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Objective <p>To investigate the effects of Bushen Huoxue Granule on the ubiquitin-proteasome system (UPS) in an <i>in vitro</i> model of Parkinson’s disease.</p> Methods <p>After treated with 1-methyl-4-phenylpyridinium (MPP<sup>+</sup>, 1 mmol/L) for 24 h, the cells were incubated with drug-free serum, Madopar-containing serum or Bushen Huoxue Granule-containing serum (BCS, 5%, 10%, and 20%) for another 24 h. The levels of α-synuclein (α-syn), tyrosine hydroxylase (TH) and UPS-related proteins were detected by Western blot. The expression levels of α-syn in PC12 cells were also analyzed by Western blot after treated with proteasome inhibitor MG132 and WT-α-syn plasmid transfection, respectively, as well as the alterations induced by subsequent BCS intervention. Immunocytochemistry was performed to determine the changes in α-syn phosphorylation at serine 129 (pSer129-α-syn) expression. The 20S proteasome levels were measured by enzyme-linked immunosorbnent assay.</p> Results <p>BCS (volume fraction ⩽20%) intervention could alleviate the MMP<sup>+</sup>-induced cell viability decrease (<i>P</i>&lt;0.05). In the MPP<sup>+</sup> treated cells, α-syn was up-regulated, while TH and proteins of UPS such as ubiquitin (Ub), Ub binding with Ub-activating enzyme (UBE1), Parkin and Ub C-terminal hydrolase-1 (UCHL-1) were down-regulated (<i>P</i>&lt;0.05). BCS intervention could attenuate the above changes (<i>P</i>&lt;0.05). The activity of BCS on blocking α-syn accumulation was weakened by MG132 (<i>P</i>&lt;0.05). While α-syn level was significantly increased in cells transfected with plasmid, and reduced by BCS intervention (<i>P</i>&lt;0.05). pSer129-α-syn was increased in MPP<sup>+</sup>-induced PC12 cells, whereas decreased by later BCS intervention (<i>P</i>&lt;0.05). The 20S proteasome activity of MPP<sup>+</sup>-induced PC12 cells was decreased, but increased after BCS intervention (<i>P</i>&lt;0.05).</p> Conclusion <p>BCS intervention protected UPS function, increased 20S proteasome activity, promoted pathological α-syn clearance, restored cell viability, and reversed the damage caused by MPP<sup>+</sup> in the <i>in vitro</i> model of Parkinson’s disease.</p>

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Effect of Bushen Huoxue Granule on Clearance of Pathological α-Synuclein in MPP+-Induced PC12 Cells

  • 期刊论文

  • 发表日期：

    2025年3月25日

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  • Zhen-xian Luan,
  • Xiang-lin Tang,
  • Fei-ran Hao,
  • Min Li,
  • Shao-dan Li,
  • Ming-hui Yang

摘要

Objective

To investigate the effects of Bushen Huoxue Granule on the ubiquitin-proteasome system (UPS) in an in vitro model of Parkinson’s disease.

Methods

After treated with 1-methyl-4-phenylpyridinium (MPP+, 1 mmol/L) for 24 h, the cells were incubated with drug-free serum, Madopar-containing serum or Bushen Huoxue Granule-containing serum (BCS, 5%, 10%, and 20%) for another 24 h. The levels of α-synuclein (α-syn), tyrosine hydroxylase (TH) and UPS-related proteins were detected by Western blot. The expression levels of α-syn in PC12 cells were also analyzed by Western blot after treated with proteasome inhibitor MG132 and WT-α-syn plasmid transfection, respectively, as well as the alterations induced by subsequent BCS intervention. Immunocytochemistry was performed to determine the changes in α-syn phosphorylation at serine 129 (pSer129-α-syn) expression. The 20S proteasome levels were measured by enzyme-linked immunosorbnent assay.

Results

BCS (volume fraction ⩽20%) intervention could alleviate the MMP+-induced cell viability decrease (P<0.05). In the MPP+ treated cells, α-syn was up-regulated, while TH and proteins of UPS such as ubiquitin (Ub), Ub binding with Ub-activating enzyme (UBE1), Parkin and Ub C-terminal hydrolase-1 (UCHL-1) were down-regulated (P<0.05). BCS intervention could attenuate the above changes (P<0.05). The activity of BCS on blocking α-syn accumulation was weakened by MG132 (P<0.05). While α-syn level was significantly increased in cells transfected with plasmid, and reduced by BCS intervention (P<0.05). pSer129-α-syn was increased in MPP+-induced PC12 cells, whereas decreased by later BCS intervention (P<0.05). The 20S proteasome activity of MPP+-induced PC12 cells was decreased, but increased after BCS intervention (P<0.05).

Conclusion

BCS intervention protected UPS function, increased 20S proteasome activity, promoted pathological α-syn clearance, restored cell viability, and reversed the damage caused by MPP+ in the in vitro model of Parkinson’s disease.