Animal models

All experiments were approved by the Ethics Committee of Zhengzhou University (2023-KY-0305-002). Fifty male Sprague-Dawley rats (8 wk) weighting 200–250 g were purchased from Shanghai Slac Laboratory Animal Co., Ltd. (Shanghai, China) (SCXKI (Hu) 2017-0005). All rats were maintained 1 wk to understand specific pathogen-free conditions on a 12-h light/cycle with temperature 20–25℃ and free access to food and water. Forty-two rats were randomly selected to construct models of IVDD according to Zhao et al. (2021). Briefly, the rats were anesthetized 3% sodium pentobarbital (30 mg/kg). The IVDD rat models were constructed by surgically excising the paraspinal musculature and the supraspinous and interspinous ligaments from neck 2–7 (C2–C7). Specific operation steps can be found in the previous research (Yu et al. 2020). There were two rats sacrificed during modeling. The other eight rats were treated with sham surgery as the control group.

Ginsenoside Rg1 (> 98%) were purchased from the National Institutes for Food and Drug Control  (Beijing, China). A total of 40 rats with cervical IVDD were successfully constructed, and randomly divided into four groups (10 rats): IVDD group (same amount of saline), low-dose (L-Rg1) group (intraperitoneal injection of 20 mg/kg/d ginsenoside Rg1), medium-dose (M-Rg1) group (intraperitoneal injection of 40 mg/kg/d ginsenoside Rg1), and high-dose (H-Rg1) group (intraperitoneal injection of 80 mg/kg/d ginsenoside Rg1). After 8 wk of administration, all of the rats were euthanized, and the IVD tissues were collected for the subsequent experiments.

Hematoxylin-eosin (HE) staining

Firstly, the soft tissues surrounding the IVD tissue were removed. Then, the IVD tissue of rats was fixed in 4% paraformaldehyde for 48 h, and decalcified in 15% ethylenediaminetetraacetic acid (EDTA) solution. After dehydrated with gradient ethanol, the tissues were embedded whole in paraffin and cut into the slices of thickness 5 μm. After deparaffinization, the sections were stained with hematoxylin for 5 min staining and eosin solution for 30 s. Then, the sections were dehydrated with ethanol, cleared with xylene, and sealed with neutral resin. The morphology of IVD tissues was observed under a microscope (Olympus, Tokyo, Japan), with at least three sections for each rat.

Safranin O-fast green staining

The IVD samples embedded in paraffin were cut into sections of 5 μm as described previously: After deparaffinization, the sections were stained with the Weigert solution for 3 min. After rinsing with tap water for 10 min, the sections were stained with fast green for 3 min, and safranin O for 5 min. Sections were mounted with neural resin after they were rinsed with ethanol and xylene. The morphology of IVD tissues was observed under a microscope (Olympus), with at least three sections for each rat. And the degree of cartilage degeneration was determined by the modified Mankin score, according to Go et al. (2022). Three visual fields were selected to calculate the averaged Mankin score for each section.

Immunofluorescence

After being dewaxed and rehydrated, the sections were placed in boiling citric acid buffer for 10 min to retrieve the antigen and then incubated in 3% H2O2 at 37℃ for 15 min to block endogenous peroxidase activity. After being blocked with 5% goat serum for 1 h, the sections were incubated with primary antibody against NF-κB p65 (1:200, Abcam, Waltham, MA) at 4℃ overnight. And the secondary antibody (FITC-conjugated) was incubated at 37℃ for 1 h. The sections were mounted with antifade mounting medium with DAPI. And the sections, at least three sections for each rat, were observed and the fluorescent images were obtained using a fluorescence microscope (DM2500, Leica, Wetzlar, Germany).

Rat NP cell isolation, culture, and identification

According to the previous study (Yu et al. 2020), SD rats were euthanized with 3% pentobarbital sodium. After removing the cervical skin, the cervical intervertebral disc of the rats was separated under the anatomical microscope, and immersed and disinfected in 75% ethanol for 5–10 min. Then, the gel-like NP tissue from the cervical intervertebral disc was separated and washed with sterile PBS twice. Then, NP tissues were cut into small fragments, and released and isolated with 0.25% trypsin for 10 min and 0.2% collagenase II for 5 h at 37℃ in DMEM/F12 containing 0.1% of fetal bovine serum and 1% of penicillin-streptomycin. After filtration by sterile cell sieves, the cells were washed with PBS and collected by centrifugation (1000 r/min for 5 min).

After isolation, NP cells were resuspended in the DMEM/F12 medium with 10% FBS (Gibco, Billings, MT), and 1% penicillin-streptomycin and cultured in an incubator at 37℃ with 5% CO2. The medium was changed every 3 d. The NP cells from 2 to 3 generations were used for the following experiments. And the morphology of rat NP cells was observed under an inverted microscope and identified by toluidine blue staining.

