Taurochenodeoxycholic Acid Activates Calcium Signaling to Protect Against Fuchs’ Endothelial Corneal Dystrophy

Abstract

Purpose

Fuchs’ endothelial corneal dystrophy (FECD) is the leading cause of corneal endothelial dystrophy. This study aimed to investigate the protective effects and mechanism of taurochenodeoxycholic acid (TCDCA) in FECD.

Methods

TCDCA levels were quantified in aqueous humor from patients with FECD and ultraviolet A (UVA)-induced FECD mice. Corneal endothelial cell (CEC) morphology and function were evaluated by optical coherence tomography (OCT) and ZO-1 staining following TCDCA treatment. In vitro, UVA-induced human corneal endothelial cells (HCECs) were treated with TCDCA, and cell viability, mitochondrial membrane potential, ATP, and reactive oxygen species (ROS) levels were measured. RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were used to explore molecular mechanisms, and the role of Ca²⁺ signaling was validated using the inhibitor 2-APB in vivo.

Results

Analysis of FECD aqueous humor revealed significantly elevated TCDCA levels. In UVA-induced mouse model, TCDCA administration ameliorated corneal endothelial dysfunction, as evidenced by reduced corneal thickness, increased endothelial cell density, and a lower percentage of abnormal cells. Further, in vitro studies revealed a concentration-dependent effect of TCDCA, with 100 µM TCDCA significantly enhancing cell viability, reducing ROS production, restoring mitochondrial membrane potential, and promoting ATP synthesis. RNA-seq and functional studies identified that TCDCA exerts its beneficial effects on corneal endothelial function by activating the calcium signaling pathway.

Conclusions

TCDCA demonstrated a protective effect on corneal endothelial cells during the pathogenesis of FECD. Therefore, TCDCA may be a promising novel therapeutic target for attenuating the progression of FECD.

The cornea is an avascular, transparent tissue composed of five distinct layers: the corneal epithelium, Bowman’s layer, stroma, Descemet’s membrane, and corneal endothelium (CE). The CE, located at the inner surface of the cornea, consists of a monolayer of hexagonal cells with limited regenerative capacity in vivo. Damage or loss of corneal endothelial cells (CECs) often lead to corneal edema and visual impairment.^1,2 Fuchs endothelial corneal dystrophy (FECD) is a hereditary ocular disease characterized by progressive CEC loss and abnormal thickening of Descemet’s membrane, predominantly affecting the middle-aged and elderly population. As the disease advances, endothelial dysfunction results in corneal swelling and blurred vision. Currently, corneal transplantation remains the primary clinical treatment; however, donor shortages and postoperative complications limit its widespread application.³ Therefore, pharmacological strategies to repair or delay CEC damage have become a significant research focus.

TCDCA is a conjugated bile acid formed by the combination of chenodeoxycholic acid (CDCA) and taurine, and is classified as a secondary bile acid in the human body. TCDCA plays a critical role in modulating bile flow, dissolving cholesterol crystals, and regulating hepatoenteric metabolis.^4,5 In recent years, TCDCA has been increasingly recognized for its diverse biological activities, including involvement in inflammatory responses,^6,7 mediation of apoptosis,⁸ modulation of oxidative stress,² and protection of mitochondrial function.^9,10 Tauroursodeoxycholic acid (TUDCA), a C-7 epimer of TCDCA, has garnered considerable attention in ophthalmic research. Previous studies have demonstrated that TUDCA reduces apoptosis and delays retinal degeneration by inhibiting endoplasmic reticulum stress and oxidative stress, while preserving mitochondrial membrane stability.¹¹ Additionally, TUDCA promotes corneal epithelial cell viability and restores epithelial homeostasis in dry eye conditions.¹² It has also been shown to improve the barrier function of diabetic retinal endothelial cells by suppressing endoplasmic reticulum (ER) stress and reducing O-GlcNAcylation.¹³ Therefore, as a physiologically derived and relatively safe metabolite, TCDCA may represent a promising corneal endothelial protectant and offer a safe and effective pharmacological strategy for the treatment of FECD and other corneal endothelial disorders.

