Long Noncoding RNA ANPODRT Overexpression Protects Nucleus Pulposus Cells from Oxidative Stress and Apoptosis by Activating Keap1-Nrf2 Signaling

Oxidative stress and subsequent nucleus pulposus (NP) cell apoptosis are important contributors to the development of intervertebral disc degeneration (IDD). Emerging evidences show that long noncoding RNAs (lncRNAs) play a role in the pathogenesis of IDD. In this study, we investigated the role of lncRNA ANPODRT (anti-NP cell oxidative damage-related transcript) in oxidative stress and apoptosis in human NP cells. We found that ANPODRT was downregulated in degenerative NP tissues and in NP cells treated with tert-butyl hydroperoxide (TBHP, the oxidative stress inducer). ANPODRT overexpression alleviated oxidative stress and apoptosis in NP cells exposed to TBHP, while ANPODRT knockdown exerted opposing effects. Mechanistically, ANPODRT facilitated nuclear factor E2-related factor 2 (Nrf2) accumulation and nuclear translocation and activated its target genes by disrupting the kelch-like ECH-associated protein 1- (Keap1-) Nrf2 association in NP cells. Nrf2 knockdown abolished the antioxidative stress and antiapoptotic effects of ANPODRT in NP cells treated with TBHP. Collectively, our findings suggest that ANPODRT protects NP cells from oxidative stress and apoptosis, at least partially, by activating Nrf2 signaling, implying that ANPODRT may be a potential therapeutic target for IDD.


Introduction
Low back pain (LBP), one of the most common health issues, is a leading cause of disability worldwide and results in an enormous global economic and public health burden [1]. Intervertebral disc (IVD) degeneration (IDD) and secondary pathomorphological changes in spine structure are widely acknowledged as the major causes of LBP [2,3]. The IVD consists of three interrelated structures: the nucleus pulposus (NP), the annulus fibrosus, and the cartilaginous endplates. The centrally situated NP allows the IVD to maintain a high water content and withstand different patterns of mechanical stress [4]. Excessive NP cell apoptosis disrupts normal metabolic activity within the NP, thus disrupting the normal IVD structure and physiological function, which is a key contributor to IDD initiation and progression [5,6].
Oxidative stress results from an imbalance between the production of reactive oxygen species (ROS) and scavenging capacity of the antioxidant system [7]. Oxidative stress leads to the damage of nucleic acids, lipids, and proteins through the production of high levels of ROS, which leads to cytotoxicity. Recent studies have verified the presence of oxidative stress and increased concentrations of oxidation products in degenerated discs [8,9]. Furthermore, oxidative stress and its induced mitochondrial pathway play an important role in NP cell apoptosis and IDD [10][11][12][13]. We and others have previously reported that amelioration of oxidative stress and subsequent NP cell apoptosis has a therapeutic effect on IDD progression [14][15][16]. Therefore, an understanding of the underlying pathway regulating oxidative stress and subsequent NP cell apoptosis would significantly benefit IDD treatment.
Antioxidant system plays an important role in the occurrence of oxidative stress. The Keap1-Nrf2 signaling cascade is considered a central hub that neutralizes ROS and restores cellular redox balance [17][18][19][20]. Under resting conditions, Nrf2 activity is tightly restricted through its interaction with Keap1 in the cytoplasm. Keap1 reportedly serves as a substrate scaffold for Cul3-containing E3 ubiquitin ligase, which can induce ubiquitin-proteasome degradation of Nrf2 [17]. Upon dissociation from Keap1, Nrf2 is translocated into the nucleus, where it initiates the transcription of a battery of antioxidative and cellular defense targets to counteract oxidative stress and modulate redox balance [20]. In a mouse model of IDD, degenerative changes in IVDs in Nrf2knockout mice were more severe than those in wild-type mice [3]. We previously reported that the activation of the Nrf2 cascade is an effective strategy to protect human disc cells from oxidative damage and prevent IDD progression [14,15]. Therefore, Nrf2 signal plays an important role in IDD characterized by oxidative stress.
Many lncRNAs dysregulated in IDD have been previously identified through a lncRNA microarray analysis [33]. Among these differentially expressed lncRNAs, lncRNA AC068196.1 was significantly downregulated in IDD. We named lncRNA AC068196.1 as anti-NP cell oxidative damage-related transcript (ANPODRT). This study investigated the role of ANPODRT in oxidative stress and apoptosis in human NP cells. We showed that ANPODRT alleviates oxidative stress and apoptosis in human NP cells, and the activation of the Keap1-Nrf2 signaling cascade is involved in this process. Our results provide novel insights into the mechanism of oxidative stress and apoptosis in NP cells, with therapeutic implications for treating IDD.

