Glioprotective Effects of Lingonberry Extract Against Altered Cellular Viability, Acetylcholinesterase Activity, and Oxidative Stress in Lipopolysaccharide-Treated Astrocytes

Abstract

Altered astrocytic function is a contributing factor to the development of neurological diseases and neurodegeneration. Berry fruits exert neuroprotective effects by modulating pathways involved in inflammation, neurotransmission, and oxidative stress. The aim of this study was to examine the effects of the lingonberry extract on cellular viability and oxidative stress in astrocytes exposed to lipopolysaccharide (LPS). In the reversal protocol, primary astrocytic cultures were first exposed to 1 µg/mL LPS for 3 h and subsequently treated with lingonberry extract (10, 30, 50, and 100 μg/mL) for 24 and 48 h. In the prevention protocol, exposure to the lingonberry extract was performed before treatment with LPS. In both reversal and prevention protocols, the lingonberry extracts, from 10 to 100 μg/mL, attenuated LPS-induced increase in reactive oxygen species (around 55 and 45%, respectively, P < 0.01), nitrite levels (around 50 and 45%, respectively, P < 0.05), and acetylcholinesterase activity (around 45 and 60%, respectively, P < 0.05) in astrocytic cultures at 24 and 48 h. Also, in both reversal and prevention protocols, the lingonberry extract also prevented and reversed the LPS-induced decreased cellular viability (around 45 and 90%, respectively, P < 0.05), thiol content (around 55 and 70%, respectively, P < 0.05), and superoxide dismutase activity (around 50 and 145%, respectively, P < 0.05), in astrocytes at both 24 and 48 h. Our findings suggested that the lingonberry extract exerted a glioprotective effect through an anti-oxidative mechanism against LPS-induced astrocytic damage.

Keywords: Acetylcholinesterase; Astrocyte; Cellular viability; Lingonberry extract; Lipopolysaccharide; Oxidative stress.

Fig. 1

Experimental scheme of the two protocols used in the study. Astrocytes were obtained from primary cultures and maintained in standard conditions for 20 days. In the reversal protocol, astrocytes were exposed to lipopolysaccharide (LPS, 1 µg/mL) for 3 h and subsequently treated with lingonberry extract. In the prevention protocol, cells were first treated with lingonberry extract for 3 h and subsequently exposed to LPS

Fig. 2

Effects of lingonberry extract on cellular viability in primary astrocytic cultures after 24 h (a) and 48 h (b). Cellular viability of astrocytes treated with lipopolysaccharide (LPS) and lingonberry extract (reversal protocol) after treatment for 24 h (c) and 48 h (d). Cellular viability of astrocytes treated with lingonberry extract and LPS (prevention protocol) after treatment for 24 h (e) and 48 h (f). Cellular proliferation of astrocytes treated with LPS and lingonberry extract (reversal protocol) after treatment for 24 h (g) and 48 h (h). Cellular proliferation of astrocytes treated with LPS and lingonberry extract (prevention protocol) after treatment for 24 h (i) and 48 h (j). Values represent mean ± standard error of the mean. The experiments were performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001, different from control cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, different from LPS group

Fig. 3

Representative phase-contrast microphotographs of astrocytes exposed to lipopolysaccharide (LPS) and treated with the lingonberry extract after 48 h (images were taken using an Olympus inverted microscope; magnification 40×). Arrows indicate astrocytes evidencing the different morphologies found between the groups

Fig. 4

Acetylcholinesterase (AChE) activity in primary astrocytic culture exposed to lipopolysaccharide (LPS) and lingonberry extract after 24 and 48 h in both reversal and prevention protocols. Values represent the mean ± standard error from three independent experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, different from control cells; ^#P < 0.05, ^##P < 0.01, different from LPS group

Fig. 5

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in primary astrocytic culture exposed to lipopolysaccharide (LPS) and lingonberry extract after 24 and 48 h in the reversal protocol. Values represent the mean ± standard error from three independent experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001, different from control cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, different from LPS group

Fig. 6

Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in primary astrocyte culture exposed to lipopolysaccharide (LPS) and lingonberry extract after 24 and 48 h in the prevention protocol. Values represent the mean ± standard error from three independent experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, different from control cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001, different from LPS group

Fig. 7

Mechanisms involved in the glioprotective actions of lingonberry extract in lipopolysaccharide (LPS)-induced damage in astrocytes. AChE acetylcholinesterase, iNOS inducible nitric oxide synthases, ROS reactive oxygen species, SH thiol content, SOD superoxide dismutase