This study identified elevated TCDCA in patients with FECD aqueous humor according to metabolomics analysis and used ultraviolet A (UVA)-induced mouse model to explore TCDCA's role in CEC homeostasis. TCDCA administration prevented and reversed UVA-induced CEC damage in vivo, enhanced proliferation and mitochondrial function in vitro, and acted via Ca²⁺ signaling, with inhibition reversing its effects. These results highlight TCDCA's therapeutic potential for FECD and related disorders.

Methods

Aqueous Humor Metabolomic Analysis

The clinical samples used in this study followed the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital with the informed consent of all patients. Aqueous humor from age-related patients with cataract (control) and patients with FECD was collected and centrifuged to remove debris (Table 3). Metabolite profiling was performed by liquid-chromatography tandem mass spectrometry (LC-MS/MS; PTM BIO, Hangzhou, China) using a high-resolution mass spectrometer. Data were processed using standardized workflows. All procedures complied with ethical and quality control standards.

Table 3.

Patient ID

Patients	Gender	Age
Fuchs’ endothelial dystrophy
1	Female	66
2	Female	69
3	Male	66
4	Female	72
5	Male	70
6	Female	61
Age-related cataract
1	Male	70
2	Female	71
3	Female	69
4	Male	63
5	Female	68
6	Male	60
7	Female	65
8	Female	69

Animals

Female C57BL/6 mice (7 to 8 weeks old) produced by Beijing Vital River Laboratory Animal Technology Co., Ltd. were used in the experiment. The mice were housed in a specific pathogen-free environment at the Shandong Eye Institute animal facility. All animals were treated in accordance with the guidelines established by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Public Health Policy on Humane Care and Use of Laboratory Animals (US Public Health Review), and this study was approved by the Ethics Committee of Shandong First Medical University under the ethical approval number W2024022700029.

Cell Culture

Human corneal endothelial cells (HCECs) were cultured in flasks coated with 10 mg/mL laminin and 10 mg/mL chondroitin sulfate (Sigma-Aldrich, USA). Cells were maintained in human endothelial serum-free medium (Creative Bioarray, USA) supplemented with 3% fetal bovine serum (ExCell Bio, China) and 1% penicillin-streptomycin (Millipore, USA) at 37°C in a 5% CO₂ incubator. Upon reaching 90% confluence, the cells were passaged with trypsin and used for experiments under the same conditions.

Measurement of TCDCA Concentration

TCDCA were measured using the TCDCA-ELISA detection kit (F0351-0B, Fankewei, China) according to the manufacturer's instructions. The concentrations of TCDCA were determined in serum samples from patients with FECD and aqueous humor from UVA irradiation mice using a microplate spectrophotometer.

UVA Irradiation of Mouse Cornea and HCECs

Based on the established methodology from Professor Jurkunas’ prior research,¹⁴ a UVA-induced murine corneal endothelial damage model was established by delivering 500 J/cm² UVA (398 mW/cm², Thorlabs, m365LP1) to a 4 mm corneal area for 20 minutes and 57 seconds. Corneal morphology and central corneal thickness (CCT) were evaluated at baseline and on days 2, 4, and 6 using slit-lamp (BQ900, Haag-Streit, Switzerland) and OCT (OPTOVUE, USA); tissues were harvested on day 7. In vitro, HCECs were exposed to 365 nm UVA (14.77 mW/cm², Analytik Jena, XX-15L) at a distance of 10 cm for 55 minutes and 30 seconds (50 J/cm²) and incubated in serum-free medium for 24 hours before analysis.

