δ-Opioid Receptor Activation Inhibits Ferroptosis by Activating the Nrf2 Pathway in MPTP-Induced Parkinson Disease Models

Introduction Recent studies suggest the involvement of ferroptosis in the pathogenesis of Parkinson disease (PD). δ-Opioid receptors (DORs) have neuroprotective effects in PD. It is not known whether the neuroprotective effects of DORs in PD are attributable to the inhibition of ferroptosis. Therefore, we aimed to investigate the role of DORs in ferroptosis in MPTP-induced PD models. Methods To identify the influence of DORs on ferroptosis in MPTP-induced PD models, we measured the malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels, analyzed the levels of ferroptosis-related proteins (GXP4 and SLC7a11) and Nrf2 expression by using western blotting, and assessed mitochondrial dysfunction by using JC-1 staining and transmission electron microscopy. Results DOR activation reduced the 4-HNE and MDA levels, increased the GXP4 and SLC7a11 levels, and ameliorated mitochondrial dysfunction in MPTP-induced PD models. These neuroprotective effects of DORs could be blocked by Nrf2-siRNA. Thus, the effects of DORs on ferroptosis in PD models were partially controlled by Nrf2, which regulated GXP4 and SLC7a11 synthesis. Conclusion DORs exert neuroprotective effects in PD models by inhibiting ferroptosis partially via activating the Nrf2 pathway.


Introduction
Parkinson disease (PD) is the second most common neurodegenerative disease in the world [1]. PD predominantly occurs in elderly patients. Te clinical manifestations of PD include both motor and nonmotor symptoms that gradually progress over the course of the disease and ultimately lead to the loss of self-care ability, which increases family and social burden. Although it is known that the main pathological change in PD is the loss of dopaminergic neurons in the substantia nigra and striatum, the precise mechanism underlying this change is not yet clear. Te cause of this neuronal loss is closely related to the process of cell death, specifcally, ferroptosis and other types of programmed cell death, which have now become a focus of PD research [2].
Ferroptosis [3] is an iron-dependent, erastin-specifcinduced programmed cell death characterized by changes such as shrinkage of mitochondria, loss of mitochondrial crests, exhaustion of reduced glutathione (GSH), and accumulation of reactive oxygen species (ROS). Inhibitors of programmed cell death do not inhibit ferroptosis, though some iron chelators do reverse it. Te key to ferroptosis is GSH. Under the catalytic action of glutathione peroxidase 4 (GPX4), GSH can help prevent the abnormal accumulation of cellular ROS. Te targeted inhibition of GPX4 leads to the accumulation of lipid peroxides and induces ferroptosis [4]. Studies indicate that ferroptosis is an early and characteristic type of cell death in PD [5][6][7][8]. Dj-1, a protein linked to earlyonset PD, inhibits ferroptosis [9]. An iron-chelating agent was shown to exert a signifcant protective efect against PD in mice [10]. Te previous fndings ofer new research avenues to develop strategies for the early treatment of PD.
Opiates are a group of diverse neurotransmitters that include endorphins and enkephalins. Opioid receptors also exhibit multiple polymorphisms (e.g., μ, δ, and κ receptors) and are widely distributed in the central nervous system. Delta opioid receptors (DORs) are mainly distributed in the striatum. DORs play an important neuroprotective role [11,12]. Our previous studies showed that the activation of DORs increases the phosphorylation of cAMP response element-binding (CREB) protein, stabilizes mitochondrial membrane potential [13], and weakens the overexpression/ aggregation of synuclein [14], which plays a neuroprotective role in PD models. Te functions of opioid receptors are yet to be fully characterized.
Nrf2 protein is a leucine zipper transcription factor that regulates the expression of multiple genes and serves a variety of physiological functions. Studies have shown that the activation of opioid receptors can activate the Nrf2 pathway [15][16][17][18][19] and that Nrf2 is a key transcriptional regulator of ferroptosis [20,21]. Terefore, we hypothesized that the activation of DORs exerts a neuroprotective efect by activating Nrf2 to inhibit ferroptosis in PD models. Tus, the aim of the present study is to further explore this potential mechanism underlying the neuroprotective efect of DORs and provide a theoretical basis for the development of new anti-PD drugs.

Open Field Test.
Tis approach has been used by our research team before [22]. Each mouse was placed in the center of the open-feld apparatus. Te center zone was defned as a square, 10 cm away from the walls. Te distance traveled and time spent in the center zone by each animal was recorded for 10 min by using a video-imaging system (EthoVisionXT; Noldus Information Technology, Wageningen, Te Netherlands).

