Fifteen 8-week-old male C57BL/6 mice (20–22 g) were obtained from the Animal Experiment Center of Kunming Medical University and housed under standard conditions (23 $\pm$ 1 °C, 12 h light/dark cycle) for one week. The mice were randomly assigned to three groups (n = 5 per group): control, MPTP, and MPTP + lutein groups. The lutein dosage was determined according to the methods of Nataraj et al. [19]. Lutein (SL8650; Solarbio, Beijing, China) was dissolved in 4% dimethyl sulfoxide (DMSO) (ST038; Beyotime, Shanghai, China) and administered intraperitoneally at 10 mg/kg/day beginning 1 h before MPTP injection and continuing throughout the MPTP treatment period. MPTP (30 mg/kg, dissolved in saline; HY-15608; MedChemExpress, Monmouth Junction, NJ, USA) was injected intraperitoneally for 7 consecutive days to induce PD. Control mice received an equal volume of solvent. After 14 days of lutein treatment, behavioral tests were conducted. After the completion of behavioral tests, mice were acclimated in the procedure room for at least 15 min to minimize stress. Euthanasia was then performed by intraperitoneal injection of an overdose of pentobarbital sodium (P3761,Sigma‑Aldrich, St. Louis, MO, USA; 150 mg/kg). Each mouse was gently restrained by an experienced researcher to ensure a rapid and accurate injection. This dose induces rapid unconsciousness, followed by respiratory depression and cardiac arrest, leading to death. Death was confirmed by the absence of the pupillary reflex and cessation of breathing before tissue collection. All procedures were designed and performed to minimize animal distress.

Human neuroblastoma cells (SH-SY5Y) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (SCSP-5014; Shanghai, China). All cell lines used in this study were rigorously authenticated and confirmed to be free of mycoplasma contamination. Authentication was performed by short tandem repeat (STR) profiling, and mycoplasma testing was conducted regularly using the polymerase chain reaction (PCR)-based method; all results were negative. Cells were cultured in DMEM/F12 (11320033; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (A5256701; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (15070063; Gibco, Grand Island, NY, USA) at 37 °C under 5% CO₂. To establish a PD cellular model, cells were treated with 1 mM MPP⁺ (D048, Sigma-Aldrich, St. Louis, MO, USA) for 24 h. For lutein pretreatment, cells were incubated with 10 µM lutein for 2 h prior to MPP⁺ exposure.

TRIM31 overexpression (OE-TRIM31) and knockdown (si-TRIM31) plasmids, along with their corresponding negative controls (OE-NC, si-NC), were purchased from GenePharma (Shanghai, China). SH-SY5Y cells were seeded in 6-well plates and transfected at 70–80% confluence using Lipofectamine 3000 (L3000150; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Transfection efficiency was confirmed by Western blotting.

Rotarod test: Mice were placed on an accelerating rotarod (5–40 rpm), and the latency to fall was recorded. If a mouse did not fall within 5 min, it was recorded as 300 s. Each mouse was tested five times at 30 min intervals. Adhesive removal test: A small adhesive label was placed on each mouse’s forepaw, and the time to remove it was recorded. Three trials were performed per mouse at 30 min intervals. Pole test: Mice were placed on a vertical pole (1 cm in diameter, 60 cm in height), and the time to descend to the floor was recorded. The average of three trials was calculated.

Brain tissues were fixed, paraffin-embedded, and sectioned at 5 µm. For hematoxylin and eosin (H&E) staining, sections were deparaffinized, stained with hematoxylin and eosin (G1120; Solarbio), and imaged under a light microscope. For immunohistochemistry, antigen retrieval was performed using citrate buffer. Endogenous peroxidase activity was blocked with 3% H₂O₂, and nonspecific binding was blocked with 5% goat serum (C0265; Beyotime). Sections were incubated overnight at 4 °C with anti-TH antibody (1:500, ab137869; Abcam, Cambridge, UK), followed by an horseradish peroxidase (HRP)-conjugated secondary antibody (1:500, ab97051; Abcam) and 3,3’-diaminobenzidine (DAB) development (DA1010; Solarbio). Finally, the samples were observed using an optical microscope (BX53; Olympus, Tokyo, Japan) and photographed for record.

Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay (C0038; Beyotime). SH-SY5Y cells were seeded in 96-well plates, treated as indicated, and incubated with CCK-8 reagent for 1 h. Absorbance was measured at 450 nm. Apoptosis was detected by flow cytometry using a fluorescein Isothiocyanate-conjugated Annexin V (Annexin V-FITC)/propidium iodide (PI) staining kit (556547, BD Biosciences, San Jose, CA, USA). After treatment, the cells were incubated in the dark at 25 °C for 15 min. Finally, the apoptosis rate of the cells was detected using a flow cytometer (FACScan; Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA).

Proteins were extracted from tissues or cells using radio-immunoprecipitation assay (RIPA) lysis buffer (P0013, Beyotime), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00010; Merck Millipore, Burlington, MA, USA). After blocking with 5% skim milk, membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies (1:4000, ab97051; Abcam or 1:5000, ab205719; Abcam). Protein bands were visualized using an enhanced chemiluminescence (ECL) kit (34075; Thermo Fisher Scientific, Waltham, MA, USA) and quantified with ImageJ (V1.8.0.112, National Institutes of Health, Bethesda, MD, USA). Primary antibodies used included Bcl-2-associated X protein (Bax) (1:1000, ab32503; Abcam), B-cell lymphoma-2 (Bcl-2) (1:1000, ab196495; Abcam), cleaved caspase-3 (1:1000, #9661S; Cell Signaling Technology, Danvers, MA, USA), Drp1 (1:1000, ab184247; Abcam, Cambridge, UK), TRIM31 (1:1000, PA5-40961; Thermo Fisher Scientific, Waltham, MA, USA), and $\beta$-actin (1:1000, ab8226; Abcam, Cambridge, UK).

Mitochondrial reactive oxygen species (ROS) levels were measured using MitoSOX Red (S0061S; Beyotime). Cells were stained for 25 min at 37 °C and imaged by fluorescence microscopy. Mitochondrial membrane potential was assessed using JC-1 staining (C2006; Beyotime), and the ratio of red to green fluorescence was calculated. ATP content was determined using a commercial ATP assay kit (A095-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and a microplate reader. Complex I activity was measured using a spectrophotometric assay kit (BC0515; Solarbio).

Cells were lysed in NP-40 lysis buffer (P0013F; Beyotime, Shanghai, China) supplemented with a protease inhibitor “cocktail” (539134; Merck Millipore). Lysates were then centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected and incubated with a Drp1 antibody (1:1000, ab184247; Abcam) at 4 °C for 6 h. After incubation, protein A/G agarose magnetic beads (#78609; Thermo Fisher Scientific) were added, and the mixture was rotated and incubated overnight at 4 °C. The immune complexes bound to the beads were washed three times with immunoprecipitation (IP) buffer. Finally, the attached proteins were extracted by boiling in loading buffer and analyzed by Western blotting.

Cell supernatants were prepared as described previously. Protein samples were incubated overnight at 4 °C with an anti-TRIM31 antibody (1:100, PA5-40961; Thermo Fisher Scientific), followed by incubation for 1 h at 4 °C with protein A/G magnetic beads (30 µL/sample). As a negative control, supernatants were incubated with rabbit IgG (1:500, 98136-1-RR; Proteintech Group Inc., Rosemont, IL, USA). The magnetic beads were subsequently triple-rinsed with lysis buffer, and the immunoprecipitated proteins were eluted using loading buffer for Western blot analysis.