Immunohistochemistry

Corneal tissues were fixed in 4% paraformaldehyde, dehydrated, embedded, sectioned, and subjected to deparaffinization, rehydration, and antigen retrieval. After blocking endogenous peroxidase and nonspecific binding with hydrogen peroxide and 5% BSA, sections were incubated overnight at 4°C with anti-Farnesoid X Receptor (FXR) antibody (1:200, Proteintech, #25055-1-AP), followed by HRP-conjugated secondary antibody (Gene Tech, China) for 2 hours at room temperature. DAB was used for visualization, and slides were counterstained with hematoxylin, dehydrated, and imaged (OLYMPUS-BX60, China).

Immunofluorescence

After enucleation, corneas were fixed in 4% paraformaldehyde for 10 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked for 1.5 hours. Tissues were incubated overnight at 4°C with primary antibodies against ZO-1 (1:200, Thermo Fisher, #40-2200) and Na⁺/K⁺-ATPase (1:200, Millipore, #05369), followed by incubation with secondary antibodies (1:200, Abcam, #150076 and #150077) for 2 hours at room temperature. Nuclei were stained with DAPI for 5 minutes. Fluorescence images were obtained using a confocal microscope (Zeiss LSM800).

Quantitative Real-Time PCR

Total RNA was extracted from HCECs and reverse-transcribed into cDNA. The qRT-PCR was performed using SYBR Master Mix (Vazyme) on a Rotor-Gene Q system (Qiagen), with β-actin as the internal control. Experiments were run in triplicate; primer sequences are listed in Tables 1 and 2³ (delohaida; Qingdao, China).

Table 1.

Human Primer Sequences Used for qRT-PCR

Gene	Forward Primer	Reverse Primer
ACTIN	GGGAAATCGTGCGTGACATT	GGAACCGCTCATTGCCAAT
DRP1	GCGGCAAATCAAACGTCTAGAA	TTTGTAACAGGCAACCTTTTACGA
MFN1	CGTTTTTCCCTGGGCTGGT	TGGCGTTGCTGGAGTGGT
MFN2	TGGAGCTCTTGGCTCAAGACTATAA	GGACTACTGGAGAAGGGTGGAAG
OPA1	GCTCTGCAGGCTCGTCTCA	AGCTGGGTGCTCCTCATTACAT
TFAM	TTTACCGAGGTGGTTTTCATCTG	CGCTGGGCAATTCTTCTAATTAG
NRF1	TCAAGTACTCTACAGGTCGGGGA	TTCCCGCCCATGCTGTTTA
PGC-1α	TTTTCTCGACACAGGTCGTGTT	TCTCACATACAAGGGAGAATTTCG
ND1	CAAAGGCCCCAACGTTGTAG	AGAAGAGCGATGGTGAGAGCTAA
ND6	CATACTCTTTCACCCACAGCACC	AGGGGGAATGATGGTTGTCTTT
CACNA1D	CTGGCCTGCGTTGCTGTATA	ATGACAAAGCCCACAAAGATGTT
PTGER3	CCGCTCCTGATAATGATGTTGA	GGTAAACCCAAGGATCCAAGATC
FGF18	GACGATGTGAGCCGTAAGCA	ACCGAAGGTGTCTGTCTCCACTA
PTGFR	ATGACAAAGCCCACAAAGATGTT	AGATTTGATTCCATGTTGCCATT
PLCG2	GCGTCTACCCAAAGGGACAA	TGCGCCCATTGAGAGAAAA
BMP2	CCCCCTACATGCTAGACCTGTATC	CACTCGTTTCTGGTAGTTCTTCCAA
WNT11	AAGTTTTCCGATGCTCCTATGAAG	GATGGAGCAGGAGCCAGACA
CCND2	CGCAACTGGAAGTGTGGGA	CCTCAATCTGCTCCTGGCAA
TCF7	GAACATTTCAACAGCCCACATC	CAGCTCACAGTGTGGGGGA
FZD8	CAAGACAGGCCAGATCGCTAAC	AGCGCTCCATGTCGATAAGG
CACNA2D1	TGCAGCCAGGGATATTGAGAA	TCGAGATCATCCTTTGCATTGTAG
NGFR	CGACAACCTCATCCCTGTCTAT	CCACTGTCGCTGTGGAGTTTT
GADD45G	CCTCACTGCCGGCGTCTA	CGTTCTCGCAGCAGAAAGC

Table 2.

