Regulation of endoplasmic reticulum stress on autophagy and apoptosis of nucleus pulposus cells in intervertebral disc degeneration and its related mechanisms

Cell culture

Primary rat nucleus pulposus (NP, cat. no. BH-Y420) cells that purchased from Bohui Biotechnology Co., Beijing, China were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12, Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) with 5% CO₂ at 37 °C. Hypoxic treatment conditions were 2% O₂, 5% CO₂ and 93% N₂. The NP cells in hypoxia group (Hypo) were placed in a three-gas incubator for 24 h of hypoxia. In serum deprivation (SD) group, NP cells were treated with serum deprivation and cultured for 24 h. NP cells in Hypo+SD group were treated with serum deprivation and hypoxia for 24 h. NP cells in LPS group were treated with serum deprivation and hypoxia for 24 h, and then NP cells were induced by lipopolysaccharide (LPS, 1 μg/mL, MedChemExpress, Princeton, NJ, USA) for 24 h to construct the IVDD cell model (Zhou et al., 2022). In this article, ERS inhibitor (4-phenylbutyric acid, 4-PBA, MedChemExpress, Princeton, NJ, USA) and ERS activator (Thapsigargin, TP, MedChemExpress, Princeton, NJ, USA) were used to explore the mechanism. NP cells with 2–5 passages were used for the study according to the product instructions.

Cell counting kit-8 assay

NP cells were injected into 96-well plates (8 × 10³cells/well) incubated for 24 h. Subsequently, 10 µL cell counting kit-8 (CCK-8) solution (Beyotime, Jiangsu, China) was added to each well and the cells were incubated for another 2 h. Then, cells were detected by enzyme-labelled instrument (Thermo Fisher Scientific, Waltham, MA, USA) with a wavelength at 450 nm. The cell survival rate was calculated as follows: cell survival rate = [(experimental OD - blank OD)/(control OD - blank OD)] × 100%.

Immunofluorescence

NP cells with indicated treatment were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.25% Triton X-100 for 10 min. After that, NP cells were then incubated with anti-glucose regulated protein 78 (GRP78; cat. no. ab21685; 1:200, Abcam, Cambridge, UK) and anti-GAPDH (cat. no. ab181602; 1:500, Abcam, Cambridge, UK) at 4°C overnight, following which was the incubation with appropriate secondary antibodies (Alexa Fluor 488; cat. no. ab150077; 1:200; Abcam, Cambridge, UK) for 1 h. The fluorescence images were acquired using a fluorescent microscope (Olympus FluoView^™ FV1000, Tokyo, Japan) with an excitation wavelength of 488 nm and an emission wavelength of 519 nm. Positive cells in all fluorescence images were counted using ImageJ to calculate the positive rate.

Green fluorescent protein-light chain 3 immunofluorescence

Transfection of green fluorescent protein (GFP)-light chain 3 (LC3) was performed according to the manufacturer’s instructions (Genomeditech, Shanghai, China). NP cells with indicated treatment were fixed with 5% paraformaldehyde for 10 min at room temperature, permeabilized with 0.3% Triton-X for 5 min, and stained with 4′-6-diamidino-2-phenylindole (DAPI) for 3 min. Finally, NP cells that washed by double distilled water (ddH₂O) were mounted on tissue slides with mounting medium (Thermo Fisher Scientific, Waltham, MA, USA). A fluorescence microscope (Nikon Corporation, Tokyo, Japan) was used to capture and analyze fluorescence images. Positive cells in all fluorescence images were counted using ImageJ to calculate the positive rate.

Flow cytometry

The Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (Beyotime, Jiangsu, China) was used to quantify the apoptosis of NP cells. NP cells that washed with PBS were resuspended in binding buffer and then incubated with FITC-labeled Annexin V and propidium iodide (PI) in the dark for 15 min. Cells were analyzed using the FACScan flow cytometry (BD Biosciences, San Jose, CA, USA). According to the permeability of cell membranes to FITC and PI under different conditions, cells with mechanical damage are shown in the upper left quadrant as (FITC−/PI+), live cells are shown in the lower left quadrant as (FITC−/PI−), non-live cells are shown in the upper right quadrant, which are necrotic cells or late apoptotic cells, as (FITC+/PI+), and early apoptotic cells are shown in the lower right quadrant as (FITC+/PI-).