Total RNA was extracted with TRIzol (15596026; Invitrogen, Carlsbad, CA, USA), and cDNA was synthesized using a reverse transcription kit (P4202; Genenode, Wuhan, China). qPCR was performed with SYBR Green (SR1110; Solarbio) on a 7900HT Fast Real-Time PCR System. The 2^{-Δ⁢Δ⁢Ct} method was used for quantification, with $\beta$-actin as the internal control. Primer sequences are listed in Table 1.

All data were analyzed using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA) and are presented as the mean $\pm$ standard deviation (SD). Prior to group comparisons, datasets were assessed for normality using the Shapiro-Wilk test and for homogeneity of variance using the Brown-Forsythe test. Comparisons between two groups were performed using an independent samples t-test (if parametric assumptions were met) or the Mann-Whitney U test (if not). For comparisons among multiple groups, one-way or two-way analysis of variance (ANOVA) was applied according to the experimental design. If ANOVA indicated a significant difference, post hoc comparisons were conducted using Tukey’s test. All experiments clearly distinguished between biological replicates (independent animals or independent cell culture batches) and technical replicates (multiple measurements within the same sample). The sample size (n) reported throughout refers to the number of biological replicates. A p value 0.05 was considered statistically significant.

First, we investigated the potential protective effect of lutein in a PD mouse model. H&E staining revealed marked focal encephalomalacia and neuronal structural damage in MPTP-treated mice, whereas lutein intervention significantly ameliorated these pathological alterations (Fig. 1A). Behavioral tests further demonstrated that MPTP induction led to motor dysfunction characterized by prolonged pole climbing time, shortened retention time, and delayed label removal, all of which were effectively reversed by lutein treatment (Fig. 1B–D). Immunohistochemical analysis revealed a notable decrease in TH-positive neurons in the MPTP group compared with the control group, whereas lutein treatment markedly increased the number of TH-positive neurons (Fig. 1E). Further in vitro validation of lutein’s role demonstrated that compared with the negative control (NC) group, the MPP⁺ group exhibited significantly reduced cell viability and markedly increased apoptosis. Lutein treatment effectively reversed these changes (Fig. 1F,G). Western blot analysis of apoptosis-related proteins revealed that compared with the NC group, the MPP⁺ group had significantly lower Bcl-2 expression and markedly higher Bax and cleaved caspase-3 expression. Lutein treatment partially reversed these changes in protein expression levels (Fig. 1H) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). These results suggest that lutein can alleviate PD progression in mice and inhibit neuronal cell damage in vitro.

Fig. 1.

Lutein attenuated PD progression in mice and inhibited neuronal injury in vitro. (A) Pathological damage was detected through H&E staining, scale bar: 50 µm. (B) Rotarod assessment for motor ability. (C) Adhesive removal test to assess motor function. (D) Pole-climbing test to assess motor function. (E) IHC analysis of TH expression, scale bar: 50 µm. (F) CCK-8 assay of cell viability. (G) Flow cytometry identification of apoptosis. (H) Western blot analysis of apoptosis-associated proteins Bcl-2, Bax, and cleaved caspase-3. ^* p 0.05, ^** p 0.01, ^*** p 0.001 (A–E: n = 5; F–H: n = 3). PD, Parkinson’s disease; H&E, hematoxylin and eosin; IHC, immunohistochemistry; TH, tyrosine hydroxylase; CCK-8, Cell Counting Kit-8; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; FITC, fluorescein Isothiocyanate-conjugated; NC, negative control; MPP⁺, 1-methyl-4-phenylpyridinium ion.

Previous studies have shown that mitochondrial dysfunction is strongly associated with PD [20, 21]. Consequently, we evaluated the effect of lutein on MPP⁺-induced alterations in mitochondrial function in neurons. Measurements of ROS levels and mitochondrial membrane potential revealed that, compared with the NC group, MPP⁺ treatment significantly increased mitochondrial ROS levels and significantly decreased the mitochondrial membrane potential. In contrast, lutein treatment reduced ROS levels and increased the mitochondrial membrane potential (Fig. 2A,B). Treatment with MPP⁺ reduced ATP concentrations, whereas lutein markedly reversed this effect and restored ATP levels (Fig. 2C). In addition, lutein treatment significantly increased the activity of mitochondrial respiratory chain complex I (Fig. 2D). These results suggest that lutein can improve mitochondrial dysfunction induced by MPP⁺ in neuronal cells.

