1.1 Animals,grouping and modeling
36 SPF male SD rats(4 weeks old and weigh 230±20g),provided by the experimental animal center of the Second Affiliated Hospital of Harbin Medical University, were randomly divided into three groups: Control group, Sham group and OA group and 12 in each group. All experiments met the relevant criteria in the declaration of Helsinki, and this study has been approved by the ethics committee of the Second Affiliated Hospital of Harbin Medical University.
OA modeling: The rats were anesthetized with 1% pentobarbital sodium (40mg/kg) intraperitoneally,fixed in the supine position on the operating table.The right lower extremity were sterilized, and the knee joint cavity were exposed.An incision of about 1.5cm was made from the medial tibial tubercle of the lower limb to the upper margin of the patella. After cutting through all layers of tissue, the knee cavity was exposed. Dislocate the lateral patella and expose the anterior cruciate ligament. Cut off the anterior cruciate ligament.Compound patella joint,close knee joint cavity and suture the incision and disinfection. In the sham group, only skin tissue was cut open to expose the knee cavity and then sutured.
1.2 Sampling and materials
6 rats were killed in each group at 4 weeks and 16 weeks after modeling respectively, separated right lower extremity knee cartilage tissue and fixed overnight in 4% neutral formaldehyde solution, decalcificated for 6h with 10% EDTA and continuous sliced after paraffin embedding by slice machine(5μm). Another part of cartilage tissue was washed twice with PBS and 0.1% DEPC water respectively and stored in liquid nitrogen.
Rabbit anti-rat Bax,Bcl-2,LC3B,Beclin-1,mTOR,p-mTOR primary antibodies were purchased from Wuhan Sanying Biotechnology Co. LTD(Proteintech Group, Inc). GDC-0349 was purchased from Selleck(China). All plasmids were designed and synthesised by Shanghai Sangon Biotech.
1.3 HE staining
Dried slices were dewaxed in xylene, hydrated in gradient alcohol and then stained in Hematoxylin dye solution for 15 min, added with 1% ethanol hydrochloride for 30s, flushed and stained in 1% Eosin solution for 10 min and rapidly bleached in 1% acetic acid. After the gradient alcohol was dehydrated in turn, the slices were transparented in the xylene and sealed with neutral gum after air drying. Each sample randomly selected 5 visual fields and observed under high magnification microscope. Assessed the degree of articular cartilage lesion (0-14 points) according to the Mankin scoring criteria, the higher the score, the more serious it is.
1.4 Observation on the cell apoptosis of OA cartilage tissue
Paraffin sections of cartilage tissue were dewaxed 10min×2 in xylene, hydrated in radient alcohol, dropped 20 µg/ml protease without DNase K(Beyotime), incubated in 37℃ constant temperature for 25 min and washed in PBS for three times. The TUNEL detection solution containing TdT enzyme and fluorescent labeling solution was added and mixed well.The number and proportion of positive cells were observed under fluorescence microscope.
1.5 Western blot
The cartilage tissue was ground to homogenate, 1×RIPA Buffer and PMSF were added into on the ice bath,transferred to 1.5mL EP tube, centrifuged at 4 ℃ and 12000r/min for 5min, the supernatant was taken and centrifuged at 4℃ and 12000r/min for 20min and then the supernatant protein extract was taken. Concentration and quantification of protein were detected by BCA method. Electrophoresis, membrane transfer and sealing were carried out, and the corresponding first antibody(Abcam,UK) were added for incubation overnight according to the routine method. Added the second antibody and incubate for 2 hours. Added ECL luminescent agent to develop in darkroom. The Quatity-One software was used for analysis.
1.6 Detection of GAS5 expression by qRT-PCR
Samples of cartilage tissue were ground into powder in liquid nitrogen environment, added 1ml Tritol of lysate (100g/mL), homogenized and incubated at room temperature for 5 min. Total RNA extracted using Trizol kit (Invitrogen). After reverse transcription (reaction system: 16℃30min, 42℃30min, 85℃5min), the expressions of GASS5 were detected by Real-Time quantitative PCR.Reaction conditions: pre-denaturation at 95 ℃ for 5min, 10s at 95 ℃, 30s at 60 ℃, 40 cycles; 15s at 95 ℃, 60s at 60 ℃. 2-ΔΔ CT method was used for analysis.
1.7 Isolation and culture of chondrocytes
The cartilage tissue of knee joint of OA model group rats after 16 weeks of modeling were obtained, cutted into pieces (about 1mm3) and placed in 0.25% trypsin. After digestion at 37 ℃ for 1h, the supernatant was discarded and the tissues were washed three times. After centrifugation at 1200r/min for 15 min, the supernatant was discarded and the primary chondrocytes were obtained. The collected primary chondrocytes were resuspended in RPMI1640 complete culture medium containing 10% fetal bovine serum,100U/mL penicillin and 100U/mL streptomycin, cultured in 37 ℃ 5% CO2 incubator and the culture medium was changed every two days. They were subcultured when the cells fused to 80%.
1.8 Transfection
The second generation OA chondrocytes were divided into BC(Blank control) group, NC(Negative control) group, si-GAS5 group and GAS5 group. The empty plasmid, negative control, siRNA-GAS5 and GAS5 plasmids(Shanghai sangon) were transfected with Lipofectamine 2000 transfection reagent respectively, and the transfection effects were verified by qRT-PCR.
1.9 Detection of apoptosis by flow cytometry
The cells were cultured for 48h after transfection. After the cells were centrifuged at 1500r /min for 5 min, 500 μL of Binding Buffer was added and then resuspended. The cell suspension concentration was adjusted to 1× 106 cells/mL. After Annexin V-FITC 5μL was added and mixed well, the cell suspension was incubated at 4℃ in dark for 15 min.After 5μL of PI dye solution was added and mixed well and incubated at 4 ℃ in dark for 5 min. Apoptosis rate was detected by flow cytometry.
1.10 Detection of autophagy by transfection of GFP-LC3 plasmid
The plasmids of GFP-LC3 were diluted with Opti-MEM and then mixed with Lipofectamine 2000. Chondrocytes were adjusted to 3×10^5/ Well and inoculated into 6-well plates, 2mL serum-free medium was added and cultured for 24 hours ,the medium was discarded. Chondrocytes were washed in PBS for three times and 2mL Opti-MEM was added, and then the mixture containing GFP-LC3 was added. After 6h of continuous culture, the chondrocytes were replaced with complete medium. After 24 hours of continuous culture, the fluorescence distribution were observed under inverted fluorescence microscope.
1.11 Luciferase reporter assay
Through the analysis of Starbase and miRTarBase tool,the miRNA and the downstream target gene of GAS5 cound be predicted preliminarily.It was finally found that miR-144 could target to bind GAS5 and regulate autophagy and apoptosis related protein mTOR. In order to verify its targeting relationship, PCR amplified the fragments of the binding site with miR-144 in GAS5 and mTOR respectively, and constructed the corresponding psiCHECK-2 vector. The OA chondrocytes were divided into two groups and transfected with miR-34a mimic and its negative control mimic plasmids respectively,and then each group was transfected with GAS5-WT/GAS5-MUT plasmids or mTOR-WT/mTOR-MUT plasmids. Then the fluorescence intensity in each group was determined by Dual Luciferase reporter gene Kit (Invitrogen, USA).
1.12 Statistical analysis
The statistical software (IBM,USA) was used to process all data in this study. All data were expressed as mean ± SD.Independent sample t-test was used for comparison. When P< 0.05 the difference was statistically significant.