The effects of levofloxacin on rabbit fibroblast-like synoviocytes in vitro

Abstract

It is widely accepted that tendon and cartilage are adversely affected with the toxic effects of quinolones. However, the effects of quinolones on synovium have not been deciphered completely. In this study, our main objective was to investigate the effects of levofloxacin, a typical quinolone antibiotic drug, on fibroblast-like synoviocytes (FLSs) in vitro. FLSs of rabbits were treated with levofloxacin at different concentrations (0, 14, 28, 56, 112 and 224 μM). The possible cytotoxic effects of levofloxacin on FLS were determined. Levofloxacin significantly reduced the cell viabilities, gene expression of hyaluronan synthase-2 (HAS-2), and the level of hyaluronan in FLSs. Moreover, levofloxacin-induced concentration-dependent increases of apoptosis and active caspase-3 were determined in this study. Ultrastructural damages of FLSs were observed by electron microscopy. The mRNA expression levels of matrix metalloproteinase (MMP)-3 and MMP-13 were increased in FLSs treated with levofloxacin. In addition, levofloxacin played a role in suppressing the expression of interleukin (IL)-1 and IL-6. Our data suggest that the cytotoxic effects of levofloxacin on FLS were shown to be able to affect cell viability and HA synthesis capacity. The potential mechanisms of the cytotoxic effects may be attributed to the apoptosis and increased expression of MMPs.

Introduction

Quinolone antibacterial agents have excellent antibacterial activities, good tissue penetration, and high bioavailability (Oliphant and Green, 2002). Therefore, they are widely used in clinical practice. However, recent studies have reported that quinolones induce adverse effects on the musculoskeletal system, including myalgia, Achilles tendon rupture, and arthralgia (O-Lee et al., 2005, Tsai and Yang, 2011). In fact, clinical reports about tendinopathies associated with quinolone therapy are not rare in recent years (Corrao et al., 2006, De Sarro and De Sarro, 2001). Sode's research indicated that quinolone use might triple the risk of Achilles tendon rupture (Sode et al., 2007). The toxic effects of quinolones on tendon and cartilage have been well investigated in animals (Kappel et al., 2002, Kato et al., 1995). In our recent study, we found that levofloxacin, one of the quinolones, triggered cytotoxic effects on anterior cruciate ligament cells of rabbits (Deng et al., 2011). The experimental studies all considered that apoptosis and matrix metalloproteinases (MMPs) might be responsible for the cytotoxic effects of quinolones. Based on these findings, the scope of knowledge pertaining to the toxicities of the quinolones on locomotor system has expanded. While the researchers focus on the structures that withstand direct physiological stress force. Nevertheless, as the tissue that is in close relation to cartilage, ligament and tendon, synovium may not be ignored because changes in the microenvironment in synovium may directly or indirectly influence the function of the aforementioned structures. The influences of synovitis to articular cartilage degradation in rheumatoid arthritis and osteoarthritis are good examples for their relation (Scanzello and Goldring, 2012). Presently, there is limited data to indicate the toxicity of quinolone on synovial tissue. According to a morphological study, mild edema and an increased number of mononuclear cells were observed in the synovial membrane and tendon sheath (sheath is histologically a kind of synovial tissue) of rats treated with pefloxacin and levofloxacin (Kashida and Kato, 1997). However, such investigation has not been reported in later studies.

Histologically, synovial tissue is a layer of loose connective tissue which produces synovial fluid to lubricate and supply nutrient to the target tissue. Synovial tissue plays an indispensable role in maintaining the normal function of the structures it encapsulates (Edwards, 1994). Although the adverse effects of quinolones on cartilage, tendon, and ligament have been extensively studied till date, no in-depth research has paid attention to synovium or synoviocytes. Dysfunction of synovial tissue caused by any factor may adversely affect the function of joints and tendon sliding in the sheath, thereby causing corresponding clinical disorders. Moreover, MMPs and cytokines, including interleukin-1 (IL-1) and IL-6, produced and released from synoviocytes diffuse from the synovial fluid to create an impact on cells and matrix in the target tissue. Therefore, the current study focuses on the effects of levofloxacin on rabbit fibroblast-like synoviocytes (FLSs).

Levofloxacin, a typical kind of quinolone, is a potential cause of tendonitis and tendon rupture (Lado et al., 2005). Moreover, levofloxacin-induced pathological changes of synovial membrane were preliminarily observed in an animal experiment (Kashida and Kato, 1997). Therefore, the goal of the present study was to test the hypothesis that levofloxacin would cause cell damage to FLSs in vitro. We examined the cell viability, ultrastructural changes and hyaluronan (HA) synthesis capacity in rabbits FLSs treated with levofloxacin. Moreover, apoptosis was determined by flow cytometry, and characteristic apoptotic marker of active caspase-3 was analyzed using immunocytochemical methods. mRNA expression levels of matrix metalloproteinase enzymes (MMP-3, MMP-13) and cytokines (IL-1, IL-6) were also determined in this study for exploring the probable mechanism.