Bioinformatic analysis
First, miRNAs related to PD were identified with the help of existing literature, and subsequently, the PD-associated miRNA expression dataset GSE16658 deposited in the GEO database was extracted and analyzed using the R language. There were 32 samples in the GSE16658 dataset, comprising 13 normal samples and 19 PD samples. The enrichment of miRNA in various EVs was detected by the EVmiRNA database. Furthermore, Diana TOOLS (miTG score >0.35) and microRNA (energy < −15, mirsvr_score < −0.6) were adopted to predict the miRNA downstream genes, which were intersected with the human transcription factors obtained from hTFtarget, Cistrome, and JASPAR databases to determine the downstream transcription factors of miRNA. A protein-interaction network was then constructed with String (minimum required interaction score: 0.4), and the core degree was calculated using Cytoscape to determine the most critical transcription factors. In addition, the binding sites of key transcription factors targeted by miRNA were obtained from DIANA TOOLS. Moreover, the downstream pathway of transcription factors was determined by Coexpression analysis from MEM and existing literature.
Cell culture and transfection
Human neuroblastoma cell line SH-SY5Y was procured from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM (30030, Gibco, Carlsbad, California, USA) supplemented with 100 U/mL penicillin and 100 U/mL streptomycin (15070063, Gibco) and 10% fetal bovine serum (FBS, 16140089, Gibco). The cells were cultured in a humid incubator containing 5% CO2 at 37 °C.
SH-SY5Y cells (4 × 105 cells/well) were subsequently seeded in a 6-well plate, and transduced using Lipofectamine 2000, and plasmids were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, China). After culturing for 48 h, 6-OHDA was added at the specified concentration for 24 h. Cells in the designated groups were treated with p38 inhibitor SB203580 (10 μmol/L, Sigma-Aldrich, St. Louis, USA) [31]. In the subsequent groups, a dosage of 100 μM was selected for the 6-OHDA treatment, and the cells were divided into the following groups: blank (no treatment), 6-OHDA (6-OHDA treatment), 6-OHDA + NC mimic (6-OHDA treatment + transduced with NC mimic), 6-OHDA + miR-181a–2–3p mimic (6-OHDA treatment and transduced with miR-181a–2–3p mimic), NC mimic (transduced with NC mimic), miR-181a–2–3p mimic (transduced with miR-181a–2–3p mimic), NC inhibitor (transduced with NC inhibitor), miR-181a–2–3p-inhibitor (transduced with miR-181a–2–3p inhibitor), 6-OHDA + NC inhibitor + si-NC (6-OHDA treatment + transduced with NC inhibitor and si-NC), 6-OHDA + NC inhibitor + si-EGR1 (6-OHDA treatment + transduced with NC inhibitor and si-EGR1), 6-OHDA + miR-181a–2–3p inhibitor + si-EGR1 (6-OHDA treatment + transduced with miR-181a-2-3p inhibitor and si-EGR1), 6-OHDA + oe-NC + si-NC (6-OHDA treatment)+ transduced with oe-NC + si-NC, 6-OHDA + oe-NC + si-NOX4 (6-OHDA treatment + transduced with oe-NC + si-NOX4), 6-OHDA + oe-EGR1 + si-NOX4 (6-OHDA treatment + transduced with oe-EGR1 + si-NOX4), 6-OHDA + oe-NC + DMSO (6-OHDA + DMSO treatment + transduced with oe-NC), 6-OHDA + oe-NC + SB203580 (6-OHDA + SB203580 treatment + transduced with oe-NC), 6-OHDA + oe-NOX4 + DMSO (6-OHDA + DMSO treatment + transduced with oe-NOX4), 6-OHDA + oe-NOX4 + SB203580 (6-OHDA + SB203580 treatment + transduced with oe-NOX4), 6-OHDA + NC mimic + oe-NC (6-OHDA treatment + transduced with NC mimic + oe-NC), 6-OHDA + miR-181a–2–3p mimic + oe-NC (6-OHDA treatment + transduced with miR-181a–2–3p mimic + oe-NC), and 6-OHDA + miR-181a–2–3p mimic + oe-NOX4 (6-OHDA treatment + transduced with miR-181a–2–3p mimic + oe-NOX4) groups.
