Taurochenodeoxycholic acid alleviates MPP⁺/MPTP-induced neurotoxicity in vitro and in vivo by suppressing ferroptosis via TGR5/cGAS/STING signaling pathway

Abstract

Parkinson's disease (PD) is characterized by progressive loss of dopaminergic neurons in the substantia nigra, with ferroptosis emerging as critical pathogenic mechanisms. Recent evidence suggests that STING activation can induce neuronal ferroptosis through autophagic degradation of GPX4. Taurochenodeoxycholic acid (TCDCA), a naturally occurring bile acid, has demonstrated neuroprotective properties through activation of Takeda G protein-coupled receptor 5 (TGR5). However, whether TCDCA can improve PD by modulating the cGAS-STING-ferroptosis axis remains unexplored. We investigated the effects of TCDCA treatment on motor function, dopaminergic neuronal survival, oxidative stress markers, ferroptosis-related proteins (GPX4, SLC7A11, ACSL4), and cGAS-STING signaling components in the substantia nigra of male mice subjected to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration and in MPP⁺-treated SH-SY5Y cells. Behavioral assessments demonstrated that TCDCA significantly improved motor dysfunction in both open field and pole tests. TCDCA treatment markedly increased tyrosine hydroxylase-positive neurons and reduced oxidative stress markers including malondialdehyde and ferrous iron levels while restoring superoxide dismutase activity and glutathione content in the substantia nigra. Results showed that TCDCA upregulated TGR5 expression and concurrently suppressed cGAS and STING activation in both in vivo and in vitro PD models. Importantly, TCDCA treatment significantly enhanced the expression of anti-ferroptotic proteins GPX4 and SLC7A11 while reducing pro-ferroptotic ACSL4. These neuroprotective effects were associated with TGR5 upregulation and cGAS-STING pathway suppression. Our findings demonstrate that TCDCA alleviates PD-related neurodegeneration by inhibiting cGAS-STING-mediated ferroptosis through TGR5 activation, suggesting that TCDCA holds promise as a candidate drug for the treatment of PD.

Graphical Abstract

1. Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide, affecting approximately more than 2% of individuals over 65 years of age, with incidence rates continuing to rise as the global population ages (Chen et al., 2022; Eo et al., 2024). The disease is pathologically characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of intracellular α-synuclein aggregates known as Lewy bodies (Bonanno et al., 2022; Prieto Huarcaya et al., n.d.). This neuronal loss leads to dopamine depletion in the striatum, resulting in the cardinal motor symptoms of PD including resting tremor, bradykinesia, rigidity, and postural instability (Salemi et al., 2022). Currently, levodopa (L-DOPA) remains the gold standard pharmacological treatment for managing PD symptoms; however, long-term use is associated with motor complications such as dyskinesia and the "on-off" phenomenon, as well as diminishing therapeutic efficacy over time (Kolmančič et al., 2022; Pérez-Arancibia et al., 2023). Moreover, existing treatments are primarily symptomatic and do not halt or reverse the underlying neurodegenerative process (Hiller et al., 2022). Therefore, there is an urgent need to identify novel therapeutic agents that can protect dopaminergic neurons and address the multiple pathological mechanisms driving PD progression.

Accumulating evidence suggests that neuroinflammation and ferroptosis play critical roles in the pathogenesis of PD (Trist et al., 2019; Yu et al., 2023). Mitochondrial dysfunction, a hallmark feature of PD, leads to the release of mitochondrial DNA (mtDNA) into the cytoplasm, where it acts as a danger-associated molecular pattern (Li et al., 2021a). Cytosolic mtDNA is recognized by cyclic GMP-AMP synthase (cGAS), which catalyzes the synthesis of cyclic GMP-AMP (cGAMP), subsequently activating stimulator of interferon genes (STING) (Jiménez-Loygorri et al., 2024). The cGAS-STING pathway triggers the production of type I interferons and pro-inflammatory cytokines, exacerbating neuroinflammation and accelerating neurodegeneration in PD (Dhapola et al., 2025). Importantly, recent studies have revealed that STING activation can directly induce neuronal ferroptosis, a form of regulated cell death characterized by iron-dependent accumulation of lipid peroxides, through mechanisms including autophagic degradation of glutathione peroxidase 4 (GPX4), the central negative regulator of ferroptosis (Woo et al., 2024). Neuronal STING activation leads to GPX4 degradation and subsequent cell iron death, providing a mechanistic link between neurodegenerative disease and iron death.