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 16 IDD patients (3 males and 2 females, grade II; 2 males and 1 female, grade III; 2 males and 2 females, grade IV; 1 male and 3 females, grade V). Nucleus pulposus cell isolation and culture were carried out as previously described [34]. Five human NP tissues (grade II) were used for NP cell isolation. Briefly, the NP tissue samples were separated, cut into small pieces, and treated with 0.25% trypsin (Gibco) for 30 min and then 0.2% type II collagenase (Invitrogen) for 4 h at 37°C. After isolation, the NP cells were resuspended in DMEM/F12 containing 15% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin and incubated at 37°C in a humidified 5% CO 2 atmosphere. The culture medium was replaced every three days. When the NP cells grew to 80% confluence, they were detached by trypsinization and passed for expansion. Fluorescently labeled antibodies against NP cell markers (CD24 and KRT18) were used to identify the phenotype of NP cells, as described previously [35]. Cells from the second passage were used in subsequent experiments.

Cell
Transfection. The siRNA targeting ANPODRT, Keap1, Nrf2, and appropriate negative controls were obtained from RiboBio. The overexpression plasmids containing ANPODRT and the matched negative control plasmid were obtained from GeneChem. Cell transfections were performed with lipofectamine 2000 (Invitrogen) based on provided instructions.

Cell Viability Assay.
According to the manufacturer's instructions, the viability of human NP cells was assessed using a cell counting kit (CCK-8; Dojindo). Briefly, the cells were seeded in 96-well plates and exposed to corresponding treatments. 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).

Measurement of ROS and Malondialdehyde (MDA)
Level. After corresponding treatment, the ROS and MDA level in human NP cells were measured using kit for ROS (Beyotime) and MDA (Beyotime), respectively, as per manufacturer instructions.
2.5. Flow Cytometry. Apoptosis levels in human NP cells from each treatment group were assessed using an Annexin V-APC/7-AAD Apoptosis Detection Kit (Yeasen) as previously described. After labelling, samples were examined using a FACSCalibur flow cytometer (BD Biosciences).

Western Blotting and Coimmunoprecipitation (Co-IP).
After cell treatments, protein samples from NP cells were extracted using commercial kits (Beyotime) according to the manufacturer's instructions. Protein concentrations were determined with a BCA Protein Assay Kit (Beyotime). Equal amounts of protein from each sample were separated using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 7.5% nonfat milk for 1 hour and then incubated at 4°C overnight with the following primary antibodies: cytochrome c, cleaved caspase-3, Nrf2, and Keap1. GAPDH and LaminB were used as internal controls. The membranes were subsequently incubated with the respective secondary antibodies at room temperature for 1 hour. Protein bands were visualized by the enhanced chemiluminescence method (Amersham Biosciences) according to 2 Oxidative Medicine and Cellular Longevity the manufacturer's instructions. Band intensities were quantified using the ImageJ software (NIH). For Co-IP analysis, anti-Keap1 or control IgG was used as the primary antibody, and then the antibody-protein complex was following incubated with Protein A/G PLUS-Agarose. The agarose-antibody-protein complex was collected and then analyzed by Western blotting.

RNA Extraction and Quantitative
Real-Time PCR (qRT-PCR). After treatments of human NP cells in the different groups, total RNA was extracted using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Takara Biotechnology, Otsu, Japan). qRT-PCR was conducted using SYBR Green Kit Master Mix (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 7500 Sequencing Detection System, according to the manufacturer's instructions. GAPDH was used as an internal control.

