All experimental procedures were approved by the Animal Care and Ethics Committee of Zhengzhou University. The primary NP cells were were isolated and cultured as previously described [18, 19]. The male Sprague-Dawley rats (3 months, 250–300 g) were purchased from Experimental Animal Center of Zhengzhou University. Briefly, the NP tissue of each IVD was obtained with ophthalmic forceps and cutted into 1 mm${}^3$ fragments. The isolated fragments were digested with 0.25% type II collagenase (Sigma, USA) at 37 ${}^{\circ }$C for 30 minutes and filtered through a 70 $\mu$m filter to remove debris. The obtained NP cells were cultured in complete culture medium Dulbecco’s modified Eagle’s medium/ham’s F-12 (DMEM/F-12, Gibco, USA) and 20% fetal bovine serum (FBS, Gibco, USA) supplemented with 1% penicillin/streptomycin (Sigma, USA). When the cells reached 80–90% confluence, they were digested with 0.25% tripsinase (Beyotime, China). Due to the small amount of NP cells in the lumbar IVD, we extracted 8 rats each time for primary cell culture, and extracted NP cells 12 times. The total number of rats used in this article is about 96. The second generation of NP cells were used throughout the following experiments.

TNF-$\alpha$ and IL-1$\beta$ are the extremely broad studied inflammatory factors in IVD degeneration [30, 31, 32, 33]. Referring to previous research, the intervention concentration of TNF-$\alpha$ or IL-1$\beta$ is 50 ng/mL, 20 ng/mL respectively [30, 31, 32, 33], the treatment time periods are 0, 24, 48, 72 hours, and choose the appropriate action time on this basis. The RIPK1 inhibitor necrostatin-1 (Nec-1, Sigma, USA), RIPK3 inhibitor GSK872 (Merck, Germany) and MLKL inhibitor necrosulfonamide (NSA, Sigma, USA) were applied to experimental groups, while the control groups were given isopyknic dimethylsulfoxide (DMSO, Sigma, USA).

To evaluate NP cells viability, the cell counting kit-8 (CCK-8, Dojindo, Japan) was employed. NP cells were seeded at a density of 5 $\times$ 10${}^3$ cells per well in 96-well culture plates and then incubated for 24 hours. At the appropriate inflammation treatment time periods, following themanufacturer’s instructions, 10 $\mu$L of CCK-8 solution (Dojindo, Japan) and 100 $\mu$L of DMEM-F12 were added to each well and incubated for 2 hours at 37 ${}^{\circ }$C. The cell viability was quantified by detecting the absorbance at 450 nm using a spectrophotometer (ELx808 Absorbance Microplate Reader, Bio-Tek, USA).

The PI positive ratio (indicating necrotic cells) of NP cells were determined using PI single-staining (Nanjing Keygen Biotech, China). At each time point, the cells were harvested, stained according to the manufacturer’s instructions, and then analyzed by flow cytometry. The PI staining positive ratio allowed us to quantify the necrotic cells (PI positive).

According to the manufacturer’s (Beyotime, China) instructions, the release of LDH in culture medium was utilized to detect NP cells cytotoxicity under inflammatory condition. In brief, the LDH release activity is presented as the release of LDH in the culture medium relative to the total cellular LDH.

The NP cells were exposed to TNF-$\alpha$ or IL-1$\beta$ for 48 hours. The Nec-1, GSK872 or NSA were applied to observe the effect of necroptosis on morphological changes of NP cells during inflammatory condition. At the given time points, cells were photographed using phase-contrast microscopy (Olympus, Tokyo, Japan).

NP cells were seeded in 24-well culture plates and treated as described above. Following 48 hours treatment, the NP cells were washed twice with PBS, and then incubated with Calcein-AM (2 $\mu$M), a membrane permeable probe to label the live cells, at 37 ℃ in the dark for 20 minutes. After being gently rinsed three times in PBS, the cells were stained with 5 $\mu$mol/L PI according to manufacturer’s instructions. Under the blue light excitation, the living cells appeared green, and nuclei of dead NP cells displayed red fluorescence. Finally, the stained NP cells were observed under a laser scanning confocal microscope (LSM, Heidelberg, Germany).

To analyze the MMP changes in NP cells under inflammatory conditions, JC-1 fluorescent probes (Beyotime, China) was introduced into this study. Briefly, the NP cells were incubated with the JC-1 solution in the dark for 20 minutes, and then the MMP in NP cells was evaluated under flow cytometry. Finally, the evaluation of MMP is expressed as the ratio of red relative to green fluorescence intensity.