Cytotoxicity and Cell Viability Assessments.
Cytotoxicity was quantifed using lactate dehydrogenase (LDH) activity measurement kits (KeyGEN BioTECH, Nanjing, China) according to the manufacturer's instructions. Cell viability was assessed using CCK-8 kits (TransGen Biotech, Beijing, China) according to the manufacturer's instructions. Te absorbance value (optical density, 450 nm) was measured using a microplate reader (BioTek Instrumentals Inc., Winooski, VT, USA).

2.7.
Immunofuorescence. For immunofuorescence analysis, tissue samples from the substantia nigra region were fxed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and incubated with 10% bovine serum albumin for 1 h at room temperature. Primary antibodies were added, and the incubation was continued at 4°C overnight. Te sections were then washed with PBS and incubated with fuorescent labeled secondary antibodies (mouse monoclonal antityrosine hydroxylase antibody, 1 : 2000; Sigma-Aldrich). Finally, the sections were examined using confocal microscopy (TCS SP8; Leica, Solms, Germany). Immunofuorescence was quantifed using ImageJ software.
2.9. Western Blot Analysis. Samples from PC12 cells or the mouse models were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions and then transferred to polyvinylidene difuoride membranes. Nonspecifc binding was blocked using a blocking bufer. Te membranes were then incubated overnight at 4°C with specifc primary antibodies diluted in Tris-bufered saline supplemented with 0.1% Tween20 (TBST) and 1% nonfat dry milk. After being washed 3 times with TBST, the membranes were incubated for 1 h at room temperature with goat antirabbit IgG diluted to a ratio of 1 : 5000 in 1% nonfat dry milk. Te membranes were again washed 3 times with TBST, and immunoreactive bands were observed using Pierce ® ECL Western Blotting Substrate (TermoFisher Scientifc, Uppsala, Sweden). For quantitative analysis of proteins, the membranes were stripped using Restore ™ Western Blot Stripping Bufer (TermoFisher Scientifc, Uppsala, Sweden) and reprobed with anti-β-actin antibody (1 : 5000).

Mitochondrial Membrane Potential Measurement.
Te mitochondrial membrane potential (ΔΨm) of PC12 cells was measured using fuorescence microscopy and the JC-1 Mitochondrial Membrane Potential Assay Kit (Abcam Co., St. Louis, MO, USA) according to the manufacturers' instructions. Healthy cells containing JC-1 aggregates were identifed under a fuorescence microscope at excitation and emission wavelengths of 540 and 570 nm, respectively. Unhealthy cells containing JC-1 monomers were identifed by detecting fuorescein isothiocyanate under a fuorescence microscope at excitation and emission wavelengths of 485 and 535 nm, respectively.

Transmission Electron Microscopy.
Substantia nigra samples from the mice were cut into 60-80-nm-thin sections on an ultramicrotome and imaged using a transmission electron microscope (JEM-1400plus; JEOL, Tokyo, Japan).

Statistical Analysis.
All values were presented as the mean ± standard error. One-way analysis of variance followed by the Bonferroni test was used to conduct multiple pairwise comparisons to identify signifcant diferences. P < 0.05 was deemed to indicate statistical signifcance.
Next, we assessed the role of DORs in the mouse model of MPTP-induced PD by conducting an open feld test. After 1 week of treatment with DADLE, the movement distance and mean speed of the MPTP + DADLE-treated mice were signifcantly greater than those of the MPTP-treated mice (P < 0.01, Figures 2(a) and 2(b). Similar results were obtained for ferrostatin-1. Tese fndings suggest that DADLE and ferrostatin-1 aided the recovery of motor activity in mice with MPTP-induced PD.
To determine whether DORs help protect against MPTP-induced damage to nigrostriatal dopaminergic neurons, we performed immunofuorescence analysis of tyrosine hydroxylase, a marker for dopaminergic neurons, in tissue samples of the substantia nigra and striatum region collected from mice with PD. Te results showed that DADLE and ferrostatin-1 decreased the MPTP-induced loss of dopaminergic neurons and nerve fbers in the substantia nigra (Figures 3 and 4).

