- Molecular Biology
- Molecular Medicine
- General Medicine
- Biochemistry
Background:
The pathology of Parkinson's disease (PD) indicates that iron deposition
exists in dopaminergic neurons, which may be related to the death of cellular lipid iron peroxide.
The extracellular autophagy adaptor SQSTM1(p62) of dopamine (DA) neurons can activate the
intracellular Keap1-Nrf2-ARE signaling pathway to inhibit ferroptosis, which has a protective effect
on DA neurons.
Objective:
The objective of this study was to investigate the protective mechanism of the Keap1-
Nrf2-ARE antioxidant pathway against iron death in dopaminergic neurons.
Methods:
The experiment was divided into a control group (Control group), 1-methyl-4-
phenylpyridiniumion control group (MPP+ Control group), p62 overexpression group (MPP+OVp62),
and p62 overexpression no-load group (MPP+ OV-P62-NC). The inhibitors brusatol and
ZnPP inhibited the activation of NF-E2-related factor 2(Nrf2) and Heme oxygenase-1(HO-1),
respectively, and were divided into brusatol group (MPP+OV-p62+brusatol) and ZnPP group
(MPP+OV-p62+ZnPP). RT-qPCR was used to detect transfection efficiency, and Cell Counting
Kit-8 (CCK8) was used to detect cell activity. FerroOrange, 2,7-Dichlorodihydrofluorescein
diacetate (DCFH-DA), and Liperfluo probes were used to detect intracellular iron, reactive oxygen
species (ROS), and lipid peroxidation (LPO) levels. Western Blotting detected the levels of Nrf2,
HO-1, Kelch-like ECH-associated protein1 (Keap1), and their downstream Glutathione peroxidase
4(GPX4) and Acyl-CoA synthetase long-chain family member 4(ACSL4). The levels of LGlutathione
(GSH) and Malondialdehyde (MDA) were detected by GSH and MDA kits, and the
activation of Keap1-Nrf2-ARE pathway was verified at the cellular level to have an antioxidant
protective effect on iron death in dopaminergic neurons.
Results:
(1) The results of RT-qPCR showed that compared with the control group, the
expression of the p62 gene was significantly increased in the MPP+OV-p62 groups (P = 0.039),
and the p62 gene was significantly increased in the brusatol and ZnPP groups, indicating
successful transfection (P =0.002; P=0.008). (2) The immunofluorescence probe flow results
showed that compared to the normal control group, the contents of three kinds of probes in MPP+
model group were significantly increased (P =0.001; P <0.001; P<0.001), and the contents of
three kinds of probes in MPP+OV-p62 group were decreased compared to the MPP+ model
group (P =0.004). The results indicated that the levels of iron, ROS, and LPO were increased in
the MPP+ group and decreased in the MPP+OV-p62 group. (3) Compared with the control group,
the expressions of Nrf2, HO-1, and GPX4 in the MPP+OV-p62 group were increased (P =0.007;
P =0.004; P=0.010), and the expressions of Keap1 and ACSL4 in MPP+p62 overexpression
group were decreased (P =0.017; P =0.005). Compared with the MPP+ control group, Nrf2 and
GPX4 were increased in the MPP+OV-p62 group, and ACSL4 was decreased in the MPP+OVp62
group (P =0.041; P <0.001; P <0.001). The results of the GSH and MDA kit showed that
compared with the normal control group, the content of GSH in the MPP+ control group was
decreased (P < 0.01), and the content of MDA was increased (P < 0.01). Compared with the
MPP+ model group, GSH content was increased (P = 0.003), and MDA content was decreased in
the MPP+OV-p62 group (P < 0.001). Nrf2, HO-1, and GPX4 increased in the MPP+p62
overexpression group but decreased in the brusatol group and ZnPP group (P < 0.001).
Conclusion:
Based on the transfection of P62 plasmid, it was found that P62 plasmid can inhibit
the lipid peroxidation of iron death in dopaminergic nerve cells by activating the Nrf2 signaling
pathway, thus playing a protective role in dopaminergic nerve cells.
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