Velvet Antler Methanol Extracts Ameliorate Parkinson's Disease by Inhibiting Oxidative Stress and Neuroinflammation: From C. elegans to Mice

Abstract

Velvet antler is the traditional tonic food or medicine used in East Asia for treating aging-related diseases. Herein, we try to dissect the pharmacology of methanol extracts (MEs) of velvet antler on Parkinson's disease (PD). Caenorhabditis elegans studies showed that MEs decreased the aggregation of α-synuclein and protected oxidative stress-induced DAergic neuron degeneration. In vitro cellular data indicated that MEs suppressed the LPS-induced MAPKs and NF-κB activation, therefore inhibiting overproduction of reactive oxygen species, nitric oxide, tumor necrosis factor-α, and interleukin-6; blocking microglia activation; and protecting DAergic neurons from the microglia-mediated neurotoxicity. In vivo MPTP-induced PD mouse investigations found that MEs prevented MPTP-induced neuron loss in the substantia nigra and improved the behavioral rotating rod performance in MPTP-treated mice by increasing the expression level of tyrosine hydroxylase (TH) and downregulating α-synuclein protein expression. In all, these results demonstrate that MEs ameliorate PD by inhibiting oxidative stress and neuroinflammation.

Figure 1

MEs decreased the aggregation and toxicity of α-synuclein in NL5901 worms. (a) The survival curve of NL5901 worms treated with MEs. (b) The endpoint microscope fluorescence image of α-synuclein aggregates in NL5901 worms treated with or without MEs on days 3 and 5 past the adult stage. (c) The quantitative analysis of the α-synuclein aggregates in NL5901 worms shown in (b). All the worms in the experiment were synchronized to the young adult stage and subsequently started to be exposed to 100 μg/mL MEs. Error bars represented the SEM of three independent replicates of total worms.

Figure 2

MEs protected against neuron injury induced by 6-OHDA in C. elegans. (a) The endpoint microscope fluorescence image of DAergic neurons in BZ555 worms treated with or without MEs on days 1, 2, and 3 past the adult stage. The BZ555 worms synchronized at the L4 larval stage were exposed to 6-OHDA and subsequently started to be exposed to 100 μg/mL MEs for 1-3 days. (b) The quantitative analysis of the intensity of DAergic neurons shown in (a). (c) The difference of the average number of body bends per 20 s between N2 worms in OP50 seeded plates and plates without food. N2 worms synchronized at the L4 larval stage were exposed to 6-OHDA and subsequently started to be exposed to 100 μg/mL MEs for 1-3 days. Data were expressed as the mean ± SEM.

Figure 3

MEs inhibit ROS generation in the activated microglia. (a) The endpoint microscope fluorescence image of intracellular ROS of BV2 cells. Intracellular ROS generation was measured by fluorescence staining with H₂DCF-DA. LPS (200 ng/mL)-activated BV2 cells were treated with MEs (20 and 40 μg/mL), scale bar = 100 μm. The quantitative analysis of the fluorescence intensity of DCF shown in (a). (b) TUNEL staining. LPS (200 ng/mL)-activated BV2 cells were treated with MEs (20 and 40 μg/mL) for 24 h. DNA fragmentation was detected by TUNEL assay. Scale bar = 100 μm.

Figure 4

MEs inhibit proinflammatory factors by suppressing MAPKs and NF-κB activation in the activated microglia. (a–c) MEs inhibited LPS-induced NO (a), TNF-α (b), and IL-6 (c). BV2 cells were treated with LPS (200 ng/mL) and indicated concentrations of MEs. After 24 h treatment, NO, TNF-α, and IL-6 in the supernatant were measured; data are expressed as mean ± SD. (d) The endpoint microscope immunofluorescence image of Iba-1 in BV2 cells. BV2 cells were treated with LPS (200 ng/mL) and indicated concentration of MEs for 24 h. Iba-1 immunofluorescence staining was subsequently performed. Scale bar = 100 μm. (e, f) Effect of MEs on the LPS-induced phosphorylation of p65, ERK, JNK, and p38. BV2 cells were treated with LPS (200 ng/mL) and indicated concentrations of MEs for 1.5 h. Cell lysates were prepared, and the protein samples were analyzed by western blot analysis. TAK-242 (1 μM), a classic TLR4 antagonist, was used as the control. Results are representative of those obtained from three independent experiments.

Figure 5

MEs improve parkinsonism in MPTP-treated mice. (a) Experimental timeline for the construction of the MPTP-induced PD mouse model and the administration of MEs. C57BL/6 mice (male, 7-8-week-old) were injected MPTP intraperitoneally at 30 mg/kg/day for five days, and MEs (30 mg/kg/day) were injected intraperitoneally for 5 days since the administration of MPTP. On day 6, the rotarod test was performed. On day 7, mice were sacrificed and tissues were prepared for immunohistochemical (IHC) and western blotting. (b) Rotarod behavioral performance of MPTP-induced PD mice after ME treatment. Data are presented as the mean ± SD (n = 6). (c) Immunohistochemistry for tyrosine hydroxylase (TH) in the substantia nigra (scale bar = 100 μm) and striatum (scale bar = 1000 μm). (d) Expression of α-syn by western blot analysis. The blots were reprobed to detect GAPDH as the internal control. (e) Effect of MEs on the LPS-induced phosphorylation of Akt, p65, and p38 in the striatum. The protein in obtained tissue was analyzed and quantified by western blotting. (f) Effect of MEs on the nuclear translocation of NF-κB in substantia nigra (scale bar = 100 μm). C57BL/6 mice (male, 7-8-week-old) were injected MPTP intraperitoneally at 30 mg/kg/day for five days, and MEs (30 mg/kg) were injected intraperitoneally for 5 days since the administration of MPTP. On day 7, mice were sacrificed and substantia nigra tissues were collected, and immunofluorescence was performed.

Figure 6

Acute toxicity of MEs in different organs. C57BL/6 mice (male, 7-8-week-old) were treated with indicated concentrations of MEs for 7 days. Mice were sacrificed, and different organs (heart, liver, spleen, lung, and kidney) were collected for hematoxylin and eosin (H&E) staining. Scale bar = 100 μm.