DOR Activation Inhibits Ferroptosis in MPTP-Induced PD
Models. Ferroptosis may be an early pathological change in PD, and increased lipid peroxide content is an important marker of ferroptosis. GPX4 and SLC7a11 are key proteins that regulate ferroptosis, and mitochondrial dysfunction plays a key role in ferroptosis. Terefore, after the activation of DORs in MPTP-induced PD models, we measured the MDA and 4-HNE contents, the protein expression levels of GPX4 and SLC7a11, and mitochondrial dysfunction. We found that MPTP induced MDA and 4-HNE accumulation induced mitochondrial dysfunction and decreased the protein expressions of GPX4 and SLC7a11, all of which indicated that ferroptosis is involved in PD (Figures 5 and 6). Treatment of PC12 cells and PD mouse models with ferrostatin-1 or DADLE remarkably reduced the accumulation of MDA and 4-HNE (Figures 5(a) and 6(a)), decreased the protein expressions of GPX4 and SLC7a11 (Figures 5(b) and 6(b)), and restored the mitochondrial membrane potential (Figure 5(c), Supplementary Figure S3A). In addition, transmission electron microscopy showed that the mitochondrial membranes appeared intact, and the mitochondrial cristae were restored after the DADLE and ferrostatin-1 treatments (Figure 6(c)). Collectively, the previous results show that the activation of DORs had an inhibitory efect on ferroptosis in MPTP-treated PC12 cells and mice.

DOR Activation Enhances Nrf2 Protein Expression in
MPTP-Induced PD Models. As Nrf2 is a crucial factor regulating ferroptosis, we next investigated whether the activation of DORs by DADLE or ferrostatin-1 could increase the expression of Nrf2. Western blot analysis revealed that after treatment with DADLE or ferrostatin-1, Nrf2 expression was upregulated in PC12 cells and PD mouse models (Figures 5(b) and 6(b)), which demonstrated that DORs increased Nrf2 synthesis in MPTP-induced PD models.

DORs Regulate Ferroptosis via the Nrf2 Pathway in MPTP-
Treated PC12 Cells. To further analyze the neuroprotective and antiferroptosis efects of DOR, we transfected PC12 cells with anti-Nrf2 siRNA. We found that in PC12-siNrf2 cells, Nrf2 expression was remarkably reduced to 42.16% ± 4.44% of the expression in PC12 cells (P < 0.05; Supplementary Figure S2). Furthermore, the neuroprotective and antiferroptosis efects of DORs were preserved in MPTP-treatedPC12-scrambled siRNA cells but drastically diminished in MPTP-treatedPC12-siNrf2 cells (Figure 7,

