All experimental animals utilized in this study were male C57BL/6 J mice (23–25 g, 8–10 weeks old), procured from the Laboratory Animal Center of Nantong University. The mice were housed in a specific pathogen-free (SPF) environment under a 12-h light/dark cycle with ad libitum access to food and water. All experimental procedures involving animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals, with experimental protocols approved by the Animal Ethics Committee of Nantong University (approval number: S20240308-009). All experimental procedures followed recommendations in the ARRIVE guidelines.

Mice were randomly assigned to Control, MPTP, or MPTP + OMT groups. Mice in the MPTP group received intraperitoneal injections of MPTP·HCl (20 mg/kg, M0896, Sigma-Aldrich) four times in a single day at 2-h intervals, following an established protocol for the MPTP model (Jackson-Lewis and Przedborski, 2007). Mice in the MPTP + OMT group were administered OMT (20 mg/kg, PHL89748, Sigma-Aldrich) intraperitoneally 2 h before the first MPTP injection and received another dose of OMT 2 h after the fourth MPTP injection. Starting from day 2, OMT was administered once daily until tissue collection on day 7. Control mice received intraperitoneal injections of an equal volume of 0.9% NaCl.

Following anesthesia with isoflurane inhalation, mice received intracranial injections into the bilateral SNpc (stereotaxic coordinates relative to Bregma: anteroposterior −3.28 mm, mediolateral ±1.2 mm, dorsoventral −4.7 mm) of either 0.5 nmol miR-141-3p agomir, miR-141-3p agomir negative control (NC agomir), miR-141-3p antagomir, or miR-141-3p antagomir negative control (NC antagomir) (OBiO Technology Shanghai Corp., Ltd.), dissolved in 2.5 μL PBS. A 10-μL Hamilton syringe was used for injection, delivered over 10 min via a syringe pump (Harvard Apparatus). The needle was left in place for an additional 10 min post-injection before slow withdrawal. Mice were kept warm until fully recovered.

A rotarod apparatus (Harvard Apparatus) was used to assess motor coordination and balance. Prior to formal testing, mice underwent a three-day training period on the rotarod at a constant speed of 5 rpm/min. On the fourth day, an accelerated rotarod test was performed in which the rotation speed increased from 5 rpm/min to 40 rpm/min over a 5- min period. Only mice capable of remaining on the rod for 3–5 min were selected for subsequent experiments. Seven days after MPTP injection, each mouse was tested three times, and the average latency to fall was recorded as a measure of motor function. A maximum latency of 300 s was assigned if a mouse did not fall within the 5-min test period.

The open field test was conducted using an arena (40 cm × 40 cm × 40 cm) equipped with an automated data acquisition system (Shanghai Xinruan Information Technology Co. Ltd.). Mice were individually placed in the open arena and allowed to freely explore for 5 min before a 15-min recording session. To get rid of the odor from each previous mouse, the instruments were cleaned with 75% ethyl alcohol. The total distance traveled, average speed, time spent in the center zone, and distance moved in the center zone were collected. Calculated.

After euthanizing the mice (24–27 g) with excessive inhalation isoflurane, mice were immediately subjected to transcardiac perfusion fixation. Whole brains were carefully extracted and post-fixed in 4% PFA at 4 °C for 12 h, followed by dehydration in 30% sucrose until sedimentation occurred. These samples were reserved for subsequent immunofluorescence staining. Another group of brains was promptly dissected to isolate the ventral midbrain containing SNpc (stereotaxic range: anteroposterior −2.92 to −3.88 mm from Bregma), which were immediately snap-frozen in liquid nitrogen for subsequent analyses including real-time PCR, Western blot, and ELISA.

Brain tissue sections (20 μm) were cut on a cryostat (Leica) and floated in PBS for three washes. Sections were then blocked with 3% BSA containing 0.1% Triton X-100 for 1 h, followed by incubation at 4 °C for 24 h with the following primary antibodies: Rabbit anti-AIF-1/Iba1 Antibody (1:1000, 019–19,741, FUJIFILM Wako Pure Chemical Corporation) and Goat anti-Tyrosine Hydroxylase Antibody (1:500, ab317785, abcam). After PBS washes, sections were incubated overnight at 4 °C in the dark with secondary antibodies: Donkey anti-Rabbit IgG Alexa Fluor™ 488 (1:500, A21206, Thermo Scientific) and Donkey anti-Goat IgG Alexa Fluor™ 568 (1:500, A11053, Thermo Scientific). Following another PBS wash, nuclei were stained with Hoechst.

