Protective effect of Humanin on rotenone-induced dopamine neuron toxicity

Abstract

Objective To investigate the mechanism and protective effect of Humanin (HN) on rotenone (Rot)-induced toxic damage for dopamine neurons. Methods The Rot-poisened PC12 cell model was constructed, and the control group, the Rot poisening group, the HN pretreated Rot poisening group, and the HN treatment group were set up. ELISA was used to detect the content of HN inside and outside of Rot-infected cells, CCK-8 assay was used to detect cell viability, and ATP detection kit was used to detect the intracellular ATP content. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay was used to detect the level of reactive oxygen species (ROS) in cells. Western blotting was performed to detect the expression level of mitochondrial autophagy regulatory proteins Pink1, Parkin, p62, LC3, mitochondrial biogenesis regulatory protein PGC1α, division/fusion regulatory proteins OPA1, MFN2, DRP1, p-DRP1 and antioxidant stress regulatory proteins Keap1 and Nrf2. HBAD-mcherry-EGFP-LC3 adenovirus transfected cells was used to observed the number of autophagosomes and autophagolysosomes. Results The results showed that the intracellular concentration of HN in PC12 in the Rot poisening group was significantly higher than that in the control group (P < 0.05);Compared with the control group, the Rot poisening group had significantly decreased activity of PC12 cells, decreased ATP content and increased production of ROS. After the poisen of Rot in PC12 cells, the expression of Pink1 and p-Parkin, the ratio of LC3Ⅱ/LC3Ⅰ and the expression of p-DRP1 in mitochondrial fusion protein was increased, while the expression of p62, the expression of mitochondrial biogenesis protein PGC1α, mitochondrial fusion proteins MFN2 and OPA1, and antioxidant stress proteins Keap1 and Nrf2 were decreased (all P < 0.05). The number of autophagosomes and autophagolysosomes in PC12 cells in the Rot poisening group was higher than that in the control group (P < 0.05), and HN pretreatment (20 μmol/L) could significantly improve the changes mentioned above caused by Rot poisening (P < 0.05). Conclusion HN ameliorates Rot-induced toxic damage for dopamine neurons by inhibiting mitophagy and mitochondrial division and promoting mitochondrial biogenesis and fusion, and anti-oxidative stress.

Keywords

• humanin
• rotenone
• dopamine neuron
• mitochondria