Discussion
Te current treatment for PD consists of increasing the neurotransmission of dopamine to provide symptomatic relief. Although dopaminergic drug therapy can delay the progression of the disease and improve symptoms, a high concentration of dopamine may have neurotoxic efects. A specifc dopaminergic neuroprotective drug is still lacking [23]. A better understanding of disease-related pathological mechanisms and their relationship with the cell death processes driving dopaminergic neuronal loss is required for the discovery of novel therapeutic targets for the treatment of PD. PD-associated neuronal cell death was previously attributed to apoptosis. Recently, however, ferroptosis was reported to be a possible mechanism of neuronal cell death in PD [8]. Some pathological features of PD, such as iron overload, increased lipid peroxidation, decreased GSH levels, DJ-1 consumption, and decreased coenzyme Q10, are known to be involved in ferroptosis, which strongly implicates this programmed cell death process in PD [7,24,25]. Although MPTP-induced death and ferroptosis shared some features, such as occurrence of lipid peroxidation and inhibition by Fer-1, MPTP-induced death seemed to be distinct from ferroptosis because MPTP-induced death was accompanied by ATP depletion and mitochondrial swelling [26]. Further investigation of other indirect regulated factors may be useful for understanding the mechanisms of neuronal loss and for treatment of Parkinson's disease.
Ferroptosis is a type of programmed cell death that is activated when iron-dependent lipid peroxidation reaches lethal levels, and the only unique morphological feature is wrinkling of the mitochondrial membrane. Te peroxidation substrates in ferroptosis are phospholipid-bound polyunsaturated fatty acids in the cell membrane; the lipid peroxides decompose into toxic derivatives such as 4-HNE Evidence-Based Complementary and Alternative Medicine and MDA [27]. GPX4 is a key enzyme in the ferroptosis pathway [28]. GPX family members such as GPX1-GPX8 have been found in mammals [29]. Direct GPX4 inactivation by RAS-selective lethal 3 (RSL3) is commonly used to induce ferroptosis under experimental conditions. GPX4 provides electrons for prototype glutathione (GSH), releasing oxidized glutathione [30]. GSH is synthesized in cells from glutamate and cysteine, of which the latter is the ratelimiting substrate. Cysteine is synthesized from methionine through the transsulfuration pathway or absorbed as an oxidized cystine dimer by the XcT (encoded by SLC7A11) reverse transporter, which is required for intracellular cysteine transport. XcT also plays an important role in regulating ferroptosis [31]. It is generally accepted that the levels of GPX4, MDA, 4-HNE, and SLC7A11 serve as powerful indices of the level of ferroptosis.
Several studies have demonstrated the neuroprotective functions of DORs [11,[32][33][34][35][36]. In cultured cortical neurons, DOR activation signifcantly reduces glutamate-induced neurotoxicity [37], and the neuroprotective efects of DORs are mediated through the regulation of ion channels [38,39]. Some studies have suggested that opioid receptor agonists can be used as dopamine alternatives in the treatment of PD [40]. DOR agonists, such as DADLE and UFP-512, have displayed the ability to delay neuronal death in PD [13,35,[41][42][43][44]. However, the mechanism underlying the neuroprotective efects of DORs is not completely understood. Some studies have shown that DORs can promote the expression and nuclear translocation of Nrf2 [19]. Nrf2 is the main transcription factor regulating antioxidant reactions and has an antiferroptosis efect [20,21]. In the presence of oxidative stress, Nrf2 is translocated to the nucleus, which induces the expression of endogenous antioxidant proteins that prevent lipid peroxidation [45]. Persistent exposure to erastin results in the upregulation of the Nrf2-dependent enzyme cystathionine-β-synthase, which is responsible for the biosynthesis of cysteine, which fghts cell death [46]. Nrf2 also controls NAD(P)H, several iron-metabolizing proteins, GPX4, and other key proteins for the biosynthesis of GSH (e.g., XcT and . Te quantitative analysis of the TH-positive regions was performed using ImageJ. All statistical diferences were tested using a t-test. Data are expressed as the mean ± SEM (n � 5/group). Te signifcance is expressed as ## P < 0.01 vs. control group. * * P < 0.01 vs. MPTP group. ns, not signifcant vs. MPTP group. DOR, δ-opioid receptor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson disease; DADLE, [d-Ala 2 , d-Leu 5 ]-enkephalin; ns, not signifcant. glutathione synthetase). Te oxidative stress-induced Nrf2 response is not specifc to ferroptosis, as Nrf2 inhibition is also associated with apoptotic cell death. Tus, the role of Nrf2 in PD may be related to multiple types of cell death.
In summary, we have explored the neuroprotective and antiferroptosis efects of DORs in PD models and clarifed the mechanism underlying these efects of DORs (Figure 8). Te main fndings of our study are as follows: (1) DORs confer neuroprotection in MPTP-induced PD models, (2) DOR activation inhibits ferroptosis in MPTP-induced PD models, (3) DOR activation enhances Nrf2 protein expression in MPTP-induced PD models, and (4) DORs regulate ferroptosis via the Nrf2 pathway in MPTP-treated PC12 cells.
Our study fndings indicate that DORs suppress the decline in cell viability and mitigate ferroptosis in MPTPinduced PD by activating the Nrf2 pathway. Tus, DORbased therapy and antiferroptosis agents represent novel therapeutic approaches that might be developed for the treatment of PD. In addition, we look forward to the DOR exerting a successful therapeutic efect and might be administered either as an adjuvant in combination with other therapeutic strategies. Especially, the combined utilization of the DOR-based therapy with other traditional treatment such as supplement of dopamine. In future studies, our results can be verifed in Nrf2 knockout mice, and the mechanism underlying the neuroprotective efects of DORs can be further investigated. In addition, our investigation    sought to elucidate the mechanisms of Nrf2 alteration, especially in nucleus and cytoplasm separately.

Data Availability
Te dates of this study are stored at the Hainan Medical University. Te datasets supporting the conclusion of this study are available from the corresponding author on reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
BC worked on conceptualization, writing-original draft, and writing-review and editing. LZ worked on conceptualization and writing-original draft. TC did the supervision and writing-review and editing. YD did the supervision and writing-review and editing, QX did the supervision and writing-review and editing. All authors contributed to the article and approved the submitted version. Benchi Cai and Lifan Zhong contributed equally to this work and share frst authorship.