The quantitative statistical analysis used the ratio of the number of Iba1-positive microglia to the area of TH-positive neurons. This analytical approach was adopted because that simply counting the number of IBA1-positive microglia and the area of TH-positive neurons would vary due to different sections. Sections with higher TH-positive neuron density exhibit proportionally greater microglial proliferation following neuronal loss, and vice versa. Therefore, the microglia number-to-DA neuron area ratio provides a more accurate representation of pathological changes. All immunohistochemical analyses were conducted on SNpc-region sections selected from mouse brain atlases, with focus on sections displaying maximal microglial density per sample.

The microglial BV2 cells were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37 °C in a humidified 5% CO₂ incubator. One day prior to transfection, cells were seeded in and transfection was performed when cells reached approximately 60% confluence. BV2 cells were transfected with 100 nM miR-141-3p mimics or miR-141-3p mimics negative control (NC mimics) using LipofectMax transfection reagent for 6 h, followed by replacement with fresh DMEM containing 10% FBS for 48 h. Subsequently, the transfected cells were stimulated with 1 mM MPP⁺ (Sigma-Aldrich) for 24 h.

Bioinformatic analysis via TargetScan predicted a complementary binding site between miR-141-3p and the 3’-UTR of HMGB1 mRNA. To validate this interaction, the mutant reporter vector pMIR-REPORT Luciferase-HMGB1 3’-UTR (MUT) and wild-type reporter vector pMIR-REPORT Luciferase-HMGB1 3’-UTR (WT) were co-transfected with either miR-141-3p mimics or negative control (NC mimics) (OBiO Technology Shanghai Corp., Ltd.) into U251 cell lines. Luciferase activity was measured using a dual-luciferase reporter assay system 48 h post-transfection.

Total RNA from cells and animal tissues was extracted using the Rapid RNA Extraction Kit and TRIzol™ Reagent (Vazyme Biotech Co., Ltd.), respectively, followed by reverse transcription with the HiScript III RT SuperMix for qPCR Kit. Quantitative real-time PCR was performed using a BioRad CFX96TOUCH Real-Time PCR instrument. Gene expression levels normalized to 18S was conducted with the 2^−△△CT method. The primer sequences are listed in Supplementary Table 1.

Levels of HMGB1, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in BV2 cell culture supernatants or the ventral midbrain containing the SNpc were tested by ELISA kits (USCN Life Science Inc). Tissue homogenates were sonicated until clarification, followed by centrifugation at 4 °C for 5 min, with the supernatant aspirated as test samples. A volume of 100 μL of each standard or test sample was added to the wells and incubated at 37 °C for 1 h. After aspiration, 100 μL of detection antibody was added and incubated for 1 h at 37 °C. The plate was then washed, and 100 μL of horseradish peroxidase (HRP)-conjugated secondary antibody was added, followed by incubation for 30 min at 37 °C. After five washes, a substrate solution was added and incubated for 20 min at 37 °C in the dark. The reaction was stopped, and absorbance was measured at 450 nm.

The ventral midbrain containing the SNpc or BV2 cell were lysed using RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail (Roche). Following the protein concentration was determined by BCA assay, equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk for 1 h, the membranes were incubated overnight at 4 °C with the following primary antibodies: β-actin monoclonal antibody (1:8000, A5441, Sigma-Aldrich) and anti-HMGB1 antibody (1:25000, ab79823, abcam). Subsequently, membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies: goat anti-mouse IgG HRP (1:8000, A3682, Sigma-Aldrich) and goat anti-rabbit IgG HRP (1:8000, A0545, Sigma-Aldrich). Protein bands were visualized using enhanced chemiluminescence, and images were captured with an automated chemiluminescence imaging system (Tanon). Band intensities were quantified using ImageJ software, and normalized to β-actin levels.

