4,4'-disulfanilyl-2,2'-stilbenedisulfonic acid disodium salt for use in the prevention, delay and / or treatment of blue light-induced retinal damage

Abstract

This invention discloses the application of disodium 4,4'-diisothiocyanate-2,2'-stilbene sulfonate in the preparation of drugs for the prevention, delay, and / or treatment of blue light-induced retinal damage. It belongs to the field of biomedical technology. This invention demonstrates for the first time that sodium DIDS can effectively protect photoreceptor cells from blue light damage: at the cellular level, sodium DIDS can restore mitochondrial membrane potential, increase ATP levels, reduce reactive oxygen species accumulation, and inhibit mitochondrial-dependent apoptosis; at the animal level, intravitreal injection of sodium DIDS can improve vision in mice exposed to blue light, restore the amplitude of a-waves and / or b-waves in electroretinograms, and protect the outer segment length and outer nuclear layer thickness of cone cells. Sodium DIDS provides a novel therapeutic strategy for blue light-induced retinal photodamage through multi-target synergistic protection of mitochondrial function.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the use of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate in the preparation of medicaments for the prevention, delay and / or treatment of blue light-induced retinal damage. Background Technology

[0002] The eye is a sophisticated organ capable of perceiving light. Its core function is to convert external light signals into neural electrical signals, which are then transmitted via the optic nerve to the brain's visual center, ultimately forming vision. The retina, as the starting point of this process, is a highly ordered neural tissue located in the inner layer of the eyeball wall. Its neural circuits are composed of five main types of neurons: photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, and ganglion cells. These neurons work together through a delicate synaptic network to receive, process, and transmit light signals.

[0003] In recent years, with the widespread use of digital devices (such as smartphones, computers, and televisions) and LED lighting, the duration and intensity of human exposure to high-energy blue light have increased significantly, raising serious public concerns about potential retinal damage. Blue light, especially blue light with wavelengths in the 435–445 nm band, can penetrate the refractive media of the eye and reach the retina due to its short wavelength and high energy. It is efficiently absorbed by chromophores in retinal pigment epithelial cells and photoreceptor cells, thereby inducing photochemical reactions. Prolonged or high-intensity exposure to blue light can lead to photochemical damage to the retina, with the main pathological features being: inducing excessive production of reactive oxygen species, triggering oxidative stress; subsequently triggering mitochondrial dysfunction, endoplasmic reticulum stress, and inflammatory responses; ultimately leading to photoreceptor cell degeneration and death, resulting in irreversible vision loss.

[0004] In-depth research has shown that mitochondrial dysfunction is the core mechanism of photoreceptor cell degeneration in blue light injury. Mitochondria are particularly abundant in photoreceptors, which have extremely high energy demands. Blue light exposure directly damages the structure and function of mitochondria in photoreceptor cells, leading to a decrease in mitochondrial membrane potential, depletion of adenosine triphosphate (ATP), and a large accumulation of reactive oxygen species, triggering abnormal opening of the mitochondrial permeability transition pore (mPTP), ultimately activating the mitochondrial apoptosis pathway. Voltage-dependent anion channels (VDACs), porins located on the outer mitochondrial membrane and an important component of mPTPs, undergo abnormal oligomerization under stress, leading to increased permeability of the outer mitochondrial membrane, promoting the release of apoptosis factors such as cytochrome c, thereby activating the apoptosis cascade. In models of diabetic retinopathy and ischemia-reperfusion injury, inhibition of VDAC1 oligomerization has shown neuroprotective effects.

[0005] Disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate is the sodium salt of DIDS, a small organic molecule compound known as a chloride channel blocker and capable of inhibiting the abnormal oligomerization of VDAC1. However, whether DIDS and its sodium salt can combat blue light-induced photoreceptor cell damage has not yet been reported.

