The nucleus pulposus samples were collected from patients undergoing surgery from Jan 2016 to May 2018. The magnetic resonance classification of IDD was performed according to a previous report (10). Written informed consents were obtained from patients and counterparts and all the experimental protocols were approved by the Ethic Committee of Honghui Hospital affiliated to Xi'an Jiaotong University (approval no. 2019-12).

The NP tissues were dissected into pieces of 1.0 mm³ and digested with 0.25% protease at 37˚C for 1 h, followed by 0.2 mg/ml of collagenase type II for 4 h, prior to filtration through a sterilized mesh with 70-µm pore size and centrifugation at 300 x g, at 4˚C for 5 min. Cells were resuspended and maintained in DMEM/F12 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Sigma-Aldrich; Merck KGaA) at 37˚C in 5% CO₂. The medium was replaced every 2-3 days and the primary culture was sub-cultured when cells grew to ~80˚C confluence. NPCs were incubated with 10 ng/ml TNF-α (Sigma-Aldrich; Merck KGaA) or equal amount of DMEM medium as a negative control for 24 h before further assessment.

The fresh umbilical cord was obtained from in a full term neonate, followed by being washed with pre-cold PBS to remove the blood clot. Afterwards, the Wharton's jelly was isolated from umbilical cord and cut into 1 mm³ fragments, which were incubated with type II collagenase (0.2 mg/ml, Sigma-Aldrich; Merck KGaA) at 37˚C for 18 h and then digested using 2.5% trypsin for 30 min with agitation. Finally, the cells were fixed with 2 ml FBS for 2 h and then maintained in 8 ml of L-DMEM medium (HyClone; GE Healthcare Life Sciences) containing 1% penicillin-streptomycin, 50 µg/ml L-ascorbic acid and 2 mM glutamine at 37˚C in a humidified atmosphere of 5% CO₂. The culture medium was replaced every 2 days. The cells were passaged when the confluence reached 80-90%. WJ-MSCs from passage 3 were used for determination of cell surface antigens. The identification of MSCs is illustrated in Fig. 1 and MSCs differentiate into NP-like cells, with elevated expression of NP-related marker genes, when co-cultured with NPCs.

The content of released proteins in cell culture supernatant was measured using specific ELISA kits (R&D Systems). Briefly, the wells of a high protein binding 96-well plate (Corning Costar, Inc.) were coated overnight with 2 ng of monoclonal antibody against IL-1β, IL-6 or IL-8. Wells were then washed 4 times with PBS and blocked for at least 1 h with PBS containing 10% FBS, followed by the addition of clear cell supernatants and standard dilutions of recombinant human IL-1β, IL-6 or IL-8, prior to 2-h incubation at 37˚C. After washing thoroughly with a buffer containing PBS and 0.05% Tween-20, each well was incubated with biotinylated antibody for 60 min and then washed 3 times. After staining with streptavidin-HRP for 30 min, substrate working solution was added to each well and incubated for 15-20 min in the dark. The reaction was then stopped by the addition of 100 µl of 2 M H₂SO₄, and the OD₄₅₀ was measured with an ELISA plate reader (Model 550; Bio-Rad Laboratories, Inc.). Each experiment was independently repeated at least three times.

Primary polyclonal antibodies against human p-p38, p38, ASK-1, MKK-3, MKK-6, MMP-3, MMP-13, aggrecan, and actin were purchased from Abcam. Proteins were extracted from NP tissues or cell lysates with RIPA lysis buffer supplemented with protease inhibitors and phosphorylase inhibitor. The protein concentration was measured using the BCA assay (Pierce) following the manufacturer's instructions. The total proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) before being transferred to polyvinylidene fluoride (PVDF) membrane, which were blocked with 5% silk milk for at least 1 h and incubated overnight at 4˚C with specific primary antibodies (1:1,000 diluted). After rinsing with TBST, the membrane was incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit IgG (1:3,000 diluted). Finally, the labeled proteins were visualized with ECL substrates (EMD Millipore) before quantitative analysis using AlphaView software (ProteinSimple). Each experiment was independently repeated at least three times.

