The mitochondria‐targeted anti‐oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance

Abstract Objective Mitochondrial dysfunction, oxidative stress and nucleus pulposus (NP) cell apoptosis are important contributors to the development and pathogenesis of intervertebral disc degeneration (IDD). Here, we comprehensively evaluated the effects of mitochondrial dynamics, mitophagic flux and Nrf2 signalling on the mitochondrial quality control, ROS production and NP cell survival in in vitro and ex vivo compression models of IDD and explored the effects of the mitochondria‐targeted anti‐oxidant MitoQ and its mechanism. Material and methods Human NP cells were exposed to mechanical compression to mimic pathological conditions. Results Compression promoted oxidative stress, mitochondrial dysfunction and NP cell apoptosis. Mechanistically, compression disrupted the mitochondrial fission/fusion balance, inducing fatal fission. Concomitantly, PINK1/Parkin‐mediated mitophagy was activated, whereas mitophagic flux was blocked. Nrf2 anti‐oxidant pathway was insufficiently activated. These caused the damaged mitochondria accumulation and persistent oxidative damage. Moreover, MitoQ restored the mitochondrial dynamics balance, alleviated the impairment of mitophagosome‐lysosome fusion and lysosomal function and enhanced the Nrf2 activity. Consequently, damaged mitochondria were eliminated, redox balance was improved, and cell survival increased. Additionally, MitoQ alleviated IDD in an ex vivo rat compression model. Conclusions These findings suggest that comodulation of mitochondrial dynamics, mitophagic flux and Nrf2 signalling alleviates sustained mitochondrial dysfunction and oxidative stress and represents a promising therapeutic strategy for IDD; furthermore, our results provide evidence that MitoQ might serve as an effective therapeutic agent for this disorder.


| INTRODUC TI ON
Intervertebral disc (IVD) degeneration (IDD) is widely acknowledged to be the primary cause of low back pain (LBP)-a chronic, expensive and common musculoskeletal disorder that is prevalent in developed and developing countries. 1 The IVD consists of three inter-related structures: the nucleus pulposus (NP), the annulus fibrosus (AF) and the cartilaginous endplate. The centrally situated NP allows the IVD to maintain a high water content and thereby withstand mechanical impact. 2 Excessive apoptosis of NP cells can trigger metabolic disorders within the NP extracellular matrix, resulting in the destruction of normal IVD structure and physiological function that eventually leads to IDD. 2,3 Thus, determination of the key molecular mechanisms of NP cell apoptosis would be of great significance to the management of IDD.
The maintenance of a healthy and functional mitochondrial network is critical during development as well as for physiological adaptations and responses to stress in the body. 4,5 Mitochondria are not only the main cellular source of reactive oxygen species (ROS), they are also particularly susceptible to oxidative injury. 6 Recently, studies have verified the presence of oxidative stress and increased concentrations of oxidation products in degenerated discs. [7][8][9] Additionally, oxidative stress and subsequent mitochondrial dysfunction participate in the intrinsic pathway of cellular apoptosis, which have been confirmed in NP cell death and IDD induced by various risk factors. [10][11][12][13][14][15] Moreover, mitochondrial dysfunction induced by ROS can further enhance the production of ROS, leading to a feed-forward vicious cycle between mitochondria and ROS that causes sustained oxidative damage. 16 Thus, in addition to direct intervention targeting oxidative stress, the role of appropriate mitochondrial quality control in NP cell survival under pathological conditions is also worth studying.
Mitochondria are highly dynamic organelles that continuously undergo fission and fusion, known as mitochondrial dynamics. 17 The subtle equilibrium of mitochondrial dynamics is conducive to the maintenance of a healthy pool of mitochondria. 18 Destruction of this equilibrium is implicated in various human diseases including cancer, type 2 diabetes and osteoarthritis. 4,19,20 In addition to mitochondrial dynamics, the timely and selective removal of damaged mitochondria through an autophagic process termed mitophagy is important for maintaining mitochondrial quality. 5 Impairment of mitophagic flux results in the accumulation of dysfunctional mitochondria and ROS associated with various diseases. 21,22 It has been shown that mitophagy and mitochondrial dynamics are inter-related yet distinct processes. During mitochondrial fission, the damaged daughter mitochondria are first separated then targeted by the lysosome for elimination, preventing damaged mitochondria from being incorporated back into the active and healthy mitochondrial pool via fusion. 4 The occurrence of oxidative stress caused by ROS overproduction is inseparable from anti-oxidant system defects. Nuclear factor E2-related factor 2 (Nrf2) is a key redox-sensitive transcription factor that regulates the anti-oxidant defence system by activating the expression of a number of cytoprotective genes in response to oxidative stress. 23 In addition, there is evidence that Nrf2 modulates mitochondrial function and metabolism. 24 Therefore, the present study investigated the maintenance of mitochondrial homeostasis via the simultaneous regulation of mitochondrial dynamics, mitophagy and Nrf2 signalling using a pharmacological method to rescues mitochondrial dysfunction and NP cell apoptosis under pathological conditions.