[0006] Therefore, how to develop new drugs that can treat blue light-induced retinal damage is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned problems existing in the prior art and provides the use of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate in the preparation of medicaments for the prevention, delay and / or treatment of blue light-induced retinal damage.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The primary objective of this application is to provide the use of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate in the preparation of medicaments for the prevention, delay, and / or treatment of blue light-induced retinal damage.

[0010] As a preferred technical solution, the disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate exerts a therapeutic effect by protecting photoreceptor cells damaged by blue light and blue light-induced retinal damage.

[0011] As a more preferred technical solution, the protection of photoreceptor cells damaged by blue light is achieved through one or more of the following methods:

[0012] 1) Restore the mitochondrial membrane potential of photoreceptor cells;

[0013] 2) Increases the level of adenosine triphosphate (ATP) in photoreceptor cells;

[0014] 3) Reduce the accumulation of reactive oxygen species in photoreceptor cells;

[0015] 4) Inhibits the activation of mitochondrial-dependent apoptosis pathways.

[0016] As a more preferred technical solution, the disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate achieves protection against blue light-induced retinal damage through one or more of the following methods:

[0017] 1) Protect the visual function of animals exposed to blue light, including improving vision and / or restoring the amplitude of a-waves and / or b-waves in electroretinograms;

[0018] 2) Protect the retinal structures of animals exposed to blue light, including protecting the length and / or number of outer segments of cone cells, and / or maintaining the thickness of the outer nuclear layer of the retina.

[0019] As a preferred technical solution, the dosage form of the drug is an ophthalmologically acceptable dosage form, including eye drops, ophthalmic gels, ophthalmic ointments, microemulsions, liposomes, nanoparticles, ophthalmic inserts, or intravitreal injections.

[0020] As a preferred technical solution, the drug is administered via intravitreal injection, subconjunctival injection, eye drops, or ophthalmic application.

[0021] Another object of this application is to provide a pharmaceutical composition for preventing, delaying or treating blue light-induced retinal damage, comprising a therapeutically effective amount of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate and one or more ophthalmologically acceptable excipients.

[0022] Another object of this application is to provide a method for protecting photoreceptor cells from blue light damage for non-therapeutic purposes, comprising the step of contacting the photoreceptor cells with an effective concentration of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate.

[0023] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) Clear mechanism of action, targeting the core link of mitochondrial dysfunction: This invention reveals for the first time that sodium DIDS exerts a retinal protective effect by regulating mitochondrial function through multiple targets. Specifically, it includes: (a) inhibiting the upregulation of VDAC expression and maintaining the integrity of the outer mitochondrial membrane; (b) protecting the mitochondrial membrane potential; (c) improving cellular energy metabolism and counteracting blue light-induced ATP depletion; and (d) reducing oxidative stress and lowering reactive oxygen species levels. This multi-link, multi-target synergistic protective network makes the protective effect more comprehensive and lasting.

[0025] (2) It fills a technological gap in a specific application area: In the prior art, DIDS has only been reported for use in models such as diabetic retinopathy and ischemia-reperfusion injury. This invention is the first to extend the application of sodium salt of DIDS to the field of blue light-induced photoreceptor cell damage, filling this technological gap and providing a new candidate drug for these diseases for which there are currently no effective treatments.

[0026] (3) Significant advantages compared to existing protection strategies: Currently, the main methods to mitigate blue light-induced retinal damage include supplementing with antioxidants (such as vitamin C, vitamin E, lutein, etc.). Compared with these existing technologies, the present invention has the following advantages: (a) Direct administration without the need for complex nanocarrier encapsulation; (b) As a small molecule compound, it has good cell membrane penetration and rapid onset of action; (c) Multi-target synergistic protection, rather than single-target scavenging of reactive oxygen species, resulting in better protective effects; (d) The pharmacological characteristics of DIDS sodium salt are well-defined, with low drug-likeness risk, which is conducive to accelerating clinical translation.

