1. Introduction
Low back pain (LBP), which has become a serious public health problem, ranking
sixth in the global burden of disease, is a very common clinical symptom, and
more than 80% of people have experienced LBP torture during their lifetime
[1, 2, 3]. Intervertebral disc (IVD) degeneration is the main pathogenic factor of
LBP [4]. IVD is a fibrocartilage disc located between two adjacent vertebral
bodies. It is composed of three parts: the gel-like nucleus pulposus (NP), the
inner and outer fibrous annulus (AF), and the upper and lower cartilage endplates
(CEP). The NP degeneration is the primary factor of IVD degeneration and plays a
central role in the degeneration process [5, 6], which is closely related to NP
cells death [7, 8]. Among them, apoptosis and autophagic death are often reported
in previous researches. However, no ideal regulatory target has
been found to efficiently prevent NP cells death during multiple stages of disc
degeneration.
Necroptosis is a type of death with necrosis-like morphological
characteristics. Unlike apoptosis, necroptosis exhibits caspase-independent
characteristics; unlike traditional necrosis, it is a precisely regulated form of
death. Due to that it breaks the traditional view that necrosis cannot be
controlled, it has been listed as a newest type of programmed death following
apoptosis and autophagic cell death [9, 10]. It has made important breakthroughs
in tumors, brain injury, inflammatory diseases, etc., and is expected to become a
new target for the treatment of many clinical diseases [11, 12, 13]. In most cases,
the receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein
kinase 3 (RIPK3)/mixed series protein kinase-like domain (MLKL) axis is the
classic signaling pathway to initiate and mediate necroptosis [14, 15]. However,
literature has reported that activating the RIPK3/MLKL signal axis can also
effectively mediate necroptosis, which does not depend on RIPK1
activation [16]; in addition, study has even confirmed that RIPK1 can
inhibit RIPK3/MLKL-mediated necroptosis [17]. In other words, the
regulatory mechanism of necroptosis is a complex process involving the expression
and regulation of a series of molecules. Chen et al. [18] reported for
the first time that necroptosis mediated by the RIPK1/RIPK3/MLKL pathway is
closely involved in compression-induced NP cells death. Then, in Jun 2018 they
once again reported that RIPK1-mediated mitochondrial dysfunction is closely
related to compression-induced NP cells necroptosis and apoptosis [19],
which provides a new direction for the study of related mechanisms of IVD
degeneration.
The IVD degeneration is accompanied by inflammation, and the inflammation
further intensifies the degeneration, forming a cascade amplification effect
similar to the “inflammation waterfall” [20, 21]. The infiltration of the IVD
mediated by inflammation is also an important pathological basis for the
occurrence and development of disc degeneration and discogenic LBP [22, 23]. Thus
far, there is no report about inflammation-mediated necroptosis of NP cells. In
view of the fact that inflammation plays a key role in the occurrence of
necroptosis, necroptosis also closely regulate the occurrence and development of
inflammation; therefore, further study whether inflammatory conditions can
mediate necroptosis of NP cells and clarify the precise molecular regulation
mechanism, is expected to open up a new idea for effectively inhibition of NP
cells death.
Regarding the underlying mechanism of necroptosis, the mitochondrial signaling
pathway is a hot research topic [24]. Mitochondrial division and fusion are not
only pivotal for the maintenance of mitochondrial inheritance and its own
functions, but also for energy metabolism, aging and cell death [25, 26].
Mitochondrial is the regulatory center of apoptosis, autophagy and necroptosis.
Literature reported that mitochondrial dysfunction caused by excessive
mitochondrial division, such as increased opening of mitochondrial membrane pores
(MPTP), decreased mitochondrial membrane potential (MMP), increased mitochondrial
ROS production, and ATP depletion, are closely involved in necroptosis [27, 28].
However, there is still study implying that necroptosis does not depend on
mitochondrial function damage such as increased opening of MPTP and decreased MMP
[29]. Mitochondrial signaling pathways and necroptosis are intricate, and more
researches are needed to clarify.
In the present study, we systematically addressed whether
inflammation-induced NP cells necroptosis. This study is also aimed
at exploring the precise mechanism of its occurrence, such as mitochondrial
dysfunction and oxidative stress, and ultimately providing a brand new and more
efficient strategy to prevent NP cells death.
2. Materials and Methods
2.1 NP Cells Isolation and Culture
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 fragments. The isolated fragments were digested with
0.25% type II collagenase (Sigma, USA) at 37 C for 30 minutes and
filtered through a 70 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.
2.2 Inflammatory Stimulation was Applied to Rat NP Cells
TNF- and IL-1 are the extremely broad studied inflammatory
factors in IVD degeneration [30, 31, 32, 33]. Referring to previous research, the
intervention concentration of TNF- or IL-1 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).
