Daphnetin-mediated mitophagy alleviates intervertebral disc degeneration via the Nrf2/PINK1 pathway

Abstract

Intervertebral disc degeneration (IDD) is a major cause of low back pain (LBP), and effective therapies are still lacking. Reactive oxygen species (ROS) stress induces NLRP3 inflammasome activation, and this, along with extracellular matrix metabolism (ECM) degradation in nucleus pulposus cells (NPCs), plays a crucial role in the progression of IDD. Daphnetin (DAP) is a biologically active phytochemical extracted from plants of the Genus Daphne, which possesses various bioactivities, including antioxidant properties. In the present study, we demonstrate that DAP significantly attenuates tert-butyl hydroperoxide (TBHP)-induced ECM degradation, oxidative stress and NLRP3 inflammasome activation in NPCs. Furthermore, DAP could facilitate mitophagy to increase the removal of damaged mitochondria, consequently reducing mitochondrial ROS accumulation and alleviating NLRP3 inflammasome activation. Mechanistically, we unveil that DAP activates mitophagy by stimulating the Nrf2/PINK1 signaling pathway in TBHP-induced NPCs. In vivo experiments further corroborate the protective effect of DAP against IDD progression in a rat model induced by disc puncture. Accordingly, our findings reveal that DAP could be a promising therapeutic candidate for the treatment of IDD.

Introduction

Intervertebral disc degeneration (IDD) is a prominent contributor to the occurrence of low back pain (LBP) and is a leading cause of disability worldwide [1]. Existing IDD care options focus primarily on pain relief and often fall short of providing lasting therapeutic outcomes [2]. The peripheral annulus fibrosus (AF), the core nucleus pulposus (NP), and the cartilaginous endplates (CEP) are the three distinct components that make up the intervertebral disc (ID). Among these, nucleus pulposus cells (NPCs) play crucial roles in regulating extracellular matrix metabolism (ECM) and maintaining the equilibrium between proteoglycan and type II collagen formation [3].

NPCs inhabit a microenvironment characterized by hypoxia and limited nutrient availability as a result of the absence of a direct blood supply. This condition makes them particularly vulnerable to the accumulation of inflammatory agents and reactive oxygen species (ROS) [4], which can further impact ECM anabolism and catabolism [5]. Emerging evidence suggests that mitigating oxidative stress in NPCs represents a promising therapeutic approach for treating IVDs [6].

Previous studies have suggested that stimulation of the nucleotide-binding oligomerization domain-like pyrin domain-containing protein 3 (NLRP3) inflammasome, accompanied by interleukin-1 beta (IL-1β) secretion and excessive caspase-1 generation, substantially influences the development of IVD pathogenesis [ 7– 9] . Additionally, NLRP3 inflammasome activation has been linked to endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and ROS in IDD [10], all of which have the potential to modulate the synthesis of diverse inflammatory cytokines, hence exacerbating IDD [ 8, 11] . Recent research suggests that the NLRP3 inflammasome might act as a diagnostic indicator for IVD [ 7, 9, 12] .

Daphnetin, also called 7,8-dihydroxy coumarin (DAP), is a naturally occurring derivative of coumarin that is derived from plants of the Daphne genus. It has been used clinically for conditions such as rheumatoid arthritis and coagulation disorders [13]. Numerous studies have indicated that DAP can inhibit NLRP3 inflammasome activation in models of tert-butyl hydroperoxide (TBHP)-induced cell death and acute liver failure [ 14, 15] . Moreover, previous studies have demonstrated that activating mitophagy can hinder NLRP3 inflammasome activation, and DAP has been shown to restore mitochondrial function [ 14, 15] . However, whether the protective effects of DAP extend to IDD remains uncertain.

Nevertheless, whether DAP can suppress NLRP3 inflammasome activation by increasing mitophagy has not been definitively established. Consequently, our study aimed to determine the impacts of DAP on TBHP-induced NPCs responses and investigate the underlying mechanisms involved in mitophagy. Additionally, we assessed the preventive effect of DAP against IDD in a rat model.

Materials and Methods

Ethical statement

Protocols for all surgical procedures, therapies, and postoperative care administered to the animals were performed in compliance with the criteria outlined in the Helsinki Declaration and approved by the Animal Care and Use Committee of Wenzhou Medical University (approval No. wydw2023-0513).

Reagents and antibodies

DAP with a purity > 98%, Mito Q, and cyclosporin A were obtained from MCE (Monmouth Junction, USA). TBHP and type II collagenase were acquired from Sigma-Aldrich (St. Louis, USA). The hematoxylin‒eosin (HE) staining kit and the Safranin O-fast green (SO) staining kit were purchased from Beijing Solarbio Science and Technology (Beijing, China). Antibodies (Abs) targeting Collagen II (ab307674), MMP13 (ab315267), Aggrecan (ab3378), NLRP3 (ab263899), and IL-1β (ab283818) were acquired from Abcam (Cambridge, USA). Primary Abs against Caspase 1 (#2225), LC3B (#2775), P62 (#5114), Tom20 (#42406), and Keap1 (#4678) were supplemented by Cell Signaling Technology (Danvers, USA). Primary Abs against β-actin (AC038), ADAMTS5 (A23125), PINK1 (A24745), Nrf2 (A21176), and Histone H3 (A17562) were from ABclonal Technology (Wuhan, China). Goat Abs against rabbit and mouse IgGs conjugated with HRP were acquired from Bioworld (Nanjing, China). Alexa Fluor 594- and 488-conjugated secondary Abs were acquired from Abcam. The chemical compound 4′,6-diamidino-2-phenylindole (DAPI) was obtained from Beyotime (Shanghai, China).

