Male mice have more consistent physiological and metabolic responses, which helps reduce variability in experimental results [33]. C57BL/6 male mice were used to control for sex variables and improve the consistency and comparability of results. As effect sizes and standard deviations were not readily available directly from the existing literature and were difficult to estimate, the “resource equation method” was used to determine sample sizes [34, 35]. This study was approved by the Experimental Animal Ethics Committee of Southern Medical University (Approval No. L2021119). Eighty-five eight-week-old male C57BL/6 mice (20–22 g) from Southern Medical University’s Animal Center (License No. SYXK[Yue]2021-0041) were acclimated for one week in a controlled environment (22 $\pm$ 2 °C, 12-hour light-dark cycle) with food and water ad libitum. To assess EA’s effect on MPTP-induced PD, mice were randomized into control (n = 11) and treatment (n = 44) groups. Treatment group mice were injected intraperitoneally with MPTP (30 mg/kg, Sigma-Aldrich, St Louis, MO, USA, dissolved in normal saline) once daily for seven consecutive days, while controls were administered equal volumes of saline (i.p.) [36]. The mice in the treatment group were then randomly and equally assigned to the MPTP, EA, drug and sham EA groups and received the corresponding treatments. Following behavioral analysis, mice were deeply anesthetized with 5% isoflurane (No. R510-22-10, RWD Life Science, Shenzhen, China) at a flow rate of 0.8 L/min and decapitated, after which brain tissues were collected for immunohistochemical, Western blot (WB) and transmission electron microscopy (TEM) assessments.

To further investigate EA’s influence on MAMs in the SN, mice were randomly divided into three groups (10 mice/group): control, MPTP and EA groups. Treatment and intervention were consistent with prior protocol, mice were then euthanized and the brain tissues were analyzed by WB, immunofluorescence, transcriptome analysis and RT-qPCR. The procedure is given in Fig. 1A.

Fig. 1.

Time axis of experimental procedures and positioning of acupoints. (A) Timeline outlining the experimental phases, including adaptation, PD model establishment, intervention, and brain collection, with behavioral tests scheduled at key time-points. (B) Photograph showing the setup of a mouse undergoing electroacupuncture treatment. (C) Schematic diagram depicting the selected electroacupuncture acupoints in a mouse: GV20 (middle of the parietal bone) and GB34 (lateral calf, in the depression below the anterior aspect of the fibular head). PD, Parkinson’s disease; GV20, governing vessel 20; GB34, gallbladder meridian 34.

EA treatment was conducted at the GV20 (middle of the parietal bone) and GB34 (lateral calf, in the depression below the anterior aspect of the fibular head) acupoints for 20 minutes daily following the induction of MPTP (Fig. 1B,C). GB34 on both sides was alternated daily. Acupuncture needles (0.16 mm $\times$ 5 mm; Suzhou Global Acupuncture Medical Equipment Co., Ltd., Suzhou, China) were inserted three mm deep at GV20 and unilateral GB34. The needles were then connected to electrodes delivering sparse-dense waves (5/100 Hz, 1 mA). Sham EA involved needle insertion at the same points without electrical stimulation. The entire treatment lasted 14 days [37].

The drug group received levodopa (D9628, Sigma-Aldrich, dissolved in normal saline) and benserazide (B7283, Sigma-Aldrich, dissolved in normal saline) via intraperitoneal injection at a dose of 20 mg/kg and 5 mg/kg, respectively, once daily for 14 consecutive days.

Bilateral SN of four mice in each group were homogenized in lysis buffer prepared with a protein extraction kit (KGB5303-100, Nanjing KeyGen Biotech Co., Ltd., Nanjing, Jiangsu, China), followed by centrifugation and collection of supernatant. Protein concentration was measured using a Bicinchoninic Acid Protein Assay Kit (P0009, Shanghai Beyotime Biological Co., Ltd., Shanghai, China). Total lysates were denatured in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (NA0007, Beijing Leagene Bio-Technology Co., Ltd., Beijing, China) at 95 °C for eight minutes. 20 µg of protein were separated on 6%–12.5% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (ISEQ00010, Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk in Tris-buffered saline with 0.05% Tween 20 for two hours at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: anti-IP3R (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-377518), anti-Mfn2 (1:10,000; Proteintech, Wuhan, Hubei, China; 12186-1-AP), anti-VAPB (1:6000; Proteintech, 14477-1-AP), anti-TH (1:5000; Proteintech, 25859-1-AP), anti-$\alpha$-syn (1:5000; Proteintech, 10842-1-AP) and anti-GAPDH (1:2000, Proteintech, 10494-1-AP). The following day, the PVDF membranes were washed and incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (1:40,000; Proteintech, SA00001-2) for one hour at room temperature. After washing, immunoblot detection was performed using an enhanced chemiluminescence Western blotting detection kit (WBKLS0500, Millipore, Bedford, MA, USA). Densitometry analysis was performed with ImageJ software (version 2.0.0, Rawak Software Inc., Stuttgart, Germany).