2.3 Cell Viability Detection
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 10 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 L of CCK-8 solution (Dojindo, Japan) and 100 L of
DMEM-F12 were added to each well and incubated for 2 hours at 37 C. The
cell viability was quantified by detecting the absorbance at 450 nm using a
spectrophotometer (ELx808 Absorbance Microplate Reader, Bio-Tek, USA).
2.4 Propidium Iodide (PI) Positive Ratio Assay
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).
2.5 Lactate Dehydrogenase (LDH) Release Ratio
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.
2.6 The Morphological Changes of NP Cells
The NP cells were exposed to TNF- or IL-1 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).
2.7 Live and Dead Cell Staining
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 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 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).
2.8 Mitochondrial Membrane Potential (MMP) Evaluation
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.
2.9 Measurement of Mitochondrial Permeability Transition Pore (MPTP)
Opening
The MPTP of NP cells was measured by MPTP Fluorescence Assay Kit (Genmed,
China). At each time point, cells were collected, then 500 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 C for 20 minutes. Finally, the sample was
resuspended in Reagent A and analyzed using flow cytometry and LSM.
2.10 Detection of Reactive Oxygen Species (ROS)
ROS levels in the NP cells were analyzed using the ROS-specific fluorescent
probe 2′,7′-dichlorofluorescin diacetate (HDCF-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
M HDCF-DA at 37 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.
2.11 Transfection of Small Interfering RNA
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 cell using
lipofectamine RNAi MAX (invitrogen). 24 hours later, the transfected cells were
digested and recultivated for subsequent experiments.
2.12 Immunofluorescence Staining
Following 0, 24, 48, 72 hours TNF- or IL-1 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 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.
2.13 Quantitative Real-Time Polymerase Chain Reaction (RT-PCR)
Analysis
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 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′.
2.14 Western Blotting Analysis of the Protein Expression
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 C for 20
minutes, sonicated for 1 minute, and centrifuged at 12000 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 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).
2.15 Statistical Analysis
The data are expressed as the mean values 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.
3. Results
3.1 The Activation of the RIPK1/RIPK3/MLK Pathway of NP Cells is
Positively Correlated with the Inflammation Treatment Time
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- or
20 ng/mL IL-1 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- or 20 ng/mL IL-1 treatment for 24, 48 and 72 hours (Fig. 1C,D). Also, through immunofluorescence detection, we intuitively observed that
with the prolongation treatment of TNF- or IL-1, 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- or 20 ng/mL
IL-1 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- or 20 ng/mL IL-1
for 0, 24, 48 and 72 hours. (E) The fluorescence photomicrograph of p-MLKL
expression detected by immunofluorescence staining. Scale bars = 50 M.
Values are expressed as mean SD from three independent experiments
(*p 0.05, ** p 0.01, ***p 0.001 vs.
control).
3.2 Necroptosis is Closely Involved in Inflammation-Induced NP Cells
Death
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- treatment or 20 ng/mL
IL-1 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- or 20 ng/mL IL-1 treatment for 24, 48 and
72 hours, which were effectively inhibited by 20 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- or 20 ng/mL IL-1 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 M), GSK872 (5 M) and NSA (5 M) to perform the
follow-up experiments. Supplementary material is the detailed screening process
(Supplementary Material). As expected, Nec-1 (20
M) can effectively inhibit the increased PI positive rate of NP cells
induced by TNF- and IL-1 (Fig. 2E–G). After systematic
analysis and comparison, we chose 50 ng/mL TNF- or 20 ng/mL
IL-1 inflammatory factors treatment for 48 hours throughout the
following experiments.
Fig. 2.
The protective effects of Nec-1 (20 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- or 20 ng/mL
IL-1 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- or 20 ng/mL
IL-1 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 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 M) and MLKL specific inhibitor NSA
(5 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- or IL-1 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 M), GSK872 (5 M) or NSA (5 M),
the cell viability of NP cells exposed to 50 ng/mL TNF- or 20 ng/mL
IL-1 for 48 hours was measured using the CCK-8 assay. (B) The
cytotoxicity of NP cells exposed to 50 ng/mL TNF- or 20 ng/mL
IL-1 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 SD from three independent experiments
(*p 0.05, ** p 0.01, *** p 0.001
vs. control).
3.3 The Morphological Changes of NP Cells Treated
with Necroptosis Specific Inhibitors
After 48 hours exposure of NP cells to TNF- or IL-1, 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- or IL-1 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- or 20 ng/mL
IL-1 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 M.
3.4 The Inflammation-induced NP Cells Death is
Inhibited by SiRIPK3 or SiMLKL, but Aggravated by SiRIPK1
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- or IL-1
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- or
IL-1 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- or 20 ng/mL IL-1 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- or 20 ng/mL IL-1 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- or 20 ng/mL IL-1 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
SD from three independent experiments (*p 0.05, **
p 0.01, *** p 0.001 vs. control).
