1,25(OH)2D3 Mitigates Oxidative Stress-Induced Damage to Nucleus Pulposus-Derived Mesenchymal Stem Cells through PI3K/Akt Pathway

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain. The local environment of the degenerated intervertebral disc (IVD) increases oxidative stress and apoptosis of endogenous nucleus pulposus-derived mesenchymal stem cells (NPMSCs) and weakens its ability of endogenous repair ability in degenerated IVDs. A suitable concentration of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been certified to reduce oxidative stress and cell apoptosis. The current study investigated the protective effect and potential mechanism of 1,25(OH)2D3 against oxidative stress-induced damage to NPMSCs. The present results showed that 1,25(OH)2D3 showed a significant protective effect on NPMSCs at a concentration of 10−10 M for 24 h. Protective effects of 1,25(OH)2D3 were also exhibited against H2O2-induced NPMSC senescence, mitochondrial dysfunction, and reduced mitochondrial membrane potential. The Annexin V/PI apoptosis detection assay, TUNEL assay, immunofluorescence, western blot, and real-time quantitative polymerase chain reaction assay showed that pretreatment with 1,25(OH)2D3 could alleviate H2O2-induced NPMSC apoptosis, including the apoptosis rate and the expression of proapoptotic-related (Caspase-3 and Bax) and antiapoptotic-related (Bcl-2) proteins. The intracellular expression of p-Akt increased after pretreatment with 1,25(OH)2D3. However, these protective effects of 1,25(OH)2D3 were significantly decreased after the PI3K/Akt pathway was inhibited by the LY294002 treatment. In vivo, X-ray, MRI, and histological analyses showed that 1,25(OH)2D3 treatment relieved the degree of IVDD in Sprague–Dawley rat disc puncture models. In summary, 1,25(OH)2D3 efficiently attenuated oxidative stress-induced NPMSC apoptosis and mitochondrial dysfunction via PI3K/Akt pathway and is a promising candidate treatment for the repair of IVDD.


Introduction
The prevalence of low back pain (LBP) is 11.9%, as reported in the Lancet, and the peak prevalence ranges from 28% to 42% in people of middle-to old-age (40~69 years) [1]. Intervertebral disc degeneration (IVDD) is a usual cause of LBP in aging people and increases the socioeconomic burden [2,3]. Regrettably, conventional treatments mainly relieve symptoms instead of repairing or regenerating the structure and function of the degenerative intervertebral disc (IVD) [4]. In recent years, multiple types of mesenchymal stem cells (MSCs), including bone marrow-derived MSCs (BMSCs), adipose-derived MSCs, umbilical cord-derived MSCs, and other types of MSCs have been used to treat IVDD [5][6][7][8][9].The separation and identification of endogenous nucleus pulposus-derived MSCs (NPMSCs) in 2007 rendered a possibility for the endogenous restoring of IVDD [10]. Our previous study also confirmed that NPMSCs exist in normal and degenerated IVDs [11]. Interestingly, compared with MSCs from other sources, NPMSCs have better tolerance in the hyperosmolar and acidic microenvironment of deteriorated IVDs [12]. However, pathological factors, including annulus fibrosus (AF) rupture, inflammation, and oxidative stress, may induce the generation of reactive oxygen species (ROS) [13]. The generated ROS enhances the senescence and apoptosis of nucleus pulposus (NP) cells and NPMSCs, which are primary characteristics of IVDD [14,15].
The proliferation and migratory ability of BMSCs were enhanced after treatment with 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) via activation of thePI3K/Akt pathway [16]. Vitamin D was found can participate in the proliferation, aging, apoptosis, inflammation, oxidative stress, and extracellular matrix expression of NP cells [17]. Moreover, previous studies including our study found that the phosphatidylinositide 3-kinases/protein kinase B (PI3K/ Akt) pathway is closely related to NPMSC apoptosis [18][19][20]. It has also been reported that 1,25(OH) 2 D 3 inhibits cell apoptosis through the PI3K/Akt pathway [21,22]. Therefore, the purpose of this study was to explore whether 1,25(OH) 2 D 3 protects NPMSCs from H 2 O 2 -induced oxidative damage via PI3K/Akt pathway and to provide a basis for its clinical application in the treatment of IVDD.