NPC isolation and culture

NP tissue was collected from male Sprague–Dawley (SD) rats (Animal Center of Wenzhou Medical University) ranging from 200–250 g and aged four weeks. The NP tissues were dissected into 1-mm ³ fragments under aseptic conditions and cleaned with phosphate-buffered saline (PBS). The NP tissues were subsequently digested via a 0.1% collagenase type II solution in DMEM/F12 culture medium (Gibco, Grand Island, USA) and maintained at 37°C for 4 h. Following the washing and suspension steps, the NPCs were cultivated in DMEM/F12 supplemented with 1% antibiotics (penicillin/streptomycin) and 15% fetal bovine serum (FBS; Gibco). The cultivation process took place in a controlled environment with a 5% CO ₂ at 37°C. NPCs were collected from the culture when the cell density reached approximately 70% confluence. The collection process included the use of 0.25% trypsin-EDTA. The NPCs were subsequently transferred to 6-well culture plates for further cultivation. The subsequent set of cellular units was used for experimental purposes, with the culture media being refreshed every three days.

Cell viability assay

The measurement of cell viability was conducted using a Cell Counting Kit-8 (CCK-8; Dojindo Co., Kumamoto, Japan). In line with the manufacturer’s recommendations, NPCs were seeded in 96-well plates at a density of 8 × 10 ³ cells/well, and subsequently subjected to treatment with various amounts of DAP according to the experimental plan. Following the completion of the treatment, the cells were washed with PBS. Subsequently, 90 μL of DMEM and 10 μL of CCK-8 solution were added to each well and incubated at 37°C for 1.5 h. The absorbance was measured at a wavelength of 450 nm with a microplate reader (Thermo Fisher Scientific, Rockford, USA).

Live/dead cell staining

NPCs were plated at a density of 5 × 10 ⁵ cells per well in a 6-well plate. Following the indicated treatments, NPCs were stained with a mixture of dyes for 30 min at 37°C, according to the protocol provided by the Live/Dead Viability Assay Kit (Beyotime). The assay utilizes red and green fluorescence to distinguish nonviable and viable cells, respectively.

Real-time PCR

The PCR experiment was accomplished using a previously published method [16]. Total RNA was extracted from the cells via the TRIzol reagent (Invitrogen, Carlsbad, USA). The synthesis of complementary DNA was performed, followed by amplification via the PrimeScript-RT reagent kit and SYBR Green Master Mix (TaKaRa, Kyoto, Japan). The 2 ^–ΔΔCt method was used to calculate the relative mRNA level of each target gene. The primer sequences for the relevant genes are shown in Supplementary Table S1.

Western blot (WB) analysis

Total protein was extracted from NPCs and disrupted with cold radioimmunoprecipitation assay (RIPA) buffer supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). Afterward, the lysate was centrifuged at 12,000 g for 10 min at 4°C. The quantification of protein levels was carried out with a bicinchoninic acid (BCA) protein assay kit (Beyotime). The protein samples were subsequently separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). Next, the membranes were blocked in a solution containing 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at ambient temperature. The membranes were subsequently probed using primary Abs that corresponded to the target proteins (anti-β-actin, 1:3000 dilution; anti-Collagen II, Aggrecan, ADAMTS5, MMP13, NLRP3, Caspase 1, IL-1β, LC3B, P62, PINK1, Nrf2, Histone H3, and Keap1, 1:1000 dilution) at 4°C overnight. The membranes were washed with TBST and subsequently incubated with their corresponding secondary Abs for 2 h. Finally, the ChemiDoc XRS+Imaging System and ImageLab 3.0 software (Bio-Rad, Hercules, USA) were used to observe and measure the protein bands.

Immunofluorescence and confocal microscopy

The NPCs that underwent treatment were subjected to a washing step with PBS, followed by fixation with a 4% paraformaldehyde solution for 15 min. Subsequently, permeabilization was achieved by treating the NPCs with a 0.1% Triton X-100 solution diluted in PBS for 3 min. Afterwards, the samples were treated for a 1 h at 37°C with 5% bovine serum albumin to suppress nonspecific binding. The samples were subsequently exposed overnight to primary Abs targeting Collagen II (1:100), MMP13 (1:100), IL-1β (1:100), LC3B (1:100), Tom20 (1:100), and Nrf2 (1:100) at 4°C. On the following day, the NPCs were washed and then exposed to secondary Abs that were tagged with either Alexa Fluor 488 or Alexa Fluor 594. The exposure lasted for a duration of 1 h at 37°C. Additionally, the NPCs were stained with DAPI to label the nuclei, with a staining period of 5 min. Finally, sample images were acquired under a confocal laser scanning microscope (Leica Microsystems, Heidelber, Germany).

