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Procyanidin C1 ameliorates acidic pH stress induced nucleus pulposus degeneration through SIRT3/FOXO3-mediated mitochondrial dynamics

Journal of Translational Medicine volume 22, Article number: 1071 (2024) Cite this article

Abstract

Intervertebral disc degeneration (IVDD) is a common cause of low back pain. Procyanidin C1 (PCC1) has been demonstrated to exert a protective effect on nucleus pulposus (NP) cells, and therefore, plays a critical role in the prevention and therapy of IVDD. Clarifying the pathophysiological characteristics and molecular mechanisms of IVDD may be helpful in establishing novel preventive and therapeutic strategies. This study aimed to investigate the probable mechanisms underlying the protection against acidic pH stress induced human NP cell injury. In vitro, acidic pH stress induced degeneration, mitochondrial dynamics imbalance, mitophagy, and mitochondria-mediated apoptosis in NP cells, all of which were ameliorated by PCC1. Autophagy inhibition partially eliminated the protective effects of PCC1 on mitochondrial homeostasis in NP cells. Moreover, PCC1 activated the sirtuin 3 (SIRT3)/forkhead box O3 (FOXO3) signaling pathway, a pivotal signaling pathway involved in the regulation of mitochondrial homeostasis in NP cells. In vivo, PCC1 ameliorated IVDD in a rat model and preserved the extracellular matrix of NP cells. Consequently, the protective effects of PCC1 on NP cells may inhibit IVDD progression via regulation of the SIRT3/FOXO3 signaling pathway. Therefore, regulation of the SIRT3/FOXO3 signaling pathway may be a novel preventive and therapeutic strategy for IVDD.

Introduction

Low back pain is an extremely prevalent musculoskeletal disorder, that affected 619 million people globally in 2020, and is also one of the leading causes of high years lived with disability worldwide [1, 2]. The intervertebral disc (IVD) consists of a gelatinous nucleus pulposus (NP) surrounded by a laminar annulus fibrosus and sandwiched by cartilage endplates [3]. IVD degeneration (IVDD), which commonly results in a progressive inflammatory response, stenosis, and even spinal deformity, is a well-known cause of low back pain [4,5,6]. However, the pathophysiology of IVDD remains to be elucidated, including the observed extracellular matrix deterioration, inflammatory cytokine release, resident cell death, secondary disc height, and motility changes [7,8,9,10].

Mitochondria play critical roles in a variety of cellular processes, including calcium homeostasis, energy metabolism, redox balance, stress resistance, and organelle communication, and they have been implicated in the pathogenesis and progression of IVDD [11,12,13,14,15]. Mitochondrial homeostasis is important for maintaining disc integrity and is closely associated with the pathogenesis and progression of IVDD [13, 15]. The mitochondrial surveillance system, which includes mitochondrial dynamics and mitophagy, regulates the health mitochondrial quality, thereby influencing the survival, proliferation and extracellular matrix production of NP cells [11, 13, 16]. Disruption of mitochondrial dynamics via aberrant mitochondrial fusion or fission is closely related to disruption of mitochondrial homeostasis, which results in IVDD progression [11, 13, 17, 18]. Mitophagy is involved in the selective removal of defective mitochondria and the maintenance of mitochondrial quality control [11]. Therefore, the targeted regulation of mitophagy and mitochondrial quality control may have therapeutic potential in inflammatory or degenerative diseases, such as IVDD [13, 19, 20].

Sirtuin 3 (SIRT3), which is located in the mitochondria, is essential f fission and fusion, and plays critical roles in regulating oxidative stress, inflammation, autophagy and apoptosis [21,22,23]. As one of the most conserved and widely recognized human longevity genes, forkhead box O3 (FOXO3) has a cytoprotective effect on various tissues, is important in the regulation of mitochondrial homeostasis, and plays a critical role in the treatment of age-related diseases, such as IVDD [24,25,26].

Procyanidin C1 (PCC1), a polyphenolic component extracted from grape seeds, increases lifespan and health span by regulating senescent cells [27, 28]. PCC1 plays a potent role in inhibiting inflammatory activity, resulting in a significant decrease in prostaglandin E2 and cyclooxygenase-2 levels [29]. PCC1 may be essential for the regulation of mitochondrial homeostasis [27]. A variety of signaling pathways, including the mitogen-activated protein kinase signaling pathway, nuclear factor-kappa-B (NF-κB) signaling pathway, and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, have been proposed to be activated by PCC1 [29, 30]. In addition, PCC1 mediates the nuclear translocation of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and increases the expression of heme oxygenase [31]. However, the molecular mechanisms by which PCC1 slows the progression of IVDD are not fully understood.