Materials and Methods
2.1. Isolation and Culture of NPMSCs. Sprague-Dawley (SD) rats (weight, 250-350 g; age, 2.5-3.5 months) were purchased from the Laboratory Animal Center of Yangzhou University (License No. SYXK (Su) 2021-0027). The Ethical Committee of the Clinical Medical College of Yangzhou University approved the experimental studies. The separation method of NP tissues was the same as described in our previous studies [11,18,[23][24][25][26]. Briefly, the SD rats were euthanized with an anesthesia overdose, and the collected NP tissues from the SD rats were separated under aseptic conditions and washed carefully with phosphate-buffered saline (PBS) containing 1% penicillin-streptomycin (Gibco, USA). The NP tissues were cut into fragments of 1 mm 3 and digested by collagenase type II (Gibco, USA) for 12 h at 37°C. After being washed with normal saline and centrifuged at 1000 rpm for 4 min, the cells were resuspended in MSC complete medium (Cyagen, USA) complemented with 10% fetal bovine serum (FBS; HyClone, USA) and 1% penicillin-streptomycin and cultured at 37°C under 5% CO 2 . The culture medium was replaced every three days. Each primary culture was digested and subcultured at a ratio of 1 : 3 when the adherent cells met 80% confluence. A microscope (Olympus, Japan) was used to observe and photograph the cells. Third passage cells were gained for subsequent experiments.

Surface Marker Identification of NPMSCs.
Surface markers, including CD34, CD45, CD73, CD90, and CD105 have been reported to identify MSCs according to the stan-dards proposed by the International Society for Cellular Therapy (ISCT) [27]. Immunofluorescent staining was used to examine these surface markers. Briefly, NPMSCs were inoculated on 25 mm diameter polylysine-containing cell slides in a 12-well plate and cultured in the MSC complete medium. NPMSCs were then fixed with 4% paraformaldehyde for 15 min and washed twice with PBS containing 0.5% Triton X-100 for 15 min. After being blocked with 10% bovine serum albumin for 1 h at 37°C, the cells were incubated with primary antibodies (1 : 100) at 4°C overnight. The cell slides were washed twice with TBST and then incubated with a conjugated secondary antibody (1 : 500; Abcam, United Kingdom, catalog Nos. ab150077, ab150078, ab150080, and ab175471) for 2 h at room temperature. After being treated with the antifade mounting medium containing 4 ′ ,6-diamidino-2 ′ -phenylindole (DAPI) for 10 min, the cell slides were observed and photographed using a fluorescence microscope (Leica, Wetzlar, Germany).

Multilineage Differentiation Ability of NPMSCs.
Osteogenic, adipogenic, and chondrogenic differentiation of NPMSCs was induced to identify its multilineage differentiation potential. Briefly, NPMSCs were seeded in 6-well plates, and multilineage differentiation kits (Cyagen Biosciences, China) were used following the manufacturer's instructions to induce cell differentiation when the cells reached 80% confluence. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min when the required days for induction were reached. The cells were then stained with Alizarin red (Sigma, USA), Oil Red O (Sigma, USA), and Alcian blue (Sigma, USA) and recorded under a microscope.

