Protopanaxadiols Eliminate Behavioral Impairments and Mitochondrial Dysfunction in Parkinson's Disease Mice Model

Abstract

Currently, there are no effective therapies to cure Parkinson's disease (PD), which is the second most common neurodegenerative disease primarily characterized by motor dysfunction and degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Protopanaxadiols (PPDs), including 20 (R)- protopanaxadiol (R-PPD) and 20 (S)- protopanaxadiol (S-PPD), are main metabolites of ginsenosides. The role of ginsenosides in neurodegenerative diseases has been thoroughly studied, however, it is unknown whether PPDs can attenuate behavioral deficits and dopaminergic neuron injury in PD model mice to date. Here, we administered PPDs to MPTP-induced PD model mice and monitored the effects on behavior and dopaminergic neurons to investigate the effects of R-PPD and S-PPD against PD. Our results showed that R-PPD and S-PPD (at a dose of 20 mg/kg, i.g.) treatment alleviated MPTP (30 mg/kg, i.p.) induced behavioral deficits. Besides, R-PPD and S-PPD protected MPP⁺-induced neuron injury and mitochondrial dysfunction, and reduced the abnormal expression of Cyt C, Bax, caspase-3 and Bcl-2. These findings demonstrate that R-PPD and S-PPD were potentially useful to ameliorate PD.

Keywords: Behavioral impairments; Mitochondrial dysfunction; Parkinson’s disease; Protopanaxadiols.

Fig. 1

PPDs improve motor behavior and the reduction of TH protein in MPTP-treated mice. Behavioral parameters were measured on D 7 (n = 9) and D 14 to assess motor function in different treatment groups (n = 6). A Timeline of the experimental procedure in the MPTP-induced mouse PD model. B and C Day 7 open field test. D and E Day 7 pole test. F and G TH expression on Day 7 (n = 3). H and I Day 14 open field test. J and K Day 14 pole test. L and M TH expression on Day14 (n = 3). Quantified data are normalized to the control group (the control group value is equal to 1). Data are expressed as means ± S.E.M, (n = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ^#P < 0.05, ^##P < 0.01 represents control vs. MPTP group, *P < 0.05, **P < 0.01 represents MPTP vs. MPTP + protopanaxadiols or L-DOPA

Fig. 2

The effects of PPDs on MPP⁺-induced neuronal injury and mitochondrial deficits. A R-PPD. B S-PPD. A and B Primary neurons were pretreated with R-PPD and S-PPD for 2 h, followed by MPP⁺ treatment for 24 h, and the cell viability was measured by CCK8. C Mitochondrial membrane potential was measured by JC-1. In healthy cells, JC-1 monomers aggregate to form polymers, and mitochondria exhibit intense red fluorescence. JC-1 is present as a monomer in apoptotic or necrotic cells, and mitochondria are strongly green fluorescence. Scale bars, 25 μm. D The absorbance of JC-1 was detected by fluorescent microplate reader. The ratio of red fluorescence signal to green fluorescence signal was calculated to judge the health of mitochondrial membrane potential. E The DCFH-DA method measures intracellular reactive oxygen species (ROS) accumulation and is monitored by fluorescence microscopy. Scale bars, 200 μm. F ROS fluorescence intensity was analyzed and quantified. Quantified data are normalized to the control group (the control group value is equal to 1). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ^#P < 0.05, ^##P < 0.01 represents control vs. MPP⁺ group, *P < 0.05, **P < 0.01 represents MPP⁺ vs. MPP⁺  + protopanaxadiols

Fig. 3

Effect of PPDs on MPTP-induced mitochondria-mediated apoptosis proteins. A–E The expressions of Bcl-2, Bax, caspase-3 and Cyt C were detected by western blot on Day7. F–J The expressions of Bcl-2, Bax, caspase-3 and Cyt C were detected by western blot on Day14. Quantified data are normalized to the control group (the control group value is equal to 1). Data are expressed as means ± S.E.M, (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ^#P < 0.05, ^##P < 0.01 represents control vs. MPTP group, *P < 0.05, **P < 0.01 represents MPTP vs. MPTP + protopanaxadiols or Levodopa

Fig. 4

Effect of PPDs on MPP⁺-induced mitochondria-mediated apoptosis proteins. A–E The expression of apoptosis proteins Bcl-2, caspase-3, Bax and Cyt C in primary neurons was detected by western blot. Quantified data are normalized to the control group (the control group value is equal to 1). Data are expressed as means ± S.E.M, (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc analysis where ^#P < 0.05, ^##P < 0.01 represents control vs. MPP⁺ group, *P < 0.05, **P < 0.01 represents MPP⁺ vs. MPP⁺  + protopanaxadiols