Fig. 1
figure 1

Cytotoxic effects of PCC1 and different pH level on human NP cells, and protective effects of PCC1 against the acidic pH stress induced changes of extracellular matrix metabolism. (A) Cell viability after treatment with different pH level for 24 h. (B) Cell viability after treatment with PCC1 for 24 h. (C) The expression levels of COL2A1, COL1A2, aggrecan and MMP3 were determined by qRT-PCR in NP cells after treatment with PCC1 for 24 h before exposure to acidic pH for 8 h. (D and E) COL2A1 and COL1A2 protein levels were confirmed by immunofluorescence staining (magnification: 400×; scale bar: 20 μm). Cell viability is expressed as mean ± standard deviation, with the value of control group set to 100%. Control, NP cells were treated by PBS; PCC1 0µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (0µM); PCC1 10µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (10µM); PCC1 20µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (20µM); PCC1 40µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (40µM). Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Therefore, we explored the mechanisms underlying the protective effects of PCC1 against acidic pH stress induced imbalance in mitochondrial dynamics in NP cells and the potential roles of the SIRT3/FOXO3 signaling pathway in the regulation of mitochondrial dynamics in NP cells. Our findings demonstrate that PCC1 protects against mitochondrial dynamics imbalance and apoptosis, both in vitro and in vivo, and that targeting PCC1 may be a novel therapeutic and preventive strategy for IVDD.

Materials and methods

Isolation and culture of primary human NP cells

Human NP tissue samples were obtained from patients who underwent spinal surgery for idiopathic scoliosis, lumbar disc herniation, or vertebral fracture. Magnetic resonance images (MRI) were collected from patient medical records to evaluate the degree of disc degeneration according to the MRI-based Pfirrmann grading system [32]. Informed consent was obtained from all participants in this study. Ethical approval was obtained from the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. S214).

Primary human NP tissues were collected from patients and stored in Hank’s balanced salt solution for transportation. NP tissues were cut into pieces and used to seed and expand NP cells via culture in Dulbecco’s modified Eagle’s medium supplemented with F12 nutrient mixture (DMEM/F-12; Gibco, Grand Island, NY, USA) and 15% fetal bovine serum (Gibco) at 37 °C in 5% CO2 [13, 15]. Human NP cells from the second passage were used for subsequent experiments.

Stimulation of human NP cells

Human NP cells were seeded, incubated overnight, and serum-starved in 1.0% fetal bovine serum for 24 h. The NP cells were then incubated alone or pretreated with PCC1 (Aladdin, Shanghai, China; 10, 20, and 40 µM) for 24 h, followed by incubation alone or subjection to acidic pH stress (pH 7.1–7.3, pH 6.5–6.7, and pH 5.9–6.1) for 8 h. An autophagic inhibitor 3-MA (MedChemExpress, NJ, USA; 5 µM) was used to regulate the signaling pathway.

Cell viability analysis

Cell viability was assayed using a cell counting kit-8 (CCK-8; Dojindo Co, Kumamoto, Japan) assay according to the manufacturer’s protocol. After treatment with acidic pH stress (pH 7.1–7.3, pH 6.5–6.7, and pH 5.9–6.1) or PCC1 (10, 20, and 40 µM), human NP cells were washed with phosphate-buffered saline (PBS), followed by the addition of 100 µL of DMEM containing 10 µL of CCK-8 solution and incubation for approximately 1 h. The absorbance of the cells was measured at 450 nm using a Spectra MAX microplate reader (Multiskan MK3; Thermo Fisher Scientific, Waltham, MA, USA).

Flow cytometry

Cell death was evaluated using an Annexin V-FITC /propidium iodide (PI) Apoptosis Kit (KeyGen Biotech, Jiangsu, China). Human NP cells were collected and resuspended in 400 µL of binding buffer. Subsequently, 5 µL of Annexin V-FITC and 2 µL of PI were added to the samples, followed by incubation for 15 min. Finally, fluorescence was measured by flow cytometry (Beckman Coulter, Brea, CA, USA).

Transmission electron microscopy

Human NP cells were collected, washed twice with PBS, and centrifuged at 300 × g for 15 min. The cells were subjected to fixation using 2.5% glutaraldehyde at 25 °C for 2 h and 1% osmium tetroxide at 25 °C for 2 h, and then dehydrated using ascending series of ethanol solutions, followed by permeabilization with acetone. Finally, the pellets were submerged in an embedding medium and subjected to ultrathin sectioning. Uranyl acetate and lead citrate were used to provide contrast between regions. The mitochondrial state was examined using a transmission electron microscope (Hitachi, Tokyo, Japan).

Quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from human NP cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcriptase (TaKaRa, Tokyo, Japan) was used to generate complementary DNA (cDNA) from total RNA. The qRT-PCR was conducted using a SYBR Premix Ex Tag kit (TaKaRa). The cycling conditions consisted of 40 cycles of denaturation at 95℃ for 5 s, followed by amplification at 60℃ for 24 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The PCR primer sequences are listed in Table 1.

Table 1 List of primers used in quantitative real-time polymerase chain reaction (qRT-PCR)
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Western blotting

Total protein was extracted from human NP cells using RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors. Proteins were separated using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride (PVDF) membranes using a consistent electrical current of 400 mA for 2 h, followed by membrane blocking with 5% non-fat milk at 25 °C for 1 h. The membranes were then incubated with diluted primary antibodies against serine/threonine PTEN-induced putative kinase (PINK1), Parkin, Nix, Bnip3, LC3, mitofusin-2 (MFN-2), dynamin-related protein-1 (DRP-1), optic atrophy-1 (OPA-1), mitochondrial fission 1 (FIS1), SIRT3, FOXO3, Bax, Bcl-2, cleaved caspase-3, caspase-9, and GAPDH at 4 °C overnight. The dilutions of the primary antibodies are listed in Table 2. The membranes were subsequently incubated with 1:3000 diluted secondary antibodies at 25 °C for 1 h. Protein expression levels were evaluated using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and visualized using the Chemi-DocMP Imaging System (12003154, Bio-Rad, Hercules, CA, USA).

Fig. 2
figure 2

PCC1 alleviates apoptosis of NP cells caused by acidic pH stress. (A) The protein levels of Bax, Bcl-2, cleaved caspase-3, and caspase-9, were confirmed by western blotting. (B) The expression levels of Bax, Bcl-2, cleaved caspase-3, and caspase-9, were determined by qRT-PCR. (C) NP cells stained with Annexin V-FITC and PI and then analyzed by flow cytometry. Cells positively stained for Annexin V-FITC and negatively stained for PI were scored as early apoptotic. Cells positively stained for both Annexin V-FITC and PI were scored as late apoptotic or necrotic. (D) Apoptotic rates of each group; values are expressed as mean ± standard deviation. (E-F) According to the results of TUNEL staining, the rate of TUNEL positive cells increased in the acidic pH stimulated group, while the rate of TUNEL positive cells decreased in the PCC1 pretreated group. Control, NP cells were treated by PBS; PCC1 0µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (0µM); PCC1 10µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (10µM); PCC1 20µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (20µM); PCC1 40µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (40µM). Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Table 2 Primary antibodies for Western blotting
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Immunofluorescence

After treatment, human NP cells were washed thrice with PBS, followed by fixation using 4% paraformaldehyde for 15 min. Permeabilization was performed by adding a solution containing 0.3% Triton X-100 for 20 min. Thereafter, the cells were blocked with 1% (w/v) bovine serum albumin at 25 °C for 1 h and incubated overnight at 4 °C with the primary antibody. Subsequently, the sample was incubated with the secondary antibody at 25 °C for 1 h. The dilutions of the primary and secondary antibodies are listed in Table 3. The cytoskeleton and nucleus were labeled with TRITC-phalloidin and 4’,6-diamidino-2-phenylindole (DAPI) staining solution (5 mg/mL), respectively. The fluorescence signals were visualized using a fluorescence microscope (Olympus IX51, Olympus, Tokyo, Japan).

Table 3 Primary and secondary antibodies for immunofluorescence
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Fig. 3
figure 3

PCC1 alleviates excessive mitophagy caused by acidic pH stress. (A) The protein levels of mitophagic indicators, such as PINK, Parkin, Nix, Bnip3, LC3-I and LC3-II, were confirmed by western blotting. (B) The expression levels of PINK, Parkin, Nix, Bnip3, and ratio of LC3-II/LC3-I, were determined by qRT-PCR. (C) Transmission electron microscopy demonstrated that PCC1 ameliorated the generation of mitophagic vesicles that contained impaired mitochondria induced by acidic pH stress. Control, NP cells were treated by PBS; PCC1 0µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (0µM); PCC1 10µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (10µM); PCC1 20µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (20µM); PCC1 40µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (40µM). Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Fig. 4
figure 4