Intracellular oxidative stress determination (DCFH-DA, MDA, and SOD2 activity)

The intracellular level of ROS was assessed using a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) ROS test kit (Beyotime) in accordance with the manufacturer’s guidelines. The concentrations of malondialdehyde (MDA) were assessed via commercially accessible test kits for total superoxide dismutase and malondialdehyde (Byotime). The enzymatic activity of manganese superoxide dismutase (MnSOD) was measured via a MnSOD test kit with WST-8 (Byotime).

Enzyme-linked immunosorbent assay (ELISA)

Samples of supernatant from cultured cells were collected after the application of stimuli. IL-1β quantification in the supernatants of cell cultures was performed via mouse IL-1β ELISA kits (EM004-96; ExCell Bio, Taicang, China) according to the manufacturer’s instructions.

Mitochondrial membrane potential (MMP) assay

The evaluation of the MMP was conducted with MitoTracker Red CMXRos (Molecular Probes™; Thermo Fisher Scientific), a fluorescent dye that selectively labels the mitochondria of viable cells. The staining process included incubating the cells with 100 nM dye for 30 min at 37°C. Afterward, the nuclei of the NPCs were stained with DAPI for 15 min at 37°C. The images were obtained with a Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan), and the quantification of fluorescence intensity was conducted with ImageJ software 2.1 (NIH, Bethesda, USA).

MitoSOX Assay

Intracellular superoxide anion levels were evaluated via the use of MitoSOX, a red fluorescent dye from Yeasen (Shanghai, China) that is specifically designed to stain superoxide anions within cells. In accordance with the manufacturer’s protocol, 5 μM MitoSOX was applied for a 45-min incubation at 37°C. Next, the nuclei were subjected to Hoechst/DAPI staining for 15 min at 37°C while being shielded from any exposure to light. The samples were examined with a Nikon ECLIPSE Ti microscope (Nikon), and the quantification of fluorescence intensity was performed with ImageJ software 2.1 (NIH).

siRNA transfection

The siRNA molecules targeting the Nrf2 and PINK1 genes were acquired from GenePharma (A01003; Shanghai, China). Lipofectamine 2000 siRNA transfection reagent (Thermo Fisher Scientific) was used to introduce the negative control or siRNA molecules into the NPCs following the guidelines provided by the manufacturer. Following 24 h of transfection, the culture media were replaced by new media. After reaching confluence, the transfected NPCs were subcultured to conduct further tests. The sequences of the siRNAs are provided in Supplementary Table S2.

Molecular modeling

The three-dimensional conformation of DAP was generated via Discovery Studio 2016 software and then subjected to energy minimization via the CHARMM force field. The structure of the Keap1-Nrf2 complex (PDB ID: 4XMB) was acquired from the PDB repository ( https://www.rcsb.org/) and then loaded into Discovery Studio 2016. The Keap1-Nrf2 complex underwent a series of operations, including cleaning, preparation, water removal, and hydrogen addition. Ultimately, CDOCKER software, which offers partial flexibility, was selected to carry out the docking process, taking into account the designated binding locations and receptor radius. The evaluation of the molecular docking data was conducted by considering the CDOCKER energy scores, interaction sites, and interaction force types [17].

Rat IDD model

Forty-eight 8-week-old male SD rats were acquired from the Animal Center of Wenzhou Medical University. To minimize selection bias, we employed a random number generator (the RAND function in Microsoft Excel) to randomly assign experimental animals to different experimental and control groups. Throughout the experiment, the researchers were blinded to the treatment received by the animals. Similarly, analysts were unaware of the sample treatment groups during the data analysis phase [18]. Additionally, we ensured that all the experimental animals were maintained under identical or similar environmental conditions, including temperature, humidity, diet, and light cycle. Following a previously described procedure [19], after an intraperitoneal injection of pentobarbital sodium at 2% (w/v) and a dosage of 40 mg/kg was administered, the Co7/8 rat tail disc was precisely discovered by digitally palpating the coccygeal vertebrae. A needle (21 G) was employed to penetrate the epidermis of the tail discs vertically, creating an aperture in the complete layer of the AF. The puncture depth was limited to 5 mm according to preliminary experiments. Following the surgical procedure, the rats were subjected to random allocation into three distinct groups, namely, the sham, IDD, and IDD+DAP groups. In the IDD+DAP experimental cohort, the rats were subjected to treatment with 100 mg/kg DAP. DAP was administered via the intragastric route on a daily basis [20]. The rats were subjected to daily monitoring to ensure their survival, according to the guidelines outlined in the Rat Health Guide ( http://ratguide.com). In accordance with the AVMA Guidelines for Animal Euthanasia, the rats were sacrificed at two time points after surgery, namely, at 4 and 8 weeks.

