Application of taurochenodeoxycholic acid in preparation of medicine for preventing and / or treating Fuchs corneal endothelial dystrophy

Abstract

The invention provides application of taurochenodeoxycholic acid in preparation of a medicine for preventing and / or treating Fuchs corneal endothelial dystrophy, and particularly belongs to the technical field of medicine preparation. The invention provides an application of taurochenodeoxycholic acid in preparation of a medicine for preventing and / or treating Fuchs corneal endothelial dystrophy. Test results show that taurochenodeoxycholic acid can realize prevention and / or treatment of corneal endothelial cell injury caused by UVA; the number reduction, activity reduction, oxidative stress, ROS level rise and mitochondrial membrane potential drop of corneal endothelial cells caused by UVA are relieved; mitochondrial biological energy deficiency caused by UVA is relieved; and the prevention and / or treatment of the Fuchs corneal endothelial dystrophy are / is realized by activating a Ca < 2 + > signal channel.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the application of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy (FECD). Background Technology

[0002] Taurochenodeoxycholic acid (TCDCA) is a conjugated bile acid formed by the combination of chenodeoxycholic acid (CDCA) and taurine. It is one of the secondary bile acids synthesized in the liver of humans and mammals. TCDCA plays an important role in regulating bile flow, dissolving cholesterol crystals, and modulating enterohepatic metabolism. In recent years, it has been found to possess various biological activities, including participation in inflammatory responses, mediating apoptosis, oxidative reactions, and protecting mitochondrial function. Tauroursodeoxycholic acid (TUDCA) is the C-7 epimer of TCDCA. Previous studies have shown that TCDCA, as a physiologically derived and relatively safe metabolite, has anti-inflammatory, antioxidant, and mitochondrial protective effects; however, there are no reports on the effects of TCDCA on corneal endothelial cells. Summary of the Invention

[0003] The purpose of this invention is to provide the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy. Taurine chenodeoxycholic acid can achieve the prevention and / or treatment of Fuchs' corneal endothelial dystrophy, and also the prevention and / or treatment of corneal endothelial cell damage.

[0004] This invention provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy.

[0005] The present invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of corneal damage.

[0006] The present invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of corneal endothelial cell damage.

[0007] The present invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of UVA-induced corneal endothelial cell damage.

[0008] The present invention also provides the application of taurine chenodeoxycholic acid in the preparation of a drug that has the function of alleviating one or more of the following: reduction in the number and vitality of corneal endothelial cells, oxidative stress, increase in ROS levels and decrease in mitochondrial membrane potential caused by UVA.

[0009] The present invention also provides the application of taurine chenodeoxycholic acid in the preparation of drugs to alleviate mitochondrial bioenergy deficiency caused by UVA.

[0010] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

[0011] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of medicaments for the prevention and / or treatment of corneal damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

[0012] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of corneal endothelial cell damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

[0013] This invention also provides activation of Ca 2+ The use of agents that activate signaling pathways in the preparation of drugs for the prevention and / or treatment of UVA-induced corneal endothelial cell damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