[0027] (4) Sufficient experimental evidence and in vivo and in vitro model verification: This invention not only confirmed the mitochondrial protective effect of sodium DIDS in the 661W photoreceptor cell line, but also fully confirmed the significant protective effect of sodium DIDS on visual function and retinal structure in vivo in the classic blue light exposure mouse model through multi-dimensional indicators such as visual behavior (optic motor response), electroretinography, and histology (outer nuclear layer thickness, cone cell outer segment length).

[0028] In summary, this invention effectively reduces blue light-induced photoreceptor cell damage by protecting mitochondrial function and mitigating oxidative stress, exhibiting significant advantages such as a clear mechanism, multi-target synergy, convenient administration, and good drug-like properties. This provides a new technical solution and a solid theoretical foundation for developing protective agents against blue light-induced retinal damage and other oxidative retinal diseases. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the experimental design route for this invention.

[0031] Figure 2 This study investigated blue light-induced photoreceptor damage and mitochondrial dysfunction. The findings included: A. Morphological images of 661W cells after different durations of blue light exposure; B. Cell viability assay using the CCK-8 assay; C. ATP concentration detection; D. TMRE staining and fluorescence imaging of mitochondrial membrane potential; and E. Quantitative analysis of mean TMRE fluorescence intensity. ATP concentration and TMRE intensity were normalized to the control group mean. Data are expressed as mean ± standard error; * p < 0.05; ** p < 0.01; *** p < 0.001. n = 3–9.

[0032] Figure 3 This study aimed to investigate the upregulation of apoptosis proteins in 661W cells after different durations of blue light exposure. A. Western blot analysis of the expression levels of Cleaved Caspase-3 (CC3), Bax, Bcl-2, Cleaved Caspase-9 (CC9), and the internal control Tublin. B. Statistical analysis of the quantified protein bands of Bax, Bcl-2, Cleaved Caspase-3, and Cleaved Caspase-9. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; n = 3.

[0033] Figure 4 The study aimed to investigate the upregulation of VDAC protein expression in 661W cells after blue light exposure. A. Western blot analysis of VDAC1, VDAC2, and the internal control Tublin expression levels at different time points after blue light irradiation; B. Statistical analysis of VDAC1 and VDAC2 protein band quantification in each group; C. Fluorescence images of the control and blue light exposure groups. CS, citrate synthase. *** p<0.001 vs. control; n=3.

[0034] Figure 5 To assess the effectiveness of sodium DIDS in rescuing blue light-induced photoreceptor cell damage, the study included: A. cytotoxicity assay of sodium DIDS; B. effect of sodium DIDS on the survival rate of blue light-damaged cells; C. images of 661W cells stained with TMRE (top) or DHE (bottom) in the control group, blue light exposure group, and blue light exposure combined with 300 μM sodium DIDS treatment group; D. quantification of mean fluorescence intensity by TMRE; E. quantification of mean fluorescence intensity by DHE; and F. quantification of ATP concentration. All values ​​were normalized relative to the control group. Data are expressed as mean ± standard error; * p < 0.05; ** p < 0.01; *** p < 0.001, compared with the control group or the group connected by the line.

[0035] Figure 6 To reduce the expression of apoptosis proteins in 661W cells after blue light damage using sodium DIDS; A. Western blot analysis was used to detect changes in the expression levels of Bax, Bcl-2, CC3, CC9, and the internal control Tublin; B. Statistical results were obtained after quantifying the protein bands of Bax, Bcl-2, CC3, and CC9 in each group. *** p<0.001; n=3.

[0036] Figure 7Administration of DIDS sodium salt inhibited the increased expression of CS, VDAC1, and VDAC2 in photoreceptor cells.