Mouse Primer Sequences Used for qRT-PCR

Gene	Forward Primer	Reverse Primer
Actin	ACGGCCAGGTCATCACTATTG	AGAGGTCTTTACGGATGTCAACGT
Cacna1d	CCTTCCAGGAACAGGGAGAAA	ATTCGAAAGGCGAGGAGTTCA
Fgf18	CAAGTCCTGGGCCGTAGGA	GAGCTTGCCTTTTCGGTTCA
Ptger3	TGGTCGCCGCTATTGATAATG	CCAGGGATCCAAGATCTGGTT
Plcg2	CTCGGACTTGGTTGTATACTGCAA	GCCCTTCTGATTGTATCGCAAT

Live/Dead Staining

HCECs were seeded in 6-well plates (5 × 10⁵ cells/well) and irradiated with UVA at 80% confluency. After 24 hours in serum-free medium, cells were treated with TCDCA (50, 100, and 200 µM) for another 24 hours. Cell viability was assessed using a Live/Dead Double Staining Kit (Abbkine, USA) following the manufacturer's instructions. Stained cells were imaged under an inverted fluorescence microscope.

Reactive Oxygen Species Production

Intracellular reactive oxygen species (ROS) levels were measured using the ROS Assay Kit (S0033S; Beyotime, China) according to the manufacturer's instructions. After UVA irradiation and TCDCA treatment, HCECs were washed twice with PBS, and 500 µL of DCFH-DA working solution was added to each well in a 6-well plate. Cells were incubated at 37°C for 30 minutes. Fluorescent signals indicating ROS production were observed and captured using an inverted fluorescence microscope.

JC-1 Staining

Mitochondrial membrane potential (ΔWm) was assessed using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2006, Beyotime, China) following the manufacturer's instructions. After UVA irradiation and treatment with TCDCA, HCECs were washed twice with PBS. Subsequently, 500 µL of JC-1 working solution was added to each well of a 6-well plate, and the cells were incubated at 37°C for 30 minutes. Fluorescence signals indicating JC-1 monomers (green) and aggregates (red) were observed and captured using an inverted fluorescence microscope.

Mitochondrial Respiration Analysis

Cellular metabolic activity was evaluated using the Seahorse XFp Analyzer with the XF Cell Mito Stress Test and Real-Time ATP Rate Assay Kits (Agilent Technologies, USA). HCECs were seeded in 8-well Seahorse plates (20,000 cells/well) and incubated with Seahorse assay medium supplemented with glucose and sodium pyruvate. Mitochondrial modulators—oligomycin, FCCP, and rotenone/antimycin A—were sequentially injected. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and normalized to total protein content determined by BCA assay (Beyotime, China).

ATP Production

Intracellular ATP levels were measured using the ATP Assay Kit (S0027; Beyotime, China). After UVA irradiation and treatment with TCDCA, HCECs were washed twice with PBS and lysed with cell lysis buffer. According to the manufacturer's protocol, the lysates were mixed with the ATP detection working solution, and luminescence was measured using a microplate luminometer.

RNA-Seqencing

RNA was extracted from HCECs in UVA and UVA + TCDCA groups and subjected to RNA-seq using the Illumina NovaSeq 6000 platform by OE Biotech (Shanghai, China). Raw reads were processed with fastp, aligned using HISAT2, and quantified by HTSeq-count. PCA and DEG analysis (DESeq2, Q < 0.05, |fold change| > 2) were performed in R software, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. For both the UVA and UVA + TCDCA groups, fifth-passage HCECs were seeded in 6-well plates at 4:00 PM on day 1 and allowed to adhere completely before receiving respective treatments the following morning.

Statistical Analysis

Data analysis was performed using SPSS 25.0 statistical software and GraphPad Prism 7 software (GraphPad Software, USA). All data are expressed as means ± standard errors (SEs). The 2-tailed t-test was used to analyze relationships between the 2 groups. Any P value < 0.05 was considered statistically significant.