The MPTP of NP cells was measured by MPTP Fluorescence Assay Kit (Genmed, China). At each time point, cells were collected, then 500 $\mu$L preheated cleaning solution (Reagent A) and isopyknic working solution containing neutralization solution and staining solution (Reagent B) were added into cell suspension. Next they were mixed gently and incubated in the dark at 37 ${}^{\circ }$C for 20 minutes. Finally, the sample was resuspended in Reagent A and analyzed using flow cytometry and LSM.

ROS levels in the NP cells were analyzed using the ROS-specific fluorescent probe 2′,7′-dichlorofluorescin diacetate (H${}_2$DCF-DA) (Sigma, USA). Briefly, at each time point, the culture medium was discarded, and the NP cells were harvested. Then, the cells were resuspended and incubated with 20 $\mu$M H${}_2$DCF-DA at 37 ${}^{\circ }$C in the dark for 20 minutes. Subsequently, serum-free medium was used to rinse the cells twice. Finally, the mean fluorescence intensity (MFI) was quantified by flow cytometry. Furthermore, in order to evaluate the intracellular ROS level in situ, the cells were examined under the LSM.

The Rat SiRNA-RIPK1, SiRNA-RIPK3 and SiRNA-MLKL were designed and manufactured by Biomics (Biomics Biotechnologies Co. Ltd, China) according to current guidelines. The effective siRNA sequences for SiRNA-RIPK1, SiRNA-RIPK3 and SiRNA-MLKL were used as follows. SiRNA-RIPK1: 5′-GUCUUCGCUAACACCACUAdTdT-3′, 5′-UAGUGGUGUUAGCGAAGACdTdT-3′; SiRNA-RIPK3: 5′-CAUGUCAGUACAACCGAGAdTdT-3′, 5′-TCTCGGTTGTACTGACATGdTdT-3′; SiRNA-MLKL: 5′-CUGGAGGCUACCAAGUAAATTdTdT-3′, 5′-UUUACUUGGUAGCCUCCAGTTdTdT-3′. The NP cells were transfected with above effective sequence at a concentration of 100 pmol/10${}^5$ cell using lipofectamine RNAi MAX (invitrogen). 24 hours later, the transfected cells were digested and recultivated for subsequent experiments.

Following 0, 24, 48, 72 hours TNF-$\alpha$ or IL-1$\beta$ treatment, NP cells were washed three times in PBS and fixed in 4% paraformaldehyde at room temperature for 15 minutes. Next, the cells were blocked in 5% bovine serum albumin diluted with 0.3% Triton X-100 for 30 minutes. Then, cells were incubated with p-MLKL primary antibody (Abcam, USA) at a 1:100 dilution overnight at 4 ${}^{\circ }$C in the dark. After washing, the NP cells were incubated with fluorophore-conjugated secondary antibody for 60 minutes. Finally, the stained samples were visualized and photographed under LSM.

At each time point of inflammation treatment, the total RNA of NP cells was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions and then transcribed into complementary DNA (cDNA). Quantitative RT-PCR was performed using a standard PCR kit and SYBR Green/Fluorescein qPCR Master Mix (2X) (Fermentas, Canada) on an ABI Prism 7900HT sequence detection system (Applied Biosystems, USA). The GAPDH was used as house-keeping gene (control), and relative mRNA expression levels of target genes were subjected to analysis of amplification curve, and the data were calculated using the 2${}^{-△△\text{CT}}$ method. The primer sequences used for RT-PCR were designed and synthesized as follows: RIPK1: 5′-AGGAGGAAAGGAAGCGAAGG-3′, 5′-GGTTGTGCTGGGATAAGGAAGA-3′; RIPK3: 5′-ATGTCTAAACTCTCAGCCGTA-3′, 5′-ATTGAGCCATAACTT GACAGA-3′; MLKL: 5′-TCTCCCAACATCCTGCGTAT-3′, 5′-TCCCGAGTGGTGTAACCTGTA-3′; GAPDH: 5′-CGCTAACATCAAATGGGGTG-3′, 5′-TTGCTGACAATCTTGAGGGAG-3′.