PCC1 sustains a balance in mitochondrial dynamics in NP cells. (A) The protein levels of SIRT3, FOXO3, MFN-2, DRP-1, OPA1, FIS1, were confirmed by western blotting. (B) The expression levels of SIRT3, FOXO3, MFN-2, DRP-1, OPA1, FIS1, were determined by qRT-PCR. (C) Immunofluorescence staining showed that downregulated SIRT3 protein levels caused by acidic-pH was alleviated by PCC1 administration. Control, NP cells were treated by PBS; PCC1 0µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (0µM); PCC1 10µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (10µM); PCC1 20µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (20µM); PCC1 40µM, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (40µM). Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

After 1 h of fixation in paraformaldehyde (4%), the NP cells were incubated for 10 min with 0.1% Triton X-100 and 3% hydrogen peroxide. After washing with PBS, TUNEL inspection fluid and DAPI were used to co-stain the cells. A fluorescence microscope (Olympus IX51) was used to randomly examine three slides. The apoptotic index was calculated by dividing the number of dead cells by the total number of cells, as described previously [15].

Fig. 5
figure 5

Autophagy inhibitor reverses the protective effect of PCC1. (A) Transmission electron microscopy showed that PCC1 ameliorated the generation of mitophagic vesicles that contained impaired mitochondria under acidic pH, while the generation of mitophagic vesicles were downregulated by autophagy inhibitor (3-MA). (B) The protein expressions of PINK, Parkin, Nix, Bnip3, LC3-I and LC3-II, were confirmed by western blotting. (C) The protein expressions of Bax, Bcl-2, cleaved caspase-3 and caspase-9, were confirmed by western blotting. pH 6.5, NP cells were treated by acidic pH stress (pH 6.5–6.7); pH 6.5 + 3-MA, NP cells were treated by acidic pH stress (pH 6.5–6.7) and 3-MA (5µM); pH 6.5 + PCC1, NP cells were treated by acidic pH stress (pH 6.5–6.7) and PCC1 (40µM); pH 6.5 + PCC1 + 3-MA, NP cells were treated by acidic pH stress (pH 6.5–6.7), PCC1 (40µM) and 3-MA (5µM). Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Animal models and surgical procedures

Sprague–Dawley rats (male, 3 months old, n = 24) were purchased from the Laboratory Animal Center of Huazhong University of Science and Technology. After anesthetization with 2% (w/v) pentobarbital (40 mg/kg) via intraperitoneal injection, tail discs (Co6/7 and Co7/8) were confirmed by trial radiograph, and surgeries were performed as described previously [13, 15, 21, 33]. Following surgery, PCC1 was injected intraperitoneally at a dose of 8 mg/kg per day until the rats were euthanized. The rats were divided into four groups: the sham-operated group (control), the PBS group (which underwent surgery and received PBS treatment), the PUN group (which underwent surgery and received no treatment), and the PCC1 group (which underwent surgery and received PCC1 treatment). Eight weeks post-surgery, rats were euthanized, and IVD tissues were collected for histological analysis.

Fig. 6
figure 6

Inhibition of SIRT3 attenuates the protective effects of PCC1. (A) The protein levels of SIRT3, FOXO3, MFN-2, DRP-1, OPA-1, FIS1, were confirmed by western blotting. (B) The protein expressions of PINK, Parkin, Nix, Bnip3, LC3-I and LC3-II, were confirmed by western blotting. (C) The protein expressions of Bax, Bcl-2, cleaved caspase-3 and caspase-9, were confirmed by western blotting. (D-F) aggrecan, COL2A1, and MMP3 protein levels were further confirmed by immunofluorescence staining (magnification: 400×; scale bar: 20 μm). PCC1 + pH 6.5, NP cells were treated by PCC1 (40µM) and acidic pH stress (pH 6.5–6.7); PCC1 + pH 6.5 + NC, NP cells were treated by PCC1 (40µM), acidic pH stress (pH 6.5–6.7), and PBS; PCC1 + pH 6.5 + SIRT3 OE, NP cells were treated by PCC1 (40µM), acidic pH stress (pH 6.5–6.7), and SIRT3 overexpression; PCC1 + pH 6.5 + SIRT3 siRNA, NP cells were treated by PCC1 (40µM), acidic pH stress (pH 6.5–6.7), and SIRT3 siRNA; PCC1 + pH 6.5 + SIRT3 siRNA2, NP cells were treated by PCC1 (40µM), acidic pH stress (pH 6.5–6.7), and SIRT3 siRNA2. Three independent experiments were performed. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Radiological examination

Before modeling and 8 weeks after puncture, radiography was performed on the rats. All rats were anesthetized by intraperitoneal injection of 2% (w/v) pentobarbital (40 mg/kg) and fixed in a prone position using an X-ray machine (MULTIX Impact, SIEMENS, Germany). The disc height index was determined according to published methods [33,34,35].

