Use of ptgds inhibitor in preparation of drug for treating cataracts

Abstract

The use of a PTGDS inhibitor in the preparation of a drug for treating cataracts. AT-56 is an effective and selective PTGDS inhibitor, and competitively inhibits the generation of PGD2 by occupying a catalytic site of PTGDS, and PTGDS leads to oxidative stress damage of human lens epithelial cells by catalyzing the synthesis of PGD2, thereby promoting the occurrence and development of aging and opacity of a lens, so that AT-56 can effectively alleviate the level of cataracts by means of reducing apoptosis caused by oxidative stress damage.

Description

Application of PTGDS inhibitors in the preparation of drugs for treating cataracts Technical Field

[0001] The present invention relates to the technical field of cataract treatment, and in particular to an application of a PTGDS inhibitor in the preparation of a drug for treating cataract. Background Art

[0002] Cataracts are partial or complete clouding of the lens, leading to progressive vision loss. Cataracts are the leading cause of blindness. According to statistics, cataracts account for one-third of all causes of blindness in my country and are the second leading cause of severe and moderate visual impairment, second only to uncorrected refractive error. Among all cataract types, age-related cataracts are the most common. Several large-scale population-based studies have shown that the prevalence of age-related cataracts increases with age, from 3.9% in those aged 55-64 years to 92.6% in those aged 80 years and older. With population growth and an aging population, the number of cataract patients will increase, placing a greater burden on individuals and society. Currently, the most effective treatment for age-related cataracts is surgery, but it carries the risk of intraoperative and postoperative complications, such as posterior capsule rupture, secondary cataracts, and corneal endothelial decompensation. Therefore, understanding the molecular mechanisms of lens opacification associated with aging may provide potential targets for drug treatment of age-related cataracts. This patent aims to alleviate and delay the occurrence and development of cataracts by developing drugs.

[0003] The lens is composed of epithelial cells (LECs) and fiber cells. LECs are closely attached to the inner surface of the anterior lens capsule. During lens growth, LECs proliferate in the lens germinal zone and then differentiate into lens fiber cells at the lens equator. LECs are the most metabolically active site in the lens and play a crucial role in lens growth, differentiation, and repair, maintaining the transparency and internal stability 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. This technology can map gene expression profiles in individual cells and reveal the regulatory relationships between complex and rare cell populations and genes. This technology comprehensively and unbiasedly identifies disease-associated cell populations and can explore the impact of genes on disease at the cellular level, thus paving the way for widespread application in disease research. It is undeniable that single-cell transcriptome sequencing offers unprecedented opportunities for identifying therapeutic targets. Technical issues

[0005] However, there are currently no studies in the literature using single-cell transcriptome sequencing to explore the molecular mechanisms associated with lens opacity in aging. Therefore, this study aimed to perform single-cell transcriptome sequencing on epithelial cells from aged, opacified and clear lenses, identifying significantly differentially expressed genes and pathways between the two samples through bioinformatics analysis. This work was then validated using an in vitro lens epithelial cell culture model and an isolated rat lens organ culture.

[0006] To date, the only drug that has demonstrated clinical efficacy in the treatment of cataracts is lanosterol. Studies of two families with congenital cataracts have revealed that lanosterol, present in the normal lens, is key to regulating the abnormal aggregation and disaggregation of lens proteins. This represents a significant breakthrough in the treatment of cataracts. However, because this data does not incorporate data on age-related cataracts, current clinical trials have shown some shortcomings, such as the lack of significant efficacy for age-related nuclear cataracts. Technical Solutions

[0007] In order to solve the technical defect of the shortage of existing cataract drugs, the present invention provides an inhibitor of PTGDS for use in the preparation of a drug for treating cataracts, which can delay and reverse the occurrence and development of cataracts.

[0008] The technical solution adopted by the present invention is: application of a PTGDS inhibitor in the preparation of a drug for treating cataracts, wherein the PTGDS inhibitor is AT-56.

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

[0010] The cataract treatment drug is a drug for treating age-related cataracts.