The substantia nigra is particularly vulnerable to ferroptosis due to its high iron content, elevated levels of polyunsaturated fatty acids, and substantial metabolic demands (Li et al., 2023a). In PD patients and animal models, increased levels of lipid peroxidation markers such as malondialdehyde (MDA) and elevated ferrous iron (Fe²⁺) accumulation have been consistently observed in the substantia nigra (Guo et al., 2021; Zhang et al., 2024). Dysregulation of ferroptosis-related proteins such as solute carrier family 7 member 11 (SLC7A11), which facilitates cystine uptake for GSH synthesis, and acyl-CoA synthetase long-chain family member 4 (ACSL4), which promotes lipid peroxidation, contributes to neuronal vulnerability (Ingold et al., 2018; Zhou et al., 2025). Inhibition of the cGAS-STING pathway has been shown to reduce ferroptosis, protect dopaminergic neurons, and ameliorate motor deficits in PD models, suggesting that targeting ferroptosis may offer therapeutic benefits (Woo et al., 2024). Accordingly, therapeutic strategies that could effectively suppress STING-mediated ferroptosis in PD remain highly regarded.

Bile acids, traditionally known for their roles in lipid digestion and absorption, have emerged as important signaling molecules with diverse biological functions beyond the gastrointestinal system. Taurochenodeoxycholic acid (TCDCA), a taurine-conjugated bile acid, has demonstrated anti-inflammatory, antioxidant, and cytoprotective properties in various disease models (Khalaf et al., 2022). TCDCA exerts its effects primarily through activation of Takeda G protein-coupled receptor 5 (TGR5), a membrane-bound receptor widely expressed in various tissues including the brain (Qi et al., 2020). TGR5 activation has been shown to suppress inflammatory responses by inhibiting nuclear factor-κB (NF-κB) signaling and to enhance mitochondrial function through modulation of energy metabolism pathways (Guo et al., 2016). Importantly, TGR5 is expressed in neurons, astrocytes, and microglia within the central nervous system, suggesting its potential role in neuroprotection (Yanguas-Casás et al., 2017). Previous studies have reported that TGR5 agonists can ameliorate neurological deficits in models of stroke and Alzheimer's disease (Liang et al., 2021; McMillin et al., 2015). However, whether TCDCA can palliate dopaminergic neuronal loss in PD by modulating the cGAS-STING-ferroptosis axis and the underlying mechanisms have not been investigated.

Given the critical role of the cGAS-STING pathway in mediating ferroptosis in PD pathogenesis, and the established anti-oxidation and cytoprotective properties of TCDCA, we hypothesized that TCDCA may exert neuroprotective effects in PD by suppressing cGAS-STING activation and consequently inhibiting ferroptosis through TGR5 activation. To test this hypothesis, we employed both in vivo and in vitro PD models using MPTP-treated mice and MPP⁺-exposed SH-SY5Y neuroblastoma cells. Our study aimed to evaluate the therapeutic effects of TCDCA on motor dysfunction and dopaminergic neuronal damage in MPTP-induced PD mice, investigate the impact of TCDCA on the cGAS-STING signaling pathway and ferroptosis markers in both animal and cellular models, examine whether TCDCA-mediated suppression of cGAS-STING contributes to its anti-ferroptotic effects, and determine the role of TGR5 in mediating the neuroprotective actions of TCDCA. Our findings demonstrate that TCDCA significantly ameliorates motor deficits, protects dopaminergic neurons, suppresses cGAS-STING activation, and inhibits ferroptosis through TGR5-dependent mechanisms, suggesting that TCDCA may represent a promising therapeutic candidate for PD treatment by targeting ferroptosis.

2. Materials and methods

2.1. Animals

All animal experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the Life Ethics Review Committee of the Henan Academy of Sciences (Ethics number: HNAS.EC202507a003). Male C57BL/6 mice aged 8–10 weeks were used for all experiments. House male C57BL/6 mice under standard laboratory conditions with a 12 h light/dark cycle at 22 ± 2 °C with 55 ± 5% relative humidity and provide free access to food and water.

2.2. Animal model

After one week of acclimatization, randomly divide mice into six groups with 12 mice per group: Control group receiving saline treatment, MPTP group receiving MPTP plus saline vehicle, MPTP plus TCDCA 50 mg/kg group, MPTP plus TCDCA 100 mg/kg group, MPTP plus TCDCA 200 mg/kg group, and MPTP plus L-DOPA group serving as positive control. To establish the PD mice model, intraperitoneally inject MPTP hydrochloride dissolved in sterile saline at 30 mg/kg body weight once daily for five consecutive days (Ni et al., 2025). Administer equivalent volumes of sterile saline to control mice using the same injection schedule. Beginning on day 6 after the first MPTP injection, administer TCDCA at doses of 50, 100, or 200 mg/kg and L-DOPA at doses of 75 mg/kg once daily for 14 consecutive days. Administer saline vehicle by gavage to control and MPTP groups following the same schedule. Monitor body weight throughout the experimental period. Conduct behavioral assessments on days 19–20 following the initiation of MPTP treatment. Sacrifice mice on day 21 for tissue collection and subsequent analyses (Fig. 1A).

Fig. 1.

TCDCA improves motor function in MPTP-induced PD mice. (A) Schematic diagram of the experimental design showing MPTP injection, drug administration, and behavioral testing timeline. (B) Total distance traveled in the OFT over 5 min. (C) Time required to climb down the pole in the pole test. Data are presented as mean ± SEM (n = 9). Compared with Control group, ###P < 0.001; compared with MPTP group, *P < 0.05, **P < 0.01.

