IL-1β promotes ADAMTS enzyme-mediated aggrecan degradation through NF-κB in human intervertebral disc

The study was approved by the Shanghai First People’s Hospital Ethics Committee. All patients and their next of kin signed an informed consent form allowing the researchers to use IVD tissues obtained during spinal surgery. All donor patients underwent spinal fusion surgery, which requires the intervertebral disc to be resected. Patients were aged between 19 and 45 years old; mean age was 30.52 years old. Only patients aged between 18 and 45 years old were included in this study. According to the Pfirrmann grading scale [22], the discs were non-degenerative (Pfirrmann < grade III) and degenerative IVD tissue samples. All IVD tissues used in this study would have been discarded as medical waste and would not have been used in the patient’s treatment. Part of the tissues was stored in liquid nitrogen for later use; the second part was then directly sent to the cell culture laboratory super clean bench for primary culture of the nucleus pulposus cells. NP tissues were washed three times with phosphate-buffered saline (PBS; Gibco, USA), minced into small fragments, and digested in 0.25 % trypsin (Gibco, USA) and 0.2 % type II collagenase (Gibco, USA) and then placed in PBS for approximately 3 h at 37 °C in a gyratory shaker. Cells were filtered through a 70-μm mesh filter (BD, USA), and primary NP cells were cultured with growth medium (Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 Nutrient Mixture [DMEM-F12; Gibco, USA], 20 % fetal bovine serum [FBS; Gibco, USA], 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco USA)) in a 100-mm culture dish in a 5 % CO₂ incubator. The cells were treated in trypsin at approximately 80 % confluence and subcultured in a 60-mm culture dish (2.5 × 10⁵ cells/well). All experiments in this study used first- or second-generation cells.

Human non-degenerative NP cells were grown to confluence in 60-mm culture dishes, underwent serum starvation overnight to synchronize the cell cycles, and were stimulated with 0~20 ng/mL recombinant human IL-1β (Peprotech, London, UK) for 24 h, and the catabolic response and NF-κB activation were observed. To justify the effectiveness of the NF-κB inhibitor, cells were pretreated with various concentrations of BAY11-7082 (Sigma-Aldrich, St. Louis, MO, USA) (2.5 μM, 5.0 μM) for 1 h, followed by treatment for 48 h with IL-1β (10 ng/mL) and BAY11-7082. Cells were then harvested for messenger ribonucleic acid (mRNA) and protein analysis.

The first-generation NP cells were plated in flat-bottom 24-well plates (5 × 10³/well) and were fixed with 4 % paraformaldehyde, permeabilized with 0.2 % Triton-X 100 in PBS for 10 min, blocked with PBS containing 5 % FBS, and incubated with antibodies against p65 (1:200) at 4 °C overnight. As a negative control, cells were reacted with isotype IgG under similar conditions. After washing, the cells were incubated with anti-rabbit secondary antibody (Jackson, USA) at a dilution of 1:200 for 1 h at room temperature. Cells were imaged using a laser scanning confocal microscope (Olympus Fluoview, Japan).

Lentivirus (8 × 10⁸ TU/mL) packaging of green fluorescent protein (GFP) LV-shp65 and negative control (NC) were constructed in Genechem (Shanghai, China). Cells (1 × 10³) were plated onto 96-well plates 48 h before the LV-shp65 and negative control were added to infected NP cells at various volumes to determine the best MOI value at which concentrations have no virus toxicity effect on cells. Ninety-six hours after infection, the GFP gene expression was observed under a fluorescence microscope, and the infected cells were collected for silencing treatment. Then, cells were harvested for protein extraction at 5 days after viral transduction. The experiment was repeated three times.

Total RNA was extracted from the NP cells and NP tissues by TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The mRNA was analyzed with real-time polymerase chain reaction (PCR) using the ABI 7500 Fast real-time PCR system (Applied Biosystems, Carlsbad, CA). cDNA was then reverse transcribed (Thermo, USA) according to the manufacturer’s instructions. Real-time PCR reactions were done in triplicate in 96-well plates using a SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) with a final volume of 20 μL. All primers (Table 1, obtained from Sangon, Shanghai, China) were designed based on coding sequences. The cycle threshold values were obtained, and data were normalized to β-actin expression using the 2^−ΔΔCt method [23].

The SPSS 17.0 software (SPSS Inc., Chicago, USA) was used for statistical calculations. Student’s t test and analysis of variance were used for comparisons between the different groups. All experiments were repeated three times with cells from different IVD tissues, and for each experimental condition, the experiment was repeated three times; the data is represented as mean ± standard deviation. The findings were considered to be statistically significant when P < 0.05.