Annexin V-FITC/PI
Staining. An Annexin V-FITC/PI apoptosis detection kit (KeyGen, China) was used to analyze cell apoptosis. After being washed with PBS, the NPMSCs of each group were collected by trypsinization and resuspended in 500 μl of 1x binding buffer. Then, 5 μl PI and 5 μl Annexin-V were added in cell suspension, and the cells were incubated in the dark at room temperature for 15 min. The percentage of cells positively labeled by Annexin V-FITC and PI was analyzed using flow cytometry (BD Company, USA).
2.9. TUNEL Staining. The level of DNA damage was evaluated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining (Beyotime, China). The NPMSCs from the four groups were fixed in freshly prepared 4% paraformaldehyde for 30 min at 37°C, permeabilized with 0.1% Triton X-100 for 5 min, and washed with PBS 3 times at each step. Afterward, the cells were stained with TUNEL according to the manufacturer's instructions, and a fluorescence microscope and ImageJ software were used to observe and analyze the number of TUNEL-positive cells.
2.10. JC-1 Assay for Mitochondrial Membrane Potential (MMP). The MMP of NPMSCs was detected using a fluores-cent probe iodide 5,5 ′ ,6,6 ′ -tetrachloro-1,1 ′ ,3,3 ′ -tetraethylbenzimidazolcarbocyanine (JC-1) detection kit (KeyGen, China). In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer, which emits intense red fluorescence (polarized). In damaged mitochondria, JC-1 exists in the cytoplasm in the form of a monomer and produces green fluorescence (depolarization) due to the decrease or loss of MMP. After treatment according to the manufacturer's instructions, the NPMSCs in each group were observed and photographed using a fluorescence microscope. The ratio of green to red fluorescence intensity was analyzed using the ImageJ software.
2.11. ROS Assay. A ROS detection fluorescent probe dihydroethidium (DHE) kit (KeyGen, China) was used to measure the generation of ROS in NPMSCs. The NPMSCs were inoculated into a 6-well plate and treated as described above. The NPMSCs were then washed twice with PBS and incubated with 10 μM DHE for 30 min at 37°C. A fluorescence microscope and ImageJ software were used to observe and analyze the intracellular ROS fluorescence.
2.12. Immunofluorescence Assay. The intracellular expression of the target proteins was detected using immunofluorescence staining. Briefly, NPMSCs were inoculated on 25 mm diameter polylysine-containing cell slides in a 12-well plate and treated as described above. The NPMSCs were then fixed in 4% paraformaldehyde for 15 min and washed twice with PBS containing 0.5% Triton X-100 for 15 min at room temperature. The NPMSCs were then blocked with QuickBlock™ Blocking Buffer for Immunol staining (Beyotime, China) for 1 h. The cells were incubated with primary antibodies (including p-Akt (bs-0876R), Bioss, China; Akt (#4691), Cell Signaling Technologies, USA; p21 (10355-1-AP) and p53 (10442-1-AP), Proteintech, USA; and Caspase-3 (A0214), Bax (A0207), and Bcl-2 (A11025), ABclonal, USA; 1 : 100) overnight at 4°C in a dark wet chamber. Afterward, the cells were washed twice with TBST and incubated with a conjugated goat antirabbit IgG secondary antibody at 37°C for 2 h. Subsequently, after being stained with DAPI for 10 min, the stained NPMSC samples were visualized and photographed using a fluorescence microscope or a laser scanning confocal microscope (Zeiss LSM 710, Germany) and analyzed using the ImageJ software.

Western Blot Assay.
A Whole Cell Lysis Assay Kit (KeyGen, China) was used to extract the total proteins, and a BCA protein assay kit (Beyotime, China) was then used to measure the protein concentration. Equal protein samples from each group were separated by 10% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked in 5% nonfat milk 2 h on a shaker at room temperature and incubated with primary antibodies overnight at 4°C. The membranes were then washed with TBST three times and incubated with secondary antibodies for 2 h at room temperature. Afterward, protein signals were visualized using an 3 Oxidative Medicine and Cellular Longevity enhanced chemiluminescence system and analyzed using the ImageJ software. The loading control was β-actin.
2.14. Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR) Assay. The mRNA expression levels of Caspase-3, Bcl-2, Bax, and β-actin were quantified. The total RNA of the NPMSCs was extracted using TRIzol reagent (Invitrogen, USA). According to the manufacturer's instructions, reverse transcription from the whole RNA to complementary DNA (cDNA) was performed using Prime Script-RT reagent kit (Vazyme Biotech, China), and amplification of cDNA was performed using SYBR Premix Ex Taq (Vazyme Biotech, China). The comparative Ct method was used to calculate the expression levels of target genes in the different groups. β-Actin expression levels were used to normalize relative gene expression levels. The primers were designed according to the sequences in GenBank using the Prime 5.0 software and are listed in Table 1.