Mitoquinone (MitoQ)-a mitochondria-targeted anti-oxidant-
consists of co-enzyme Q10 and a TPP cation that can easily accumulate several 100-fold within the mitochondria, thereby making it more powerful than untargeted anti-oxidants in preventing mitochondrial oxidative damage. 25 In vitro and in vivo studies have demonstrated that MitoQ is protective against many oxidative damage-related diseases. [26][27][28][29][30][31][32][33] In addition, daily oral doses of 40 or 80 mg MitoQ have been shown to protect against liver damage in hepatitis C patients 34 and can be safely administered to Parkinson's disease patients for up to 1 year. 35 There is evidence that the protective effect of MitoQ is partially mediated by the modulation of mitochondrial dynamics, mitophagy and Nrf2 signalling. 33,36 These potential therapeutic benefits are encouraging and warrant further investigation of MitoQ as an intervention to prevent the development of IDD.
As a load-absorbing structure of the spine, the IVD is subjected to various magnitudes of mechanical compression throughout daily life. [37][38][39] Inappropriate or excessive compression applied to IVDs is an important contributing factor in causing IDD. 10,[40][41][42][43][44] Therefore, the in vitro and ex vivo models of IDD induced by compression are more in line with the pathogenesis of IDD. Studies have reported that ROS overproduction, mitochondrial dysfunction and apoptosis were induced by compression at a magnitude of 1.0 MPa in NP cells, all of which were closely related to IDD. 10,11,45,46 Thus, we for the first time systematically investigated the changes in mitochondrial dynamics, mitophagy and Nrf2 signalling in human NP cells under 1.0 MPa compression. In addition, we studied the effect of MitoQ on human NP cells, as well as the roles of mitochondrial dynamics, mitophagy and Nrf2 signalling in the action of MitoQ. An ex vivo compression model of rat tail disc degeneration was used to confirm the results of the in vitro experiments. Our aim was to provide novel insights for the development of effective therapeutic strategies to inhibit IDD progression.

| Tissue specimens and cell culture
This study was approved by the Ethics Committee of Tianjin Medical University General Hospital, and written informed consent was obtained from each donor. Nucleus pulposus specimens were collected from 10 IDD patients (6 males and 4 females, 41-69 years of age) suffering from cervical spondylotic myelopathy who received anterior cervical discectomy and fusion at Tianjin Medical University General Hospital. Nucleus pulposus cell isolation and culture were carried out as previously described. 47 During passaging, no significant changes in morphology were observed between primary (passage 0) and later-passage (passage 2) cells.
Therefore, we used second-passage cells cultured in a monolayer for experiments.

| Compression treatment
IVD organs or NP cells were exposed to continuous high pressure in a previously described compression apparatus. 10,48 In brief, the tissues or cells were placed in cell culture plates on the bottom of the compression apparatus, which contained a small amount of distilled water to maintain the moisture level. The compression apparatus was placed in an incubator at 37°C. A mixture of 0.5% CO 2 and 99.5% compressed air was pumped into the apparatus until the pressure reached 1.0 MPa. The control group samples were incubated at 37°C without any compression under the same culture conditions.

| Cell viability assay
According to the manufacturer's instructions, the cytotoxic effect of compression on human NP cells was assessed using a cell counting kit (CCK-8; Dojindo). Briefly, the cells were seeded in 96-well plates and treated with MitoQ or compression. Subsequently, the cells were incubated with 10 μL CCK-8 solution at 37°C for 2 hours. The absorbance at 450 nm was measured using a spectrophotometer (BioTek).

| Western blotting
After cell treatments, total, cytoplasmic and mitochondrial proteins from NP cells were extracted using commercial kits (Beyotime) ac-    (Invitrogen), respectively, as previously described. 15 After labelling, samples were examined using a FACSCalibur flow cytometer (BD Biosciences).

| Measurement of malondialdehyde (MDA) levels
MDA levels were measured using assay kits for MDA (Beyotime) as per manufacturer's instructions.

