
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39256449
71810
10.1038/s41598-024-71810-8
Article
HDAC/H3K27ac-mediated transcription of NDUFA3 exerts protective effects on high glucose-treated human nucleus pulposus cells through improving mitochondrial function
Zheng Cheng 1
Guo Dongshuai 2
Zhang Tong 2
Hu Weiran 2
Zhang Bo 2
Feng Hang 2
Gao Yanzheng 2
Yang Guang dryangguang@zzu.edu.cn

2
1 https://ror.org/038hzq450 grid.412990.7 0000 0004 1808 322X Xinxiang Medical University, 601 Jinsui Avenue, Hongqi District, Xinxiang City, Henan Province, Xinxiang, 453003 China
2 grid.414011.1 0000 0004 1808 090X Department of Spinal and Spinal Surgery, Henan Key Laboratory for Intelligent Precision Orthopedic Medicine, Henan Provincial People’s Hospital, People’s Hospital of Zhengzhou University, No.7, Wei Wu Road, Jinshui District, Zhengzhou, 450003 China
10 9 2024
10 9 2024
2024
14 211659 1 2024
30 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Diabetes mellitus (DM) is a well-documented risk factor of intervertebral disc degeneration (IVDD). The current study was aimed to clarify the effects and mechanisms of NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) in human nucleus pulposus cells (HNPCs) exposed to high glucose. NDUFA3 was overexpressed in HNPCs via lenti-virus transduction, which were co-treated with high glucose and rotenone (a mitochondrial complex I inhibitor) for 48 h. Cell activities were assessed for cell viability, cell apoptosis, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) ratio, oxygen consumption rate (OCR) and mitochondrial complexes I activities. High glucose decreased cell viability, increased apoptotic cells, increased ROS production, decreased MMP levels and OCR values in HNPCs in a dose-dependent manner. Rotenone co-treatment augmented the high glucose-induced injuries on cell viability, apoptosis, ROS production and mitochondrial function. NDUFA3 overexpression counteracted the high glucose-induced injuries in HNPCs. HDAC/H3K27ac mechanism was involved in regulating NDUFA3 transcription. NDUFA3 knockdown decreased cell viability and increased apoptotic cells, which were reversed by ROS scavenger N-acetylcysteine. HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries through suppressing cell apoptosis, eliminating ROS, improving mitochondrial function and oxidative phosphorylation. This study sheds light on candidate therapeutic targets and deepens the understanding of molecular mechanisms behind DM-induced IVDD.

Keywords

Mitochondrial complex I
Mitochondrial function
ROS
Oxidative phosphorylation
Intervertebral disc degeneration
Subject terms

Cell biology
Molecular biology
Physiology
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Intervertebral disc degeneration (IVDD) is defined as a chronic musculoskeletal degenerative disease attributed to a disrupted balance between anabolic and catabolic processes occurred in the intervertebral discs, contributing to low back pain1,2. Based on symptoms of patients, the current available therapeutic strategies for IVDD include conservative interventions, surgical section as well as biotherapy3. A significant body of literature has demonstrated that oxidative stress, mitochondrial dysfunction, and nucleus pulposus (NP) cells apoptosis are critical mechanisms underlying the pathogenesis of IVDD4,5. Diabetes mellitus (DM) is a well-established key contributor to IVDD through inducing degenerative changes, such as cell apoptosis and matrix degradation of NP cells6. Comprehensively illuminating the molecular mechanisms of IVDD is essential for the determination of potential therapeutic targets.

NADH: ubiquinone oxidoreductase subunit A3 (NDUFA3) is one supernumerary subunit required for the formation of extra-membrane arm of human mitochondrial respiratory chain complex I, affecting assembly and stability of the matrix arm7. A recent study provides bioinformatics findings in support of the close association of NDUFA3 with mitochondria dysfunction in airway epithelial cells8. Another report concerning brain damage mechanism reveals that NDUFA3 is an important regulatory protein related to oxidative stress, cell apoptosis, and inflammatory response in cerebral cortex of rats with middle cerebral ischemia/reperfusion9. However, there are rare reports with regard to the biological functions of NDUFA3 in the pathogenesis of IVDD.

