Involvement of the ERK pathway in the protective effects of glycyrrhizic acid against the MPP+-induced apoptosis of dopaminergic neuronal cells

Abstract

Glycyrrhizic acid (GA), a major compound separated from Radix Glycyrrhizae, has been shwon to exert various biochemical effects, including neuroprotective effects. In the present study, we investigated the protective effects of GA against 1-methyl-4-phenylpyridinium (MPP+)‑induced damage to differentiated PC12 (DPC12) cells. Compared with the MPP+-treated cells, GA markedly improved cell viability, restored mitochondrial dysfunction, suppressed the overexpression of cleaved poly(ADP-ribose) polymerase (PARP), and suppressed the overproduction of lactate dehydrogenase (LDH) and intracellular Ca2+ overload. The protective effects of GA on cell survival were further confirmed in primary cortical neurons. GA markedly increased the expression of phosphorylated extracellular signal-regulated kinase (p-ERK), as well as its migration from the cytoplasm to nucleus. PD98059, an inhibitor of ERK, blocked GA-enhanced ERK activation and reduced cell viability. However, pre-treatment with GA had no effects on the expression of phosphorylated AKT (p-AKT) and total AKT (t-AKT). These results indicate that the GA-mediated neuroprotective effects are associated with its modulation of multiple anti-apoptotic and pro-apoptotic factors, particularly the ERK signaling pathway. This study provides evidence supporting the use of GA as a potential therapeutic agent for the treatment of neurodegenerative diseases and neuronal injury.

Figure 1

Effects of glycyrrhizic acid (GA) against 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurotoxicity in differentiated PC12 (DPC12) cells. Cells were pre-treated with 5–100 μM GA for 3 h, followed by exposure to 4 mM MPP⁺ for 24 h. Compared with the MPP⁺-treated cells, pre-treatment with GA (A) enhanced cell viability, (B) reduced the release of intracellular LDH and (C) decreased the apoptotic rate. Data are expressed as a percentage of the corresponding control cells and are presented as the means ± SD (n=3). ^#P<0.05, ^###P<0.001 vs. untreated cells; ^*P<0.05 and ^**P<0.01 vs. MPP⁺-exposed cells. CTRL, control.

Figure 2

Glycyrrhizic acid (GA) exerts protective effects on primary cortical neuronal cell survival against 1-methyl-4-phenylpyridinium (MPP⁺) neurotoxicity. (A) The purity of primary cortical neuronal cells was determined by β3-tubulin staining. Cells were pre-treated with 5–100 μM GA for 3 h, followed by exposure to 0.5 mM MPP⁺ for 24 h. Pre-treatment with GA (B) promoted cell viability and (C) prevented the morphological changes induced by MPP⁺. Data are expressed as a percentage of the corresponding control cells and are presented as the means±SD (n=6). ^###P<0.001 vs. untreated cells; ^**P<0.01 vs. MPP⁺-exposed cells. CTRL, control.

Figure 3

Effects of glycyrrhizic acid (GA) on (A) mitochondrial dysfunction (×20 magnification; scale bar, 100 μm) and (B) intracellular Ca²⁺ overload (×40 magnification; scale bar, 20 μm) induced by 1-methyl-4-phenylpyridinium (MPP⁺). Cells were pre-treated with 25 μM GA for 3 h, followed by exposure to 4 mM MPP⁺ for (A) 12 or (B) 3 h. Data are expressed as a percentage of the corresponding control cells and are presented as the means ± SD (n=3). ^###P<0.001 vs. untreated cells, ^**P<0.01 vs. MPP⁺-exposed cells. CTRL, control.

Figure 4

Glycyrrhizic acid (GA) suppressed the high expression of cleaved PARP caused by 1-methyl-4-phenylpyridinium (MPP⁺). Differentiated PC12 (DPC12) cells were pre-treated with GA for 3 h and then co-treated with 4 mM MPP⁺ for 24 h. Data are expressed as a percentage of the corresponding control cells and are presented as the means ± SD (n=3). ^#P<0.05 vs. untreated cells; ^*P<0.05 vs. MPP⁺-exposed cells. CTRL, control.

Figure 5

The extracellular signal-regulated kinase (ERK) signaling pathway contributes to the glycyrrhizic acid (GA)-mediated neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP⁺) neurotoxicity. (A) Differentiated PC12 (DPC12) cells were treated with 4 mM MPP⁺ and collected at 0, 10, 30, 60 and 180 min. (B) DPC12 cells were pre-treated with 25μM GA for 3 h, and then collected at 0, 10, 30, 60 and 180 min. (C) Following pre-treatment with 25 μM GA for 3 h, cells were collected at 0, 10, 30, 60 and 180 min after exposure to 4 mM MPP⁺. (D) Quantification data of the expression of phosphorylated ERK (p-ERK) were normalized to the corresponding values of total ERK (t-ERK) and expressed as a percentage of corresponding cells collected at 0 min. (E) GA enhanced the migration of p-ERK from the cytoplasm to the nucleus. DPC12 cells were pre-treated with or without 25 μM GA for 3 h and then co-treated with MPP⁺ for 1 h. Data are expressed as the means ± SD (n=3) and analyzed by one-way ANOVA. ^**P<0.01 and ^***P<0.001 vs. non-treated cells. CTRL, control.

Figure 6

AKT signaling pathway is not involved in the glycyrrhizic acid (GA)-mediated neuroprotective effects against 1-methyl-4-phenylpyridinium (MPP⁺). (A) Differentiated PC12 (DPC12) cells were collected at 0, 10, 30, 60 and 180 min after exposure to 4 mM MPP⁺. (B) DPC12 cells were pre-treated 25 μM GA for 3 h, and then collected at 0, 10, 30, 60 and 180 min. (C) Following pre-treatment with 25 μM GA for 3 h, cells were collected at 0, 10, 30, 60 and 180 min after exposure to 4 mM MPP⁺. (D) Quantification data of the expression of phosphorylated AKT (p-AKT) were normalized to the corresponding values of total AKT (t-AKT) and expressed as a percentage of corresponding cells collected at 0 min. Data are the means ± SD of 3 replicate values in 3 separate experiments and analyzed by one-way ANOVA.

Figure 7

Promoting effects of glycyrrhizic acid (GA) on (A) the phosphorylaiton of extracellular signal-regulated kinase (ERK) and (B) cell viability were blocked by PD98059 (ERK inhibitor). Cells were pre-treated with or without 10 μM PD98059 for 30 min, and then exposed to 25 μM GA for 3 h and incubated with 4 mM 1-methyl-4-phenylpyridinium (MPP⁺) for (A) 1 or (B) 24 h. Data are expressed as the means ± SD (n=3) and analyzed using one-way ANOVA. ^###P<0.001 vs. control group; ^**P<0.01 vs. MPP⁺-treated group; ^$$P<0.01 vs. GA-treated group. CTRL, control.