Primary chondrocytes
Sprague-Dawley (SD) rats were purchased from the Model Animal Research Institute of Nanjing University and raised in the SPF Animal Center of Scientific Research Center. Animal experiment operations and animal welfare standards were approved by the Ethics Committee of the People's Hospital of Longhua District, Shenzhen. Before the experiment, 4 rats were randomly selected from 18 rats, and the cartilage tissue on the surface of the rat's femoral condyle was cut by sterile surgery, and the fragments were rinsed and trimmed in PBS containing penicillin sodium/streptomycin double antibody. The rat articular chondrocytes were extracted by a modified two-step enzymatic digestion method[16], filtered with a 40 μm cell strainer, and resuspended in a 10% fetal bovine serum high-glycemic DMEM. The medium was changed for the first time on the third day, and then every 2 days. The cells were passaged after the culture flask was 95% full, and the first passage chondrocytes were used as seed cells. When culturing the cells of subsequent generations, add chondrocyte induction medium (DMEM high glucose medium containing 2% fetal bovine serum, 1% penicillin-streptomycin solution, 1% ITS, 50 ml ascorbic acid, 100nmol/L ground Cells were resuspended with dexamethasone and 10ng/mL TGF-β), centrifuged and cultured in a 37ºC, 5% CO2 incubator. The medium was changed per 48h for 2 weeks.
Shear force model establishment
In this study, the Allegro XRS 20 Bioreactor System (Pall Corporation, US) was used to give cells a quantitative shear force stimulation, and the assemble of equipment's component and the experimental operation methods refer to the official instructions. Briefly, the shear force was calculated according to the Poiseuille equation (s = 6Qu /wH2)[17] to determine the strength of the fluid shear force used in this experiment. Among these parameters, Q represents the liquid flow rate, u represents the liquid viscosity coefficient (UDMEM=0.03 dynes /cm2 [18]), w represents the width of the fluid chamber (w=3.0 cm), and H represents the height of the fluid chamber (2.0 cm). During the experiment, the fluid shear stress was determined by adjusting the liquid flow in the formula. Establish 5 shear force gradients of 0 dyn/cm2, 6 dyn/cm2, 12 dyn/cm2, 18 dyn/cm2, 24 dyn/cm2, and duration gradients of 0 min, 30 min, 45 min, 60 min, and 90 min, orthogonally. Each gradient was used to stimulate 10 thousand chondrocytes separately (repeat experiment n=3). The cells stimulated by shearing force were transferred to T25 culture flask and cultured in 37℃ 5% CO2 environment for 24h.
Cell proliferation curve
The cell proliferation ability was determined by the Cell Counting Kit-8 (CCK-8, GlpBio Technology, USA). The cell collection, measurement and data analysis methods were carried out with reference to the standard instructions. Briefly, remove 100 μL of cell suspension to 96 well plate, and add 10 μL CCK-8 solution. Incubate the culture plate in an incubator for 2 hours, and measure the absorbance at 450 nm using a microplate reader. Calculate the increase in cell number based on the previously established standard proliferation curve. All data were calibrated with the proliferation capacity of the 0 dyn/cm2, 90 min group to obtain the relative proliferation percentage.
RT-qPCR and Western Blot
The expression of genes related to cartilage proliferation and development was determined by reverse transcription-qPCR and Western Blot. The cells were collected by centrifugation, the supernatant was removed, and the total RNA was extracted using the TRIzol Total RNA Extraction Kit (BioTeke, China), and the Advantage RT-for-PCR Kit (TAKARA, Japan) was used to reverse transcription of RNA into cDNA. Subsequently, quantitative PCR was performed using 12 pairs of primers (Table 1) shown in Table 1 with Premix Ex Taq (TAKARA, Japan), according to PCR temperature in official instruction and annealing temperature corresponding to each pair of primers. PCR reaction was conducted and fluorescence signal was collected with a Thermal Cycler Dice Real Time System III (TaKaRa, Japan), while GAPDH was used as an internal reference for the experiment and the delta-delta CT (ΔΔCt) of each sample were calculated[19].