Western blot

NP cells were lysed in radioimmunoprecipitation assay (RIPA) Lysis Buffer (Beyotime, Jiangsu, China) on the ice to extract total proteins. The protein concentration was detected with a bicinchoninic acid (BCA) protein assay kit (Beyotime, Jiangsu, China). A total of 30 μg proteins were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes, which were blocked with 5% bovine serum albumin (BSA), were incubated with primary antibodies specific to LC3 (cat. no. ab192890; 1/2,000; Abcam, Cambridge, UK), GRP78 (cat. no. ab108615; 1/1,000; Abcam, Cambridge, UK), C/EBP homologous protein (CHOP; cat. no. #AF5280; 1/1,000; Affinity Biosciences, Cincinnati, OH, USA), spliced X-box binding protein 1 (XBP-1s; cat. no. #AF5110; 1/1,000; Affinity Biosciences, Cincinnati, OH, USA), Bcl-2 associated X (Bax; cat. no. ab32503; 1/1,000; Abcam, Cambridge, UK), B cell lymphoma-2 (Bcl-2; cat. no. ab194583; 1/1,000; Abcam, Cambridge, UK), Cleaved-caspase3 (cat. no. #AF7022; 1/1,000; Affinity Biosciences, Cincinnati, OH, USA), Caspase3 (cat. no. ab184787; 1/2,000; Abcam, Cambridge, UK), Caspase12 (cat. no. ab315271; 1/1,000; Abcam, Cambridge, UK), Beclin-1 (cat. no. ab207612; 1/2,000; Abcam, Cambridge, UK), p62 (cat. no. ab109012; 1/10,000; Abcam, Cambridge, UK), receptor-interacting protein 1 (RIP1; cat. no. #AF7877; 1/1,000; Affinity Biosciences, Cincinnati, OH, USA) and receptor-interacting protein 3 (RIP3; cat. no. #AF7942; 1/1,000; Affinity Biosciences, Cincinnati, OH, USA) at 4 °C overnight. GAPDH is a feasible House-keeping gene as internal references in the disc cells (Yurube et al., 2011). Therefore, GAPDH (cat. no. ab181602; 1/10,000; Abcam, Cambridge, UK) was used for normalization. On the next day, the membranes were incubated with appropriate secondary antibodies (cat. no. ab6721; 1/2,000; Abcam, Cambridge, UK). Finally, the protein blots were visualized using the enhanced chemiluminescence (ECL) reagent (Beyotime, Jiangsu, China). ImageJ was used to analyze the gray value of the target protein and the GAPDH. Calculate the relative expression level of the protein using the formula: Relative expression level of protein = grayscale value of the target protein/grayscale value of the GAPDH.

Enzyme-linked immunosorbent assay

The production of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), inducible nitric oxide synthase (iNOS) and interleukin-6 (IL-6) in the supernatant of NP cells were evaluated with ELISA kits (Abcam, Cambridge, UK) according to the manufacturer’s instructions. The absorbance values of each well were measured at 450 nm using an enzyme marker, and the average absorbance values of the standards and samples were calculated for each set of replicates. The concentration of the standard was taken as the horizontal coordinate and the A450 value as the vertical coordinate, and a smooth line was used to connect the coordinate points of each standard. The corresponding concentrations of the samples were calculated from the absorbance values of the samples and the standard curve.

Statistical analysis

All data that expressed as means ± standard deviation were analyzed with GraphPad Prism 5. All experiments were independently repeated in triplicate and all experimental data were biologically repeated in triplicate. Statistical comparisons between two groups were conducted by Two-Tailed Student’s t test and comparisons among three or more groups were conducted with one-way analysis of variance followed by the Tukey’s post hoc test. P < 0.05 was considered statistically significant.