2.3. Assessment of motor function using open field test

Perform the open field test (OFT) to evaluate spontaneous locomotor activity and exploratory behavior (Li et al., 2021b). Transport mice to the testing room and allow acclimatization for at least 1 h prior to testing. Use a square white arena measuring 50 cm × 50 cm × 50 cm with walls to prevent escape. Ensure uniform illumination at approximately 50 lux across the arena. Place each mouse individually in the center of the arena and allow free exploration for 5 min. Measure total distance traveled as the primary outcome parameter. Calculate the data automatically using the tracking software.

2.4. Evaluation of motor coordination using pole test

Conduct the pole test to assess motor impairment by securing a wooden pole (1 cm diameter, 50 cm length) vertically to the ground with a small ball attached to the top, wrapping both the pole and ball completely with gauze to prevent slipping (Ahuja et al., 2024). After adaptation training, place each mouse on the ball at the top of the pole and record the total time required to climb down to the bottom, testing each mouse three times with intervals of at least 1 h between trials and calculating the average as the final measurement.

2.5. Immunofluorescence staining

Section cryoprotected brains coronally at 20 μm thickness using a cryostat and collect sections containing the substantia nigra. Wash sections three times with PBS, permeabilize with 0.3% Triton X-100 for 15 min, and block with 5% BSA in PBS containing 0.1% Triton X-100 for 1 h at room temperature. Incubate with primary antibodies (anti-TH 1:500 or anti-GPX4 1:200) overnight at 4 °C, wash three times with PBS, then incubate with fluorophore-conjugated secondary antibodies (1:500) for 2 h at room temperature in the dark. Counterstain with DAPI (1:1000) for 10 min, wash, and mount with anti-fade mounting medium. Capture images using a fluorescence microscope or confocal microscope, and quantify fluorescence intensity using image analysis software with background subtraction.

2.6. Western blot analysis

Homogenize frozen brain tissue in RIPA lysis buffer containing protease and phosphatase inhibitors, incubate on ice for 30 min, and centrifuge at 12,000 × g for 15 min at 4 °C to collect supernatants. Determine protein concentration using the BCA assay and load equal amounts onto 10–12% SDS-PAGE gels. Transfer proteins to PVDF membranes, block with 5% non-fat milk in TBST for 1 h, and incubate with primary antibodies (anti-TH, anti-GPX4, anti-SLC7A11, anti-ACSL4, anti-TGR5, anti-cGAS, anti-STING at 1:1000; anti-β-actin at 1:2000) overnight at 4 °C. After washing, incubate with HRP-conjugated secondary antibodies (1:5000) for 1 h, detect bands using ECL reagent, and quantify band intensities using densitometry software with normalization to β-actin.

2.7. Measurement of oxidative stress markers

Homogenize brain tissue samples in ice-cold PBS (1:9, w/v), centrifuge at 3000 × g for 10 min at 4 °C, and collect supernatants for biochemical analyses. Homogenize brain tissue samples in ice-cold PBS (1:9, w/v), centrifuge at 3000 × g for 10 min at 4 °C, and collect supernatants for biochemical analyses. Measure MDA, SOD, GSH, and Fe²⁺ levels according to the manufacturer's instructions of the respective assay kits, normalizing all measurements to protein concentration determined by the BCA assay.

2.8. Cell culture and treatment

Culture human neuroblastoma SH-SY5Y cells in DMEM supplemented with 10% FBS and antibiotics at 37 °C in 5% CO₂. Seed cells at appropriate densities (2 × 10⁵ cells/well in 6-well plates and allow overnight attachment. Expose cells to MPP⁺ (0.5 mM) for 24 h to establish the cellular injury model, with TCDCA pretreatment (50, 100, or 200 μM) for 2 h before MPP⁺ exposure and maintained throughout the treatment period.

2.9. Cellular oxidative stress measurement

Assess cell viability using the CCK-8 assay by adding 10% CCK-8 solution to fresh medium, incubating for 2 h at 37 °C, and measuring absorbance at 450 nm to calculate viability as percentage of control. Detect intracellular ROS using DCFH-DA (10 μM) by incubating cells for 30 min at 37 °C in the dark, washing three times with serum-free medium, and measuring fluorescence at excitation 488 nm and emission 525 nm. Harvest cells by trypsinization, wash twice with ice-cold PBS, and lyse by three freeze-thaw cycles. Centrifuge lysates at 12,000 × g for 10 min at 4 °C, collect supernatants, and measure MDA, SOD, GSH, and Fe²⁺ levels using the same assay kits described for tissue samples, normalizing all measurements to protein concentration.

2.10. Statistical analysis

Present all data as mean ± SEM from at least three independent experiments and perform statistical analyses using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons, with P < 0.05 considered statistically significant.

