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Scientific RepoRts | 7:41067 | DOI: 10.1038/srep41067
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IL-1β induces apoptosis and
autophagy via mitochondria
pathway in human degenerative
nucleus pulposus cells
Jieliang Shen, Shengxi Xu, Hao Zhou, Huzhe Liu, Wei Jiang, Jie Hao & Zhenming Hu
IL-1β has been reported highly expressed in degenerative intervertebral disc, and our previous study
indicated IL-1β facilitates apoptosis of human degenerative nucleus pulposus (NP) cell. However,
the underlying molecular mechanism remains unclear. We here demonstrate that IL-1β played a
signicantly pro-apoptotic eect under serum deprivation. IL-1β decreased Bcl-2/Bax ratio and
enhanced cytochrome C released from mitochondria to cytosol, which proved mitochondria-meidated
apoptosis was induced. Subsequently, mitochondria damage was detected under IL-1β stimualtion.
In addition, IL-1β-mediated injuried mitochondria contributes to activate autophagy. However,
pretreatment with the autophagy inhibitor 3-methyladenine showed the potential in further elevating
the apoptosis rate induced by IL-1β in NP cells. Our results indicated that the mitochondrial pathway
was involved in IL-1β-induced apoptosis of NP cells. Meanwhile, the damaged mitochondria-induced
autophagy played a protective role against apoptosis, suggesting a postive feedback mechanism under
inammatory stress.
Intervertebral disc degeneration (IVDD) is an age-dependent molecular degenerative process, and its associ-
ated back pain generates a heavy economic burden on the aging society
1
. e inner nucleus pulposus (NP) tis-
sue changes most during degeneration, including cell death enhancement, extracellular matrix destruction and
inammatory factors accumulation, which result in reduction of the spinal biomechanics and cause back pain
2
.
Interleukin (IL)− 1β is considered to be the most important cytokine involved in multiple pathological processes
of IVDD
3,4
. Our previous work has indicated that IL-1β promotes the human degenerative NP cell apoptosis via
its downstream signaling target NF-κ B
5
. However, the underlying mechanism of IL-1β -induced apoptosis in
degenerative NP cells remains enigmatic.
Progressive accumulation of damaged macromolecules leading to cell dysfunction and death is a major char-
acteristic of age-related diseases
6
. Mitochondria are master subcellular organelles that produce and supply energy
to maintain intracellular homeostasis. Under stressed conditions, dysregulated mitochondria release a set of mol-
ecules to activate downstream mitochondrial apoptotic pathway
7
. Recent evidence has suggested IL-1β induces
excessive accumulation of ROS in bovine NP cells, which causes oxidative stress
8
. However, there is no direct evi-
dence whether IL-1β could induce mitochondria-mediated apoptosis in human NP cells. In addition, autophagy
is found to be activated by damaged mitochondria to maintian intercellular homeostasis, and regulate cellular loss
against apoptosis
9
. Our previous work also conrmed that promoting autophagy could inhibit apoptosis in human
NP cells
10
. Up to date, no study has concerned the role of IL-1β on the apoptosis and autophagy in degenerative
NP cells. In the present study, we set out to investigate whether IL-1β induced apoptosis via mitochondria path-
way, if so, whether the damaged mitochondria would further activate autophagy. We believe to clarify the apop-
tosis and autophagy responding to IL-1β stress is important for better understanding the mechanism of IVDD.
Results
IL-1β expression and cell apoptosis in situ detection. First, we evaluated the relationship between
IL-1β expression and apoptosis incedence in NP tissues. Representative MRI scans of patients with LVF and
Department of Orthopaedic Surgery, the First Aliated Hospital of Chongqing Medical University, Chongqing, China.