X-ray imaging

X-ray imaging was conducted on each rat at 4 and 8 weeks postsurgery via X-ray irradiation equipment (Kubtec, Stratford, USA). All the rats were immobilized in a prone posture. The ID height was measured via ImageJ software (NIH). The disc height index (DHI) was then calculated to represent the alterations in ID height, as outlined in a prior investigation. [21] The DHI variation of punctured IDD cases was expressed as DHI% (DHI% = postpunctured DHI/prepunctured DHI × 100%).

Histopathologic analysis and immunohistochemical examination

The rats were euthanized at two specific time points, namely, 4 and 8 weeks following the surgical procedure, via the administration of an intraperitoneal overdose of pentobarbital. The tails were subsequently collected for additional analysis. The samples were subjected to fixation, decalcification, and subsequent embedding in paraffin. Next, the embedded tissue was cut into slices measuring 5 μM in thickness for further studies. To conduct histological evaluation, slides containing each disc sample were subjected to HE and SO staining. The morphology of the NP and AF cells was observed via a microscope (Olympus, Tokyo, Japan), followed by impartial evaluation via a team of proficient histology researchers. On the basis of the aforementioned grading standard, the histologic grade attributed to a normal disc was 5, whereas moderately deteriorated discs were assigned scores ranging from 6 to 11. On the other hand, severely degenerated discs were assigned scores ranging from 12 to 15 [ 19, 22] .

After deparaffinization, each slice was subjected to incubation in a 3% H ₂O ₂ solution for 10 min. Subsequently, the sections were rinsed with PBS. The sections were subsequently blocked with a 1% (w/v) solution of goat serum albumin for 1 h at 37°C, followed by incubation with the primary antibodies (anti-Nrf2, anti-LC3B, and anti-NLPR3, 1:200) at 4°C overnight. The following day, the sections were incubated with secondary Abs conjugated with HRP for 1 h at 37°C. A minimum of three sections from each sample were examined, and the resulting images were subjected to analysis via Image-Pro Plus software, version 6.0 (Media Cybernetics, Rockville, USA).

Statistical analysis

Data were presented as the mean ± standard deviation (SD), and GraphPad Prism (Version 8.0.0) was used for the statistical analysis. The analysis of data comparisons was conducted by t-test (two groups) or one-way analysis of variance (ANOVA) followed by Tukey′s post hoc test. P  < 0.05 was considered statistically significant.

Results

DAP impacts the NPCs survival

Figure 1A depicts the chemical structure of DAP. Initially, NPCs were exposed to various concentrations of DAP (0, 5, 10, 20, 40, 80, 160 and 320 μM) for a period of 24 h. Subsequently, we conducted a CCK-8 assay to evaluate the impact of DAP on NPC survival. The results indicated that at concentrations of up to 40 μM DAP had no discernible effect on NPC viability compared with the untreated control group. However, at a concentration of 80 μM, DAP decreased cell viability ( Figure 1B). To cause oxidative stress and mitochondrial dysfunction in NPCs, TBHP was employed, as previously reported in other studies [23]. Subsequent experiments revealed that TBHP resulted in a dose-dependent decrease in NPC viability, which was subsequently ameliorated through DAP treatment ( Figure 1C,D). Live/dead staining also demonstrated a similar protective effect of DAP ( Figure 1E,F). These findings indicate that DAP is not toxic to NPCs at concentrations less than 40 μM and has a dose-dependent protective effect against TBHP-induced cell death.

Figure 1 .

Impact of DAP on NPC survival

(A) The chemical structure of DAP. (B) The assessment of DAP cytotoxicity in NPCs was conducted via CCK8 assay after exposure to different doses of DAP (0, 5, 10, 20, 40, 80, 160 and 320 μM) for 24 h. (C) The cytotoxicity of TBHP in NPCs was assessed via CCK8 assay after exposure to various concentrations (0, 25, 50, 100, and 200 μM) of TBHP for 24 h. (D) NPCs were treated with DAP (20 and 40 μM) and TBHP (50 μM) for 24 h. Cell viability was determined via CCK-8 assay. (E,F) Images and quantification of live/dead staining of NPCs. PI (red) was used to stain dead cells, and calcein AM (green) was used to stain live cells. Scale bar: 100 μm. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 6 for the CCK8 assay and n = 3 for live/dead cell staining.

DAP alleviates TBHP-induced ECM degradation in NPCs

IDD progression is positively correlated with the ECM metabolism of NPCs. Consequently, we assessed the protective effects of DAP on ECM metabolism by investigating ECM-related genes and proteins through PCR, WB analysis, and immunofluorescence staining. Our PCR ( Figure 2A,B) and WB ( Figure 2C,D) analyses revealed that application of TBHP resulted in a decrease in the protein expression levels of Collagen II and Aggrecan while simultaneously increasing the expression levels of a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinase-13 (MMP13). Remarkably, DAP effectively reversed the detrimental effects of TBHP on ECM metabolism in NPCs. Furthermore, our immunofluorescence staining results demonstrated that DAP significantly mitigated TBHP-induced Collagen II downregulation and MMP13 upregulation, consistent with the PCR and WB analysis data ( Figure 2E,F). These findings collectively show that DAP has a pivotal function in regulating the ECM balance in TBHP-induced NPCs.