All statistical analyses were performed using GraphPad Prism version 9.5.0. Shapiro–Wilk normality test and Levene’s test were used to assess data normality and homogeneity of variance, respectively. Comparisons between two groups were analyzed using Student’s t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) plus Tukey’s post hoc test was employed. Data are presented as mean ± SEM. Statistical significance was set at p < 0.05.

A mouse model of PD was established using MPTP. Results revealed a significant downregulation of miR-141-3p mRNA expression in the SNpc region of the mice (Figure 1A), concurrently with a marked upregulation of HMGB1 mRNA expression (Figure 1B). In vitro experiments were performed using the mouse microglial cell line BV2. After stimulation with MPP⁺, consistent results were obtained, showing an inverse relationship between miR-141-3p and HMGB1 expression (Figures 1C,D). Bioinformatics analysis using TargetScan software predicted that miR-141-3p binds complementarily to the 3’-UTR of HMGB1 mRNA (Figure 1E). This prediction was further verified by dual-luciferase reporter gene assays. The mutant (MUT) or wild-type (WT) HMGB1 mRNA 3’-UTR reporter gene was cloned into the dual-luciferase reporter vector (pMIR-REPORT). In U251 cells transfected with miR-141-3p mimics, the luciferase activity of the pMIR-REPORT Luciferase-HMGB1 3’-UTR (WT) reporter gene was significantly reduced. In contrast, when the predicted miR-141-3p binding site in the HMGB1 mRNA 3’-UTR was mutated, luciferase activity remained unaltered (Figure 1F). Western blot analysis in U251 cells confirmed that transfection of miR-141-3p mimics led to a decrease in HMGB1 protein expression, while transfection of miR-141-3p inhibitor had no significant effect on HMGB1 protein levels (Figure 1G). The above experimental results demonstrate that miR-141-3p specifically binds to the 3’-UTR of HMGB1 and effectively downregulates its expression.

To further assess the functional impact of miR-141-3p on HMGB1, in vitro experiments were conducted using miR-141-3p mimics. MPP⁺ stimulation was found to promote the HMGB1 mRNA expression in BV2 cells. However, upon transfection of miR-141-3p mimics into BV2 cells (miR-141-3p was overexpressed after transfection with miR-mimics as shown in Supplementary Figure S1), a significant reduction in HMGB1 mRNA expression was observed compared to the MPP⁺ group (Figure 2A). Additionally, the mRNA expressions of multiple pro-inflammatory factors, including IL-6, TNF-α, CXCL2, IL-1α, CCL3, CCL4, and IL-17, were significantly downregulated relative to the MPP⁺ group (Figure 2A). At the protein level, compared with the MPP⁺ group, the miR-141-3p mimics + MPP⁺ group exhibited a significant decrease in HMGB1 protein expression, with statistical significance (Figure 2B). ELISA quantification of HMGB1, TNF-α and IL-6 in the supernatant of BV2 cells revealed that the expression levels of these inflammatory factors were also significantly reduced in the miR-141-3p mimics + MPP⁺ group, compared to the MPP⁺ group (Figure 2C). These results indicate that miR-141-3p mimics can effectively reduce the expression and release of HMGB1, the release of pro-inflammatory factors from microglia at both the mRNA and protein levels.

To further validate the conclusion obtained from the in vitro experiments, stereotactic injections were performed on C57BL/6 J mice. The mice were then divided into three groups: NC agomir + 0.9% NaCl (referred to as NC agomiR), NC agomir + MPTP (referred to as MPTP), and miR-141-3p agomir + MPTP. Seven days later, behavioral tests were conducted on each group of mice. In the rotarod test, mice in the miR-141-3p agomir + MPTP group remained on the rotarod for a significantly longer period compared to the MPTP group. This finding suggests that miR-141-3p agomir enhances the balance and coordination abilities of MPTP model (Figure 3A). In the open field test, although the movement parameters did not fully return to normal levels, the total distance traveled and average speed of the miR-141-3p agomir + MPTP group were significantly higher than those of the MPTP group. Moreover, in the central area of the nine-grid, the movement distance and time of the miR-141-3p agomir + MPTP group were also significantly greater than those of the MPTP group. These data suggest that miR-141-3p agomir improves the spontaneous activity and exploration behavior of MPTP model (Figure 3B).

