Reagents
ASP was purchased from Shanghai Yilin Biotech. Co., Ltd. (Shanghai, China). The purity of ASP is approximately 92%. The component sugars are glucose, galactose, arabinose, rhamnose, mannose, and xylose. The average molecular weight of ASP is 85.0kDa. ASP was dissolved in PBS and diluted with DMEM-F12 for the experiments. Collagenase II (Worthington Biochemical Corp., Lakewood, NJ, USA) was dissolved in DMEM at 2.5 mg/ml to digest articular cartilage. Sodium nitroprusside(SNP) was purchased from Dandong Medical and Pharmaceutical Co., Ltd. (Heilongjiang, China), reconstituted in sterile normal saline at 40mg/ml and stored at 4℃ avoiding light. CQ, 3-MA, P276-00 and SCH772984 were purchased from Selleckchem (Houston, TX, USA).
Isolation and culture of osteoarthritis articular Chondrocyte.
Cartilage tissue specimens were obtained from OA patients during joint replacement surgery in Changzhou Second People’s Hospital. All participants had signed a written informed consent prior to the subjects entering the study. In addition, the study was approved by the Ethics Committee of Nanjing Medical University. All the tissues were carefully minced and digested with 2.5 mg/ml collagenase II in serum-free Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) for 4-6 hrs at 37°C, filtered through a 70μm cell strainer (BD, Durham, NC, USA), extensively washed with blank DMEM and finally cultured in DMEM/F12 supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA), 100 U penicillin, and 100 μg/ml streptomycin in a standard cell culture chamber containing 5% CO2. Non-adherent cells were removed after 3 days. Adherent cells were split at a ratio of 1:2 until they grew to 90% confluence. Chondrocytes were used from passages 3 to 5 in subsequent experiments.
Determination of Cell viability and proliferation by MTS assay
Cell viability and proliferation assays were performed using the tetrazolium compound-based CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay (Promega, Madison, WI, USA). OA chondrocytes were seeded at approximately 5000 cells per well in 96-well plates in triplicate for 7 days under regular growth conditions (DMEM-F12 with 10% FBS). After seeding for 24 hrs, ASP was added in the media, and then the MTS assay was performed daily according to the manufacturer’s instructions for the subsequent 6 days. Generally speaking, 20µl of MTS solution reagent was pipetted into each well of the 96-well assay plate containing the chondrocytes in 100µl of fresh culture medium. Then the plate was incubated at 37°C for 2 hours in a humidified, 5% CO2 atmosphere, and the absorbance at 490nm was recorded using an absorbance microplate reader (Elx808™ Bio-Tek Instruments, Winooski, VT)
Cell apoptosis detection by DAPI staining
Chondrocytes were seeded on sterile glass slides coated with gelatin, and then treated with SNP alone or with ASP for indicated time. Cells were fixed and nuclei were stained with DAPI (Sigma-Aldrich, MO, USA) in the dark for 5 min and the fluorescence (Nikon Eclipse Ti, Japan) was observed.
Detection of cell apoptosis rate by flow cytometry: AnnexinV/PI staining
2*105 chondrocytes were seeded in 6-well plates. Cell apoptosis rates were detected by Annexin V-FITC/PI kit (Vazyme Biotech Co., Ltd. , Nanjing, China) according to the manufacturer’s instructions. Generally speaking, the cells were washed with ice-cold PBS and trypsinized. Removing the supernatant after centrifugation, the cells were resuspended in 100 µL binding buffer, and incubated with 5 µL Annexin V-FITC for 10 min at room temperature avoiding direct light. Then 5 µL PI and 400 µL binding buffer were mixed into the flow tube.
The apoptosis ratio was assessed with a flow cytometer (BD, Biosciences, San Jose, CA, USA), and the results were analyzed and assembled by FlowJo software(Tree Star, Inc., USA).
Immunofluorescence
2-5×104 chondrocytes were seeded on sterile glass slides precoated with gelatin. After the indicated treatment, cells were fixed in 4% paraformaldehyde at 4℃ for 15 min and blocked with PBS containing 5% normal goat serum and 0.3% Triton X-100 for 1 h at room temperature. Staining of the treated cells with LC3A/B (D3U4C) XP® Rabbit mAb (Alexa Fluor® 488 Conjugate, CST, USA) at 1/100 dilution was performed overnight at 4 °C in PBS containing 1% BSA and 0.3% Triton X-100. Nuclei were counterstained with DAPI in the dark for 5 min and the fluorescence (Nikon Eclipse Ti, Japan) was observed.
Western blot analysis
Cultured chondrocytes were lysed with RIPA buffer and boiled. SDS-polyacrylamide gel electrophoresis was conducted on a polyacrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane. All antibodies, purchased from Cell Signaling Technology were used to detect the autophagy levels, apoptosis, proliferation and signaling pathways. Rabbit anti-human β-actin polyclonal antibody was used to detect the Actin signal as an internal control and relative expression levels were quantified by running the Quantity One software. Antibodies information were performed in Table1.
Table1
antibodies
|
manufacturer
|
isotypes
|
catalog
|
Atg5 Antibody
|
CST
|
Rabbit IgG
|
#2775
|
Bax Rabbit mAb
|
CST
|
Rabbit IgG
|
#5023
|
Beclin-1 Rabbit mAb
|
CST
|
Rabbit IgG
|
#3495
|
Bcl-2 Antibody
|
CST
|
Rabbit IgG
|
#4223
|
CyclinD1 Rabbit mAb
|
CST
|
Rabbit IgG
|
#2978
|
p21 Rabbit mAb
|
CST
|
Rabbit IgG
|
#2947
|
Phospho-p44/42MAPK(ERK1/2) Rabbit mAb
|
CST
|
Rabbit IgG
|
#4377
|
P62 Antibody
|
CST
|
Rabbit IgG
|
#5114
|
ERK1/2 Rabbit mAb
|
CST
|
Rabbit IgG
|
#4695
|
Ras Antibody
|
CST
|
Rabbit IgG
|
#3965
|
Raf Antibody
|
CST
|
Rabbit IgG
|
#9422
|
p-MEK1/2 Rabbit mAb
|
CST
|
Rabbit IgG
|
#9154
|
MEK1/2 Mouse mAb
|
CST
|
Mouse IgG
|
#4694
|
LC3B Antibody
|
CST
|
Rabbit IgG
|
#2775
|
β-Actin Rabbit mAb
|
CST
|
Rabbit IgG
|
#4097
|
|
|
|
|
Lists of antibodies.
mRFP-GFP-LC3 analysis
Chondrocytes were seeded in precoated slides with a density of 5 × 104 cells. One day after seeding, cells were infected with mRFP-GFP-LC3-labeled adenovirus (Genechem, Shanghai, China) according to the manufacturer’s instructions. The virus expresses the monomeric RFP-GFP-tagged LC3 (tfLC3) as an autophagic flux reporter comprised of LC3 protein fused with monomeric red fluorescent protein (mRFP) and green fluorescent protein (GFP). The GFP signal would quenched within the lysosome lumen by the acidic and/or proteolytic environment. Yellow puncta which is consist with colocalized GFP (green) and mRFP (red) fluorescent signals in the cytoplasm indicate early autophagosomes, while the mRFP signals alone (red) represent late autolysosomes.
Statistical analysis
Statistical analyses were performed using Prism (GraphPad Software, San Diego, CA, US). Unpaired Student’s t test was used for two groups and one-way ANOVA for more than two groups. The symbols *, **, ***, and # indicated p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively. All quoted P values were 2-tailed, and those less than 0.05 were considered statistically significant.