Statistical
Analysis. Data are presented as the mean ± standard deviation ðSDÞ of at least three independent experiments and were analyzed using the SPSS version 18.0 soft-ware (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 < 0:05 was considered statistically significant.

ANPODRT Was Downregulated in Degenerative NP
Tissues and TBHP-Stimulated NP Cells. ANPODRT is significantly downregulated in degenerative NP tissues. To investigate the association between ANPODRT and IDD, NP tissues of patients with different degrees of degeneration were harvested to determine the ANPODRT expression levels through qRT-PCR. As shown in Figure 1(a), in this study, ANPODRT was downregulated in NP tissues with the degree of disc degeneration. Since oxidative stress is an essential contributor to IDD pathophysiology, TBHP, an exogenous ROS donor, was applied to establish an IDD model in vitro. The CCK-8 assay revealed that TBHP inhibited NP cell viability ( Figure 1(b)). Notably, TBHP treatment downregulated ANPODRT in a dose-and time-dependent manner in human NP cells (Figures 1(c) and 1(d)). These results indicate that ANPODRT is potentially associated with IDD.  Oxidative Medicine and Cellular Longevity

ANPODRT Overexpression Attenuates TBHP-Induced
Oxidative Stress and Apoptosis in Human NP Cells. To determine whether ANPODRT plays a role in the TBHP-induced IDD model in vitro, we used an ANPODRT expression vector to overexpress ANPODRT in TBHP-treated human NP cells. qRT-PCR confirmed the overexpression efficiency ( Figure 2(a)). Oxidative stress and its induced mitochondrial pathway-mediated apoptosis of NP cells play an important role in IDD pathogenesis. Therefore, indicators of oxidative stress and mitochondrial apoptotic pathway were subsequently assessed. The CCK-8 assay revealed that ANPODRT overexpression inhibited the reduction in NP cell viability induced by TBHP (Figure 2(b)). Furthermore, ROS and MDA levels were determined to evaluate oxidative stress levels in human NP cells. As shown in Figures 2(c) and 2(d), TBHP treatment elevated the ROS and MDA levels, which was suppressed by ANPODRT overexpression. Moreover, flow cytometry revealed that ANPODRT overexpression alleviated the TBHP-induced increase in NP cell apoptosis (Figure 2(e)). Mitochondrial cytochrome c (cyt c) release and caspase-3 activation induced by cyt c are important features of mitochondrial pathway-mediated apoptosis. We found that protein levels of cytoplasmic cyt c and cleaved caspase-3 were significantly increased in human NP cells treated with TBHP, and these TBHP-induced alterations were prevented through ANPODRT overexpression (Figures 2(f)-2(h)).

ANPODRT Knockdown Aggravates TBHP-Induced
Human NP Cell Injury. To investigate the role of ANPODRT in the TBHP-induced IDD model in vitro, ANPODRT expression was knocked down using siRNA in TBHP-treated human NP cells. As shown in Figure 3(a), qRT-PCR analysis revealed that ANPODRT downregulation induced by TBHP in human NP cells was aggravated through transfection with si-ANPODRT. Functional studies revealed that contrary to ANPODRT overexpression, ANPODRT knockdown further exacerbated TBHP-induced oxidative stress and apoptosis in human NP cells, as manifested by a reduction in cell viability (Figure 3

ANPODRT Activates Nrf2 Signaling in Human NP Cells.
Nrf2 is a key regulator of the cellular antioxidative defense system.   ). Therefore, we speculated that ANPODRT potentially disrupts the Keap1-Nrf2 association. To investigate this possibility, we analyzed the association between Keap1 and Nrf2 in human NP cells after ANPODRT overexpression or knockdown. Co-IP assays revealed that ANPODRT overexpression reduced the association between Keap1 and Nrf2 in human NP cells ( Figure 5(o)). In contrast, the association between Keap1 and Nrf2 was increased upon ANPODRT knockdown ( Figure 5(p)). Together, these findings suggest that ANPODRT induces the dissociation of the Keap1-Nrf2 complex, thus activating the Nrf2 cascade in human NP cells.