Isolation and culture of mouse primary neuron cells
The whole body of mice was wiped with alcohol, the head was cut off, and the scalp and skull were carefully peeled off to collect the intact brain tissues, which were placed in ice-cold PBS. Subsequently, the meninges were carefully peeled off using pointed tweezers, and the blood vessels in the cerebellum and brain were collected in EP tubes. The tissues were repeatedly cut and incubated with prewarmed trypsin for 20 min. After the digestion was completed, serum-containing DMEM was added to terminate digestion, and pipetting was repeated several times. The tissue mass was removed using a 200-mesh sieve. The following day, the medium was renewed to neuron culture medium and added with 5-fluoro-deoxyuridine after 48 h.
Extraction and identification of MSC–EVs
Healthy human umbilical cord mesenchymal stem cells (hucMSCs) (Yiyan Biotechnology, Shanghai, China) were cultured overnight in DMEM without EVs. Upon reaching 80–90% cell confluency, the supernatant was centrifuged and precipitated in a serum-free DMEM containing 25 mM HEPES (pH = 7.4), and centrifuged again. The obtained precipitate was stored at −80 °C.
TEM was adopted to identify EVs. Briefly, 10 μL of EVs were allowed to stand for 1 min. Next, the EVs were counter-stained with 30 μL of PTA (pH = 6.8) for 5 min, and an incandescent lamp was used to bake-dry the EVs, and photographs were taken under the TEM.
In short, the Zetasizer Nano-ZS90 instrument (Malvern Instruments, Worcestershire, UK) was adopted. The EV sample was diluted with 0.15 M NaCl (1:50) for detection.
The EVs were dissolved in RIPA and quantitatively identified. The antibodies used were purchased from Abcam (Cambridge, UK): CD9 (ab92726, dilution ratio of 1:1000), CD63 (ab134045, dilution ratio of 1:1000), TSG101 (ab125011, dilution ratio of 1:2000), and calinexin (ab22595, dilution ratio of 1:1000).
EV-uptake assay
Purified EVs were mixed and incubated with PHK67 (MINI67, Sigma-Aldrich) for 5 min. The staining was stopped with the addition of 2 mL of 10% bovine serum albumin (BSA). Subsequently, EV-free serum medium was supplemented to 8.5 mL, and 1.5 mL of 0.971 M sucrose was slowly added from the bottom of the test tube to ensure that PKH-67-EVs were on top of the sucrose solution. Following centrifugation, EV pellets were resuspended in PBS. Next, SH-SY5Y cells were cultured, the medium was renewed for 48 h, and added with PKH67-labeled EVs at 37 °C for 6 h. Later, the cells were fixed with 4% polytetrafluoroethylene for 15 min, stained with 0.1 g/mL DAPI (C1002, Beyotime, Nantong, China) for 5 min, and then SH-SY5Y cells were placed in a fluorescence microscope (Olympus, Tokyo, Japan) to detect the fluorescence expression.
Cy3-labeled miR-181a–2–3p transfection
The pre-miR miRNA precursor (miR-181a–2–3p, Ambion, Austin, Texas, USA) was labeled with Label IT provided by siRNA Tracker Cy3 kits (Mirus, Madison, Wisconsin). The MSCs (1 × 105) were transfected with 10 nM Cy3-labeled pre-miR miRNA precursor. After that, the medium was renewed with fresh serum-free AIM V medium (Invitrogen) and used to prepare EVs one day after cultivation.
Absolute quantitative detection of miRNA
The concentration of miRNA, measured with the help of nucleic acid A260 nm spectrophotometry, was converted into copy number to prepare a standard curve. Following RT-qPCR, its PCR signal (crossover point, Cp) was interpolated into the standard curve using the LightCycler®480 software 1.5 (Roche, Basel, Switzerland) to determine the concentration of miRNA. The miRNA presenting with the highest degree of enrichment was selected, and the copy number of each explant was quantified using the RT-qPCR absolute-quantification method. In addition, synthetic nonhuman Xaer-miR-39 was added during RNA-isolation process. The copy of cel-miR-39 was quantified using absolute quantification of RT-qPCR to evaluate the similar recovery efficiency of cel-miR-39 in the test samples. As a result, there was no deviation in the quantification of miR-181a–2–3p. Afterward, the number of explant particles in each formulation was measured by means of nanoparticle-tracking analysis (NTA) or qNano. We chose two independent quantifications to overcome the lack of optimal quantification criteria for explant particles.