One hemisphere was separated and immersed in 4% paraformaldehyde for 24 hours, paraffin-embedded and sectioned into 6-µm-thick slices. Immunohistochemistry was performed following kit instructions (PV-9000, Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). Brain sections underwent xylene deparaffinization and ethanol solutions (from 100% to 75%) and were washed with phosphate-buffered saline (PBS). Antigen retrieval was performed by boiling sections in Tris-EDTA buffer (pH 9.0) in a microwave oven for 15 minutes. Endogenous peroxidase blocking reagent was added dropwise to each section and incubated for 10 minutes at room temperature. After washing with PBS, the sections were incubated with the primary antibodies: anti-tyrosine hydroxylase (TH) (1:5000; Proteintech, 25859-1-AP) and anti-$\alpha$-syn (1:500; Proteintech, 10842-1-AP) overnight at 4 °C. The next day, an appropriate amount of secondary antibody enhancement solution was added and incubated at 37 °C for 20 minutes. After washing with PBS, the sections were incubated with a biotinylated goat anti-mouse/rabbit IgG polymer at 37 °C for 20 minutes. Finally, the sections were incubated with the DAB substrate kit (ZLI-9018, Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) at room temperature for five minutes and counterstained with hematoxylin (DH0006, Beijing Leagene Bio-Technology Co., Ltd., Beijing, China) and Nissl staining (DK0022100, Beijing Solaibao Technology Co., Ltd., Beijing, China). Images were captured using a pathology slide scanner (KF-PRO-005-EX, Ningbo Jiangfeng Biological Information Technology Co., Ningbo, Zhejiang, China).

Brains were perfused transcardially with 4% paraformaldehyde, fixing the entire organ. Post-fixation, hemispheres were separated, and one hemisphere was processed for immunofluorescence and sliced into 20-micron frozen sections. These sections were immersed in Frozen Sections Antigen Repair Agent (P0090, Shanghai Beyotime Biological Co., Ltd.), followed by three washes and incubation in 0.3% Triton X-100 (T9284, Sigma-Aldrich) for 30 minutes. After washing with PBS, sections were blocked with PBS solution containing 5% goat serum (16210064, Gibco, New York, NY, USA) for 30 minutes. Sections were then incubated with primary antibodies overnight at 4 °C: anti-translocase of outer mitochondrial membrane 20 (TOM20) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA; SC-17764) and anti-calreticulin (1:500; Proteintech,10292-1-AP). After washing,slices were dark incubated for two hours with goat anti-rabbit Alexa Fluor 488-conjugate IgG (1:500; Abcam, Cambridge, UK; ab150077) and goat anti-mouse Alexa Fluor 647-conjugate IgG (1:500; Abcam, ab150115). Finally, slices were mounted using Fluoroshield with 4^′,6-diamidino-2-phenylindole (DAPI) (F6057, Sigma-Aldrich) and imaged using a Leica BX53 fluorescent microscope.

The SN tissue on one side of three mice in each group was taken as a sample and fixed in 2.5% glutaraldehyde overnight. Samples were then rinsed in buffer, post-fixed in 1.0% osmium tetroxide for one hour, rinsed again, and dehydrated through graded ethanol series. After resin embedding, the samples were made into ultrathin sections. These sections were viewed on a TEM (H-7500, Hitachi, Tokyo, Japan).

