Bavachalcone’s protective effects in a mouse model of Parkinson’s disease linked to gut-brain interaction and body metabolism

Eight-week-old male C57BL/6 mice (23 ± 2 g) were obtained from Shanghai Model Organisms Center, Inc. The mice were maintained in a standard environment (temperature, 22 ± 2 °C; humidity, 50% ± 10%) under specific pathogen-free conditions. All animal experiments were approved by the Animal Ethics Committee of Shaoxing People's Hospital (Approval no. 2024Z078). The mice were randomly assigned to four groups (n = 6 per group): (1) Control group, (2) MPTP model group (MPTP), (3) low-dose bavachalcone + MPTP group (Bavac-L, 30 mg/kg), (Aladdin, China; Cat. No. 28448–85-3), and (4) high-dose bavachalcone + MPTP group (Bavac-H, 60 mg/kg).

All animals underwent daily oral gavage for seven consecutive days. The Bavac-L and Bavac-H groups were administered bavachalcone, respectively, whereas the Control and MPTP groups received phosphate-buffered saline (PBS, pH 7.4). On day 7 post-treatment, mice in the MPTP, Bavac-L, and Bavac-H groups were intraperitoneally injected with MPTP (25 mg/kg/d, Sigma-Aldrich, USA; Cat. No. M0896) for seven consecutive days. An equivalent volume of isotonic sodium chloride solution was administered to the Control group.

All surgical procedures were performed under anesthesia induced by intraperitoneal injection of 0.3% pentobarbital sodium (50 mg/kg; Sigma-Aldrich, USA; Cat. No. 4390-16-3), with rigorous measures taken to minimize animal suffering. The study protocol was approved by the Institutional Animal Care and Use Committee of Shaoxing People's Hospital (Shaoxing, China; ID number: 2024Z078) and strictly complied with China's Regulations for the Administration of Affairs Concerning Experimental Animals. All experimental procedures conformed to the ARRIVE guidelines for preclinical research.

Brain tissues from the striatum were rapidly isolated and stored at −80 °C. Tissues were homogenized in 1 × RIPA lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The homogenates were centrifuged at 12,000 g for 15 min at 4 °C, and the supernatants were collected for protein analysis.

Equal amounts of total protein from each group were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred onto 0.45 μm polyvinylidene difluoride membranes. Membranes were blocked with 5% (w/v) skim milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature to prevent nonspecific binding.

Subsequently, membranes were incubated overnight at 4 °C with primary antibodies, including mouse anti-tyrosine hydroxylase (TH) antibody (Sigma, USA; Cat. No. AB152; 1:1000) and mouse anti-GAPDH antibody (Proteintech, China; Cat. No. 60004-1-Ig; 1:1000). After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse secondary antibodies (Cell Signaling Technology, USA; 1:1000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection kit and imaged using an Invitrogen iBright 1,500 imaging system.

Brain samples were collected and fixed in 4% paraformaldehyde at −4 °C for subsequent preparation of paraffin-embedded sections. Mouse brain tissue sections were deparaffinized using environmentally friendly dewaxing reagents and dehydrated using anhydrous ethanol. Sections were incubated overnight at 4 °C with a rabbit monoclonal anti-TH primary antibody (Sigma, USA; Cat. No. AB152; 1:200). Following three washes with PBS, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Proteintech, China; Cat. No. SA00001-2; 1:200) for 50 min at room temperature, then washed three times with PBS. TH immunoreactivity was visualized using DAB chromogenic substrate, and images were acquired using a light microscope.

Sections were deparaffinized in xylene and rehydrated using a graded ethanol series. Antigen retrieval was performed by boiling the sections in 10 mM citrate buffer (pH 6.0) for 15 min. After cooling, sections were blocked with 5% normal goat serum containing 0.3% Triton X-100 for 1 h at room temperature.

Primary antibodies against GFAP (Abcam, UK; Cat. No. ab7260; 1:1000) and IBA1 (Wako, Japan; Cat. No. 019–19,741; 1:500) were applied overnight at 4 °C. After washing with PBS, the sections were incubated with Alexa Fluor 594- or Alexa Fluor 488-conjugated secondary antibodies (Invitrogen, USA; Cat. No. 8890 & 4,412; 1:1000) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI (Proteintech, China; Cat. No. 28718-90-3; 1 μg/mL). Images were captured using a fluorescence microscope.

Whole blood samples were collected from the mice via cardiac puncture and allowed to clot at room temperature. Serum was separated by centrifugation at 3000 rpm for 10 min at 4 °C and stored at −80 °C until analysis.

Serum levels of TNF-α, IL-1β, and IL-6 were quantified using commercial ELISA kits (Byabscience Biotechnology Co., Ltd., China; Cat. No. BY-EM220852, BY-EM220174 and BY-EM220188) according to the manufacturer's protocols. Absorbance was measured at 450 nm using a microplate reader, and cytokine concentrations were calculated based on standard curves.

Fresh fecal samples were collected from mice and immediately stored at −80 °C. Total bacterial genomic DNA was extracted using a commercial extraction kit according to the manufacturer's instructions. The V3–V4 hypervariable regions of the bacterial 16S rRNA gene were amplified using PCR with specific primers. Amplicons were purified, quantified, and sequenced on an Illumina MiSeq platform.