3. Results

3.1. TCDCA ameliorates motor dysfunction in MPTP-induced PD mice

To evaluate the therapeutic potential of TCDCA in PD, we first assessed motor function using behavioral tests. The OFT revealed that MPTP-treated mice exhibited significantly reduced total distance traveled within 5 min compared to Control (P < 0.001, Fig. 1B). Treatment with TCDCA at medium (100 mg/kg) and high (200 mg/kg) doses, as well as L-DOPA (positive control), significantly increased locomotor activity compared to the MPTP group (P < 0.05, P < 0.01). In the pole test, MPTP-treated mice showed a marked increase in the total time required to climb down the pole (P < 0.0001, Fig. 1C), indicating impaired motor coordination. Both TCDCA treatment (100 and 200 mg/kg) and L-DOPA significantly reduced climbing time compared to the MPTP group (P < 0.05), demonstrating improved motor coordination. These results indicate that TCDCA effectively ameliorates MPTP-induced motor deficits in PD mice.

3.2. TCDCA protects dopaminergic neurons in the substantia nigra and striatum of PD mice

Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, serves as a marker for dopaminergic neurons (Ma and Rong, 2022). To assess the neuroprotective effects of TCDCA, we examined TH expression in the substantia nigra (SN) and striatum. Immunofluorescence staining revealed that MPTP administration significantly reduced the number of TH-positive neurons in the SN compared to control (P < 0.001, Fig. 2A–B). Treatment with TCDCA (100 and 200 mg/kg) and L-DOPA significantly increased the number of TH-positive neurons (P < 0.05), suggesting protection against dopaminergic neuronal loss. Western blot analysis confirmed these findings, showing that TCDCA (100 and 200 mg/kg) and L-DOPA treatment significantly elevated TH protein expression in the SN compared to the MPTP group (Fig. 2C–D, P < 0.05, P < 0.01, P < 0.001). These results demonstrate that TCDCA exerts neuroprotective effects by preserving dopaminergic neurons in both the SN and striatum of MPTP-induced PD mice.

Fig. 2.

TCDCA protects dopaminergic neurons in MPTP-induced PD mice. (A) Representative immunofluorescence images of TH-positive neurons (red) in the substantia nigra. Scale bar = 100 μm. (B) Quantification of TH fluorescence intensity in the substantia nigra. (C) Western blot analysis of TH protein expression in the substantia nigra. (D) Quantification of TH protein levels normalized to β-actin. Data are presented as mean ± SEM (n = 3). Compared with Control group, ##P < 0.01; compared with MPTP group, *P < 0.05.

3.3. TCDCA alleviates oxidative stress in the brain tissue of PD mice

To investigate whether TCDCA exerts antioxidant effects, we measured oxidative stress markers in brain tissue. MPTP treatment significantly increased malondialdehyde (MDA) levels (P < 0.001, Fig. 3A), a marker of lipid peroxidation. TCDCA treatment at all doses (50, 100, and 200 mg/kg) and L-DOPA significantly reduced MDA levels (P < 0.01). Conversely, MPTP significantly decreased the activities of antioxidant enzymes superoxide dismutase (SOD) (P < 0.001, Fig. 3B) and reduced glutathione (GSH) levels (P < 0.001, Fig. 3C), while increasing ferrous iron (Fe²⁺) content (P < 0.01, Fig. 3D). Treatment with TCDCA (200 mg/kg) and L-DOPA significantly restored SOD activity (P < 0.05). Furthermore, TCDCA (100 and 200 mg/kg) and L-DOPA significantly elevated GSH levels (P < 0.05, P < 0.01), while TCDCA (200 mg/kg) and L-DOPA reduced Fe²⁺ accumulation (P < 0.05). Given that excessive iron accumulation and lipid peroxidation are hallmark features of ferroptosis, and GSH depletion sensitizes cells to ferroptotic cell death, these findings prompted us to further investigate whether TCDCA protects against ferroptosis in PD mice (Cao et al., 2024).

Fig. 3.

TCDCA alleviates oxidative stress in MPTP-induced PD mice. (A) MDA levels in brain tissue. (B) SOD activity in brain tissue. (C) GSH levels in brain tissue. (D) Fe²⁺ content in brain tissue. Data are presented as mean ± SEM (n = 4). Compared with Control group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared with MPTP group, *P < 0.05, **P < 0.01.

