Patients and samples resources

This study was conducted under the guidance of the Ethics Committee of the Haikou People's Hospital (ethics approval no. 2024–89). All animal experiments were conducted in strict compliance with the IACUC guidelines.

Cell culture

HT22 cells were purchased from Sigma-Aldrich and cultured in accordance with the manufacturer’s instructions. OM-MSCs were collected and cultured from the olfactory mucosa of mice. In this study, we collected samples from the root of the mouse middle turbinate and washed the samples with an antibiotic–antimycotic solution (Invitrogen, CA). The samples were cut into pieces with a length of 2 mm and thickness ranging from 300 to 400 μm. The sample pieces were placed in a dish containing Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, CA) containing 10% fetal bovine serum (FBS). The tissue slides were cultured at 37 °C and 5% CO2. The medium was changed every three days during the culture process.

Isolation and identification of OM-MSCs exosome

The Exosomes were isolated from the OM-MSCs medium through ultracentrifugation using previously reported methods (Xun et al. 2020; Théry et al. xxxx). FBS was depleted with exosomes by ultracentrifugation at 100,000 × g for 18 h, and the supernatant was filtered with a 0.22 μm syringe filter (Millipore, USA). The 90% confluent OM-MCS cultures were washed twice with PBS and incubated in growth medium containing DMEM/F12 supplemented with 10% exosome-depleted FBS for two days. After that, cells were removed from conditioned medium (CM) together with dead cells and cells fractured under 300 g/10 min, 2000 g/10 min, and 10,000 g/30 min, respectively, at 4 °C. Then, the CM was filtered with a 0.22 μm syringe filter. The supernatant was first centrifuged at 100,000 g for 70 min at 4 °C using an SW32Ti rotor (Beckman Coulter, Netherlands). The exosome-containing pellet was washed with PBS and centrifuged at 100,000 g for 70 min. Finally, the pellet was resuspended in PBS (0.1 mL. The protein concentration was measured using a BCA kit (Beyotime, China).

Cell transfection

HT22 cells were cultured in 6-well plates until attachment. To inhibit lncRNA A2M-AS1, TP53INP1, or IGF2BP1, short hairpin RNA (shRNA) against the corresponding genes, and noncoding shRNA (sh-NC) were obtained from GeneCopoecia (Guangzhou, China). In addition, Lnc A2M-AS1, TP53INP1, or IGF2BP1 sequences were inserted into the pcDNA3.1, vector (termed oe-Lnc A2M-AS1, oe-TP53INP1, or oe-IGF2BP1). An empty pcDNA3.1 vector (oe-NC) as a control was obtained from RiboBio (Guangzhou, China). All plasmids were transfected into HT22 cells using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions, followed by co-culture with OM-MSCs derived exosomes as an experimental group or medium as a control. The corresponding cells were used for further experiments after 48 h of incubation.

Ingestion of PKH67-labeled exosomes by recipient HT22 cells

The Exosome pellets were isolated as described above. The pellets were labeled with PKH67(Merck, United States) according to a previously described protocol (Pužar Dominkuš et al. 2018). The cells were then placed on an eight-well chamber slide. After 24 h incubation for quantitation, 8 μg of labeled exosomes was added to the media of HT22 cells and incubated together at 37 °C for 24 h. HT22 cells added with same volume of exosome-free medium were used as blank controls. The cells of the experiment group and the control group were fixed with 4% paraformaldehyde over a period of 20 min and stained with DAPI. Next, the uptake of exosomes was observed and evaluated using confocal microscopy.

qPCR analysis

Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Hilden, Germany). mRNA was extracted from total RNA using the Dynabeads mRNA DIRECT kit (61,006, Thermo Fisher Scientific) according to the manufacturer’s protocol. cDNA was synthesized from mRNA using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The PCR process was then conducted using the virous primers purchased from OriGENE company (USA) for corresponding genes including LC3, A2M-AS1 and TP53INP1 with SYBR Green PCR Master Mix kit (Takara, Japan) and 0.5 μL of DNA template under the instruction given by the manufacturer. Then the mixture was put into thermal cycling under these conditions: 95 °C for 3 min following by 95 °C for 30 s, 52 °C for 30 s and 72 °C for 50 s for 30 cycles. To quantify the expression level, the relative mRNA expression levels were defined with the fluorescence intensity observed.

