Use of inhibitors of ptgds for the manufacture of a medicament for the treatment of cataracts

Abstract

The application of the inhibitor of PTGDS in the preparation of the drug for treating cataract, AT-56 is an effective and selective inhibitor of PTGDS, AT-56 competitively inhibits the generation of PGD2 by occupying the catalytic site of PTGDS, and PTGDS causes oxidative stress damage of human lens epithelial cells by catalyzing the synthesis of PGD2, thereby promoting the occurrence and development of lens aging turbidity, and by reducing the cell apoptosis caused by oxidative stress damage, the degree of cataract can be effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of cataract treatment technology, specifically to the application of a PTGDS inhibitor in the preparation of drugs for treating cataracts. Background Technology

[0002] Cataracts are a condition where the lens of the eye becomes partially or completely cloudy, leading to progressive vision loss. Cataracts are a leading cause of blindness. Statistics show that cataracts account for one-third of all causes of blindness in my country, and are also the second leading cause of severe and moderate visual impairment, after uncorrected refractive errors. Among all types of cataracts, age-related cataracts are the most prevalent. Large-scale population-based studies have shown that the prevalence of age-related cataracts increases with age, rising from 3.9% in those aged 55-64 to 92.6% in those over 80. With increasing population growth and aging, the number of cataract patients will continue to rise, placing a greater burden on individuals and society. Currently, the most effective treatment for age-related cataracts is surgery, but intraoperative and postoperative complications are possible, such as posterior capsule rupture, secondary cataracts, and corneal endothelial decompensation. Therefore, understanding the molecular mechanisms of age-related lens opacity could provide potential targets for drug treatment of age-related cataracts. This patent aims to reduce and delay the onset and progression of cataracts through the development of drugs.

[0003] The lens is composed of lens epithelial cells (LECs) and fibroblasts. The lens epithelial cells are tightly attached to the inner surface of the anterior lens capsule. During lens growth, the lens epithelial cells proliferate in the lens germinal zone and then differentiate into lens fibroblasts at the lens equator. Lens epithelial cells are the most metabolically active part of the lens, playing a crucial role in its growth, differentiation, and damage repair. They also play a vital role in protecting the transparency and homeostasis of the entire lens.

[0004] Single-cell transcriptome sequencing is a revolutionary technology that has emerged in recent years. Compared to traditional bulk transcriptome sequencing, single-cell transcriptome sequencing offers unbiased, reproducible, high-resolution, and high-throughput transcriptome analysis of individual cells. It can map gene expression profiles of single cells, revealing complex and rare regulatory relationships between cell populations and genes. This technology comprehensively and unbiasedly identifies disease-related cell populations and mines the impact of genes on diseases at the cellular level, thus enabling its wide application in disease research. Undeniably, single-cell transcriptome sequencing provides unprecedented opportunities to identify therapeutic targets.

[0005] However, current literature lacks studies exploring the molecular mechanisms related to lens aging and opacity using single-cell transcriptome sequencing. Therefore, this study aims to perform single-cell transcriptome sequencing on epithelial cells of both aging and opaque lenses and clear lenses. Bioinformatics analysis will be used to identify significantly different genes and related pathways between the two samples, and these findings will be validated using an in vitro lens epithelial cell culture model and rat isolated lens organ culture.

[0006] To date, lanosterol is the only drug that has shown some clinical efficacy in the treatment of cataracts. Studies of two families with congenital cataracts have revealed that lanosterol, present in the normal lens, is key to regulating the abnormal aggregation and deaggregation of lens proteins, representing a significant breakthrough in cataract drug treatment. However, because this data does not integrate data on age-related cataracts, current clinical trials have also shown some limitations, such as the drug's lack of significant effect on age-related nuclear cataracts. Summary of the Invention

[0007] To address the technical shortcomings of existing cataract drug treatments, this invention provides the application of a PTGDS inhibitor in the preparation of cataract treatment drugs, which can delay and reverse the occurrence and development of cataracts.