Figure 2 .

The effects of DAP on TBHP-induced ECM degradation in NPCs

(A,B) Acan,Col2a1,Adamts5, and Mmp13 gene expressions were quantified via PCR in NPCs treated with or without DAP (20 and 40 μM) combined with TBHP (50 μM) for 24 h. (C,D) The protein expressions Aggrecan, Collagen II, ADAMTS5, and MMP13 in NPCs were measured via WB analysis. (E,F) Representative confocal microscopic images of Collagen II and MMP13. Scale bar: 100 μm. Data are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.

DAP inhibits TBHP-induced ROS generation and NLRP3 inflammasome activation in NPCs

To examine the impact of DAP on the production of ROS after treatment with TBHP, we measured the ROS levels, MDA concentrations, and SOD2 activity in NPCs. DCFH-DA was employed in our study as a means to accurately quantify ROS via its conversion into the fluorescent chemical DCF. These effects significantly hindered ROS generation in TBHP-induced NPCs following DAP treatment ( Figure 3A,B). Additionally, DAP treatment significantly reduced MDA levels and restored SOD2 activity in TBHP-induced NPCs ( Figure 3C,D). These findings indicate that DAP effectively reduces ROS production and enhances the antioxidant capacity of TBHP-induced NPCs.

Figure 3 .

Effects of DAP on TBHP-induced ROS generation and NLRP3 inflammasome stimulation in NPCs

(A,B) ROS assays were conducted in NPCs treated with or without DAP (20 and 40 μM) in combination with TBHP (50 μM) for 24 h. (C) The MDA intracellular level was evaluated via an MDA assay kit. (D) Intracellular SOD2 levels were evaluated via a SOD2 assay kit. (E,F) The protein expressions of NLRP3, Caspase 1, and IL-1β in NPCs were measured via WB analysis. (G) Representative confocal microscopic images of IL-1β-producing cells. Scale bar: 100 μm. (H) ELISA of IL-1β in the supernatants from NPCs. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.

Given that ROS accumulation can activate the NLRP3 inflammasome, a key factor in IDD development [24], we subsequently investigated the impact of DAP on TBHP-induced NLRP3 inflammasome activation in NPCs. WB analyses revealed that TBHP significantly increased the expression levels of NLRP3 and related inflammatory proteins, including those associated with NLRP3-dependent caspase-1 stimulation and IL-1β maturation. However, this effect was concentration-independently suppressed by DAP treatment ( Figure 3E,F). The immunofluorescence staining results also revealed that TBHP treatment led to an increase in the IL-1β level, which was subsequently reversed by DAP treatment ( Figure 3G). ELISA results confirmed the inhibitory impact of DAP on IL-1β maturation and secretion ( Figure 3H). These results collectively demonstrate that DAP protects against NLRP3 inflammasome stimulation in NPCs.

DAP enhances mitophagy and hinders mitochondrial ROS generation in TBHP-treated

NLRP3 inflammasome activation can induce mitochondrial oxidative stress damage, subsequently promoting NLRP3 inflammation activation [25]. Damaged mitochondria are normally removed through mitophagy [26]. Therefore, we investigated whether mitophagy contributes to the protective effects of DAP. The WB analysis results in Figure 4A,B demonstrate an increase in the microtubule-associated protein light chain 3 (LC3)-II/I protein ratio and mitophagy-related protein PINK1 expression, alongside with a hindrance in P62 expression in the DAP-treated groups, in contrast to the TBHP-treated group. Mitophagy was further assessed by co-immunostaining LC3B with the mitochondrial outer membrane marker Tom20. The results indicated that, compared with TBHP treatment, DAP treatment caused a greater number of LC3B-containing aggregates that were located either along or close to the mitochondria ( Figure 4C). To evaluate mitochondrial dysfunction and ROS production, we conducted Mitotracker and MitoSOX assays. The results revealed that DAP suppressed TBHP-induced mitochondrial ROS production and mitigated the loss of the mitochondrial membrane potential (MMP), similar to the effect of the mitochondrial-specific antioxidant Mito Q ( Figure 4D,E). These findings suggest that DAP alleviates oxidative stress-induced mitochondrial dysfunction and ROS accumulation by enhancing mitophagy.

Figure 4 .

Effects of DAP on mitophagy and mitochondrial ROS generation in TBHP-treated NPCs

(A,B) The expressions of LC3B I/II, P62, and PINK1 in NPCs treated with or without DAP (40 μM) with TBHP (50 μM) for 24 h were measured via WB analysis. (C) Representative confocal microscopic images of the intracellular distribution of LC3B and mitochondria (Tomm20) in NPCs. Scale bar: 20 μm. (D,E) TBHP-induced NPCs were treated with DAP (40 μM) or Mito Q (1 μM, a mitochondria-specific antioxidant) for 24 h. Representative confocal microscopic images of MitoSOX and MitoTracker. Scale bar: 100 μm for MitoSOX and 50 μm for MitoTracker. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.

