Pharmaceutical composition for prevention or treatment of steroid-induced cataract

Abstract

The present invention relates to a pharmaceutical composition comprising a material inhibiting chondroitin sulfate proteoglycan 5 (CSPG5) or a transcription factor thereof, thereby being capable of inhibiting the expression and mobility of an epithelial-mesenchymal transition-related protein induced by a steroid in lens epithelial cells, and thus enabling the reduction of steroid-induced cataract.

Description

Pharmaceutical composition for the prevention or treatment of steroid-induced cataract

[0001] The present invention relates to a pharmaceutical composition for preventing or treating steroid-induced cataracts.

[0002]

[0003] Cataracts cause vision loss in more than 30% of people worldwide. Cataract surgery can improve vision, but it is expensive and carries several side effects, including dry eye and cystoid macular edema. Known risk factors for cataracts include age, diabetes, trauma, ultraviolet exposure, use of medications such as steroids, and inflammatory eye diseases such as uveitis. Steroid-induced cataract (SIC) is a side effect of long-term corticosteroid use. With increasing life expectancy and the increasing prevalence of diseases requiring corticosteroid treatment, the incidence of SIC is expected to increase. Current treatment approaches for inflammatory conditions such as allergies and rheumatic diseases have reportedly increased the risk of SIC. Unlike diabetes or age-related cataract (ARC), SIC can develop at a young age and, due to its location, can cause vision loss in its early stages. However, the precise mechanisms and primary effects of steroids on the lens are poorly understood.

[0004]

[0005] The present invention aims to provide a pharmaceutical composition having excellent efficacy against steroid-induced cataracts.

[0006]

[0007] 1. A pharmaceutical composition for preventing or treating steroid-induced cataracts, comprising a substance that inhibits CSPG5 (Chondroitin Sulfate Proteoglycan 5) or its transcription factor.

[0008] 2. A pharmaceutical composition for preventing or treating steroid-induced cataracts, wherein the transcription factor in 1 above is EZH2 (Enhancer of zeste homolog 2) or B-myb (Myb-related protein B).

[0009] 3. A pharmaceutical composition for preventing or treating steroid-induced cataracts, wherein the substance that inhibits CSPG5 in the above 1 is CSPG5 shRNA.

[0010] 4. A pharmaceutical composition for preventing or treating steroid-induced cataract, wherein the substance that inhibits the transcription factor in the above 1 is EI1 (CAS No. 1418308-27-6) or A490.

[0011] 5. A pharmaceutical composition for preventing or treating steroid-induced cataracts, wherein the steroid in the above 1 is cortisol, hydrocortisone, prednisolone, methyltrednisolone or dexamethasone.

[0012]

[0013] The pharmaceutical composition of the present invention can improve steroid-induced cataracts by inhibiting the expression and motility of epithelial-mesenchymal transition-related proteins induced by steroids in lens epithelial cells.

[0014]

[0015] Figure 1. Effect of dexamethasone administration on CSPG5 expression. HLE-B3 cells were cultured in medium containing dexamethasone (0.1 mg / mL) and analyzed at various time points (8, 24, 48, and 72 hours). (A) Expression of growth factor genes was analyzed using the RT2 Profiler PCR Array. (B) Expression of the CSPG5 gene. Data are presented as mean Delta-CT values. Bars represent mean ± % CV (n = 3). (C) Representative Western blot images of CSPG5 expression in human corneal epithelial, HLE-B3, and ARPE-19 cells. (D) Graph showing relative quantification of CSPG5 protein expression levels (n = 4). (E) CSPG5 protein expression in human anterior capsule from a patient was detected by immunofluorescence (red). DAPI was used as a nuclear marker (blue). Scale bar, 50 μm. Scale bar in enlarged images, 10 μm (bottom). (F) Protein expression ratios determined using ImageJ (n = 4). (G) Relative quantification graph of mRNA expression levels of CSPG5 analyzed by qRT-PCR (n = 5). Bars represent mean ± SEM. p < 0.05 was considered significant. ARC; age-related cataract; SIC; steroid-induced cataract.

