Isolation and culture of primary mouse chondrocytes
The immature C57BL/6 mice (10 days) were euthanized with an overdose of sodium pentobarbital. The knee cartilages of mice were collected under aseptic conditions, and the tissues were treated with 2 mg/ml (0.1%) collagenase II (Solarbio Science & Technology Beijing, China) for 4 h at 37 °C. Next, the digested cartilage tissues were suspended and seeded into tissue culture flasks. Chondrocytes grow in DMEM/F12 (Gibco, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (FBS; Thermo Scientifc, Logan, UT, USA) and 1% penicillin/streptomycin antibiotics (Gibco, Invitrogen) in the incubator maintained at 5% CO2 at 37 °C. After 24 h of incubation, the medium was first changed. When up to 80% to 90% confluence, the cells were harvested by using 0.25% Trypsin-EDTA (Gibco, Invitrogen). Then, cells were replanted into new culture plates at the appropriate density and cultured in the incubator maintained at 5% CO2 at 37 °C. All of our tests were conducted using chondrocytes from the second passage.
Application of cyclic stretch
Chondrocytes were seeded on a 6-well collagen-coated BioFlex plate (Flexercell International, McKeesport, PA, USA) containing a flexible silicone elastomer substratum and cultured for 3-5 days to 80% confluence. The plates were then placed in a Flexercell Strain Unit (Flexercell International, McKeesport) and subjected to 20% surface elongation at a frequency of 6 cycles/min, each cycle consisting of 3 seconds stretch alternating with 3 seconds of relaxation with a computer-controlled vacuum stretch apparatus (FX-4000T Tension Plus System; Flexercell International, McKeesport). Chondrocytes were harvested after 6, 12 and 24 h, respectively; while control cells were cultured on similar plates and kept in the same incubator without mechanical stress.
siRNA transfection
siRNAs for the mouse Piezo1 gene and mouse cPLA2 gene were obtained from GeneChem (Shanghai, China). Chondrocytes were seeded on a 6-well plate and cultured for 24 h to 60–70% confluence. The cells were transfected with 50 nM negative control or siRNA duplexes using GP-transfect-Mate (GeneChem). The transfection efficacies were measured via RT-qPCR or western blot experiments.
Cell treatment
To investigate the effect of LMP on the apoptosis and cartilage degeneration in chondrocyte during cyclic stretch, chondrocytes were pretreated with CA074 (20 mM, Med Chem Express, Monmouth Junction, NJ, USA) and pep A (12.5 mg/ml, Med Chem Express, Monmouth Junction) 1 h before mechanical stretch for 24 h. To investigate the Piezo1 activation in chondrocyte, chondrocytes were treated with different concentrations of Yoda1 (0, 1 5 10, 25, 50 µM; Med Chem Express, Monmouth Junction) respectively for 6, 24 and 48 h. In order to determine the effect of Piezo1 in chondrocyte during cyclic stretch, chondrocytes were treated with cyclic stretch or Yoda1 (25µM) for 24 h in the absence or presence of GsMTx4 (3 µM; Med Chem Express, Monmouth Junction) pretreatment. Besides, chondrocytes transfected si-Piezo1 or si-cPLA2 to downregulate Piezo1 or cPLA2 expression respectively, followed by cyclic stretch or Yoda1 (25µM) treatment for 24 h.
Western blotting analysis
The total protein extracted from chondrocytes was isolated using RIPA lysis buffer. The protein concentration was determined via a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF film (Bio-Rad, California, USA). After blocking with 10% non-fat milk for 2 h, the films were incubated with primary antibodies at 4°C overnight, and followed by incubation with the respective secondary antibodies (goat anti-mouse IgG (H+L)-HRP, Bioworld Technology Minneapolis, MN, USA and goat anti-rabbit IgG (H+L)-HRP, Bioworld Technology Minneapolis, MN, USA) for 1.5 h at room temperature. Bands signals were visualized using and quantified by Image Lab 3.0 software (Bio-Rad, California, USA) via Western HRP Substrate (Zen Bioscience, Chengdu, China). The information regarding diluted concentrations of primary antibodies is indicated in Table 1.
