Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

Abstract Ferroptosis, a novel type of cell death mediated by the iron‐dependent lipid peroxidation, contributes to the pathogenesis of the intervertebral disc degeneration (IDD). Increasing evidence demonstrated that melatonin (MLT) displayed the therapeutic potential to prevent the development of IDD. Current mechanistic study aims to explore whether the downregulation of ferroptosis contributes to the therapeutic capability of MLT in IDD. Current studies demonstrated that conditioned medium (CM) from the lipopolysaccharide (LPS)‐stimulated macrophages caused a series of changes about IDD, including increased intracellular oxidative stress (increased reactive oxygen species and malondialdehyde levels, but decreased glutathione levels), upregulated expression of inflammation‐associated factors (IL‐1β, COX‐2 and iNOS), increased expression of key matrix catabolic molecules (MMP‐13, ADAMTS4 and ADAMTS5), reduced the expression of major matrix anabolic molecules (COL2A1 and ACAN), and increased ferroptosis (downregulated GPX4 and SLC7A11 levels, but upregulated ACSL4 and LPCAT3 levels) in nucleus pulposus (NP) cells. MLT could alleviate CM‐induced NP cell injury in a dose‐dependent manner. Moreover, the data substantiated that intercellular iron overload was involved in CM‐induced ferroptosis in NP cells, and MLT treatment alleviated intercellular iron overload and protected NP cells against ferroptosis, and those protective effects of MLT in NP cells further attenuated with erastin and enhanced with ferrostatin‐1(Fer‐1). This study demonstrated that CM from the LPS‐stimulated RAW264.7 macrophages promoted the NP cell injury. MLT alleviated the CM‐induced NP cell injury partly through inhibiting ferroptosis. The findings support the role of ferroptosis in the pathogenesis of IDD, and suggest that MLT may serve as a potential therapeutic approach for clinical treatment of IDD.


