Reagents and antibodies

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), Rhap and MPP+ (1-methyl-4-phenylpyridinium-iodide) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies: TH (F-11, sc-25269; Santa Cruz); p-STING (72971 S), STING (13647T), p-TBK1 (5483T), TBK1 (3504T), p-IRF3 (37829T), IRF3 (4302T), p-NF-κB p65 (3033 S), Alexa Fluor® 555-conjugated anti-rabbit (4413 S) and anti-mouse IgG (4409 S; Cell Signaling Technology); Iba1 (ab289874), LC3B (ab150135; Abcam); GAPDH (60004-1), NF-κB p65 (10745-1-AP), PINK1 (23274-1), Parkin (14060-1), DRP1 (12957-1), cGAS (26416-1), TOM20 (11802-1), P62 (18420-1), DYKDDDDK (20543-1), His tag (84814-1; Proteintech); DNA (690014; Progen). DyLight 488-conjugated goat IgG (70-GAM4882; Multi Sciences) was used. Kits: JC-1 mitochondrial membrane potential (C2006), mitochondria isolation (C3601), HRP-conjugated secondary antibodies (Beyotime Biotechnology, Shanghai), TNF-α/IL-4 ELISA (Invitrogen), and IFN-β1 ELISA (Multi Sciences).

PD mouse model construction and drug treatment

A total of 60 male (8-week-old) C57/6J mice were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). The mice were raised in a temperature-controlled room with an adequate diet and regular lighting over a 12 h/12 h light/dark cycle. Saline, corn oil, MPTP (25 mg/kg) and Rhap (50 mg/kg or 100 mg/kg), alone or together, were used to treat the mice in several groups. The chronic PD model induced by MPTP was established according to our previously described protocol [34]. The mice received intraperitoneal injections of MPTP (25 mg/kg daily) for 5 consecutive weeks, whereas the control (Ctrl) group of mice was administered an equivalent volume of saline. Rhap was initially dissolved in DMSO to prepare a 50 mg/ml stock solution, which was subsequently diluted to working concentrations. The effects of Rhap on both PD model mice and normal mice were investigated after the compound was administered via oral gavage at doses of 50 mg/kg and 100 mg/kg every other day for 5 consecutive weeks. Behavioral assessments were systematically conducted within 7 days following treatment cessation, and brain tissue was isolated for subsequent experimental testing. The animal care and experimental procedures of this study were approved by the Animal Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (Reference number: 2022364).

Behavioral testsOpen field test (OFT)

The mice were placed in the open field center and monitored for 15 min via EthoVision XT (Beijing, China). Movement trajectories were analyzed for total distance, speed, and central zone entries.

Pole-climbing test

A 9 mm × 75 cm metal rod was horizontally positioned. The mice were placed head-down at the apex, and latency (s) to descend was recorded. Five trials per mouse were averaged to calculate the final latency.

Rotarod test

The mice underwent 3 days of training (10 rpm) and formal testing (4–40 rpm acceleration over 5 min) via a rotarod (Ugo Basile SRL, Gemonio, VA, Italy). Fall latency was recorded across three trials with ≥ 1 h intertrial intervals.

Grasping test

The grip test was used to evaluate muscle grip strength. The mice were suspended on a 1 mm horizontal wire (30 cm height) for 10 s. The scores were as follows: 3 (both hind paws), 2 (single hind paw), 1 (no grasp), and 0 (fall). Three trials were performed with 30 min intervals.

BV2 cell culture, drug treatment, and SiRNA transfection

BV2 microglia (purchased from the American Type Culture Collection, ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% FBS/1% P/S under standard conditions (37 °C/5% CO₂), and the medium was changed every 48 h. Neuroinflammatory cell models were established via BV2 cells treated with MPP+. The cells were seeded into 6-well plates at a density of 5 × 105 cells/ml. MPP+ was dissolved in 10 mM PBS and diluted to a working concentration (500 µM). Rhap was dissolved in 500 mM DMSO and diluted to a working solution concentration of 40 µM. MPP+ and Rhap were cocultured with the cells for 24 h to explore the neuroprotective effects of Rhap. Three PINK1-specific small interfering RNAs (siRNA-1: 5′-CACGCTTAGCTGCAAATGT-3′; siRNA-2: 5′-CCAAGCGCGTGTCTGACCC-3′; and siRNA-3: 5′-GCACACTGTTCCTCGTTAT-3′) along with a negative control siRNA were custom designed and synthesized by RiboBio (Guangzhou, China). RNA interference-mediated Pink1 knockdown was performed via the use of Lipofectamine 3000 transfection reagent (Invitrogen) in accordance with the manufacturer’s protocol.