Nrf2 Activation Is Required for ANPODRT-Induced
Human NP Cell Protection against Oxidative Injury. The present results show that ANPODRT overexpression activates the Nrf2 cascade and protects human NP cells from oxidative injury. To confirm whether Nrf2 mediates the protective effects of ANPODRT, rescue experiments were performed through siRNA-mediated Nrf2 silencing. Western blotting confirmed the Nrf2 knockdown efficiency (Figures 6(a) and 6(b)). Importantly, the protection of ANPODRT overexpression against TBHP-induced oxidative stress and apoptosis in human NP cells was reversed through Nrf2 knockdown, as indicated through the results of ROS levels ( Figure 6(c)), MDA production ( Figure 6(d)), apoptosis rate (Figure 6(e)), cytoplasmic cyt c levels (Figures 6(f) and 6(g)), and cleaved caspase-3 levels (Figures 6(f) and 6(h)). Together, these results indicate that the effects of ANPODRT overexpression-induced antioxidative injury

Discussion
Although numerous studies have reported the important roles and clinical significance of lncRNAs in various diseases, the functional roles of most of these molecules are unknown, particularly in the context of IDD [36][37][38]. In this study, we investigated the expression and function of ANPODRT in IDD. We observed a decreasing trend for ANPODRT expression during IDD pathogenesis, and the well-recognized oxidative stress inducer TBHP downregulated ANPODRT in human NP cells in a dose-and time-dependent manner. Gain-of-function and loss-of-function assays indicated that ANPODRT overexpression suppressed oxidative stress and apoptosis in TBHP-treated human NP cells, while ANPODRT knockdown exerted opposing effects. Mechanistically, these results show that ANPODRT disrupted the Keap1-Nrf2 association, inducing Nrf2 accumulation and nuclear translocation and the expression of Nrf2 target genes in human NP cells. Nrf2 knockdown abolished lncRNAinduced antioxidative stress and antiapoptotic effects in TBHP-treated human NP cells. These data indicate that ANPODRT inhibits oxidative stress and apoptosis in human NP cells, thus highlighting it as a potential target for IDD treatment.
Accumulating evidence indicates that degenerated IVDs exhibit increased ROS levels, which contributes to IDD pathogenesis. Furthermore, TBHP has several advantages over H 2 O 2 , such as high stability and gradual release. Accordingly, previous reports support the in vitro TBHPinduced IDD model used herein [39,40]. Apoptosis can be initiated by the extrinsic death receptor pathway and the intrinsic mitochondrial apoptosis pathway. The latter has been confirmed to be deeply involved in the increased NP cell death in IDD pathogenesis [16,39]. Oxidative stress-induced dysfunctional mitochondria can release proapoptotic proteins to form the apoptosome and activate caspase cascades for NP cell apoptosis. Cytoplasmic translocation of mitochondrial cyt c and caspase-3 activation induced by cyt c are characteristics of NP cell apoptosis through the mitochondrial pathway [41]. Several lncRNAs reportedly participate in oxidative stress and the mitochondrial apoptosis pathway. For example, Niu et al. reported that lncRNA Oip5-as1 suppresses microRNA-29a, thus activating the SIRT1/AMPK/PGC1αpathway, which attenuates oxidative stress and mitochondria-mediated apoptosis 13 Oxidative Medicine and Cellular Longevity during myocardial ischemia/reperfusion injury [42]. Li et al. reported that lncRNA H19 overexpression attenuates H 2 O 2 -induced cardiomyocyte injury by suppressing micro-RNA-877-3p/Bcl-2 pathway-mediated mitochondrial apoptosis [43]. However, the role of ANPODRT in oxidative stress and its induction of the mitochondrial apoptosis pathway in human NP cells remain unknown. Therefore, we performed a functional analysis of ANPODRT to determine whether it is involved in oxidative stress and human NP cell apoptosis during IDD. The present results show that ANPODRT was downregulated during IDD pathogenesis, and oxidative stress induced by TBHP downregulated ANPODRT. Notably, ANPODRT overexpression attenuated the accumulation of ROS and MDA, suppressed the upregulation of cytoplasmic-cyto-c and cleaved caspase-3, and inhibited the elevation of the apoptotic rate in human NP cells stimulated with TBHP. These effects were reversed through ANPODRT inhibition. These findings suggest that ANPODRT inhibits oxidative stress and apoptosis in human NP cells.