Western blotting

The Western blotting was performed according to classic instructions. The cells were collected, and total protein was extracted with RIPA lysis buffer (Thermo Fisher Scientific, USA). The quantity of total protein was measured using the Pierce BCA Protein Assay Kit (Thermofisher, China). Proteins were extracted from the medium and quantified using the same method. Then, 150 μL of the lysate protein mixture was mixed with 50 μL of 4 × loading buffer and boiled for 5 min. Total protein at Equal amounts were separated by 10% SDS (sodium dodecyl sulfate–polyacrylamide) gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Beyotime, China). The membranes were immunoblotted using the following primary antibodies: CD9(1:1000, ab307085, Abcam), CD63(1:1000, ab315108, Abcam), calnexin (1:1000, ab22595, Abcam), LC3(1:2000, ab192890, Abcam), p62(1:10,000, ab109012, Abcam), COX IV (1:2000, ab202554, Abcam), TP53INP1(1:2000, ab202026, Abcam), IGF2BP1(1:1000, ab290736, Abcam), and TH (1:5000, ab137869, Abcam). After incubation at 4 °C for 8 h, the membranes were blocked with skimmed milk at a percentage of 5%. The secondary antibodies linked to horseradish peroxidase (Beyotime Institute of Biotechnology) were applied to the membrane, and the protein bands were visualized using ECL reagent (Thermo Fisher Scientific, USA) using a chemiluminescence system (Bio-Rad, USA). Total protein was semiquantified based on the ratio of GAPDH for each protein on each gel. In the present study, ImageJ software (1.53 version, United States) was used to quantify the results of Western Blotting.

RNA Pull-Down and RNA immunoprecipitation (RIP) assay

RNA pull-down analysis was conducted as previous protocol described (Chopra et al. xxxx). the RNA–protein complex was immunoprecipitated using streptavidin magnetic beads (MedChemExpress, United States). The complex was then separated for PCR and western blotting. RIP was performed using an RIP kit (Merck, United States), according to the manufacturer’s instructions. RNA was purified using TRIzol (Thermo Fisher, United States) for qPCR analysis.

RNA stability test

The Cells transfected with oe-A2M-AS1 and/or sh-IGF2BP1(short hairpin RNA) were treated with actinomycin D(ActD) (Merck, United States) at a dose of 5 μg/mL for 0, 3, and 6 h to inhibit transcription. The cells were then subjected to qPCR to measure level of TP53INP1 mRNA for the evaluation of the stability of TP53INP1 mRNA.

Animal model

Neurotoxity was induced in C57BL/6 mice at an age of 10–12 weeks by giving MPTP through intraperitoneal injection at a dose of 20 mg/kg/d for 14 days to establish PD animal models. Mice injected with saline of the same dose during the same period were set as blank control.

To examine the inhibition effect of OM-MSC and the mechanism behind this, other two experiment groups were set with PD mice models injected OM-MSC-Exosomes and sh-Lnc A2M-AS1 together were set as other two experiment groups. Mice in each experimental group received 1.4 × 10 (Williamson et al. 2023) nanoparticles of OM-MSC exosomes, which were extracted in previous steps, with or without sh-Lnc A2M-AS1 nanoparticles of same dose, respectively, through stereotactic injection into the right lateral ventricle one week after the final MPTP injection at coordinates which was suggested by Zhuo et al. in a previous study (Zhuo et al. 2024). The control group received saline of same amount as a blank control with identical injection method. Each experimental group and the control group consisted of 6 mice.

Animal behavior tests

The mice were taken for behavior experiments including open field test and APO rotation test at the 4th to 6th weeks, and sacrificed for autopsy analysis after 6 weeks. In the open field test, the mice were placed in the middle of an experiment case with a size of 50 × 50 × 40 cm, and the bottom of the box was divided into squares of same area when enclosed by an opaque black box. Then the black box was removed after 30 s and the behavior of mice was recorded by video for 3 min. The video images were analyzed using Image OF software (O’Hara & Co., Ltd., Japan). In the rotation-induction test, the mice from each group were injected APO (Sigma-Aldrich, United States) of 0.5 mg/kg. The number of rotations of induced PD rats within 30 min was compared with the model and blank control, and the improvement of motor function of rats in the experimental group was observed.

H&E and nissl staining

For H&E staining, after unfrozen with water at room temperature, the tissues were first stained with Harris hematoxylin solution (Merck, United States) for 1 min, Then the sample slides were rinsed with tap water for 15 min and placed in distilled water and 95% alcohol for 30 s, respectively. The samples were counterstained with eosin Y solution (Merck, United States) for 1 min. Finally, the slides were dehydrated and mounted with resinous mounting medium (Merck, United States).

For Nissl staining, after the samples were hydrated, the slides were placed in a cresyl violet acetate solution (Merck, United States) for 5 min. Then After rinsed in distilled water for 3 times, the slides were dehydrated with 95% alcohol and mounted with resinous mounting medium.

Statistical analysis

All data presented in this study were analyzed with consistent values and expressed as mean ± standard deviation (SD). Data were analyzed using SPSS software (version 26.0; IBM Inc.), differences in gene expression levels were analyzed using paired t-tests, and correlations between data were analyzed using Pearson correlation analysis. p < significance was defined as p < 0.05.