The SN tissue from the remaining side of three mice in each group was used for transcriptomic analysis. Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China) performed all transcriptomic procedures—including RNA purification, reverse transcription, library preparation (TruSeqTM RNA Kit, Illumina; 1 µg total RNA), and Illumina platform sequencing—according to the Illumina’s instructions (San Diego, CA, USA). Principal component analysis of gene expression profiles was performed using the platform (https://www.majorbio.com). The DEseq2 algorithm based on the platform was used to identify DEGs among the control, MPTP and EA groups. The fold change threshold was set at $|$log₂fold change$|$ $\ge$1.5 and the adjusted p-value was set to 0.05. A Venn diagram was used to identify the key DEGs related to the effect of EA, focusing on those that showed expression levels closer to the controls. The expression levels of DEGs were visualized with a heatmap based on the platform. The gene sets related to MAMs were downloaded and compared with DEGs on the platform. Furthermore, functional enrichment analysis including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome was conducted to identify significantly enriched DEGs with a Bonferroni-corrected p-value $\le$ 0.05, compared to that of the whole-transcriptome.

Total RNA from the SN of four mice in each group was extracted using TRIzol reagent (15596026CN, Invitrogen, Carlsbad, CA, USA). A BioTek spectrophotometer was used to detect the concentration and purity of RNA, which was then diluted in a proportion appropriate to make a final concentration of 1000 ng/µL. The Evo M-MLV Reverse Transcription Kit (AG11705, Accurate Biotechnology, Changsha, Hunan, China) was used to synthesize cDNA by reverse transcription of total RNA. The target gene expression levels were determined with a synergetic binding reagent (SYBR) RT-PCR kit (AG11739, Accurate Biotechnology, Changsha, Hunan, China). RT-qPCR was performed using a Roche LightCycler 96. Primers were procured from TSINGKE (Beijing, China) and the sequences are shown in Table 1. GAPDH was considered as the housekeeping gene to normalize the expression levels of targeted genes, three parallel holes were set for every sample and the relative expression of genes was analyzed according to the 2^{-Δ⁢Δ⁢Ct} method.

To analyze data, the average of several groups of data was calculated and outliers with scores greater than 1.5 interquartile ranges from the first and third quartiles of the data distribution were eliminated. Additionally, after excluding outliers, to ensure consistency in the number of animals per group, an equal number of animals were randomly selected from each group for evaluation. Statistical analysis was conducted using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). Data are reported as mean $\pm$ standard error of the mean. Normally distributed data were compared using one-way analysis of variance (ANOVA) followed by a Bonferroni multiple comparison test (variances equal) or the Dunnett T3 test (variances unequal) [42] with statistical significance assumed for p 0.05. Non-normally distributed data were analyzed using the Kruskal-wallis test. Graphs were obtained from GraphPad Prism 9.0.

To determine whether EA can increase motor function in the MPTP model, pole climbing, rotarod, wire hang and open field tests were performed. Following successful establishment of the PD mouse model on day seven, behavioral assessments revealed that MPTP-treated mice exhibited significant motor impairments across all tests (Fig. 2), confirming the successful replication of Parkinsonian motor dysfunction characteristics and providing a reliable model for subsequent intervention studies. Moreover, there were no statistical differences between the treatment groups, which excludes the interference of factors on the results that may have existed prior to the implementation of the intervention, thus avoiding false-positive results post-intervention. After 14 days of counterpart interventions, one-way ANOVA showed significant differences among the groups in the behavioral tests. In the pole climbing test, the total landing time of the MPTP group mice was significantly prolonged (p 0.001 vs. control, Fig. 2A). The scores of the MPTP group were significantly reduced in the pole climbing and wire hang tests (p 0.001 vs. control, as shown in Fig. 2B,E). The MPTP group also exhibited a markedly diminished latency to fall and a lower falling speed in the rotarod test (p 0.001 vs. control, as shown in Fig. 2C,D). Furthermore, the total distance and average speed of mice in the MPTP group in the open field test were also found to be significantly reduced (p 0.001 vs. control, Fig. 2F,G). Representative track maps for each group demonstrated the same trend (Fig. 2J,K). Meanwhile, mice in the MPTP group exhibited a significant reduction in both the time spent in the central zone and the number of entries into the central zone. These findings suggest that MPTP-treated PD mice display anxiety-like behaviors, characterized by a marked decrease in exploratory activity (p 0.001 vs. control, Fig. 2H,I). In contrast, the EA and drug groups demonstrated functional recovery as evidenced by their performance in the behavioral tests, while the sham EA group showed no recovery (Fig. 2). These results clearly demonstrate that EA exhibits significant effects in promoting MPTP-induced motor function recovery in Parkinsonian mice. A reduction in tyrosine hydroxylase (TH) activity leads to diminished DA production, which impairs neuronal function and may contribute to the pathogenesis of PD [43], while $\alpha$-syn is a pathological marker of PD [44]. The neuroprotective effect of EA on MPTP-induced neuronal death was evaluated by measuring TH levels in STR and SN and $\alpha$-syn level in SN. As depicted in Fig. 3, a comparison of the optical density of TH-positive cells in the SN revealed a significant reduction in the MPTP and sham EA groups relative to controls (p 0.001, Fig. 3A,F); however, the optical density was markedly higher in the EA and drug groups than in the MPTP and sham EA groups (p 0.01, p 0.001, respectively). A comparable trend was observed in the optical density of the STR (Fig. 3A,G). Furthermore, $\alpha$-syn levels were elevated in the MPTP and sham EA groups (p 0.001 vs. control, Fig. 3B,H), whereas EA and drug treatment resulted in a significant reduction in $\alpha$-syn levels (p 0.001 vs. MPTP group). The WB results offer further corroboration of the morphological findings (Fig. 3C–E, the original WB figures for Fig. 3C are provided in the Supplementary Materials). Based on the results of IHC and WB experiments, it can be concluded that EA effectively alleviates the accumulation phenomenon of $\alpha$-syn in the SN.