3.5 The Inflammation-Induced MMP Loss and mPTP Opening in NP Cells
Were Largely Reversed by Necroptosis Specific Inhibitors
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- or IL-1, 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- or IL-1 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- or 20 ng/mL IL-1 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- or 20 ng/mL
IL-1 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 M, 50 M. Values are expressed as mean SD from four
independent experiments (*p 0.05, ** p 0.01,
*** p 0.001 vs. control).
3.6 The Inflammation-Induced Oxidative Stress in NP Cells Were
Largely Reversed by Necroptosis Specific
Inhibitors
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- or IL-1
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- or IL-1 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- or 20 ng/mL IL-1 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 M.
4. Discussion
The IVD degeneration not only involve a large population of middle-aged and
elderly people, but also show a rising trend among young people [34, 35]. The
conservative therapy and surgical therapy are currently used in clinical
practice. Conservative therapy can alleviate clinical symptoms to a certain
extent, but cannot reverse the biological function of the degenerated IVD; while
the surgical therapy represented by discectomy and fusion have problems such as
the inability to restore the normal height and weight-bearing capacity of IVD,
and even aggravate problems such as degeneration of adjacent segments [36, 37].
Therefore, in-depth research on the precise molecular mechanism of IVD
degeneration and exploring new ways to prevent and treat IVD degeneration is a
major demand in the health field.
The interaction between NP cells death and inflammation plays a key role in the
occurrence and development of IVD degeneration [38, 39]. Necroptosis is generally
considered to be a death mode that aggravates the development of inflammation.
For example, in a study of TNF--mediated kidney inflammation and injury
[40], inhibiting inflammation can restore kidney function to a large extent,
which is closely related to the down-regulation inflammation can largely inhibit
macrophage polarization and necroptosis of kidney tissue. In another study of
cerebral ischemia-reperfusion model, microglioma cells can produce a certain
concentration of TNF-, which in turn mediates endothelial cell
necroptosis and finally breaks the blood-brain barrier, aggravates brain damage,
and further promotes the development of inflammation and brain tissue necrosis
[41]. Contrary to the above effects, necroptosis can also limit and eliminate
inflammation. For example, when inflammatory factors such as TNF- and
Fas mediate necroptosis [42], if cells do not undergo death, they can synthesize
and release more inflammatory factors. At this time, inhibiting necroptosis can
aggravate the synthesis and release of inflammatory factors [42]. In the process
of skin damage caused by staphylococcus epidermidis infection [43], activation of
RIPK1/RIPK3/MLKL signal axis-mediated necroptosis effectively inhibit the
IL-1 expression in epidermal cell and excessive inflammation activation,
while down-regulation of inflammation can in turn produce a certain degree of
negative feedback on necroptosis, and the two work together to repair skin
damage. To sum up: there is a close relationship between necroptosis and
inflammation, but their regulatory effects are diversified, depending on
different environments or different intervention methods.
In current study, following 50 ng/mL TNF- treatment or 20 ng/mL
IL-1 treatment for 24, 48 and 72 hours, the Western blot results
demonstrated that the expression levels of RIPK1, pRIPK1, RIPK3, pRIPK3, MLKL,
and pMLKL, especially pRIPK1, pRIPK3, and pMLKL, were all notably increased. The
gene expression trend of RIPK1, RIPK3 and MLKL is highly consistent with the
protein expression level. Besides, through immunofluorescence detection, we
intuitively observed that with the prolongation treatment of TNF- or
IL-1, the expression level of necroptosis downstream core molecule pMLKL
gradually increased. In order to further systematically verify that necroptosis
is involved in NP cells death induced by inflammatory factors, we also employed
RIPK1 specific inhibitor Nec-1, RIPK3 specific inhibitor GSK872 and MLKL specific
inhibitor NSA into current study. During the condition of TNF- or
IL-1 treatment for 48 hours, the results demonstrated that Nec-1, GSK872
or NSA could markedly reverse the decrease of NP cells activity, meanwhile,
Nec-1, GSK872 or NSA efficiently reduced the inflammation-mediated LDH release
and cell death ratio. Taken together, these results indicated that
RIPK1/RIPK3/MLKL-mediated necroptosis was closely involved in
inflammation-induced NP cells death. In view of the in vitro findings
that inflammation-mediated necroptosis of NP cells plays a critical role in NP
cells death. We speculate that in an in vivo animal model of
inflammation-induced IVD degeneration, regulation of NP cells necroptosis is
expected to largely inhibit disc degeneration process.
