The role of melatonergic system in intervertebral disc degeneration and its association with low back pain: a clinical study

Introduction

The prevalence of low back pain (LBP) disorder is estimated to affect approximately 540 million individuals globally, which poses a substantial and intricate health concern (Knezevic et al., 2021). The etiology of LBP is multifaceted, while the intervertebral disc degeneration (IVDD) is still one of the predominant underlying factors (Hartvigsen et al., 2018). The precise cause of IVDD and its pathogenesis remain elusive, with potential associated factors encompassing age, smoking, diabetes, genetic alterations (Teraguchi et al., 2017; Miller, Schmatz & Schultz, 1988) and apoptosis (Yang et al., 2020). Furthermore, the progression of IVDD may be influenced by mechanisms involving inflammation and pain signaling (Liu et al., 2021).

Melatonin (N-acetyl-5-methoxytryptamine) is synthesized in mitochondria, therefore, almost all cells including bone marrows have the capacity to produce melatonin (Tan et al., 2023). This molecule plays an important role in the regulation of cellular functions, including antioxidant (Tan et al., 2015), anti-inflammatory (Hardeland, 2018), anti-degenerative (Sarlak et al., 2013) and anti-apoptotic activities (Yang et al., 2015) etc. Turgut et al. (2003), Turgut et al. (2006) first reported the potential association between melatonin and IVDD. They observed that pinealectomy which diminishes the circulating melatonin accelerated process of IVDD in chicken while melatonin treatment improved the condition of IVDD model of rats. Subsequently, the accumulated evidence has shown that melatonin plays significant roles in the IVDD process, including protecting vertebral endplate chondrocytes against apoptosis and calcification (Zhang et al., 2019), preventing nucleus pulposus (NP) cells apoptosis induced by oxidative stress (He et al., 2018) or by excessive mitophagy (Chen et al., 2019) and suppressing NF-κB signaling pathway which is associated with the increased proinflammatory cytokines (Chen et al., 2020). We previously also demonstrated that melatonin was an important promotor in chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) (Gao et al., 2014). Additionally, melatonin effectively hinders the proliferation of NP cells through its receptors MT1 and MT2 mediated action (Li et al., 2017). Although the pathogenesis of IVDD remains poorly defined, the result from a controlled clinical trial has shown that the standard therapy combined with melatonin increases the treatment efficacy in LBP (Kurganova & Danilov, 2015).

Based on these evidences mentioned above, in the current study, we will examine whether melatonergic system alterations involve in LBP in individuals with IVDD by analyzing their clinical specimens. If so, the fucus will be given to the correlation between the melatonin receptors expression and NP tissue apoptosis since a variety of biological functions of melatonin are mediated by its receptors.

Materials & Methods

Results

Demographic information and health characteristics

First, the Pfirrmann classification was applied to MRI images of human lumbar intervertebral discs, and the respresentative results were showed in Fig. 1. Then, the degrees of the IVDD were classived with the method reported by Teraguchi et al. (2014). The results show that among 107 patients, 59 (55.1%) exhibited degenerative discs, while 48 (44.9%) had normal discs. Those patients with degenerative discs were significantly older as well as had lower LBP-VAS score than patients with normal discs (p < 0.001). However, no significant differences were observed in LP-VAS score along with BMI, history of diabetes, and smoking history between the groups (p > 0.05) based on the current sample size as shown in Table 1.

Table 1:

Demographic information and health characteristics between normal and degeneration groups.

Characteristics	Total	Normal	Degeneration	P value
Patients(n%)	107	48(44.9%)	59(55.1%)	–
Gender	107	48	59	0.069
Male	70	36	34
Female	37	12	25
Age(years)	43.0 ± 14.9	36.5 ± 13.9	48.4 ± 13.7	<0.001*
BMI	23.4 ± 3.4	23.3 ± 3.8	23.5 ± 3.1	0.726
Height(m)	1.66 ± 0.84	1.67 ± 0.07	1.66 ± 0.09	0.376
Weight(kg)	65 ± 12.34	65.08 ± 11.87	64.92 ± 12.82	0.947
Diabetes	107	48	59	0.063
Yes	5	0	5
No	102	48	54
History of smoking	107	48	59	0.655
Yes	5	2	3
No	102	46	56
VAS, back	107	5.5 ± 1.1	3.2 ± 1.4	<0.001*
VAS, leg	107	3.1 ± 1.1	3.1 ± 1.3	0.852

DOI: 10.7717/peerj.17464/table-1

Notes:

Values are expressed as mean ± SD or n.