[0014] This invention provides the application of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy. The invention was validated in vivo using techniques such as immunofluorescence and real-time quantitative PCR; and in vitro using techniques such as cell proliferation assay, ROS level assay, ATP level assay, mitochondrial function assay, and mitochondrial membrane potential assay. Experimental results show that taurine chenodeoxycholic acid can prevent and / or treat UVA-induced corneal endothelial cell damage; alleviate UVA-induced reduction in corneal endothelial cell number, decreased viability, oxidative stress, increased ROS levels, and decreased mitochondrial membrane potential; alleviate UVA-induced mitochondrial bioenergy deficiency; and activate Ca2+. 2+ Signaling pathways enable the prevention and / or treatment of Fuchs' corneal endothelial dystrophy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The diagram shows the results of increased TCDCA content in the aqueous humor of patients with Fuchs corneal endothelial dystrophy provided by this invention; wherein, A is a metabolomics flowchart; B is a graph of bile acid-related metabolite content; C is a graph of TCDCA content in humans and mice; D is a graph of FXR expression in human cornea; and E is a graph of FXR expression in mouse cornea. Figure 2 The diagrams provided by this invention illustrate the results of intraperitoneal administration of TCDCA in preventing UVA-induced corneal endothelial cell damage. A represents the flowchart of intraperitoneal administration to mice; B represents the thickness of the ocular surface and cornea in mice; C represents the immunofluorescence of ZO1 and Na+ / K+-ATPase in the mouse cornea; D represents the central corneal thickness in mice; E represents the statistical graph of the number of corneal endothelial cells in mice; F represents the statistical graph of the average cell area of ​​corneal endothelial cells in mice; and G represents the statistical graph of the coefficient of variation of corneal endothelial cells in mice. Figure 3 The diagrams provided by this invention illustrate the results of intraperitoneal administration of TCDCA in treating UVA-induced corneal endothelial cell damage. A represents a flowchart of intraperitoneal administration to mice; B represents the thickness of the ocular surface and cornea in mice; C represents the immunofluorescence of ZO1 and Na+ / K+-ATPase in the mouse cornea; D represents the central corneal thickness in mice; E represents the statistical graph of the number of corneal endothelial cells in mice; F represents the statistical graph of the average cell area of ​​corneal endothelial cells in mice; and G represents the statistical graph of the coefficient of variation of corneal endothelial cells in mice. Figure 4 The following diagrams illustrate the results of TCDCA improving the function of human corneal endothelial cell lines provided by this invention. Figures A and B show bright-field and live / dead staining images of HCECs in the normal, UVA, UVA+T50, UVA+T100, and UVA+T200 groups, respectively. Figure C shows the ROS content of HCECs in the normal, UVA, UVA+T50, UVA+T100, and UVA+T200 groups. Figure D shows the JC-1 staining of HCECs in the normal, UVA, UVA+T50, UVA+T100, and UVA+T200 groups. Figure E is a statistical graph of the fluorescence intensity of the JC-1 monomer based on the experimental results in Figure D. Figure F is a statistical graph of the fluorescence intensity of the JC-1 monomer aggregate based on the experimental results in Figure D. Figure 5 The diagram provided by this invention illustrates the effect of TCDCA in regulating corneal endothelial cell damage repair by improving mitochondrial function; where A represents HCECs in the UVA group and the UVA+TCDCA group. DRP1 , MFN1 , MFN2 , OPA1 , TFAM , NRF1 and PGC-1a A) Relative expression level; B) Relative expression level of ND1 and ND6 in the UVA group and UVA+TCDCA group; C) Oxygen consumption rate of HCECs in the UVA group and UVA+TCDCA group; D) ATP content of HCECs in the UVA group and UVA+TCDCA group. Figure 6 The TCDCA provided by this invention significantly alters the transcriptomics of corneal endothelial cells by activating Ca2+. 2+ The results of signaling pathways promoting corneal endothelial cell function recovery are shown in the figure; where A is a single-cell transcriptomics flowchart of HCECs; B is a volcano plot of differentially expressed genes in HCECs of the UVA group and UVA+TCDCA group; C is a KEGG enrichment analysis plot of HCECs of the UVA group and UVA+TCDCA group; D is a plot of relative expression levels of genes related to the Hippo signaling pathway; E is a plot of relative expression levels of genes related to the MAPK signaling pathway; F is a plot of Ca 2+ Relative expression levels of genes related to signaling pathways; Figure 7 The TCDCA provided by this invention activates Ca 2+ The results of the signaling pathway protecting corneal endothelial cell function are shown in the following figures: A is a flowchart of intraperitoneal administration in mice; B is a graph of ocular surface and corneal thickness in mice; C is a graph of central corneal thickness in mice; D is an immunofluorescence graph of ZO1 and Na+ / K+-ATPase in mouse corneas; E is a statistical graph of the number of corneal endothelial cells in mice; F is a statistical graph of the average cell area of ​​corneal endothelial cells in mice; and G is a statistical graph of the coefficient of variation of corneal endothelial cells in mice. Detailed Implementation

[0017] This invention provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy. This invention does not specifically limit the source of taurine chenodeoxycholic acid; conventional commercially available products are acceptable, such as those purchased from MedChemExpress (MCE), catalog number HY-N2027. This invention discovers that taurine chenodeoxycholic acid can achieve the prevention and / or treatment of Fuchs' corneal endothelial dystrophy.