IVDD Model Induction.
Eighteen SD rats were randomly divided into three groups (n = 6 per group): control group (no operation), IVDD group (punctured), and 1,25(OH) 2 D 3 group (punctured and treated with 1,25(OH) 2 D 3 ). The IVDD model was established as reported previously [28]. Briefly, the rat was placed in a prone position after anesthesia with an intraperitoneal injection of 1% pentobarbital sodium at 0.1 mg/kg, and a percutaneous needle puncture was performed with a 21G needle in the coccygeal intervertebral disc (Co 6-7). The needle was carefully inserted in the middle of the disc, perpendicular to the skin and parallel to the endplate, rotated 180°, and held for 5 s. After removal of the needle, the wound was covered with gauze and the rats underwent standard postoperative procedures. The 1,25(OH) 2 D 3 was dissolved in DMSO (10 -3 M/L) and further diluted in saline immediately before intraperitoneal administration. Two weeks of puncture, the 1,25(OH) 2 D 3 group received 100 ng/kg/day 1,25(OH) 2 D 3 [29]. Meanwhile, the control group and IVDD group received an equal amount of saline supplemented with the required volume of DMSO.
2.16. X-Ray and Magnetic Resonance Imaging (MRI) Assay. X-ray and MRI scans were taken prepuncture and at 6 weeks after the puncture, respectively. X-ray was used to measure the disc height index (DHI) [30]. A 7.0-T MRI scanning system (Philips Intera Achieva 7.0 MR, Netherlands) was used, and sagittal T2-weighted images were used to assess the Pfirrmann grade of IVDD based on signal intensity and intervertebral disc height [31].
2.17. Histologic Assay. All rats were euthanized by an anesthesia overdose after 4 weeks of treatment. The harvested IVD specimens were fixed with 4% paraformaldehyde, decalcified with a 10% ethylenediaminetetraacetic acid solution, and embedded in paraffin, successively. The specimens were then cut into 5 μm slices, and the slices were stained with hematoxylin-eosin (HE), toluidine blue, and safranin-O/fast green (S-O). The histologic grading scale criteria reported by Norcross et al. [32] was adopted to evaluate the histologic images.
2.18. Statistical Analysis. Analyses were performed using IBM SPSS 19.0, and GraphPad Prism 8 was used to make statistical graphs. All measurements were performed in triplicate, and the results are expressed as the mean ± standard deviation (mean ± SD). The Kolmogorov-Smirnova test or Shapiro-Wilk test is used to check whether the data conformed to a normal distribution. One-way analysis of variance (ANOVA) was used to analyze the data from multiple groups. The Student t-test was applied to analyze the differences between the two groups. P < 0:05 was considered statistically significant.

Results
3.1. Identification of NPMSCs. The separated NPMSCs gathered into a chrysanthemum shape and formed into a long spindle shape after passage (Figure 1(a)). Similar to our previous studies [18,23,33], the MSC-associated surface markers CD73, CD90, and CD105 in NPMSCs presented high fluorescence expression, whereas the surface markers CD34 and CD45 showed presented low fluorescence expression ( Figure 1(b)). The results of osteogenic, adipogenic, and chondrogenic induction in vitro confirmed the multipotential differentiation of NPMSCs (Figure 1(c)). These results demonstrated that the NPMSCs isolated from the IVD corresponded to the criteria of MSC proposed by ISCT. Additionally, cell proliferation was appropriately inhibited when treated with 100 μM H 2 O 2 for 6 h, which was the same result as our previous study [24,33] (Figure 2(b)). Therefore, this condition was used to induce oxidative stress damage in NPMSCs in the following experiments.

Ideal Incubation
3.3. 1,25(OH) 2 D 3 Enhanced Cell Proliferation. The effect of 1,25(OH) 2 D 3 on the proliferation of NPMSCs was detected using an EdU staining kit. As shown in Figures 2(c) and 2(d), the number of EdU-labeled NPMSCs in the H 2 O 2 group was significantly lower than that in the control group (P < 0:05), and the number of EdU-labeled NPMSCs was partially increased in the 1,25(OH) 2 D 3 group (P < 0:05). However, LY294002 significantly weakened the increased proliferation effect of 1,25(OH) 2 D 3 (P < 0:05).
3.4. 1,25(OH) 2 D 3 Inhibited Cell Senescence. The cell senescence was evaluated using SA-β-Gal staining. Aging cells presented high SA-β-gal activity and stained blue. The percentage of SA-β-gal-positive NPMSCs in the H 2 O 2 group was higher than that in the control group (P < 0:05). However, the percentage of SA-β-gal-positive NPMSCs was inhibited after 1,25(OH) 2 D 3 pretreatment (P < 0:05). Incubation with LY294002 significantly weakened the protective effect of 1,25(OH) 2 D 3 , and the percentage of SA-β-Gal-positive cells was increased (P < 0:05, Figures 3(a) and 3(b)). 4 Oxidative Medicine and Cellular Longevity To further explore the active therapeutic effect of vitamin D on cell aging, we detected the expression of senescence-related proteins (p21 and p53) using immunofluorescence and western blotting. As shown in Figures 3(c)-3(f), the intracellular fluorescence and expression levels of p21 and p53 in NPMSCs of the H 2 O 2 group were higher than those in the control group (P < 0:05), whereas the high fluorescence and increased protein expression levels of p21 and p53 were significantly reversed by pretreatment with 1,25(OH) 2 D 3 (P < 0:05). However, pretreatment with LY294002 decreased the protective effect of 1,25(OH) 2 D 3 by upregulating the expression levels of p21 and p53 in NPMSCs (P < 0:05). These results demon-strate that 1,25(OH) 2 D 3 can inhibit the senescence of NPMSCs that is induced by H 2 O 2 .