[0037] Figure 8 To illustrate the protective effect of sodium DIDS on the retinal structure and function of mice against blue light damage, the study included: A. Schematic diagram of the experimental procedure for CD1 mice receiving intravitreal injection of DIDS before blue light exposure; B. Schematic diagram of the optokinetic response system (left) and visual acuity measured by the system (right); C. Example electroretinogram waveforms of mice in different treatment groups responding to flash stimuli of different intensities under dark adaptation conditions; D. Mean amplitudes of a wave (left) and b wave (right); E. Images of retinal sections stained with opsin (labeled extracellular cone cell layer) and DAPI; FG. Length (F) and number (G) of extracellular cone cell layer (opsin-labeled) in each group of mice; H, I. Mean thickness of the outer nuclear layer across the entire region (H) and thickness of the outer nuclear layer at different centrifugal positions along the retinal section (I). * p<0.05; ** p<0.01; *** p<0.001. Detailed Implementation

[0038] 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 some embodiments of the present invention, and not all embodiments. 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.

[0039] Example 1

[0040] Establishment of a cell culture and blue light damage model ( Figure 1 )

[0041] This embodiment is used to verify the damaging effects of blue light exposure on photoreceptor cells and the protective effect of sodium DIDS.

[0042] Cell line and culture conditions: The 661W photoreceptor cell line was kindly provided by Dr. Jing Zhuang (Zhongshan Ophthalmic Center, Sun Yat-sen University). Cells were cultured in a humidified incubator at 37°C with 5% CO2, using DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. When the cells reached 80%-90% confluence, they were digested with 0.25% trypsin-EDTA, centrifuged at 900 rpm for 5 minutes, and seeded at an appropriate density for subsequent experiments.

[0043] Establishment of the blue light damage model: After sterilizing the LED array lamp panel (spectral range 450–480 nm), it was placed in a CO2 incubator. The light intensity at the cell plane was measured using an illuminometer and adjusted to 5000 ± 200 Lux. After the cells were seeded and cultured to approximately 70% confluence, the old culture medium was discarded, and fresh culture medium with or without sodium DIDS was added. The cells were then placed under the LED lamp panel for blue light exposure for specified durations (0, 3, 6, 12, and 24 hours).

[0044] Experimental grouping: The in vitro experiment was divided into three groups: (1) Control group: normal culture, no blue light exposure, no drug treatment; (2) Blue light group: blue light exposure, no drug treatment; (3) DIDS group: pretreated with sodium DIDS (4,4'-diisothiocyanate-2,2'-stilbenesulfonate disodium salt, CAS No.: 67483-13-0) for 1 hour before blue light exposure (sodium DIDS was present throughout the blue light exposure).

[0045] Results analysis:

[0046] 1) Time-dependent damage to photoreceptor cells caused by blue light exposure

[0047] Exposure of 661W cells to blue light (5000 Lux) resulted in progressive morphological degeneration and loss of viability. During blue light exposure of 3 to 6 hours, cells maintained normal spindle morphology and firm adherence; however, prolonged exposure (12 to 24 hours) induced cell swelling, cytoplasmic shrinkage, and significant cell detachment. Figure 2 (A in the middle).

[0048] Cell viability was assessed using a CCK-8 assay kit. Cells were cultured at 1 × 10⁶ cells / year. 5 Cells were seeded at a density of 250 μL / well in 48-well plates and incubated overnight at 37°C. After treatment, cells were washed 2-3 times with preheated HBSS, then 250 μL of DMEM containing 25 μL CCK-8 reagent was added, and the cells were incubated at 37°C for 30 minutes. Absorbance was measured at 450 / 630 nm using a microplate reader. Comparison with the control group was performed using one-way ANOVA followed by Dunnett's post-hoc test.

[0049] The formula for calculating cell viability is: Viability (%) = [(OD experimental group − OD blank) / (OD control group − OD blank)] × 100%.

[0050] The CCK-8 assay results were consistent with this: compared with the control group, cell viability did not decrease significantly after 3 to 6 hours of exposure, but viability began to decline after 12 hours of exposure, and decreased significantly by 24 hours. Figure 2 (B in the middle).