High glucose promotes programmed cell death of human nucleus pulposus cells (HNPCs) to accelerate degeneration of intervertebral discs10–12. Thus high-glucose environment has been widely used to establish in vitro model to decipher the pathogenic mechanisms of IVDD13,14. In the present study, NDUFA3 was overexpressed in primary HNPCs treated with high glucose, in combination with rotenone, an mitochondrial complex I inhibitor, or not. Effects of NDUFA3 on high glucose-treated HNPCs were evaluated in terms of cell viability, cell apoptosis, reactive oxygen species (ROS) accumulation, mitochondrial function, and oxidative phosphorylation to analyze its anti-apoptotic, anti-oxidative and protective potential. Besides, further experiments were performed to clarify the regulatory mechanisms behind NDUFA3 transcription. Our study would provide novel insights for developing therapeutic strategies targeting mitochondrial dysfunction to treat IVDD.

Materials and methods

Clinical sample collection

The study was approved by the medical ethics committee of Henan Provincial People's Hospital (approval number MR-41–23-046,547 on 22 September 2023) and was conducted in accordance with the Declaration of Helsinki. Written informed consents were obtained from all participants. Degenerative nucleus pulposus (NP) samples were collected from 30 patients with intervertebral disc degeneration (IVDD). IVDD was assessed using the Pfirrmann grades including II, III, and IV15, who underwent operations at Hospital (Table 1). Non-degenerative NP samples were collected as controls from ten volunteers. Table 1 Human IVDD specimen information.

Donor	IVDD level	Age	Gender	Symptoms	Duration of symptoms prior to surgery (months)	Pfirrmann grade	
1	L2-L3	27	M	Radiculopathy: LBP, LP	3	II	
2	L2-L3	30	M	Radiculopathy: LBP, LP	4	II	
3	L2-L3	25	M	Radiculopathy: LBP, LP	2	II	
4	L3-L4	17	F	Radiculopathy: LBP, LP	6	II	
5	L3-L4	25	M	Radiculopathy: LBP, LP	3	II	
6	L3-L4	18	M	Radiculopathy: LBP, LP	4	II	
7	L3-L4	23	F	Radiculopathy: LBP, LP	4	II	
8	L4-L5	26	F	Radiculopathy: LBP, LP	5	II	
9	L4-L5	16	F	Radiculopathy: LBP, LP	6	II	
10	L4-L5	20	F	Radiculopathy: LBP, LP	3	II	
11	L4-L5	53	M	Radiculopathy: LBP, LP and palsy	6	III	
12	L4-L5	48	F	Radiculopathy: LBP, LP and palsy	10	III	
13	L4-L5	54	M	Radiculopathy: LBP, LP and palsy	8	III	
14	L4-L5	42	F	Radiculopathy: LBP, LP and palsy	12	III	
15	L4-L5	52	M	Radiculopathy: LBP, LP and palsy	8	III	
16	L4-L5	60	M	Radiculopathy: LBP, LP and palsy	9	III	
17	L5-S1	44	F	Radiculopathy: LBP, LP	12	III	
18	L5-S1	48	F	Radiculopathy: LBP, LP	8	III	
19	L5-S1	55	F	Radiculopathy: LBP, LP	9	III	
20	L5-S1	73	M	Radiculopathy: LBP, LP	12	III	
21	L4-L5	63	M	Radiculopathy: LBP, LP and palsy	10	IV	
22	L4-L5	60	F	Radiculopathy: LBP, LP	10	IV	
23	L4-L5	56	F	Radiculopathy: LBP, LP and palsy	18	IV	
24	L4-L5	64	F	Radiculopathy: LBP, LP	20	IV	
25	L4-L5	61	M	Radiculopathy: LBP, LP and palsy	14	IV	
26	L5-S1	45	M	Radiculopathy: LBP, LP	20	IV	
27	L5-S1	52	M	Radiculopathy: LBP, LP and palsy	18	IV	
28	L5-S1	44	F	Radiculopathy: LBP, LP	21	IV	
29	L5-S1	72	F	Radiculopathy: LBP, LP and palsy	24	IV	
30	L5-S1	55	M	Radiculopathy: LBP, LP	18	IV	
F female, M male, IVDD intervertebral disc degeneration, L lumbar, LBP low back pain, LP leg pain.