[0018] This invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of corneal damage. Experimental results show that taurine chenodeoxycholic acid can achieve the prevention and / or treatment of corneal damage.

[0019] This invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of corneal endothelial cell damage. Experimental results show that taurine chenodeoxycholic acid can achieve the prevention and / or treatment of corneal endothelial damage.

[0020] This invention also provides the use of taurine chenodeoxycholic acid in the preparation of medicaments for the prevention and / or treatment of UVA-induced corneal endothelial cell damage. Experimental results show that taurine chenodeoxycholic acid can achieve the prevention and / or treatment of UVA-induced corneal endothelial cell damage.

[0021] This invention also provides the application of taurine chenodeoxycholic acid in the preparation of a drug that alleviates one or more of the following effects induced by UVA: decreased number and viability of corneal endothelial cells, oxidative stress, increased ROS levels, and decreased mitochondrial membrane potential. Experimental results show that taurine chenodeoxycholic acid can alleviate these effects.

[0022] This invention also provides the application of taurine chenodeoxycholic acid in the preparation of drugs to alleviate UVA-induced mitochondrial bioenergy deficiency. Experimental results show that taurine chenodeoxycholic acid can alleviate UVA-induced mitochondrial bioenergy deficiency.

[0023] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy; the activation of Ca 2+ The reagents involved in the signaling pathway included taurine chenodeoxycholic acid. Experimental results showed that taurine chenodeoxycholic acid activates Ca2+... 2+ Signaling pathways enable the prevention and / or treatment of Fuchs' corneal endothelial dystrophy.

[0024] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of medicaments for the prevention and / or treatment of corneal damage; the activation of Ca 2+ The reagents involved in the signaling pathway included taurine chenodeoxycholic acid. Experimental results showed that taurine chenodeoxycholic acid activates Ca2+... 2+ Signaling pathways enable the prevention and / or treatment of corneal damage.

[0025] This invention also provides activation of Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of corneal endothelial cell damage; the activation of Ca 2+ The reagents involved in the signaling pathway included taurine chenodeoxycholic acid. Experimental results showed that taurine chenodeoxycholic acid activates Ca2+... 2+ Signaling pathways enable the prevention and / or treatment of corneal endothelial cell damage.

[0026] This invention also provides activation of Ca 2+ The use of agents that activate signaling pathways in the preparation of drugs for the prevention and / or treatment of UVA-induced corneal endothelial cell damage; the activation of Ca 2+The reagents involved in the signaling pathway included taurine chenodeoxycholic acid. Experimental results showed that taurine chenodeoxycholic acid activates Ca2+... 2+ The signaling pathway enables the prevention and / or treatment of UVA-induced corneal endothelial cell damage. In a specific embodiment, the taurine chenodeoxycholic acid can be administered via intraperitoneal injection.

[0027] To further illustrate the present invention, the application of taurine chenodeoxycholic acid provided by the present invention in the preparation of a medicament for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0028] Example 1 Aqueous humor metabolomics analysis: Clinical samples used in this study were collected in accordance with the principles of the Declaration of Helsinki, approved by the Ethics Committee of Shandong Eye Hospital, and with informed consent from all patients. Aqueous humor was collected from elderly patients with cataracts (control group) and FECD patients, and centrifuged to remove debris. Metabolite analysis was performed using LC-MS / MS (PTM BIO, Hangzhou, China) with a high-resolution mass spectrometer. Standardized workflows were used for data processing. All procedures complied with ethical and quality control standards.

[0029] Animals: Female C57BL / 6 mice (7-8 weeks old) manufactured by Beijing Vital River Laboratory Animal Technology Co., Ltd. were used in the experiment. These mice were housed in a specific pathogen-free environment at the Shandong Eye Institute animal facility. All animals were handled in accordance with the ARVO Statement on the Use of Animals for Ophthalmological and Visual Research and the Public Health Policy on Humane Care and Use of Laboratory Animals (US Public Health Review). This study was approved by the Ethics Committee of Shandong First Medical University, with ethics approval number W2024022700029.