[0016] Figure 2. Effect of Dexa on the viability of HLE-B3 cells. HLE-B3 cells were cultured in medium containing various concentrations of Dexa (0.1 and 0.5 mg / ml) for 48 hours. (A) The effect of Dexa on mitochondrial dehydrogenase activity in HLE-B3 cells was measured using the CCK-8 assay (n=8). The viability of control cells was set to 100%. Viability was expressed as a percentage of control cells. A significance threshold of p < 0.05 was used. (B) The effect of Dexa on cell morphology was detected by F-actin staining (green). DAPI was used as a nuclear marker (blue). Scale bar, 25 μm. Protein expression ratios were determined using ImageJ software (n=3). Data are presented as mean ± SEM.

[0017] Figure 3. Effect of CSPG5 downregulation on dexa-induced cell migration. (A) CSPG5-knockdown HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 48 h. The effect of CSPG5 inhibition on mitochondrial dehydrogenase activity in dexa-treated HLE-B3 cells was measured by the CCK-8 assay (n = 8). The viability of control cells was set as 100%. Viability was expressed as a percentage of control cells. (B) The effect of CSPG5 inhibition on cell morphology was detected by F-actin staining (green). DAPI was used as a nuclear marker (blue). Scale bar, 25 μm. Protein expression ratios were determined using ImageJ (n = 3). (C) CSPG5-knockdown HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 8 h. The effect of CSPG5 inhibition on dexa-induced cell migration was measured using a wound healing assay. The relative healing rate was expressed as the ratio of the initial wound area at 0 h compared to that at 8 h (n = 3). (D) CSPG5-knockdown HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 48 h. Representative Western blot images of dexa-induced EMT marker expression in CSPG5-knockout HLE-B3 cells. Arrowheads indicate CSPG5. Graphs showing relative quantification of protein expression levels of (E) fibronectin, (F) CSPG5, (G) Vimentin, and (H) α-SMA (n = 3). Bars represent the mean ± SEM. p < 0.05 was the threshold for significance.

[0018] Figure 4. Effect of dexa on CSPG5-related transcription factors in HLE-B3 cells treated with dexa. (A) HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 4 h. Graph showing relative quantification of mRNA expression levels of EZH2, (B) B-MYB was analyzed by qRT-PCR (n=7). (C) HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 24 h. Representative Western blot images of dexa-induced transcription factor expression in HLE-B3 cells. Graph showing relative quantification of total protein expression levels of (D) EZH2 and (E) B-MYB (n=3). (F) HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 24 h. Representative Western blot images of the localization of transcription factors induced by dexa in HLE-B3 cells. Graphs showing relative quantification of total protein expression levels of (G) EZH2 and (H) B-MYB (n = 5). (I, J) HLE-B3 cells were cultured in medium containing dexa (0.1 mg / mL) for 48 h. The effect of dexa on transcription factor expression was detected by immunofluorescence (EZH2 and B-MYB; red). DAPI was used as a nuclear marker (blue). Scale bar, 25 μm. Protein expression ratios were determined using ImageJ (n = 3). Bars represent the mean ± SEM. p < 0.05 was the threshold for significance.

[0019] Figure 5. Inhibitory effects of CSPG5-related transcription factors in dexa-induced cells. (A) HLE-B3 cells were pretreated with EI1 and A490 (10 μM) and cultured in medium containing dexa (0.1 mg / mL) for 4 h. The mRNA expression level of CSPG5 was analyzed by qRT-PCR (n = 7). (B) HLE-B3 cells were pretreated with EI1 (10 μM) and cultured in medium containing dexa (0.1 mg / mL) for 24 h. Representative Western blot images of EMT marker expression in transcription factor-suppressed HLE-B3 cells. Arrowheads indicate CSPG5. (C) Graph showing the relative quantification of protein expression levels of fibronectin, CSPG5, vimentin, and α-SMA (n = 3). (D) HLE-B3 cells were pretreated with A490 (10 μM) and then cultured in medium containing dexa (0.1 mg / mL) for 24 h. Representative Western blot images of EMT marker expression in transcription factor-suppressed HLE-B3 cells. (E) Graph showing the relative quantification of protein expression levels of fibronectin, CSPG5, vimentin, and α-SMA (n=3). (F) HLE-B3 cells were pretreated with EI1 (10 μM) and then cultured in medium containing dexa (0.1 mg / mL) for 8 h. The effect of EZH2 inhibition on dexa-induced cell migration was measured by a wound-healing assay. (G) Relative healing is expressed as the ratio of the initial wound area at 0 h compared to 8 h (n=3). (H) HLE-B3 cells were pretreated with A490 (10 μM) and then cultured in medium containing dexa (0.1 mg / mL) for 8 h. The inhibitory effect of B-MYB on dexa-induced cell migration was measured using a wound healing assay. (I) Relative healing rate is expressed as the ratio of the initial wound area at 0 h to that at 8 h (n = 3). Data are presented as mean ± SEM. p < 0.05 was the threshold for significance. EI1, EZH2 inhibitor.A490, a B-MYB inhibitor.