Serum deprivation induces autophagy and apoptosis and hypoxia down-regulates autophagy of NP cells

Firstly, the present study examined the effects of Hypo and SD induction on NP activity, autophagy, ERS and apoptosis. The CCK-8 results showed a significantly lower level of cell activity in the SD group as compared to the Normal group (Fig. 1A). And compared with the Normal group, SD treatment significantly increased the expression of ERS biomarker GRP78 (Fig. 1B) and the proportion of NP cell apoptosis (Fig. 1C). In addition, SD treatment significantly increased the expression of autophagy-related protein LC3-II/LC3-I but downregulated p62 (Figs. 1D and 1E). However, compared with the Normal group, Hypo treatment had no significantly effect on NP cell activity (Fig. 1A), GRP78 expression (Fig. 1B), apoptosis (Fig. 1C) and LC3-II/LC3-I expression, but significantly increased p62 expression (Figs. 1D and 1E). Compared with Hypo group, cell activity and p62 expression in Hypo + SD group was decreased, while the expression of GRP78, proportion of apoptosis, and the expression of LC3-II/LC3-I was increased. However, compared with the SD group, the activity and p62 expression of NP cells in the Hypo + SD group were increased, while the apoptosis rate, GRP78 expression and LC3 expression were decreased. Moreover, treatment with chloroquine further decreased LC3 expression and further increased p62 expression in NP cells compared to the Hypo + SD group (Figs. 1D and 1E). These results indicate that the autophagy level of normal cultured NP cells in vitro is low, and serum deprivation can further induce autophagy and apoptosis. Hypoxia alone could down-regulate the level of autophagy in NP cells.

Effect of ERS on autophagy and apoptosis in IVDD model cells

Inflammation is an important pathological mechanism of IVDD (Chen et al., 2022). Therefore, in this study, the model of IVDD in vitro was established by inducing NP cells with LPS under hypoxic and serum deprivation condition. The mechanism of ERS on autophagy and apoptosis in IVDD model cells was further investigated using the ERS inhibitor 4-PBA and activator TP, which inhibit or promote ERS by inhibiting histone deacetylase (HDAC) and microsomal Ca2+-ATPase, respectively. The results showed that 4-PBA (0–200 μM) and TP (0–1 μM) had no significant effect on the viability of NP cells (Figs. 2A and 2B). However, the cell activity (Fig. 2C) was significantly decreased compared with Control group, and the expression of GRP78 (Fig. 2D), the proportion of apoptosis (Fig. 2E), and the expression of LC3 (Fig. 2F) were significantly decreased. Compared with LPS group, LPS + 4-PBA treatment increased cell viability, decreased apoptosis level, and downregulated the expression of GRP78 and LC3. While LPS + TP treatment showed an opposite effect to LPS + 4-PBA treatment.

Next, we examined the expression of autophagy-, apoptosis- and ERS-related proteins and the results as shown in Fig. 3. LPS induced an increase in the expression of ERS-related proteins GRP78, CHOP and XBP-1S (Fig. 3A), and apoptosis-related proteins Bax, Cleaved caspase3 and Caspase12, and a decrease in Bcl-2 expression (Fig. 3B). Also, compared with the Control group, the expressions of autophagy-related proteins LC3II/I and Beclin-1 were significantly increased by LPS stimulation (Fig. 3C). In comparison with LPS group, the expression of GRP78, CHOP, XBP-1S, Bax, Cleaved caspase3, Caspase12, LC3II/I and Beclin-1 were significantly decreased, and the expression of Bcl-2 was significantly increased in the LPS + 4-PBA group. In contrast, the expression trend of these proteins in the LPS + TP group was opposite to that in the LPS + 4-PBA group.