Inhibiting mitophagy reverses the protective effect of DAP on TBHP-treated NPCs

To further investigate whether mitophagy is a key mechanism underlying the protective effects of DAP, we employed the mitophagy inhibitor cyclosporin A. WB analysis of the LC3B-II/I protein ratio and P62 and PINK1 expression was conducted, revealing that pretreatment of NPCs with cyclosporin A abrogated the protective effect of DAP ( Figure 5A,B). Additionally, we observed that the protective effects of DAP on mitochondrial damage, including the preservation of the MMP, as detected by Mitotracker and the reduction in ROS production, as detected by MitoSOX, were significantly attenuated by cyclosporin A ( Figure 5C,D). Furthermore, the inhibitory effect of DAP on TBHP-induced NLRP3 inflammasome stimulation, as shown by the reduced protein expressions of NLRP3, Caspase 1, and IL-1β in NPCs according to the WB analysis results, was significantly compromised by cyclosporin A ( Figure 5E,F). To conduct a more in-depth examination of the involvement of mitophagy in the control of ECM equilibrium by DAP, immunofluorescence staining was performed, revealing that the protection of DAP from TBHP-induced ECM degradation was significantly abrogated by cyclosporin A ( Figure 5G,H). Collectively, these results indicate that DAP suppresses NLRP3 inflammasome activation and ECM degradation by enhancing mitophagy to mitigate mitochondrial ROS stress.

Figure 5 .

Inhibiting autophagy reverses the protective effects of DAP on THBP-induced mitochondrial injury, the NLRP3 inflammasome, and the degradation of the ECM in NPCs

(A,B) The expressions of LC3B I/II, P62, and PINK1 were measured via WB analysis in NPCs treated with Cyclosporin A (1 μM, a mitophagy inhibitor) for 1 h prior to DAP administration (40 μM) with TBHP (50 μM) for 24 h. (C,D) Representative confocal microscopic images of MitoSOX and MitoTracker. Scale bar: 100 μm for MitoSOX and 50 μm for MitoTracker). (E,F) The protein expressions of NLRP3, Caspase 1, and IL-1β in NPCs were measured via WB analysis. (G,H) Representative confocal microscopic images of Collagen II and MMP13. Scale bar: 100 μm. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.

Molecular docking predicts the interaction between DAP and the Keap1-Nrf2 complex

On the basis of previous research and considering the anti-ROS effects of DAP, we hypothesized that DAP could interact with the Keap1-Nrf2 complex. The molecular docking results demonstrated a robust binding affinity between DAP and the Keap1-Nrf2 complex, as shown by a binding energy of –6.7 kcal/mol ( Figure 6A–C). DAP forms six pairs of robust hydrogen bonds with active amino acid residues, including ASN-382, SER-363, and ARG-380, at distances of 2.2 Å, 2.5 Å, 2.6 Å, 2.3 Å, 2.5 Å, and 1.9 Å, respectively, all of which are within the typical range of traditional hydrogen bonds (≤ 3.5 Å). These interactions play a vital role in stabilizing small-molecule ligands. Additionally, the phenyl ring of DAP engaged in two pairs of strong π-π conjugate interactions with the TYR-334 amino acid within the active pocket of DAP. Collectively, these interactions contribute to the stability of DAP within the Keap1-Nrf2 complex, suggesting that DAP exerts its effects through Nrf2.

Figure 6 .

DAP increases the level of mitophagy by activating the Nrf2/PINK1 pathway in NPCs

(A) Model of DAP. (B) Ribbon model of the Keap1-Nrf2 complex. (C) 3D docking analysis between DAP and the Keap1-Nrf2 complex. (D,E) The nuclei was isolated from NPCs, and the Nrf2 concentration in the nucleus and the concentration of Keap1 in whole cells were assessed via WB analysis. The cells were treated with or without the injection of DAP (40 μM) together with TBHP (50 μM) for 24 h. (F) Representative confocal microscopic images of Nrf2. Scale bar: 50 μm. (G,H) Nrf2 and PINK1 expressions were downregulated by siRNAs. The expressions of Nrf2, LC3B I/II, P62, and PINK1 in NPCs were measured via WB analysis. (I) Representative confocal microscopic images of the intracellular distribution of LC3B and Tomm20 in NPCs. Scale bar: 20 μm. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3.

DAP promotes mitophagy via the Nrf2/PINK1 pathway

To further confirm the contribution of the Nrf2 pathway to the impact of DAP on PINK1-mediated mitophagy in TBHP-induced NPCs, we conducted the following experiments. First, we assessed the Nrf2 protein levels in the nucleus and Keap1 in entire cells via WB analysis. The results demonstrated that DAP significantly increased Nrf2 nuclear translocation while reducing Keap1 expression in the whole cells ( Figure 6D,E). We also performed immunofluorescence staining of Nrf2 to visualize its nuclear translocation in NPCs. As expected, the results showed that DAP facilitated the intranuclear accumulation of Nrf2 ( Figure 6F). Furthermore, we used siRNAs to downregulate the expressions of Nrf2 and PINK1 to assess their effects on DAP-mediated mitophagy. We initially validated the effects of si-Nrf2 and si-PINK1 and found that the siRNAs significantly downregulated the target proteins ( Supplementary Figure S1). The WB analysis results indicated that the effects of DAP, including the upregulation of Nrf2 in the nucleus and the promotion of mitophagy (including increased PINK1 expression), were significantly blocked by Nrf2 knockdown ( Figure 6G,H).