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

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

[0013] The beneficial effects of the present invention are as follows: the present invention provides an application of a PTGDS inhibitor in the preparation of a drug for treating cataracts. AT-56 is an effective and selective PTGDS inhibitor. AT-56 competitively inhibits the production 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, thereby promoting the occurrence and development of lens aging opacities. By reducing cell apoptosis caused by oxidative stress damage, the degree of cataracts can be effectively alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1A shows the overall results of single-cell transcriptome sequencing; Figure 1B shows the expression of up-regulated and down-regulated genes in PTGDS in cataract samples relative to clear lens samples, as shown in violin plots; Figure 1C shows GSEA of 21,884 genes using the KEGG database; Figure 1D shows the visualization of differentially expressed genes in the Arachidonic acid metabolism signaling pathway.

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

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

[0017] FIG4 shows GSEA of the arachidonic acid signaling pathway in each subgroup.

[0018] Figure 5 shows the visualization of differentially expressed genes in the arachidonic acid signaling pathway of each subgroup.

[0019] Figure 6 shows A. the relative expression of PTGDS between the two samples; B. the expression of PTGDS in the lens epithelial cell line; C. the expression of PGD2 after adding different AT-56 concentrations; D. the difference in cell activity among the groups after adding different AT-56 concentrations.

[0020] Figure 7 shows ROS fluorescence staining in A. Control (DMSO group); B. E. PTGDS after addition 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 shows ROS fluorescence staining after the addition of DMSO (A); PGD2 at concentrations of 0.1 nM, 0.4 nM, 0.7 nM, and 1.0 nM (B, B, and C). Scale bars: A = B = C = D = E = 50 μM.

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

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

[0024] Figure 11 shows A. DMSO was added; B. 200 μM H2O was added 2， C shows the addition of 200 μM H₂O₂ and 10 μM AT-56. Red arrows indicate apoptotic lens epithelial cells. Scale bar: A = B = C = 50 μM.

[0025] Figure 12 shows A: blank control group; B: 200 μM H2O2 treatment for 12 h; C: 200 μM H2O2 + 7 μM AT-56 treatment for 12 h; D: 200 μM H2O2 + 10 μM AT-56 treatment for 12 h; E: 200 μM H2O2 treatment for 12 h; F: 200 μM H2O2 treatment for 12 h, followed by AT-56 7 μM treatment for 24 h; G: 200 μM H2O2 treatment for 12 h, followed by AT-56 10 μM treatment for 24 h. Modes for Carrying Out the Invention

[0026] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] The present study discovered that the PTGDS gene and the arachidonic acid pathway in which it resides exhibit significant differences between aged, opaque and clear lenses. PTGDS was purified from rat brain by Urade Y et al. in 1985. In the arachidonic acid signaling pathway, PTGDS acts as the prostaglandin H2 (PGH2) D-isomerase, catalyzing the isomerization of the 9,11-ring peroxide group of PGH2 to produce prostaglandin D2 (PGD2) with 9-hydroxy and 11-keto groups. Arachidonic acid (AA) is the most abundant, active, and widely distributed polyunsaturated essential fatty acid in the human body. It possesses extremely potent biological activity and is closely linked to oxidative stress. Among organic inhibitors, AT-56 is a potent and selective inhibitor of PTGDS, competitively inhibiting PGD2 production by occupying the catalytic site of PTGDS.

[0028] Example 1

[0029] The overall results of single-cell transcriptome sequencing showed that among the 21,884 genes we obtained, 16,537 had p-values ​​< 0.05 (Figure 1A). Genes with an absolute fold change of 1.5 or greater in cataract samples compared to normal samples were screened: 36 genes with a log2(FC) > 1.5 and p < 0.05, indicating up-regulated genes in cataract samples relative to clear lens samples, are shown in red; 16 genes with a log2(FC) < -1.5 and p < 0.05, indicating down-regulated genes in cataract samples relative to clear lens samples, are shown in blue. The expression of PTGDS in both cataract and clear lens samples is shown in violin plots (Figure 1B). GSEA of 21,884 genes using the KEGG database (Figure 1C) revealed a normalized enrichment score (NES) of 1.52 for the arachidonic acid metabolism pathway associated with PTGDS, with a p-value < 0.05, suggesting a close association between aging-induced lens opacity and this pathway. Visualization of differentially expressed genes in the arachidonic acid metabolism pathway is shown in Figure 1D. PTGDS (5.3.99.2) was found to catalyze PGD2 synthesis within the arachidonic acid pathway. Unsupervised cluster analysis divided lens epithelial cells into four subgroups (Figure 2). Differentially expressed genes within each subgroup were screened for p < 0.05 and log2|FC| > 1.5. PTGDS met these criteria in every subgroup (Figure 3). In each subgroup, PTGDS expression was upregulated in aged-induced opacified lens samples compared to clear lens samples. Gene enrichment analysis of the two samples was performed for each subgroup. GSEA results showed that the arachidonic acid signaling pathway, where PTGDS is located, had p < 0.05 and |NES| greater than 1 in all subgroups (Figure 4), suggesting that this pathway may play a key role in the pathogenesis of lens opacities associated with age. Visualization of differentially expressed genes in the arachidonic acid signaling pathway within each subgroup revealed that PTGDS (5.3.99.2) can catalyze PGD2 synthesis. Therefore, we propose that PTGDS may promote the development of lens opacities by catalyzing PGD2 synthesis, leading to oxidative stress damage in human lens epithelial cells (LECs) (Figure 5).