tors (HanBio Technology) to evaluate the effects of compression
and MitoQ on mitophagic flux. The principle of the assay is based on differences in pH stability between the green and red fluorescent proteins. The mRFP and GFP puncta in each treatment group were detected using a laser-scanning confocal microscope (FV1000; Olympus).

| Measurement of cathepsin B (CTSB) activity, cathepsin D (CTSD) activity and lysosomal pH changes
CTSB and CTSD activities were measured using specific fluorometric assay kits for the two enzymes (BioVision) as described previously. 50 Changes in lysosomal pH were determined using LysoSensor Green DND-189 (Yeasen) as described previously 51 ; this reagent exhibits a pH-dependent increase in fluorescence intensity in acidic environments.

| Assessments of IVD ex vivo compression model
The rat disc tissues from each group were harvested. The specimens were fixed in formaldehyde, decalcified, dehydrated, embedded in paraffin and sectioned at a thickness of 4 μm. The sections were stained with haematoxylin and eosin (HE) and safranin O-fast green (SO), and the histological grades of the specimens were determined to quantify damage based on a previously described method. 52 For TUNEL staining, the sections were handled using a TUNEL Apoptosis Assay Kit (Beyotime) and samples were imaged with a fluorescence microscope (Olympus IX71). Immunohistochemistry was performed as described previously. 53 For immunohistochemical analysis, sections were incubated with primary antibodies against cleaved

| Statistical analysis
Data are presented as the mean ± standard deviation (SD) of at least three independent experiments and were analysed using SPSS version 18.0 software (SPSS Inc, Chicago, IL, USA). Differences between groups were evaluated using Student's t test or one-way analysis of variance (ANOVA) followed by Tukey's test. P < .05 was considered statistically significant.

| Effects of MitoQ on compression-induced cytotoxicity in human NP cells
Compression was used to establish an IDD model in vitro. As shown in Figure

| Effects of MitoQ on compression-induced ROS accumulation, mitochondrial dysfunction and apoptosis in human NP cells
Next, we investigated whether MitoQ protects NP cells from com-

| Effects of MitoQ on compression-induced alterations in mitochondrial dynamics in human NP cells
Migration of Drp1 from the cytoplasm to mitochondria is a pre-requisite for mitochondrial fission. As shown in Figure 3A,B, compression treatment enhanced the translocation of Drp1 to the mitochondria, as evidenced by the stronger colocalization of Drp1 with mitochondria in compression-exposed cells compared to that in control cells,

| Effects of MitoQ-maintained mitochondrial dynamics balance on compression-induced damage in human NP cells
We next investigated whether these beneficial effects of MitoQ are

| Effects of MitoQ on PINK1/Parkin-mediated damaged mitochondrial clearance in human NP cells exposed to compression
We first investigated whether the PINK1/Parkin signalling pathway was activated in human NP cells exposed to compression. Western blot results showed that the total protein expression of PINK1 and Parkin increased after compression treatment ( Figure  Tom20 in human NP cells revealed an elevated colocalization of Parkin and Tom20 in compression-exposed cells compared to control cells ( Figure 5G,H). Next, we assessed the effect of compression in the next step of mitophagy, the engulfment of mitochondria by autophagosomes.
The immunofluorescence results showed that compression treatment promoted colocalization of LC3 and Tom20 ( Figure 5I,J). Moreover, Western blot analysis indicated that the protein level of mitochondrial LC3-II was increased in compression-exposed cells compared with control cells ( Figure 5K,L). These results suggested that compression activates PINK1/Parkin pathway-mediated mitophagy.
Dysfunctional mitochondria are delivered to lysosomes for degradation following their engulfment in autophagosomes, a process that requires undamaged mitophagic flux. Western blot results indicated that compression treatment markedly increased the p62 protein level, but did not decrease the LC3-II/I ratio and Beclin-1 protein level ( Figure 6A,B). This finding was confirmed by transfection of a tandem mRFP-GFP-LC3 construct into human NP cells. As compared with the control group, the compression group showed an increase in yellow but not red puncta ( Figure 6C,D). These data suggested that the mitophagic flux was impaired in compression-exposed human NP cells.
Next, we explored the mechanism underlying mitophagic flux blockade by compression. The activities of CTSB and CTSD-key proteases that degrade trapped cargo in lysosomes-were measured to evaluate lysosomal degradation ability in the treated human NP cells. As shown in Figure 6E,F, compression decreased the activity levels of both enzymes. Low lysosomal pH is required for the maturation and activation of most lysosomal enzymes; thus, we measured the lysosomal pH by LysoSensor Green DND-189 staining. As shown in Figure 6G, compression treatment induced an obvious decrease in fluorescence intensity.
Further, we investigated mitophagosome-lysosome fusion based on LC3-LAMP1 colocalization. Compression led to a significant decrease in LC3-LAMP1 colocalization when compared to the control groups ( Figure 6H,I). The colocalization of Tom20 and LAMP1 was decreased in compression-exposed cells compared to control cells ( Figure 6J,K), suggesting that dysfunctional mitochondria engulfed in mitophagosomes would not be able to enter lysosomes in compression-exposed human NP cells. These data suggested that compression impairs lysosomal function and mitophagosome-lysosome fusion. ( Figure 6E-K). Collectively, these findings suggested that MitoQ promotes PINK1/Parkin-mediated mitophagy and repairs defective mitophagy flux in human NP cells exposed to compression.