HNPCs isolation, culture and treatment

The non-degenerative or degenerative NP tissues were removed from the disc, washed 3 × phosphate buffer saline, and cut into small pieces. Then, the fragment was digested with 0.25% type II collagenase (Sigma‐Aldrich). HNPCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under a 5% CO2 and 5% oxygen atmosphere at 37 °C. When the HNPCs grew to 80% confluence, they were split once. HNPCs of passage 2 were used for the subsequent experiments. HNPCs were cultured in DMEM supplemented with 10% FBS (25 mM glucose, control group) or 10% FBS medium with a high glucose concentration (50, 100, or 200 mM, experiment group) for 48 h. High glucose concentration was referred to the previous studies10,16. To assess the effect of rotenone under HG condition, HNPCs were co-treated with 200 mM glucose and 1 μM rotenone (a mitochondrial complex I inhibitor) for 48 h. To inhibit ROS generation, cells were transduced with NDUFA3 short hairpin RNA (shRNA) lentivirus, followed by 1 mM N-acetylcysteine (NAC; a ROS scavenger) treatment for 48 h. To inhibit HDAC activity, cells were pretreated with 20 nM trichostatin A (TSA; HDAC inhibitor) for 2 h, followed by an incubation in 200 mM glucose for 48 h. To examine the dose-dependent effects of rotenone, NAC or TSA against high glucose- or NDUFA3 knockdown-induced decrease in cell viability, HNPCs were exposed to various concentrations of rotenone (0.2, 1 and 5 μM), NAC (0.25, 0.5 and 1 mM), or TSA (5, 10 and 20 nM).

Production of lentiviral vectors and cell transduction

To overexpress NDUFA3, the specific coding sequence was synthesized and cloned into pLVX-Puro plasmids (Clontech Laboratories). To knock down NDUFA3, the specific shRNA sequences targeting NDUFA3 (shNDUFA3-1 5′-CAAGAATGCCTGGGACAAGGA-3′; shNDUFA3-2, 5′-GCTGGTCGTGTCCTTCGTCGT-3′; and shNDUFA3-3, 5′-CTACTTCAAGTACTCCGTCAT-3′) or scramble shRNA (5′-TCGAGGCTGGTGCAGCATCAT-3′) were synthesized by Sangon Biotech (Shanghai, China) and cloned into linearized pLKO.1 plasmids (Addgene). The human embryonic kidney 293 T (HEK-293 T) cell line was used as a host for virus packaging17. The recombinant plasmids (11 µg) mixed along with the packaging plasmids psPAX2 (8.5 µg) and pMD2G (2.5 µg; Addgene) were co-transfected into 293 T cells in a 6-well plate (1 × 105 cells/well) for 6 h at 37 °C using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Subsequently, 48 h after transfection, the recombinant lentivirus in the cell supernatant was collected by centrifugation at 5,000 × g for 5 min and the purification and titration of recombinant lentivirus was performed as previously described18. HNPCs were plated in a 6-well plate (5 × 105 cells/well) and infected with the recombinant lentivirus-transducing units at an multiplicity of infection of 20 in the presence of 8 µg/ml polybrene (Sigma-Aldrich) for 24 h at 37 °C. The medium was changed after 12 h and the cells were analyzed 48 h post-transduction. Cells transduced with pLKO.1-scramble shRNA (shNC) or blank pLVX-Puro (Vector) were used as negative control. To exclude off-target effects of RNA interference, consistent findings were confirmed using three shRNAs with different sequences in all experiments19.

CCK-8 assay

HNPCs viability was analyzed by cell counting kit-8 (CCK-8) assay as previously described12. Briefly, HNPCs (2 × 103 cells per well) in a 96-well plate were incubated with 10 μL CCK-8 solution (Dojindo, Kumamoto, Japan) for 2 h at 37ºC. The absorbance was measured at 450 nm using an Epoch2 microplate reader (Biotek, Winooski, VT, USA).

Flow cytometry

To detect cell apoptosis by flow cytometry, the cells were first stained with Annexin-V-FITC solution (5 μL) at 4 °C for 15 min, were then stained with propidium iodide (5 μL) for 15 min20.