Moreover, si-PINK1 significantly downregulated PINK1 expression and abrogated the effects of DAP, similar to the effects in the si-Nrf2 group, except for the upregulation of Nrf2 in the nucleus. These results indicate that PINK1 is downstream of Nrf2 and that DAP might regulate PINK1-mediated mitophagy through Nrf2 ( Figure 6G,H). Additionally, the colocalization of the fluorescence signals of LC3 and Tom20 further supported the protective role of Nrf2/PINK1 in DAP-induced mitophagy, which is consistent with the WB analysis results ( Figure 6I). These findings indicate that DAP may protect NPCs by stimulating Nrf2/PINK1-mediated mitophagy.

DAP improves the IDD process in a puncture-induced rat model

Building upon the findings from our in vitro experiments, we proceeded to determine the impact of DAP in an in vivo setting, utilizing a rat tail needle-punctured IDD model. To measure the degree of disc degeneration among the various treatment groups, X-ray imaging was performed at both the 4 ^th and 8 ^th weeks postpuncture. The results clearly revealed a significant reduction in the DHI in the IDD group compared with the sham group. In contrast, the DAP-treated group presented a notably greater DHI than the IDD group ( Figure 7A,B). The in vivo protective effects of DAP were further substantiated through histological analysis. H&E staining and SO staining of rat ID tissue, which were conducted at the 4 ^th and 8 ^th weeks postpuncture, provided compelling evidence. In the IDD group, we observed a notable reduction in the number of NPCs, accompanied by their replacement by fibrochondrocytes and the collapse of the cartilaginous endplate. However, treatment with DAP effectively mitigated these degenerative changes ( Figure 7C,D).

Figure 7 .

Impact of DAP on IDD in a rat model induced with puncture

(A,B) ID X-ray images of rats from the various experimental groups (sham, IDD, and IDD + DAP groups) at the 4th and 8th week. White arrows: the loss of DHI. (C) Representative HE and SO staining images of punctured discs in various groups (scale bar: 1 mm). (D) The evaluation of histological scores was performed at the 4th and 8th week postsurgery. (E,F) Immunohistochemical staining for Nrf2, LC3B, and NLPR3 in the NPCs of the discs in the different groups. Scale bar: 50 μm. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 8.

Furthermore, immunohistochemical staining for Nrf2, LC3B, and NLRP3 in the puncture-induced rat model supported our in vitro findings. The results indicated that DAP not only increased Nrf2 expression but also promoted autophagy while concurrently inhibiting NLRP3 inflammasome activation ( Figure 7E,F). This consistency with our in vitro results further emphasizes the therapeutic potential of DAP in the context of IDD.

Discussion

LBP significantly impacts quality of life, with IDD being a primary cause. Unfortunately, pharmacological choices for IDD treatment are scarce. Currently, IDD patients often rely on nonsteroidal anti-inflammatory drugs (NSAIDs) or muscle relaxants to alleviate symptoms [ 27, 28] . Nevertheless, these medications are ineffective in preventing IDD or slowing its progression. Consequently, there is a pressing need for more efficacious drugs to both prevent and treat IDD. DAP, a natural coumarin derivative from plants in the Daphne genus, represents a promising candidate [29]. Medicinal plants, which are abundant sources of bioactive chemicals such as those that produce DAP, possess a wide range of pharmacological actions and minimal side effects. DAP has been reported to exert protective effects on various diseases, including rheumatoid arthritis, cardiovascular diseases, neurological disorders, and various cancers [ 30– 33] . Our study revealed that DAP mitigates TBHP-induced ECM degradation, reduces ROS generation, and inhibits NLRP3 inflammasome activation in NPCs. Furthermore, it hinders the progression of IDD by enhancing Nrf2/PINK1-mediated mitophagy. These results provide robust evidence that DAP is a very effective therapeutic drug for treating IDD ( Figure 8).

Figure 8 .

Schematic illustration of DAP-mediated mitophagy regulation in inhibiting oxidative stress and NLRP3 inflammasome activation in NPCs

DAP is a mitophagy promoter that inhibits oxidative stress and prevents the accumulation of mitochondrial ROS, which also alleviates NLRP3 inflammation activation and results in the degradation of the ECM in NPCs, consequently suppressing IDD progression.