Radioactive dopamine-uptake test
Cells seeded in a 24-well plate were incubated with 16 nM [3H]dopamine (32,6 Ci/mmol Perkin Elmer, Waltham, MA). The uptake reaction was then stopped. In order to release the ingested [3H]dopamine, the cells were incubated with 500 μL of 96% (v/v) ethanol at 37 °C for 30 min. Subsequently, an aliquot of 5 mL scintillation fluid was added to the vial to evaluate the radioactivity in a scintillation counter.
MTT assay
Cells (8 × 104 cells/well) seeded in a 24-well plate were incubated with 0.5 mg/mL MTT reagent for 2 h at humid atmosphere containing 5% CO2 at 37 °C. After the medium was removed, the formazan crystals were dissolved with 200 μL of solubilization buffer [20% SDS (pH 4.7)] within necessary time. Afterward, the chromaticity was measured with a spectrophotometer at a wavelength of 570 nm.
Detection of intracellular oxidation level
The H2DCFDA probe was adopted to assess the cellular oxidant content, which could pass through the membrane, and the probe could be converted into a fluorescent compound after oxidation. Following treatment, the culture medium was renewed with DMEM medium containing 10 μM H2DCFDA. After incubation, the cells were lysed in a buffer containing PBS and 1% NP-40. Afterward, the fluorescence in the lysate (λex = 495, λem = 530) was measured using a Fluoroskan Ascent FL microplate fluorometer.
Lactate-dehydrogenase (LDH) release assay
After cell treatment, the medium was centrifuged at 1000 × g for 10 min. Subsequently, LDH activity in the obtained supernatant was determined using spectrophotometry with LDH-P UV AA kits, according to the manufacturer’s instructions. In short, the conversion of reduced nicotinamide adenine dinucleotide to oxidized nicotinamide adenine dinucleotide was tracked at a wavelength of 340 nm. The results were expressed as a percentage of the control.
Isolation and quantification of RNA
Total RNA content was extracted from SH-SY5Y cells or EVs using the TRIzol reagent (Catalog 16096020, Thermo Fisher, Austin, Texas, USA). Reverse-transcription kits (RR047A, Takara, Otsu, Shiga, Japan) were adopted for mRNA detection. Poly-A tailing detection kits (B532451, Sangon, Shanghai, China; containing universal PCR primer R and U6 universal PCR primer R) was further used for miRNA. SYBR® Premix Ex TaqTM II kit (DRR081, Takara) were used for sample loading, and RT-qPCR was performed in fluorescent quantitative PCR instrument. U6 mRNA levels were used as the internal control. For miRNA in EVs, syn-cel-miR-39 was employed as an endogenous control for data normalization. The primer sequences are listed in Supplementary Table 1. The 2−ΔΔCt method was used.
Western blot
Total protein content was extracted from SH-SY5Y cells or EVs using RIPA kits (R0010, Solarbio, Beijing, China). Briefly, 40 µg of each sample was extracted, separated with 10% SDS-PAGE, and electrotransferred onto a PVDF membrane (Merck Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked and incubated with the following primary antibodies: EGR1 (dilution ratio of 1:1000, #4154, CST, Beverly, MA, USA), NOX4 (dilution ratio of 1:2000, ab109225, Abcam), p–p38 (dilution ratio of 1:1000, #4511, CST), P38 (dilution ratio of 1:1000, #8690, CST), TH (dilution ratio of 1:5000, ab137869, Abcam), 4-HNE (dilution ratio of 1:1000, ab46545, Abcam), and GAPDH (dilution ratio of 1:10000, ab128915, Abcam). Afterward, the membrane was incubated with IgG (dilution ratio of 1:10000, ab97051, Abcam). Later, the membrane was developed using an electrogenerated ECL, and then the membrane was exposed to light using the Image Quant LAS 4000 C gel imager (GE Company, Schenectady, NY, USA) and analyzed with the ImageJ software (1.48 u, National Institutes of Health, Maryland).
Chromatin-immunoprecipitation (ChIP) assay
ChIP assay was carried out with the help of ChIP kits (Merck Millipore). Briefly, SH-SY5Y cells were taken from each group, and added with 1% formaldehyde upon reaching 70–80% cell confluency. Next, the cells were fixed to make intracellular DNA and protein cross-linking, and then the fixing was stopped with the addition of glycine. Subsequently, the complex was lysed at 4 °C and randomly fragmented into 500–1000 bp fragments by means of ultrasonification. The obtained supernatant was collected and divided into three tubes. The positive-control antibody RNA polymerase II, negative-control antibody normal rabbit IgG, and rabbit anti-EGR1 (dilution ratio of 1: 50, #4154, CST) were added. Protein Agarose/Sepharose was used to precipitate endogenous DNA–protein complexes, which was de-cross-linked at 65 °C. DNA fragments were purified by phenol/chloroform. NOX4 promoter expression was tested using RT-qPCR and primers are shown in Supplementary Table 2.