Table 1
The primer pairs used in qPCR
Gene ID
|
Ref.Accession
|
Primers
|
Product length
|
ACAN
|
L07049.1
|
F: 5'- CAGATGGCACCCTCCGATAC -3'
|
151 bp
|
R: 5'- GACACACCTCGGAAGCAGAA -3'
|
COL1A2
|
NM_007743.3
|
F: 5'- CCCAGAGTGGAACAGCGATT -3'
|
449 bp
|
R: 5'- TTTTGGAGCAGCCATCGACT -3'
|
PTGS2
|
NM_011198.4
|
F: 5'- CATCCCCTTCCTGCGAAGTT -3'
|
178 bp
|
R: 5'- CATGGGAGTTGGGCAGTCAT -3'
|
Sox9
|
FJ790141.1
|
F: 5'- CCACCATCTGCATCCCTACC -3'
|
983 bp
|
R: 5'- GGTCAACTGAAGCGGAGTGA -3'
|
Runx2
|
DQ458792.1
|
F: 5'- AAGCCACAGTGGTAGGCAGT -3'
|
604 bp
|
R: 5'- GCCACTTGGGGAGGATTTGT -3'
|
PGE2
|
BC004846.1
|
F: 5'- ATACTTGCCACCCAACGACC -3'
|
451 bp
|
R: 5'- GGAGTTCTGATGGTCAGCGT -3'
|
col10a1
|
NM_009925.4
|
F: 5'- AAGGCCATGAATGACCAGGG -3'
|
183 bp
|
R: 5'- TGTTCGGTACACGTTGGGAG -3'
|
RhoA
|
JN971019.1
|
F: 5'- CTTGCTAGCCCCAAGACACA -3'
|
973 bp
|
R: 5'- CTGGAATGCCATGGTTCCCT -3'
|
LIMk1
|
NM_010717.3
|
F: 5'- TGCGGCCTCTCTCTTATCCA -3'
|
375 bp
|
R: 5'- ACAAGATGAGGCACCCAGAC -3'
|
Cofilin
|
D00472.1
|
F: 5'- TGCACCCCTCAAGAGCAAAA -3'
|
375 bp
|
R: 5'- ATACGGAGTAGGGGTGTCGTT -3'
|
ERK5
|
AB019373.1
|
F: 5'- GCGCATTAAGGAGGCCATTG -3'
|
682 bp
|
R: 5'- AGGAGTACTAGTGGGCTGGG -3'
|
FAK
|
NM_007982.2
|
F: 5'- AAAATCCAGCCAGCTCCTCC -3'
|
435 bp
|
R: 5'- CACGGGCTACAGAGGCTAAG -3'
|
Another set of harvested cells were subjected to western blot analysis for quantification of ERK5, PGE2, COX-2 and phosphorylated ERK5 (pERK5). The primary antibodies were Anti-ERK5 antibody (ab40809, Abcam, USA), Anti-PEG2 antibody (ab2318, Abcam, USA), Anti-COX2 antibody (ab15191, Abcam, USA) and Anti-GAPDH antibody (ab8245, Abcam, USA). After each incubation step, the NC films were washed with TBST (pH=7.0, 0.5% Tween-20) for 30 min, and incubated with the enhanced HRP-DAB substrate color development kit PA110 (Beyotime Biotechnology, China) for 1min. Images were captured with an AlphaImager HP imaging system (ProteinSimple LLC., USA). The relative quantification of each sample in the western blot was normalized to that of GAPDH.
Enzyme linked immunosorbent assay (ELISA)
The cell mass was dissociated by trypsinization, and the suspended cells were collected by centrifugation. The total cell density was counted by a Countstar BioTech cell counter (Countstar, China). Take 20 thousand cells and add 200 μL RIPA cell lysate (Beyotime, China) for cell lysis. Marker proteins related to cartilage differentiation (Col1A2, GAG, SOX9) was quantitative determined through ELISA. Rat collagen type I α2 (Col1A2) was tested with Rat COL1A2 ELISA detection kit (JL34211-48T, Jonln Bio, China). The GAG protein content in the cell samples was detected by the rat glycosaminoglycan (GAG) ELISA detection kit (SenBeiJia Biological Technology, China). SOX-9 ELISA kit (BNCC, China) was used for the quantitative experiment of SOX-9 protein. The pipetting, color development and data conversion of the kit were performed according to the kit instructions and standard curves.
siRNA oligos and transfection
The expression level of ERK5 protein is knocked down by RNA interference (RNAi). Three small interfering RNAs (siRNA) were designed for the 573th bp, 648th bp, and 789th bp sites of Rat ERK5 mRNA through the online software Ambion siRNA target finder (www.ambion.com), and synthesized chemically from Sangon Biotech, China. 25 pmol of each siRNA oligo was transfected to 10 thousand chondrocytes pre-cultured in 6 well plate with Lipofectamine™ RNAiMAX Transfection Reagent (Thermo Fisher, USA) according to user manual. A set of mimic siRNA (Invitrogen Silencer Negative Control No. 1 siRNA, Thermo Fisher, USA) that does not affect the expression of ERK5 was set up as a negative control.
Statistical analysis
All data in the experiment were recorded in the format of mean ± standard deviation (SD). The statistical analysis software is Origin 2018 (OriginLab Corporation, Massachusetts, USA). One-way analysis of variance (ANOVA) is used to compare whether there is a difference between multiple groups; *P <0.05 indicates a statistical difference, **P <0.01 indicates a Very obvious difference. In terms of experimental parallel testing, rat primary chondrocytes were derived from 4 parallel experimental animals and used after mixing. In the grouping of shear force model and cell experiment, each group set up 3 parallel groups (n=3); CCK-8 cell count, qPCR, Western Blot and ELISA set up 5 parallel groups (n=5).