Fig. 7
figure 7

PCC1 attenuates IVDD in a rat model. (A) Digital X-ray image of intervertebral disc from different experimental groups. (B) Quantification of narrowing of disc space, the disc height indexs in four groups (six rats at each time point for each group). (C and D) Representative HE staining and SO staining of disk samples from different experimental groups post-surgery (original magnification × 40, scale bar: 100 μm). Three sections were randomly selected for quantification, with a representative example shown. (E) The histological grades evaluated post-surgery in three groups (six rats per group). Control, the sham-operated group; PCC1 group, the group which underwent surgery and received PCC1 treatment; PBS group, the group which underwent surgery and received PBS treatment; PUN group, the group which underwent surgery and received no treatment. ⁎p < 0.05, ⁎⁎p < 0.01 versus control

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Histopathologic analysis

The rats were sacrificed at 8 weeks after puncture. Discs were fixed in formaldehyde (4%, pH 7.4) for 12 h, decalcified in 10% formic acid, dehydrated, and embedded in paraffin. The specimens were sectioned at 4 μm. Sections were stained with hematoxylin and eosin (HE) and Safranin O-Fast Green (SO). The cellularity and morphology of cells were examined under a microscope (Olympus).

Fig. 8
figure 8

Schematic graph of Procyanidin C1 (PCC1) ameliorates acidic pH stress induced nucleus pulposus degeneration through SIRT3/FOXO3-mediated mitochondrial dynamics

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Statistical analysis

The results are presented as mean ± standard deviation. Statistical analyses were performed using SPSS (version 22.0; IBM Corp., Armonk, NY, USA). Normal distribution of the data was assessed using the Kolmogorov–Smirnov test, between-group comparisons were evaluated by paired-sample t-tests, and comparisons among three or more groups were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s test. Nonparametric data were analyzed using the Mann–Whitney U test. P < 0.05 was considered statistically significant.

Results

PCC1 ameliorates acidic pH stress induced NP degeneration

When human NP cells were exposed to different pH levels for 24 h, cell viability decreased as the pH became more acidic. Treatment with pH 7.1–7.3 or pH 6.5–6.7 for 24 h did not result in significant cytotoxicity to NP cells, while treatment with pH 5.9–6.1 significantly reduced cell viability by more than 50%; therefore, pH 5.9–6.1 was used for the subsequent experiments (Fig. 1A). Additionally, when human NP cells were exposed to PCC1 at concentrations of 10, 20, 40, 80, and 120 µM for 24 h, those treated with 10, 20, and 40 µM exhibited high viability; therefore, these concentrations were used in the subsequent experiments (Fig. 1B).

To determine the protective effects of PCC1 on the regulation of extracellular matrix metabolism homeostasis in the IVD, human NP cells were pretreated with PCC1 with or without acidic pH stress. The expression levels of COL2A1, COL1A2, aggrecan, and MMP3 were determined using qRT-PCR. The expression of COL2A1 and aggrecan was downregulated by acidic pH but upregulated by PCC1 in a dose-dependent manner (Fig. 1C). Furthermore, the expression of COL1A2 and MMP3 was significantly upregulated by acidic pH stress but was downregulated by PCC1 in a dose-dependent manner (Fig. 1C). The effects of acidic pH stress and PCC1 pretreatment on COL1A2 and MMP3 protein levels were confirmed by immunofluorescence staining (Fig. 1D-E).

PCC1 alleviates NP cell apoptosis caused by acidic pH stress

Downregulation of Bcl-2 and upregulation of Bax, cleaved caspase-3, and caspase-9 were observed in the acidic pH stimulated group. However, PCC1 reversed these changes in a dose-dependent manner. Thus, PCC1 appears to protect against acidic pH stress induced mitochondrial apoptosis (Fig. 2A-B). The rate of apoptosis in NP cells rose to nearly 50% in response to treatment with acidic pH, significantly higher than the rate in the control group (p < 0.01). Pretreatment with PCC1 could attenuate this increase in a dose-dependent manner (Fig. 2C-D). TUNEL staining also demonstrated that the number of TUNEL-positive cells increased in the acidic pH stress group but decreased in the PCC1 treatment group (Fig. 2E-F).

PCC1 alleviates excessive mitophagy caused by acidic pH stress

We next determined the protective effects of PCC1 against mitophagy in NP cells treated with acidic pH stress. The expression of mitophagy indicators, such as PINK, Parkin, Nix, and Bnip3, and the LC3-II/LC3-I ratio decreased in NP cells treated with PCC1 in a dose-dependent manner (Fig. 3A-B). Moreover, transmission electron microscopy demonstrated that PCC1 ameliorated mitophagic vesicle generation after acidic pH stress treatment (Fig. 3C).

