Multitarget effects of Korean Red Ginseng in animal model of Parkinson's disease: antiapoptosis, antioxidant, antiinflammation, and maintenance of blood-brain barrier integrity

Abstract

Background: Ginsenosides are the main ingredients of Korean Red Ginseng. They have extensively been studied for their beneficial value in neurodegenerative diseases such as Parkinson's disease (PD). However, the multitarget effects of Korean Red Ginseng extract (KRGE) with various components are unclear.

Methods: We investigated the multitarget activities of KRGE on neurological dysfunction and neurotoxicity in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. KRGE (37.5 mg/kg/day, 75 mg/kg/day, or 150 mg/kg/day, per os (p.o.)) was given daily before or after MPTP intoxication.

Results: Pretreatment with 150 mg/kg/day KRGE produced the greatest positive effect on motor dysfunction as assessed using rotarod, pole, and nesting tests, and on the survival rate. KRGE displayed a wide therapeutic time window. These effects were related to reductions in the loss of tyrosine hydroxylase-immunoreactive dopaminergic neurons, apoptosis, microglial activation, and activation of inflammatory factors in the substantia nigra pars compacta and/or striatum after MPTP intoxication. In addition, pretreatment with KRGE activated the nuclear factor erythroid 2-related factor 2 pathways and inhibited phosphorylation of the mitogen-activated protein kinases and nuclear factor-kappa B signaling pathways, as well as blocked the alteration of blood-brain barrier integrity.

Conclusion: These results suggest that KRGE may effectively reduce MPTP-induced neurotoxicity with a wide therapeutic time window through multitarget effects including antiapoptosis, antiinflammation, antioxidant, and maintenance of blood-brain barrier integrity. KRGE has potential as a multitarget drug or functional food for safe preventive and therapeutic strategies for PD.

Keywords: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Korean Red Ginseng extract; Multitarget effect.

Fig. 1

KRGE attenuates MPTP-induced locomotor (neurological) impairment. To determine the most effective dose, mice were orally treated with KRGE (37.5 mg/kg, 75 mg/kg, and 150 mg/kg) once daily from 1 h before the first MPTP intoxication (20 mg/kg, every 2 h × 4). (A) Pole test 7 days post MPTP. (B) Performance on the rotarod test 3 days post MPTP. (C) Nest-building behavior 1 day post MPTP scored after 18 h in the cage with a square of cotton Nestlet. (D) Survival rate scored 7 days post MPTP. To investigate its therapeutic time window, mice were orally treated with KRGE (150 mg/kg) once daily from 1 h before the first MPTP intoxication (pretreatment), and 24 h (post 24h), 72 h (post 72h), and 120 h (post 120h) after the last MPTP intoxication. (E) Pole test.(F) Performance on the rotarod test. (G) Nest-building behavior test. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 and **p < 0.01 versus MPTP group. ANOVA, analysis of variance; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 2

KRGE attenuates MPTP-induced nigrostriatal dopaminergic neuronal damage. Brain sections were prepared 7 days after MPTP intoxication. (A–F) The prepared brain sections were stained with cresyl violet. (G–R) The brain sections were immunostained with TH antibody.(S–U) Quantification. (S) Quantification was carried out by measuring the density of cresyl violet–stained cells. (T) Quantification was carried out by counting the number of TH-immunoreactive cells of the SNpc or striatum.(U) Quantification was carried out by measuring the density of TH immunoreactivity. The SNpc and striatum were sampled 7 days after MPTP intoxication, accomplished by Western blot analysis using TH antibody, and quantified. (V) SNpc. (W) Striatum. The densities were displayed as relative value to that from sham group. Scale bar = 100 μm. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 versus MPTP group. ANOVA, analysis of variance; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 3

KRGE attenuates apoptosis in the SNpc and striatum after MPTP intoxication. (A–H) Brain sections from 7 days after MPTP intoxication were stained using a TUNEL kit. The number of apoptotic cells was manually counted. (I) In SNpc. (J) In striatum. (K–N) The brain was sampled 7 days after MPTP intoxication; immunoblot was performed using cleaved caspase-3 and p-protein kinase B (AKT) antibodies and quantified. The band of GAPDH in graph M and N was shared with graphs K and L. Scale bar = 50 μm. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 and **p < 0.01 versus MPTP group. ANOVA, analysis of variance; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, substantia nigra pars compacta.

Fig. 4

KRGE attenuates the activation of microglia and inflammatory mediators in the SNpc and striatum after MPTP intoxication. Brain sections obtained 7 days after MPTP intoxication were immunostained using Iba-1 antibody. (A–D) SNpc. (E–H) Striata. SNpc and striata from 7 days after MPTP intoxication were analyzed. (I and J) By Western blot. (K–T) By RT-PCR. The band of GAPDH in graphs O and T was shared with graphs K–N and P–S, respectively. CD11b (I and J), IL-1β (K and P), IL-6 (L and Q), TNF-α (M and R), iNOS (N and S), and COX-2 (O and T). Scale bar = 50 μm. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 and **p < 0.01 versus MPTP group. ANOVA, analysis of variance; COX, cyclooxygenase; IL, interleukin; iNOS, inducible nitric oxide; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; RT-PCR, reverse transcription polymerase chain reaction; SNpc, substantia nigra pars compacta; TNF, tumor necrosis factor.

Fig. 5

KRGE contributes to the maintenance of the BBB after MPTP intoxication. (A–D) SNpc sections from 7 days after MPTP intoxication were immunostained using GFAP antibody. (E–H) Striatal sections from 7 days after MPTP intoxication were immunostained using GFAP antibody. SNpc and striata were analyzed and quantified. (I and J) Western blot. (K–N) Real-time PCR. (K) VCAM-1. (L) ICAM-1. (M) ZO-1. (N) Claudin-5. Scale bar = 100 μm. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 and **p < 0.01 versus MPTP group. ANOVA, analysis of variance; BBB, blood–brain barrier; GFAP, glial fibrillary acidic protein; ICAM, intercellular adhesion molecule; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PCR, polymerase chain reaction; SNpc, substantia nigra pars compacta; VCAM, vascular cell adhesion molecule; ZO-1, zona occludens-1.

Fig. 6

KRGE activates Nrf2 pathway and inhibits MAPKs and NFκB signaling pathways after MPTP intoxication. Striata from 7 days after MPTP intoxication were used for Western blot. (A) Nrf2. (E) p-ERK. (F) p-JNK. (G) p-p38. (H) p-NF-κB. (I) p-IκB. The band of GAPDH in graph I was shared with graphs E–H. Striata from 7 days after MPTP intoxication were used for real-time PCR analysis. (B) HO-1. (C) NQO1 (D) GCLs. ANOVA test; #p < 0.05 and ##p < 0.01 versus Sham group. *p < 0.05 versus MPTP group. ANOVA, analysis of variance; ERK, extracellular signal–regulated kinase; GCLs, γ-glutamate cysteine ligase regulatory subunit; JNK, Jun N-terminal kinase; KRGE, Korean Red Ginseng extract; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Nrf2, nuclear factor erythroid 2–related factor 2; NQO1, NAD(P)H quinone oxidoreductase 1; PCR, polymerase chain reaction.