[0008] The technical solution adopted in this invention is: the application of PTGDS inhibitors in the preparation of drugs for treating cataracts, wherein the PTGDS inhibitor is AT-56.

[0009] The concentration of AT-56, the PTGDS inhibitor, in the cataract treatment drug is 7-10 μM.

[0010] The cataract medication mentioned is for treating age-related cataracts.

[0011] The inhibitor of PTGDS is AT-56, which is used in the preparation of drugs to treat oxidative stress damage to lens epithelial cells.

[0012] The concentration of AT-56, an inhibitor of PTGDS, in the drug for treating oxidative stress damage to lens epithelial cells is 10 μM.

[0013] The beneficial effects of this invention are as follows: This invention provides an application of a PTGDS inhibitor in the preparation of drugs for treating cataracts. AT-56 is an effective and selective PTGDS inhibitor. AT-56 competitively inhibits the generation of PGD2 by occupying the catalytic site of PTGDS. PTGDS catalyzes the synthesis of PGD2, thereby causing oxidative stress damage to human lens epithelial cells, which promotes the occurrence and development of lens aging and opacity. By reducing cell apoptosis caused by oxidative stress damage, the degree of cataracts can be effectively reduced. Attached Figure Description

[0014] Figure 1 A shows the overall results of single-cell transcriptome sequencing; Figure 1 B represents the expression of upregulated and downregulated genes in PTGDS in cataract samples compared to clear lens samples, as shown in the violin diagram. Figure 1 C represents the GSEA analysis of 21,884 genes using the KEGG database; Figure 1 D is a visualization of differentially expressed genes in the Arachidonic acid metabolism signaling pathway.

[0015] Figure 2 A. Four epithelial cell subpopulations of the overall sample. The number of each subpopulation in BE corresponds to the top 10 differentially expressed genes of the 0-3 lens epithelial cell subpopulations.

[0016] Figure 3 The image shows the volcano plot (AD) of differentially expressed genes in each subgroup, and EH represents the expression level of PTGDS in each subgroup of the two samples.

[0017] Figure 4 GSEA for the arachidonic acid signaling pathway in each subgroup.

[0018] Figure 5 Visualize differentially expressed genes in the arachidonic acid signaling pathway for each subgroup.

[0019] Figure 6 The following are the findings: A. Relative expression of PTGDS between the two samples; B. Expression of PTGDS in lens epithelial cell lines; C. Expression of PGD2 after adding different concentrations of AT-56; D. Differences in cell activity among the groups after adding different concentrations of AT-56.

[0020] Figure 7 A. Control group (DMSO group); B and E correspond to ROS fluorescence staining after adding PTGDS at concentrations of 100 pg / mL, 400 pg / mL, 700 pg / mL, and 1000 pg / mL, respectively. Scale bar: A=B=C=D=E=50 μM.

[0021] Figure 8 A. Added DMSO; B and C correspond to ROS fluorescence staining after adding PGD2 at concentrations of 0.1 nM, 0.4 nM, 0.7 nM, and 1.0 nM, respectively. Scale bar: A=B=C=D=E=50 μM.

[0022] Figure 9This diagram illustrates the inhibition of ROS production in lens epithelial cells by AT-56 in an H2O2-induced epithelial cell oxidation model. 9A represents the control group (DMSO group); 9B shows ROS fluorescence staining in the epithelial cell oxidation model with 200 μM H2O2; and 9C shows ROS staining with 200 μM H2O2 and 10 μM AT-56. ROS: Green fluorescent staining. Scale bar: A=B=C=100 μM.

[0023] Figure 10 A represents the MDA content of different concentrations of PTGDS; 10B represents the MDA content of different concentrations of PGD2; 10C represents the MDA content of AT-56 added to 200 μM H2O2.