1,25(OH) 2 D 3 Decreased H 2 O 2 -Induced Cell Apoptosis.
The protective effect of 1,25(OH) 2 D 3 against H 2 O 2 -induced apoptosis was evaluated using Annexin V/PI staining. Flow cytometry showed that more cells appeared in the Q2 and Q3 quadrants in the H 2 O 2 group, which indicates that the apoptotic rate of NPMSCs in the H 2 O 2 group was significantly higher than that in the control group (P < 0:05). The apoptotic rate of NPMSCs was partially decreased after pretreatment with 1,25(OH) 2 D 3 (P < 0:05), but the presence of   Immunofluorescence, western blotting, and qRT-PCR were adopted to assess the intracellular fluorescence, proteins, and mRNA expression, respectively, of the antiapoptotic molecule Bcl-2 and the proapoptotic molecules Bax and Caspase-3. The results showed that compared with the control group, Bax and Caspase-3 were significantly upregulated, but Bcl-2 was downregulated in NPMSCs of the H 2 O 2 group (P < 0:05), whereas the fluorescence intensity, proteins, and mRNA expression levels of Bax and Caspase-3 were downregulated and that of Bcl-2 was upregulated in NPMSCs of the 1,25(OH) 2 D 3 group compared with those in the H 2 O 2 group (P < 0:05). However, the protective effect of 1,25(OH) 2 D 3 on H 2 O 2 -induced apoptosisrelated protein and mRNA expression was eliminated in the presence of LY294002 (P < 0:05, Figure 5).  6(b)). In addition, the excessive production of ROS can damage mitochondrial homeostasis. As shown in Figures 6(c) and 6(d), compared with the control group, the level of intracellular ROS in the H 2 O 2 group was significantly increased (P < 0:05). However, the excessive production of ROS was significantly suppressed by preincubation with 1,25(OH) 2 D 3 (P < 0:05), and the weakening effect of 1,25(OH) 2 D 3 on ROS levels was decreased by preincubation with LY294002 (P < 0:05). These results represent that 1,25(OH) 2 D 3 effectively alleviates H 2 O 2 -induced oxidative stress by diminishing the level of ROS in NPMSCs, and this effect was weakened by LY294002 implicating PI3K is involved in this process.

Effects of 1,25(OH) 2 D 3 on the PI3K/Akt Pathway.
To further explore the mechanism of 1,25(OH) 2 D 3 on NPMSC apoptosis, immunofluorescence and western blotting were used to evaluate the expression levels of Akt and p-Akt in the cytoplasm. The fluorescence intensity and protein expression of Akt among the four groups were not significantly different (P > 0:05). However, the fluorescence intensity and protein expression levels of p-Akt/Akt in the H 2 O 2 group were significantly decreased compared with those in the control group (P < 0:05). Interestingly, this effect was significantly reversed by pretreatment with 1,25(OH) 2 D 3 (P < 0:05), but pretreatment with LY294002 decreased the protective effect of 1,25(OH) 2 D 3 by downregulating p-Akt    Oxidative Medicine and Cellular Longevity in NPMSCs (P < 0:05, Figure 7). Our results demonstrate that the protective action of 1,25(OH) 2 D 3 is related to activation of the PI3K/Akt pathway.

Radiographic and MRI
Evaluation. X-ray was performed to evaluate DHI, and no significant difference in DHI was found between the three groups before puncture. However, compared with the control group (0:0983 ± 0:037), the DHI of the IVDD group (0:050 ± 0:004) was significantly lost at 6 weeks postpuncture (P < 0:01). Furthermore, the DHI of the 1,25(OH) 2 D 3 group (0:070 ± 0:003) was higher than that of the IVDD group (P < 0:01) (Figures 8(a) and  8(b)). Moreover, the degenerative grade of IVD as measured by MRI according to Pfirrmann classification showed that the Pfirrmann grade of the three groups before puncture was no statistically significant difference. However, at sixweek postpuncture, the grade of the IVDD group was significantly more serious than that of the control group (P < 0:05), and the grade of the 1,25(OH) 2 D 3 group was slighter than that of the IVDD group (P < 0:05, Figures 8(c) and 8(d)).
The results showed that 1,25(OH) 2 D 3 intervention could delay the progression of IVDD in vivo.
3.9. Histological Analysis. HE, toluidine blue, and S-O staining of the control group showed well-structured inner gel-like NP and outer concentric ring-like AF tissues. In contrast, the NP tissue almost disappeared, and the wellstructured IVD tissue was damaged in the IVDD group; the histological score was also significantly lower than that of the control group (P < 0:01). Interestingly, there was still some NP tissue in the 1,25(OH) 2 D 3 group, and the histological score was higher than that of the IVDD group (P < 0:01, Figure 9).