ROS generation in HNPCs was analyzed using a ROS assay kit (S0033; Beyotime Institute of Biotechnology). After indicated treatments, HNPCs were stained with 10 μM DCFH-DA in darkness for 30 min and then the fluorescence intensity of DCFH-DA was analyzed.

Mitochondrial membrane potential (MMP) ratio was calculated as red (JC-1 aggregates)/green (JC-1 monomers) fluorescence intensity using JC-1 assay kit (C2006, Beyotime Institute of Biotechnology)21. Flow cytometry was conducted on CytoFLEX flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Extracellular flux analysis

Oxygen consumption rate (OCR) level was estimated using Seahorse XF24 Extracellular Flux Analyzer as previously described22. Subsequently, 1 μM oligomycin (ATP synthase inhibitor), 1.5 μM FCCP (uncoupler) and mixture of antimycin A (complex III inhibitor; 0.5 μM) & rotenone (complex I inhibitor; 0.5 μM) were added into “A” well, “B” well, and “C” well of Seahorse gauging plate using Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies).

Measurement of mitochondrial complex I activity

The activities of mitochondrial complexes I were measured by MitoCheck Complex I activity assay kit (BC0515; Beijing Solarbio Science & Technology Co., Ltd, China) according to the manufacturer’s instructions.

Quantitative real-time PCR (qRT-PCR)

RNA was converted into cDNA using a reverse transcription cDNA synthesis PrimeScript kit (Takara Biotechnology, Dalian, China). And qRT-PCR was done by a SYBR Green PCR Master Mix kit (Applied Biosystem, Thermo). The sequences of primers are listed below: NDUFA3-F: 5ʹ-CTACAACTACCCAGTGCCCG-3ʹ; NDUFA3-R: 5ʹ-CACACACATGCTCACGTTCG-3ʹ; actin beta, F: 5ʹ-AGGATTCCTATGTG GGCGAC-3ʹ; R: 5ʹ-ATAGCACAGCCTGGATAGCAA-3ʹ. The value of gene expression was determined after normalization to β-actin23. The relative amounts of mRNA were calculated using the comparative Ct (2−ΔΔCt) method.

Western blot

After cell lysate extraction, the blots were transferred onto polyvinylidene fluoride membranes (Millipore)24. The target proteins were blotted with indicated primary antibodies as following: NDUFA3 (17,257–1-AP; Proteintech), H3K27ac (ab4729; abcam), and β-actin (81,115–1-RR; Proteintech). The membranes were soaked with HRP-conjugated secondary antibodies (ZB-2301, ZB-2305; ZSGB-BIO, Beijing, China). Expressions of target proteins were determined using an Enhanced Chemiluminescence Detection kit (Pierce Biotechnology, Rockford, IL, USA) and images were analyzed using ImageJ sofware for subsequent statistical processing. Data was normalized by β-actin, a loading control for each experiment23.

Chromatin immunoprecipitation (ChIP) assay

HNPCs were fixed with 1% formaldehyde and sonicated for chromatin fragmentation. The chromatin solutions were then incubated overnight with anti-H3K27ac (Cell Signaling Technology, Danvers, MA, USA; 8173) or control IgG antibody (Cell Signaling Technology; 3900) at 4 °C. H3K27ac binding in the NDUFA3 promoter region was quantified by qRT-PCR. All values were normalized to the input25. Data were analyzed by the Ct method and plotted as % input, calculated by the following formula: ΔCt[normalized IP] = Ct[IP] − Ct[Input] + Log2(Input dilution factor), and % input = 2^(−ΔCt[normalized IP]) × 100%.

Statistical analysis

All experiments were performed in triplicate. All data were presented as mean ± standard deviation (SD). GraphPad Prism software 8.4.2 (GraphPad Software, La Jolla, CA, USA) was used for statistics analyses. The normality of the distribution of variables was assessed using the Shapiro–Wilk test. Owing to the normal distribution of variables, statistical analyses were performed using the two-sided unpaired Student’s t-test (for comparison between two groups) or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test (for multi-group comparisons). A confidence level of at least 95% (p < 0.05) was set.