Raw sequencing data were quality-filtered, merged, and clustered into operational taxonomic units at a 97% similarity threshold using QIIME software. Alpha diversity indices, including Shannon, Chao1, and ACE, were calculated to assess species richness and diversity. Beta diversity was analyzed using Principal Coordinate Analysis and nonmetric multidimensional scaling based on Bray–Curtis distances. Differential microbial taxa among groups were identified using LEfSe analysis, and the taxonomic composition was visualized using community bar plots at various taxonomic levels (family, genus, and species). Data analysis was performed using majorbio tools created by Major-BIO Co. Ltd. (Shanghai, China) at https://cloud.majorbio.com/↗.

Untargeted metabolomic profiling was performed using liquid chromatography–tandem mass spectrometry with a high-resolution mass analyzer (Thermo Fisher Q Exactive). Chromatographic separation was achieved using a C18 reverse-phase column with gradient elution. Data acquisition was performed in both positive and negative ion modes.

Raw data were processed for peak alignment, normalization, and identification using Compound Discoverer software. Multivariate statistical analyses—including principal component analysis, partial least squares discriminant analysis (PLS-DA), and orthogonal PLS-DA—were conducted to identify global metabolic differences. Differential metabolites were selected based on a variable importance in projection value >1.0 and p < 0.05. KEGG pathway enrichment analysis was performed to explore the biological pathways involved.

All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA, USA). At least three independent replicates were used to ensure accuracy. Statistical analyses were performed using GraphPad Prism 5. Student's t-test was used to compare two groups, and one-way analysis of variance with Tukey's or Bonferroni's multiple comparison test was used to assess the significance in more than two groups. Data are presented as the mean ± standard error of the mean. Statistical differences between groups were evaluated, and p-values < 0.05 were considered statistically significant. Significance levels are indicated as follows: p < 0.05, *p < 0.01, **p < 0.001, and ***p < 0.0001.

To investigate the protective effects of bavachalcone, a bioactive compound derived from Psoralea corylifolia, in a mouse model of PD, mice were randomly assigned to four groups: Control, MPTP model, Bavac-L, and Bavac-H. Bavachalcone was administered via oral gavage for seven consecutive days, followed by intraperitoneal MPTP injection to induce the Parkinsonian phenotype. After model induction, behavioral assessments—including the open field test, pole test, and rotarod test—were performed to evaluate the therapeutic effect of bavachalcone on motor deficits (Figure 1A).

The open field test was used to assess spontaneous locomotor activity, a general indicator of motor performance. Mice in the MPTP group showed a significant reduction in total distance traveled and less complex movement trajectories compared with those in the Control group, indicating impaired locomotor function. By contrast, mice treated with bavachalcone exhibited increased total distance traveled and more complex movement patterns, suggesting that bavachalcone mitigated MPTP-induced motor impairment (Figures 1B,C).

The pole and rotarod tests were used to assess bradykinesia and motor coordination, respectively. MPTP treatment significantly increased the time required to descend the pole, whereas bavachalcone treatment significantly reduced this latency (Figure 1D). In the rotarod test, mice in the MPTP-treated group exhibited significantly reduced travel distance, whereas those administered bavachalcone demonstrated markedly increased travel distance on the rotating rod, indicating improved motor coordination (Figure 1E). Collectively, these behavioral results demonstrate that bavachalcone attenuates MPTP-induced motor dysfunction in mice.

To evaluate dopaminergic neuronal damage, TH protein expression in the striatum was assessed using western blotting. TH levels were significantly reduced in the striatum of MPTP-treated mice compared with those in the control group. Notably, bavachalcone treatment partially restored TH expression levels relative to those in the MPTP group (Figures 2A,B), suggesting a neuroprotective effect.

Immunohistochemical staining was performed to detect TH-positive neurons in the substantia nigra (Figures 2C,D). Compared with the control group, the MPTP group showed a significant reduction in the number of TH-positive dopaminergic neurons. However, bavachalcone administration resulted in a notable increase in the number of TH-positive neurons relative to that in the MPTP group.

These findings indicate that bavachalcone effectively mitigates MPTP-induced dopaminergic neurodegeneration in the nigrostriatal pathway in the mouse model of PD.

To determine whether bavachalcone alleviates inflammation in mice with PD, serum levels of proinflammatory cytokines were measured using ELISA. MPTP-treated mice exhibited significantly elevated levels of TNF-α, IL-1β, and IL-6 compared with those in the control group. Notably, bavachalcone treatment significantly reduced the levels of these cytokines in both low- and high-dose groups (Figures 3A–C).

To assess glial activation in the substantia nigra, immunofluorescence staining was performed to detect GFAP and IBA1 expression (Figures 3D–F). MPTP administration substantially increased GFAP and IBA1 expression, indicating significant activation of astrocytes and microglia, respectively. Bavachalcone effectively attenuated glial activation in a dose-dependent manner. These results indicate that bavachalcone mitigates both central and peripheral inflammatory responses in the MPTP-induced PD model.