For co-culture without direct cell-cell contact, NPCs (1x10⁶ cells/well) were seeded on the bottom of transwell 6-well plates, and WJ-MSC cells were placed onto the apical compartment of a 6-well polyester track-etched cell culture insert with a membrane pore size of 0.4-µm (Thermo Fisher Scientific, Inc). Sequentially, cells were grown in DMEM/F12 medium at 37˚C in a humidified atmosphere of 5% CO₂. NPCs were cultured on common plates as a control and subjected to further analysis.

Data are presented as means ± standard deviation (SD). All in vitro experiments included at least 3 replicates per group. Groups were compared using the two-tailed Student's t-test for parametric data. All statistical analyses were carried out using SPSS 19.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

Pro-inflammatory cytokine TNF-α was used to mimic the inflammatory milieu of IDD, and the expression profiles of several inflammation markers in NPCs were evaluated. As shown in Fig. 2A, the levels of IL-1β, IL-6 and IL-8 were dramatically increased upon TNF-α stimulation. We also observed remarkable augment in the expression of MMP-3 and MMP-13. Inversely, aggrecan level synthesized by NPCs was significantly declined after cytokine treatment (Fig. 2B), suggestive of aggrecan fragmentation and breakdown of hydrated ECM within the nucleus.

TNF-α-treated NPCs were co-cultured with WJ-MSCs to determine the effect of WJ-MSCs on the intracellular inflammatory response and the involved signaling transduction. As displayed in Fig. 2A, WJ-MSCs evidently compromised the increased production of IL-1β, IL-6 and IL-8 by NPCs. In fact, there was no significant difference in the expression of these markers within MSCs, in the presence and absence of TNF-α stimulation (data not shown). Additionally, expression levels of both MMP-3 and MMP-13 were suppressed in NPCs that co-cultured with WJ-MSCs in comparison to cells exposed to TNF-α alone, whereas aggrecan production was elevated in the presence of WJ-MSCs (Fig. 2B). Therefore, WJ-MSCs can ameliorate inflammatory status within the co-cultured NPCs without direct contact.

Following TNF-α stimulation, the phosphorylation of apoptosis signal regulating kinase-1 (ASK-1) was significantly enhanced (Fig. 3). Moreover, the MAPK kinases including MKK-3 and MKK-6 were also activated after incitement by the pro-inflammatory cytokine TNF-α, hence promotion of p38 phosphorylation. The above indicate that the p38 MAPK signaling was activated, which might contribute to the promotion of inflammatory responses during the degenerative process.

Notably, the phosphorylated forms of the above-mentioned molecules implicated in p38 MAPK pathway were found decreased due to indirect co-culture between NPCs and WJ-MSCs, suggesting that WJ-MSCs may exert the protective role by virtue of the activation of p38 MAPK pathway.

To verify this observation, p38 MAPK signaling inhibitor SB-203580 was utilized and, as expected, it was found that this led to an obvious decrease in the phosphorylation forms of p38 (Fig. 4). Although TNF-α-induced MMP-3 and MMP-13 was decreased due to WJ-MSC co-culture, their levels synthesized by NPCs that were co-cultured with WJ-MSCs were further reduced in the presence of SB-203580, whereas aggrecan showed the opposite results. Furthermore, when cells were treated with SB-203580, NPCs co-cultured with WJ-MSCs showed no significant difference from those cultured alone, which suggested that blockage of p38/MAPK pathway protected NPCs against ECM breakdown, regardless of the analogous function of WJ-MSCs. Considering IL-1β, IL-6 and IL-8 protection, NPCs exposed to SB-203580 displayed values lower than those observed in the co-cultured cells devoid of SB-203580. Also there was barely detectable difference between NPCs cultured alone and the co-cultured cells when they were subjected to SB-203580 treatment. Thus, we concluded that WJ-MSCs might suppress TNF-α-mediated inflammation in NPCs by partially inactivating p38/MAPK signaling.