Differential Activation of Glioprotective Intracellular Signaling Pathways in Primary Optic Nerve Head Astrocytes after Treatment with Different Classes of Antioxidants

Abstract

Optic nerve head astrocytes are the specialized glia cells that provide structural and trophic support to the optic nerve head. In response to cellular injury, optic nerve head astrocytes undergo reactive astrocytosis, the process of cellular activation associated with cytoskeletal remodeling, increases in the rate of proliferation and motility, and the generation of Reactive Oxygen Species. Antioxidant intervention has previously been proposed as a therapeutic approach for glaucomatous optic neuropathy, however, little is known regarding the response of optic nerve head astrocytes to antioxidants under physiological versus pathological conditions. The goal of this study was to determine the effects of three different antioxidants, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (Mn-TM-2-PyP), resveratrol and xanthohumol in primary optic nerve head astrocytes. Effects on the expression of the master regulator nuclear factor erythroid 2-related factor 2 (Nrf2), the antioxidant enzyme, manganese-dependent superoxide dismutase 2 (SOD2), and the pro-oxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), were determined by quantitative immunoblotting. Furthermore, efficacy in preventing chemically and reactive astrocytosis-induced increases in cellular oxidative stress was quantified using cell viability assays. The results were compared to the effects of the prototypic antioxidant, Trolox. Antioxidants elicited highly differential changes in the expression levels of Nrf2, SOD2, and NOX4. Notably, Mn-TM-2-PyP increased SOD2 expression eight-fold, while resveratrol increased Nrf2 expression three-fold. In contrast, xanthohumol exerted no statistically significant changes in expression levels. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake and lactate dehydrogenase (LDH) release assays were performed to assess cell viability after chemically and reactive astrocytosis-induced oxidative stress. Mn-TM-2-PyP exerted the most potent glioprotection by fully preventing the loss of cell viability, whereas resveratrol and xanthohumol partially restored cell viability. Our data provide the first evidence for a well-developed antioxidant defense system in optic nerve head astrocytes, which can be pharmacologically targeted by different classes of antioxidants.

Antioxidant Effects of the Prenylated Flavonoid, Xanthohumol, on Corneal Epithelial Cells in Experimental Dry Eye Disease

“…Intriguingly, Xanthohumolencapsulating PLGA NP had a much larger effect on 8-OHdG labeling in the cornea, reducing density of immunolabel by ~50% (Figure 9), compared with an ~25% reduction elicited by Mn-TM-2-PyP [17]. Given that the antioxidant potential of Mn-TM-2-PyP is significantly greater than that of Xanthohumol ( [17,31]), this finding may suggest that Xanthohumol-encapsulating PLGA NP are not only able to be retained at the ocular surface for a prolonged period of time despite their negative surface charge, but also achieve sustained activation of the endogenous antioxidant system.…”

“…Human corneal epithelial cells (HCE-T; RIKEN BioResource Research Center, Tsukuba, Japan) were cultured as described by us previously [17,[31][32][33]. Cultures of passages 79 to 95, were used for experiments.…”

An endogenous PI3K interactome promoting astrocyte-mediated neuroprotection identifies a novel association with RNA-binding protein ZC3H14

“…A variety of homeostatic and prosurvival astrocyte activities have recently been described, strengthening the concept of key glioprotective central nervous system mechanisms ( 2 , 52 , 53 , 54 , 55 ). In previous work, we established that the astrocyte secretome contains potent neuroprotective factors that ameliorate metabolic and excitotoxic neuronal injury in in vitro and in vivo models ( 12 ).…”

New Highlights of Resveratrol: A Review of Properties against Ocular Diseases

“…In fact, many studies have demonstrated that RSV can reduce ROS in glaucoma models (in vitro and in vivo) through several pathways ( Figure 4 ) [ 114 , 115 , 116 ]. This downregulation of ROS could be due to the modulation of the endogenous antioxidant system [ 117 ], such as induction of nuclear factor erythroid-2-related factor 2 (Nrf2) translocation into the nucleus, where it binds to antioxidant response elements (ARE). This latter leads to the production of heme oxygenase-1 (HO-1), an anti-oxidant enzyme [ 117 , 118 ].…”