PCC1 sustains a balance in mitochondrial dynamics in NP cells

Western blot and qRT‐PCR results demonstrated that acidic pH stimulation upregulated the expression of DRP-1 and FIS1 and downregulated that of MFN-2, OPA-1, SIRT3, and FOXO3 (Fig. 4A-B). However, PCC1 pretreatment downregulated the expression of DRP-1 and FIS1 and upregulated that of MFN-2, OPA-1, SIRT3, and FOXO3 in a dose-dependent manner (Fig. 4A-B). Moreover, immunofluorescence staining showed that the downregulation of SIRT3 protein levels caused by acidic pH stress was alleviated by PCC1 administration (Fig. 4C).

Autophagy inhibitor reverses the protective effects of PCC1

NP cells were cultured and then incubated alone or pretreated with PCC1 (40 µM) and/or 3-MA for 24 h, followed by acidic pH stress induction for 8 h. Transmission electron microscopy showed that PCC1 ameliorated the generation of mitophagic vesicles containing impaired mitochondria under acidic pH conditions, but this effect of PCC1 was inhibited by the autophagy inhibitor 3-MA (Fig. 5A). The expression of PINK, Parkin, Nix, and Bnip3, and the LC3-II/LC3-I ratio were downregulated by 3-MA (Fig. 5B). Treatment with 3-MA reversed the downregulation of Bcl-2 and the upregulation of Bax, cleaved caspase-3, and caspase-9 induced by PCC1 (Fig. 5C).

Inhibition of SIRT3 attenuates the protective effects of PCC1

To further investigate the role of SIRT3 signaling, SIRT3 overexpression, SIRT3 siRNA, and SIRT3 siRNA2 were applied. SIRT3 overexpression upregulated the expression of SIRT3, FOXO3, MFN-2, and OPA-1, downregulated the expression of DRP-1 and FIS1, whereas SIRT3 siRNA and SIRT3 siRNA2 downregulated the expression of SIRT3, FOXO3, MFN-2, and OPA-1, upregulated the expression of DRP-1 and FIS1 (Fig. 6A). SIRT3 overexpression downregulated the expression of PINK, Parkin, Nix, and Bnip3 and the LC3-II/LC3-I ratio, whereas SIRT3 siRNA and SIRT3 siRNA2 upregulated the expression of PINK, Parkin, Nix, and Bnip3 and the LC3-II/LC3-I ratio (Fig. 6B). SIRT3 overexpression upregulated the expression of Bcl-2, downregulated the expression of Bax, cleaved caspase-3 and caspase-9, whereas SIRT3 siRNA and SIRT3 siRNA2 downregulated the expression of Bcl-2, upregulated the expression of Bax, cleaved caspase-3 and caspase-9 (Fig. 6C). Immunofluorescence staining showed that SIRT3 overexpression upregulated aggrecan, COL2A1 protein levels, and downregulated MMP3 protein level; whereas SIRT3 siRNA and SIRT3 siRNA2 downregulated aggrecan, COL2A1 protein levels, and upregulated MMP3 protein level (Fig. 6D-E).

PCC1 attenuates rat IVDD in vivo

To determine the therapeutic effects of PCC1 on IVDD in vivo, we constructed a Sprague-Dawley rat model. Digital radiographs of IVDs were obtained for the different experimental groups (Fig. 7A-B). HE and SO staining confirmed the therapeutic effects of PCC1 on IVDD in the degenerative model. In the control group, oval-shaped NP tissues occupied the major portion of the disc height in the midsagittal cross-section of the slides, with a high glycosaminoglycan content in the NP area. Compared with that of the control group, the disc height in the PBS and PUN groups collapsed, with a significant loss of cells, an increase in tissue fibrillation in the NP area, and a tear pattern in annulus fibrosus tissues (Fig. 7C-D). Nevertheless, PCC1 treatment delayed these histopathological changes in the presence of more NP cells. The histological score of the punctured discs was lower in the PBS and PUN groups than in the PCC1 group; therefore, PCC1 may ameliorate IVDD in vivo (Fig. 7E).

Discussion

It has been demonstrated that IVDD involves a variety of pathogenic factors, including biomechanical changes, genetic susceptibility, trauma, oxidative stress, acidic pH stress, and inflammation [15, 36, 37]. Mitochondrial dysfunction plays a critical role in the pathogenesis and progression of IVDD [15]. Once excessive reactive oxygen species are produced in the mitochondria, an imbalance in mitochondrial homeostasis is induced or mitochondrial quality control is aggravated, and then mitochondria-mediated apoptosis is induced in NP cells [15].

