Cell culture

The lumbar IVD tissues of 8-week-old male rats were obtained and processed for further experiments. The tissues were initially treated with 0.2% pronase at 37 °C for 1 h, followed by 15 min of exposure to 2.5% collagenase II at the same temperature. The NPCs were then collected from the digested tissues and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. The cells were maintained at 37 °C, 5% CO2, and 20% O2, with the medium being changed every two days until they reached 80–90% confluence12,13.

Cell viability analysis

We seeded 96-well plates with 2 × 103 cells per well for 24 h before adding increasing concentrations of phillyrin (0, 10, 20, 40, 80, 160 and 320 µM in DMSO; Med Chem Express, MCE, China). Following phillyrin treatment, the cells were cultured in 10 µL of CCK-8 reagent(Med Chem Express, MCE, China) in fresh complete media for 1 h at 37 °C. In the simulated control, untreated cells were compared to cells in control medium and treated with CCK-8 reagents in the blank control. In this study, optical density readers were used to measure the absorbance at 450 nm.

Western blot analysis

We utilized RIPA buffer containing 1% proteinase inhibitor and 1% phosphotransferase inhibitor (Cwbio, Jiangsu, China) to lyse the NPC samples. Total protein extraction was then carried out through centrifugation.

Subsequently, western blot analysis was performed on each sample, utilizing 20 µl of protein. To account for variations in protein weights, the preboiled protein samples and loading buffer went through electrophoresis for 90 min on a 10% or 12% SDS-PAGE gel. The gel particles were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were cut horizontally to identify proteins of various sizes. For antibody detection, we employed the following antibodies at the indicated dilutions: anti-MMP3 (1:1000), anti-MMP9 (1:1000) from Abcam; anti-p38 (1:1000), anti-p-p38 (1:1000) from CST; anti-MMP13 (1:1000), anti-Aggrecan (1:1000), anti-Bcl2 (1:1000), anti-Bax (1:1000), anti-Caspase-3(1:1000), anti-p65 (1:1000), and anti-p-p65 (1:1000) from Immunoway; anti-GAPDH (1:5000) and anti-Tubulin (1:5000) from Cwbio.

Following incubation with the primary antibodies, the membranes were washed using TBST and subsequently incubated with secondary antibodies for one hour. Following three rounds of washing, the membranes were incubated with secondary antibodies for one hour. Signals were detected and analyzed using an ECL imager (Syngene G: BOX ChemiXT4, United Kingdom).

Immunofluorescence analysis

The cells underwent three washes with PBS, were then fixed with 4% paraformaldehyde, and washed with 0.5% Triton X-100 for 5 min. To prevent any nonspecific binding, a solution of bovine serum albumin (1%) was applied and left for 1 h at 37 °C. Following this step, the cells were washed with PBS and subsequently incubated with primary antibodies overnight at 4 °C. The cells were subjected to three consecutive washes with PBS, after primary antibody incubation, then incubated with the secondary antibody (Alexa Fluor® 488/594 conjugated, at a concentration of 1:100) for 1 h. Additionally, DAPI staining was performed for 5 min at 37 °C. A microscope, specifically the Olympus BX63 (NY, USA), was used to capture images after three further washes with PBS.

ROS flow cytometry

In order to assess the presence of intracellular reactive oxygen species (ROS) at the individual cell level, we utilized a staining technique. Specifically, we treated the cells with a solution containing 10µM DCFH-DA in DMEM lacking fetal bovine serum (FBS) for 30 min. This treatment occurred at 37℃, within an environment enriched with 5% CO2. Subsequently, we detached the cells by applying a trypsin solution and subjected them to centrifugation in 1.5 ml tubes at a force of 500 × g for 4 min. Following the removal of the supernatant, we resuspended the resulting cell pellet in PBS (phosphate-buffered saline). By employing a specialized instrument, we collected flow cytometry data, which was then analyzed utilizing Flow Jo software to determine the geometric mean fluorescence intensity. For comparison, we included unstained and untreated samples as controls in our analysis.

Surgical procedures

Approval for all animal experiments conducted in this study was obtained from the Institutional Animal Care and Use Committee (SYSU-2022-G0104) at Sun Yat-Sen University. Sprague–Dawley rats, were procured from Sun Yat-sen University’s Laboratory Animal Center, and they were free access to food and water for a week before to the experiment. They were randomly divided into three distinct groups, namely the control, IDD, and phillyrin groups. To ensure compliance with ethical guidelines, rats in the IDD and phillyrin groups were administered 2% pentobarbital (50 mg/kg) for anesthesia and subsequently punctured using a 21G needle through the skin. Following a 30-second interval, 10µM of phillyrin was precisely injected into the discs of rats within the phillyrin group, while an equal volume of DMSO was injected into the discs of rats within the IDD group. The experiment was conducted in a controlled environment, ensuring the rats’ well-being by providing them with ample water and food and maintaining suitable temperature, humidity, and light conditions.

Effect of phillyrin on an ex vivo IVD culture model

The IVDs, having intact endplates, were extracted from the rats and subjected to culture in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. As per prior investigations, the specimens were cultured under conditions of 37 °C and 5% oxygen while maintaining optimal humidity levels [14]. During the culture process, IL-1β and phillyrin were administered to the IVDs. The medium was routinely replaced every 48 hours.

Magnetic resonance imaging method

4 weeks after surgery, we utilized MRI to investigate disc degeneration in rats. Intraperitoneal injection of an overdose of pentobarbital (50 mg/kg) was employed to euthanize each rat. The rat tails were then examined using a 3.0 T clinical magnet (Philips Intera Achieva 3.0MR) to detect the signal and structures in sagittal T2-weighted images [15]. We evaluated the degeneration of intervertebral discs according to the system [16, 17].

Histological and immunohistochemical (IHC) staining

The specimens were preserved by immersing them in a solution of 4% paraformaldehyde for 48 h. Following this, a decalcification process lasting over 30 days was performed, after which the specimens were embedded in paraffin. In order to evaluate disc degeneration, the sections were subjected to routine staining techniques utilizing Haematoxylin-eosin stain and Safranin O-fast green-staining. Subsequently, the sections underwent incubation with primary antibodies targeting MMP3 (1:200) and BAX (1:200), and the disc samples were then exposed to secondary antibodies. ImageJ software was used in visualizing the images, as well as calculating the histology scores based on an established grading system [18, 19].

Statistical analysis

We repeated each experiment at least three times, and expressed the results as the mean ± SD. Statistical analysis was conducted using GraphPad Prism 8 software (La Jolla, CA, USA). The Student’s t-test and two-way ANOVA were employed to assess statistical significance, with P values below 0.05 considered statistically significant.