Fig. 2.

Lutein improves MPP⁺-induced mitochondrial dysfunction in neuronal cells. (A) Mitochondrial ROS identified via MitoSOX Red staining, scale bar: 10 µm. (B) Mitochondrial membrane potential measured using JC-1 staining, scale bar: 50 µm. (C) ATP concentration in SH-SY5Y cells measured with a kit. (D) Activity of mitochondrial respiratory chain complex I. ^*** p 0.001 (n = 3). ROS, reactive oxygen species.

Drp1 plays a crucial role in mitochondrial fission. Previous studies have shown that Drp1 expression is upregulated in PD [22] and that its ubiquitination is crucial for regulating mitochondrial fission [23]. Western blot analysis indicated that compared with NC treatment, MPP⁺ treatment markedly increased Drp1 expression and decreased its ubiquitination (Fig. 3A,B). Lutein treatment increased ubiquitinated Drp1 levels (Fig. 3C) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). These results indicate that Drp1 ubiquitination is decreased in PD and that lutein can promote its ubiquitination.

Fig. 3.

Lutein promotes the ubiquitination of Drp1. (A) Western blot analysis of Drp1 expression. (B) Detection of ubiquitinated Drp1 levels. (C) The effect of lutein treatment on Drp1 ubiquitination. ^** p 0.01 (n = 3). Drp1, dynamin-related protein 1; IB, Immunoblotting; Ub, Ubiquitin; IP, immunoprecipitation.

Previous studies have shown that TRIM31 is enriched in mitochondria [12]. Consequently, our research focused on the role of TRIM31 in controlling mitochondrial activity in neurons. Analysis of the Gene Expression Omnibus (GEO) database (GSE160299) revealed that TRIM31 expression is reduced in PD patients (Fig. 4A,B). Furthermore, TRIM31 expression tended to decrease in MPTP or MPP⁺-induced animal and cellular models (Fig. 4C,D). As previously noted, TRIM31 can regulate Drp1 expression in response to mitochondrial damage caused by cerebral ischemia [11]. Co-immunoprecipitation experiments further verified the interaction between TRIM31 and Drp1 (Fig. 4E). To investigate whether TRIM31 regulates neuronal mitochondrial function through Drp1, we overexpressed TRIM31. Western blot analysis demonstrated a significant increase in TRIM31 expression (Fig. 4F). Ubiquitination assays showed that TRIM31 overexpression significantly promoted Drp1 ubiquitination (Fig. 4G) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). CCK-8 and flow cytometry assays revealed that overexpression of TRIM31 significantly promoted SH-SY5Y cell proliferation and reduced apoptosis (Fig. 4H,I). Further analysis of indicators related to mitochondrial function revealed that overexpression of TRIM31 decreased mitochondrial ROS levels, increased mitochondrial membrane potential and ATP content, and elevated mitochondrial respiratory chain complex I activity (Fig. 4J–M). These findings suggest that TRIM31 overexpression improves mitochondrial function in neuronal cells by promoting Drp1 ubiquitination.

Fig. 4.