[0030] 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 survived in serum-free human endothelial culture 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. When confluence reached 90%, cells were passaged using trypsin digestion and experiments were performed under the same conditions.

[0031] Measurement of TCDCA concentration: TCDCA was detected using a TCDCA-ELISA kit (F0351-0B, Fankewei, China) according to the manufacturer's instructions. The concentrations of TCDCA in serum samples from FECD patients and in the aqueous humor of UVA-irradiated mice were determined using a microplate spectrophotometer.

[0032] UVA irradiation of mouse corneas and HCECs: by 500 J / cm 2 UVA (398mW / cm) 2 A UVA-induced mouse model of corneal endothelial injury was established by delivering a slit lamp (BQ900, Haag-Streit, Switzerland) to a 4 mm corneal region for 20 min 57 s. Corneal morphology and central corneal thickness (CCT) were assessed at baseline and on days 2, 4, and 6 using optical coherence tomography (OCT) (OPTOVUE, USA); tissue was harvested on day 7. In vitro, HCECs were exposed to 365 nm UVA (14.77 mW / cm²). 2 Under Analytik Jena (XX-15L) conditions, at a distance of 10cm, for 55 minutes and 30 seconds (50J / cm), the concentration was 50J / cm. 2 The samples were cultured in serum-free medium for 24 hours and then analyzed.

[0033] Immunohistochemistry: Corneal tissue was fixed with 4% paraformaldehyde, dehydrated, embedded, sectioned, degummed, replenished with fluid, and antigen recovered. After blocking endogenous peroxidase and nonspecifically binding to hydrogen peroxide and 5% BSA, the tissue was incubated overnight at 4°C with anti-farnesoid X receptor (FXR) antibody (1:2000, Proteintech, #25055-1-AP), followed by incubation with enzyme-labeled secondary antibody (GeneTech, China) at room temperature for 2 hours. DAB was used for visualization; sections were reverse stained with hematoxylin, dehydrated, and imaged (OLYMPUS-BX60, China).

[0034] Immunofluorescence: Corneas were fixed with 4% paraformaldehyde for 10 min at room temperature, infiltrated with 0.1% Triton X-100 for 5 min, and blocked for 1.5 h. The tissues were then subjected to immunofluorescence assays targeting FXR (1:1000, Proteintech, #25055-1-AP), ZO-1 (1:2000, Thermo Fisher, #40-2200), and Na+. + / K +Primary antibody against ATPase (1:2000, Millipore, #05369) was incubated overnight at 4°C, followed by incubation with secondary antibodies (1:2000, Abcam, #150076 and #150077) at room temperature for 2 h. Cell nuclei were stained with DAPI for 5 min. Fluorescence images were obtained using a confocal microscope (Zeiss LSM800).

[0035] qPCR: Total RNA was extracted from HCECs and reverse transcribed into cDNA. qPCR was performed using SYBR Master Mix (Vazyme) on the Rotor-Gene Q system (Qiagen), with β-actin as an internal control. The experiment was performed three times; primer sequences are listed in Table 1 (delohaida, Qingdao, China).

[0036] Table 1 qPCR primer sequences

[0037] Live / dead cell staining: HCECs were seeded in 6-well plates (5 × 10⁻⁶). 5 Cells were irradiated with UVA (number of cells per well), and the groups included: Normal, UVA, UVA+T50, UVA+T100, and UVA+T200. When cell confluence reached 80%, cells were irradiated with UVA. Except for the Normal group, the other four groups were cultured in serum-free medium for 24 hours after UVA irradiation, followed by treatment with TCDCA (50 μM, 100 μM, and 200 μM) for 24 hours. Cell viability was assessed using a live / dead double staining kit (Abbkine, USA) according to the manufacturer's instructions. Stained cells were imaged under an inverted fluorescence microscope.

[0038] ROS 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 of a 6-well plate. The plates were incubated at 37°C for 30 minutes. The fluorescence signal generated by ROS was observed and captured using an inverted fluorescence microscope.