Fig. 2.

Behavioral analysis. (A) Time to landing in the pole climbing test (Day 7 F = 7.645, p 0.001; Day 21 F = 15.396, p 0.001). (B) Score in pole climbing test (E, Day 7 F = 4.106, p = 0.009; Day 21 F = 14.49, p 0.001). (C) Latency to fall in the rotarod test (Day 7 F = 21.235, p 0.001; Day 21 F = 13.74, p 0.001). (D) Falling speed in the rotarod test (Day 7 F = 21.262, p 0.001; Day 21 F = 15.955, p 0.001). (E) Score in the wire hang test (Day 7 F = 47.845, p 0.001; Day 21 F = 50.77, p 0.001). (F–I) Parameters measured in open filed test (Mean speed, Day 7 F = 359.523, p 0.001; Day 21 F = 66.39, p 0.001; Total distance, Day 7 F = 360.298, p 0.001; Day 21 F = 60.63, p 0.001; Entries in Center, Day 7 F = 15.545, p 0.001; Day 21 F = 3.401, p = 0.021; Center duration, Day 7 F = 10.309, p 0.001; Day 21 F = 1.533, no significant difference). (J) Representative track maps of each group in open field test Day 7. (K) Representative track maps of each group in open field test Day 21. Data were presented as mean $\pm$ standard error of the mean. *p 0.05, **p 0.01, ***p 0.001 vs. Control; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs. MPTP; n = 7, one-way analysis of variance with Bonferroni post hoc test used. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; EA, electroacupuncture.

Fig. 3.

EA ameliorated neuropathological alterations produced by MPTP in PD mice. (A) Representative immunohistochemistry analysis of TH for SN and STR. Magnifications of $\times$40 were taken. Top row enlarges the original image by $\times$5 (scale bar: SN 200 μm; STR 625 μm), the bottom row enlarges the original image by $\times$40 (scale bar: SN 50 μm; STR 50 μm). (B) Representative immunohistochemistry analysis of $\alpha$-syn, magnification as above (scale bar: top row 400 μm; the bottom row 50 μm). (C) Western blot of TH, $\alpha$-syn and GAPDH expression in the SN. (D,E) Quantitative analysis of Western blots for $\alpha$-syn ((D), F = 8.188, p = 0.003) and TH ((E), F = 13.43, p = 0.005). (F,G) Optical density of TH⁺ fiber immunoreactivity in SN ((F), F = 47.15, p 0.001) and STR ((G), F = 22.25, p 0.001). (H) Quantitative results of $\alpha$-syn-positive aggregates (F = 13.22, p = 0.0005). SN, substantia nigra; STR, striatum; TH, tyrosine hydroxylase; $\alpha$-syn, $\alpha$-synuclein; Data presented as mean $\pm$ standard error of the mean. **p 0.01, ***p 0.001 vs. Control; ^#p 0.05, ^#⁢#p 0.01 vs. MPTP; n = 3, one-way analysis of variance with Bonferroni post hoc test was used.