BMI

body mass index

VAS

Visual analog scale

Student’s t test or Pearson’s chi-square test.

*P < 0.05.

The association of melatonin receptors expression and IVDD

The potential association of melatonin receptors MT1, MT2 and IVDD was also investigated by using IHC staining. The results showed that both melatonin receptors MT1 and MT2 were present in NP specimens (Fig. 2A). The ODI analysis of the images indicated the variable expression of melatonin receptors among samples correlated to IRS values (Figs. 2B and 2C). MT2 were highly expressed in NP specimens of all individuals without significant differences between degeneration and normal groups (p = 0.688). However, MT1 staining score was significantly higher in patients in degeneration group than those in normal group (p < 0.001) (Table 3).

Analysis of the association between apoptosis in NP and the Pfirrmann grade

Finally, the association between the apoptosis of NP cells and IVDD was investigated and the results were illustrated in Fig. 3. The cell apoptosis rate of NP cells was determined across various Pfirrmann grades and the results revealed that higher Pfirrmann grade exhibits increased apoptosis rate of NP cells, i.e., significantly elevated apoptosis in NP cells was associated with the severely degenerated intervertebral discs compared to the normal intervertebral discs.

Discussion and Conclusions

To the best of our knowledge, this report probably represents the pioneering attempt to investigate the potential associations among melatonin receptors, IVDD and LBP in patients by utilizing clinical data including radiographic images of spine and NP specimens of patients.

It has been known that the incidence of IVDD is strongly correlated with age. For example, under the age of 50, the prevalence of IVDD was 71% in males and 77% in females but it would surpass 90% in both genders over the age of 50 (Teraguchi et al., 2014). Consistent with previous reports, we also observed that patients with IVDD were significantly older than those individuals without IVDD. However, no correlation of IVDD with BMI, diabetes or smoking were detected, respectively.

Some degenerative intervertebral discs can cause discogenic LBP, others are painless depending on the location and severity. In addition, the presence or absence of morphological changes including disc herniation, narrowing, or Schmorl’s nodes contribute to the differentiating factors of symptomatic or asymptomatic in patients (Gopal et al., 2012). Son et al. (2021) proposed that the intervertebral disc height (DH) discrepancy ratio, as determined by standing and supine positions with/without axial loading, could serve as a screening index for discogenic LBP in patients with IVDD. However, they failed to find any significant difference in the degree of IVDD between the LBP (VAS ≥7.0) and the control groups. Nevertheless, other research showed the close relationship between DH and IVDD with disc space narrowing being identified as a characteristic of IVDD (Dolan & Adams, 2001). In light with this, we conducted a comparison of the DH discrepancy ratio among patients with varying Pfirrmann grades. Our findings revealed that patients with severe IVDD displayed a higher DH discrepancy ratio and lower LBP-VAS score. This inconsistence may be attributed to the inclusion of non-surgeied individuals with discogenic LBP in the study of Son et al. (2021), as well as the relatively limited sample size. In addition, the specificity or sensitivity of the Pfirrmann grade based on the imaging of MRI sometimes is not sufficient to confirm a diagnosis of discogenic LBP and has some shortcomings to fully identify the sources of LBP in those with IVDD (Ito & Creemers, 2013). Therefore, the Pfirrmann grades used to classify IVDD can not completely match the clinical symptoms.

Lumbar instability is another parameter to evaluate the severity of IVDD. It indicates the degenerative changes in the intervertebral disc and surrounding structures leading to alterations in the mechanical properties of the functional spinal units (Galbusera et al., 2014). It serves as an important risk factor contributing to the occurrence of LBP and often serves as a decision-making index for surgical intervention involving spinal fusion and decompression (Leone et al., 2007). We should mention that the debate regarding the clinical definition and diagnostic criteria of lumbar instability remains and it still lacks universally accepted standard. In this study, we adopted widely accepted criteria of lumbar instability, i.e., sagittal rotation value of 10° and sagittal translation value of 4 mm (Leone et al., 2007). Our results indicated a significantly higher prevalence of lumbar instability among individuals with IVDD compared to the control.