Lysosomes staining with LysoTracker and LysoSensor
LysoTracker red DND-99 (Yeasen Biotechnology, Shanghai, China) and pH-sensitive LysoSensor Blue DND-167 (Thermo Fisher Scientific, L7533) probes were used to visualize the lysosomes and determine lysosomal activity, respectively. Chondrocytes were incubated with LysoTracker (50 nM) and LysoSensor (1 µM) at 37°C for 30 min. Live-imaging was captured using confocal microscopy (Nikon ECLIPSE 80i, Tokyo, Japan) under the same settings parameters. The fluorescence intensity was measured using Image J.
Acridine orange (AO) release experiment
The distribution of AO dye was used to evaluate LMP, as previously reported42. Chondrocytes were incubated with 10 mg/ml AO dye (Yuanye Bio-Technology, Shanghai, China) at 37◦C for 15 min. Live-imaging was captured using confocal microscopy (Nikon ECLIPSE 80i) under the same settings parameters. The fluorescence intensity was measured using Image J.
Immunofluorescence staining
Chondrocytes were seeded on 24 well plates containing coverslips and cultured for 1-2 days. The mitochondria were stained with MitoTracker Deep Red FM (Thermo Scientific Madison, WI, USA). Cells were fixed in 4% paraformaldehyde for 10 min. After blocking with 5% bovine serum albumin (BSA) at 37°C for 30 min, the cells were incubated with primary antibodies at 4°C overnight, and followed by incubation with the respective secondary antibodies (goat anti-rabbit IgG H&L, Alexa Fluor® 488, and goat anti-mouse IgG H&L, Alexa Fluor® TRITC). The immunofluorescence staining was also performed in vivo studies. After deparaffinization and rehydration, sections were restored by high-pressure antigen retrieval. Then, the sections were blocked with 5% BSA, incubated with primary antibodies, and followed with the respective secondary antibodies. The nuclei were stained with DAPI. The images were captured using microscope (Olympus, Tokyo, Japan) or confocal microscopy (Nikon ECLIPSE 80i) under the same settings parameters. Images for co-localization analysis were assessed using ImageJ (Fiji). The information regarding diluted concentrations of primary antibodies is indicated in Table 1.
Cytosolic CTSB Activity Measurement
Cytosolic CTSB activity was measured to detect LMP43. Firstly, a series of digitonin concentrations (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, and 200 mg/ml) were used to determine an appropriate concentration for the selective permeabilization of the plasma membrane. The appropriate concentration of the digitonin dilution was determined to be the one that generated the best possible permeabilization of the plasma membrane (LDH release, detected by LDH assay (Beyotime Institute of Biotechnology)) with minimal cathepsin release from the lysosomes. 200 µg/mL digitonin was used for the complete permeabilization of the cell membrane. Briefly, after the desired treatment, the digitonin at the appropriate concentration was added to each appropriate well of the plate. The cells were then incubated on ice for 10 min on a rocking table (70 rpm). The lysis buffer above the cells (cell-free extract) was transferred to a new 96 well plate. 75 µL of 5 mM DTT (Sigma-Aldrich Chemical Company, Milwaukee, WI, USA) and 30 µM cathepsin-specific fluorogenic substrate Z-FR-AMC (219392, Millipore) was added to the wells of the plate. The kinetics of cathepsin activity were measured 30 min at 37◦C with a Varioskan Flash microplate reader (Thermo Scientific) at an excitation wavelength of 370 nm and an emission wavelength of 460 nm. The extent of LMP in each group was determined by the percentage of cytosolic CTSB activity compared to the total activity at the appropriate concentration of digitonin (15 mg/ml) used to extract the cytosolic fraction.
TUNEL staining
The TUNEL Apoptosis Detection Kit was used to determine the extent of DNA damage (Alexa Fluor 488, Yeasen Biotechnology). Chondrocytes or cartilage sections were stained with TUNEL kit according to the manufacturer’s instructions at 37 °C for 30 min. The nuclei were stained with DAPI. The images were captured using microscope (Olympus) under the same settings parameters to count TUNEL positive cells.