MAMs in the SN were observed through TEM. As shown in Fig. 4, compared with controls MAMs in the MPTP and sham groups were loose, with significant distance between mitochondria and ER, reducing the percentage of mitochondrial-ER contact in the whole mitochondria (p 0.01 vs. control, Fig. 4F). In contrast, the MAMs in the EA and drug groups were tighter (p 0.01 vs. MPTP group). To further explore the specific effects of EA intervention on MAMs, immunofluorescence and WB were performed on control, MPTP and EA groups. The double-labelling immunofluorescence technique was employed according to [45], using translocase of outer mitochondrial membrane 20 (TOM20) and calreticulin (CRT), to observe and analyze the level of intracellular mitochondrial-ER co-localization. Results showed that the mitochondrial-ER co-localization coefficient decreased in the MPTP group when compared to control, but increased in the EA group compared to the MPTP (Fig. 5A,C). IP3R, Mfn2 and VAPB are essential components of highly dynamic protein complexes and the collection of functional regulatory proteins that make up MAMs. Once the expression of these proteins is reduced, it leads to the structure of MAMs becoming loose and triggers their functional disorder [46, 47]. WB results showed IP3R, Mfn2 and VAPB protein levels in SN tissue of the MPTP group decreased (Fig. 5B, p 0.05, p 0.05 vs. control, respectively; the original WB figures for Fig. 5B are provided in the Supplementary Materials). In the EA group, these protein levels significantly increased (p 0.01, p 0.05, p 0.01 vs. MPTP group, respectively, Fig. 5D–F). These results strongly suggest that EA in PD significantly improves the morphology and function of MAMs in SN neurons.

Fig. 4.

Results of endoplasmic reticulum membranes in the SN in each group of mice were observed under transmission electron microscope. (A–E) Representative transmission electron micrographs for each group, the arrowheads with loops show regions of association. (F) The percentage of the mitochondria surface closely apposed to endoplasmic reticulum (defined as $\le$30 nm proximity) in the whole mitochondria of different groups (F = 21.41, p = 0.0019). Data were presented as mean $\pm$ standard error of the mean. **p 0.01 vs. Control; ^#⁢#p 0.01 vs. MPTP; n = 3, one-way analysis of variance with Bonferroni post hoc test used. Magnification: 40,000× , scale bar: 500 nm.

Fig. 5.

Effect of EA on MPTP-induced changes of MAMs in PD mice. (A) Co-localization of ER and mitochondria in each group of substantia nigra neurons detected by immunofluorescence. DAPI marks the nucleus blue. TOM20 stains the mitochondria red. CRT marks the endoplasmic reticulum, shown as green. The overlapping area of TOM20 and CRT represents the MAMs (magnification, $\times$200). (B) Western blots of IP3R, Mfn2, VAPB and GAPDH expression in the SN. (C) Quantified colocalization of CRT with TOM20 assessed by the Pearson coefficient (F = 176.881, p 0.001). (D–F) Quantitative results of IP3R (F = 60.58, p 0.001), Mfn2 (F = 9.558, p = 0.013), VAPB (F = 14.38, p = 0.005). DAPI, 4^′,6-Diamidino-2^′-phenylindole; TOM20, translocase of outer mitochondrial membrane 20; CRT, calreticulin; IP3R, inositol1,4,5-trisphosphate receptors; Mfn2, mitofusin 2; VAPB, vesicle associated membrane protein associated protein B; ER, endoplasmic reticulum. Data given as mean $\pm$ standard error of the mean. *p 0.05, **p 0.01, ***p 0.001 vs. Control; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs. MPTP; n = 3, one-way analysis of variance with Bonferroni post hoc test used.

To explore EA’s mechanism in PD, gene expression profiling was completed for different groups. A volcano map revealed 1008 DEGs in the MPTP vs. control (658 up, 350 down). Between the MPTP and EA groups, 869 DEGs were identified (364 up, 505 down). Heat maps visualized expression patterns of up-regulated and down-regulated genes for the control, MPTP and EA groups. As Fig. 6A shows, red and blue colors respectively illustrate higher and lower expression of genes. DEGs overlaps were then analyzed with Venn diagrams (Fig. 6B). EA downregulated 231 of the 658 up-regulated DEGs and upregulated 98 of the 350 down-regulated DEGs in the MPTP group. The 329 DEGs may be key EA targets against PD, as shown in the heat map (Fig. 6C).

