Antioxidant activity of hyaluronic acid investigated by means of chemiluminescence of equine neutrophil bursts and electron paramagnetic resonance (EPR) spectroscopy

The activated polymorphonuclear neutrophils (PMNs), the reactive oxygen and nitrogen species (ROS/RNS) released by PMNs and the derived inflammatory processes are strictly interlinked factors involved in the pathogenesis and progression of human inflammatory airways diseases such as chronic obstructive pulmonary disease (COPD), asthma and airway infections. Similar diseases are also present in horses which suffer from recurrent airway obstruction (RAO) and inflammatory airway disease (IAD). In order to decrease oxidative stress during inflammation and over‐regulated PMN respiratory bursts, a rational strategy is to increase antioxidant defenses by administering agents with antioxidant activity. This approach offers more practical therapeutic possibilities and various antioxidant molecules are available. Hyaluronic acid, a naturally occurring high molecular weight non‐sulfated glycosaminoglycan (GAG) composed of repeating disaccharide units containing glucuronic acid and N‐acetylglucosamine, has numerous roles in the activation and modulation of the inflammatory processes, including the antioxidant activity. The aim of this study was to examine whether a preparation of hyaluronic acid (MW 900.000 Da) interferes with ROS and RNS during the course of equine PMN respiratory bursts. To establish the lowest concentration at which this preparation of hyaluronic acid still has antioxidant activity, luminol amplified chemiluminescence (LACL) was applied. Electron paramagnetic resonance (EPR) spectroscopy was also used to investigate the direct antiradical (scavenger) activity of the preparation of hyaluronic acid. The hydroxyl radical was significantly scavenged in a hyaluronic acid concentration‐dependent way from 2.5 to 0.15 mg/ml. Superoxide anion, Tempol radical and the ABTS●+ were significantly inhibited from 2.5 to 0.62 mg/ml. Studying the LACL of stimulated equine neutrophils we observed that hyaluronic acid induced a statistically significant concentration‐effect reduction of the LACL from 5 mg/ml to the last significant active concentration of 1.25 mg/ml. These findings were confirmed also when L‐Arg was added in order to investigate the inhibition of the resulting peroxynitrite anion. Our findings indicate that, in addition to the human use, hyaluronic acid can be adopted also for horses to antagonize the oxidative stress generated by free radicals. In order to maximize the therapeutic options a possible use in horse respiratory diseases through direct aerosol administration could be hypothesized in analogy with the same use in humans. This last possibility should be further evaluated.