[0051] To reveal the effects of blue light on mitochondrial function, this study examined ATP levels and mitochondrial membrane potential in cells after different blue light exposure durations, as detailed below:

[0052] Intracellular ATP assay: Intracellular ATP levels were quantified using a luciferin-luciferase-based ATP assay kit. Cells were lysed with ATP lysis buffer (1 / 10 the volume of the culture medium) and centrifuged at 12,000 × g for 5 minutes at 4°C. ATP standards were serially diluted with lysis buffer to 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM. The ATP assay working solution was prepared by mixing the ATP assay reagent and diluent at a 1:9 ratio. For assays, 100 μL of the working solution was added to each well and incubated at room temperature for 3–5 minutes to quench background luminescence. Then, 20 μL of sample or standard was quickly added, mixed thoroughly, and the luminescence value was recorded using a microplate reader. ATP concentration was calculated using a standard curve.

[0053] Mitochondrial membrane potential assessment: Mitochondrial membrane potential was assessed using the TMRE fluorescent probe. Preparation of TMRE working solution immediately before use: Dilute 1000× TMRE stock solution 1:1000 with assay buffer and store protected from light. Wash cells 2-3 times with HBSS and incubate with TMRE working solution (half the original culture medium volume) at 37°C for 30 minutes. After washing twice with preheated culture medium, images were taken using a fluorescence microscope (excitation wavelength: 550 nm; emission wavelength: 575 nm), and fluorescence intensity was quantified using ImageJ software. Comparison with the control group was performed using one-way ANOVA followed by Dunnett's post-hoc test.

[0054] Results analysis: Blue light exposure led to a time-dependent decrease in ATP levels, with significant reductions observed at 6, 12, and 24 hours post-exposure. Figure 2 (C in the text). Meanwhile, TMRE staining showed that mitochondrial membrane potential progressively decreased from 6 hours post-exposure and continued to worsen until 24 hours (…). Figure 2 (D and E in the text). In summary, these data indicate that blue light can induce progressive photoreceptor damage characterized by mitochondrial dysfunction.

[0055] 2) Blue light irradiation promotes photoreceptor cell apoptosis.

[0056] Protein extraction and immunoblotting analysis: For cell samples, pretreatment with complete culture medium containing 250 μM EGS at 37°C for 15 minutes before lysis was performed. Protein samples were quantified using the BCA method and then separated by SDS-polyacrylamide gel electrophoresis. The stacking gel concentration was 5%, and the separating gel concentration was 12%. Electrophoresis conditions were constant voltage 80 V (30 minutes) followed by 120 V (90 minutes). Proteins were then transferred to methanol-activated PVDF membranes under constant current of 300 mA for 0.5–1.5 hours, depending on the target protein molecular weight. After transfer, the membranes were blocked with 5% skim milk at room temperature for 2 hours, followed by overnight incubation with the corresponding primary antibody at 4°C. The next day, after washing, the membranes were incubated with HRP-labeled secondary antibody at room temperature for 2 hours. After chemiluminescence staining, the bands were analyzed for grayscale values ​​using ImageJ software.

[0057] The changes in apoptosis factors in 661W cells after blue light irradiation were detected using the Western blotting method described above. The results showed that after blue light irradiation for different durations, the expression levels of Bax / Bcl-2, Cleaved Caspase-3, and Cleaved Caspase-9 in 661W cells were significantly higher than those in the normal control group (e.g., ...). Figure 3 This suggests that blue light irradiation may activate apoptosis signaling pathways, leading to apoptosis in 661W cells. This change is closely related to the duration of blue light irradiation, exhibiting a time-dependent alteration, with Bax / Bcl 2, Cleaved Caspase 3, and Cleaved Caspase 9 showing a time-dependent increase.

[0058] 3) Blue light damage increases the expression of VDAC in photoreceptor cells.

[0059] The expression changes of VDAC1 and VDAC2 in photoreceptor cells after blue light damage were detected using the aforementioned Western blotting method. The results showed that the expression level of VDAC1 after 24 hours of blue light irradiation tended to be upregulated compared to the control group, while the expression level of VDAC2 increased with the extension of blue light irradiation time, and the expression level significantly increased at 24 hours (e.g., ...). Figure 4 As shown in A and B in the diagram, blue light irradiation may increase the expression levels of VDAC1 and VDAC2, and this change is closely related to the duration of blue light irradiation, showing a time-dependent increase.

