Parkinson’s disease (PD), the second most common neurodegenerative disease¹⁾, mainly caused by the death of dopaminergic neurons in substantia nigra²⁾. Dopamine signals used to enhance the decision-making network can be used to enhance and coordinate motor performance³⁾. This leads to dopamine deficiency in the basal ganglia, which leads to movement disorders characterized by the typical motor symptoms of PD⁴⁾.

Dopamine neurons are projected onto the striatum to control movement, cognition, and motivation via faster dopamine, glutamate, and GABA synaptic actions, which can convey the temporal information from dopamine neuron firing, as well as slower volume transfer¹⁾. For that reason, particularly important neural network for the development of PD is a neural in which dopaminergic neurons of substantia nigra are connected to nerve cells in the striatum region. The dopamine agonist (DA) receptors’ subclass, D1 and D2 mediate opposing valence behaviors, so that expression of DA D1 receptors stimulate the direct pathway neuron activation, and expression of DA D2 stimulates the indirect pathway neuron activation. Therefore considering endogenous DA mainly stimulates D1-receptor expressing neurons and inhibits D2-receptor expressing neurons⁵⁾, PD, in which substantia nigra dopamine neurons degenerate, decreases dopamine secretion to the striatum, decreases the activity of nerve cells which cause movement, and conversely, increases the activity of nerve cells which suppress movement, resulting in PD behavioral abnormalities.

Acupuncture stimulation studies in animal models of PD have shown that acupuncture treatment is a neuroprotective treatment that increases the release of various neuroprotective agents, such as brain-derived neurotrophic factor, cyclophilin A, and glial cell line-derived neurotrophic factor⁶⁾. It has also been shown that acupuncture not only regulates neurotransmitter balance in the basal ganglion circuit, but can also protect dopamine neurons from degeneration via antioxidant stress, anti-inflammatory, and anti-apoptotic pathways⁷⁾. Recent studies have already confirmed that acupuncture can delay the progression of the disease in the early stages of PD⁸⁾.

This study observed changes in protein expression in the striatum, the lesion site of PD. Through this, the goal was to identify factors involved in the mechanism of the effectiveness of acupuncture treatment in PD.

1. Animal and MPTP model

PD animal models were fabricated using neurotoxic compounds such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)⁹⁾. The animal models were divided into four groups: control (CTL), group that underwent MPTP treatment only (MPTP), group that underwent MPTP and acupuncture at LR3 and GB34 (MPTP-A), and group that underwent MPTP and acupuncture in non-acupoint (MPTP-NA), and we used 6-week-old male inbreeding C57BL/6 mice (Samtaco, Korean), which were divided into these four groups and weighed 20∼25 g.

Control mice were injected intraperitoneally with 0.9% (100 μl) of saline solution once a day for 28 days. Mice in the MPTP group were injected intraperitoneally with MPTP- HCl (free base 20 mg/kg) dissolved in 0.9% (100 μl) saline solution at intervals of 24 hours for 28 days, resulting in a persistent chronic model of PD. The day after the final MPTP treatment, cold 0.05 M sodium phosphate buffer was perfused percutaneously. The Sangji University’s Animal Experiments Committee approved all animal protocols that used in this study (protocol #2024-5). Reagents used but not mentioned in the study were purchased from Sigma (USA).

2. Acupuncture administration

Acupuncture was performed manually 2 hours after the first MPTP injection, followed by a total of 14 sessions, 48 hours apart. Mice in the acupuncture group were hand-fixed 2 hours after MPTP administration. Acupuncture needles were inserted bilaterally to a depth of 1 mm at LR3, located on the dorsum of the foot, in the depression proximal to the 1st metatarsal space, and 3 mm at GB34, situated on the lateral side of the lower leg in the depression anterior and inferior to the head of the fibula. The needle rotated at a rate of 2 revolutions per second for 15 seconds. In the non-acupuncture group, needles were inserted at a depth of 3 mm on both sides of the hip, performing the same procedure as described above.

3. Proximity extension assay (PEA)

The sacrifice of mice anesthetized using Alfaxan was performed and transcardial perfusion was also conducted with cold PBS (n=4/genotype). The bilateral striatum regions were extracted from the brains perfused with cold PBS were collected in low-affinity polypropylene tubes, snap-frozen in liquid nitrogen and stored at −80℃ until further analysis. Levels of 92 proteins in striatum were analyzed using Target 96 Mouse Exploratory Panel with PEA technology, according to manufacturer’s instructions (Olink Proteomics). This dual recognition technique is based on matched pairs of antibodies labeled with complementary DNA oligonucleotide tags, which creates a unique DNA barcode for each antibody pair that is amplified by qPCR. The protein signal values of Normalized Protein eXpression (NPX) are calculated on a logarithmic base 2 (Log₂) scale.

