Animals
Healthy 8-week-old male Sprague-Dawley rats, weighing ~180 g, were purchased from the Shanghai Laboratory Animal Commission (Shanghai, China) under license number SCXK 2013–012. The experimental procedures involving animals were performed according to the guidelines for the Care and Use of Laboratory Animals 2010 by the Ministry of Science and Technology of the People's Republic of China. The present study was approved by the ethics committee of Shandong Jimin No. 1 People's Hospital (Shandong, China). Osteoarthritis (OA) was induced in the rats by the methods described by Ying et al (23). Briefly, rats were anesthetized with ether, the right knee was exposed and the patella was dislocated laterally. Subsequently, the right knee was fully flexed, followed by anterior crucial ligament transection and medial meniscus resection using micro-scissors.
Treatment strategy
The rats in the treatments groups were administered intragastrically with 0.25, 0.30, 0.35, 0.40, 0.45 and 0.50 mg/kg doses of tanshinone IIA for 28 days. The rats in the normal control and ACLT + MMx groups received normal saline for the same duration. The animals were housed under a 12-h light/dark cycle in a humidity-controlled (60–64%) and sterilized room at 25°C with access to fresh water and standard laboratory food ad libitum.
Histological analysis
On day 29 following the completion of treatment, the animals were sacrificed following anesthetization with halothane. The bones (tibia and femur) were removed and then subjected to decalcification using EDTA. The paraffin-embedded bone was cut into thin sections of 2 µm, which were de-paraffinized in boiling xylene. The sections were subjected to hematoxylin and eosin staining, followed by histopathological examination using a Mankin scale, in which 0 indicated normal cartilage and 12 indicated full disintegration (24). Changes in the synovial lining were determined using the Image-Pro Plus 6.0 image analysis system (Media Cybernetics, Inc., Rockville, MD, USA).
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay for the detection of apoptotic cells
For the analysis of apoptotic cells in the cartilage sections, the sections were washed with 1% PBS/BSA and then fixed in 4% paraformaldehyde for 20 min. The sections were permeabilized using 0.1% Triton-X 100 for 15 min on ice. A TUNEL assay using fluorescein isothiocyanate (FITC)-conjugated dUTP, and an Apoptosis Detection System kit (Roche Diagnostics GmbH, Mannheim, Germany) were used for the analysis of apoptosis, according to the manufacturer's instructions.
Flow cytometric analysis
The chondrocytes of the rats belonging to the ACLT + MMx and Tanshinone IIA treatment groups were examined for apoptosis using Annexin V binding and propidium iodine (PI) staining, followed by flow cytometry. The cells were washed with ice-cold PBS and double-stained with FITC-conjugated Annexin V protein and PI for 30 min. Subsequently, 488-nm laser flow cytometry coupled to a cell sorter (FACSCalibur; BD Biosciences, San Jose, CA, USA) was used for analysis of the stained cells.
Enzyme-linked immunosorbent assay (ELISA) analysis
For determination of inflammatory cytokines, blood samples (4 ml) were collected from the aorta of the abdominal region of the rats, and serum was separated by centrifugation at 3,000 × g for 10 min at 4°C. The levels of IL-1β (cat. no. EK0393), tumor necrosis factor-α (TNF-α; cat no. EK0526) and inducible nitric oxide synthase (iNOS; cat no. EK0472) were quantified using commercially available ELISA kits (ScienCell Research Laboratories, Carlsbad, CA, USA).
Western blot analysis
The cartilage of the rats was washed and placed into lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with phenylmethylsulfonyl fluoride and aprotinin for 4 h at 4°C. The content of protein was determined using a detergent compatible protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of extracted protein samples (50 µg) was mixed with 2X SDS buffer, and were separated on a 10% polyacrylamide gel by electrophoresis. The proteins were then transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.) followed by incubation in blocking buffer (PBS with 7.5% non-fat dry milk, 2% BSA and 0.1% Tween-20) for 2.5 h at 4°C. The protein expression levels of MMP-13 and TIMP-1 were determined following incubation at 4°C overnight with the following primary antibodies: Mouse monoclonal anti-MMP-13 (cat. no. MA5-14247, 1:400 in blocking buffer; Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA), mouse monoclonal anti-TIMP-1 (cat. no. MA1-773, 1:500 in blocking buffer; Pierce; Thermo Fisher Scientific, Inc.) and anti-β-actin (cat. no. 2791, 1:800 in blocking buffer; OriGene Technologies, Inc., Beijing, China). The membranes were washed with PBS and 0.1% Tween-20, followed by incubation with secondary horseradish peroxidase-conjugated goat polyclonal anti-rabbit antibodies (cat. no. 31460, 1:10,000 dilution in blocking buffer; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at 4°C. The membranes were then washed in PBS and developed using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Uppsala, Sweden).
Statistical analysis
Data are expressed as the mean ± standard deviation. The experiments were repeated at least three times, with the mean of the results presented. The data were analyzed using one-way analysis of variance followed by Student's t-test using SPSS software version 21.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.