Sal003 was obtained from Selleck Chemicals (S7437, Houston, TX, United States). DMSO was used to dissolve Sal003. It was then stored at −20°C with a stock solution of 40 mM.

Cervical dislocation was performed in six-week-old male Sprague-Dawley (SD) rats. The rats were then rinsed with 75% ethanol for 30 min. We obtained NP tissues from their intervertebral discs (Co1–Co6) and soaked them in 1% collagenase II for 2 h. The cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovin serum and 1% penicillin-streptomycin under routine conditions (37°C with 5% CO²), followed by centrifugation (37°C, 300 × g for 5 min) and suspension.

The Cell Counting Kit-8 Assay (CCK-8 kit, E606335, Sangon Biotech Co. Ltd., Shanghai, China) was used to measure cell toxicity and proliferation. To determine cell toxicity, cells were cultured in 96-well plates by 1*10⁴ before they were administered with different doses of Sal003 (0, 1.25, 2.5, 5, 10, 20, and 40 μM) for 24 h. To ascertain proliferation, the culture of cells was performed in 96-well plates at 2.5 × 10³ with previous concentrations and were administered Sal003 for 24, 48, and 72 h. Fresh complete media containing 10 μL of CCK-8 reagent was added into plates according to specific time periods, and the cells were incubated at 37°C for 2 h. Absorbance at 450 nm (optical density values, OD) was assessed using an Infinite M200 Pro reader (Tecan Life Sciences, Männedorf, Switzerland).

NP primary cells were pre-administered Sal003 (5 μM) for 2 h following which Tg (1 μM) was used to stimulate cells for 24 h. Afterwards, extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer’s instructions. Complementary DNA (cDNA) was obtained from RNAs using a cDNA Synthesis Kit. Real-time quantitive PCR (RT-qPCR) was performed using the TB Green Premix Ex Taq Kit (Takara Bio) in a Real-Time PCR System. NCBI BLAST was used to design specific primer pairs. Primer information is provided in Table 1. The target gene expression was adjusted and normalized to that of β-actin. Relative gene expression levels were quantified using the comparative 2^−ΔΔCT method.

To determine changes in the matrix metalloproteinase (MMP) family, apoptosis, and long-term activated signaling at the protein level, primary NP cells were treated as described previously. After washing twice with 1 × phosphate-buffered saline (PBS), RIPA mixed with phosphatase and protease inhibitors (Roche, Basel, Switzerland) was used for the extraction of total cellular proteins. The proteins in the supernatant were collected for further quantification after centrifugation at 12,000×g for 15 min using a BCA protein quantification kit (23,227, Thermo Fisher Scientific, Waltham, MA, United States). After dissolving them in SDS-sample loading buffer, the proteins (approximately 25 μg) were separated on 4%–20% SDS-PAGE gels and electroblotted onto 0.22-μm PVDF membranes (Merck-Millipore, CA, United States). Membranes were blocked with 5% skim milk at room temperature (RT) for 2 h and then incubated with primary antibodies (diluted 1:1,000 in 5% BSA–TBST) overnight at 4°C. Primary antibodies against MMP3 (ab52915), MMP9 (ab228402), MMP13 (ab39012), aggrecan (ab3773), Bax (ab32503), and Bcl-2 (ab196495) were purchased from Abcam (Cambridge, United Kingdom). Other antibodies used, such as β-actin (D6A8), Cleaved Caspase-3 (ASP175), Bip (3183S), CHOP (L63F7), XBP-1s (E9V3E), and IRE1α (14C10) were purchased from Cell Signaling Technology (Danvers, MA, United States). Membranes were washed three times with TBST for 10 min and then incubated with anti-rabbit or anti-mouse secondary antibodies (1:10,000 dilution) for 1 h with protection from light exposure. After washing three times with TBST, the protein immunoreactivity of the membranes was analyzed using a fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, United States). Measurements of OD values were performed using ImageJ software (National Institutes of Health, United States).

For immunofluorescence detection, slides were used to seed cells in a six-well plate at a density of 4×10⁵ cells per well for 8 h, waiting for attachment and proliferation. NP cells were pre-administered with or without Sal003 (5 μM) for 2 h and subsequently treated with Tg (1 μM) for 24 h. NP cells were fixed with paraformaldehyde (4%) for 15 min at RT. For permeation of the membranes, 0.25% Triton X-100 was used for 10 min and then washed three times with PBS. After 2 h of blocking with 5% BSA at RT, cells were incubated with primary antibodies against MMP13, Aggrecan, CHOP, XBP-1s (diluted 1:200, Abcam) overnight at 4°C. PBS was used to wash the cells thrice the next day. Alexa Fluor 488 or 594 secondary antibody (anti-rabbit, Cell Signaling Technology) was used for incubation of the cells for 1 h after which they were washed three times. After incubation for 15 min with DAPI and washing, the images were obtained using a fluorescence microscope (Leica SP8 confocal microscope, Germany).