Dual-luciferase reporter gene assay
A dual-luciferase reporter gene assay was adopted to verify whether EGR1 could be targeted by miR-181a–2–3p, and human EGR1 3′UTR WT fragments containing miR-181a–2–3p binding sites were synthesized. Endonuclease sites, SpeI and Hind III, which introduced pMIR reporter, were obtained from Huayueyang Biotechnology, Beijing, China. MUT sites were designed based on the EGR1 WT 3′UTR and miR-125a-5p binding site, and the target fragment was subsequently inserted into the pMIR-reporter plasmid. Lipofectamine 3000 (Invitrogen) was employed to cotransfect the plasmids with miR-181a–2–3p mimic or NC-mimic into HEK293T cells. The luciferase activity at a wavelength of 570 nm was detected using a dual-luciferase reporter system (Promega, Madison, WI) and Glomax20/20 luminometer fluorescence detector (Promega). Relative luciferase activity = Firefly luciferase activity/Renilla luciferase activity
CCK-8 assay
SH-SY5Y cells (at 1 × 103 cells/well) were seeded in a 96-well plate and cultured for 1–5 days, and cell-proliferation activity of SH-SY5Y in different groups was subsequently tested according to the manufacturer’s instructions of the CCK-8 kit (K1018, Apexbio, Boston, MA, USA). Afterward, 10 μL of CCK-8 solution was added and incubated for 1 h, and absorbance was measured at a wavelength of 450 nm.
Flow cytometry
AnnexinV/PI apoptosis-detection kits (BD Biosciences, Franklin Lakes, NJ) were adopted to detect apoptosis rate. Briefly, the cells were resuspended in binding buffer and incubated with 5 μL of fluorescein isothiocyante (FITC)–Annexin V and 5 μL of PI for 15 min, and the FACScan flow-cell flow system (Becton Dickinson, San Diego, CA, USA) was used for apoptosis detection.
ROS detection
Different kits provided by Jiancheng Bioengineering Institute (Nanjing, China) were employed for the detection of SOD levels (Cat. No.: A001-3) and MDA levels (Cat. No.: A003-1) in cell culture medium. Cells in each group were seeded in 24-well plates, incubated with 200 μL of serum-free DCFH-DA, and fluorescence was subsequently detected by means of flow cytometry.
Construction of PD mouse models
Injections of 6-OHDA into the right striatum were used to establish a PD mouse model. Briefly, the mice were placed in a stereotaxic device (Neurostar, Tubingen, Germany) and anesthetized with 2% isoflurane. The coordinates of the same insertion point and different depths in the right striatum were as follows: AP–0.2 mm, ML–3 mm, DV–4.5 mm, AP–0.2 mm, ML–3 mm, and DV–5.5 mm. Subsequently, the mice were stereotactically injected with 6-OHDA (3 μL of 5 μg/μL 6-OHDA solution containing 0.2% ascorbic acid). Meanwhile, the sham operated mice were injected with 0.2% ascorbic acid normal saline (n = 8). APO-induced rotation is regarded as an ideal predictor of maximum dopamine consumption in the unilateral striatum to evaluate and screen 6-OHDA-induced PD mice [35]. On the 4th week post operation, the mice were injected with 0.5 mg/kg APO intraperitoneally. After 5 min of APO administration, rotation data were recorded continuously for 30 min. The mice with 7 laps per min were regarded as successfully modeled mice [36]. The successfully modeled mice were then randomly divided into three groups (n = 8), and treatment was initiated at the 4th week after the operation as follows: (1) in the 6-OHDA + saline group, each mouse was administered 0.5 mL of normal saline through the tail vein, which exceeded 5 min and the flow rate did not exceed 0.1 mL/min; (2) the 6-OHDA + MSC–EV–NC mimic (6-OHDA-induced PD mice were injected with MSC–EV via tail vein, with a concentration of 200 g/0.5 mL saline, and a flow rate not exceeding 0.1 mL/min) group; (3) the 6-OHDA + MSC–EV–miR-181a–2–3p mimic group (6-OHDA-induced PD mice were injected with MSC–EV–miR-181a–2–3p mimic with a concentration of 200 µg/0.5 mL saline and a flow rate not exceeding 0.1 mL/min). The mice were treated as above-mentioned once every 3 days for a duration of 8 weeks. The Animal Ethics Committee of Henan Provincial People’s Hospital, (Zhengzhou University People’s Hospital) (2019-067 A) also provided ethical approval for animal experimentation in the current study.