[0020] Figure 6. Effect of dexa on EMT markers in primary human lens epithelial cells. (A) Primary human lens epithelial cells were cultured in medium containing dexa (0.1 mg / mL) for 8 hours. Representative Western blot images of dexa-induced CSPG5 expression in primary human lens epithelial cells. Arrowheads indicate CSPG5. (B) Graph showing relative quantification of CSPG5 protein expression (n = 3). (C) Representative Western blot images of dexa-induced CSPG5 expression using chondroitinase. (D) Graph showing relative quantification of CSPG5 protein expression levels with chondroitinase (n = 6). (E) Representative Western blot images of dexa-induced EMT marker expression in primary human lens epithelial cells. Graph showing relative quantification of protein expression levels of (F) fibronectin and (G) α-SMA (n = 3). Bars represent mean ± SEM. p < 0.05 was the significance threshold.

[0021]

[0022] The present invention is described in detail below.

[0023]

[0024] The present invention relates to a pharmaceutical composition for preventing or treating steroid-induced cataracts.

[0025] Steroid-induced cataracts are cataracts associated with systemic or topical glucocorticoid steroids, abnormal migration of lenticule endothelial cells (LECs) from the lens equator to the posterior lens pole, and a restricted central area within the posterior subcapsular region. These steroids can be, for example, cortisol, hydrocortisone, prednisolone, methyltrednisolone, or dexamethasone.

[0026] The present inventors confirmed that the expression of CSPG5 is significantly increased in the lens epithelium of patients with steroid-induced cataract, and that downregulation of CSPG5 or inhibition of its transcription factor suppresses the expression and motility of steroid-induced epithelial-mesenchymal transition-related proteins in lens epithelial cells, thereby improving steroid-induced cataract. Accordingly, the pharmaceutical composition of the present invention comprises a substance that inhibits CSPG5 (Chondroitin Sulfate Proteoglycan 5) or its transcription factor.

[0027] A substance that inhibits CSPG5 (Chondroitin Sulfate Proteoglycan 5) can inhibit its mRNA or protein (e.g., activity, function, expression, level, etc.). This can be, for example, CSPG5 shRNA.

[0028] The transcription factor for CSPG5 may be, for example, EZH2 (Enhancer of zeste homolog 2) or B-myb (Myb-related protein B).

[0029] A substance that inhibits the transcription factor CSPG5 may inhibit (e.g., activity, function, expression, level, etc.) its mRNA or protein. This may be, for example, EI1 (CAS No. 1418308-27-6) or A490.

[0030] The composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier refers to a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, which transports the active ingredient from one organ or part of the body to another. Examples of the carrier include, but are not limited to, saline solution, buffered saline solution, water, glycerol, and ethanol, and any suitable formulation known in the art may be used.

[0031] The composition of the present invention can be administered orally or parenterally.

[0032] In the present invention, the term “administration” means introducing the pharmaceutical composition of the present invention to a subject of administration by any appropriate method.

[0033] Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration.

[0034] In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalers using methods known in the art.