[0039] JC-1 Staining: Mitochondrial membrane potential (ΔΨm) was assessed using the JC-1 Mitochondrial Membrane Potential Assay Kit (C2006, Beyotime, China) according to the manufacturer's instructions. HCECs were washed twice with PBS after UVA irradiation and TCDCA treatment. Subsequently, 500 µL of JC-1 working solution was added to each well of a 6-well plate, and cells were incubated at 37°C for 30 min. Fluorescence signals of JC-1 monomers (green) and aggregates (red) were observed and captured using an inverted fluorescence microscope.

[0040] Mitochondrial stress assay: Cellular metabolic activity was assessed using a Seahorse XFp analyzer, XF Cell Mito stress assay, and real-time ATP rate assay kit (Agilent Technologies, USA). HCECs were seeded in 8-well hippocampal plates (20,000 cells / well) and incubated in hippocampal experimental medium supplemented with glucose and sodium pyruvate. Mitochondrial regulators—oligomycin, FCCP, and rotenone / antimycin a—were injected sequentially. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured and normalized to the total protein content determined by the BCA method (Beyotime, China).

[0041] ATP production: Intracellular ATP levels were detected using the ATP Assay Kit (S0027; Beyotime, China). After UVA irradiation and TCDCA treatment, HCECs were washed twice with PBS and lysed with cell lysis buffer. The lysate was mixed with the ATP assay working solution according to the manufacturer's protocol, and luminescence was measured using a microplate photometer.

[0042] RNA-seq: RNA was extracted from HCECs in the UVA and UVA+TCDCA groups and sequenced using the Illumina NovaSeq 6000 platform from OE Biotech (Shanghai, China). Raw reads were processed with FASTP, aligned with HISAT2, and quantified using HTSeq-count. PCA and DEG analyses were performed in the R region (DESeq2, Q<0.05, |foldchange|>2), followed by KEGG pathway enrichment. For the UVA and UVA+TCDCA groups, 5th generation HCECs were seeded into 6-well plates at 4:00 PM on day 1 and processed the following morning after complete adhesion.

[0043] Statistical analysis: Data analysis was performed using SPSS 25.0 and GraphPad Prism 7 software (GraphPad software, USA). All data are expressed as mean ± standard error (SEs). Two-tailed t-tests were used to analyze the relationship between the two groups. P < 0.05 was considered statistically significant.

[0044] I. Increased TCDCA levels in the aqueous humor of patients with Fuchs' corneal endothelial dystrophy: Metabolomics analysis was first performed on the aqueous humor of 8 patients with age-related cataracts and 6 patients with FECD ( Figure 1In contrast to patients with senile cataracts, FECD patients exhibit altered aqueous humor metabolites. Metabolomics analysis of the aqueous humor revealed enrichment of numerous metabolites along the bile acid metabolic pathway, with TCDCA showing the most significant abundance change compared to other bile acids. Figure 1 (B in the original text). Considering its endogenous origin and reported bioactivity in other systems, this invention selected TCDCA as the target metabolite for further research. Serum levels of TCDCA were significantly elevated in FECD patients compared to serum from elderly cataract patients, and in the aqueous humor of UVA-irradiated mice compared to normal unirradiated mice. Figure 1 (C in the original text). To investigate its potential functional relevance in corneal endothelial cells, this invention used immunohistochemistry and immunofluorescence to detect the expression of the known TCDCA receptor FXR. FXR is expressed in the cornea of ​​normal humans and the corneal endothelium of normal C57 mice. Figure 1 D and Figure 1 (E in the text). In summary, these findings suggest that TCDCA may be involved in the regulation of corneal endothelial cell repair after injury.