3.4. TCDCA inhibits ferroptosis and regulates the cGAS-STING signaling pathway in PD mice

Immunofluorescence analysis revealed that MPTP treatment significantly reduced GPX4 expression in the SN (P < 0.001, Fig. 4A–B), a central regulator that prevents ferroptosis by reducing lipid peroxides. TCDCA treatment (100 and 200 mg/kg) and L-DOPA significantly restored GPX4 expression (P < 0.01), which was further confirmed by Western blot analysis (Fig. 4C–D). Additionally, MPTP decreased the expression of SLC7A11 and increased ACSL4 protein levels (P < 0.05, P < 0.001). TCDCA (200 mg/kg) and L-DOPA significantly increased SLC7A11 expression, while TCDCA (100 and 200 mg/kg) and L-DOPA reduced ACSL4 levels. Ferroptotic cell death is often accompanied by mitochondrial damage and mtDNA release, which can activate the cGAS-STING innate immune pathway (Lan et al., 2024). MPTP treatment significantly reduced TGR5 expression while increasing cGAS and STING protein levels (P < 0.05, P < 0.01, Fig. 4E–F). TCDCA treatment (100 and 200 mg/kg) significantly upregulated TGR5 expression (P < 0.05) and, notably, TCDCA (200 mg/kg) and L-DOPA significantly suppressed cGAS and STING expression (P < 0.05, P < 0.01). These results suggest that TCDCA ameliorates motor dysfunction and dopaminergic neuronal damage in PD mice by inhibiting cGAS-STING-mediated ferroptosis through TGR5 activation.

Fig. 4.

TCDCA inhibits ferroptosis and modulates cGAS-STING signaling in PD mice. (A) Representative immunofluorescence images showing GPX4 expression (green) in the substantia nigra. DAPI (blue) indicates nuclei. Scale bar = 100 μm. (B) Quantification of GPX4 fluorescence intensity. (C) Western blot analysis of ferroptosis-related proteins (SLC7A11, GPX4, ACSL4). (D) Quantification of SLC7A11, GPX4, and ACSL4 protein levels. (E) Western blot analysis of TGR5, STING, and cGAS. (F) Quantification of TGR5, STING, and cGAS protein levels normalized to β-actin. Data are presented as mean ± SEM (n = 3). Compared with Control group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared with MPTP group, *P < 0.05, **P < 0.01, ***P < 0.001.

3.5. TCDCA protects SH-SY5Y cells against MPP⁺-induced cytotoxicity and oxidative stress

To further investigate the neuroprotective mechanisms of TCDCA at the cellular level, we employed the MPP⁺-induced SH-SY5Y neuronal cell model. First, we determined the safe concentration range of TCDCA by exposing cells to concentrations ranging from 25 to 400 μM for 24 h. Cell viability assays showed that TCDCA at concentrations up to 200 μM did not exhibit cytotoxicity (Fig. 5A). Next, we established that MPP⁺ at 0.5 mM significantly reduced cell viability (P < 0.01, Fig. 5B), which was selected for subsequent experiments. As shown in Fig. 5C, MPP⁺ treatment significantly decreased cell viability, while TCDCA (100 and 200 μM) significantly improved cell survival (P < 0.05, P < 0.01). Furthermore, MPP⁺ significantly elevated ROS production, MDA levels, and Fe²⁺ content, while reducing GSH and SOD levels compared to control cells. TCDCA treatment dose-dependently reversed these changes, with the most pronounced effects observed at 100 and 200 μM concentrations (Fig. 5D-H). These results indicate that TCDCA protects neuronal cells against MPP⁺-induced oxidative injury and cytotoxicity.

Fig. 5.

TCDCA protects SH-SY5Y cells from MPP⁺-induced injury. (A) Cell viability of SH-SY5Y cells treated with various concentrations of TCDCA (0–400 μM) for 24 h. (B) Cell viability of SH-SY5Y cells exposed to different concentrations of MPP⁺ (0–2 mM) for 24 h. (C) Cell viability of SH-SY5Y cells pretreated with TCDCA followed by MPP⁺ exposure. (D) ROS levels measured by fluorescence. (E) MDA levels. (F) GSH levels. (G) SOD activity. (H) Fe²⁺ content. Data are presented as mean ± SEM (n = 4). Compared with Control group, ##P < 0.01, ###P < 0.001; compared with MPP⁺ group, *P < 0.05, **P < 0.01, ***P < 0.001.

3.6. TCDCA inhibits ferroptosis and suppresses cGAS-STING signaling activation in MPP⁺-treated SH-SY5Y cells

To validate our in vivo findings, we examined ferroptosis markers and the cGAS-STING pathway in MPP⁺-treated SH-SY5Y cells. Western blot analysis revealed that MPP⁺ significantly increased ACSL4 expression while reducing GPX4 and SLC7A11 levels compared to control cells. TCDCA treatment dose-dependently reversed these changes (Fig. 6A–B, P < 0.05, P < 0.01), confirming its anti-ferroptotic effects in vitro. Regarding the cGAS-STING signaling pathway, MPP⁺ treatment significantly decreased TGR5 expression while elevating STING and cGAS protein levels. TCDCA treatment restored these alterations in a dose-dependent manner (Fig. 6C–D, P < 0.05, P < 0.01, P < 0.001). Notably, TCDCA at 200 μM most effectively suppressed cGAS and STING activation. Taken together, these findings indicate that TCDCA protects SH-SY5Y cells against MPP⁺-induced ferroptotic cell death by suppressing the cGAS-STING pathway through TGR5 activation, corroborating our in vivo observations.