Following 0, 24, 48, 72 hours treatment, the NP cells sample were lysed in a RIPA lysis buffer (Beyotime, China) containing protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Beyotime, China) at 4 ${}^{\circ }$C for 20 minutes, sonicated for 1 minute, and centrifuged at 12000$\times$ g for 15 minutes, and then the supernatant was collected. The protein concentration was quantified using an enhanced BCA protein assay kit (Nanjing Keygen Biotech, China). Protein samples were separated by 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to the nitrocellulose membranes, which were incubated with primary antibodies overnight at 4 ${}^{\circ }$C and then incubated with a horseradish peroxi-dase-conjugated secondary antibody for 2 hours according to the manufacturer’s instruction. Finally, the protein was developed using the enhanced chemiluminescence (ECL) method as previously described. The primary antibodies were used as follows: RIPK1 (1:500, CST, USA), phospho-PKA substrate (1:1000, CST, USA), RIPK3 (1:500, Abcam, UK), pRIPK3 (phosphoS232, 1:1000, Abcam, UK), MLKL (1:500, Abcam, UK), GAPDH (1:5000, Abcam, UK).

The data are expressed as the mean values $\pm$ standard deviation (SD) of at least three independent experiments. The data analysis was performed using SPSS 22.0 software package (Boao Yijie, Beijing Technology Co., Ltd, Beijing, China). Differences between groups were determined by Student’s t-test or one-way analysis of variance (ANOVA), followed by the Bonferroni post hoc test. The probability of p 0.05 was considered statistically significant.

To investigate whether necroptosis was involved in inflammation-induced NP cells death, we first examined the expression of necroptosis-associated target molecules. The Western blot results demonstrated that the expression level of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL and p-MLKL, especially p-RIPK1, p-RIPK3 and p-MLKL, were all increased following 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ treatment for 24, 48 and 72 hours (Fig. 1A,B). Similarly, through the RT-PCR detection, the gene expression trend of RIPK1, RIPK3 and MLKL is highly consistent with the protein expression level following 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ treatment for 24, 48 and 72 hours (Fig. 1C,D). Also, through immunofluorescence detection, we intuitively observed that with the prolongation treatment of TNF-$\alpha$ or IL-1$\beta$, the expression level of necroptosis downstream core molecule p-MLKL gradually increased (Fig. 1E).

Fig. 1.

Effects of inflammatory factor on protein and gene expression of necroptosis-associated target molecules in rat NP cells. (A,B) Representative western-blot graphs of RIPK1, p-RIPK1, RIPK3, p-RIPK3, MLKL, p-MLKL and GAPDH in NP cells subjected to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 0, 24, 48 and 72 hours. Data from treated groups have been normalized to GAPDH. (C,D) The mRNA level of RIPK1, RIPK3 and MLKL measured by RT-PCR in NP cells subjected to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 0, 24, 48 and 72 hours. (E) The fluorescence photomicrograph of p-MLKL expression detected by immunofluorescence staining. Scale bars = 50 $\mu$M. Values are expressed as mean $\pm$ SD from three independent experiments (*p 0.05, ** p 0.01, ***p 0.001 vs. control).

To further confirm the involvement of necroptosis in inflammation-induced NP cells death, we used necroptosis specific inhibitor Nec-1 to treat NP cells under inflammatory conditions. The results of CCK-8 assays showed that Nec-1 markedly improved the decreased activity of NP cells caused by 50 ng/mL TNF-$\alpha$ treatment or 20 ng/mL IL-1$\beta$ treatment for 24, 48 and 72 hours (Fig. 2A,B). The LDH release into the culture media from damaged cells is positively correlated with cell damage and cytotoxicity. The LDH release was increased in NP cells under 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ treatment for 24, 48 and 72 hours, which were effectively inhibited by 20 $\mu$M Nec-1 (Fig. 2C,D). Moreover, the PI positive (cell death) ratio was detected to synthetically evaluate the NP cells survival capacity. Following exposure to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 24, 48 and 72 hours, the positive rate of PI showed a clear trend of gradual increase (Fig. 2E–G). We then carried out inhibitor concentration gradient correlation detection to screen the optimal necroptosis inhibitor intervention concentration. On this basis, we selected Nec-1 (20 $\mu$M), GSK872 (5 $\mu$M) and NSA (5 $\mu$M) to perform the follow-up experiments. Supplementary material is the detailed screening process (Supplementary Material). As expected, Nec-1 (20 $\mu$M) can effectively inhibit the increased PI positive rate of NP cells induced by TNF-$\alpha$ and IL-1$\beta$ (Fig. 2E–G). After systematic analysis and comparison, we chose 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ inflammatory factors treatment for 48 hours throughout the following experiments.