IVDD is characterized by aggravated intradiscal acidosis and progressive loss of extracellular matrix [38]. The pH value of discs has been shown to range from 7.1 in healthy discs to 6.5 in degenerated discs and 5.7 in severely degenerated discs [39]. Consequently, glycolysis in hypoxic tissues occurs together with lactate accumulation and acidosis. In NP cells, acidic pH lower than 6.5 induces catabolic metabolism, exacerbating the breakdown of proteins in the extracellular matrix and decreasing glycolytic flux [38, 39]. Similarly, our findings suggest that acidic pH stress induces apoptosis, excessive mitophagy, and degeneration of NP cells.

Naturally derived procyanidins have several properties, including anti-inflammatory, anti-arthritic, anti-allergic, and anticancer activities [27,28,29,30,31, 40, 41]. Oxygen-free radicals can be scavenged by procyanidins [27]. PCC1, a natural senotherapeutic agent, is able to inhibit the formation of the senescence-associated secretory phenotype [27]. PCC1 impairs the functional integrity of the mitochondria, suppressing oxidative damage and inflammation, and being involved in depleting senescent cells [27]. Our findings suggest that PCC1 attenuates acidic pH stress induced apoptosis, excessive mitophagy, and degeneration of NP cells.

Mitochondrial dynamics refers to the regulation of mitochondrial fission, fusion, mitophagy, and transport, determining mitochondrial morphology, function, and subcellular distribution [17, 22, 42,43,44]. Disrupted mitochondrial dynamics is important for the pathogenesis of various diseases, including IVDD [23, 45, 46]. Mitochondrial dynamics is regulated by the expression and activities of mitochondrial fusion and fission proteins [22, 23, 42]. The fusion of the outer and inner mitochondrial membranes is mediated by MFN-1/2 and OPA-1, respectively [17, 44, 47,48,49]. Mitochondrial fission occurs at contact sites between the endoplasmic reticulum and mitochondria and is primarily regulated by DRP-1 [17, 44, 47,48,49,50]. It involves the recruitment of DRP-1 by various outer mitochondrial membrane adapter proteins, including mitochondrial fission factor [43, 51]. An imbalance in mitochondrial dynamics is closely associated with the progression of neurodegenerative disorders, metabolic diseases, cardiac diseases, cardiovascular diseases, cancers, and other diseases [17, 43, 44]. Our findings reveal upregulated expression of DRP-1 and FIS1 and downregulated expression of MFN-2 and OPA-1 in degenerative NP cells. Therefore, disrupted mitochondrial dynamics, with excessive mitochondrial fission and suppressed mitochondrial fusion, is closely associated with mitochondrial dysfunction and IVDD progression. However, the changes in the expression of mitochondrial dynamics associated proteins, including mitochondrial fusion and fission proteins, were reversed by PCC1 in this study.

Mitophagy plays a critical role in the regulation of mitochondrial homeostasis by selective elimination of damaged mitochondria, maintaining mitochondrial quality and quantity [11, 20, 52, 53]. PINK1 is usually eliminated constitutively after being imported into the inner mitochondrial membranes, and Parkin is diffusely distributed in the cytosol in its autoinhibited form [53]. Once PINK1 importation into the mitochondria is blocked, PINK1 accumulates at the outer mitochondrial membranes and then recruits and activates Parkin via phosphorylation at S65 in the ubiquitin-like domain of Parkin. Under oxidative stress, Parkin is converted to an active E3 and ubiquitinates many different outer mitochondrial membrane proteins, such as MFN-1/2 and PINK1, which further phosphorylate these ubiquitinated proteins to recruit more Parkin to the mitochondria [53]. When mitochondrial substrates on the outer mitochondrial membranes are ubiquitinated by Parkin, LC3 attached to the phagophores is recruited via autophagy adaptors [53]. After being engulfed by autophagosomes and fusing with lysosomes, mitochondria are destroyed [53]. However, overactivation or insufficient mitophagy has also been shown to induce mitochondrial dysfunction and cellular senescence [53]. Thus, suitable mitophagy is beneficial for the regulation of mitochondrial health and tissue integrity, while excessive or defective mitochondrial fission may result in widespread mitochondrial disruption and even detrimental mitophagy [13, 44, 54,55,56]. Enhanced mitophagy was also observed in NP cells exposed to oxidative stress, with exacerbated NP cell dysfunction [57]. Our findings suggest that overactivated mitophagy is induced by acidic pH stress and that these changes are reversed by PCC1.