Fig. 6.

TCDCA inhibits ferroptosis and cGAS-STING signaling in MPP⁺-treated SH-SY5Y cells. (A) Western blot analysis of ferroptosis-related proteins (ACSL4, SLC7A11, GPX4) in SH-SY5Y cells. (B) Quantification of ACSL4, GPX4, and SLC7A11 protein levels. (C) Western blot analysis of cGAS-STING pathway proteins (cGAS, STING, TGR5). (D) Quantification of TGR5, STING, and cGAS protein levels normalized to β-actin. Data are presented as mean ± SEM (n = 3). Compared with Control group, #P < 0.05, ##P < 0.01; compared with MPP⁺ group, *P < 0.05, **P < 0.01, ***P < 0.001.

4. Discussion

In the present study, we demonstrated that TCDCA exerts neuroprotective effects in MPTP-induced PD mice and MPP⁺-treated SH-SY5Y cells through multiple mechanisms. Our findings reveal that TCDCA ameliorates motor dysfunction, protects dopaminergic neurons in the substantia nigra, suppresses the cGAS-STING pathway, inhibits ferroptosis, and alleviates oxidative stress. These beneficial effects appear to be mediated primarily through activation of TGR5, a G protein-coupled bile acid receptor.

Motor dysfunction is the hallmark clinical manifestation of PD, resulting from progressive loss of dopaminergic neurons in the substantia nigra pars compacta and consequent dopamine depletion in the striatum (Ardizzone et al., 2022; Rabaneda-Lombarte et al., 2022). Our behavioral assessment using the OFT and pole test demonstrated that MPTP administration significantly impaired locomotor activity and motor coordination in mice, consistent with previous reports(Ni et al., 2025). Treatment with TCDCA at doses of 100 and 200 mg/kg markedly improved both spontaneous locomotor activity and motor coordination, achieving efficacy comparable to L-DOPA. These behavioral improvements were accompanied by increased expression of TH, the rate-limiting enzyme in dopamine synthesis and a specific marker for dopaminergic neurons, in both the substantia nigra and striatum (Kim et al., 2023). Immunofluorescence analysis revealed that TCDCA treatment significantly increased the number of TH-positive neurons in the substantia nigra, suggesting that TCDCA protects dopaminergic neurons from MPTP-induced degeneration rather than merely enhancing dopamine synthesis in surviving neurons. The neuroprotective effects of TCDCA are particularly noteworthy given that most current PD therapies, provide only symptomatic relief without addressing the underlying neurodegenerative process (He et al., 2020). While L-DOPA effectively replenishes dopamine levels in the short term, its long-term use is associated with motor complications and diminishing efficacy (Pike et al., 2022). In contrast, agents that can preserve dopaminergic neuronal integrity may offer more sustained therapeutic benefits and potentially slow disease progression. Our findings suggest that TCDCA may belong to this latter category of disease-modifying agents.

TCDCA exerts its biological effects primarily through two types of receptors: the membrane-bound G protein-coupled receptor TGR5 and the nuclear receptor FXR. FXR is predominantly expressed in the liver and intestine, where it regulates bile acid synthesis and enterohepatic circulation; its expression in the CNS is relatively limited (Zhang et al., 2026). In contrast, TGR5 is widely distributed in the CNS, including in neurons, microglia, and astrocytes, and its activation attenuates neuroinflammation and neuronal injury in models of depression, EAE, and PD (Huang et al., 2022; Tao et al., 2024; Xu et al., 2023). The present study therefore focused on TGR5-mediated mechanisms, consistent with its predominant neuroprotective role. The potential contribution of other bile acid receptors, including FXR, to TCDCA's neuroprotective effects warrants further investigation in future studies.

Although the present study focused on neuronal ferroptosis, it is important to note that TCDCA also exerts neuroprotective effects through modulation of glial cell function. Our previous study demonstrated that TCDCA activates autophagy and suppresses microglial neuroinflammation in MPTP-induced PD mice through TGR5-mediated AMPK/mTOR, AKT/NF-κB, and Pink1/Parkin signaling pathways (Ni et al., 2025). In addition, TCDCA attenuates astrocyte-mediated neuroinflammation via the TGR5/AKT/NF-κB pathway in a mouse model of EAE (Xu et al., 2023). The present study extends this body of work by identifying a novel neuronal mechanism—suppression of cGAS-STING-mediated ferroptosis—through which TCDCA confers neuroprotection, thereby providing a more comprehensive picture of its CNS actions. The cGAS-STING pathway is activated by cytosolic DNA, including mtDNA released from damaged mitochondria (Bi et al., 2020). Upon binding to cytosolic DNA, cGAS synthesizes the second messenger cGAMP, which activates STING, thereby affecting the downstream signaling pathway (Zhang et al., 2020). Our results demonstrated that MPTP treatment significantly upregulated cGAS and STING expression in the substantia nigra, while TCDCA treatment dose-dependently suppressed the expression of both cGAS and STING. Importantly, we observed that TCDCA-mediated upregulation of TGR5 was associated with concurrent suppression of cGAS-STING activation, suggesting that TGR5 may negatively regulate this pathway. TGR5, upon activation, increases intracellular cAMP levels and activates protein kinase A (PKA), which has been shown to exert effects through multiple pathways (Qi et al., 2020). Our previous study demonstrated that TCDCA activated autophagy and inhibited microglia-mediated neuroinflammation through regulation of AKT/NFκB, AMPK/mTOR, and Pink1/Parkin signaling pathways via TGR5 activation (Ni et al., 2025). The observation that MPTP treatment decreased TGR5 expression while increasing cGAS-STING activation, and that TCDCA reversed both changes, provides correlative evidence for TGR5-mediated suppression of the cGAS-STING pathway.