IDD is a chronic age-related degenerative condition characterized by ECM degeneration, an inflammatory response, and mitochondrial impairment, including the accumulation of ROS [ 34, 35] . Elevated ROS levels disrupt cellular homeostasis, leading to increased DNA damage and dysfunctional mitochondria, particularly in degenerative disorders with compromised antioxidant defenses [36]. The sustained release of ROS induces oxidative stress, further activating the NLRP3 inflammasome [ 34, 35] and ultimately resulting in an imbalance in the ECM of NPCs [37]. TBHP is an exogenous inducer of oxidative stress with the advantage of high stability and is commonly used as a source of ROS [38]. The use of TBHP-induced oxidative stress models allows for consistent induction of oxidative stress in cell culture experiments [ 39, 40] . Therefore, in this study, we utilized TBHP to cause oxidative damage in vitro [41]. As anticipated, DAP effectively reversed TBHP-induced ECM degradation. This effect was evident through the upregulation of Collagen II and Aggrecan expression and the downregulation of MMP13 and ADAMTS5 expression, underscoring the protective effects of DAP on NPCs exposed to TBHP. Furthermore, our rat model of puncture-induced IDD substantiated the outcomes observed in our in vitro experiments.

Inflammasomes are cytosolic multiprotein complexes that play pivotal roles in various bone diseases [42]. Among these, the NLRP3 inflammasome has been thoroughly researched and shown to have a significant function in the development of bone disorders, including osteoarthritis, osteomyelitis, and metabolic osteopathy [9]. Additionally, NLRP3 inflammasome activation involves multiple triggering signals throughout the IDD process, making it a potential therapeutic target for IDD [ 7, 43] . Previous research has shown that supplementation with MFG-E8 can inhibit H ₂O ₂-induced oxidative stress, mitochondrial dysfunction, pyroptosis, and ECM degradation in NPCs through the Nrf2/TXNIP/NLRP3 axis [37]. Previous studies have established the ability of DAP to inhibit NLRP3 inflammasome activation in conditions such as acetaminophen-induced hepatotoxicity and cigarette smoke-induced chronic obstructive pulmonary disease (COPD) [ 44, 45] . In our study, we observed that DAP effectively suppressed the accumulation of intracellular ROS induced by TBHP, along with the upregulation of the expressions of NLRP3 inflammasome-related proteins, including NLRP3, caspase-1, and IL-1β, in NPCs.

Numerous regulatory methods have been investigated to weaken the assembly of the NLRP3 inflammasome, with a focus on mitophagy-mediated clearance of damaged mitochondria [ 46, 47] . Mitophagy selectively removes dysfunctional and senescent mitochondria, thus maintaining mitochondrial quality and homeostasis [48]. Dysfunctional mitochondria release signals, including mitochondrial ROS that can activate NLRP3 inflammasome assembly [49]. Given these insights, we postulated that encouraging the removal of impaired mitochondria could reduce the formation of NLRP3 complexes. Our findings demonstrate that DAP stimulates mitophagy, which improves the elimination of damaged mitochondria and reduces the buildup of mitochondrial ROS in TBHP-induced NPCs.

Moreover, inhibiting mitophagy via Cyclosporin A attenuated the inhibitory effect of DAP on NLRP3 inflammasome activation and ECM degradation. These results indicate that DAP suppresses NLRP3 inflammasome assembly in NPCs through a mitophagy-dependent mechanism. Previous studies have highlighted the ability of DAP to ameliorate mitochondrial dysfunction [14]. Our study is the first to emphasize the crucial function of mitophagy in mediating the protective effects of DAP.

Recent research has shown that Nrf2 may initiate mitophagy via the Nrf2/PINK1 signaling pathway [50]. Additionally, DAP protects against carcinogenesis and oxidative damage by upregulating Nrf2 expression [ 44, 51] . To investigate the protective effect of DAP on mitophagy associated with Nrf2, we observed that DAP significantly promoted Nrf2 nuclear translocation while reducing Keap1 expression in total cell lysates. Furthermore, previous research has highlighted the role of PINK1 in mediating mitophagy by regulating Parkin [52]. Interestingly, certain antioxidants, such as MitoQ, have been shown to enhance PINK1-mediated mitophagy by activating the Nrf2 signaling pathway [50]. To further confirm the functions of Nrf2 and PINK1 in DAP-mediated mitophagy, we used siRNAs to knock down Nrf2 and PINK1. Our results confirmed that DAP-mediated mitophagy is indeed regulated by the Nrf2/PINK1 signaling pathway.

Nevertheless, our research has several limitations. First, we did not investigate the precise mechanism through which DAP affects the Nrf2-Keap1 complex. Second, while PINK1 typically promotes mitophagy through Parkin, we did not conduct further experiments to knock down Parkin and explore its role in our study. Finally, while DAP has several clinical uses, further clinical research is needed to understand its effects on IDD.

In conclusion, the present research provides evidence supporting the beneficial impacts of DAP in IDD treatment. This study demonstrates that DAP has a beneficial effect on oxidative stress, ECM degradation, and NLRP3 inflammasome activation caused by TBHP. This effect is achieved by increasing Nrf2/PINK1-mediated mitophagy in NPCs. Furthermore, the therapeutic efficacy of DAP in delaying IDD in rats was shown via in vivo tests. These results provide novel perspectives for understanding the potential of DAP in the therapeutic management of IDD.

Supplementary Data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 82172494, 82372461, and 82202757), the Basic Scientific Research Project of Wenzhou (No. Y2023034) and the Basic Research Projects of Wenzhou Medical University (No. KYYW202310).