| INTRODUC TI ON
Intervertebral disc degeneration (IDD) is a leading cause of low back pain, which affects almost 80% of the global population at least one time during their life. 1 Although the pathogenesis has not been clearly defined, it is accepted that the progression of IDD is initiated and accelerated by the depletion of nucleus pulposus (NP) cells and degradation of extracellular matrix (ECM). Therefore, it will be of great clinical significance to elucidate the pathogenetic mechanisms underlying IDD via investigating NP cells. NP cell, a main type of cell resident in the NP of the intervertebral disc (IVD), is critically important for maintaining the physiological function of the IVD. NP cells are responsible for the synthesis and decomposition of the ECM, which, in turn, maintains the normal structural and functional properties of the IVD.
A previous study demonstrated that the anabolism/catabolism molecules of ECM like collagen type II α1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase-13 (MMP-13), thrombospondin type 1 motif 4 (ADAMTS4) and thrombospondin type 1 motif 5 (ADAMTS5) are changed in the degenerated IVD tissue. 2 Additionally, the degree of oxidative stress in the degenerative IVD is aggravated and the levels of oxidative stress-associated markers, including reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH), are also altered in the degenerative IVD. 3 While the exact aetiology of IDD remains undetermined, the loss of NP cells and the subsequent degradation of the ECM are considered as the major pathological characteristics during the development of IDD. 4 Oxidative stress not only triggers inflammation and matrix degradation, but also promotes the decrease in the number of viable and functional cells in the micro-environment of the IVD. Moreover, increasing evidence has confirmed that the loss of NP cells due to the apoptosis, autophagic and necrosis plays a critical role in the pathological development of IDD. 5,6 Ex vitro studies have demonstrated that the biologic interactions between NP cells and activated macrophages formed a positive feedback loop, leading to severe inflammation, which subsequently induced the synthetic and degraded imbalance of ECM and finally exacerbated the IDD development. 7 Therefore, amelioration of NP cell injury via targeting the inflammatory macrophages and the modulation of cell death of NP cells, might be a potential and attractive therapeutic strategy for the treatment of IDD in clinic.
Ferroptosis, discovered by Stockwell et al. in 2012, is an emerging new type of programmed cell death, which is morphologically, biochemically and genetically distinct from apoptosis, autophagy and necrosis. 8 Mechanistically, ferroptosis is caused by the elevation of intracellular iron levels and the accumulation of lipid ROS. 8 Studies have shown that ferroptosis was involved in the pathological process of various diseases, including carcinogenesis, stroke, intracerebral haemorrhage, traumatic brain injury, ischemia-reperfusion injury, kidney injury and degenerative diseases such as Parkinson's, Huntington's, and Alzheimer's diseases. 9 More importantly, clear evidence has consistently demonstrated that ferroptosis contributed to the pathogenesis of IDD. 10,11 In clinic trials, the proteins markers of ferroptosis, such as the levels of glutathione peroxidase 4 (GPX4) expression and solute carrier family 7 member 11 (SLC7A11) were significantly decreased, while acyl-CoA-synthetase long-chain family number 4 (ACSL4) was increased in the degenerated IVD tissue compared with the normal tissue. 10,11 Animal studies showed that the degree of disc degeneration was attenuated in the iron chelator deferoxamine (DFO) group and ferroptosis inhibitors ferrostatin-1 (Fer-1) relative to the saline group. 11 Therefore, key factors in ferroptosis-related pathways like SLC7A11, GPX4, ACSL4 and lysophosphatidylcholine acyltransferase 3 (LPCAT3) played an important role in multiple diseases including tissue injury, inflammation and IDD. 12 Melatonin (N-acetyl-5-methoxytryptamine, MLT), is an indoleamine hormone, which displays a variety of biological properties, including anti-oxidant, 13,14 anti-inflammatory, 15,16 antiapoptosis, 17,18 anti-cancer, 19 anti-diabetic 20 and immunomodulation. 21,22 Recently, clinical and animal studies suggested the role of MLT in the IDD. 3,[23][24][25][26][27][28][29] In patients with IDD, the level of plasma MLT concentration was significantly lower in comparison with that of healthy control group, which was correlated with the disease duration, severity and the levels of inflammatory cytokines. 24 Animal studies revealed the importance of MLT in IDD, that exogenous MLT administration activated the recovery process in the degenerated IVD tissue in rats 3 and rabbits 29 ; whereas surgical pinealectomy significantly reduced serum MLT levels in chicken, resulted in the accelerated deterioration of IDD. 30 Additionally, MLT was an effective inhibitor of ferroptosis and its anti-ferroptosis provided a potential therapeutic target for treating traumatic brain injury and acute sleep deprivation-induced cognitive impairment. 31

| Preparation of CM
To activate the polarisation of macrophages, macrophages were stimulated by lipopolysaccharide (LPS). 33

| MTT assay
The cytotoxicity of MLT on NP cells was evaluated by the MTT assay (Sigma-Aldrich). MLT was purchased from Rhawn and dissolved in dimethyl sulphoxide (DMSO). NP cells were seeded in a flat-bottom 96-well plate at an initial density of 6 × 103 cells/well and incubated at 37°C, and 5% CO 2 conditions. After 24 h, cells were treated with 0.01, 0.1, and 1 μM concentration of melatonin respectively. Cells treated with only 0.1% DMSO served as a negative control. Such treated NP cells were incubated in a humidified environment with 5% CO 2 at 37°C for 24 h. The MTT reagent (10 μL) was then added to each well and incubated at 37°C for 4 h. Then the medium in each well was removed after centrifuge and followed by adding 200 μL DMSO solution before being gently agitated for 10 min at room temperature. The absorbance at 490 nm was read using a microplate reader (Thermo Scientific, Vario Skan Flash).

| ROS, MDA and GSH assays
Intracellular ROS levels of NP cells were detected using a ROS detection assay kit (Beyotime) according to the manufacturer's instructions. NP cells were seeded at a density of 2 × 105 cells/well in a six-well cell culture plate and were treated with the CM from RAW for GSH detection. The absorbance of each well at the wavelength of 412 nm was determined using a microplate reader (Rayto).

| Intracellular iron determination
The intracellular iron content was determined using the colorimetric Iron Assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. Briefly, 0.5 mL of cell lysate was mixed with 1.5 mL of the determination buffer and was heated for 5 min in a boiling water bath. Then the mixture was cooled to room temperature with flowing water before being centrifuged at 3500× rpm for 10 min. The OD of the final mixtures was measured at a wavelength of 570 nm using a microplate reader (Rayto).