Melatonin has been reported to have a protective effect on intervertebral disc cells (Zhang et al., 2019; Chen et al., 2019), including preventing oxidative stress-induced NP cells apoptosis (He et al., 2018), and potentially alleviate chronic pain, particularly LBP (Danilov & Kurganova, 2016). Our previous investigation provided evidence of the presence of MT1 and MT2 in the IVDD tissue (Li et al., 2017). The involvement of melatonin receptors in pain modulation has been observed in various models of inflammation and neuropathic pain. Tang et al. (2022) reported the alleviative effect of melatonin on radicular pain induced by lumbar disc herniation in rats, primarily through its binding to the MT2. The MT2 mediated pain relief activity was also reported in osteoarthritis related pain in rats (Liu et al., 2022). Notably, the diminished expression of MT1 weakens the regulatory activity of melatonin on pain perception in rats (Huang et al., 2013). In current study, it was found that NP tissues from the patients with IVDD had a significantly upregulated MT1 expression, whereas MT2 expression was not significantly difference between groups. A substantial correlation was observed between increased levels of MT1 and MT2 expression and a significant reduction in LBP-VAS score among patients. Based on the definition of minimum clinically important difference (MCID), a difference exceeding 3.0 points in the LBP-VAS score is considered to have clinical significance (Ma et al., 2011). The results showed that the patients with high MT1 staining score from 3 to 12 had the average difference in LBP-VAS score of 3.3 points. In addition, highly elevated NP cells apoptosis found in the severe IVDD suggests that apoptosis plays a role in the progression of IVDD, consistent with previous study (Miyazaki et al., 2015). It is well documented that melatonin is a strong antiapoptotic molecule which is at least partially mediated by its MT1/2 receptors (Radogna et al., 2007).

Based on all the evidence, we hypothesize that during the initial phases of IVDD, as an adaptive response, NP cells start to upregulate its MT2 expression against this pathological alteration. As IVDD progression, the resultant LBP as a chronic stressor stimulates the expression of MT1. The elevated MT1 level can maximize the analgesic effect of melatonin on LBP as well as inhibits apoptosis in NP cells while the expression of MT2 remains relatively stable throughout this progression. This may be the reason that highly expressed MT2 was observed in all patients without significant differences between groups while significantly high level of MT1 expression was only detected in patients with severe IVDD compared to the control patients. Previous studies have revealed the ubiquitous presence of both MT1 and MT2 across various anatomical regions in the human body, including the brain, retina and other internal organs (Ahmad et al., 2023), indicating the important physiopathological roles of melatonergic system in human body. In addition, the three-dimensional structures of the human melatonin receptors MT1 and MT2 unveiled that many melatonin structure-like compounds exhibited a specific affinity towards the MT1 rather than MT2 (Stauch et al., 2019; Johansson et al., 2019). This characteristic of MT1 implays its relatively high efficiency to modulate LBP in patients with IVDD compared to the MT2. Our discovery provides better opportunity to develop the novel MT1 agonists to protect against IVDD and its associated LBP with less adverse effects.

In conclusion, in this clinical study, we have identified the highly elevated MT1 expression in NP tissues of patients with IVDD and LBP compared to the control patients, which may be a compensiting response of the body to the pathological alteration of the IVDD and LBP. These findings offer novel perspectives on the therapeutic potential to use MT1 specific agonists to treat IVDD and LBP.

Limitations

The study had limitations common to clinical research, being a single-center observational study, so our findings should be confirmed by others. Healthy human intervertebral disc samples were difficult to obtain, so we used Teraguchi et al.’s (2014) criteria to classify discs with Pfirrmann grade 3 or lower as the control group. The subjective nature of the Pfirrmann grade system may introduce bias in assessing IVDD, but no better methodology currently exists. This issue should be addressed in the future.