[0030] Example 2

[0031] PTGDS was expressed in both age-related cataract and clear lens samples, with significantly increased PTGDS gene expression in the former, as shown in Figure 6A. Western blot confirmed that the lens HLEC-B3 cell line expressed PTGDS in vitro (Figure 6B). AT-56 was added to the cell line. We first performed a PGD2 ELISA assay and found that 10 μM AT-56 significantly inhibited PGD2 production compared to other concentrations. CCK8 assay results further confirmed that the addition of 7 μM and 10 μM AT-56 resulted in greater cell viability compared to the addition of DMSO.

[0032] Both PTGDS and PGD2 increase ROS production in lens epithelial cells in a concentration-dependent manner. As shown in Figure 7, A: Control (DMSO group); B: ROS fluorescence staining after addition of 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. As shown in Figure 8, A: ROS fluorescence staining after addition of DMSO; B: ROS fluorescence staining after addition of 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.

[0033] AT-56 inhibited ROS production in lens epithelial cells in the H2O2 epithelial cell oxidation model (Figure 9). A: Control (DMSO group); B: ROS fluorescence staining in the epithelial cell oxidation model with 200 μM H2O2; C: ROS: Green fluorescence staining in the epithelial cell oxidation model with 200 μM H2O2 and 10 μM AT-56. Scale bar: A = B = C = 100 μM.

[0034] Example 3

[0035] PTGDS / PGD2 increased MDA, and AT-56 effectively reduced MDA production

[0036] MDA is an important indicator of oxidative stress, and its level reflects the degree of oxidative stress damage. MDA levels were measured after adding different concentrations of PTGDS (Figure 10A) and PGD2 (Figure 10B), revealing an increase in MDA with increasing concentrations. Adding AT-56 to H2O2 (Figure 10C) effectively reduced MDA production.

[0037] Example 4

[0038] AT-56 reduces cell apoptosis caused by oxidative stress

[0039] When lens epithelial cells are damaged by oxidative stress, excessive ROS accumulate in the cells, leading to apoptosis and ultimately cataract formation. As shown in Figure 11, the addition of 200μM H2O2 to the lens epithelial cell culture resulted in a significant increase in apoptosis. In another group, the addition of 10μM AT-56 to 200μM H2O2 effectively mitigated apoptosis.

[0040] Example 5

[0041] The rat lens in vitro oxidative opacity model verified that AT-56 can alleviate lens opacity caused by H2O2 oxidative stress

[0042] In rat lens culture in vitro, it was found that the addition of AT-56 alleviated the progression of cataracts (Figure 12A-D). At the same time, after cataracts were induced by H2O2, the addition of AT-56 could effectively reduce the degree of cataracts (Figure 12E-G).

[0043] Technical personnel should note: Although the present invention has been described according to the above specific implementation methods, the inventive concept of the present invention is not limited to this invention. Any modification using the inventive concept will be included in the scope of protection of this patent.

Claims

1. The use of a PTGDS inhibitor in the preparation of a drug for treating cataracts, characterized in that: The PTGDS inhibitor is AT-56.

2. The use 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 use according to claim 1, characterized in that: The cataract treatment drug is a drug for treating age-related cataracts.

4. The application of AT-56, an inhibitor of PTGDS, in the preparation of drugs for treating oxidative stress damage of lens epithelial cells.

5. The use according to claim 4, characterized in that: The concentration of AT-56, an inhibitor of PTGDS, in the drug for treating oxidative stress damage of lens epithelial cells is 10 μM.

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