IL, USA), NaF (5 mM), and Na3VO4 (200 mM). The protein concentration of the lysate was measured using the BCA protein assay kit (Thermo Fischer). Nuclear or total cell proteins were resolved on 8% to 12% SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% bovine serum albumin reagent (Beyotime, Jiangsu, China) and incubated overnight at 4 °C with primary antibody dilution buffer (the dilution followed the specification; Abcam) and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) for 2 h. Afterward, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA) and exposed to X-ray film. The bands were analyzed with Gel-Pro Analyzer 4.0 (Bio-Rad, Hercules, CA, USA).
Biomechanical test
Biomechanical tests were performed 4 weeks after the surgical reconstruction. Mutilation was performed at the lower end of the humerus. The supraspinatus and its insertion were carefully retained; the other muscles, except for the supraspinatus, were removed. The supraspinatus-humerus structure was obtained. The specimen was immediately placed in 4% paraformaldehyde solution, and the shoulder joint tissue was firmly fixed on an INSTRON biomechanical tester (Instron, Boston, MA, USA). The instrument was loaded, and a displacement velocity of 5 mm/min was applied to test the maximum tensile load of the supraspinatus tissue. The loading load when the supraspinatus tendon was broken was observed and recorded in detail as the maximum breaking load (N). The linear slope of the load-displacement curve was taken as the stiffness.
Enzyme-linked immunosorbent assay
IL-1RA was determined by enzyme-linked immunosorbent assay (ELISA) using a commercially available ELISA set (Sagon Biotech, Shanghai, USA). ELISA was performed according to the manufacturer’s instructions. All samples and standards were measured in duplicate.
Statistical analysis
SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. The measurements are presented as the mean ± standard deviation. The data were analyzed using a one-way analysis of variance followed by Bonferroni’s post-hoc test for multiple comparisons (p < 0.05 was considered to indicate a statistically significant difference).