Ganoderma lucidum Protects Dopaminergic Neuron Degeneration through Inhibition of Microglial Activation

Abstract

Abundant evidence has suggested that neuroinflammation participates in the pathogenesis of Parkinson's disease (PD). The emerging evidence has supported that microglia may play key roles in the progressive neurodegeneration in PD and might be a promising therapeutic target. Ganoderma lucidum (GL), a traditional Chinese medicinal herb, has been shown potential neuroprotective effects in our clinical trials that make us to speculate that it might possess potent anti-inflammatory and immunomodulating properties. To test this hypothesis, we investigated the potential neuroprotective effect of GL and possible underlying mechanism of action through protecting microglial activation using co-cultures of dopaminergic neurons and microglia. The microglia is activated by LPS and MPP(+)-treated MES 23.5 cell membranes. Meanwhile, GL extracts significantly prevent the production of microglia-derived proinflammatory and cytotoxic factors [nitric oxide, tumor necrosis factor-α (TNF-α), interlukin 1β (IL-1β)] in a dose-dependent manner and down-regulate the TNF-α and IL-1β expressions on mRNA level as well. In conclusion, our results support that GL may be a promising agent for the treatment of PD through anti-inflammation.

Figure 1

The morphology of rat microglia cells labeled with OX-42. Rat microglia were incubated for 24 h with vehicle ((a) 100x; (b) 400x), LPS 0.25 μg/ml ((c) 100x; (d) 400x). Microglia were transformed into an amoeboid morphology after treated with LPS. Scale bar represents 100 μm.

Figure 2

LPS and CF increase cytokines through activating microglia. Microglial activation was determined by measuring the levels of TNF-α, IL-1β, NO and superoxide in cells after exposure to LPS (0.25 μg/ml) and CF (150 μg/ml). The levels of TNF-α, IL-1β, NO and superoxide in vehicle controls are 106.55 pg/ml, 119.09 pg/ml, 0.6 μM and 5.22 U/ml, respectively. Levels were expressed as fold increase as compared to concentrations of the control. All cytokines were increased significantly (*P < .05).

Figure 3

GL protects against LPS or CF induced production of NO in a dose-dependent fashion. Cultures were treated with GL at indicated concentration 30 min prior to exposure with 0.25 μg/ml LPS or 150 μg/ml CF. Culture supernatants were collected and assayed for NO. Data are expressed as fold increase of control group and presented as means ± SD of two experiments performed in triplicate. *P < .01 compared with LPS only treated cultures and **P < .05 compared with CF only treated cultures.

Figure 5

GL protects against LPS or CF induced production of superoxide in a dose-dependent fashion. Cultures were treated with GL at indicated concentration 30 min prior to exposure with 0.25 μg/ml LPS or 150 μg/ml CF. Superoxide generation was measured with the SOD assay kit-WST. Data are expressed as fold increase of control group and presented as means ± SD of two experiments performed in triplicate. *P < .001 compared with LPS only treated cultures and **P < .05 compared with CF only treated cultures.

Figure 6

GL protects against LPS or CF induced production of TNF-α (a) and IL-1β (b) in a dose-dependent fashion. Cultures were treated with GL at indicated concentration 30 min prior to exposure with 0.25 μg/ml LPS or 150 μg/ml CF. TNF-α and IL-1β levels were determined as described in Methods section. Data are expressed as means ± SD of two experiments performed in triplicate. *P < .05 and **P < .001 compared with LPS and CF only treated cultures for TNF-α. ^# P < .001 and ^## P < .001 compared with LPS and CF only treated cultures for IL-1β, respectively.

Figure 7

GL protects against MPP⁺-induced reduction of [³H] DA uptake in MES 23.5 cell cultures with or without microglia. The cultures were treated with vehicle or 100 μM MPP⁺, and 400 μg/ml GL. GL was given 30 min before the MPP⁺ exposure. Uptake of [³H] DA was assessed as described in Methods section. Data were generated from cell samples in duplicated experiments, and are expressed as percent of the vehicle group. *P < .001 compared with corresponding MES 23.5 in the absence or presence of microglia cultures without exposure to MPP⁺; **P < .05 compared with the MPP⁺-treated MES 23.5 cultures without microglia; ^# P < .001 compared with corresponding MES23.5 in the absence or presence of microglia cultures without exposure to GL.

Figure 4

GL protects against LPS-induced reduction of [³H] DA uptake in MES 23.5 cell cultures with or without microglia. The cultures were treated with vehicle or 0.25 μg/ml LPS, and 400 μg/ml GL. GL was given 30 min before the LPS exposure. Uptake of [³H] DA was assessed as described in Methods section. Data were generated from cell samples in duplicated experiments, and are expressed as percent of the vehicle group. *P < .05 compared with corresponding MES 23.5 in the absence or presence of microglia cultures without exposure to LPS; ^# P < .01 compared with corresponding MES 23.5 and microglia co-cultures without exposure to GL.

Figure 8

GL protects against LPS or CF induced overexpression of mRNA levels of TNF-α (a) and IL-1β (b) in a dose-dependent fashion. Cultures were treated with GL at indicated concentration 30 min prior to exposure with 0.25 μg/ml LPS or 150 μg/ml CF. Total RNA was extracted and then subjected to real-time PCR as described in Methods section. Data are expressed as percentage of the control group (LPS or CF only treated group, respectively) calculated from the average threshold cycle values and presented as the mean ± SD. Independent RNA preparations from different sets of cultures were prepared and determinations were performed in triplicate from the RNA samples of a set of experiment. *P < .05 compared with LPS or CF only treated cultures, respectively.

Figure 9

The molecular mechanisms between microglial activation and neuron death. Microglia can be activated by inflammatory trigger, such as LPS and other toxins and consequently produce proinflammatory factors and cytokines which on one side can cause auto-implication of ROS, NO and superoxide radicals to form highly oxidizing peroxynitrite species and also activate other resting microglia. TNF-dependent microglia activation in the SN creates an environment of oxidative stress through activation of NADPH oxidase. IL-1β can disrupt the blood brain barrier and facilitate the infiltration of leukocytes into CNS. All these factors can activate NF-κB, which can up-regulate pro-apoptotic genes leading to neuronal death. These events form a vicious circle leading to progressive neuronal degeneration. GL can inhibit the generation of microglia-derived toxic factors through its anti-inflammatory effect.