*3.3. Neutral or Beneficial E*ff*ects of Melatonin in RA*

Notably, a cross-sectional study from Iran has reported finding significantly higher morning serum levels of melatonin in patients with RA compared with healthy controls, but no correlation between melatonin and RA disease activity score or other disease characteristics, including age, disease duration, medications, gender, or season of sampling [36]. The study also reported finding higher serum melatonin values in newly diagnosed patients compared with patients with established RA, which needs further investigation [36].

Matrix metalloproteinases (MMPs) are a family of endopeptidases primarily responsible for catalyzing the degradation of the extracellular matrix (ECM) [46]. MMPs play important roles in RA. Elevated levels of circulating MMP-3, MMP-8 and MMP-9 are associated with disease progression in

RA [47]. In particular, MMP-2 and MMP-9 are expressed in synoviocytes, CD34<sup>+</sup> endothelial cells, monocytes and macrophages of rheumatoid synovium, indicating that both molecules are critical to pannus formation and invasion in RA progression [47]. Interestingly, melatonin reportedly directly inhibits secreted MMP-9 by binding to the active site and significantly reducing the catalytic activity of MMP-9 in both in vitro and cultured cells, in a dose- and time-dependent manner [46]. Thus, melatonin could have an important role in the prevention of joint destruction in RA.

Research demonstrating that melatonin dose-dependently inhibits the proliferation of RA fibroblast-like synoviocytes (FLS) through the activation of the ERK/P21/P27 pathway suggests that inhibiting the invasion of RA FLS through cartilage and into bone may have important implications in the treatment of RA [48]. Blocking NF-κB signaling appears to be the way in which melatonin protects cells from oxidative stress [28] and largely explains how melatonin suppresses proinflammatory cytokines such as IL-1β and TNF-α [20]. Other pathways and molecules associated with inflammation that are modulated by melatonin include the mitogen-activated protein kinase (MAPK) and nuclear erythroid 2-related factor 2 (Nrf2) pathways, as well as Toll-like receptors [15].

In a clinical trial involving RA patients, six months of melatonin treatment (10 mg/day) was associated with a general decrease from baseline in concentrations of peroxidation markers [35]. Conversely, ESR and neopterin concentrations were increased from baseline with melatonin and significantly higher at six months than concentrations in the placebo-treated cohort, which experienced a significant downward trend in these inflammatory indicators during the trial [35]. Paradoxically, neither the elevations in ESR and neopterin concentrations nor the decrease in tissue peroxidation associated with melatonin translated into significant differences from the placebo group in terms of patients' symptoms, or in the concentrations of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) [35]. The study researchers concluded that melatonin does not appear to be beneficial in RA [35].

The findings of Forrest et al. are discussed by Maestroni et al., who argue that because melatonin enhances the production of Th1-type and inflammatory cytokines in RA, upregulates cell-mediated and humoral responses, and also exacerbates CIA in mice, melatonin likely promotes RA disease and is inappropriate for therapeutic use [49]. Maestroni et al. also emphasized the high blood melatonin concentrations (280 pg/mL) observed 12 h after dosingin theRA cohort [49], in relation to the notion that higher blood melatonin concentrations, especially in the early morning, may be responsible for morning stiffness and joint swelling experienced by patients with arthritis [6,43]. Maestroni et al. conjecture that autoreactive T cells in RA patients synthesize and release melatonin, thereby worsening the disease process [49].

Nevertheless, Korkmaz [50] has defended Forrest et al., pointing out that melatonin has shown strong anti-inflammatory activity in studies using known inflammatory agents, such as zymosan [51], lipopolysaccharide [52,53], and carrageenan [54]. Korkmaz speculated that the high blood melatonin concentrations in the RA cohort may have been a compensatory response to RA inflammation and were comparable to the high levels of melatonin in cerebrospinal fluid and its metabolites in meningitis populations and in pediatric patients with epilepsy [50]. Korkmaz maintained that melatonin is an appropriate adjunctive therapy for RA [50].

Melatonin appears to play an important role in microRNA (miRNA) expression in RA. miRNAs are small, non-coding RNAs that post-transcriptionally mediate protein expression by targeting protein-coding genes implicated in cancer cell proliferation, differentiation, apoptosis, and migration [55]. A recent study found that melatonin appears to inhibit miR-590-3p expression and induce apoptosis in human osteoblasts [55]. In another study, melatonin treatment effectively downregulated TNF-α and IL-1β production in human RA synovial fibroblasts (the MH7A cell line) by suppressing PI3K/AKT, ERK, and NF-κB signaling and upregulating miR-3150a-3p expression [20]. Those investigations confirmed that the MT1 receptor mediates the anti-inflammatory effects of melatonin and that melatonin not only inhibits inflammatory cytokine release in mice with CIA-induced arthritis, but also attenuates CIA-induced cartilage degradation and bone erosion [20]. This evidence suggests that melatonin targets miRNAs, which could be explored in clinical trials examining the efficacy of melatonin in the treatment of RA. The following figure (Figure 1) and table (Table 1) illustrate the processes through which melatonin exerts its therapeutic effects.

**Figure 1.** Neutral or beneficial effects of melatonin in rheumatoid arthritis (RA). The dotted line represents the uncertainty over the effects of melatonin upon macrophages and monocytes.



#### **4. Summary**

Various research suggests that melatonin has disease-promoting effects in RA and that it could increase the severity of RA, in contradiction to the beneficial effects of melatonin in other autoimmune inflammatory diseases. However, in the past decade, some studies have demonstrated that melatonin can alleviate RA through the inhibition of RA synovial fibroblast proliferation, TNF-α and IL-1β expression, as well as MMP-9 activity. The anti-inflammatory character of melatonin in RA is associated with regulation of microRNAs (such as miR-3150a-3p). More investigations are therefore warranted to explore the possible double-edged effects of melatonin in RA. The use of melatonin in patients with RA needs thorough consideration by clinical physicians.

**Author Contributions:** Conceptualization, C.-H.T.; literature collection and preparation, I.J.M., C.-C.H.; writing—original draft preparation, I.J.M., S.-C.L.; writing—review and editing, I.J.M., C.-C.H.; supervision, C.-H.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from the National Science Council of Taiwan (MOST108- 2320-B-039-065; MOST 108-2320-B-039-064-); China Medical University Hospital (DMR-109-208); and China Medical University, Taichung, Taiwan (CMU108-MF-114).

**Conflicts of Interest:** The authors declare no conflict of interest.
