Chinese Herbal Complex 'Bu Shen Jie Du Fang' (BSJDF) Modulated Autophagy in an MPP+-Induced Cell Model of Parkinson's Disease

Abstract

Autophagy plays an important role in the development of Parkinson disease (PD). Previous studies showed that autophagy could protect cells from α-synuclein toxicity and promote functional coupling of mitochondria. But it is still a question whether modulating autophagy can be used to treat PD. In traditional Chinese medicine, a specific Chinese herbal complex called Bu Shen Jie Du Fang (BSJDF) has a long history of treating motor impairments similar to Parkinson disease, while its mechanism is still unclear. As a pilot study, we aimed to evaluate the efficacy and its mechanism of Bu Shen Jie Du Fang in an MPP⁺-induced cell model of Parkinson's disease. And the phase contrast microscope (PCM) revealed that the BSJDF group had the greatest surviving cell counts compared with all other treated cell groups except the normal group. And Cell Counting Kit 8 (CCK8) assays showed a similar result. In BSJDF group, 3.7 ×10⁷ cells/dish was identified by hemocytometer counts, which was significantly higher than other groups except the normal cells (p<0.05). In the BSJDF group, autophagy can be observed by transmission electron microscopy (TEM). Protein expression of Atg12 and LC3 in the BSJDF group was upregulated compared to the PD model group (p<0.05). Atg12 mRNA expression was also upregulated in the BSJDF group (p<0.05). In conclusion, our study indicated that the therapeutic mechanisms of BSJDF may be mediated by stimulating autophagy, and modulating autophagy can be used to treat PD.

Figure 1

The cells were reviewed under PCM enlarged 100× and 1000×. (a) The normal group which is untreated PC12 cells; (b) the rapamycin group treated with MPP⁺ for 24 h and then rapamycin for 24 h; (c) the PD model group treated with MPP⁺ for 48 h; (d) the BSJDF group treated with MPP⁺ for 24 h and then BSJDF serum for 24 h; (e) the 3-MA group treated with MPP⁺ for 24 h and then 3-MA for 24 h; (f) the NH₄CL group treated with MPP⁺ for 24 h and then NH₄Cl for 24 h. The cell number in the BSJDF group is larger than the others except the normal group.

Figure 2

The survival cells counted by hemocytometer. A, the normal group, the survival cells were 7.3 × 10⁷; B, the rapamycin group, the survival cells were 2.8 × 10⁷; C, the PD model group, the survival cells were 1.8 × 10⁷; D, the BSJDF group, the survival cells were 3.4 × 10⁷; E, the 3-MA group, the survival cells were 1.6 × 10⁷; F, the NH₄CL group, the survival cells were 1.6 × 10⁷; the number of cells surviving in the BSJDF group was larger than other groups except the normal group (p<0.05), ∗p<0.05 and ∗∗p<0.01.

Figure 3

The correlation between the counts and OD (optical density) (R² = 0.9998).

Figure 4

The OD results in different groups by CCK8. A, the normal group; B, the rapamycin group; C, the PD model group; D, the BSJDF group; E, the 3-MA group; F, the NH₄CL group. The number of cells surviving in the BSJDF group has more cell counts than the others, except the normal group (p<0.05), ∗p<0.05 and ∗∗p<0.01.

Figure 5

The formation of autophagosomes in treated cells was evaluated by TEM. (a) the normal group; (b) the rapamycin group; (c) the PD model group; (d) the BSJDF group; (e) the 3-MA group; (f) the NH₄CL group. The arrowheads autophagy (scale bar: 2.0 μm). “Red arrow” indicates autophagosomes.

Figure 6

1, the normal group; 2, the rapamycin group; 3, the PD model group; 4, the BSJDF group; 5, the 3-MA group; 6, the NH₄CL group. GAPDH was used as a loading control. (a) Atg12 western blot densitometry in ST. (b) Beclin-1 western blot densitometry in ST. (c) LC3 western blot densitometry in ST. ∗p<0.05 and ∗∗p<0.01.

Figure 7

1, the normal group; 2, the rapamycin group; 3, the PD model group; 4, the BSJDF group; 5, the 3-MA group; 6, the NH₄CL group. GAPDH was used as a loading control. (a) Atg12 mRNA expression level in RT-qPCR. (b) Beclin-1 mRNA expression level in RT-qPCR. (c) LC3 mRNA expression level in RT-qPCR. ∗p<0.05 and ∗∗p<0.01.