[0035] In the case of preparations for oral administration, the composition of the present invention can be formulated into powders, granules, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. using methods known in the art. For example, oral preparations can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture to obtain a tablet or dragee. Examples of suitable excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; fillers such as gelatin and polyvinylpyrrolidone. Additionally, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrating agent, depending on the case. Furthermore, the composition of the present invention may additionally include an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier, a preservative, and the like.

[0036] The total daily dosage of the composition of the present invention can be determined by a treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a specific subject is preferably applied differently depending on various factors such as the type and degree of response to be achieved, the specific composition including whether other formulations are used in some cases, the age, weight, general health condition, sex, and diet of the subject, the time of administration, the route of administration, and the excretion rate of the composition, the treatment period, drugs used together or concurrently with the specific composition, and similar factors well known in the medical field. Therefore, the preferred dosage of the composition of the present invention can be determined in consideration of the above-mentioned matters and can be appropriately selected by a person skilled in the art.

[0037] The subject to which the composition of the present invention can be applied may be any animal that can develop such a disease, and the animals include not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs, and cats.

[0038]

[0039] The present invention will be described in more detail with reference to the following examples.

[0040]

[0041] Example

[0042] 1. Materials and Methods

[0043] 1.1. Participant Recruitment

[0044] The study was conducted by recruiting cataract patients who gave informed consent from the Department of Ophthalmology, Gyeongsang National University Hospital. The study was approved by the Gyeongsang National University Hospital Institutional Review Board (GNUH2019-05-014), and all procedures involving human data were performed in accordance with the current ethical standards of the institutional and national research committees. All procedures involving human materials were performed in accordance with the current ethical standards of the institutional and national research committees, the Declaration of Helsinki and its later amendments, or comparable ethical standards. The anterior capsules of cataract patients were collected during phacoemulsification by a single surgeon (SJK). Serial circular capsule separation was performed using forceps measuring 5.0–5.5 mm in diameter. The capsules were rinsed with balanced salt solution to remove viscoelastic and collected under sterile conditions.

[0045]

[0046] 1.2. Definition of cataract

[0047] An experienced ophthalmologist used the cataract classification system, LOCS III, to classify opacity into grading scales of increasing severity based on photographic criteria of six nuclear colors (NC1-NC6), six opalescent colors (NO1-NO6), five cortical colors (C1-C5), and posterior subcapsular colors (P1-P5). ARC was defined as a cataract occurring in an individual aged ≥40 years, with a LOCS III grading of NO2 and NC2 or greater, and P3 or less, and without the presence of diabetes mellitus, glaucoma, shallow anterior chamber, uveitis, high myopia (axial length <27.0 mm), pseudoexfoliation, traumatic cataract, subluxation cataract, previous ocular surgery, ocular disease (e.g., retinitis pigmentosa), or systemic / intraocular steroid use. SIC is defined as a cataract associated with systemic or topical glucocorticoid activity, abnormal migration of LECs from the lens equator to the posterior lens pole, and a restricted central area in the posterior subcapsular area, according to previous studies.

[0048]

[0049] 1.3. Human lens epithelial cell (LEC) culture

[0050] In the operating room, human anterior lens capsules were extracted into e-tubes or cell culture medium and washed with PBS for further analysis. For primary human LEC culture, the lens epithelial monolayer was peeled, small pieces were attached to the bottom of a 6-well plate, and the medium was changed once a week. This procedure was also approved by the Gyeongsang National University Hospital Institutional Review Board (GNUH2019-05-014).

[0051]

[0052] 1.4. Cell culture and processing

[0053] HLE-B3 cells, human corneal epithelial cells, and human adult retinal pigment epithelial (ARPE)-19 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained and grown to 80–90% confluency in MEM medium (Cat # 11095080; Thermo Fisher Scientific, Waltham, MA, USA). The medium contained 1% antimicrobial solution and 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA) and was used at 37°C and 5% CO2. Cells were treated with 0.1 mg / mL dexa over time, and additionally treated with 10 μM EI1 and A490 at the indicated time points.

