Sample collection

The degenerated cartilage tissues were obtained from the knee joints of 40 patients (age, 57.54 ± 8.9 years) who underwent total knee arthroplasty. The normal cartilage tissues were collected from 40 volunteers (age, 38.94 ± 4.2 years) with the femoral neck fracture with no history of rheumatoid arthritis or OA. The body mass index (BMI) of all selected patients is 28.5 ± 3.1 kg/m2 which meets the obesity diagnostic criteria of greater than or equal to 23 kg/m2. These cartilage samples were immediately snap-frozen and stored in the liquid nitrogen for further experiments. The present study was approved by the Clinical Research Ethics of China-Japan Union Hospital of Jilin University.

Animals and experimental group

Seven-week-old C57BL/6 male wild-type (WT) mice were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). After one week of acclimatization, the mice were randomly divided into four groups of 12 mice. Three test groups were fed with a diet composed of 60.0% kcal from fat, 20.0% kcal from carbohydrates, and 20.0% kcal from protein to constitute the high-fat diet (HFD) groups, and the remaining group was fed with a composed of 10.0% kcal from fat, 70.0% kcal from carbohydrates, and 20.0% kcal from protein to constitute the normal control diet (NCD) group from week 1 to week 21. The HFD and NCD used in this study were provided by HFK Bioscience Co., LTD (Beijing, China). After the HFD induction for 12 weeks, the mice were randomly divided into three groups: HFD + Sham group (surgery with an opened knee-joint capsule without ACLT + MMx) and two OA groups (HFD + OA + Vechicle and HFD + OA + Sal B; surgery with ACLT + MMx). One day after the operation, the mice in HFD + OA + Sal B group were received an intraperitoneal-injection of Sal B (25 mg/kg) daily for 10 weeks, whereas the mice in HFD + OA + Vechicle group were injected with DMSO. Mice were housed in chambers with natural light at controlled temperature of 24 ± 1 °C and 40–60% humidity. Body weights were measured once a week for a total of 21 weeks, and the widths of the knee joints were measured weekly by calipers from 13 to 21 week. Mice were euthanized with CO2 at the end of 20 week. Immediately, blood samples were collected and centrifuged for 30 min at 10,000 × g at 4 °C. Then, the obtained supernatant was stored at − 80 °C for further experiments. And the knees of mice were dissected after all tests were completed.

All of the experimental procedures involving animal care and use met the Guidelines set forth by the Chinese National Institutes of Health, and was approved by the local Institutional Animal Care Ethics Committee for animal studies at China-Japan Union Hospital of Jilin University.

Osteoarthritis animal model

The surgery with ACLT/MMx were performed to establish the rat model of OA as described by period studies [31]. After anesthetization with 3% pentobarbital sodium (Tocris, Avonmouth, UK), the hair on the right knee was clipped. Right knee was subsequently exposed before an incision was made in the medial aspect of the joint capsule (anterior to the medial collateral ligament), then the anterior cruciate ligament was transected, and the medial meniscus was completely resected in a manner that did not injure the articular cartilage. Subsequently, the joint was irrigated with normal saline, the capsule was sutured with 4–0 chromic catgut, and the skin was closed with 4–0 nylon mattress sutures. And the mice were allowed to move, eat and drink freely after surgery. The control group (Sham group) received sham operations involving an arthrotomy but without transecting anterior cruciate ligament and removing medial meniscus.

Histological analysis

The collected knee joints were fixed in brown vials with 4% paraformaldehyde for 2 days, then decalcified with 10% ethylenediaminetetraacetic acid (EDTA, PH 7.2) for 4 weeks. After decalcification, the joints were embedded in paraffin blocks and sagittally cut into sections at a thickness of 5 mm. The sections were then dewaxed in xylene and hydrated with graded ethanol series. Hematoxylin/eosin (H&E) staining (Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China) was then used to examine the morphological changes. The histological examinations of cartilage were blindly evaluated according to the grading of Osteoarthritis Research Society International (OARSI) scoring system [11]. The cartilage matrix loss width, the cartilage degeneration score, the total and significant cartilage degeneration widths, and the zonal depth ratio of the lesions were specifically evaluated.