In order to further clarify the role of RIPK1/RIPK3/MLKL pathway in the process
of inflammation-induced NP cells necroptosis, NP cells were treated with
effective SiRIPK1, SiRIPK3 and SiMLKL sequences respectively. Contrary to Nec-1,
SiRIPK1 treatment reduced the activity of NP cells and exacerbated NP cells death
under inflammatory conditions. As expected, both SiRIPK3 and SiMLKL notably
restrained TNF- or IL-1 induced NP cells death and markedly
up-regulated NP cells activity. That is to say, the inflammation-induced NP cells
death is inhibited by SiRIPK3 and SiMLKL but aggravated by SiRIPK1. Due to that
RIPK1 has various functions, and its different expression levels or the effects
of phosphorylation at different sites vary greatly. After carefully analysis, we
speculate that overexpression of RIPK1 significantly increase cell death, and
moderate expression is extremely necessary to promote cell survival. We will
intensify the exploration of the exact molecular mechanism of SiRIPK1 in
promoting inflammation-induced NP cells death in follow-up studies.
Classically, necroptosis signaling was thought to involve mitochondrial
dysfunction and increased oxidative stress level mainly originated from the
mitochondria in the execution of cell death [44, 45]. The close association
between necroptosis and mitochondrial dysfunction is illustrated in many studies.
For example, the latest literature report that SIRT3 deficiency aggravate
hyperglycemia-induced mitochondrial damage, increased ROS accumulation, promote
necroptosis, possibly activate the NLRP3 inflammasome, and ultimately exacerbate
diabetic cardiomyopathy in the mice [46]. Mitochondrial dysfunction can greatly
promote the occurrence of necroptosis involving a variety of mechanisms,
including production of mitochondrial ROS [47], activation of mitochondrial
phosphatase phosphoglycerate mutase family member 5 [48], or promotion the
opening of MPTP [49]. However, there is still study showing that the occurrence
of necroptosis does not depend on mitochondrial function damage such as increased
opening of MPTP and decreased MMP [29]. The current study demonstrates that,
under inflammation treatment conditions, Nec-1, GSK872, or NSA may via inhibition
mPTP opening and MMP loss to alleviate NP cells necroptosis; meanwhile, the
results suggest that the TNF- or IL-1 induced oxidative stress
in NP cells were largely reversed by necroptosis specific inhibitors. The above
results suggested that taken mitochondria as the target to improve mitochondrial
function, which can effectively inhibit inflammation-mediated NP cells death by
inhibiting mitochondrial dysfunction-mediated necroptosis.
At present, the efficacy of these inhibitors are mainly reflected by cell
experiments, and there are few studies focus on in vivo animal
experiments to verify the effects of these drugs. In animal model studies and
clinical studies related to IVD degeneration, there is no report about that
necroptosis inhibitors can directly improve IVD degeneration. This is the key
content that our research group is currently working on. In our study, several
limitations need to be pointed out. First, no in vivo experiments were
conducted. The drug concentrations of related inhibitors screened by cell
experiments are difficult to apply to animal models. Hence, we intend to
investigate whether inflammatory stimulation mediates NP cells necroptosis
through mitochondrial function disfunction and oxidative stress pathway
in vivo in subsequent studies. Only in this way can we better translate
to animal models and clinical research. Second, the disc microenvironment is
intricate in nature. In addition to biomechanical loading, low nutrient levels,
hypoxia, high aciditye, high osmolarity, etc., also play crucial role in NP cells
death and IVD degeneration. We are currently considering a composite model to
better model the actual microenvironment of IVD degeneration. Therefore,
in vivo animal experiments need to be strengthened in future
research.
5. Conclusions
In conclusion, this study confirmed that mitochondrial dysfunction and
oxidative stress act as a crucial role in NP cells necroptosis under inflammation
condition. This finding introduces a new perspective to inhibition NP cells
death, and it is extremely expected to offer a more efficient
strategy of delaying or even retarding IVD degeneration.
Author Contributions
CC, ZMS and JZ designed the research. CC, ZMS, ZDL and
SFC performed the experiments. CC, MZ and ZYM acquired
and analyzed the data. CC, YHG and SLC contributed to writing
of the manuscript. Finally, all authors have reviewed
and approved the final submitted manuscript. The integrity
of this work is guaranteed by CC and JZ.
Ethics Approval and Consent to Participate
All experimental procedures were approved by the Animal Care and Ethics
Committee of Zhengzhou University (Ethic approval code is AF/SC-08/04.0).
Acknowledgment
Not applicable.
Funding
This study was supported by the Ministry of Science and Technology National Key
Research and Development Program (122300411149), the Natural Science Foundation
of Henan Province (182300410349), and the Joint Construction Project of Henan
Provincial Health Committee and Ministry of Health (201701017).
Conflict of Interest
The authors declare no conflict of interest.