[0054]

[0055] 1.5. Measurement of cell viability

[0056] 5 x 10 HLE-B3 cells 4Cells were cultured in 24-well plates at a density of 10 cells / well. After 24 h, the cells were cultured in media treated with dexamethasone (0.1 mg / mL) for 48 h, and cell viability was measured using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan). Cells were cultured in 10 L of CCK-8 solution in a humidified atmosphere at 37°C and 5% CO2 for 1 h. The amount of formazan dye produced by cellular dehydrogenase was determined by measuring the absorbance at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cells were observed using an inverted microscope (Olympus, Tokyo, Japan) to detect phenotypic differences between the substance-treated cells and the control group.

[0057]

[0058] 1.6. shRNA transfection

[0059] 4 x 10 HLE-B3 cells 6 Cells were seeded into 6-well plates at a density of 10 cells / well and cultured for 24 h. Cells were transfected with CSPG5 shRNA or control shRNA (SHCLNG-NM013884; Sigma-Aldrich) according to the manufacturer's instructions. The empty vector was transfected with Lipofectamine 3000, Opt. MEM TM Cells were transfected using a medium (Life Technologies, Carlsbad, CA, USA). Cells were cultured for an additional 24 h after transfection.

[0060]

[0061] 1.7. Wound healing analysis

[0062] HLE-B3 cells were seeded at 1 x 10 in 6-well plates. 6Cells were seeded at a density of 10 cells / well, and after 24 h, the monolayer was scraped with a sterile 200 μL pipette tip. The cells were treated with 0.1 mg / mL dexa. In some experiments, cells were co-treated with 10 μM EI1 and A490. After 8 h, cell migration was observed using an inverted microscope (Olympus).

[0063]

[0064] 1.8. RT 2 profiler PCR array

[0065] RT 2 First-strand cDNA was synthesized from 0.5 μg of each RNA sample according to the First Strand Kit (Qiagen, Hilden, Germany) manual. Samples were prepared using RT-PCR according to the device type. 2 SYBR ® The mixture was mixed with Green Master Mix and aliquoted into PCR array wells. After PCR, the relative expression level was analyzed using the △△Ct method.

[0066]

[0067] 1.9. Extraction of cytoplasmic and nuclear proteins

[0068] Nuclei and cytoplasm were extracted according to the manual of NE-PER Nuclear / Cytoplasmic Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA). After removing the medium, cells were washed with ice-cold phosphate-buffered saline (PBS). The cell pellet was resuspended in ice-cold CER I buffer and incubated on ice for 20 min. The cytoplasmic extract was isolated by centrifugation (13,600 × g, 10 min, 4°C). The pellet was washed with ice-cold PBS and resuspended in CER II containing protease inhibitors and phosphatase inhibitors. After incubation on ice for 30 min, the nuclear extract was isolated by centrifugation (13,600 × g, 20 min, 4°C). β-Actin (1:10,000, Sigma-Aldrich) was used as a loading control, α-tubulin (1:10,000, Sigma-Aldrich) as a cytoplasmic protein marker, and lamin A / C (1:1000, Santa Cruz, CA, USA) as a nuclear protein marker.

[0069]

[0070] 1.10. Immunofluorescence

[0071] Extracted human anterior lens capsules were flattened on poly-D-lysine-coated 24-well plates for 7 days. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and blocked with 5% normal donkey serum for 2 h. The cells were then incubated with anti-CSPG5 (1:500, Abcam, Cambridge, UK) at 4°C. Subsequently, the tissues were treated with donkey anti-rabbit immunoglobulin G secondary antibodies and detected under a fluorescence microscope (Olympus). Data were analyzed using Olympus FLUOVIEW Fv10 Asw 4.2 software (Olympus, Tokyo, Japan).

[0072]

[0073] 1.11. Quantitative real-time PCR

[0074] Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA), and 1 g of RNA was used for cDNA synthesis according to the iCycler thermocycler (Bio-Rad Laboratories, Hercules, CA, USA) manual. qRT-PCR was performed using the iQ SYBR Green Supermix kit (Bio-Rad Laboratories) and Light Cycler 480 II (Roche Life Science, Indianapolis, IN, USA). PCR primers were designed based on the cDNA sequences in the NCBI data bank (Table 1).

