Dangguijakyak-San Protects against 1-Methyl-4-phenyl-1,2,3,6,-tetrahydropyridine-Induced Neuronal Damage via Anti-Inflammatory Action

Abstract

Dangguijakyak-san (DJS), a famous traditional Korean multiherbal medicine, has been used to treat gynecological and neuro-associated disease. Recent studies demonstrated that DJS has multiple bioactivities including neuroprotection. In the present study, we were to investigate the effect of DJS and its mechanism in an in vitro and in vivo model of Parkinson's disease (PD). In primary mesencephalic culture system, DJS attenuated the dopaminergic cell damage induced by 1-methyl-4-phenylpyridine toxicity, and it inhibited production of inflammatory factors such as tumor necrosis factor α (TNF- α ), nitric oxide (NO), and activation of microglial cells. Then, we confirmed the effect of DJS in a mouse PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the pole test, DJS at 50 mg/kg/day for 5 days showed increase of motor activity showing shortened time to turn and locomotor activity compared with the MPTP only treated mice. In addition, DJS significantly protected nigrostriatal dopaminergic neuron from MPTP stress. Moreover, DJS showed inhibition of gliosis in the substantia nigra pars compacta. These results have therapeutic implications for DJS in the treatment of PD via anti-inflammatory effects.

Figure 1

Protective effect of DJS against MPP+ neurotoxicity in primary mesencephalic cells. After cells were treated with DJS and 15 μM MPP+, dopaminergic neurons were stained with a TH antibody. The number of TH-positive cells was counted (a) and the representative images were shown (b). Scale bar = 100 μm. Each column represents the mean ± SEM from four replications. Data are expressed as percentages relative to the controls. ^### P < 0.001 significantly different from the control group. ***P < 0.001 significantly different from the MPP+ only treated group.

Figure 2

Inhibitory effect of DJS on MPP+-induced gliosis in primary mesencephalic cells. After cells were treated with DJS and 15 μM MPP+, microglia cells were stained with a Mac-1 antibody. The number of Mac-1-positive cells was counted (a), and the representative images were shown (b). Scale bar = 200 μm. Each column represents the mean ± SEM from four replications. Data are expressed as percentages relative to the controls. ^### P < 0.001 significantly different from the control group. *P < 0.05 significantly different from the MPP+ only treated group.

Figure 3

Inhibitory effects of DJS on MPP+-induced NO and TNF-α productions in primary mesencephalic cells. After cells were treated with DJS and 15 μM MPP+, the supernatants were activated with Griess reagent and rat TNF-α ELISA kit. The nitrite contents (a) and TNF-α level (b) were determined. Each column represents the mean ± SEM from four replications. Data are expressed as percentages relative to the controls. ^### P < 0.001 significantly different from the control group. ***P < 0.001, **P < 0.01, and *P < 0.05 significantly different from the MPP+ only treated group.

Figure 4

Inhibitory effect of DJS on MPTP-induced bradykinesia in a mouse model of PD. DJS at 50 mg/kg/day for 5 days was administered, and MPTP at 20 mg/kg was injected four times at 2 h interval on the day of last DJS treatment. On the first day of MPTP injection, the pole test was performed. The times to turn (T-LA) and the times to completely downward and to arrive at the floor (b) were recorded. Each column represents the mean ± SEM from ten replications. Data are expressed as percentages relative to the controls. ^### P < 0.001 significantly different from the control group. ***P < 0.001, *P < 0.05 significantly different from the MPTP only treated group.

Figure 5

Protective effect of DJS on MPTP-induced dopaminergic neuronal damage in a mouse model of PD. DJS at 50 mg/kg/day for 5 days was administered, and MPTP at 20 mg/kg was injected four times at 2 h interval on the day of last DJS treatment. On the seventh day of MPTP injection, dopaminergic neurons and fibers in the SNpc and the ST, respectively, were stained with a TH antibody. The number of TH-positive cell bodies was counted (a), and the optical density of TH-positive fibers was measured (b). The representative images were shown (c). Scale bar = 100 μm. Each column represents the mean ± SEM from six replications. Data are expressed as percentages relative to the controls. ^### P < 0.001 significantly different from the control group. ***P < 0.001, *P < 0.05 significantly different from the MPTP only treated group.

Figure 6

Inhibitory effect of DJS on MPTP-induced gliosis in a mouse model of PD. DJS at 50 mg/kg/day for 5 days was administered, and MPTP at 20 mg/kg was injected four times at 2 h interval on the day of last DJS treatment. On the first day of MPTP injection, microglia and astrocyte in the SNpc were stained with Mac-1 and GFAP antibodies, respectively, and the photographs were shown.