RNA-seq

Total RNA was isolated from mouse substantia nigra brain tissues, and RNA sequencing was performed by Biomarker Technologies Co., Ltd. (Beijing, China). Subsequent data analysis was conducted through the Bioinformatics Pipeline Tool BMKCloud (www.biocloud.net) online platform of Biomarker Technologies Co., Ltd.

Molecular Docking analysis

The 3D structure of Rhap (SDF format) was retrieved from PubChem and energy-minimized in MOE 2022. The PINK1 and DRP1 sequences were sourced from UniProt and structurally optimized via the MOE Amber 10: EHT force field. Molecular docking (MOE-DOCK) between Rhap and murine PINK1 was performed via the TriangleMatcher algorithm and London ΔG scoring, and the top 10 poses according to binding energy (kcal/mol) were selected. The minimal ΔG conformation was analyzed for 3D interactions. A full-length PINK1-DRP1 complex was modeled via AlphaFold3 multimer prediction. Interface analysis identified key binding sites (hydrogen bond networks, hydrophobic clusters) and critical residues.

Cellular thermal shift assay (CETSA)

Soluble protein lysates from BV2 microglia were pretreated with Rhap (40 µM) or DMSO at room temperature for 2 h prior to CETSA heat pulse. Aliquots were transferred to PCR tubes and heated at specified temperatures (40–80 °C) for 3 min via a thermal cycler (Applied Biosystems, USA), followed by cooling at 4 °C. After centrifugation (20,000 × g, 4 °C, 15 min), soluble supernatants were mixed with loading buffer and subjected to Western blotting.

Western blotting

Total protein was extracted from mouse substantia nigra and striatum tissues by adding RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime Biotechnology). Proteins were quantified with a BCA kit (Beyotime Biotechnology), and the proteins were denatured at 100 °C after loading buffer was added. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes with different pore sizes. The samples were then blocked with 5% fetal bovine serum and incubated overnight at 4 °C with the corresponding primary antibody, after which the secondary antibody was added. The chemiluminescence imaging system GeneGnome XRQ (Gene Company, Hong Kong, China) was used along with ECL ultrasensitive luminescent solution, and the results were processed via ImageJ software.

Immunohistochemistry and Immunofluorescence staining

Mouse brains were perfusion-fixed with 4% PFA, cryoprotected in 20–30% sucrose, and sectioned (30 μm) via a Leica cryostat. The sections were blocked before incubation with primary antibodies (4 °C, overnight). For immunofluorescence, species-matched fluorescent secondary antibodies and DAPI nuclear counterstain were applied, and imaging was performed via Leica confocal microscopy. For immunohistochemistry, the sections were incubated with biotinylated secondary antibodies, developed with DAB, and imaged with a Leica brightfield system. ImageJ facilitated the quantitative analysis.

Quantitative RT-PCR

Total RNA was extracted from brain tissue and BV2 cells via TRIzol. The RNA was then quantified to establish a uniform concentration, after which it was reverse transcribed into single-stranded cDNA. The cDNA template was then amplified via the SYBR Green Master Mix kit. The sequences of primers used were listed below: Tnf-α: forward (5′-GCCTCTTCTCATTCCTGCTTGTGG-3′) and reverse (5′- GTGGTTTGTGAGTGTGAGGGTCTG-3′); Ifn-α1: forward (5′- AAGCCATCCCTGTCCTGAGTGAG-3′) and reverse (5′- GCTGCTGGTGGAGGTCATTGC-3′); Il4: forward (5′- TACCAGGAGCCATATCCACGGATG-3′) and reverse (5′- TGTGGTGTTCTTCGTTGCTGTGAG-3′); Il10: forward (5′- TCCCTGGGTGAGAAGCTGAAGAC-3′) and reverse (5′- CACCTGCTCCACTGCCTTGC-3′); Ifn-β1: forward (5′- GCGTTCCTGCTGTGCTTCTCC-3′) and reverse (5′- TGAAGTCCGCCCTGTAGGTGAG-3′); Gapdh: forward (5′- GAAGGTCGGTGTGAACGGAT-3′) and reverse (5′- CAATCTCCACTTTGCCACTGC-3′). Gapdh was used as an internal reference for normalization, and the final results were quantified via the 2−∆∆Ct method.