Results

Involvement of TCDCA in the Pathogenesis of FECD

We first conducted metabolomic profiling of aqueous humor from eight age-related patients with cataract patients and six patients with FECD (Fig. 1A). Metabolomic analysis of the aqueous humor revealed that numerous metabolites were enriched in the bile acid metabolism pathway, among which TCDCA exhibited the most prominent alteration in abundance compared with other bile acids (Fig. 1B). Considering its endogenous origin and reported biological activities in other systems,^15–17 we selected TCDCA as the target metabolite for further investigation. TCDCA levels were significantly elevated in FECD patient serum and UVA-irradiated mouse aqueous humor (Fig. 1C). To investigate its potential functional relevance in corneal endothelial cells, we examined the expression of FXR, a known receptor of TCDCA, using immunohistochemistry and immunofluorescence. FXR was found to be expressed in the corneal endothelium of both humans and mice (Figs. 1D, 1E). Collectively, these findings suggest that TCDCA may be involved in the regulation of corneal endothelial cell repair following damage.

Figure 1.

TCDCA as a potential regulator of the pathogenesis of FECD. (A) Schematic of the experimental design. (B) Differential bile acid profiles in FECD aqueous humor (n = 6). (C) TCDCA levels in human serum (n = 5) and mouse aqueous humor (n = 4). (D) FXR immunohistochemistry in human corneal endothelium (n = 3), scale bar = 100 µm. (E) FXR immunofluorescence in murine CECs (n = 3), scale bar = 180 µm. All data are expressed as mean ± SEM. ***P < 0.001 (2-tailed t-test).

Effects of Pretreatment With TCDCA on Corneal Endothelial Cells in FECD Mice

To investigate the regulatory role of TCDCA in CEC damage repair, a UVA-induced mouse model of corneal endothelial damage was established, as previously described (Fig. 2A). Mice received intraperitoneal injections of PBS or TCDCA one day prior to 500 J/cm² UVA exposure, and on days 0, 2, 4, and 6 post-irradiation. Corneal structure was assessed by slit-lamp and OCT imaging on day 7. Compared to the PBS group, the TCDCA-treated group showed significantly improved CCT and transparency (Figs. 2B, 2D). Whole-mount immunostaining for ZO-1 and Na⁺/K⁺-ATPase revealed more regular endothelial morphology in the TCDCA group (Fig. 2C). Quantitative analysis demonstrated that TCDCA significantly improved endothelial cell density, cell area, and coefficient of variation (Figs. 2E–G). Although both subconjunctival and intraperitoneal injections improved CEC morphology (Supplementary Fig. S1), intraperitoneal injection was more effective in restoring CCT and corneal transparency. These findings indicate that intraperitoneal TCDCA administration protects against UVA-induced CEC damage.

Figure 2.

Protection against UVA-induced corneal endothelial impairment by preventive administration of TCDCA. (A) Experimental timeline of TCDCA treatment and UVA-induced damage model. (B) Slit-lamp and OCT images of mouse corneas in normal, PBS, and TCDCA groups at days 0, 2, 4, and 6 (n = 3). (C) Whole-mount immunofluorescence of ZO-1 and Na⁺/K⁺-ATPase in corneal endothelium, scale bar = 50 µm. (D) CCT measurements post-UVA (n = 3). (E–G) Quantification of endothelial cell density, cell area, and coefficient of variation based on ZO-1 staining (n = 5 or 6). All data are expressed as mean ± SEM. *P < 0.5; **P < 0.01; ***P < 0.001 (2-tailed t-test).

