Introduction Osteoarthritis OA is a prevalent disease

Introduction Osteoarthritis (OA) is a prevalent disease characterized by chronic joint degeneration, including articular cartilage loss, synovitis and osteophyte formation [1], [2], [3], [4], [5]. The cause of OA is metabolic imbalance and mechanical stress, which is coupled with inflammation, leading to a catabolic shift in cartilage homeostasis. Increased macrophage activity has been reported as a biomarker for human OA [6], and antagonizing pro-inflammatory cytokine Interleukin 1β (IL-1β) directly in the joint protects cartilage matrix in post-traumatic experimental OA, indicating a pivotal role of inflammation in OA pathogenesis [7]. IL-1β activity is mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, which upon activation induces the expression of cartilage matrix-degrading enzymes such as matrix metalloproteinases (MMPs) and subsequent matrix degradation [8]. Recently, certain antibiotics used to treat bacterial infections have been shown to alter the response of chondrocytes toward inflammatory stimuli. Tetracycline and doxycycline chelate zinc at the active sites of MMPs and inhibit experimental OA and rheumatoid arthritis [9], [10], [11], [12], [13], [14]; however, reduction in human joint pain and swelling was limited [15], [16]. The macrolide class of antibiotics, including erythromycin (EM) and azithromycin, has also been found to be anti-inflammatory. In patients with diffuse panbronchiolitis, they inhibit macrophage activation and inflammation in the lung [17], [18], [19]. Similarly, EM inhibited aseptic loosening after bone implants [20], [21]. Our group recently showed that EM inhibits inflammatory stimuli IL-1β and Lipopolysaccharides (LPS)-induced catabolic events in bovine articular chondrocytes [22]. However, the mechanism underlying this activity remains poorly understood. In the current study, we discovered that the activity of EM is mediated by the ghrelin receptor. Ghrelin is a 28 amino determining molarity peptide that is best known as the “hunger hormone,” as it increases appetite and controls blood sugar levels [23], [24], [25], [26]. The ghrelin receptor is also known as the Growth Hormone Secretagogue Receptor (GHSR1a, or abbreviated as GHSR) [27]. While a truncated variant GHSR1b also exists, it does not bind to ghrelin; therefore, ghrelin signals through its binding to GHSR1a [28]. In chondrocytes, ghrelin has been found to control fatty acid uptake and inhibit inflammation when overexpressed [29], [30], [31]. However, these are gain-of-function experiments, and it is still not known whether ghrelin signaling is truly necessary for joint maintenance under disease conditions. We identified a novel functional link between EM and ghrelin signaling in chondrocytes, since the loss of the ghrelin receptor abolished EM-mediated chondro-protection and resulted in more cartilage destruction in vivo. Administration of ghrelin to chondrocytes had a similar inhibitory activity as EM toward pro-inflammatory cytokine IL-1β-induced catabolic gene expression. This activity was diminished in ghrelin receptor null cells, suggesting that EM and ghrelin both act through the ghrelin receptor.
Materials and methods
Results
Discussion This study provides a key mechanism for the pharmacological activity of erythromycin (EM). We report that the ghrelin receptor is an essential mediator of EM chondroprotective activity, demonstrating that both ghrelin and EM can activate the ghrelin receptor and inhibit pro-inflammatory cytokine IL-1β-induced NF-κB activation and catabolic gene expression. Furthermore, the ghrelin receptor is required to resist synovitis and joint destruction in EM-treated mice in vivo, thus playing an essential role for joint maintenance under pathological conditions. Beneficial effects of ghrelin on OA cartilage has only recently been reported. In 2017, Zou et al. found that the synovial fluid of knee OA patients contained lower levels of ghrelin [61]. In the same year, Qu et al. showed that ectopic intraperitoneal injection of ghrelin could reduce surgery-induced mouse knee cartilage damage and inflammation [31]. In 2018, Liu et al. also demonstrated that supplementing ghrelin in culture media inhibited IL-1β-induced catabolic changes in human cartilage [30]. Our study is consistent with these recent findings, but we have also significantly deepened the understanding on the role of ghrelin in OA in additional ways. First, we definitively demonstrate that ghrelin signaling is critical for joint health and resistance to inflammation. Past reports focused on ectopic ghrelin treatment and did not indicate whether ghrelin signaling is required for the maintenance of the joint [30], [31]. Our study demonstrated that ghrelin signaling is essential for resisting inflammatory cytokine-induced MMP expression in vitro and delaying cartilage destruction in vivo. Second, we showed that ghrelin enhances cartilage matrix levels in human OA cartilage, even in the absence of exogenous pro-inflammatory cytokine. This has important implications for the potential use of ghrelin or its agonists to treat existing OA and halt cartilage destruction. Third, we extended our analysis in vivo to the analysis of the synovium, which plays an important role in inflammation of the joint, rather than being restricted to cartilage analysis. It is worth noting that while ghrelin administration would increase feeding and body weight [62], [63], mice lacking ghrelin have the same weight as the wild type under normal feeding conditions [64], [65]. On the other hand, mice lacking the receptor for ghrelin showed sexual dimorphism in this aspect: male mice did not exhibit any difference in body weight differences when fed with the standard chow, but female mice did [26]. Since we only used male mice, it allays the concern of body weight-associated OA in our study.