Results

High glucose led to decreased cell viability, increased apoptotic cells, excessive ROS production, mitochondrial dysfunction and impaired oxidative phosphorylation

Primary HNPCs isolated from non-degenerative NP tissues were cultured with 50, 100, and 200 mM glucose for 48 h. As shown in Fig. 1A, cell viability was decreased in response to glucose treatment in a time-dependent and concentration-dependent manner (p-value < 0.05). Apoptotic cells (Fig. 1B,C) and ROS productions (Fig. 1D,E) were markedly increased as a result of glucose treatment in a concentration-dependent manner (p-value < 0.001). MMP levels and OCRs were well-documented indicators of mitochondrial function and oxidative phosphorylation, respectively. Glucose exposure induced significant concentration-dependent decreases in MMP and OCR levels (p-value < 0.001, Fig. 1F,G). These data suggested that high glucose induces HNPCs apoptosis and mitochondrial dysfunction.Fig. 1 High glucose decreases cell viability, promotes cell apoptosis, increases ROS production, impairs mitochondria function and oxidative phosphorylation in HNPCs. HNPCs isolated from non-degenerative NP tissues were treated with 50, 100, and 200 mM glucose for 48 h, and the (A) cell viability, (B and C) cell apoptosis, (D and E) ROS production, (F) MMP levels, and (G) OCR values were measured. Data represent mean ± SD, n = 3 for each group. *P < 0.05, ***P < 0.001 versus control. Data analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Rotenone augmented the high glucose-induced effects on cell viability, apoptosis, ROS production and mitochondrial function

To examine the dose-dependent effects of rotenone against high glucose-induced decrease in cell viability, HNPCs isolated from non-degenerative NP tissues were co-treated with 200 mM glucose and rotenone (0.2, 1 and 5 μM) for 48 h. CCK-8 assay showed that 1 μM rotenone significantly inhibited the cell viability of high glucose-induced HNPCs by 27.5% while 5 μM rotenone showed decreased cell viability by 48.3% (p-value < 0.05, Fig. 2A). Therefore, 1 μM rotenone was used in our following study. It observed that co-treatment of high glucose and rotenone further induced cell apoptosis (Fig. 2B,C), promoted ROS production (Fig. 2D) and decreased MMP level (Fig. 2E) and OCRs (Fig. 2F) compared to high glucose treatment alone (p-value < 0.05). Mitochondrial complex I activity was impaired by exposure to high glucose (p-value < 0.001, Fig. 2G). Furthermore, this impairment was exacerbated by co-treatment with rotenone (p-value < 0.001, Fig. 2G). These data suggested that high glucose may induce HNPCs apoptosis and inhibit oxidative phosphorylation via the mitochondrial respiratory chain.Fig. 2 Rotenone strengthens high glucose-mediated cell viability, cell apoptosis, ROS accumulation and mitochondrial dysfunction. (A) HNPCs isolated from non-degenerative NP tissues were co-treated with high glucose (HG, 200 mM) and rotenone (0.2, 1 or 5 μM) for 48 h, and cell viability was measured. HNPCs isolated from non-degenerative NP tissues were co-treated with high glucose (HG, 200 mM) and 1 μM rotenone for 48 h, and (B and C) cell apoptosis, (D) ROS production, (E) MMP levels, (F) OCR values, and (G) mitochondrial complex I activity were measured. Data represent mean ± SD, n = 3 for each group. ***P < 0.001 versus control. #P < 0.05, ###P < 0.001 versus HG. Data analyzed using one-way ANOVA followed by Tukey’s post hoc test.