Amelioration of Therapeutic Administration of TCDCA on UVA-Induced Corneal Endothelial Damage

Building on the preventative effects of TCDCA, we further evaluated its therapeutic potential using the same UVA-induced mouse corneal endothelial damage model. Mice received intraperitoneal injections of PBS or TCDCA on days 0, 2, 4, and 6 after UVA exposure. Corneal assessments were performed by slit-lamp and OCT imaging, and samples were collected on day 7 for analysis (Fig. 3A). Consistent with the preventative study, TCDCA treatment significantly improved CCT and corneal transparency compared to PBS (Figs. 3B, 3D). Immunofluorescence staining demonstrated that the endothelial morphology in the TCDCA group was more similar to healthy controls, with significantly increased cell density, reduced cell area, and improved coefficient of variation (Figs. 3C, 3E–G). These results indicate that intraperitoneal TCDCA administration effectively promotes repair of UVA-induced corneal endothelial damage.

Figure 3.

Improvement of TCDCA on corneal endothelial damage. (A) Experimental timeline of TCDCA treatment post-UVA exposure. (B) Slit-lamp and OCT images of mouse corneas in Normal, PBS, and TCDCA groups at days 0, 2, 4, and 6 (n = 3). (C) Whole-mount immunofluorescence of ZO-1 and Na⁺/K⁺-ATPase in corneal endothelium, scale bar = 50 µm. (D) CCT measurements in PBS and TCDCA groups (n = 3). (E–G) Quantification of cell density, average cell area, and coefficient of variation based on ZO-1 staining (n = 5 or 6). All data are expressed as mean ± SEM. *P < 0.5; ***P < 0.001 (2-tailed t-test).

Restorative Effects of TCDCA on Human Corneal Endothelial Cell Dysfunction

To verify the in vitro effects of TCDCA on regulating cellular function, we established a UVA-induced damage model in HCECs. Twenty-four hours after UVA exposure, the cells were treated with TCDCA at different concentrations and analyzed after another 24 hours. Live/dead staining revealed that UVA markedly reduced cell numbers, whereas TCDCA improved cell viability in a dose-dependent manner, with the most pronounced effect observed at 100 µM (UVA + T100; Figs. 4A, 4B). In addition, ROS assays showed that UVA exposure increased oxidative stress, which was slightly reduced in the 50 µM and 200 µM groups (UVA + T50 and UVA + T200), whereas 100 µM TCDCA significantly reduced ROS levels (Fig. 4C). Consistently, JC-1 staining demonstrated a decline in mitochondrial membrane potential following UVA irradiation, which was alleviated by TCDCA, with the most notable recovery observed in the 100 µM group (Figs. 4D–F). Collectively, these results indicate that TCDCA effectively alleviates UVA-induced damage in HCECs.

Figure 4.

TCDCA ameliorates cellular viability and mitochondrial membrane potential of HCECs following UVA-induced damage . (A) Bright-field and live/dead staining images of HCECs in the normal, UVA, UVA + T50, UVA + T100, and UVA + T200 groups (green = live cells and red = dead cells; n = 3), scale bars = 550 µm and 180 µm. (B) Quantification of viable cells per group (n = 3). (C) ROS staining of HCECs from each group (n = 3), scale bar = 460 µm. (D) JC-1 staining images showing mitochondrial membrane potential (n = 3), scale bar = 180 µm. (E, F) Quantitative analysis of JC-1 monomer (E) and aggregate (F) fluorescence intensity (n = 3). All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed t-test).

Regulation of the Physiological Functions of HCECs by TCDCA Through Improvement of Mitochondrial Function

To investigate the potential biological mechanism by which TCDCA regulates CEC damage repair, we explored whether TCDCA restores the morphology and function of damaged HCECs by improving mitochondrial function, given the previously reported high bioenergetic demand of CECs. Based on the above in vitro results, 100 µM TCDCA (UVA + TCDCA) was selected as the treatment condition for subsequent experiments. First, qRT-PCR analysis showed that the expression of mitochondrial quantity- and function-related genes was significantly upregulated in the TCDCA-treated group compared to the UVA group (Figs. 5A, 5B). Next, Seahorse assays were used to evaluate mitochondrial function and cellular metabolic capacity. The results demonstrated that TCDCA treatment enhanced basal respiration, ATP production, non-mitochondrial oxygen consumption and proton leak (Fig. 5C and Supplementary Fig. S2). To further validate these findings, ATP quantification confirmed that TCDCA significantly increased ATP levels compared to the UVA group (Fig. 5D). In summary, these results indicate that TCDCA alleviates mitochondrial bioenergetic deficiency following UVA-induced damage in HCECs.