Discussion
LBP has become the most common cause of disability in adults worldwide in the past 30 years, and IVDD is an important cause of LBP in middle-aged and elderly patients. As a result, the treatment cost places a substantial burden on the family and society [2,3]. Much effort has been expended in the development of medicines and surgical treatments for IVDD, but they mainly relieve symptoms and cannot effectively alleviate or reverse the process of IVDD [34,35]. In recent years, MSC-based biological treatment has gradually

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Oxidative Medicine and Cellular Longevity exhibited advantages [6][7][8][9]25]. However, previous studies found that the local environment of the degenerated IVD, which is characterized by nutrient deficiency, hypertonicity, low pH, hypoxia, and high mechanical loading, can decrease the number and functionality of the transplanted MSCs [11,36,37]. Additionally, MSC transplantation has some potential side effects such as osteophyte formation due to MSC migration [38]. Interestingly, previous studies including ours have confirmed that endogenous NPMSCs exist in normal and degenerated IVDs [23][24][25]. The NPMSCs isolated from NP tissues in the present study also showed characteristics that fulfilled the criteria outlined by the ISCT for MSC [27]. Furthermore, NPMSCs exhibited more potent biological activity than other tissue-derived MSCs in the hypoxic environment of IVD [39]. NPMSCs may enhance the repair and regeneration ability of degenerative IVDs by differentiating into NP cells and/or inhibiting NP cell apoptosis [18,24,25], but the number of NPMSCs gradually decreases with the degeneration of the IVD, which may lead to the failure of endogenous repair [40]. Therefore, it is particularly important to maintain the number of viable and functional NPMSCs for the process of endogenous repair of IVDD.
The active metabolite of vitamin D, 1,25(OH) 2 D 3 , is a fat-soluble vitamin and a steroid compound, and the gene encoding its intracellular specific receptor (VDR) is the first reported gene that may be related to the risk of IVDD [41]. Also, 1,25(OH) 2 D 3 has shown broad protective effects such as anti-inflammatory, antioxidant, antisenescence, and antiapoptosis [42][43][44]. Thus, the present study evaluated the effect of 1,25(OH) 2 D 3 on H 2 O 2 -induced oxidative damage in NPMSCs for the first time and discussed its possible mechanism.
Although 1,25(OH) 2 D 3 has shown a broad protective effect, there is still a controversy about the effective concentration. A previous study found that 1,25(OH) 2 D 3 has a positive effect on cartilage cell proliferation at a concentration of 10 -12 M but a dose-dependent inhibitory effect at 10 -10 M and above [45]. Colombini et al. [17] and Gruber et al. [46] found that 10 −8 M 1,25(OH) 2 D 3 significantly inhibits the proliferation of AF cells and NP cells. More fascinating was the result reported by Klotz et al., where 10 −7 M 1,25(OH) 2 D 3 treatment for 72 h not only significantly inhibited BMSC proliferation but also inhibited cell apoptosis. In addition, it also delayed the development of replicative senescence in long-term culture. Moreover, BMSCs maintained clonogenic capacity, stem cell surface marker characteristics, and multipotent differentiation capacity after long-term 1,25(OH) 2 D 3 treatment [47]. Our study found that 1,25(OH) 2 D 3 showed no cytotoxic effect on cell viability   Cell senescence exhibits the characteristics of decreased cell viability and the loss of proliferation capacity. Our present results found that pretreatment with 1,25(OH) 2 D 3 can significantly decrease the ratio of oxidative stress-induced cell senescence and apoptosis, and the inhibited proliferation was also significantly mitigated, although it did not improve to the level of normal NPMSCs. Huang et al. and Zhang et al. also found that 1,25(OH) 2 D 3 inhibits NP cell apoptosis via the regulation of the nuclear factor-κB (NF-κB) signaling pathway [42,48]. Tong et al. found that 1,25(OH) 2 D 3 can activate intracellular VDR in AF cells, and activation of VDR ameliorates oxidative stress-induced apoptosis by preserving mitochondrial function [43]. Bcl-2 (antiapoptotic protein) and Bax (proapoptotic protein) are the two proteins used to evaluate cell susceptibility to apoptosis. The activity of the caspase pathway would be promoted by Bax but inhibited by Bcl-2 and inhibit cell apoptosis [49]. The present study also found that 1,25(OH) 2 D 3 significantly inhibited the upregulated expression levels of p53, p21, Caspase-3, and Bax induced by H 2 O 2 and restored the downregulated expression level of Bcl-2. Therefore, these results suggest that 1,25(OH) 2 D 3 could protect NPMSCs from H 2 O 2 -induced damage.