Ten μL NP cells at a density of 1.0×10⁷ cells/mL were seeded in 24-well plate. After 2 h of cell attachment, each well in plate was added DMEM medium with 2.5% FBS and 1% insulin transferrin selenium (ITS) (Gibco, Thermo Fisher Scientific, Waltham, MA, United States) with Tg and Sal003 as mentioned before. The medium was changed every 2 days. The cluster of NP cells was subjected to alcian blue and toluidine blue dye overnight at room temperature following fixation by 4% PFA for 10 min to assess the production of collagen synthesis after culture for 7 days.

Apoptotic NP cells were determined using the Annexin V 633 Apoptosis Detection Kit (AD11, Dojindo, Kumamoto, Japan), according to the manufacturer’s protocol. The apoptotic rate (%) in various groups was examined using flow cytometry (BD Co., United States).

Twenty male SD rats aged 12 weeks were kept under appropriate circumstances preoperatively and postoperatively. Percutaneous needle puncture was used to construct an IVDD model in rats (Han et al., 1976). Briefly, anesthesia was administered to the rats by intraperitoneal injection of pentobarbital sodium (40 mg/kg of body weight), and 75% ethanol was used to disinfect the tails. A 20G sterile needle was used to puncture the Co7/8 and Co8/9 discs from the dorsal skin into the NP center. The IVDD model was completed by a subsequent rotation of 360° and holding for 30 s in position. Eventually, re-sterilization was performed after pulling out the needle to avoid infection. 5 μL Sal003 of 5 µM was given to the Co8/9 discs every week via a microsyringe as IVDD + Sal003. The IVDD group was administered the same volume of saline weekly into the Co7/8 discs. Co6/7 discs were not administered any treatment as they were the control group.

The rats underwent X-ray examination 4 weeks postoperatively for evaluation of the intervertebral space. Digital imaging was captured in the anteroposterior axis using 21 lp/mm detectors supplied with ×5 geometric magnification (Faxitron VersaVision; Faxitron Bioptics LLC, Tucson, AZ, United States). Calculation of disc height indices (DHIs) was in accordance with a previous report in which the average IVD height (DHI) was calculated by averaging the measurements obtained from the anterior, middle, and posterior portions of the IVD and dividing it by the average of adjacent vertebral body heights. Changes in the DHI of injected discs were expressed as DHI% and normalized to the measured preoperative IVD height (DHI = 2× (D + E + F)/(A + B + C + G + H + I)% = postoperative DHI/preoperative DHI × 100) (Han et al., 2008).

IVDD progression was assessed by MRI. Four weeks postoperatively, the rats were fixed after anesthesia, and the signals of the assigned IVD section were acquired in T2-weighted images (T2WI) of the sagittal planes using a 1.5 T magnetic resonance scanner (Philips Eclipse, Cleveland, OH, United States). Subsequently, a grading system for determining IVDD levels based on MRI scan was measured by Pfirrmann grades: A homogeneous bright white structure as Grade I, inhomogeneous white structure and possible horizontal bands as Grade II, clear distinction between annulus and nucleus as Grade III, no collapsed disc space as Grade IV, and collapsed disc space as Grade V (Masuda et al., 1976).

Four% paraformaldehyde was collected and fixed in four coccygeal IVD tissues for at least 48 h. Then, paraffin was embedded in the samples, and 5 µm sections were obtained in the region of the midsagittal plane. Subsequently, sections were stained with hematoxylin and eosin (HE), alcian blue (AB), toluidine blue (TB) and safranin O-fast green (SO-FG) to assess the IVD structures. The histological score was calculated as previously described (Ji et al., 2018).

Deparaffinization and rehydration were first administered to sections, and 3% hydrogen peroxide (H₂O₂) was used for 20 min to block endogenous peroxidase. The primary antibodies were incubated overnight at 4°C, followed by blocking with 10% goat serum for 2 h at RT. Sections were then washed three times with PBS and incubated with HRP-conjugated secondary antibody for 2 h at RT the next day. Digital images were obtained using a Leica DM4000 B microscope (Leica Microsystems). The integrated optical density (IOD) was measured for semi-quantitative analysis using Image Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, MD, United States).

Apoptosis of NP cells was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using a TUNEL Kit (C1086, Beyotime Biotechnology Co., Shanghai, China). Quantification of positive cells was performed in three randomly selected fields from each sample using Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, United States). DAPI staining was used to estimate the total cell number under a fluorescence microscope.