[0045] II. Intraperitoneal administration of TCDCA can prevent UVA-induced corneal endothelial cell damage: To investigate the regulatory role of TCDCA in the repair of corneal endothelial cell (CEC) damage in mice, as mentioned above, a mouse model of UVA-induced corneal endothelial damage was established ( Figure 2 (A) In the text, mice at 500 J / cm 2 PBS or TCDCA was administered intraperitoneally one day before UVA exposure and on days 0, 2, 4, and 6 after exposure. The control group received no treatment. Corneal structure was assessed by slit-lamp imaging and OCT on day 7. Compared with the PBS group, the TCDCA-treated group showed decreased CCT and improved corneal transparency. Figure 2 B and Figure 2 (D in the text). On day 7, mouse eyeballs were removed, and the entire cornea was stained with ZO-1 and Na. + / K + - Fully adherent immunostaining with ATP showed that the endothelial morphology in the TCDCA group was more regular compared to the PBS group. Figure 2 (C in the text). Statistical analysis of the immunofluorescence staining results of ZO-1 showed that TCDCA significantly improved UVA-induced decrease in endothelial cell density, increase in cell area, and increase in coefficient of variation (C in the text). Figure 2 E~ Figure 2 (G in the text). These results indicate that intraperitoneal injection of TCDCA can prevent UVA-induced CEC damage.

[0046] III. Intraperitoneal administration of TCDCA for the treatment of UVA-induced corneal endothelial cell damage: Building upon the preventative effect of TCDCA, this invention further utilizes the same UVA-induced mouse corneal endothelial damage model to evaluate its therapeutic potential. Mice received intraperitoneal injections of PBS or TCDCA on days 0, 2, 4, and 6 after UVA exposure, while the control group received no treatment. Corneal assessment was performed using slit-lamp and OCT imaging, and samples were collected for analysis on day 7. Figure 3 (A) Consistent with preventative studies, TCDCA treatment significantly reduced CCT and increased corneal transparency compared to the PBS group. Figure 3 B and Figure 3 (D in the text). Statistical analysis of immunofluorescence staining for ZO-1 showed that the endothelial cell morphology in the TCDCA group was more similar to that in the normal group. Compared with the PBS group, the cell density was significantly increased, the cell area was significantly reduced, and the coefficient of variation was significantly decreased. Figure 3 C and Figure 3 E~ Figure 3 (G in the text). These results indicate that intraperitoneal injection of TCDCA can effectively promote the repair of UVA-induced corneal endothelial damage, thus achieving the treatment of UVA-induced corneal endothelial cell damage.

[0047] IV. TCDCA can improve the function of human corneal endothelial cell lines: To verify the regulatory effect of TCDCA on cell function in vitro, this invention established a UVA-induced HCECs damage model. After 24 hours of UVA exposure, cells were treated with different concentrations of TCDCA, and analysis was performed after 24 hours. Live / dead staining showed that UVA significantly reduced cell number, while TCDCA increased cell viability in a dose-dependent manner, with the most significant effect observed at 100 μM (UVA+T100). Figure 4 A and Figure 4 (B in the original text). Furthermore, ROS analysis showed that UVA exposure increased oxidative stress, with a slight decrease in oxidative stress in the 50 μM and 200 μM groups (UVA+T50, UVA+T200), while 100 μM TCDCA significantly reduced ROS levels ( ). Figure 4 (C in the text). Similarly, JC-1 staining showed a decrease in mitochondrial membrane potential after UVA irradiation, which was alleviated by TCDCA, with the most significant recovery observed in the 100 μM group (C). Figure 4 D~ Figure 4 (F in the text). In summary, these results indicate that TCDCA effectively alleviates UVA-induced HCECs damage.

[0048] V. TCDCA Regulates Corneal Endothelial Cell (CEC) Damage Repair by Improving Mitochondrial Function: To investigate the potential biological mechanism by which TCDCA regulates CEC damage repair, this invention explores whether TCDCA restores the morphology and function of damaged CECs by improving mitochondrial function, considering the previously reported high bioenergy requirements of CECs. Based on the above in vitro experimental results, 100 μM TCDCA (UVA+TCDCA) was selected as the treatment condition for subsequent experiments. First, qPCR analysis showed that, compared with the UVA group, the TCDCA-treated group had a lower number of mitochondria and lower levels of function-related genes (…). DRP1 , MFN1 , MFN2 , OPA1 , TFAM , NRF1 , PGC-1a The expression of ) was significantly upregulated ( Figure 5 A and Figure 5 (B in the text). Next, the hippocampus experiment was used to assess mitochondrial function and cellular metabolic capacity. The results showed that TCDCA treatment enhanced basal respiration, ATP production, and maximal respiratory capacity (B). Figure 5 (C in the text). To further validate these findings, ATP quantification confirmed that TCDCA significantly increased ATP levels compared to the UVA group (C in the text). Figure 5 (D in the text). In summary, these results indicate that TCDCA alleviates mitochondrial bioenergy deficiency following UVA-induced HCECs damage.