Moreover, it was worthwhile to mention that ERS was significantly activated by LPS in hypoxic and serum deprivation environment, accompanied by increased autophagy and apoptosis. Inhibition of ERS has a relatively significant inhibitory effect on autophagy and apoptosis in NP cells induced by LPS in hypoxic and serum deprivation environment. However, the further promotion of autophagy by activating ERS is relatively limited. Thus, the next step will be to further observe the changes that occur when apoptosis and autophagy are directly inhibited.

Effects of inhibition of apoptosis and autophagy on ERS, apoptosis and autophagy in IVDD cell models

The apoptosis inhibitor z-Val-Ala-DL-Asp-fluoromethyl ketone (Z-VAD-FMK) and the autophagy inhibitor 3-methyladenine (3-MA) were used to observe the effects of inhibiting apoptosis and autophagy on LPS-treated NP cells. They inhibit apoptosis and autophagy by blocking Caspase and phosphoinositide 3-kinase (PI3K), respectively. Interestingly, the addition of apoptosis and autophagy inhibitors did not restore NP cell activity, and the addition of apoptosis inhibitors even exacerbated the impairment of cell activity (Fig. 4A). Results obtained from IF assay showed that the expression of GRP78 was increased after z-VAD-FMK or 3-MA administration compared with LPS group (Fig. 4B). In addition, apoptosis inhibitor z-VAD-FMK significantly decreased apoptosis (Fig. 4C) and increased LC3 expression (Fig. 4D), but autophagy inhibitor 3-MA significantly increased the cell apoptosis (Fig. 4C) and decreased LC3 expression (Fig. 4D).

Meanwhile, WB experiments showed similar results to those described above. Compared with LPS group, z-VAD-FMK or 3-MA treatment increased the expressions of GRP78, CHOP and XBP-1S (Fig. 5A). Besides, by contrast with the LPS group, z-VAD-FMK treatment decreased the expressions of Bax, Cleaved caspase3 and Cleaved caspase12 and increased Bcl-2 expression. Nevertheless, in LPS+3-MA group, the expressions of Bax, Cleaved caspase3 and Cleaved caspase12 were increased while Bcl-2 expression was decreased (Fig. 5B). As Fig. 5C demonstrated, z-VAD-FMK treatment increased the expressions of LC3II/I and Beclin-1 when compared with those in LPS group, while 3-MA treatment decreased the expressions of LC3II/I and Beclin-1. The results showed that apoptosis or autophagy of LPS-induced NP cells under hypoxic and serum deprivation condition were significantly inhibited. The aforementioned results suggest that the use of apoptosis or autophagy inhibitors decreased the levels of apoptosis or autophagy in LPS-induced NP cells. However, the cell viability and the degree of ERS were not restored. This suggests that there may be other cell death mechanisms involved in the role of ERS in the occurrence and development of IVDD.

The role of ERS in the occurrence and development of IVDD is related to the regulation of necroptosis

In addition to the traditional form of cell death known as apoptosis, many new forms of cell death have been discovered in recent years such as programmed cell death that do not depend on Caspases (Liu & Lieberman, 2020). Inflammation is considered one of the main causes of IVDD (Chen et al., 2020), and necrotic apoptosis is viewed as a programmed cell death process with pro-inflammatory functions (Bertheloot, Latz & Franklin, 2021). Therefore, here we detected the expression of necroptosis related proteins. The ELISA results showed that the levels of IL-1β, TNF-α, iNOS and IL-6 were significantly increased in the LPS group when compared with the Control group. The expressions of these inflammatory cytokines were further increased after z-VAD-FMK or 3-MA administration compared with the LPS group (Fig. 6A). The WB results showed that the expressions of necroptosis related proteins receptor-interacting protein kinase 1 (RIP1) and RIP3 were significantly increased in LPS group compared with Control group. Compared with LPS group, the expressions of RIP1 and RIP3 were significantly increased after z-VAD-FMK or 3-MA administration (Fig. 6B). These results suggest that ERS can affect the occurrence and development of IVDD by regulating autophagy, apoptosis and other programmed death.