| Real-time quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from NP cells using Trizol reagent (TransGen

| Statistical analysis
Data were presented as mean ± SD. Comparison of continuous data between the two groups was performed by independent Student's t-test, which was analysed by using IBM SPSS software version 20 (IBM Corp.). Differences at p <0.05 were considered statistically significant.

| MLT displays no cytotoxicity on NP cells
To

| Reduction of oxidative stress by MLT in NP cells
To Data are expressed as mean ± standard deviation. The two groups among the five groups are compared by using an independent t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

| Protect NP cells against inflammation by the treatment with MLT
To

| Normalize the ECM production of NP cells by the treatment with MLT
To evaluate whether the ECM production of NP cells was affected by the CM treatment, the modifications of ECM production of NP cells were analysed by qPCR and Western blot, which include the syntheticassociated molecules (COL2A1 and ACAN) and ECM degradationassociated molecules (MMP-13, ADAMTS4 and ADAMTS5).

| Involvement of ferroptosis in CM-induced NP cell injury
As the unique type of cell death, ferroptosis contributes to the pathogenesis of degenerative diseases. 35 To investigate whether fer- To further investigate the association between MLT and ferroptosis, the ferroptosis inducer (erastin) and inhibitor (Fer-1) were added to CM before the culture of NP cells. As shown in Figure 5 and Figure  In the present study, we found that MLT could alleviate the CM-induced NP cell injury in a dose-dependent manner. MLT is an indoleamine hormone involved in various diseases, including lung injury, 43 renal fibrosis, 44 diabetes, 20 nonalcoholic fatty liver disease, 14 Alzheimer's disease, 45 Parkinson disease 46 and cancer. 47 The role of MLT in IDD was first discovered by Turgut  (C) TEM showed the morphological changes of mitochondria of NP cells. Data are expressed as mean ± standard deviation. The two groups among the five groups are compared by using an independent t-test. The normal mitochondria were marked with green arrows, while the abnormal mitochondria with small sizes and thick membrane were marked with red arrows. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
2 macrophages (M2) 50 and inhibiting inflammatory-related gene expressions. 51  an iron-dependent regulated cell death, which is morphologically, biochemically and genetically distinct from apoptosis, autophagy, and necrosis. 8 Iron, as a constituent of iron-sulphur proteins, hemoproteins, myoglobin and cytochrome p450 system, is essential for the accumulation of lipid peroxides in the development of ferroptosis. 52 The current study found that compared with negative control cells, the intracellular iron levels and ferroptosis's marker proteins GPX4, SLC7A11, GCLC and NRF2 expression levels significantly declined, ACSL4, LPCAT3 increased, and the microstructure of mitochondria changed in the CM-treated NP cells, which indicated the involvement of ferroptosis in IDD. In support of our result, Yang et al. studied in human IVD tissues found that the ferroptosis's marker proteins GPX4 and FTH expression levels significantly decreased, while PTGS2 was increased in the degenerated IVD tissues compared with the normal tissues. 11 In vivo studies showed that treatment with iron chelator DFO or ferroptosis inhibitors Fer-1 ameliorated IDD progression compared to the saline group. 10,11 In our study, MLT ameliorated NP cell injury and ferroptosis was involved in this process. Well the detail mechanism and pathways should further be investigated in future studies.
There were several limitations in this study. First, although multiple ferroptosis-related molecules such as GPX4, SLC7A11, ACSL4 and LPCAT3 were involved in reducing NP cell injury using MLT, the exact and detail mechanisms and pathways of ferroptosis during this process should further be explored in future studies. Second, the cell experiments in this study were performed to explore the relationship between MLT, IDD and ferroptosis, however, animal experiments should be conducted in future. In addition, this study lacked the exploration about the influence of higher concentration of MLT.
Subsequent studies on this part may be helpful to understand the effects of higher concentration of MLT in IDD. Finally, in this study, we focused on the effects of MLT treatment on NP cells other than macrophages, therefore our study did not investigate the exact components in CM from LPS-stimulated RAW264.7 macrophages, which should also be further studied in future.
In summary, current studies demonstrated that MLT treatment could markedly protect NP cells against the inflammatory injuries

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.