Mechanistically, considering that ANPODRT inhibits oxidative stress in human NP cells and that the Keap1-Nrf2 complex is an important regulator of oxidative stress, we investigated whether the Keap1-Nrf2 complex is involved in the ANPODRT-mediated protective effects in TBHPtreated human NP cells. Lv et al. reported that eriodictyolinduced retinal ganglion cell protection against high glucose-induced oxidative stress and apoptosis is associated with the activation of Nrf2 signaling [44]. Moreover, along with direct Nrf2 activation, the disruption of the Keap1-Nrf2 interaction has been widely studied. Certain internal disruptors including p62 [45], iASPP [17], and lncRNA-Sox2OT [46] reportedly interfere with the Keap1-Nrf2 complex, thus decreasing the Keap1-Nrf2 interaction and subsequently activating Nrf2 signaling, ultimately affecting the antioxidative status of the cell. Lu et al. reported that CPUY192018, a potent inhibitor of the Keap1-Nrf2 interaction, alleviates renal inflammation in mice by restricting oxidative stress and NF-κB activation [19]. The present results show that ANPODRT induced Keap1-Nrf2 disassociation, leading to Nrf2 protein accumulation and nuclear translocation and the expression of Nrf2 targets, including HO1 and NQO1, in human NP cells. Furthermore, Nrf2 knockdown almost obliterated ANPODRT-induced protection of human NP cells against oxidative stress and apoptosis. These results suggest that Nrf2 activation is required for ANPODRTinduced cytoprotective effects in TBHP-treated human NP cells.
There are several difficulties in isolating and culturing NP cells. For the isolation of NP cells, because excessive enzyme digestion will destroy the cells, we cut the NP tissue into small pieces as much as possible, so that the tissue can be in better contact with the enzyme, thus shortening the enzyme digestion time. In the process of primary cell culture, we use penicillin and streptomycin to solve the problem that primary cell culture is easy to be contaminated.
3D culture and hypoxic environment are two aspects that cannot be ignored in the study of IDD. The physiological properties of the intervertebral disc are linked to the structure of its extracellular matrix (ECM) [5]. The ECM in healthy intervertebral disc is a 3D network of natural nanoscale fibers. NP cells in vivo are surrounded by ECM. NP cells can easily lose their phenotype characteristics in monolayer growth [47]. In addition, there are steep gradients in oxygen concentrations across the avascular intervertebral disc, with O 2 falling to as low as 1% in the center of a large disc [48]. A low oxygen tension environment plays a vital role in maintaining intervertebral disc physiological function, including matrix synthesis and even cell metabolism [49]. Studies have shown that a physiological oxygen of 1% appears to promote the best NP phenotype for bovine intervertebral disc cells in alginate [48]. In addition, Pei et al. have reported that low 14 Oxidative Medicine and Cellular Longevity oxygen tension (5%) enhanced the ECM synthesis of porcine NP cells in a pellet culture system [50]. Therefore, the application of 3D culture in hypoxia can better reflect the actual environment of intervertebral disc. The role of lncRNA in IDD needs more investigations in 3D culture under hypoxia in the future. Another limitation of this study was that our experiments were performed in vitro, and in vitro results may not directly reflect the clinical settings. Thus, in vivo experiments are needed to confirm the role of ANPODRT in IDD and to increase our understanding of the molecular mechanisms underlying this effect.
In summary, this study shows that ANPODRT protects human NP cells from oxidative stress and apoptosis, at least partially, by interfering with Keap1-Nrf2 association and activating Nrf2-mediated antioxidant system. Our findings further the current understanding of the pathomechanism of IDD and suggest that ANPODRT is a potentially promising therapeutic target for IDD.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
These authors have no conflict of interest to declare.

Authors' Contributions
Liang Kang, Yueyang Tian, and Xing Guo contributed equally to this work.