| Role of mitophagic flux in the beneficial effects of MitoQ on compression-exposed human NP cells
Then, we knocked down PINK1 and Parkin using siRNAs to interfere with mitophagy activation. As shown in Figure 7A

| Effects of MitoQ on the Nrf2 pathway in compression-exposed human NP cells
The above results indicated that MitoQ markedly alleviated the oxidative stress induced by compression in human NP cells.
Therefore, it was reasonable to assume that the effect of MitoQ Further, we measured the activity of Nrf2 signalling in human NP cells exposed to compression and treated with MitoQ. As shown in Figure 8L-V, compression increased the protein expression of Nrf2 and its downstream targets and the nuclear translocation of Nrf2.
MitoQ treatment further increased the activity of Nrf2 pathway in compression-treated human NP cells ( Figure 8L-V). Overall, these findings suggested that MitoQ can activate the Nrf2 signalling pathway in compression-exposed human NP cells.

| Role of Nrf2 signalling in MitoQ-mediated anti-oxidative stress and anti-mitochondrial disorder in compression-exposed human NP cells
To determine whether and how Nrf2 mediates the protective effects  Figure 10A). With MitoQ treatment, these degenerative changes of the disc structure were alleviated ( Figure 10A).
Histological scores also indicated that MitoQ protected against IDD development ( Figure 10B). The results of TUNEL staining in IVD specimens from rats revealed that the apoptosis rate was de-

| D ISCUSS I ON
In this study, we constructed the IDD model induced by compression and found that compression leads to mitochondrial dysfunction, oxidative stress and apoptosis in human NP cells. MitoQ is a mitochondria-targeted anti-oxidant that has therapeutic effects in numerous mitochondria-related diseases. Therefore, we used MitoQ as a stabilizer of mitochondrial function to treat compression-exposed human NP cells in this study. We found that MitoQ Under normal physiological conditions, mitochondria undergo morphological changes to meet cellular energy requirements.
These changes can occur through sustained fusion and fission.
Excessive mitochondrial fission leads to fragmentation of mitochondria, resulting in mitochondrial dysfunction and ultimately cell death. 58  However, this increase in Nrf2 signalling activity was not sufficient to counteract the pathogenic events induced by oxidative stress in compression-exposed human NP cells. Moreover, MitoQ further promoted Nrf2 expression and activity in human NP cells exposed to compression. siRNA-mediated depletion of Nrf2 and HO-1 partially inhibited the protective actions of MitoQ on compression-induced oxidative stress, mitochondrial impairment and apoptosis. These results suggest that the beneficial effects of MitoQ on compression-exposed human NP cells are partially attributed to the upregulation of Nrf2 expression and activity.

| CON CLUS ION
This study demonstrated the beneficial effects of MitoQ on human NP cell apoptosis in IDD in in vitro and ex vivo models. The underlying mechanism was found to be closely associated with the maintenance of mitochondrial homeostasis and redox balance through restoration of the mitochondrial fission/fusion balance and amelioration of the mitophagic flux disturbance as well as activation of Nrf2 signalling, all of which eventually promoted the survival of human NP cells ( Figure S4). These results suggest that restoring mitochondrial functions and eradicating oxidative insults represent a promising therapeutic strategy for IDD and that MitoQ might serve as an effective therapeutic agent for this disorder.

ACK N OWLED G EM ENTS
This work was supported by the National Natural Science Foundation of China (No. 81871124).

CO N FLI C T O F I NTE R E S T
These authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.