[0024] Figure 11 A. Adding DMSO; B. Adding 200 μM H2O2; C. Adding 200 μM H2O2 and 10 μM AT-56. The red arrows represent apoptotic lens epithelial cells. D is a bar chart of the data in Figures A, B, and C. Scale bar: A=B=C=50 μM.

[0025] Figure 12 A: Blank control group; B: Treatment with 200 μM H2O2 for 12 h; C: Treatment with 200 μM H2O2 + 7 μM AT-56 for 12 h; D: Treatment with 200 μM H2O2 + 10 μM AT-56 for 12 h; E: Treatment with 200 μM H2O2 for 12 h; F: Treatment with 200 μM H2O2 for 12 h followed by treatment with 7 μM AT-56 for 24 h; G: Treatment with 200 μM H2O2 for 12 h followed by treatment with 10 μM AT-56 for 24 h. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] This study discovered significant differences in the PTGDS gene and its associated arachidonic acid pathway between aging, cloudy lenses and clear lenses. PTGDS was extracted from rat brain in purified form by Urade Y et al. in 1985. In the arachidonic acid signaling pathway, PTGDS acts as a D-isomerase of prostaglandin H2 (PGH2), catalyzing the isomerization of the 9,11-ring peroxide group of PGH2 to generate prostaglandin D2 (PGD2) with 9-hydroxy and 11-keto groups. Arachidonic acid (AA) is one of the most abundant, most reactive, and widely distributed polyunsaturated essential fatty acids in the human body. It possesses strong biological activity and is closely related to oxidative stress. Among organic inhibitors, AT-56 is an effective and selective inhibitor of PTGDS, competitively inhibiting PGD2 production by occupying the catalytic site of PTGDS.

[0028] Example 1 The overall results of single-cell transcriptome sequencing showed that among the 21,884 genes we obtained, 16,537 genes had a p-value < 0.05. Figure 1 A). Genes with an absolute fold change of 1.5 or greater than or equal to that of cataract samples relative to normal samples were screened: 36 genes with log2(FC) > 1.5 and p < 0.05 were upregulated in cataract samples relative to clear lens samples, indicated in red; 16 genes with log2(FC) < -1.5 and p < 0.05 were downregulated in cataract samples relative to clear lens samples, indicated in blue. PTGDS expression in both groups is shown in the violin diagram. Figure 1 B). Then, 21,884 genes were analyzed using the KEGG database via GSEA (Geochemical Emissions Analysis). Figure 1 C) The normalized enrichment score (NES) of the arachidonic acid metabolism signaling pathway associated with PTGDS was found to be 1.52 with a p-value < 0.05, suggesting a close relationship between lens aging and opacity and this signaling pathway. Visualization of differentially expressed genes in the arachidonic acid metabolism signaling pathway can be found in [link to visualization]. Figure 1 D, it was found that PTGDS (5.3.99.2) plays a catalytic role in PGD2 synthesis in the arachidonic acid pathway. Through unsupervised cluster analysis, lens epithelial cells were divided into four subsets ( Figure 2Differential genes in each subgroup were screened according to p < 0.05 and log2|FC| > 1.5. It was found that PTGDS met the above screening criteria in each subgroup. Figure 3 In all subgroups, PTGDS expression was upregulated in aging and cloudy lens samples compared to clear lens samples. Gene enrichment analysis was performed on the two samples in each subgroup. GSEA results showed that the arachidonic acid signaling pathway involved in PTGDS was p < 0.05 and |NES| was greater than 1 in all subgroups. Figure 4 This suggests that this pathway may play an important role in the pathogenesis of age-related lens opacities. Visualizing the differentially expressed genes in the arachidonic acid signaling pathway within each subgroup, we found that PTGDS (5.3.99.2) catalyzes PGD2 synthesis. Therefore, we propose that PTGDS may promote the development of age-related lens opacities by catalyzing PGD2 synthesis, thereby leading to oxidative stress damage in human lens epithelial cells. Figure 5 Example 2 PTGDS was expressed in both age-related cataract and clear lens samples, with a significantly increased PTGDS gene expression level in the former. Figure 6 As shown in Figure A. Western blot confirmed that the HLEC-B3 lens cell line expressed PTGDS (as shown in Figure A). Figure 6 B). Adding AT-56 to the cell line. We first used an ELISA experiment on PGD2 and found that 10 μM AT-56 significantly inhibited PGD2 production compared to other concentrations. The results of the CCK8 experiment further confirmed that adding 7 μM and 10 μM AT-56 resulted in greater cell viability compared to adding DMSO.

