Ganoderma Lucidum polysaccharides protect against MPP(+) and rotenone-induced apoptosis in primary dopaminergic cell cultures through inhibiting oxidative stress

Abstract

Oxidative stress plays a pivotal role in the progressive neurodegeneration in Parkinson's disease (PD) which is responsible for disabling motor abnormalities in more than 6.5 million people worldwide. Polysaccharides are the main active constituents from Ganoderma lucidum which is characterized with anti-oxidant, antitumor and immunostimulant properties. In the present study, primary dopaminergic cell cultures prepared from embryonic mouse mesencephala were used to investigate the neuroprotective effects and the potential mechanisms of Ganoderma lucidum polysaccharides (GLP) on the degeneration of dopaminergic neurons induced by the neurotoxins methyl-4-phenylpyridine (MPP(+)) and rotenone. Results revealed that GLP can protect dopamine neurons against MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in primary mesencephalic cultures in a dose-dependent manner. Interestingly, either with or without neurotoxin treatment, GLP treatment elevated the survival of THir neurons, and increased the length of neurites of dopaminergic neurons. The Trolox equivalent anti-oxidant capacity (TEAC) of GLP was determined to be 199.53 μmol Trolox/g extract, and the decrease of mitochondrial complex I activity induced by MPP(+) and rotenone was elevated by GLP treatment (100, 50, 25 and 12.5 μg/ml) in a dose dependent manner. Furthermore, GLP dramatically decreased the relative number of apoptotic cells and increased the declining mitochondrial membrane potential (ΔΨm) induced by MPP(+) and rotenone in a dose-dependent manner. In addition, GLP treatment reduced the ROS formation induced by MPP(+) and rotenone at the concentrations of 100, 50 and 25 μg/ml in a dose-dependent manner. Our study indicates that GLP possesses neuroprotective properties against MPP(+) and rotenone neurotoxicity through suppressing oxidative stress in primary mesencephalic dopaminergic cell culture owning to its antioxidant activities.

Keywords: Ganoderma Lucidum polysaccharides; MPP+; Parkinson’s disease; oxidative stress; rotenone.

Figure 1

Neuroprotective effect of GLP on the MPP⁺ and rotenone-induced cytotoxicity in primary mesencephalic dopaminergic cell cultures. Values were expressed as mean ± SEM with 3 wells in each group. In each well 10 randomly selected fields were analyzed. A. The mean relative survival rate of THir neurons induced by MPP⁺. B. The mean relative survival rate of THir neurons induced by rotenone. C. Microscope images of THir neurons stained with DAB in each group. D. The average length of neuritis growth of THir neurons induced by MPP⁺. E. The average length of neuritis growth of THir neurons induced by rotenone.**p < 0.01, *p < 0.05 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.

Figure 2

Effect of GLP on mitochondrial complex I activity of primary dopaminergic cell cultures treated with MPP⁺ and rotenone. Values were expressed as mean mitochondrial complex I activity with 4 samples in each group (n = 4). **p < 0.01 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.

Figure 3

Effect of GLP on necrosis of primary dopaminergic cell cultures treated with MPP⁺ and rotenone. A. Fluorescence images of primary dopaminergic cells stained with PI fluorescent at 488 nm in MPP⁺ treatment groups. B. GLP treatment reduced number of PI-positive cells in a dose-dependent manner. Values were expressed as relative proportion of PI-positive cells with 4 wells in each group. In each well 4 randomly selected fields were analyzed. **p < 0.01 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.

Figure 4

Effect of GLP on apoptosis of primary dopaminergic cell cultures treated with MPP⁺ and rotenone. A. Fluorescence images of primary dopaminergic cells stained with DAPI fluorescent at 488 nm in MPP⁺ treatment groups. Nuclei of apoptotic cells showed the features of condensed and fragmented chromatin. B. GLP treatment reduced relative number of apoptotic cells in a dose-dependent manner. Values were expressed as relative proportion of apoptotic cells with 4 wells in each group. In each well 4 randomly selected fields were analyzed. **p < 0.01 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.

Figure 5

Effect of GLP on mitochondrial membrane potential of primary dopaminergic cell cultures treated with MPP⁺ and rotenone. A. Fluorescence images of primary dopaminergic cells were merged by images taken with 488 nm and 568 nm excitation for green and red JC-1 fluorescence staining respectively in MPP⁺ treatment groups. B. GLP treatment reversed depolarized membrane potential induced by MPP⁺ and rotenone in a dose-dependent manner. Values were expressed as relative mitochondrial membrane potential with 4 wells in each group. In each well 4 randomly selected fields were analyzed. **p < 0.01 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.

Figure 6

Effect of GLP on ROS formation of primary dopaminergic cell cultures treated with MPP⁺ and rotenone. A. Fluorescence images of primary dopaminergic cells stained with C-DCDHF-DA fluorescent at 488 nm in MPP⁺ treatment groups. B. GLP treatment decreased relative ROS formation in a dose-dependent manner. Values were expressed as relative ROS fluorescence intensity with 4 wells in each group. In each well 4 randomly selected fields were analyzed. **p < 0.01 compared to cell control group, ##p < 0.01, #p < 0.05 compared to neurotoxin (MPP⁺ or rotenone) control group.