Smilagenin Protects Dopaminergic Neurons in Chronic MPTP/Probenecid-Lesioned Parkinson's Disease Models

Abstract

Current therapies for Parkinson's disease (PD) only offer limited symptomatic alleviation but fail to hamper the progress of the disease. Thus, it is imperative to establish new approaches aiming at protecting or reversing neurodegeneration in PD. Recent work elucidates whether smilagenin (abbreviated SMI), a steroidal sapogenin from traditional Chinese medicinal herbs, can take neuroprotective effect on dopaminergic neurons in a chronic model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) conjuncted with probenecid mice. We reported for the first time that SMI significantly improved the locomotor ability of chronic MPTP/probenecid-lesioned mice. SMI increased the tyrosine hydroxylase (TH) positive and Nissl positive neuron number in the substantia nigra pars compacta (SNpc), augmented striatal DA and its metabolites concentration and elevated striatal dopamine transporter density (DAT). In addition, dopamine receptor D2R not D1R was down-regulated by MPTP/probenecid and slightly raised by SMI prevention. What's more, we discovered that SMI markedly elevated striatal glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) protein levels in SMI prevented mice. And we found that SMI increased GDNF and BDNF mRNA level by promoting CREB phosphorylation in 1-methyl-4-phenylpyridimium (MPP⁺) treated SH-SY5Y cells. The results illustrated that SMI could prevent the impairment of dopaminergic neurons in chronic MPTP/probenecid-induced mouse model.

Keywords: Chinese herb; Parkinson’s disease; brain-derived neurotrophic factor; dopaminergic neurons; glial cell line-derived neurotrophic factor.

Figure 1

Time schedule of experiments in vivo.

Figure 2

Effects of smilagenin (SMI) on behavioral test of chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/probenecid-lesioned mice. (A) Time on the rod vs. rotation speed, each point was the mean ± SEM of 3 days consecutive tests. (B) Statistical comparisons of area under the curve (AUC) values between groups. (C) Total distance moved, measured as centimeters moved in 5 min, data are expressed as mean ± SEM, n = 10. *P < 0.05 and ***P < 0.001 respectively as compared with untreated model group, respectively.

Figure 3

Effect of SMI on tyrosine hydroxylase (TH)-positive and Nissl positive neuron number in chronic MPTP/probenecid-lesioned mice. Panel (A) shows the representative slices of the control mice, model mice treated with vehicle, 10 mg/kg/day, and 26 mg/kg/day SMI successively. (×100, scale bar = 100 μm). Panel (B) shows the statistical results. Panel (C) shows the representative images of Nissl-stained neurons in the substantia nigra pars compacta (SNpc) of the control mice, model mice treated with vehicle, 10 mg/kg/d, and 26 mg/kg/day SMI successively (×100, scale bar = 100 μm). Panel (D) shows quantification of Nissl-stained neurons. Number of nigral TH-positive neuron and Nissl-stained neurons in each mouse was quantified at ×100 magnification and then normalized to its corresponding control. Data are expressed as mean ± SEM, n = 3. **P < 0.01 and ***P < 0.001, respectively as compared with untreated model group, respectively.

Figure 4

Effect of SMI on striatal DA and its metabolites concentration in chronic MPTP/probenecid-lesioned mice. Panels (A–C) show amount of DA and its metabolites in striatum detected by HPLC-ECD. Panel (D) shows ratios of DAPOC/DA. Panel (E) shows ratios of HVA/DOPAC. Data are expressed as mean ± SEM, n = 7. *P < 0.05 and **P < 0.01, respectively as compared with untreated model group, respectively.

Figure 5

Effect of SMI on striatal dopamine transporter (DAT) density and protein level in chronic MPTP/probenecid-lesioned mice. Panel (A) shows representative autoradiographic images of the control mice, model mice treated with vehicle, 10 mg/kg/d, and 26 mg/kg/d SMI successively. Panel (B) shows the statistical results (n = 7). Panel (C) shows representative western blot bands of DAT. Panel (D) shows relative protein expression levels were quantified by densitometry analysis using Image J software on DAT bands (n = 4). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01, respectively as compared with untreated model group, respectively.

Figure 6

Effect of SMI on striatal dopamine D1 and D2 receptor density and protein level in chronic MPTP/probenecid-lesioned mice. (A) D1 receptor density, assessed by ³H-SCH23390 (n = 9). Panel (B) D2 receptor density, assessed by ³H-spiperone (n = 9). Panel (C) shows representative western blot bands of D1 and D2 receptor. Panels (D,E) show relative protein expression levels were quantified by densitometry analysis using Image J software on D1 and D2 receptor bands (n = 4). Data are expressed as mean ± SEM. *P < 0.05 and **P < 0.01, respectively as compared with untreated model group, respectively.

Figure 7

Effect of SMI on striatal glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) levels in chronic MPTP/probenecid-lesioned mice. (A) Striatal GDNF levels by ELISA. (B) Striatal BDNF levels by ELISA. Panels (C,E) show representative western blot bands of GDNF and BDNF. Panels (D,F) show relative protein expression levels were quantified by densitometry analysis using Image J software on GDNF and BDNF bands (n = 4). Statistical comparisons between groups, data are expressed as mean ± SEM, **P < 0.01 and ***P < 0.001, respectively as compared with untreated model group, respectively.

Figure 8

Effect of SMI on cyclic AMP responsive element binding (CREB) phosphorylation in chronic MPTP/probenecid-lesioned mice and CREB siRNA on mRNA expression of GDNF and BDNF in MPP⁺ treated SH-SY5Y cells. Panel (A) shows western blot bands of CREB and phosphorylation ofCREB (pCREB). Panel (B) shows relative expression of pCREB/CREB were quantified by densitometry analysis using Image J software on CREB and pCREB bands (n = 4). Data are expressed as mean ± SEM, *P < 0.05, **P < 0.01 and ***P < 0.001, respectively as compared with untreated model group, respectively. Panels (C,D) show relative expression of GDNF and BDNF mRNA by real time PCR. Data are expressed as mean ± SEM, ***P < 0.001.