The accumulation of ROS leads to the destruction of mitochondrial homeostasis by intensifying the opening of the mitochondrial permeability transition pore and the release of cytochrome c. Subsequently, caspase-dependent cell apoptosis was activated [50]. The formation and development of IVDD are tightly associated with the presence of ROS, which is regarded as influential intermediates in the pathogenesis of IVDD [14,24,51]. Similar to a previous study [18], our present study also found that the proliferation ability and MMP of NPMSCs were inhibited under oxidative stress induced by H 2 O 2 , and ROS production and cell apoptosis rates were increased. These results further confirm that oxidative stress is harmful to maintaining the healthy physiology of NPMSCs. A previous study found that 1,25(OH) 2 D 3 treatment significantly reduces the level of oxidative stress in degenerated IVD tissue [42]. Additionally,   [43]. Notably, our results also found that the increased ROS level and upregulated J-aggregates of MMP induced by H 2 O 2 in NPMSCs were significantly inhibited by 1,25(OH) 2 D 3 . Therefore, these results demonstrate that 1,25(OH) 2 D 3 may exert a positive effect on antioxidative stress and may regulate the function of mitochondria to protect NPMSCs. The PI3K/Akt pathway plays a key role in modulating biological processes. The activated PI3K/Akt pathway participates in the processes of cell proliferation, apoptosis, and differentiation through interactions with downstream proteins under physiological and pathological conditions [18][19][20][21][22][52][53][54]. Previous studies have found that the PI3K/ Akt pathway participates in the proliferation and apoptotic processes of NPMSCs [18][19][20]24], and 1,25(OH) 2 D 3 can modulate biological processes through PI3K/Akt pathway [21,53]. The present results also found that 1,25(OH) 2 D 3 could partially alleviate the inhibition of the PI3K/Akt pathway induced by H 2 O 2 , thereby reducing ROS production and improving the MMP. However, the positive effect of 1,25(OH) 2 D 3 on NPMSCs was weakened when the PI3K/ Akt pathway was inhibited by LY294002. Therefore, as shown in Figure 10  This study also has several limitations. First, the NPMSCs were cultured under normal oxygen conditions, which is not the actual physiological condition of IVDD.
Second, 1,25(OH) 2 D 3 was found to participate in various metabolic processes and regulate cell biological processes via multiple signaling pathways, such as nuclear factor (erythroid-derived 2)-like 2 (Nrf2), NF-κB [42,44,48]. Therefore, further study is needed to explore the other mechanisms underlying the protective effect of 1,25(OH) 2 D 3 on NPMSCs.   . (a, b) The X-ray scans and quantitative analysis of disc height index (DHI) in the different groups (control group, IVDD group, and 1,25(OH) 2 D 3 group). (c, d) The MRI images and quantitative analysis of Pfirrmann grades in the different groups. All data are the mean ± SD. * * P < 0:01 compared with the control group; # P < 0:05 and ## P < 0:01 compared with the IVDD group. 18 Oxidative Medicine and Cellular Longevity

Conclusions
In conclusion, this is the first research on the protective action of 1,25(OH) 2 D 3 against H 2 O 2 -induced oxidative damage in NPMSCs. The administration of 1,25(OH) 2 D 3 not only activated the PI3K/Akt pathway to protect mitochondrial function and mitigate the apoptosis of NPMSC in vitro but also could retard IVDD in vivo. The results support 1,25(OH) 2 D 3 as a promising treatment to repair and delay IVDD.

Data Availability
All data during the study are available from the corresponding author by request.

Disclosure
No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.