Regarding the molecular connection between TGR5 and the cGAS-STING-GPX4 axis, converging evidence from recent studies supports the biological plausibility of this signaling cascade. A study in diabetic retinopathy demonstrated that TGR5 activation suppresses cGAS/STING signaling, and that TGR5 knockdown-induced retinal damage could be rescued by a STING inhibitor, providing direct evidence that TGR5 negatively regulates STING (Li et al., 2023b). Furthermore, STING activation promotes autophagic degradation of GPX4 through the autophagy-lysosome pathway, depleting antioxidant defenses and triggering lipid peroxidation and ferroptotic cell death in models of multiple sclerosis and myocardial ischemia-reperfusion injury; these effects are reversible by STING inhibitors (Wang et al., 2025; Woo et al., 2024). Regarding TGR5 upregulation following TCDCA treatment, our prior study demonstrated that TCDCA enhances TGR5 transcriptional activity and protein expression (Ni et al., 2025). In the MPTP model, TGR5 expression is pathologically suppressed; TCDCA treatment therefore restores TGR5 toward physiological levels rather than inducing pharmacological overexpression. Taken together, we propose the following mechanistic model: TCDCA activates TGR5, which suppresses cGAS-STING signaling, thereby preventing STING-mediated autophagic degradation of GPX4 and ultimately inhibiting ferroptosis in dopaminergic neurons. We acknowledge that direct experimental validation of this cascade—for example, through TGR5 knockdown combined with STING inhibitor rescue experiments or monitoring of autophagic GPX4 flux—is required to establish causality, and this will be prioritized in future studies.

Ferroptosis, an iron-dependent form of regulated cell death that has emerged as a critical mechanism of dopaminergic neuronal loss in PD (Pham et al., 2025; Sharma et al., 2026). Recent groundbreaking studies have revealed that STING activation can directly induce neuronal ferroptosis through multiple mechanisms. STING activation promotes autophagic degradation of GPX4, the central negative regulator of ferroptosis, leading to accumulation of lipid peroxides and ferroptotic cell death (Woo et al., 2024). In our study, the pattern of molecular changes strongly supports STING-mediated ferroptosis as a key pathogenic mechanism. MPTP treatment simultaneously increased cGAS-STING expression and induced hallmark features of ferroptosis, including elevated lipid peroxidation (increased MDA), iron accumulation (elevated Fe²⁺), antioxidant depletion (decreased GSH and SOD), and altered expression of ferroptosis regulators (decreased GPX4 and SLC7A11, increased ACSL4). Crucially, TCDCA treatment suppressed cGAS-STING activation and concurrently prevented these ferroptotic changes, suggesting a causal relationship. The restoration of GPX4 levels by TCDCA is particularly noteworthy, as GPX4 requires adequate GSH availability for its function, and TCDCA enhanced both GPX4 expression and GSH content, thereby reinforcing the antioxidant defense system against ferroptosis.

Our detailed characterization of oxidative stress and ferroptosis markers provides further insights into TCDCA's neuroprotective mechanisms. Oxidative stress has long been recognized as a central pathogenic mechanism in PD, with the substantia nigra being particularly vulnerable due to its high metabolic rate, elevated iron content, and relatively low antioxidant capacity (Xiang et al., 2022). Our biochemical analyses revealed that MPTP treatment significantly increased MDA levels, a marker of lipid peroxidation, while decreasing the activities of antioxidant enzymes SOD and reducing GSH content in brain tissue. Additionally, we observed elevated Fe²⁺ levels following MPTP administration. Importantly, TCDCA treatment dose-dependently reversed these oxidative stress parameters, reducing MDA and Fe²⁺ levels while restoring SOD activity and GSH content. These antioxidant effects were also observed in our in vitro experiments using MPP⁺-treated SH-SY5Y cells, confirming that TCDCA can directly protect neurons from oxidative injury. Beyond general oxidative stress markers, we examined specific ferroptosis regulators to establish the anti-ferroptotic effects of TCDCA. SLC7A11 is a component of the system Xc⁻ cystine/glutamate antiporter, which imports cystine for GSH synthesis, thereby providing substrate for GPX4 function (Chen et al., 2021; Feng et al., 2023). ACSL4, on the other hand, preferentially catalyzes the esterification of long-chain polyunsaturated fatty acids into membrane phospholipids, promoting lipid peroxidation and ferroptosis execution(Hu et al., 2022). Our results showed that MPTP decreased SLC7A11 expression while increasing ACSL4 levels, and these changes were reversed by TCDCA treatment. The coordinate regulation of these ferroptosis markers—upregulation of GPX4 and SLC7A11 coupled with downregulation of ACSL4—provides strong evidence that TCDCA inhibits ferroptosis through multiple complementary mechanisms. Recent studies have demonstrated that ferrostatin-1, a specific ferroptosis inhibitor, has shown efficacy in animal models by attenuating neuronal damage and improving behavioral outcomes through the suppression of ferroptosis(Yi et al., 2025). However, TCDCA's ability to target both the inflammatory trigger (cGAS-STING) and the execution phase (ferroptotic machinery) may provide more comprehensive neuroprotection than agents targeting ferroptosis alone.

