Animals and ethics statement. A total of 96 Sprague-Dawley male rats (age, 3 months; weight, 250 ± 0.05 g) were obtained from the Experimental animal center of Yan'an University. The rats were randomly divided into four groups, namely the control, KOA, KOA + lentivirus-negative control (len-NC) and KOA + len-miR-27b-3p groups. Each group consisted of 24 rats, whose tissues and cells were used for reverse transcription-quantitative PCR (RT-qPCR), histological examination, western blot analysis and ELISAs. The rats were housed under a 12-h light/dark cycle. All animal experiments were approved by the Ethics Committee of Yan'an University (approval no. SXYA201901) and lasted for 2 years from design to completion.
Establishment of KOA rat model. A total of 96 rats were randomly divided into the control group, the KOA group, the KOA + len-NC group and the KOA + len-miR-27b-3p group (24 mice/group). The KOA model was established by partially removing the medial meniscus of the right knee. Prior to surgery, rats were anesthetized with 1% sodium pentobarbital (40 mg/kg), while iodine was applied as a disinfectant. Subsequently, a lateral parapatellar skin incision was performed with ophthalmic scissors along the right knee joint at 1 cm proximal to the patella under a microscope. The patella was turned outward and secured to expose the joint capsule. The medial meniscus and tibial ligament were disconnected and the partial medial meniscus was then removed. Subsequently, the patella was placed in its original position and sealed with a scalpel. Following surgery, rats were intramuscularly injected with 1.5 mg/kg ampicillin for 3 days to prevent infection. In addition, to relieve surgery-induced pain, rats were intraperitoneally injected with 0.1 mg/kg tramadol upon awakening from anesthesia. The rats were allowed to fully recover from anesthesia in warmed cages and were closely monitored for pain, infections and other surgery-related complications. After 8 weeks, all rats were euthanized by CO2 inhalation-mediated asphyxia with 10–30% volume/min gas displacement flow rate. Briefly, the rats were placed in a special closed container with air or oxygen supply. Subsequently, the concentration of CO2 was increased until cardiac arrest occurred. Finally the knee tissues were harvested for further analysis.
Chondrocyte culture and transduction with oligonucleotides. To isolate chondrocytes, the knee cartilage from 3-month-old Sprague Dawley rats was excised under aseptic conditions and 1.0 cm of the knee joint meniscus from rats in each group was collected, cut into 1 mm×1 mm sized pieces, add 0.2% I collagen enzyme with 10x body mass for 4 h digestion at 37℃ with a volume fraction of 5% CO2 saturation humidity.By 120 mesh copper mesh overfiltration, absorb filter fluid, 1500g centrifugation 5min.The obtained cells are based on a DMEM medium containing 10% fetal bovine blood, at 37℃, with a volume fraction of 5% CO2 Incubator. Change fluid every 3d once. When cells reach 80% ཞ 90% fusion, trypsin digestion 1:3 for transmission culture.Subsequently, the cartilage tissue was digested with 0.4% sterile II collagen enzym (12 ml/g of cartilage tissue) at 37˚C for 90 min followed by centrifugation at 1,400 g for 5 min. Following removal of the supernatant, the cartilage tissue was digested overnight with 0.025% sterile collagen II (12 g/ml of cartilage tissue). Reserve cartilage on liquid, centrifugal, frozen. Following counting (activity of fetal blue staining cells < 90%), cells were seeded into a 6-well plate at a density of 1x105 cells/ml in DMEM/F12 supplemented with 10% FBS and placed in an incubator at 37˚C and 5% CO2.
Once cell confluence reached 70–80%, cells were divided into four groups, namely the control, KOA, KOA + len-NC and KOA + len-miR-27b-3p groups. Cartilage chondrocytes in the KOA + len-NC and KOA + len-miR-27b-3p groups were transduced with the corresponding lentiviral particles for 24 h using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The transduction efficiency was verified by RT-qPCR.
RT-qPCR. To assess the mRNA expression levels of BDNF, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in cultured chondrocytes and those of miR-27b-3p in cartilage tissues, RT-qPCR was carried out as previously described. Briefly, chondrocytes and cartilage tissues were harvested and total RNA was extracted using TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.). The concentration of the isolated RNA was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, the cDNA of miR-489-3p was synthesized using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.), while that of BDNF, iNOS and COX-2 was synthesized using a PrimeScript RT Master mix (Takara Bio, Inc.). qPCR(Applied Biosystems, USA) was performed using a MiScript SYBR® Green PCR kit (Thermo Fisher Scientific, Inc.). The expression levels of BDNF, iNOS and COX-2 and miR-27b-3p were normalized to the internal reference genes, GAPDH and U6, respectively. The relative gene expression was calculated using the 2−ΔΔCq method. The primer sequences used are listed in Table I.