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted from chondrocytes using the High Pure RNA Isolation Kit according to the manufacturer’s protocol. According to the standard protocol of PrimeScript™ RT Master Mix (Takara Biomedical Technology, Japan, RR036A), cDNA was synthesized by reverse transcriptase. The qPCR assay was carried out using TB Green Master Reagents (Takara Biomedical Technology, Japan, RR820A). The amplification parameters were as follows: denaturation at 95°C for 30 s, annealing at 95°C for 5 s, extension at 60°C for 34 s for 40 cycles, and the signal was detected at 60°C. Finally, the target mRNAs were normalized to GAPDH mRNA. The primer sequences were as follow: for Piezo1, Forward 5′-TAACACCCTCTGTGTCATGGT-3′, Reverse 5′-GGGAGGTCTACGAAGTTCTTGAG-3′, for Pla2g4a, Forward 5′-AGGCACAGCTACATTCCCTG-3′, Reverse 5′-CATGCTGAACCGTAGGTCTGG-3′, for Gapdh, Forward 5′-AGGTCGGTGTGAACGGATTTG-3′, Reverse 5′- GGGGTCGTTGATGGCAACA-3′.
Cell Viability
Cells were seeded on a 96 well plate. After treatment, each well received 10 μL of the Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology) and was incubated at 37 °C for 1 h. Cell viability was measured using the OD450 value that had been standardized to the value of the controls.
Calcium imaging
Chondrocytes were plated on Glass Bottom Culture Dishes (NEST Biotechnology, Wuxi, China). Cells were loaded with Fluo-4 AM (Beyotime Institute of Biotechnology) at 37 °C for 30 min. Live-imaging was performed using confocal microscopy (Nikon ECLIPSE 80i). Images were recorded every 1s over 5 min and data were analyzed using NIS software (Nikon). To test the effect of Piezo1 knockdown on calcium release, the cells were pre-transfected with Piezo1 siRNA. Yoda1 (25 µM) was added 1 min from the start of the recording. Overloaded or faintly fluorescent chondrocytes (F0 < 150 AI) were excluded from the analysis as well as cells demonstrating unstable fluorescence before loading.
Mouse OA model
10-week-old C57BL/6 male mice were purchased from Hufukang Biotechnology Co., LTD, Beijing, China. The mouse OA model was induced by surgical destabilization of the medial meniscus (DMM). In brief, the mice were anesthetized with 2% (w/v) pentobarbital (40 mg/kg, ip); then the joint capsule of right knee was incised just medial to the patellar tendon and the medial meniscotibial ligament was transected with microsurgical scissors. The sham group mice were treated with an arthrotomy without the transaction of medial meniscotibial ligament. After surgery, the mice were divided into four groups: Sham, DMM, DMM + Vehicle, DMM + GsMTx4 and DMM + AACOCF3 group. 200 µl AACOCF3 (4 mM) was delivered through intraperitoneal injection twice a week for 8 weeks. 10 µl GsMTx4 (200 µM) and the equivalent amount of PBS was delivered through intraarticular injection twice a week for 8 weeks. Mice were sacrificed at 8 weeks post-OA surgery from each group, the knee joints were dissected and processed for histological evaluation.
Micro-CT
Knee joints extracted from 8-weeks DMM model mice were fixed in 4% paraformaldehyde for 24 h. Micro-CT scan was performed for the knees at 100 kV, 98 μA, 12 μm resolution on a Micro-CT scanner (Bruker Skyscan 1176, Luxembourg, Belgium). The entire joint was applied for three-dimensional (3D) reconstruction. The osteophyte was chosen as the region of interest (ROI), and the volume of ROI were analyzed.
Histopathologic analysis
Knee joints extracted from 8-weeks DMM model mice were fixed in 4% paraformaldehyde for 24 h, decalcified in 20% ethylenediaminetetraacetic acid (EDTA, Solarbio Science & Technology) decalcifying solution, and thereafter dehydrated and paraffin-embedded on the sagittal plane. Then, the tissues were cut into 5-µm sections and put on slides. The slides were stained with safranin O-fast green (S–O, Solarbio Science & Technology) and hematoxylin & eosin (H&E) dye (Solarbio Science & Technology). The images were captured using microscope (Olympus) under the same settings parameters. The OA Research Society International (OARSI) histopathology scoring system was used to detect the histologic scoring of OA in the mouse44.
Statistical analysis
The statistical tests were carried out using SPSS 19. (Chicago, IL, USA). All information was presented as mean ± SD. For significant difference testing between the two groups, unpaired Student's t-tests were conducted. To determine if there were any statistically significant differences between two groups with a sample size of three or four, one-way ANOVA with LSD (equal variances assumed) post hoc analysis or Dunnett's T3 (equal variances not assumed) approach was used. Statistics have significance at P < 0.05.