4. Statistical analysis

Student’s t-test and analysis of variance (ANOVA) in SPSS 25 (version 25.0; SPSS Inc., Chicago, IL, USA) were used for statistical analysis. Post hoc analyses were performed using the LSD test. A p-value less than 0.05 was considered statistically significant.

The rotarod test was conducted to assess the motor abilities of mice across the different experimental groups. The mice in the MPTP group exhibited a significantly earlier fall time compared to those in the CTL group (p<0.005; Fig. 1). Furthermore, the mice in the MPTP-A group demonstrated a significant improvement in motor abilities compared to the MPTP group (p<0.05; Fig. 1).

Fig. 1. The results of the rotarod test.
The motor abilities of the MPTP group significantly decreased compared to the CTL group, and the motor abilities of the MPTP_A group significantly increased compared to the MPTP group. Control (CTL) group, MPTP treatment only (MPTP) group, MPTP and acupuncture at LR3 and GB34 (MPTP-A) group, and MPTP and acupuncture in non-acupoint (MPTP-NA) group. Values are means±SE. *p<0.005 compared to CTL and ^#p<0.05, compared to MPTP were considered significant. An analysis of variance (ANOVA) was conducted for statistical analysis. Post hoc analyses were performed using the LSD test.

To investigate the proteins that are upregulated following acupuncture stimulation in a mouse model of chronic PD, an initial analysis was conducted on 92 proteins (see Fig. 2A). The NPX value presented in Figure 2B represents the expression level of the proteins, and statistical analyses were performed based on these values, leading to the subsequent results.

Fig. 2. Hierarchical clustering analysis and MDS plot.
(A) Hierarchical clustering analysis with heatmap (Euclidean distance, complete linkage) clusters the proteins and samples by their expression level (NPX value). (B) MDS plot (multidimensional scaling plot). Processed from total mean NPX values of each sample, MDS plot shows the similarity and the variability among samples in a 2D plot.

When comparing the CTL group with the MPTP group, the expression levels of Repulsive guidance molecule A (Rgma), Integrin beta-6 (Itgb6), Tumor necrosis factor receptor superfamily member 6 (Fas), V-set and immunoglobulin domain-containment protein 2 (Vsig2), Integrin beta-1-binding protein 2 (Itgb1bp2), and Platelet-derived growth factor subunit B (Pdgfb) were significantly elevated in MPTP group compared to the CTL group (Fig. 3. red line). In contrast, the MPTP-A group exhibited a significant decrease in the protein expression of Axin-1 (Axin1), Integrin beta-6 (Itgb6), Aryl hydrocarbon receptor (Ahr), and Legumain (Lgmn) when compared to the MPTP group, with a p-value of less than 0.05 (see Fig. 3, blue line; Table 1).

Fig. 3. Chord plot.
Significantly altered proteins included in GO terms related to acupuncture or MPTP treatment. The color of the circle edge boxes indicates MPTP -(red) or acupuncture- (blue) treatment.

When acupuncture was administered to mice with PD, a further analysis was conducted on the protein that exhibited a significant reduction (see Fig. 4). In the case of Axin1, the NPX value showed a slight increase in the MPTP group compared to the CTL group; however, the p-value was 0.2, indicating that this result was not statistically significant. Similarly, for Ahr and Lgmn, there were slight increases observed when comparing the CTL and MPTP groups, with p-values of 0.09 and 0.74, respectively, both of which were not significant. Notably, Itgb6 was the only protein that demonstrated a significant decrease in the MPTP group relative to the CTL group, and it also exhibited a significant increase in the MPTP-A group compared to the MPTP group.

Fig. 4. Protein expression levels (NPX value) altered by acupuncture treatment.
Values are means±SE. *p<0.05 compared to CTL, ^#p<0.05, or ^##p<0.005 compared to MPTP were considered significant. Student’s t-test was performed for statistical analysis.

The network illustrates the proteins that are altered by acupuncture treatment, as well as those associated with it (see Fig. 5), and identifies the potential pathways affected by these alterations (refer to Table 2). The involved pathways were analyzed using KEGG pathway analysis. These proteins are responsible for processing environmental information, which is crucial for signaling within the PI3K-Akt signaling pathway, and they interact with molecules in the extracellular matrix-receptor interaction pathway. Furthermore, these proteins are involved in cytoskeletal processes within muscle cells, which are essential for cellular functions such as cell motility through the regulation of the actin cytoskeleton. Additionally, they are associated with the dopaminergic synapse and the mTOR signaling pathway (see Table 2).

Fig. 5. Network of the proteins.
Network shows the proteins that are altered by acupuncture treatment, as well as those associated with it.