Collectively, these findings demonstrate that bavachalcone exerts potent anti-inflammatory effects by suppressing systemic cytokine production and central glial activation in mice with PD.

Recent studies have indicated that dysbiosis of the gut microbiota plays a critical role in the pathogenesis of PD. To investigate whether bavachalcone improves Parkinsonian phenotypes by modulating the gut microbiota, we performed 16S rDNA sequencing of fecal samples from Control, MPTP, and bavachalcone-treated mice.

The rank-abundance curve reflected high species richness and even distribution within the microbial community (Figure 4A). A curve was plotted with the amount of extracted data on the x-axis and alpha diversity index on the y-axis. Flattening of the rarefaction curves indicated that the sequencing depth was sufficient (Figure 4B). α-Diversity analysis revealed significant differences in gut microbiota richness among the control group, MPTP-treated group, and bavachalcone-intervention group (Figures 4C,D). In β-diversity analysis, both principal coordinate analysis and non-metric multidimensional scaling revealed distinct differences in gut microbiota composition among the treatment groups (Figures 4E,F).

Collectively, these results indicate that bavachalcone effectively modulates the diversity and composition of gut microbiota disrupted by MPTP treatment. Bavachalcone may help alleviate gut dysbiosis associated with PD, offering new insights into its potential therapeutic mechanisms.

The Venn diagram demonstrated partial overlap in operational taxonomic units among the three groups, with the bavachalcone group displaying a unique microbial profile, suggesting its potential for targeted microbial modulation (Figure 5A).

To further explore the specific effects of bavachalcone on gut microbiota composition, we generated community bar plots at different taxonomic levels, including phylum (Figure 5B) and genus (Figure 5C). At the phylum level, Firmicutes were enriched in the Control and bavachalcone groups but markedly decreased in the MPTP group. By contrast, the relative abundance of Bacteroidetes, Actinobacteria, and Campylobacterota was elevated in the MPTP group, indicating dysbiosis, which was partially reversed by bavachalcone treatment (Figure 5B). At the genus level, Allobaculum was highly enriched in both the Control and bavachalcone groups, whereas its abundance was significantly reduced in the MPTP group. By contrast, Ligilactobacillus was significantly elevated in the MPTP group, and this increase was suppressed by bavachalcone (Figure 5C). These results suggest that bavachalcone alleviates gut dysbiosis by reducing the abundance of harmful genera and restoring beneficial taxa.

To identify key differential taxa influenced by bavachalcone, we performed intergroup differential significance testing. At the genus level, Allobaculum was significantly enriched in the Control and bavachalcone groups but markedly reduced in the MPTP group. By contrast, Ligilactobacillus, Eubacterium xylanophilum, and Helicobacter were significantly enriched in the MPTP group and reduced in the Control and bavachalcone groups (Figure 6A). At the phylum level, Firmicutes were enriched in the Control and bavachalcone groups but significantly decreased in the MPTP group. Conversely, Bacteroidetes, Campylobacteroides, and Cyanobacteria were elevated in the MPTP group and reduced in the Control and bavachalcone groups (Figure 6B). Furthermore, the cladogram generated from phylogenetic analysis illustrated the evolutionary relations among differential taxa, suggesting that bavachalcone may exert neuroprotective effects through the modulation of specific microbial pathways (Figure 6C).

To investigate the effects of bavachalcone on systemic metabolic profiles in mice with MPTP-induced PD, serum metabolomic analysis was performed. The volcano plot showed a number of significantly altered metabolites between the bavachalcone-treated and MPTP groups, including 133 upregulated and 73 downregulated metabolites (Figure 7A). Principal component analysis demonstrated distinct clustering between the two groups, indicating treatment-related metabolic shifts (Figure 7B). Furthermore, supervised PLS-DA and orthogonal PLS-DA confirmed a clear separation between groups, suggesting that bavachalcone treatment substantially altered serum metabolite composition (Figures 7C,D).

To further identify the key metabolites contributing to group-level metabolic differences, variable importance in projection analysis based on the PLS-DA model was performed (Figure 7E). Metabolites, such as hypoxanthine, swertiamarin, riboflavin, and tiopronin, were identified as major contributors to intergroup separation. Additionally, a heatmap of the top-ranking metabolites revealed distinct expression patterns between the MPTP and bavachalcone-treated groups, with bavachalcone partially restoring the levels of several disrupted metabolites. These findings suggest that bavachalcone exerts significant regulatory effects on the serum metabolic profile, which may underlie its neuroprotective action in PD.

Further classification of the differentially abundant metabolites indicated that most belonged to compound classes, such as carboxylic acids, phospholipids, amino acids, and vitamins (Figure 8A). KEGG pathway enrichment analysis revealed that the altered metabolites were primarily associated with pyruvate metabolism, folate biosynthesis, glycolysis/gluconeogenesis, and phenylalanine metabolism—pathways closely related to the pathogenesis of PD (Figure 8B).

These results suggest that bavachalcone may exert its neuroprotective effects in PD, at least in part, by remodeling systemic metabolism and restoring key metabolic pathways disrupted by MPTP treatment.