Lichen Extracts from Cetrarioid Clade Provide Neuroprotection against Hydrogen Peroxide-Induced Oxidative Stress

Abstract

Oxidative stress is involved in the pathophysiology of many neurodegenerative diseases. Lichens have antioxidant properties attributed to their own secondary metabolites with phenol groups. Very few studies delve into the protective capacity of lichens based on their antioxidant properties and their action mechanism. The present study evaluates the neuroprotective role of Dactylina arctica, Nephromopsis stracheyi, Tuckermannopsis americana and Vulpicida pinastri methanol extracts in a hydrogen peroxide (H₂O₂) oxidative stress model in neuroblastoma cell line "SH-SY5Y cells". Cells were pretreated with different concentrations of lichen extracts (24 h) before H₂O₂ (250 µM, 1 h). Our results showed that D. arctica (10 µg/mL), N. stracheyi (25 µg/mL), T. americana (50 µg/mL) and V. pinastri (5 µg/mL) prevented cell death and morphological changes. Moreover, these lichens significantly inhibited reactive oxygen species (ROS) production and lipid peroxidation and increased superoxide dismutase (SOD) and catalase (CAT) activities and glutathione (GSH) levels. Furthermore, they attenuated mitochondrial membrane potential decline and calcium homeostasis disruption. Finally, high-performance liquid chromatography (HPLC) analysis revealed that the secondary metabolites were gyrophoric acid and lecanoric acid in D. artica, usnic acid, pinastric acid and vulpinic acid in V. pinastri, and alectoronic acid in T. americana. In conclusion, D. arctica and V. pinastri are the most promising lichens to prevent and to treat oxidative stress-related neurodegenerative diseases.

Keywords: cetrarioid clade; lichens; neuroprotection; oxidative stress.

Figure 1

Effect of methanol lichen extracts of cetrarioid clade on cell viability. SH-SY5Y cells were treated with different concentrations of extracts from 5 to 50 μg/mL for 24 h. Cell viability was determined using MTT assay. Results are expressed as mean ± standard deviation (SD) (triplicate experiments). * p < 0.05 versus control.

Figure 2

(A) Effect of methanol lichen extracts of cetrarioid clade on cytoprotection in stress oxidative models. SH-SY5Y cells were pretreated with non-cytotoxic concentrations of lichens for 24 h before H₂O₂ (250 µM, 1 h). Cell viability was determined using MTT assay. Results were expressed as mean ± SD (triplicate experiments). * p < 0.01 versus control; # p < 0.01 versus H₂O₂. (B) SH-SY5Y cells morphology after treatments.

Figure 2

(A) Effect of methanol lichen extracts of cetrarioid clade on cytoprotection in stress oxidative models. SH-SY5Y cells were pretreated with non-cytotoxic concentrations of lichens for 24 h before H₂O₂ (250 µM, 1 h). Cell viability was determined using MTT assay. Results were expressed as mean ± SD (triplicate experiments). * p < 0.01 versus control; # p < 0.01 versus H₂O₂. (B) SH-SY5Y cells morphology after treatments.

Figure 3

Effect of methanol lichen extracts of cetrarioid clade on intracellular ROS production. SH-SY5Y cells were pretreated with D. arctica (D.a), N. stracheyi (N.s.), T. americana (T.a) and V. pinastri (V.p) for 24 h before H₂O₂ (250 µM, 1 h). The levels of intracellular ROS production were measured using dichlorodihydrofluorescein diacetate (DCFH-DA) method. Results are expressed as mean ± SD (triplicate experiments). * p < 0.01 versus control; # p < 0.01 versus H₂O₂.

Figure 4

Lichen extracts from cetrarioid clade improved oxidative stress markers and antioxidant enzyme activity. SH-SY5Y cells were pretreated with Dactylina arctica (D.a), Nephromopsis stracheyi (N.s), Tuckermannopsis americana (T.a) and Vulpicida pinastri (V.p) for 24 h before H₂O₂ (250 µM, 1 h). (A) Lipid peroxidation, (B) GSH levels and (C) SOD activity, (D) CAT activity. Results are expressed as mean ± SD (triplicate experiments). * p < 0.05 versus control; # p < 0.05 versus H₂O₂.

Figure 5

Effect of lichen extracts against H₂O₂ -induced mitochondrial dysfunction in SH-SY5Y. (A) on cytosolic calcium levels. (B) on mitochondrial calcium levels. (C) on mitochondrial membrane potential. Data are expressed as means ± SD (% of control) * p < 0.001 vs. control; # p < 0.001 vs. H₂O₂).

Figure 5

Effect of lichen extracts against H₂O₂ -induced mitochondrial dysfunction in SH-SY5Y. (A) on cytosolic calcium levels. (B) on mitochondrial calcium levels. (C) on mitochondrial membrane potential. Data are expressed as means ± SD (% of control) * p < 0.001 vs. control; # p < 0.001 vs. H₂O₂).

Figure 6

Representative HPLC chromatograms (λ = 254 nm) (A) Dactylina artica (B) Tuckermannopsis americana (C) Vulpicida pinastri.