[0075]

[0076]

[0077]

[0078] 1.12. Western blot analysis

[0079] Cells were lysed in RIP Analysis buffer supplemented with Halt phosphatase inhibitor and Halt protease inhibitor (Thermo Fisher Scientific). The cell lysate was sonicated and centrifuged at 12,000 g for 10 min at 4°C to remove insoluble cell debris. The protein concentration of the cell lysate was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Whole cell lysates (15–20 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat dry milk, the blots were reacted with primary antibodies against CSPG5 (1:1000, Santa Cruz), fibronectin, vimentin, α-SMA, EZH2, and B-Myb (1:1000, Abcam), and β-actin (1:10,000, Sigma-Aldrich). Next, the membranes were reacted with HRP-conjugated anti-rabbit or anti-mouse antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA). Antibody-bound blots were detected using SuperSignal Chemiluminescent Substrate (Pierce). Images were captured with a ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA). Densitometry was performed using the ImageJ (NIH, Bethesda, MD, USA) program.

[0080]

[0081] 1.13. Chondroitinase ABC

[0082] Proteoglycan cell lysates (50 μg) were incubated on ice with 3 volumes of 95% ethanol and 1.3% potassium acetate solution. The lysates were centrifuged (10,000 × g, 4°C, 10 min). The precipitates were dissolved in Chondroitinase reaction buffer (Sigma-Aldrich) and treated with protease-free chondroitinase ABC (CHase ABC; 1 U / 50 μL, 1% BSA-supplemented PBS, Sigma-Aldrich). Proteins were first treated with CHase ABC and then reprecipitated with reaction buffer containing 3 volumes of ethanol and potassium acetate solution. The pellets were dissolved in 2x sample buffer and boiled (100°C, 3 min). After cooling to 4°C, the samples were analyzed by Western blotting (SDS-PAGE).

[0083]

[0084] 1.14. Statistical Analysis

[0085] All experimental data were analyzed using GraphPad Prism 6. Quantitative results are presented as ± the standard error of the mean (SEM) or standard deviation of the mean (SD) for at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test or one-way ANOVA followed by Tukey's multiple comparisons test. Statistical significance was determined as p <0.05.

[0086]

[0087] 2. Results

[0088] 2.1. Clinical characteristics of cataract patients

[0089] Anterior capsule samples were collected from 50 eyes of patients with ARC and 12 eyes of patients with SIC. There were no differences in sex, visual acuity, or intraocular pressure for sample acquisition. SIC patients were significantly younger than ARC patients. LOCS III showed a significantly higher cortical grade in ARC and a higher posterior subcapsular grade in SIC (Tables 2, 3, and 4).

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] 2.2. Dexa-induced upregulation of CSPG5 expression

[0098] To elucidate the effects of dexa, we analyzed changes in the expression of growth factor genes using the RT2 Profiler PCR array (Fig. 1A). Several genes, including SLCO1A2, FIGF, NODAL, IL12B, FGF11, INHA, and CSPG5, were upregulated by dexa treatment in HLE-B3 cells. In particular, CSPG5 expression showed a significant increase over 72 hours (Fig. 1B).

[0099] To analyze the intrinsic expression of CSPG5 in the absence of treatment, we compared protein levels in HLE-B3 and ARPE-19 cells with those in human anterior capsules of extracted primary human LECs. No significant differences were observed across the three cell types (Fig. 1C,D). Combined with Western blotting and qRT-PCR analyses, we found that CSPG5 protein (Fig. 1E,F) and mRNA (Fig. 1G) were significantly increased in the lens epithelium of SIC patients compared to that of ARC patients (Fig. 1F,G). These data suggest that CSPG5 is upregulated by dexa.