NDUFA3 down-regulation was observed in IVDD patients and the high glucose-treated HNPCs

NDUFA3 expression was detected in NP tissues samples isolated from IVDD patients and control subjects. NDUFA3 expression was significantly decreased at mRNA and protein level in IVDD patients with different Pfirrmann grades compared to control subjects (p-value < 0.01, Fig. 3A–C). Furthermore, among the IVDD patients with different Pfirrmann grades, the extent of decrease in NDUFA3 expression appeared to be positively related to Pfirrmann grades (Fig. 3A–C). Correlation between NDUFA3 protein expression and Pfirrmann grades are shown in Fig. 3D. Moreover, NDUFA3 expression was examined in primary HPNCs isolated from non-degenerative NP tissues treated with 50, 100, and 200 mM glucose as well. Glucose treatment at 100 and 200 mM induced significant down-regulations in NDUFA3 expression at mRNA and protein level (p-value < 0.05, Fig. 3E,F). These data suggested that NDUFA3 expression is negatively correlated with the severity of IVDD.Fig. 3 NDUFA3 expressions are increased in IVDD patients and high glucose-induced HNPCs. (A–C) mRNA levels and protein levels of NDUFA3 in NP samples from control subjects and IVDD patients. n = 10 for each group. (D) correlation between NUDFA3 expression and IVDD patients with different Pfirrmann grades. (E and F) mRNA levels and protein levels of NDUFA3 in high glucose-treated HNPCs. n = 3 for each group. Data represent mean ± SD. **P < 0.01, ***P < 0.001 versus control. Data analyzed using one-way ANOVA followed by Tukey’s post hoc test.

NDUFA3 alleviated the detrimental effects of high glucose on cell viability, cell apoptosis, ROS production, mitochondrial function and oxidative phosphorylation

In order to explore the effects of NDUFA3 in the high glucose-treated HNPCs, NDUFA3 were overexpressed in HNPCs isolated from non-degenerative NP tissues via lentivirus-mediated transduction (Fig. 4A,B), followed by 200 mM glucose treatment for 48 h. As shown in Fig. 4C–G, unsurprisingly, high glucose treatment resulted in decreases in cell viability, MMP and OCR levels, increases in apoptotic cells and excessive ROS production in HNPCs (p-value < 0.001). These high glucose-induced alterations were largely abolished by NDUFA3 overexpression (p-value < 0.001, Fig. 4C–G). These data suggested that high glucose induces HNPCs apoptosis and mitochondrial dysfunction by targeting NDUFA3.Fig. 4 NDUFA3 protects against high glucose-induced cell apoptosis, ROS production, mitochondrial dysfunction and impaired oxidative phosphorylation in HNPCs. (A and B) mRNAs and protein levels of NDUFA3 in NDUFA3-overexpressing HNPCs isolated from degenerative NP tissues. NDUFA3 were overexpressed in HNPCs isolated from non-degenerative NP tissues via lentivirus-mediated transduction, followed by high glucose (HG, 200 mM) treatment for 48 h, and (C) cell viability, (D) cell apoptosis, (E) ROS production, (F) MMP levels, and (G) OCR values were measured. Data represent mean ± SD, n = 3 for each group. ***P < 0.001 versus control. ###P < 0.001 versus HG. Data analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Rotenone offset the protective effect of NDUFA3 in HNPCs

NDUFA3-overexpressing HNPCs isolated from degenerative NP tissues were incubated with rotenone for 48 h. NDUFA3 overexpression promoted cell viability, inhibited cell apoptosis, decreased ROS production, and increased MMP and OCR levels (p-value < 0.001, Fig. 5A–E). These alterations induced by NDUFA3 overexpression were attenuated by rotenone treatment (p-value < 0.001, Fig. 5A–E). These data suggested that mitochondrial respiration may contribute to the effect of NDUFA3 on HNPCs viability and oxidative phosphorylation.Fig. 5 Rotenone compromises the protective effect of NDUFA3 in primary HNPCs. NDUFA3 were overexpressed in HNPCs isolated from degenerative NP tissue via lentivirus-mediated transduction, followed by rotenone (1 μM) treatment for 48 h, and (A) cell viability, (B) cell apoptosis, (C) ROS production, (D) MMP levels, and (E) OCR values were measured. Data represent mean ± SD, n = 3 for each group. ***P < 0.001 versus vector. ###P < 0.001 versus NDUFA. Data analyzed using one-way ANOVA followed by Tukey’s post hoc test.