TRIM31 improves neuronal cell loss and mitochondrial function by regulating Drp1 ubiquitination levels. (A) Cluster heatmap. (B) Volcano plot. (C) Western blot detection of TRIM31 expression in tissues. (D) Analysis of TRIM31 expression in cells using Western blotting. (E) Identification of the interaction between TRIM31 and Drp1 through co-immunoprecipitation. (F) Analysis of the efficiency of OE-TRIM31 transfection. (G) Identification of the ubiquitination level of Drp1. (H) Results of the CCK-8 assay for the proliferation of SH-SY5Y cells. (I) Flow cytometry for detecting apoptosis in SH-SY5Y cells. (J) MitoSOX Red staining to detect mitochondrial ROS, scale bar: 10 µm. (K) Measurement of mitochondrial membrane potential using JC-1 staining, scale bar: 50 µm. (L) Assessment of ATP concentration in SH-SY5Y cells using a kit. (M) Assessment of mitochondrial respiratory chain complex I activity. ^* p 0.05, ^** p 0.01, ^*** p 0.001 (n = 3). OE, overexpression.

These studies demonstrated that both lutein and TRIM31 can regulate ubiquitinated Drp1 levels. To further explore whether lutein affects Drp1 expression via TRIM31, TRIM31 expression was knocked down. Western blot results revealed that TRIM31 knockdown significantly decreased TRIM31 protein levels (Fig. 5A) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). RT-qPCR results showed that lutein treatment significantly increased TRIM31 mRNA levels, whereas further knockdown of TRIM31 led to a decrease in its mRNA level (Fig. 5B). In addition, Western blot results revealed that lutein treatment increased TRIM31 protein expression and decreased Drp1 protein expression, while further TRIM31 knockdown partially reversed the effects of lutein on the expression of these two proteins (Fig. 5C) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). These results suggest that lutein inhibits Drp1 expression by promoting TRIM31 transcription and translation.

Fig. 5.

Lutein indirectly regulates Drp1 expression by promoting TRIM31 expression. (A) Western blot detection of si-TRIM31 transfection efficiency. (B) TRIM31 mRNA levels in SH-SY5Y cells measured by RT-qPCR. (C) TRIM31 and Drp1 protein expression in SH-SY5Y cells assessed by Western blotting. ^* p 0.05, ^** p 0.01, ^*** p 0.001 (n = 3). RT-qPCR, reverse transcription quantitative polymerase chain reaction.

To determine whether lutein exerts its effects through the TRIM31/Drp1 signaling pathway, Drp1 was overexpressed in SH-SY5Y cells. Western blot analysis revealed that Drp1 expression was significantly upregulated in the OE-Drp1 group compared with the NC-OE group, confirming successful transfection (Fig. 6A) (The original, uncropped western blot image(s) corresponding to this figure are available in the Supplementary Material). Functional assays, including CCK-8 and flow cytometry, revealed that both lutein treatment and TRIM31 overexpression significantly increased cell viability and suppressed apoptosis, whereas further overexpression of Drp1 effectively reversed these protective effects (Fig. 6B,C). In terms of mitochondrial function, additional Drp1 overexpression led to increased mitochondrial ROS levels, decreased mitochondrial membrane potential, reduced ATP content, and decreased respiratory chain complex I activity compared with the MPP⁺ + lutein group and the MPP⁺ + OE-TRIM31 group (Fig. 6D–G). Collectively, these results indicate that lutein alleviates MPP⁺-induced neuronal loss and mitochondrial dysfunction by regulating the TRIM31/Drp1 signaling pathway.

Fig. 6.

Lutein ameliorates MPP⁺-induced neuronal damage and mitochondrial dysfunction via the TRIM31/Drp1 pathway. (A) Western blot analysis of Drp1 expression to confirm transfection efficiency. (B) CCK-8 assay for SH-SY5Y cell viability. (C) Flow cytometry for apoptosis detection in SH-SY5Y cells. (D) Measurement of mitochondrial ROS levels using MitoSOX Red staining, scale bar: 10 µm. (E) ATP concentration measured by an ATP assay kit. (F) Activity of mitochondrial respiratory chain complex I. (G) Mitochondrial membrane potential assessed by JC-1 staining, scale bar: 50 µm. ^* p 0.05, ^** p 0.01, ^*** p 0.001 (n = 3).