Fig. 2.

The protective effects of Nec-1 (20 $\mu$M) against inflammatory factor-induced viability decreased and death in rat NP cells. (A,B) The cell viability of NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 0, 24, 48 and 72 hours was measured using the CCK-8 assay. (C,D) The cytotoxicity of NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 0, 24, 48 and 72 hours was determined by LDH release. (E–G) Representative graphs and statistical analysis of PI positive ratio by flow cytometry analysis after PI staining in NP cells. Values are expressed as mean $\pm$ SD from three independent experiments (*p 0.05, ** p 0.01, *** p 0.001 vs. control).

In order to further systematically verify that necroptosis is involved in NP cells death induced by inflammatory factors, we also introduced RIPK3 specific inhibitor GSK872 (5 $\mu$M) and MLKL specific inhibitor NSA (5 $\mu$M) into this experiment. The results displayed that Nec-1, GSK872 or NSA significantly reversed the decreased activity of NP cells under 48 hours TNF-$\alpha$ or IL-1$\beta$ treatment, at the same time, Nec-1, GSK872 or NSA efficiently down-regulated the LDH release level and PI positive ratio (Fig. 3A–C). Collectively, these results indicated that RIPK1/RIPK3/MLKL-mediated necroptosis was involved in inflammation-induced NP cells death.

Fig. 3.

The viability and death changes of rat NP cells treated with necroptosis specific inhibitors. (A) Following co-treatment with necroptosis specific inhibitor Nec-1 (20 $\mu$M), GSK872 (5 $\mu$M) or NSA (5 $\mu$M), the cell viability of NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours was measured using the CCK-8 assay. (B) The cytotoxicity of NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours was determined by LDH release. (C) Representative graphs of PI positive ratio by flow cytometry analysis after PI staining in NP cells. Values are expressed as mean $\pm$ SD from three independent experiments (*p 0.05, ** p 0.01, *** p 0.001 vs. control).

After 48 hours exposure of NP cells to TNF-$\alpha$ or IL-1$\beta$, the cells gradually lost their normal morphology, became round in shape, detached from the plates, and displayed morphological changes indicating of necrosis (Fig. 4A). We further observed the survival of NP cells using the Calcein-AM/PI (live/dead) cell staining. Consistently, live/dead cell staining showed that the number of dead cells (red fluorescence) markedly increased, while the number of live cells (green fluorescence) decreased after TNF-$\alpha$ or IL-1$\beta$ treatment for 48 hours compared to that in control groups (Fig. 4B). The Nec-1, GSK872, or NSA are extremely effective in inhibiting the morphological changes of NP cells necrosis induced by inflammatory mediators (Fig. 4A,B).

Fig. 4.

The morphological changes of rat NP cells treated with necroptosis specific inhibitors. (A) The morphological changes of NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours was observed under the optical microscope. (B) Typical fluorescence photomicrograph of live/dead cell staining of NP cells. The green fluorescent signaling (Calcien-AM) indicates live cells and red fluorescent signaling (PI) indicates dead cells. Scale bars = 20 $\mu$M.

To investigate the role of SiRIPK1, SiRIPK3 and SiMLKL in inflammation-induced NP cells death, NP cells were treated with effective SiRIPK1, SiRIPK3 and SiMLKL sequences respectively according to our previous researches. The Western blot and RT-PCR results displayed that the transfected SiRNA sequences resulted in a marked decrease in protein and gene expression levels of constitutive RIPK1, RIPK3 and MLKL respectively (Fig. 5A,B,D,E,G,H). The NP cells were treated with these SiRNAs for 48 hours, prior to exposure to TNF-$\alpha$ or IL-1$\beta$ for 48 hours. Contrary to the results presented by the aforementioned RIPK1 specific inhibitor Nec-1, SiRIPK1 down-regulated the activity of NP cells under inflammatory conditions and exacerbated NP cells death (Fig. 5C,J,K). As expected, both SiRIPK3 and SiMLKL remarkedly inhibited TNF-$\alpha$ or IL-1$\beta$ induced NP cells death and notably upregulated cell activity (Fig. 5F,I,L,M,N,O). That is to say, the inflammation-induced NP cells death is inhibited by SiRIPK3 and SiMLKL, but aggravated by SiRIPK1. In follow-up research, we will further clarify its underlying mechanism.