Next, to examine the effects of NAC against NDUFA3 silencing-induced decrease in cell viability, HNPCs isolated from non-degenerative NP tissues were transduced with shNDUFA3 lentiviral vector, with the lowest NDUFA3 expression detected in cells transduced with shNDUFA3-3 (Fig. S1A,B). CCK-8 assay showed that 1 mM NAC significantly promoted the cell viability of NDUFA3 silencing-induced HNPCs compared to 0.25 and 0.5 mM NAC (p-value < 0.001, Fig. S1C). Therefore, 1 mM NAC was used in our following study. Unsurprisingly, NDUFA3 silencing led to increased apoptotic cells, excessive ROS production, and decreased MMP and OCR levels, which were largely restored by NAC treatment (p-value < 0.001, Fig. S1D–G). These data suggested that NDUFA3 silencing induces HNPCs apoptosis and mitochondrial dysfunction by producing excessive ROS.

HDAC/H3K27ac-mediated transcription of NDUFA3 protected against high glucose-induced insults to HNPCs

Results of ChIP-PCR experiments showed the binding of H3K27ac to NDUFA3 promoter (Fig. 6A). HDACs enzymes that catalyze histone acetyl participate in regulating gene transcription and protein activity26. HNPCs were treated with 20 nM TSA, a HDAC inhibitor, for 48 h. NDUFA3 mRNA levels as well as NDUFA3 and H3K27ac proteins were significantly increased by TSA treatmen at 24 h and 48 h (p-value < 0.001, Fig. 6B,C). These results indicated that NDUFA3 transcription was regulated by HDAC/H3K27ac. In order to determine whether HDAC/H3K27ac was the regulatory mechanism underlying the effects of high glucose on HNPCs, cells were pre-treated with different concentrations of TSA prior to high glucose treatment for 48 h. CCK-8 assay showed that 20 nM TSA significantly promoted the cell viability of high glucose-induced HNPCs compared to 5 and 10 nM TSA (p-value < 0.001, Fig. 6D). Therefore, 20 nM TSA was used in our following study. As mentioned above, high glucose treatment resulted in decreases in MMP and OCR levels as well as increases in apoptotic cells and excessive ROS productions (p-value < 0.001), which were largely compromised by TSA treatment (p-value < 0.001, Fig. 6E–I). Moreover, as shown in Fig. 6J,K, high glucose-induced NDUFA3 down-regulation was partly restored by TSA treatment at both mRNA and protein levels. These data suggested that HDAC/H3K27ac-mediated transcription of NDUFA3 alleviates HNPCs apoptosis and mitochondrial dysfunction induced by high glucose.Fig. 6 HDAC/H3K27ac-mediated transcription of NDUFA3 protects HNPCs against high glucose-induced injuries. (A) ChIP-PCR experiments in HNPCs isolated from non-degenerative NP tissues. (B) NDUFA3 mRNA levels and (C) protein levels of NDUFA3 and H3K27ac in HNPCs treated with 20 nM TSA. HNPCs were pretreated with 20 nM TSA for 2 h, followed by an incubation in 200 mM glucose (HG) for 48 h, and (D) cell viability, (E–F) cell apoptosis, (G) ROS production, (H) MMP levels, (I) OCR values, and (J–K) NDUFA3 mRNA and protein levels were measured. Data represent mean ± SD, n = 3 for each group. ***P < 0.001 versus IgG, 0 h or control. #P < 0.05, ###P < 0.001 versus HG. Data analyzed using (A) two-sided unpaired Student’s t-test or (B–K) one-way ANOVA followed by Tukey’s post hoc test.

Discussion

IVDD resulting in disc dehydration and disruption has been identified as a dominant etiological factor of lower back pain and disability globally, with its prevalence increasing with age, causing huge financial expenditure27. In our study, primary HNPCs were cultured in hyperglycemic environment to build an in vitro model of IVDD. Our study showed that exposure to high glucose provoked cell apoptosis, stimulated ROS accumulation, impaired mitochondrial function and disrupted oxidative phosphorylation, which are in line with previous studies28,29. Rotenone is a well-documented respiratory chain complex I inhibitor, inducing mitochondrial dysfunction and promoting ROS production30. In our study, consistently, co-treatment with rotenone aggravated the high glucose-induced injuries in HNPCs.