[0100]

[0101] 2.3. Effect of CSPG5 downregulation on Dexa-induced cell migration

[0102] To determine the appropriate dexa concentration for further experiments, cell viability was assessed at 0.1 mg / mL and 0.5 mg / mL dexa administration, and found to be unaffected for 48 hours. However, cell morphology was significantly altered (Fig. 2). To assess the function of CSPG5, HLE-B3 cells were transfected with CSPG5 shRNA to knockdown the protein. Knockdown did not affect HLE-B3 cell viability for 48 hours (Fig. 3A). However, dexa-induced F-actin expression was suppressed in CSPG5 knockdown HLE-B3 cells (Fig. 3B). Furthermore, dexa-induced wound healing was significantly inhibited (Fig. 3C). To further understand this, epithelial-mesenchymal transition (EMT) markers associated with cell migration were evaluated. The expression of fibronectin and CSPG5 was increased by dexa treatment, and this phenotype was suppressed by CSPG5 knockdown (Fig. 3D-H). These results demonstrate that CSPG5 is involved in cell migration.

[0103]

[0104] 2.4. Expression and translocation of transcription factors induced by Dexa in HLE-B3 cells

[0105] To identify the regulatory factors of CSPG5 in dexa-treated HLE-B3 cells, we focused on the transcriptional regulation of CSPG5. Given the known association between the transcription factors EZH2 and B-Myb in hepatocellular carcinoma, we analyzed the expression of these transcription factors. Dexa did not affect EZH2 mRNA expression, but increased B-Myb mRNA expression after 4 h of treatment and 24 h later (Fig. 4A-E). Comparative analysis of cytoplasmic and nuclear proteins revealed that dexa significantly increased the expression of EZH2 and B-Myb proteins in the nucleus (Fig. 4F-H). Immunofluorescence analysis further confirmed that the expression of EZH2 and B-Myb was significantly increased in the nucleus by dexa (Fig. 4I,J). These results suggest that the transcriptional regulators EZH2 and B-Myb are upregulated and translocated to the nucleus by dexa. Furthermore, dexa increased the protein expression of EZH2 and B-Myb.

[0106]

[0107] 2.5. Effect of CSPG5-related transcription factor downregulation on Dexa-induced cell migration

[0108] To elucidate the relationship between these transcription factors and CSPG5 expression, HLE-B3 cells were treated with the inhibitors EI1 and A490 to suppress EZH2 and B-Myb, respectively. Dexa-induced CSPG5 mRNA expression was suppressed by suppression of EZH2 and B-Myb (Fig. 5A). Dexa-induced fibronectin and CSPG5 protein expression was also suppressed by suppression of EZH2 (Fig. 5B,C). B-Myb suppression also exhibited a similar inhibitory effect (Fig. 5D,E). Furthermore, Dexa-induced wound healing was significantly suppressed by suppression of EZH2 and B-Myb (Fig. 5F-I). In summary, suppression of EZH2 and B-Myb inhibits Dexa-induced cell migration mediated by CSPG5 through regulation of CSPG5 gene expression.

[0109]

[0110] 2.6. Dexa-induced CSPG5 expression in primary human LECs

[0111] To confirm dexa-induced CSPG5 expression in human lens, primary human LECs were cultured with dexa. Dexa significantly increased CSPG5 protein expression in cells cultured for 8 h (Fig. 6A,B). Dexa also significantly increased CSPG5 protein expression using chondroitinase (Fig. 6C,D). Furthermore, dexa increased the protein expression of fibronectin and the EMT marker α-SMA in these cells (Fig. 6E-G). These results indicate that dexa induces CSPG5 expression and affects cell migration in primary human LECs.

Claims

1. A pharmaceutical composition for preventing or treating steroid-induced cataracts, comprising a substance that inhibits CSPG5 (Chondroitin Sulfate Proteoglycan 5) or its transcription factor.

2. A pharmaceutical composition for preventing or treating steroid-induced cataracts according to claim 1, wherein the transcription factor is EZH2 (Enhancer of zeste homolog 2) or B-myb (Myb-related protein B).

3. A pharmaceutical composition for preventing or treating steroid-induced cataracts, wherein the substance that inhibits CSPG5 in claim 1 is CSPG5 shRNA.

4. A pharmaceutical composition for preventing or treating steroid-induced cataract, wherein the substance inhibiting the transcription factor according to claim 1 is EI1 (CAS No. 1418308-27-6) or A490.

5. A pharmaceutical composition for preventing or treating steroid-induced cataract according to claim 1, wherein the steroid is cortisol, hydrocortisone, prednisolone, methyltrednisolone or dexamethasone.

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