Correspondence and requests for materials should be addressed to J.H. (email: hjie2005@aliyun.com) Z.H. (email:
spinecenter_hu@126.com)
received: 09 August 2016
accepted: 14 December 2016
Published: 25 January 2017
OPEN
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Scientific RepoRts | 7:41067 | DOI: 10.1038/srep41067
LDH were shown in Fig.1A. TUNEL assay showed that the number of TUNEL positive cells was a 37.4% and
8.2% amount in the degenerative and normal group, respectively, suggesting increased cell apoptosis was demon-
strated in degenerative NP tissues (Fig.1B). Immunological histological chemistry (IHC) for IL-1β showed that
cell clusters were formed within NP tissue in degenerative disc, meanwhile, IL-1β immunostaining was generally
observed in the cytoplasm of NP cells in all samples. However, IL-1β showed siginicantly more immunopositive
cells in the degenerative group (Fig.1C). In parallel, western blot indicated that IL-1β protein expression was
Figure 1. IL-1β expression is associated with cell apoptosis in NP tissues. (A) Representative lumbar MRI
of one patient with LVF (le) and the other with LDH (right) were classied according to Prrmann’s grading
system. e red arrows represented the grade II disc in normal group and grade IV disc in degenerative
group. (B) In situ apoptotic cells were determined by TUNEL staining in NP tissues. C and D, In situ IL-1β
protein expressions from normal and degenerative NP tissues were determined by immunological histological
chemistry and western blot. *P < 0.05 Vs. LVF group. Bars = 100 µ m.
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Scientific RepoRts | 7:41067 | DOI: 10.1038/srep41067
markedly higher in the degenerative NP tissues from LDH patients, compared to those from nondegenerative
LVF patients (Fig.1D).
IL-1β induced cell apoptosis under serum deprivation. IL-1β stimulation under serum-free medium
led to obviously morphological changes that cells turned slender with plasma membrane blebbing, and Hoechst
33258 staining showed more apoptotic cells with high bright uorescent nuclei. However, no signicant changes
were observed when NP cells were cultured under complete culture medium with 0 or 10ng/ml IL-1β (Fig.2A).
Flow cytometric analysis with Annexin-V/PI stainning indicated that serum deprivation led to a moderate
increase in cell apoptosis, but IL-1β further enhanced the number of apoptotic cells (Fig.2B). Associated with
increased apoptotic incidence, colorimetric assay revealed that the activities of caspase-3 and -9 increased to ~2.2
folds and ~1.7 folds under serum deprivation, but IL-1β signicantly enhanced this eect on caspase activation,
correspondingly up to ~3.4 folds and ~2.4 folds, compared with control group. Il-1β in complete culture medium
showed no signicant eect on caspase-3 and -9 activities (Fig.2C).
IL-1β induced mitochondria-meidated apoptosis. Since caspase-3 and -9 were found to be activated
under IL-1β treatment, the mitochondrial apoptotic pathway were rst analyzed by western blot. Results showed
that IL-1β signicantly increased pro-apoptotic protein Bax and decreased anti-apoptotic protein Bcl-2 (Fig.3A).
Simultaneously, expression of cytochrome c from mitochondria decreased and that from cytoplasm increased
under IL-1β treatment, suggesting cytochrome c was translocated from mitochondria to cytoplasm (Fig.3B).
ROS accumulation is another important mitochondrial event during apoptosis. Indeed, there is signicantly
increased ROS associated with IL-1β treatment compared to serum deprivation and control group (Fig.3C). All
these resutls indicated that the mitochondrial pathway was involved in the IL-1β induced apoptosis of NP cells.
IL-1β induced mitochondria damage. Damaged mitochondria were suggested to trigger downstream
apoptotic pathway. To directly test the role of IL-1β on the mitochondria, we rst measured mitochondrial
membrane potential (∆ Ψ m). Aer IL-1β treatment, the red/green uorescence ratio of NP cells signicantly
decreased, as observed by uorescence microscope and ow cytometry, suggesting ∆ Ψ m decreased by IL-1β
(Fig.4A). To conrm this nding, TEM was used to assess mitochondrial integrity and state. Serum deprivation
induced a few swollen mitochondira in NP cells, but accumulation of highly damaged, electron-dense mitochon-
dria were observed under IL-1β treatment (Fig.4B). Energy production is the most imporant mitochondrial
function, we analyzed the changes in ATP levels following IL-1β treatment. Serum deprivation alone seemed no
inuence on the ATP level, but exposure to IL1-β signicantly decreased ATP level (Fig.4C). All these results
demonstrated that IL-1β cuased mitochondrial damage and led to cell apoptosis in degenerative NP cells.