Mitochondria serves as a major source of energy supply as well as ROS production in HNPCs31. NDUFA3, as a critical subunit of mitochondrial complex I, has important implications in mitochondrial function and ATP production32. Yet, there is limited evidence concerning the role of NDUFA3 in IVDD. NDUFA3 was found to be down-regulated in tissue samples from IVDD patients as well as in high glucose-treated HNPCs. NDUFA3 expression was negatively correlated with IVDD severity, indicating that NDUFA3 retarded the progression of IVDD. Besides, exposure to high glucose induced dose-dependent suppressive effects on NDUFA3 expression. Excessive ROS accumulation attracts mitochondria and leads to mitochondrial dysfunction, which consequently enhances oxidative stress and triggers cell death in the pathogenesis of IVDD33. Previous data yielded from bioinformatics analysis uncovers the involvement of NDUFA3 in regulating oxidative stress and cell apoptosis9. Consistently, in the present study, NDUFA3 overexpression displayed ameliorative effects on high glucose-induced injuries in HNPCs, as evidenced by suppressed cell apoptosis, eliminated ROS accumulation, and improved mitochondrial function and oxidative phosphorylation. Moreover, silencing NDUFA3 decreased cell viability and promoted cell apoptosis. Collectively, these findings signify that NDUFA3 possesses therapeutic potential on high glucose-induced IVDD. Furthermore, the therapeutic effect of NDUFA3 overexpression on HNPCs was abolished through inhibiting mitochondrial complex I with rotenone treatment, confirming that mitochondrial complex I mediated the effects of NDUFA3 in HNPCs.

Oxidative stress is an important driver mechanism of IVDD and effectiveness of some antioxidant therapeutic strategies has been proved in palliating IVDD34. Our study showed that NDUFA3 silencing-induced cell apoptosis and decreased cell viability was suppressed by antioxidant NAC treatment. It substantiated that the effect of NDUFA3 on cell apoptosis and viability was mediated through scavenging ROS accumulation.

Histone deacetylases (HDAC) are epigenetic enzymes that facilitate histone deacetylation to repress gene transcription, affecting diverse biological functions, such as cell survival and growth35,36. Histone3 Lysin27 residue-acetylation (H3K27ac) histone modification boosts gene transcription through regulating cis-regulatory elements, and has been acknowledged as a chromatin marker of active enhancers and promoters37,38. Our study found that H3K27ac was bound to NDUFA3 promoter. HDAC inhibition counteracted the high glucose-induced effects on cell apoptosis, ROS production, mitochondrial function and oxidative phosphorylation through up-regulating NDUFA3 expression. It provides evidence that HDAC/H3K27ac is a regulatory mechanism underlying NDUFA3 transcription in HNPCs.

Despite strengths of this study, some limitations exist. First, because the NP region first exhibits degenerative changes, we mainly focused on the response of NP cells to high glucose in the present study. Second, in addition to oxidative stress and mitochondrial function, inflammation is an important pathogenic mechanism of IVDD39,40. Further studies are warranted to investigate the effects of NDUFA3 on inflammatory factors. Third, in vivo experiments are needed to validate the effect of NDUFA3 on NP cell apoptosis, oxidative stress and mitochondrial function. Fourth, the present study is just a beginning of our research plan, we will continue to decipher the downstream targets of NDUFA3 to unravel the underlying molecular mechanisms and identify promising therapeutic targets in the future study.

Conclusions

Taken together, this study demonstrated the protective effects of NDUFA3 against high glucose-induced insults to HNPCs through suppressing cell apoptosis, scavenging ROS, ameliorating mitochondrial dysfunction and improving oxidative phosphorylation. HDAC/H3K27ac regulatory mechanism was involved in regulating NDUFA3 transcription. Our study lays the ground toward understanding the biological functions of NDUFA3 in the pathophysiology of DM-induced IVDD, and towards developing potential targeted therapies. Validation of our results deserves further study.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71810-8.

Author contributions

Conceptualization: G.Y. and C.Z. Formal Analysis: D.G. and T.Z. Investigation: W.H. Methodology: B.Z. and H.F. Project Administration: Y.G. Writing—Original Draft: C.Z. Writing—Review & Editing: C.Z. and G.Y.

Funding

The funding provided by Henan Provincial Medical Science and Technology Research Program (LHGJ20230058).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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