Experimental groups

After 2–3 passages, NP cells were seeded in 6-well plates and divided into five groups. Different experimental groups were given different treatments. The control group is the group without any treatment; for the IL-1β group, NP cells were stimulated with IL-1β (10 ng/mL) for 24 h to simulate IVDD environment (Huang et al. 2022). For the 20 µmol/L Rg1 group, NP cells were co-treated with IL-1β and 20 µmol/L ginsenoside Rg1 in the DMEM/F12 medium. For the 50 µmol/L Rg1 group, NP cells were co-treated with IL-1β and 50 µmol/L ginsenoside Rg1 in the DMEM/F12 medium. For the 100 µmol/L ginsenoside Rg1 group, NP cells were co-treated with IL-1β and 100 µmol/L Rg1 in the DMEM/F12 medium. After 24 h of culture at 37℃ with 5% CO2, further experiments in cells were conducted.

Cell counting kit-8 (CCK-8) assay

CCK-8 assay (Solarbio, Beijing, China) was used to determine the effect of Rg1 on the cell viability of NP cells. For the detection of ginsenoside Rg1 and IL-1β cytotoxicity, the NP cells with or without IL-1β induced were seeded in a 96-well plate (5 × 103/well) with three replicate wells per group and intervened by various concentrations of ginsenoside Rg1 (0, 10, 20, 50, 100, 200 μmol/L). After being cultured for 24 h, the cells in each well were incubated with CCK-8 solutions for 4 h at 37℃. Then, the absorbance was measured at 450 nm on an automatic microplate reader (ELX80, Bio-Tek, Winooski, VT). As for the cell proliferation, the cells were seeded in a 96-well plate (2 × 104/well) with three replicate wells per group and cultured for 24 h, 48 h, and 72 h. Then, 10 μL of the CCK-8 solution was added in each well, and incubated at 37℃ for 4 h. The absorbance was checked as above. The experiment was repeated three times. And the proliferation curve was plotted with time point as the X-axis and A value as the Y-axis.

Flow cytometry assay

To analyze the effect of ginsenoside Rg1 on apoptosis, Annexin V/PI double staining by flow cytometric analyses was performed. Briefly, NP cells were incubated in 6-well plates at 5 × 104/well and cultured for 48 h after treatment with drugs. Then, NP cells were collected by routine digestion and centrifugation, and resuspended by binding buffer solution, and then subsequently stained with Annexin V-FITC and PI according to the protocols of Annexin V/PI Apoptosis Detection Kits (Solarbio, Beijing, China). Then, the cell apoptosis was assessed by FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ). Total apoptotic rate = early apoptotic rate + late apoptotic rate. The experiment was repeated three times.

Quantitative real-time PCR (qPCR)

The total RNA from NP tissues and NP cells of each group was isolated using TRIzol (Invitrogen). Then, cDNA was synthetized by a reverse transcriptase kit (TaKaRa, Kyoto, Japan) according to the manufacturer’s protocols. qRT-PCR detection was conducted on 7900 HT Fasting Real-Time PCR System (Applied Biosystems) with SYBR Green Mix Kit and primers of IL-1β, IL-6, TNF-α, aggrecan, collagen II, MMP3, and GAPDH. The relative expression was quantitated using the 2−ΔΔCt method, with GAPDH as normalized reference. The experiment was repeated three times.

ELISA

The contents of IL-1β, TNF-α, and IL-6 in NP tissues and NP cells were detected by ELISA kits (Solarbio, Beijing, China). The operation was performed in strict accordance with the ELISA kit instructions. The experiment was repeated three times.

Western blot

The NP tissues and NP cells cultured of 24 h were collected in each group. The total protein was extracted by cell lysis, and the protein concentration was determined by the BCA method. For immunoblotting, protein samples were separated by SDS-PAGE, and transferred into PVDF membranes (Merck Millipore, Billerica, MA). The membranes were blocked with 5% skim milk in TBST solution at room temperature for 2 h. The primary antibodies were incubated with different membranes at 4℃ overnight. The secondary antibodies, HRP-conjugated goat anti-rabbit or goat anti-mouse IgG, were incubated for 1 h. The immunoreactive proteins were visualized by electro-chemiluminescence detection system and the relation expression was calculated using ImageJ with GAPDH as the internal reference. The experiment was repeated three times.

Statistical analysis

All experiments were executed independently and repeated at least three times. All results were analyzed using statistic software SPSS 21.0. The data were demonstrated as means ± standard deviation, and Student’s t test or one-way analysis of variance (ANOVA) analysis was used to determine the significant differences between two groups or among multiple groups. p < 0.05 was considered statistically significant.