Induction of inflammatory response by different stimuli in a human lens epithelial cell line

Human lens epithelial cells, due to their ocular function, are normally exposed to radiating and oxidative stress conditions. Indeed, the lens is an environment rich in endogenous sources of reactive oxygen species (ROS), among which there are the high oxygen concentration and the chronic exposure to light, that have been linked to apoptosis in lens epithelial cells. Although multiple defence systems exist to protect the lens from the toxic effects of oxidative damage, including abundant antioxidant enzymes, reduced glutathione (GSH) and the chaperone-like functions of crystallins, increasing evidence suggest that oxidative stress may predispose the lens to cataract development. In fact, ROS can attack polyunsaturated fatty acids in membranes, causing lipid peroxidation and generating highly toxic reactive aldehydes. Among them, one of the most reactive is 4-hydroxy-2- nonenal (4-HNE) which can form adducts with DNA and proteins and, for this reason, undergoes a rapid enzymatic biotransformation that regulates its free content. The major enzymatic route of 4- HNE detoxification consists in its conjugation with glutathione (GSH) to generate 3-glutathionyl-4- hydroxynonanal (GS-HNE), an adduct that in turn can be either reduced or oxidized. It is worth underlying that the reduced form of GS-HNE, namely 3-glutathionyldihydroxynonane (GS-DHN), a product of a reaction catalyzed by both aldose reductase (AKR1B1) and carbonyl reductase (CBR1), is reported to be a mediator of cytokines, chemokines and growth factors-induced signalling pathways that trigger the inflammatory response associated with NF-κB activation, leading to apoptosis in human lens epithelial cells. In addition, AKR1B1, that catalyzes the rate limiting step of the polyol pathway, under hyperglycaemic conditions promotes, through the activation of NF-κB, the synthesis of many proteins such as proinflammatory cytokines (TNF-α, IL-1, IL-8), adhesion molecules (VCAM-1, ICAM-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), establishing a chronic pathological inflammatory response associated with the onset of diabetic complications. In summary, the aim of this thesis was providing evidence that a cultured human lens epithelial cell line, HLE-B3, could respond to both 4-HNE and D-glucose, considered as two endogenous pro-inflammatory stimuli intertwined by AKR1B1 activity, that can activate the inflammatory response associated with the transcription factor NF-κB and COX-2. The pro- inflammatory effect of both 4-HNE and hyperglycaemia was investigated in HLE-B3 cells by evaluating, through Western blotting, the increase in COX-2 expression as well as in stably transfected HLE-B3 cells by measuring NF-κB activation. Capillary electrophoresis analysis suggested the formation, in HLE-B3 cells incubated with 4-HNE, of a glutathionylated 4-HNE adduct that likely correspond to GS-HNE, indicating that the pro-inflammatory action exerted by the aldehyde could be mediated by its glutathionylated derivatives. Finally, the role played by the two enzymes potentially involved in mediating both 4-HNE- and D-glucose-induced inflammatory signalling, AKR1B1 and CBR1, was verified by assessing the anti-inflammatory effect of two their well-known inhibitors, sorbinil and rutin. Since these inhibitors appears not to significantly attenuate COX-2 expression induced by 4-HNE nor NF-κB activation triggered by hyperglycaemic conditions, probably due to methodological limitations, further experiments are needed in order to clarify the efficacy of these molecules in the anti-inflammatory response. In this regard, it has also to take into account the role of the solvent used for inhibitors solubilization, which could interfere with the performed analysis.