SIRT3 ameliorates oxidative stress-induced injury, maintaining mitochondrial homeostasis [10, 21, 22]. SIRT3 promotes the expression of OPA-1 and inhibits that of FIS1, suppressing mitochondrial fission [22, 58]. Upregulation of SIRT3 expression suppresses mitochondrial fission [23], while SIRT3 deficiency results in mitochondrial dysfunction, increasing the vulnerability of cells to oxidative stress induced apoptosis and senescence [21, 22]. SIRT3 downregulation is associated with various degenerative diseases, such as IVDD [21, 22]. Our findings suggest that the downregulation of SIRT3 is induced by acidic pH stress and that these changes are reversed by PCC1.

FOXOs are now established as longevity genes that play important roles in cellular homeostasis control, autophagy regulation, and stem cell maintenance [24, 59,60,61,62]. The activities of FOXO proteins are regulated by phosphorylation, acetylation, ubiquitination, methylation, and post-translational modifications [24]. Activation of FOXO proteins in aged IVD was found to increase autophagy and protect NP cells from stress-induced apoptosis [59]. FOXO3, one of the most prominent genes related to human longevity, belongs to a family of evolutionarily conserved transcription factors characterized by a distinct forkhead DNA-binding domain [24, 25, 39, 63]. In response to stress factors, FOXO3 translocates from the cytoplasm to the nucleus, regulating metabolic homeostasis, inflammation, oxidative stress, cell cycle regulation, cell survival, and cell death [25, 60, 63]. FOXO3 is one of the dominant isoforms involved in promoting IVD homeostasis [59]. Further, SIRT3 regulates cell energy metabolism, activating FOXO3a and participating in scavenging reactive oxygen species [64]. Our findings suggest that PCC1 ameliorates acidic pH stress induced NP degeneration through SIRT3/FOXO3-mediated mitochondrial dynamics (Fig. 8). Therefore, maintaining or restoring the expression of FOXO proteins can be a treatment strategy to promote healthy disc aging and suppress the pathogenesis of IVDD [24, 59].

This study has several limitations. First, the potential mechanisms underlying the protective effects of PCC1 against acidic pH stress induced disruption of mitochondrial dynamics in NP cells have not been fully elucidated. Second, the IVDD rat model established by tail disc puncture may not fully mimic oxidative stress induced IVDD rat models. Third, owing to the concentration differences in PCC1, the therapeutic effects of PCC1 on IVDD may also be dose-dependent, both in vitro and in vivo. Subsequent studies should focus on optimizing the dose and duration of PCC1 administration for the treatment of IVDD. Moreover, further in vitro and in vivo investigations of PCC1 may contribute to advancing its potential as a clinical treatment for IVDD.

In conclusion, we investigated the potential mechanisms underlying the protective effects of PCC1 on NP cells against acidic pH stress induced disruption of mitochondrial dynamics. Our findings suggest that PCC1 suppresses IVDD pathogenesis via the SIRT3/FOXO3 signaling pathway, indicating that the regulation of the SIRT3/FOXO3 signaling pathway may be a novel therapeutic strategy for IVDD.

Data availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Acknowledgements

We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This work was supported by the National Natural Science Foundation of China (Grant nos. 81974349, 81904020, 82205153), the Natural Science Foundation of Hubei Province (2024AFD257, 2023AFB1086), the Higher Education Teaching Research Project of Hubei Province (2021063), the Sailing Special Science and Technology Plan of Enshi Prefecture (2024048), and the Science Foundation of Union Hospital (2022xhyn036).

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  1. Wenbin Hua, Lin Xie and Chenpeng Dong contributed equally to this work.

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  1. Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China

    Wenbin Hua, Lin Xie, Chenpeng Dong, Guoyu Yang, Shouyuan Chi, Zhiqiang Xu, Cao Yang, Huiwen Wang & Xinghuo Wu

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WH and XW designed the project. WH, LX, and CD performed the experiments and statistical analysis and drafted the manuscript. WH participated in the study design and statistical analysis. CD, GY, SC, and ZX participated in the collection of NP tissue specimens and statistical analysis. WH, CY, HW, and XW assisted with revising the manuscript. All the authors reviewed the manuscript.

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Hua, W., Xie, L., Dong, C. et al. Procyanidin C1 ameliorates acidic pH stress induced nucleus pulposus degeneration through SIRT3/FOXO3-mediated mitochondrial dynamics. J Transl Med 22, 1071 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05805-4

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