Enzyme-linked immunosorbent assay (ELISA)

Isoflurane-anesthetized mice were subjected to ocular blood sampling. The blood and media extracts were then centrifuged at 5500 rpm, and the upper serum and extracts were stored at −80 °C until further analysis. The TNF-α, IFN-β1 and IL-4 protein levels were detected with the corresponding mouse ELISA kits (Thermo Fisher) according to the manufacturer’s instructions.

Coimmunoprecipitation

Protein interactions were verified by coimmunoprecipitation (Co-IP) in HEK293T cells. At 80–90% confluence, the cells were transfected with PINK1-FLAG and DRP1-HIS plasmids (Dongze Biotech) for 24 h, followed by 24 h of Rhap treatment. The cells were lysed in IP buffer (Beyotime P0013; 3 h, ice), centrifuged (13,000 rpm, 30 min), and the supernatants were quantified via a BCA assay. Protein aliquots (3 mg) were incubated with primary antibodies overnight (4 °C) and then mixed with Protein A/G beads (4 h). The beads were washed 5× with lysis buffer, resuspended in 2× loading buffer, and denatured (100 °C, 10 min).

Cytosolic MtDNA extraction and quantification

The mtDNA content in the cytoplasmic fractions was analyzed via a mitochondrial isolation kit (Beyotime Biotechnology). BV2 cells were fractionated into cytosolic, nuclear, and mitochondrial components. The cell pellets from the 10-cm dishes were resuspended in 0.5 ml of mitochondrial isolation reagent, incubated on ice (15 min), homogenized 13 times in a glass homogenizer, and centrifuged (600 × g, 4 °C, 15 min). The supernatants were subsequently subjected to secondary centrifugation (11,000 × g, 4 °C, 10 min), after which the cytoplasmic extracts were collected and stored. Cytosolic DNA was extracted via the Tengen DNA Kit. mtDNA levels were quantified by RT-qPCR/PCR with primers listed below: mt-Cytb: forward (5′- GGAGACCCAGACAACTACAT-3′) and reverse (5′- GATTGAGCGTAGAATGGCGTATGC-3′); mt-Nd1: forward (5′- CGAGCCGTAGCCCAAACAATTTC-3′) and reverse (5′- CTATGGGTCAGGCTGGCAGAAG-3′); mt-Nd2: forward (5′- GCATGAGGAGGACTTAACCAAACAC-3′) and reverse (5′- GTAGAGTGAGGGATGGGTTGTAAGG-3′); mt-Co2: forward (5′- CCGTTAAAACCATAGGGCACCAATG-3′) and reverse (5′- ACTTCTAGCAGTCGTAGTTCACCAG-3′); mt-Dloop: forward (5′- TCCTCCGTGAAACCAACAACCC-3′) and reverse (5′- CGCATTTGATGGCCCTGAAGTAAG-3′); 18sRNA: forward (5′- CGGACACGGACAGGATTGACAG-3′) and reverse (5′- TGCCAGAGTCTCGTTCGTTATCG-3′). 18sRNA was used as an internal reference for normalization, and the final results were quantified via the 2−∆∆Ct method.

Transmission electron microscopy (TEM)

BV2 cell ultrastructure was analyzed via transmission electron microscopy (TEM). The cells were fixed in electron microscopy fixative (4 °C, 30 min), washed three times with 1 M phosphate buffer, fixed in buffer (2 h), dehydrated in acetone, and embedded in 812 resins (2 h infiltration, 37 °C polymerization overnight). Ultrathin Sects. (50–80 nm) were uranium-lead stained and imaged via transmission electron microscopy (TEM).

Mitochondrial membrane potential

The mitochondrial membrane potential was detected according to the instructions of the JC-1 assay kit. The treated BV2 cells were washed three times with precooled PBS, incubated for half an hour with 1 ml of JC-1 staining solution, and then washed five times with JC-1 buffer. Images of the treated cells were acquired under a confocal microscope.

Statistics

Statistical analyses were performed with GraphPad Prism v10. Two-group comparisons were performed with Student’s t test; multigroup analyses were performed with ANOVA (one-/two-way) with Tukey’s post hoc test. The data are expressed as the means ± standard errors of the means (SEMs), with P < 0.05 considered significant.