IL-1β stimulated autophagic ux in NP cells. Autophagy is found to be triggered by mitochondrial
damage, which is a quality control process to mediate selective injuried mitochondira removal. To determine
the activation of autophagy under mitochondrial dysfunction conditions, western blot showed that IL-1β sig-
nicantly changed the autophagic marker LC-3II and P62/SQSTM1 expressions (Fig.5A). Fluorescence-based
detection of LC-3 isoforms were directly observed in NP cells, IL-1β treatment resulted in a signicant improve-
ment in the LC3 puncta, indicative of increased autophagosome formation (Fig.5B). ese results indicated that
IL-1β actually up-regulated the autophagy activity in NP cells. To assess this autophagy ux, we treated cells with
a lysosomal inhibitor, balomycin A1. e addition of balomycin A1 further increased LC3-II and p62/SQSTM1
accumulation, compared with cells treated with IL-1β only, which indicated IL-1β -mediated autophagy is not
because of reduced autophagosome turnover, but increased autophagic ux (Fig.5C). In addition, TEM obser-
vation found typical double-membraned autophagosomes in NP cells from serum deprivation group. Moreover,
mitochondrial fragmentations were observed and sections of the mitochondrion appeared to be surrounded by
double-membrane proles under IL-1β treatment (Fig.5D).
IL-1β-meidiated autophagy played pro-survival function. To evaluate the role of IL-1β -mediated
autophagy on the cell apoptosis, NP cells in serum deprived medium were pre-treated with 3MA to inhibit
the autophagy induction. 3MA treatment signicantly attenuated the LC3-II expression that indicated 3MA
decreased the autophagy incidence by IL-1β treatment (Fig.6A). Flow cytometric analysis revealed that obvi-
ously increased apoptotic ratio was observed under 3MA treatment (Fig.6B). e result suggested the autophagy
activation induced by IL-1β may be a self-protective mechanism in NP cells. Moreover, decreased ∆ Ψ m were
conrmed following autophagy inhibition by 3MA (Fig.6C). e selective degradation of mitochondria by auto-
phagy is oen based on Parkin-depend manner
11
. Western blot analysis showed that serum starvation had little
eect on the translocation of Parkin, but IL-1β recruited Parkin from cytoplasm onto depolarized mitochon-
dria (Fig.6D), which indicated that IL-1β -meidiated autophagy may be a critical homeostasis mechanism, as
mitophagy.
Discussion
e results of this study demonstrate for the rst time that IL-1β facilitates apoptosis through mitochondrial
apoptotic pathway in human degenerative NP cells. In turn, the accumulation of damaged mitochondria
enhances autophagy activation, which plays a pro-survival role against apoptosis. ese ndings suggest there
existing a functional loop between autophagy, apoptosis and IL-1β in NP cells, of which mitochondria seems to
be a key pivot.
In this study, IHC results clearly showed that in addition to immune cells, NP cells themselves are able to
produce IL-1β . Furthermore, cell apoptotic rate and IL-1β expression were found to be more prominent in the
dengerative NP tissues. In consideration of nutrition deciency for NP cells during IVDD
12
, and our nding that
appearance of cell clusters in degenerative NP tissues, these results suggested that degenerative NP cells settle in
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Scientific RepoRts | 7:41067 | DOI: 10.1038/srep41067
Figure 2. IL-1β induces cell apoptosis under serum deprivation. (A) Morphologic changes in apoptotic
NP cells. Le series is phase-contrast photomicrograph of NP cells and right series is apoptotic nuclei brightly
stained by Hoechst 33258. Amplication: 200× . (B ) Apoptotic cells were stained with Annexin V-PE and PI,
and analyzed by ow cytometry. (C) Caspase-3 and -9 activity were determined by special Caspase Activity
Assay Kits. Relative activity of caspase-3 and -9 was represented as fold changes to control (10%FBS + 0 ng/ml
IL-1β group). *P < 0.05 and
#
P > 0.05 Vs. 10% FBS group, **P < 0.05 Vs. 0% FBS group.
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Scientific RepoRts | 7:41067 | DOI: 10.1038/srep41067
Figure 3. Eect of IL-1β on apoptosis mediated through the mitochodrial pathway in NP cells. (A) Western
blot analysis for the protein expression of Bax, Bcl-2. e ratio of Bax/Bcl-2 was quantied. (B) Western blot
analysis for the protein expression of cytochrome c from mitochondria and cytoplasm, respectively. e ratio
of cytochrome c (mito)/cytochrome c (cyto) was quantied. (C) e intracellular ROS levels were measured
by ow cytometry through DCFH-DA staining. e mean uorescence intensity (MFI) of 10,000 cells was
recorded. *P < 0.05 Vs. 0% FBS group.