However, several challenges must be addressed before TCDCA can be considered for clinical application in PD. First, TCDCA is the taurine-conjugated form of CDCA, and taurine conjugation markedly increases aqueous solubility and transmembrane transport capacity compared to unconjugated bile acids. This property is analogous to TUDCA, the taurine-conjugated form of UDCA, which crosses the BBB more readily than free UDCA (Xing et al., 2023). In support of CNS bioavailability, TCDCA has been shown to suppress glioblastoma progression in vivo through the HMGCS1/HMGCR/GPX4 axis and to attenuate astrocyte neuroinflammation in EAE models via TGR5/AKT/NF-κB signaling (PMID: 37079985), both requiring CNS access (Xu et al., 2023; Xue et al., 2025). In the present study, gavage administration of TCDCA produced measurable dose-dependent changes in substantia nigra protein expression and significant behavioral improvements, providing functional evidence of CNS bioavailability. Nevertheless, direct pharmacokinetic quantification of TCDCA brain concentrations remains an important future step. Second, the mechanistic link between TGR5, cGAS-STING, and GPX4 is supported by correlative evidence and external literature, but has not been directly validated through pharmacological intervention in the current experimental system. Prior work from our group demonstrated that a TGR5 inhibitor attenuated the anti-inflammatory effects of TCDCA in LPS-induced C6 astrocytes (Xu et al., 2023), and that lentiviral silencing of TGR5 in BV2 microglia significantly abrogated TCDCA's suppression of LPS-induced neuroinflammation (Ni et al., 2025), collectively establishing TGR5 as a functional mediator of TCDCA's CNS actions. Future studies employing TGR5 antagonists or siRNA knockdown in ferroptosis models, combined with STING pathway modulators, are needed to directly establish the causal relationships proposed in the current mechanistic model. Finally, the potential contribution of other bile acid receptors, including FXR, to TCDCA's neuroprotective effects was not examined in the current study and represents an additional area for future investigation. Another important consideration is whether TCDCA would be most effective as a monotherapy or in combination with existing PD medications. Given that TCDCA acts through mechanisms distinct from L-DOPA, which primarily addresses dopamine replacement, combining TCDCA with L-DOPA might provide both symptomatic relief and disease modification. Additionally, combining TCDCA with other emerging neuroprotective agents, such as GLP-1 receptor agonists or iron chelators, might yield synergistic benefits.

5. Conclusion

In conclusion, our study establishes TCDCA as a promising disease-modifying therapeutic candidate for PD by demonstrating its ability to suppress TGR5-cGAS-STING-mediated ferroptosis. This multi-targeted mechanism results in improved motor function, dopaminergic neuronal protection, and reduced oxidative stress. Future studies are warranted to fully elucidate the molecular mechanisms involved, validate efficacy in additional PD models, optimize therapeutic regimens, and evaluate safety and effectiveness in clinical trials .

Ethics

All animal experimental procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the Life Ethics Review Committee of the Henan Academy of Sciences (Ethics number: HNAS.EC202507a003).

CRediT authorship contribution statement

Liqin Yu: Validation, Software, Project administration. Ling Chen: Software, Project administration, Formal analysis. Feifei Li: Writing – original draft, Funding acquisition, Data curation. Zhiyong He: Methodology, Formal analysis, Conceptualization. Xuefang Wang: Methodology, Investigation, Formal analysis. Ying Ni: Writing – review & editing, Writing – original draft, Visualization, Resources, Data curation. Yanli Chang: Resources, Methodology, Investigation. Yanni Ma: Validation, Software, Methodology. Lupeng Wang: Writing – original draft, Validation, Methodology, Data curation.

Fundings

This work was supported by the Henan Academy of Sciences Scientific Research Fund (240613020 and 241813071) and Henan Province Central Government's Guided Local Science and Technology Development Fund Project (Z20241471146).

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability

All raw data in this manuscript are available on request.