Figure 5.

Restoration of mitochondrial bioenergetic function by TCDCA. (A) Relative mRNA expression levels of mitochondrial-related genes (DRP1, MFN1, MFN2, OPA1, TFAM, NRF1, and PGC-1a) in HCECs from the UVA and UVA + TCDCA groups (n = 3). (B) Relative expression levels of mitochondrial DNA-encoded genes ND1 and ND6 (n = 3). (C) Seahorse analysis of OCR in the UVA and UVA + TCDCA groups (n = 3). (D) ATP levels in HCECs from the UVA and UVA + T100 groups (n = 3). All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed t-test).

Treatment of TCDCA Significantly Altered the Transcriptomic Profile of Human Corneal Endothelial Cell Lines

Based on the above in vitro findings, we performed RNA-seq analysis on HCECs from the UVA and UVA + TCDCA groups (Fig. 6A) to explore the regulatory mechanisms by which TCDCA promotes CEC repair. A total of 477 upregulated and 1096 downregulated genes were identified (Fig. 6B). KEGG enrichment analysis revealed significant upregulation of multiple signaling pathways related to cellular functions in the UVA + TCDCA group, including the Hippo, MAPK, and Ca²⁺ signaling pathways (Fig. 6C). Previous studies have demonstrated that activation of the Hippo and MAPK signaling pathways facilitates CEC migration and proliferation.^18,19 Consistent with these findings, our qRT-PCR validation (Figs. 6D, 6E) confirmed the upregulation of key components within these pathways in our experimental model. Additionally, prior research has revealed that the small GTPase Rap1A modulates store-operated calcium entry (SOCE) in lung endothelial cells, thereby influencing vascular inflammation and permeability.²⁰ To explore this possibility, we investigated the Ca²⁺ signaling pathway, given its well-established role in regulating cell proliferation, differentiation, migration, and apoptosis.^21,22 We then measured the mRNA expression of Ca²⁺ pathway-related genes, all of which were significantly upregulated in the TCDCA group (Fig. 6F). These results suggest that TCDCA may promote HCECs damage repair by activating the Ca²⁺ signaling pathway.

Figure 6.

Activation of the Ca²⁺ signaling pathway in TCDCA-treated HCECs. (A) Schematic of the experimental design. (B) Volcano plot of DEGs between the UVA and UVA + TCDCA groups (n = 4). (C) KEGG pathway enrichment analysis showing upregulation of the calcium signaling pathway. (D, E) The relative expression levels of genes related to the Hippo signaling pathway and the MAPK signaling pathway (n = 3). (F) The relative expression levels of genes related to the Ca²⁺ signaling pathway (n = 3). All data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (2-tailed t-test).

Protection of CEC Function by TCDCA Through Activation of the Ca²⁺ Signaling Pathway

Based on the previous transcriptomic analysis, to rigorously confirm the relationship between TCDCA and CEC repair after damage, we introduced the Ca²⁺ signaling pathway inhibitor 2-APB and established the TCDCA + 2-APB treatment group. The experimental timeline is shown in Figure 7A. As expected, OCT and slit-lamp images showed that the CCT and corneal clarity of the TCDCA + 2-APB group were similar to those of the PBS group (Figs. 7B, 7C). To further validate these findings, whole-mount ZO-1 staining of the cornea was performed (Fig. 7D). Consistent with the imaging results, CECs in the TCDCA + 2-APB group showed the most irregular morphology, largest average cell area, and lowest cell density, comparable to the PBS group (Figs. 7E–G). These results indicate that TCDCA promotes CEC repair after damage by activating the Ca²⁺ signaling pathway.