The experiments in our study were performed at least three times to obtain data, which are presented as means ± standard deviations. Differences between two groups were assessed by the t-test, while significant differences among groups were assessed by ANOVA. Statistical significance was set at p < 0.05. GraphPad Prism 8.3 (GraphPad Software, San Diego, CA, United States) was utilized for data analysis and creating relevant figures.

Sal003 is presented in Figure 1A.

To analyze cytotoxicity, each well of the 96-well plates was used for seeding 10,000 NP cells and cultivated for 24 h at concentrations of 0, 2.5, 5, 10, 20, and 40 μM Sal003. The cytotoxicity of Sal003 was calculated based on the 0 μM Sal003 group. Sal003 exhibited cytotoxicity in NP cells at concentrations of 10 μM or higher (Figure 1B).

To determine cell proliferation, each well of the 96-well plates was seeded by 2,500 NP cells and cultured for 24 h at concentrations of 0, 2.5, 5, 10, 20, and 40 μM Sal003. To ensure the proliferation of Sal003, the cells were subjected to a CCK-8 assay for a time period ranging from 24 to 72 h following seeding. Sal003 showed an inhibitory influence on the proliferation of NP cells at 20 μM or more. NP cells treated with 5 μM Sal003 were significantly higher than those treated with other concentrations at 48 and 72 h (Figures 1C–E). Thus, 5 μM Sal003 was used in the following experiments.

RT-qPCR, Western Blotting, and immunofluorescence were performed to determine the changes in the ECM. RT-qPCR showed upregulation of MMP3, MMP9, MMP13, and ADAMTS5, which are related to the increase in catabolic levels following stimulation by Tg. Furthermore, aggrecan (considered one of the dominant factors of ECM) levels decreased after treatment with Tg. These Tg-induced harmful factors were partially reversed by Sal003 treatment (Figure 2A).

In addition, Western Blotting demonstrated that ECM degradation, including a decrease in aggrecan and an increase in MMP3, MMP9, and MMP13 protein levels, induced by Tg, could be reversed by Sal003 (Figures 2B, D). The immunofluorescence staining results for MMP13 and aggrecan were also in accordance with the results mentioned in the study (Figure 2C). Micromass culture results were in accordance with previous outcome (Figures 2E, F).

To determine the potential protective effects of Sal003 in Tg-stimulated NP cells, flow cytometry and Western Blotting were performed to evaluate apoptotic activity. The results of flow cytometry indicated that the number of apoptotic NP cells in the Tg group was markedly higher than that in the Sal003 pretreated group (Figures 3A, B). Moreover, apoptosis-related proteins were detected using Western Blotting analysis (Figures 3C, D). The results indicated that Sal003 increased Bcl-2 and inhibited Bax and cleaved caspase-3 in NP cells. Our results suggest that ER stress-related apoptosis of NP cells caused by Tg induction is decreased following pretreatment with Sal003.

Using RT-PCR and Western Blotting, NP cells were treated with Tg to examine whether Sal003 exerted cytoprotective effects under ER stress. ATF4, ATF6, Bip, and CHOP levels were markedly higher in the Tg-treated group than those in the control group, and Sal003 partially but significantly reversed these increases (Figure 4A). In addition, Western Blotting showed that Sal003 inhibited Tg-activated ER stress by decreasing Bip, CHOP, XBP-1s, and IRE1α at the protein level (Figures 4B, C). The immunofluorescence staining results for CHOP and XBP-1s were also in accordance with the results mentioned in the study (Figures 4D, E).

Experiments were performed to determine whether Sal003 could function as a therapeutic agent for IVDD in vivo. The rat IVDD model was established by needle puncture of caudal discs. Radiography and MRI were performed 4 weeks postoperatively. HE, SOFG, AB, and TB staining were also used to assess morphological differences in IVD following Sal003 administration. After assessing histomorphology, observed findings included contraction, reduction of NP cells, disorganization at the border between the nucleus pulposus and annulus fibrosus, and extensive loss of proteoglycans in the IVDD group. However, Sal003 treatment partially mitigated these detrimental changes at the histopathological levels, demonstrating slight shrinking and loss of NP cells and proteoglycan. Histological score analysis also demonstrated the impact of Sal003 in delaying IVDD progression (Figures 5A, B). As shown in Figures 5D, E, the IVDD group showed decreased disc signal and intervertebral height after surgery. However, Sal003 partially reversed the collapsed disc height and decreased the MRI signal. TUNEL assay was also applied to determine whether Sal003 could mitigate apoptosis in vivo. Consistent with our in vitro results, Sal003 inhibited apoptosis during IVDD (Figures 5C, F). Furthermore, immunohistochemical staining of BiP, CHOP, MMP3, MMP13, Bax and cleaved caspase-3 demonstrated the mitigation by Sal003 of ECM degradation, ER stress activation and apoptosis in the rat IVDD model, which was also in agreement with the results of our ex vivo experiments (Figures 5G, H).