Our behavioral data indicate that acupuncture treatment enhances movement, as assessed by the rotarod test, in a mouse model of PD induced by MPTP. PD is a neurodegenerative disorder characterized by major symptoms such as movement disorders, including tremor, rigidity, and postural instability. Consistent with previous studies^7,10), our findings also suggest that acupuncture exerts a neuroprotective effect that ameliorates motor symptoms in this model. Furthermore, our data reveal that several proteins, including Axin1, Ahr, Lgmn, and Itgb6, are implicated in the effects of acupuncture treatment.

Axin1 is a protein encoded by the Axin1 gene in humans. The Axin1 protein has garnered significant attention in cancer research due to its role in activating the JNK/c-Jun and Smad3 signaling pathways¹¹⁾, as well as its synergistic interaction with Axin-2 in regulating developmental β-catenin signaling¹²⁾. Notably, mutations in Axin1 can promote tumor growth by aberrantly enhancing β-catenin signaling¹³⁾. Conversely, these mutations may also inhibit the proliferation of β-catenin-dependent cancers by augmenting the activity of β-catenin-destroying complexes through the application of tankyrase inhibitors.

Ahr is an electronic factor that plays a role in development, immune response, detoxification, and various syndromes by regulating the expression of drug transporters and metabolites within remote sensing and signaling networks that facilitate communication between metabolites and signaling molecules¹⁴⁾. pecifically, AHR functions as a ligand- activated transcription factor that integrates metabolic signals to govern complex transcriptional programs in a manner that is specific to the ligand, cell type, and context. In essence, Ahr serves as an environmental sensor that modulates immune responses by regulating multiple pathways involved in endogenous metabolism¹⁵⁾.

Lgmn, also referred to as δ-secretase or asparaginyl endopeptidase¹⁶⁾, is found to be overexpressed not only in tumor cells of the breast, liver, and prostate but also in macrophages that constitute the tumor microenvironment. This indicates that Lgmn plays a crucial role in regulating tumor development, invasion, and metastasis. Consequently, it serves as a significant biomarker for cancer detection and targeting, given that its expression levels in tumors or tumor- associated macrophages are markedly higher than those observed in normal cells¹⁷⁾.

The comparison of CTL to MPTP, followed by the comparison of MPTP to MPTP-A, reveals that Itgb6 is the only protein consistently identified in the context of changed protein levels. This finding suggests a significant association between the Itgb6 protein and PD. Furthermore, it implies that acupuncture treatment administered for PD may have an impact on the levels of this protein.

The significance of Itgb6 lies in its role in determining expression and availability. In addition to its involvement in long-term fibrosis, Itgb6 is directly associated with the development of cancer, periodontitis, and various potential genetic disorders¹⁸⁾. Itgb6 represents the β6 subunit of the integrin αvβ6, with β6 being intrinsically linked to the αv subunit. Consequently, the function of Itgb6 is fundamentally connected to integrin αvβ6¹⁹⁾. The primary function of αvβ6 is the activation of transforming growth factor-beta 1 (TGF-β1) that has undergone cytokine transformation²⁰⁾. αvβ6 interacts with latency-associated peptide (LAP), leading to the release of TGF-β1 through cytoskeletal mechanisms. TGF-β1 regulates numerous biological processes, including cell proliferation, differentiation²¹⁾, and immune suppression²²⁾. When these processes are combined in wound treatment, they may contribute to histopathological changes if not properly controlled.

KEGG pathway analysis indicates that the proteins activated by acupuncture treatment are involved in the pathology related to PD. These proteins are integral to the processing of environmental information, which is essential for signaling within several key pathways, including the PI3K-Akt signaling pathway²³⁾, the dopaminergic synapse²⁴⁾, and the mTOR signaling pathway²⁵⁾, all of which have been linked to PD. Furthermore, these proteins are engaged in cytoskeletal processes within muscle cells, which are critical for various cellular functions, including cell motility, through the regulation of the actin cytoskeleton²⁶⁾.

The proteins Axin1, Ahr, Lgmn, and Itgb6 demonstrate significant changes in their expression levels following acupuncture treatment. This observation has been substantiated through quantitative comparisons between the MPTP and MPTP-A models. These results suggest that acupuncture treatment is expected to be effective in treating PD by altering the expression of these proteins. However, it is essential to investigate the mechanism behind the differences observed among the other proteins (Axin1, Arhr, Lgmn), as Itgb6 is the only protein that has consistently demonstrated a change in expression levels in this context. Furthermore, it is important to identify whether there are additional, more specific biomarkers in the blood that could facilitate the use of Itgb6 protein expression changes in the treatment of PD.

None.

This research was supported by Sangji University Research Fund 2023.

The authors can provide upon reasonable request.

The authors have declared that no conflicts of interest exists.