Figure 7.

Reversal of TCDCA's therapeutic effect on CEC repair by 2-APB. (A) Experimental timeline showing intraperitoneal injections of PBS, TCDCA, and TCDCA + 2-APB with FECD model construction. (B) Slit-lamp and OCT images of mouse corneas from each group on days 4 and 6 (n = 3). (C) CCT measurements across groups on days 4 and 6 (n = 3). (D) Whole-mount ZO-1 immunofluorescence staining of CE (n = 3), scale bar = 50 µm. (E–G) Quantification of cell density, average cell area, and coefficient of variation from ZO-1 staining (n = 3). All data are expressed as mean ± SEM. ***P < 0.001 (2-tailed t-test).

Discussion

The CE maintains corneal transparency and thickness via its barrier and pump functions. However, as CECs are arrested in the G2/M phase and lack regenerative capacity in vivo, damage can lead to corneal edema and decompensation. This study explores the therapeutic potential of TCDCA, a bioactive bile acid with roles in signaling and disease,^23–26 for protecting CECs against UVA-induced injury, given its recognized bioactivities in other systems.

Previous studies have demonstrated that mitochondrial dysfunction and apoptosis are key mechanisms involved in the degeneration of CECs in FECD.²⁷ TCDCA has been reported to protect hepatocytes from Fas ligand (Fas-L)-induced apoptosis, potentially through direct effects on mitochondrial membranes.²⁸ In our study, TCDCA improved mitochondrial membrane potential and restored bioenergetic functions in UVA-damaged HCECs, including ATP production and respiratory capacity. Although these effects suggest mitochondrial protection, the role of TCDCA in apoptosis regulation requires further investigation.

Recent studies have highlighted TCDCA as a signaling molecule capable of activating multiple pathways. For instance, TCDCA activates the FXR signaling pathway in hepatic endothelial cells, subsequently triggering the Myc signaling cascade to promote neutrophil recruitment, thereby exerting therapeutic effects against obstructive cholestasis.^17,29 Additionally, TCDCA activates the TGR5 signaling pathway, mediating anti-inflammatory responses and modulating immune regulation.^7,30,31 In our study, we observed that TCDCA simultaneously activates the Hippo, MAPK, and Ca²⁺ signaling pathways. Although our subsequent experiments confirmed the critical role of the Ca²⁺ signaling pathway in this process, the potential synergistic contributions of other activated pathways require further investigation.

The Ca²⁺ signaling pathway is one of the most fundamental and ubiquitous intracellular signaling mechanisms.³² While the critical role of Ca²⁺ signaling in ocular diseases^35–39 and corneal epithelial repair is well-established,^40,41 its function in CE remains less defined. This study demonstrated the involvement of Ca²⁺ signaling in the repair of CEC damage. Nonetheless, the downstream mechanisms by which TCDCA activates Ca²⁺ signaling to mediate CEC repair remain unclear and require further investigation. Previous studies have not directly linked Ca²⁺ signaling genes (CACNA1D, PTGER3, PTGFR, FGF18, and PLGC2) to the corneal endothelium, although their roles in vascular endothelium are established—such as in regulating permeability and inflammation.^42–44 Given the shared characteristics between vascular and corneal endothelia (including barrier function and tight junctions), future studies should investigate downstream Ca²⁺ signaling pathway components in greater detail. Additionally, whereas mitochondrial Ca²⁺ signaling has been shown to regulate gene expression in neurons,⁴⁵ its potential role in corneal endothelial repair mechanisms remains unexplored and merits future study.

TCDCA demonstrates therapeutic potential for corneal endothelial disorders by mitigating UVA-induced damage and improving mitochondrial bioenergetics, with Ca²⁺ signaling implicated as a key pathway. Future work with mutation models such as COL8A2, TCF4, and SLC4A11, or human donor tissues to directly verify these findings and further assess TCDCA’s therapeutic potential for conditions such as FECD.