[0029] Both PTGDS and PGD2 can increase ROS production by lens epithelial cells in a concentration-dependent manner. Figure 7 As shown, A represents the control group (DMSO group); B and E correspond to ROS fluorescence staining after adding PTGDS at concentrations of 100 pg / mL, 400 pg / mL, 700 pg / mL, and 1000 pg / mL, respectively. Scale bar: A=B=C=D=E=50 μM. Figure 8 As shown, A represents the addition of DMSO; B and C correspond to ROS fluorescence staining after adding PGD2 at concentrations of 0.1 nM, 0.4 nM, 0.7 nM, and 1.0 nM, respectively. Scale bar: A=B=C=D=E=50 μM.

[0030] In the H2O2 epithelial cell oxidation model, AT-56 can inhibit the production of ROS by lens epithelial cells. Figure 9The model consisted of three groups: A) control group (DMSO group); B) epithelial cell oxidation model with 200 μM H2O2 and ROS fluorescence staining; and C) cells with 200 μM H2O2 and 10 μM AT-56. ROS: green fluorescence staining. Scale bar: A=B=C=100 μM.

[0031] Example 3 PTGDS / PGD2 increases MDA, while AT-56 effectively reduces MDA generation. MDA is an important indicator of oxidative stress, and the degree of oxidative stress damage is reflected by measuring the MDA content. Different concentrations of PTGDS (Plasma-to-Glucose Demand) were added. Figure 10 A) PGD2 Figure 10 B) After measuring the MDA value, it was found that MDA increased with increasing concentration. AT-56 was added to H2O2 (… Figure 10 C) can effectively reduce the generation of MDA.

[0032] Example 4 AT-56 reduces apoptosis caused by oxidative stress. Lens epithelial cells suffer from oxidative stress, leading to excessive ROS accumulation in the cells, which can cause apoptosis and ultimately result in cataracts. Figure 11 As shown, adding 200 μM H2O2 to lens epithelial cell culture resulted in significant apoptosis in the cells. In another group, adding 10 μM AT-56 to 200 μM H2O2 effectively reduced apoptosis. Example 5 A rat in vitro lens oxidative opacity model was validated, showing that AT-56 can alleviate lens opacity caused by H2O2 oxidative stress. In rat isolated lens cultures, the addition of AT-56 was found to alleviate the progression of cataracts. Figure 12 AD), and at the same time, after inducing cataracts with H2O2, the addition of AT-56 can effectively reduce the degree of cataracts (AD). Figure 12 EG).

[0033] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0034] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. The application of PTGDS inhibitors in the preparation of drugs for treating cataracts, characterized in that, The inhibitor of PTGDS is AT-56.

2. The application according to claim 1, characterized in that, The concentration of AT-56, the PTGDS inhibitor, in the cataract treatment drug is 7-10 μM.

3. The application according to claim 1, characterized in that, The cataract medication mentioned is for treating age-related cataracts.

4. The application according to claim 2, characterized in that, The PTGDS inhibitor AT-56 effectively reduces the severity of cataracts by decreasing cell apoptosis caused by oxidative stress damage.

5. The application according to claim 4, characterized in that, The concentration of AT-56, the inhibitor of PTGDS, is 10 μM.

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