Fig. 5.

The inflammation-induced rat NP cells death is inhibited by SiRIPK3 and SiMLKL, but aggravated by SiRIPK1. (A,B) The NP cells were treated with SiRIPK1 or nonspecific RNA (negative control, NC) for 48 hours, and total protein and gene expression levels were measured. (D,E) The NP cells were treated with SiRIPK3 or NC for 48 hours, and total protein and gene expression levels were measured. (G,H) The NP cells were treated with SiMLKL or NC for 48 hours, and total protein and gene expression levels were measured. The NP cells were pretreated with the selected siRNA sequence for 48 hours and then exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours. (C,F,I) Following pretreated with the selected SiRNA sequence for 48 hours, the NP cells were exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours, and cell viability of NP cells was measured using the CCK-8 assay. (J–O) Following pretreated with the selected SiRNA sequence for 48 hours, the NP cells were exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours, the representative graphs and statistical analysis of PI positive ratio by flow cytometry analysis after PI staining in NP cells. Values are expressed as mean $\pm$ SD from three independent experiments (*p 0.05, ** p 0.01, *** p 0.001 vs. control).

The normal cells stained with JC-1 exhibited abundant red along with little green fluorescence. The JC-1 aggregates were dispersed to green fluorescence when the cells suffered from damages. Following 48 hours exposure of NP cells to TNF-$\alpha$ or IL-1$\beta$, MMP loss was notably observed, as indicated by the decrease in red and increase in green fluorescence, implying mitochondrial damage occured (Fig. 6A,B). When treated with Nec-1, GSK872, or NSA, the loss of MMP in NP cells induced by inflammatory factors were efficiently rescued (Fig. 6A,B). A key feature of necroptosis is the enhanced mPTP opening. The values of relative fluorescence intensity (RFI) detected by flow cytometry evidently decreased following TNF-$\alpha$ or IL-1$\beta$ treated for 48 hours, which implyed the enhanced mPTP opening (Fig. 6C,D). In presence of Nec-1, GSK872, or NSA, the loss of fluorescence intensity was apparently alleviated, the flow cytometry and the fluorescence staining clearly confirmed this point (Fig. 6C,D). Together, these results suggested that Nec-1, GSK872, or NSA may via inhibition mPTP opening and MMP loss to alleviate NP cells necroptosis.

Fig. 6.

The inflammation-induced MMP loss and mPTP opening in rat NP cells were largely reversed by necroptosis specific inhibitors. (A) Representative dot plot of MMP in NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours were detected by flow cytometry after JC-1 staining. (B) Typical fluorescence photomicrograph of MMP loss in NP cells by LSM. (C) Typical fluorescence photomicrograph of MPTP in NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours were observed under LSM. (D) The quantitative relative fluorescence intensity (RFI) of MPTP in NP cells by flow cytometry. Scale bars = 20 $\mu$M, 50 $\mu$M. Values are expressed as mean $\pm$ SD from four independent experiments (*p 0.05, ** p 0.01, *** p 0.001 vs. control).

To investigate ROS in inflammation-mediated NP cells necroptosis, ROS generation was measured after DCFH-DA staining. Compared with 0 hour, the DCF positive ratio was increased after 48 hours TNF-$\alpha$ or IL-1$\beta$ treatment (Fig. 7A). Then we performed fluorescence detection, which was consistent with the result of flow cytometry. The fluorescence intensity of DFCH-DA markedly increased following 48 hours TNF-$\alpha$ or IL-1$\beta$ treatment (Fig. 7B). Meanwhile, under inflammatory conditions, the ROS levels were notably attenuated when treated with Nec-1, GSK872, or NSA (Fig. 7A,B). These results suggest that the inflammation-induced oxidative stress in NP cells were largely reversed by necroptosis specific inhibitors.

Fig. 7.

The inflammation-induced oxidative stress in rat NP cells were largely reversed by necroptosis specific inhibitors. (A) Representative plots of ROS in NP cells exposed to 50 ng/mL TNF-$\alpha$ or 20 ng/mL IL-1$\beta$ for 48 hours were detected by flow cytometry after the labeling of fluorescent probe DCFH-DA. (B) Visually observe the fluorescence intensity of ROS in NP cells through LSM. Scale bars = 50 $\mu$M.