Fig. 6.

Analysis of mice transcriptome samples. (A) Volcano plot for ($|$log₂FC$|$ $>$1.5, p 0.05). (B) Venn diagram of differentially expressed genes (DEGs). (C) Heat map of DEGs. (D) Gene Ontology (GO) functional annotation classification of DEGs. (E,F) Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome enrichment analysis of DEGs.

To investigate the function of the DEGs, GO classification, KEGG and Reactome annotations, analysis and enrichment analyses were performed. Following the GO database, three different ontologies were used to annotate the genes, including biological process (BP), cellular component (CC) and molecular function (MF). In GO BP terms, the DEGs were mostly enriched in catalytic activity and binding. The DEGs were mostly enriched in cell and membrane part categories according to their CC classification. MF DEGs were mostly enriched in cellular processes and biological regulation (Fig. 6D). To identify key pathways modulated by EA in PD, we analyzed DEGs via KEGG pathway enrichment. KEGG pathway analysis revealed EA modulation of the first three pathways: neuroactive ligand-receptor interactions, morphine addiction and dopaminergic synapses (Fig. 6E). Reactome analysis showed EA modulation of signaling, G protein-coupled receptor (GPCR) signaling and downstream GPCR signaling (Fig. 6F).

To explore the mechanism of EA regulation of MAMs in depth, a Venn diagram analysis was performed of 329 genes potentially regulated by EA with genes that are known to be associated with the regulation of MAMs in the GO database. 32 candidate genes most likely to be key players in the regulation of MAMs function and activity were screened by electroacupuncture (Fig. 7A). To visualize the specific changes of these genes in different experimental groups, heat maps were created to clearly show their expression differences (Fig. 7B). These DEGs were analyzed for GO and KEGG pathway enrichment to further reveal the mechanism of EA regulation of MAMs. The MF were mainly enriched in biological regulation, cellular process, and response to stimulus; the CC was enriched in cell part, membrane part and membrane, the BP was primarily enriched in binding, catalytic activity and transporter activity (Fig. 7D). Pathway analysis showed that the MAMs pathways were inhibited, such as calcium signaling pathway, cAMP signaling pathway and ECM-receptor interaction pathway (Fig. 7E). A protein-protein interaction network was constructed using the STRING database (Fig. 7C). These findings provide further evidence that fibronectin-1 (Fn1) is strongly associated with MAMs. The results indicated that EA may influence MAMs by enhancing the expression of Fn1, thereby providing a potential treatment for PD.

Fig. 7.

MAMs-related transcriptomic analysis. DEGs: (A) Venn diagram; (B) Heat map; (C) Protein-protein interaction analysis; (D) GO functional annotation classification; (E) KEGG enrichment analysis.

To validate RNA-Seq data reliability, we examined eight DEGs: adenosine a2a receptor (Adora2a), cluster of differentiation 4 (Cd4), cubilin (Cubn), dopamine receptor d1 (Drd1), fibronectin 1 (Fn1), integrin alpha 5 (Itga5), myocilin (Myoc) and proprotein convertase subtilisin/kexin type 9 (Pcsk9). Their expression trends (Fig. 8) matched RNA-Seq profiles. Three genes (Cubn, Fn1 and Myoc) were downregulated in the MPTP group (Fig. 8C,E,G) but upregulated by EA. Conversely, five genes (Adora2a, Cd4, Drd1, Itga5 and Pcsk9) were upregulated in the MPTP group but downregulated by EA (Fig. 8A,B,D,F,H).

Fig. 8.

Confirmation of RNA sequencing results by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). (A) Adora2a (F = 68.08, p 0.001). (B) Cd4 (F = 38.03, p 0.001). (C) Cubn (F = 69.89, p 0.001). (D) Drd1 (F = 29.08, p 0.001). (E) Fn1 (F = 9.999, p = 0.0012). (F) Itga5 (F = 16.60, p = 0.0036). (G) Myoc (F = 23.53, p 0.001). (H) Pcsk9 (F = 62.25, p 0.001). Data were presented as mean $\pm$ standard error of the mean. *p 0.05, **p 0.01, ***p 0.001 vs. Control; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs. MPTP; n = 3, one-way analysis of variance with Bonferroni post hoc test.