[0049] VI. TCDCA significantly alters the transcriptomics of corneal endothelial cells, by activating Ca2+. 2+ Signaling pathways promote the recovery of corneal endothelial cell function: Based on the above in vitro experimental results, this invention performed RNA-seq analysis on HCECs in the UVA and UVA+TCDCA groups (100 μM). Figure 6 The study aimed to explore the regulatory mechanism by which TCDCA promotes CEC repair. A total of 477 upregulated genes and 1096 downregulated genes were identified. Figure 6 (B in the text) KEGG enrichment analysis showed that multiple signaling pathways related to cell function were significantly upregulated in the UVA+TCDCA group, including Hippo, MAPK, and Ca2+. 2 + signal path ( Figure 6(C in the text). Previous studies have shown that activation of the Hippo and MAPK signaling pathways promotes the migration and proliferation of CECs (Ryu Y, Seo JH, Kim HS, et al. COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling / mitochondria pathway). Matrix Biol . Dec 2024;134:119-131 and HsuehYJ, Meir YJ, Lai JY, et al. Ascorbic acid ameliorates corneal endothelialdysfunction and enhances cell proliferation via the noncanonical GLUT1-ERKaxis. Biomed Pharmacother Dec 2021;144:112306. doi:10.1016 / j.biopha.2021.112306). Consistent with these findings, the qPCR validation of this invention ( Figure 6 D and Figure 6 E) in the present invention confirms the activation of the MAPK and Hippo signaling pathways in the experimental model of the present invention. The present invention detects Ca 2+ The expression of related genes in the signaling pathway was significantly upregulated in the TCDCA group. Figure 6 (F in the text). These results suggest that TCDCA may activate Ca2+. 2+ Signaling pathways promote the repair of UVA-induced HCECs damage.

[0050] VII. TCDCA activates Ca 2+ Signaling pathways protect corneal endothelial cell function: Based on previous transcriptomic analysis, this invention introduces Ca2+ signaling pathways to rigorously confirm the relationship between TCDCA and CEC repair after injury. 2 + The signaling pathway inhibitor 2-APB was used to establish a TCDCA+2-APB treatment group. The experimental timeline is as follows: Figure 7 As shown in A in the figure. 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 ( Figure 7 B and Figure 7 (C in the text). To further verify these findings, the cornea was stained with ZO-1 throughout ( ). Figure 7(D in the image). Consistent with the imaging results, the average cell area and cell density of CECs in the TCDCA+2-APB group were comparable to those in the PBS group. Figure 7 E~ Figure 7 (G in the middle). These results indicate that TCDCA activates Ca2+. 2 + Signaling pathways promote repair after CEC damage.

[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy.

2. The use of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of corneal damage.

3. The use of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of corneal endothelial cell damage.

4. The use of taurine chenodeoxycholic acid in the preparation of drugs for the prevention and / or treatment of UVA-induced corneal endothelial cell damage.

5. The application of taurine chenodeoxycholic acid in the preparation of drugs that can alleviate one or more of the following functions caused by UVA: decrease in the number and vitality of corneal endothelial cells, oxidative stress, increase in ROS levels, and decrease in mitochondrial membrane potential.

6. Application of taurine chenodeoxycholic acid in the preparation of drugs to alleviate UVA-induced mitochondrial bioenergy deficiency.

7. Activate Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of Fuchs' corneal endothelial dystrophy; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

8. Activate Ca 2+ The use of reagents targeting signaling pathways in the preparation of medicaments for the prevention and / or treatment of corneal damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

9. Activate Ca 2+ The use of reagents targeting signaling pathways in the preparation of drugs for the prevention and / or treatment of corneal endothelial cell damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

10. Activate Ca 2+ The use of agents that activate signaling pathways in the preparation of drugs for the prevention and / or treatment of UVA-induced corneal endothelial cell damage; the activation of Ca 2+ The reagents used in the signaling pathway include taurine chenodeoxycholic acid.

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