Cell culture and treatments

The mouse chondrocyte cell line ATDC5 was purchased from Shanghai Institute of Biosciences Cell Resource Center, Chinese Academy of Sciences. Cells were cultured in in Dulbecco Modified Eagle Medium/Ham Nutrient Mixture F12 (DMEM/F12; Gibco, Life Technologies, Carlsbad, CA) containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Gibco, Life Technologies). The cells were maintained in a humidified incubator with 5% CO2 at 37 °C. All these ATDC5 cells were used between the fifth and tenth passages.

Palmitic acid (PA) was purchased from Sigma-Aldrich. PA with a series of concentrations (100 µM, 200 µM, 300 µM, 400 µM and 500 µM) were used to treat ATDC5 cells for 24 h to stimulate inflammatory injury. Sal B (purity N98%) was purchased from Shanghai Winherb Medical Science Co., Ltd. (Shanghai, China), and dissolved in DMSO (≥ 99.7%, Sigma-Aldrich). ATDC5 cells were treated in the absence or presence of 25/50/or 100 μM Sal B for 24 h after PA treatment. DMSO (≥ 99.7%, Sigma-Aldrich) without Sal B was used as control group.

Cell transfection

The gene-overexpression vector (Ad-KCNQ1OT1) and the control vector (Vector) were purchased from GenScript Biotech Corp. (Nanjing, China). The small interfering RNAs against SIRT1 (SIRT1 siRNA) and negative control siRNAs (NC siRNA) were designed, synthesized and validated by Thermo Fisher Scientific (Waltham, MA, USA). MiR-128a-3p mimic, miR-128-3p inhibitor and the corresponding negative control (NC mimic and NC inhibitor) were designed and synthesized by GenePharma Corporation (Shanghai, China). All these plasmids and oligonucleotides were transfected into ATDC5 chondrocyte cells by using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA) following the guidelines of the manufacturer. At 48 h after transfection, cells were harvested for further study.

RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were extracted from treated ATDC5 cells and cartilage tissue using RNA pure Total RNA Fast Extraction Kit (Sangon, Shanghai, China), and reversely transcribed to cDNA by PrimeScript RT reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA). The quantitative analysis of KCNQ1OT1 and SIRT1 were performed by the SYBRTM Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with β -actin as an endogenous control. The levels of miR-128-3p were analyzed by SYBR PrimeScript miRNA RT‐PCR Kit (Takara Biotechnology, Dalian, China) with U6 as the internal reference. qRT-PCR was conducted on CFX96 qPCR machine (Invitrogen, Carlsbad, CA, USA) with the following steps: 10 min at 95 °C; 35 cycles of 15 s at 95 °C, 20 s at 60 °C and 15 s at 72 °C. Data were quantified using 2−ΔΔCt method [26].

Western blot analysis

Total proteins in treated ATDC5 cells or mice knee joints cartilage tissues were isolated using M-PER TM Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). BCA TM Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied for the quantification of total proteins. 25 μg of protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h at room temperature, these membranes were incubated with the following primary antibodies (Abcam, Cambridge, UK) at 4 °C overnight: cleaved-caspase-3 (ab32042, 1:500), p62 (ab91526, 1:1000), Bcl-2 (ab59348; 1:1000), LC3B (ab48394; 1:400), Bax (ab7902; 1:500), SIRT1 (ab12193; 1:2000) and β-actin (ab6276, 1:5000). After washing three times with PBS, the membranes were incubated with the secondary antibody of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (ab205718, 1:2000, Abcam) for 2 h at room temperature. Signals of proteins were captured using Bio-Rad ChemiDocTM XRS system (Bio-Rad Laboratories, Hercules, CA, USA) and the intensities of proteins were quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Cell viability assay

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay was applied to detect the cell viability. Cells were placed into the 96-well plate at a density of 5 × 103 cells/well and cultured for 24 h. After incubation in serum-free DMEM/F12 medium for 24 h, the MTT solution (5 mg/ml, 20 μl) was added to each well and incubated for 4 h in a culture environment with 5% CO2 at 37 °C. Then, the culture medium was removed and 100 μl DMSO was supplemented to each well for 10 min. Finally, the optical absorbance at 590 nm was measured by a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). All assays were performed in three triplicates.

Cell apoptosis analysis

Annexin V-FITC/PI apoptosis detection kit (Beijing Biosea Biotechnology, Beijing, China) was applied to detect the apoptotic cells. Briefly, ATDC5 cells were seeded into 6-well plate with 3 × 104 cells per well and exposure to different treatment or transfection. The treated ATDC5 cells were then washed twice with cold PBS and re-suspended in buffer. Subsequently, cells were stained using 10 μl Annexin V-FITC/PI solution for 15 min in darkness at room temperature. Finally, the stained cells were subjected to flow cytometry analysis using Guava EasyCyte flow cytometer (Beckman Coulter, Fullerton, CA, USA), and the data were analyzed by the FlowJo software (Treestar, Ashland, OR, USA).