Western blot analysis. The protein expression levels of caspase-3, caspase-9, iNOS, COX-2, BDNF, TrkB, CREB and phosphorylated (p)-CREB in cultured chondrocytes and those of BDNF, p-TrkB and p-CREB in cartilage tissues were determined by western blot analysis. Total proteins were extracted from cells and tissues with RIPA lysis buffer (Beyotime Institute of Biotechnology). The protein concentration in each group was measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). Subsequently, the protein extracts were separated by SDS-PAGE and were then transferred onto PVDF membranes. Membranes were blocked with 4% solution of BSA for 2 hrs and incubated with primary antibody at 4 ℃ overnight. Primary antibody was removed and the membranes were further incubated with respective secondary antibody for 1 hr at room temperature. Protein bands were visualized using ECL assay kit (Bio-Rad, USA). Image J software was used for the quantification of band densities.
ELISA. The samples for ELISA were prepared according to the manufacturer's instructions using the corresponding ELISA kits (DEIA048V). Briefly, 1 cm of the knee joint meniscus was removed from rats in each group. Following repeated washings with sterile PBS, the cartilage tissues were immersed in oxidizing acid aqueous solution for sterilization three times (30 min each time). Subsequently, the cartilage tissues were cut into small pieces with sterile ophthalmic scissors on a sterilized ultra-clean working bench, rinsed repeatedly in sterile distilled water, soaked in 3% hydrogen peroxide solution for 30 min and washed again with sterile distilled water for three times (30 min each time). Then, the cartilage pieces were supplemented with three volumes of sterile tri-distilled water and smashed into an articular cartilage tissue homogenate using an ultrasonic tissue pulverizer at 4˚C. The homogenate was mixed well with 10 volumes of sterile tri-distilled water in a hypotonic environment followed by 4–5 cycles of freezing at -20˚C and slowly thawing at room temperature. Following the above freezing-thawing cycles, the remaining cells were ruptured and the homogenate was centrifuged at 2,000 g for 30 min to harvest the supernatant. The isolated supernatant was centrifuged at 3,000 g for 30 min and the resulting supernatant was further subjected to centrifugation at 4,000 g for 30 min. The above steps were repeated for five cycles. Finally, the supernatant containing small particles was centrifuged at a high speed of 10,000 g for 30 min and the precipitate was used to prepare a decellularized nano-scale human articular chondrocytes-extracellular matrix (ECM) homogenate. The homogenate was transferred into Eppendorf tubes and stored at low temperature for subsequent experiments. The levels of nitric oxide, PGE2, TNF-α, IL-6, Bax, Bcl-2 and IL-6 in the above homogenates were determined using the ELISA method on a fully automatic biochemical instrument to detection of the supernatant of meniscus chondrocytes.
Flow cytometry. To evaluate the role of miR-27b-3p on chondrocyte apoptosis, a flow cytometry assay was carried out using a FITC-Annexin V apoptosis detection kit (Beyotime Institute of Biotechnology). Briefly, chondrocytes were harvested, rinsed in cold PBS and labeled with FITC-Annexin V solution for 4 min on ice. Then, the cells were rinsed in coupling solution and supplemented with a PI solution followed by incubation in the dark. Finally, the cell apoptosis rate was determined using a Fortessa flow cytometer (BD Biosciences) and the acquired data were analyzed with FlowJo software (FlowJo LLC10).
Dual luciferase reporter assay. To verify whether BDNF could be directly targeted by miR-27b-3p, a dual luciferase reporter assay was carried out. The binding sites of miR-27b-3p on the BDNF 3'-UTR were predicted using the TargetScan database(https://www.mirnet.ca/miRNet/home.xhtml). Subsequently, the BDNF 3'-UTR was subcloned into the p-MIR-GLO reporter vector (Promega Corporation) to construct the BDNF-wild-type (WT; concentration, 500 µg/µl) and BDNF-mutant (MUT; concentration, 500 ng/µl) plasmids, encompassing the WT and MUT binding sites of miR-27b-3p, respectively. The cultured chondrocytes were co-transfected with miR-27b-3p mimics/NC and BDNF-WT/BDNF-MUT using Lipofectamine 2000. Following transfection for 48 h, the relative luciferase activity was measured using a Dual Luciferase Reporter assay system (Promega Corporation).
Histological analysis. The cartilage tissues were harvested, fixed with 4% paraformaldehyde, embedded into paraffin and cut into 5-µm thick sections. The sections were then stained with Safranin O staining and images were captured under a microscope (Nikon Corporation).
Statistical analysis. All data were analyzed using GraphPad Prism 6.0 (GraphPad Software, Inc.). The results are expressed as the mean ± SD. The differences among multiple groups were analyzed using a one-way ANOVA followed by a Tukey's multiple comparison post-hoc test. P < 0.05 was considered to indicate a statistically significant difference.