Melatonin attenuates MPP⁺-induced autophagy via heat shock protein in the Parkinson’s disease mouse model

Animal grouping and modeling

Twelve SPF C57BL/6 male mice (8–10 weeks, 25–30 g) purchased from Sibeifu Biotechnology Co., Ltd., Beijing (China). Mice were maintained and bred in specific pathogen-free conditions at the Tianjin Jinke Bona Biotechnology Co., Ltd., whose animal Ethics Committee provided full approval for this research (approval number: GENINK-20230036). The mice were housed three per cage under a 12/12-h light/dark cycle at 22–23 °C with 40–70% humidity. They had free access to food and water and were acclimated for 1 week before the experiment.

They were equally divided into four groups: normal control, melatonin control, Parkinson’s model and melatonin treatment, with three mice in each group. The PD model was induced by intraperitoneal injection of MPTP (20 mg/kg) twice daily, with melatonin (10 mg/kg) administered half an hour after each MPTP injection for seven consecutive days. One hour after the last dose, the limb motor ability of the mice was measured by behavioral method. Mice was euthanized by carbon dioxide immediately followed by cervical dislocation for further ex vivo analyses. After euthanasia, the brain were harvested and cleaned with ice-cold PBS. Each mouse brain tissue were cut in half, one half fixed at 4 °C with 4% paraformaldehyde (PFA, pH 7.4) for 48 h and then embedded in paraffin for histological analysis and the remaining half snap-frozen in liquid nitrogen before stored at −80 °C for Western blot analysis.

Behavior tests

Behavioral tests began 1 day after the final drug injection following adaptive training. The open field test (OFT) was used to evaluate mice’s autonomy and exploratory behavior in response to a novel environment, reflecting anxiety levels. Mice were placed in the center of an open field box (50 × 50 × 40 cm³), divided into 16 compartments (12 peripheral and four central). They were allowed to move freely for 1 min while the VisuTrack video analysis system recorded immobility time in total and non-central regions, as well as the number and duration of entries into the central area. The box was cleaned with 75% alcohol after each experiment to remove odors. Reduced entries into the central zone, lower moving speed, and decreased total distance traveled indicated heightened anxiety.

Western blotting (WB)

The brain tissues stored at −80 °C were taken out and homogenized in a cold lysis buffer containing protease and phosphatase inhibitors (Cat# R0020; Solarbio, Beijing, China) and centrifuged at 12,000× g for 15 min at 4 °C. The supernatant was transferred to a fresh tube and measured for protein concentration using BCA assay (Cat# P0011; Beyotime, Shanghai, China). The lysate was diluted to a uniform protein concentration with lysis.

The samples were prepared for western blot analysis by mixing the diluted lysate with 5× protein loading buffer (Cat# P0015L; Beyotime, Shanghai, China), and then boiling at 100 °C for 10 min. The boiled samples were centrifuged briefly and 20 µg of each sample was taken to load into the wells of the 10% SDS-PAGE gel, along with a molecular weight marker (Cat# P1200; Solarbio, Beijing, China) for electrophoresis.

Separated proteins were transferred from SDS-PAGE gels to PVDF membranes using the wet transfer method at 100V for 1 h (Spencer et al., 2000). The membranes were then blocked in Tris-buffered saline solution with 0.1% Tween-20 (TBST) and 5% skim milk. After washing three times with TBST, the membranes were incubated with primary antibodies against HSP70 (1:1,000, CST #4872), CDK5 (1:1,000, CST #2506), LC3 II /LC3I (1:1,000, CST #4108), p62 (1:1,000, CST #5114) and GAPDH (UM4002; Tianjin Youkang Biotech, Tianjin, China, diluted to 1:2,000) for 1 h at room temperature. After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies for 0.5 h at room temperature. Finally, the immunoblots were visualized using the chemiluminescence method (Tanon 5200; Shanghai Tianneng, China). The relative protein levels were measured by Gel-Pro analyzer (Media Cybernetics, Rockville, MD, USA).

Immunohistochemistry

The PFA fixed tissues were washed with PBS and then went through dehydration by immersing in a graded ethanol series (70% ethanol, 1 h; 80% ethanol, 1 h; 95% ethanol, 1 h; and 100% ethanol, 2 h), clearing with xylene at room temperature for 2 h, infiltration with melted paraffin wax at 60 °C for 2 h. Treated brain tissues were put into a mold filled with melted paraffin and oriented appropriately to allow the paraffin to solidify at room temperature. After that, the embedded tissue blocks were stored at 4 °C until sectioning. Paraffin sections of brain tissue (4 μm thick) were stained with DAT antibody (1:400, cat#ab184451, Abcam, Cambridge, MA, USA) and incubated with horseradish peroxidase (HRP) for 50 min. The target protein was stained by the DAB staining method. Staining results were examined using an optical microscope (DP26, OLYMPUS, Tokyo, Japan). For analysis, at least six fields of view at 20× magnification were randomly selected from each section. DAT expression in brain tissue was quantitatively assessed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) by calculating the integrated optical density (IOD) per region of interest (AOI) in the positively stained areas.

Statistical analysis

All data were analyzed by either SPSS version 22.0 (IBM Corp, Armonk, New York, USA) or GraphPad Prism (GraphPad Software, San Diego, CA, USA). All values are expressed as mean ± SD. Unpaired Student’s two-tailed t test was used for two sample data comparison. Two-way ANOVA was used for multiple comparisons. The correlations were determined by calculating Pearson linear correlation coefficients or Spearman rank correlation coefficients. Significance levels were indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Effects of melatonin on behavior of PD mice

Mouse behavior analysis was performed with the open field test where mouse movement was tracked and evaluated with the VisuTrack tracking system (Fig. 1A). The average movement was significantly reduced in the Parkinson’s model group compared to the control group (t = 5.048, p < 0.05), confirming impaired motor function. Conversely, the treatment group showed a significant improvement in movement compared to the model group (t = 4.311, p < 0.05). Similarly, the number of times the mice entered the central zone was significantly lower in the model group than in the MT control group (t = 7.326, p < 0.05). However, this metric significantly increased in the treatment group compared to the model group (t = 5.500, p < 0.05), further validating the successful establishment of the Parkinson’s disease model (Figs. 1B, 1C).

Effects of melatonin on dopamine neurons in the substantia nigra of PD mice

Compared to the control group, DAT-positive nerve fibers in the substantia nigra of the MPTP group and MT treatment group were significantly reduced (t = 3.852, p < 0.01 and t = 2.919, p < 0.05). Melatonin treatment significantly increased the number of DAT-positive nerve fibers (t = 3.443, p < 0.01) (Figs. 2A and 2B).

Effects of melatonin on HSP70 protein expression in the PD mouse model

HSP70 protein expression in the MPTP group, melatonin group and melatonin treatment group were significantly decreased compared to the control group (t-test showed p < 0.01). Melatonin treatment and melatonin group significantly increased HSP70 protein expression compared to the MPTP group (t-test showed p < 0.05) (Figs. 3A and 3B).

Effects of melatonin on CDK5, LC3 II /LC3I, and p62 protein expression in the PD mouse model

Western blot analysis showed that the relative protein expressions of CDK5 protein expression was significantly inhibited in the MPTP group compared to the control group (t = 122.0, p < 0.0001), while melatonin treatment significantly increased CDK5 expression (Figs. 4A, 4B). LC3 II/LC3I and p62 in MPTP groups and melatonin treatment group were significantly higher than those in control group (t-test showed p < 0.05). while melatonin treatment significantly decreased it (t = 13.09, p < 0.01) (Figs. 4C, 4D).