Enzyme linked immunosorbent assay (ELISA)

For detecting the IL-6, TNF-α and leptin level in serum in vivo, blood samples of six mice per group were collected from the abdominal aorta, centrifuged at 1000 × g for 15 min, and then stored at − 80 °C. For in vitro assays, culture supernatant was obtained from 24-well plates after the indicated treatment. The levels of IL-6, TNF-α, PEG-2 and leptin were measured by using ELISA Kits (Abcam, Cambridge, MA) according to the manufacturers’ instructions.

Dual luciferase reporter assays

Online bioinformatic tools StarBase 2.0 (http://starbase.sysu.edu.cn/), RNA hybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) and DIANA tools (http://carolina.imis.athena-innovation.gr/diana_tools/web/) were used to predict the interactions of KCNQ1OT1 and miR-128-3p. The TargetScan (http://www.targetscan.org/vert_71/), miRDB (http://mirdb.org/) and RNA hybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid/) were used to predict the binding sites within miR-128-3p and SIRT1. The sequences of KCNQ1OT1 and SIRT1 3’-UTR that included the wild-type and mutant-type binding sites of miR-128-3p were subcloned into a pGL3 vector (Promega, Madison, WI) to create the luciferase reporter vectors WT- KCNQ1OT1, MUT- KCNQ1OT1, WT- SIRT1 and MUT- SIRT1, respectively. Then, HEK-293 cells were transfected with NC mimic or miR-128-3p mimic along with the constructed luciferase reporter vectors by using Lipofectamine® 2000. After 48 h transfection, the relative luciferase activity was measured by a Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Renilla signals were normalized to firefly signals. All experiments were performed in triplicate.

RNA immunoprecipitation (RIP) assay

RNA immunoprecipitation was performed using a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA). Briefly, ATDC5 cells were lysed in complete RIPA buffer containing a protease inhibitor cocktail and RNase inhibitor. Then, the lysates were precleared by centrifugation. The supernatants were incubated with RIP buffer supplemented with Anti-Ago2 antibody or negative control Anti-IgG beads (Millipore) overnight. After purifying the immunoprecipitated RNA in the magnetic beads, qRT-PCR analysis was subjected to detect the relative enrichment of KCNQ1OT1 and miR-128-3p.

Double-labeled adenovirus mRFP-GFPLC3 transfection and autophagy detection

ATDC5 cells were plated and cultured in confocal dishes for 4 days, and then transfected with mRFP-GFP-LC3 lentivirus (Han Heng Biology, China) according to the manufacturer’s protocol. After transfection, cells were washed with PBS twice and then fixed with 4% paraformaldehyde. Fluorescent images were observed by a laser confocal microscopy (Zeiss, Oberkochen, Germany, LSM 510). The number of yellow spots (overlay of mRFP and GFP) represents autophagic bodies and red spots (mRFP alone) represent autophagic lysosomes.

Transmission electron microscope (TEM)

After treatment, cells were digested with trypsin and centrifuged to obtain the cell masses. After removing the supernatant, cells were washed with PBS and centrifuged at 1500 r/min−1 at 4 °C for 5 min. The cell pellet was fixed with a precooled 2% glutaraldehyde solution at 4 °C for 2 h, and then post-fixed in 1% osmium tetroxide at room temperature for 2 h. After that, the cells were stained with 2% uranyl acetate solution for 2 h, dehydrated in 50%, 70%, 90% and 100% acetone, and then embedded in epoxy resin. The embedded block was cut into slices using ultramicrotome. After staining with saturated uranyl acetate and lead citrate, the slices were observed under a HITACHI, H-7500 Transmission Electron Microscope (HITACHI, H-7500, Japan).

Statistical analysis

The data is represented as mean ± standard error of mean (S.E.M.). The body weight was analyzed with a two-way analysis of variance (two-way ANOVA) followed by Dunnett’s test. The other comparisons were conducted using one-way ANOVA or Student’s test. Pearson’s correlation analysis was used to assess the correlation between KCNQ1OT1 and miR-128-3p expression. A significant difference was judged to exist at a level of P < 0.05.