HE staining
Routinely treated brain tissues from mice were sliced into 3 small sections, deparaffinized, rehydrated, and then stained with Mayer’s hematoxylin. Next, the sections were simply distinguished four times with acidic alcohol, blued, and observed under a microscope. Following observation, the sections were dehydrated, stained with eosin, and sealed.
In vivo imaging detection
Initially, 25 mg of Xenolight DiR fluorescent dye (PerkinElmer, Waltham, MA) was dissolved in 2 mL of ethanol. Next, the separated 0.5 mL MSC–EVs were diluted with 0.5 mL of PBS and incubated with DiR stock solution for 30 min. The sample was centrifuged a 100,000 × g for 60 min, the supernatant was discarded, and 300 μL of PBS was adopted to dissolve the blue precipitate in the lower layer. Subsequently, the DiR-labeled EVs were injected into mice via tail-vein injection. After 24 h, the analysis was performed using the caliper IVIS Lumina II imager.
Immunofluorescence
Paraffin-embedded brain tissues were sliced into 6 μm sections, and then subjected to immunofluorescence staining. After 5 min of antigen retrieval, the sections were incubated in 5% goat serum for 1 h and incubated with rabbit TH (dilution ratio of 1:500, GB11181, Servicebio, Wuhan, China) and 4-HNE (dilution ratio of 1:50, MA5-27570, Invitrogen), and α-SYN (dilution ratio of 1:500, ab272736, Abcam) at 4 °C overnight and with Cy3-conjugated donkey anti-rabbit antibody (dilution ratio of 1:250, GB 21403, Servicebio) or Goat Anti-Mouse Alexa Fluor 488 (dilution ratio of 1:1000, A32723, Invitrogen) for 1 h. A confocal laser microscope (Olympus) was employed to observe the immune-response cells.
Immunohistochemistry
Immunohistochemistry was routinely performed [7]. Primary antibody TH (dilution ratio of 1:500, ab137869, Abcam) was added dropwise in the refrigerator at 4 °C overnight.
TUNEL assay
SN cell apoptosis was quantitatively detected using In Situ Cell Death Detection kits (Roche Diagnostics GmbH, Mannheim, Germany). In short, dewaxing procedure was performed as mentioned above. Next, the membrane was broken using a membrane-breaking solution. Subsequently, TUNEL and TH (dilution ratio of 1:500, Servicebio) were used to determine the percentage of apoptotic dopaminergic neurons. Dopaminergic neurons were then incubated with the secondary antibody for 50 min. Following staining with DAPI, fluorescein-labeled green DNA and red dopaminergic neurons are observed under a fluorescence microscope.
Behavioral experiment
Behavior changes of mice were observed with the help of rod-climbing test and roller test. First, the rod-climbing test was performed. Briefly, a 50-cm-long and 1-cm-diameter wooden pole wese placed vertically, wrapped with gauze to prevent mice slipping off, and a 2.5-cm-diameter cork ball were fixed on the top. The mice were placed on the cork ball, and the time of climbing from the top of the ball to the top of the rod, from the top of the rod to the middle of the rod, and from the middle of the rod to the bottom of the rod were all recorded. Completion within 3 sec was recorded as 3 points, and 6 sec as 2 points. If the time exceeded 6 sec or the mouse fell from the pole, the attempt would be recorded as 1 point. Experiment results were expressed by the total score. Meanwhile, for the roller test, the mice were placed in a roller experimental device at 4 r/min for 2 min for adaption. The axis experimental device mode was set as acceleration 4 ~ 40 r/min, and the time from the acceleration of the roller to the mouse falling off the device was recorded, with the average value of three times obtained. The time interval of each measurement is 1 h.
Statistical analysis
Data analyses were performed using the GrafPad Prism software (GraphPad Software, La Jolla, CA). All quantitative data were presented as mean ± standard deviation. Comparisons between two groups were analyzed with an independent-sample t-test. One-way analysis of variance (ANOVA) with post hoc Tukey test was used for comparisons between multiple independent groups. The dual-luciferase reporter gene assay was analyzed using two-way ANOVA with Bonferroni as the post hoc. A value of p < 0.05 was regarded statistically significant.