After behavioral assessments, brain tissues from the lower half of the midbrain (including the SNpc region) were collected for analysis. Quantitative analysis of mRNA revealed that the miR-141-3p agomir + MPTP group had significantly reduced HMGB1 expression compared to the MPTP group. Additionally, the mRNA expression of IL-6, TNF-α, CXCL2, IL-1α, CCL3, CCL4 and IL-17 were significantly lower than those of the MPTP group (Figure 4A). Immunohistochemical fluorescence staining showed that compared to the NC agomiR group, the MPTP group exhibited extensive loss of TH-positive neurons in the SNpc region, accompanied by a significant increase in Iba1-positive microglial cell numbers, indicating a reactive state. In contrast, the number of TH-positive neurons in the miR-141-3p agomir + MPTP group was partially restored, and the number of Iba1-positive microglia decreased (Figure 4B, enlarged versions were shown in Supplementary Figure S2). The quantitative statistical analysis used the ratio of the number of Iba1-positive microglia to the area of TH-positive neurons. The results demonstrated a statistically significant difference between the MPTP group and the miR-141-3p agomir+MPTP group (Figure 4C). ELISA quantification demonstrated significantly reduced protein levels of pro-inflammatory cytokines IL-6 and TNF-α in the SNpc region of mice treated with miR-141-3p agomir+MPTP compared to the MPTP group (Figure 4D).

These data indicate that miR-141-3p regulates HMGB1 and modulates inflammatory and behavioral readouts in this MPTP paradigm. Building upon previous findings that OMT alleviates DA neuronal damage and microglia-mediated neuroinflammation (Gan et al., 2020), we investigated whether this effect occurs through miR-141-3p modulation. In MPTP-induced PD models, 7 days of intraperitoneal OMT treatment at varying doses increased miR-141-3p mRNA levels in the SNpc in a dose-dependent manner. (Figure 5A). Based on this result, we stereotaxically injected either NC antagomir or a miR-141-3p antagomir into the bilateral SNpc region. Ultimately, the experimental animals were divided into four groups: NC antagomir + 0.9% NaCl (referred to as NC antagomir), NC antagomir + MPTP (referred to as MPTP), NC antagomir + MPTP + OMT (referred to as MPTP + OMT), and miR-141-3p antagomir + MPTP + OMT. Behavioral assessments using the rotarod (Figure 5B) and open field tests (Figure 5C) showed that the miR-141-3p antagomir blocked the therapeutic benefits of OMT, impairing motor coordination, balance, and exploratory behavior.

Compared with the MPTP + OMT group, the expression of HMGB1 in the miR-141-3p antagomir + MPTP + OMT group was significantly increased, consistent with the trend in the MPTP group. The mRNA expression of pro-inflammatory factors (IL-6, TNF-α, CXCL2, IL-1α, CCL3, CCL4, and IL-17) showed similar upregulation patterns (Figure 6A). Iba1 and TH double-labeling in the SNpc revealed that OMT treatment attenuated the MPTP-induced loss of TH-positive neurons and reduced the number of reactive microglia. Conversely, these protective effects were abolished by miR-141-3p antagomir co-treatment, which resulted in a pronounced loss of TH-positive neurons and a concomitant increase in reactive microglia. (Figure 6B, enlarged versions were shown in Supplementary Figure S3). The Iba1 number/TH area ratio also indicated that compared with the MPTP + OMT group, the ratio in the antagomir + MPTP + OMT group significantly increased, consistent with the trend in the MPTP group (Figure 6C). ELISA quantitative detection confirmed elevated IL-6 and TNF-α protein levels in antagomir + MPTP + OMT, compared with the MPTP + OMT group (Figure 6D).

In our research results, miR-141-3p agomir and miR-141-3p antagomir were injected bilaterally into the SNpc region. To evaluate the role of miR-141-3p, we performed unilateral injections of miR-141-3p agomir or antagomir into the left SNpc, followed by MPTP administration 48 h later. Mice in the miR-141-3p agomir + MPTP group exhibited ipsilateral rotation (leftward), indicating stronger neuroprotection in the injected (left) SNpc. In contrast, mice treated with miR-141-3p antagomir + MPTP + OMT exhibited contralateral rotation (rightward), indicating a loss of OMT-mediated protection in the injected hemisphere (Supplementary Video).