[0060] We simultaneously observed changes in mitochondrial morphology and VDAC using CS (mitochondrial marker) and VDAC antibody staining. Figure 4 (C in the text). The results showed that after blue light irradiation, cells locally brightened and shrank. The fluorescence intensity of CS, VDAC1, and VDAC2 increased significantly, possibly indicating increased VDAC expression and mitochondrial shrinkage.

[0061] Example 2

[0062] Treatment with sodium DIDS can rescue photoreceptor cells damaged by blue light.

[0063] 1) Sodium DIDS treatment can salvage blue light-induced photoreceptor cell damage.

[0064] To investigate the protective potential of sodium DIDS against blue light-induced photoreceptor damage, its cytotoxic characteristics in 661W cells were first determined. Concentrations of sodium DIDS exceeding 300 μM exhibited cytotoxic effects after 24 hours of culture; therefore, 300 μM was determined as the maximum safe concentration. Figure 5 (A in the text). Subsequent dose-response experiments showed that 300 μM sodium DIDS significantly improved the viability of cells exposed to blue light. Based on this, 300 μM was used as the treatment concentration in subsequent experiments. Figure 5 (B in the middle).

[0065] Subsequently, it was verified that sodium DIDS improved blue light-induced mitochondrial dysfunction and oxidative stress.

[0066] Intracellular reactive oxygen species (ROS) detection: Intracellular ROS levels were quantitatively detected using a dihydroetylum fluorescent probe. After blue light exposure, cells were washed 2-3 times with HBSS and incubated with 5 μM dihydroetylum at 37°C for 30 minutes. After washing, images were acquired using an inverted fluorescence microscope, and fluorescence intensity was quantified using ImageJ software.

[0067] Results analysis: Sodium DIDS treatment maintained mitochondrial membrane potential, and its TMRE fluorescence intensity was higher than that of the blue light exposure group without sodium DIDS treatment. Figure 5 (C and D in the text). Simultaneously, sodium DIDS treatment significantly reduced the accumulation of intracellular reactive oxygen species, as evidenced by a significant decrease in ethidium dihydrogen fluorescein fluorescence intensity compared to the untreated blue light exposure group (C and D in the text). Figure 5 (C and E in the text). Consistent with this, after blue light exposure, the intracellular ATP level in the DIDS sodium salt treatment group was significantly higher than that in the untreated control group (C and E in the text). Figure 5 (F in the text). In summary, these results indicate that sodium DIDS effectively protects photoreceptor cells from blue light-induced damage by inhibiting oxidative stress and protecting mitochondrial function.

[0068] 2) Sodium DIDS reduces the expression of apoptosis factors in photoreceptor cells after blue light damage.

[0069] The changes in apoptosis factors in 661W cells after DIDS sodium administration were detected. Results showed that the expression levels of Bax / Bcl2 and Cleaved Caspase 9 (CC9) in 661W cells were significantly lower than those in the BL group (e.g., ...). Figure 6 The expression level of Cleaved Caspase 3 (CC3) showed a trend of being lower than that in the BL group. This suggests that sodium DIDS may inhibit the apoptosis signaling pathway, thereby suppressing apoptosis in 661W cells.

[0070] 3) DIDS sodium salt administration inhibits the increase of VDAC expression in photoreceptor cells.

[0071] After treatment with sodium DIDS, 661W cells showed decreased fluorescence intensity of VDAC 1, VDAC 2, and CS 24 hours after blue light exposure compared to the BL group, indicating reduced VDAC expression and mitochondrial shrinkage. Figure 7 ).

[0072] Example 3

[0073] Sodium DIDS has a protective effect against blue light-induced retinal damage in vivo.

[0074] After confirming the protective effect of DIDS sodium at the cellular level, this study further evaluated its in vivo therapeutic effect using albino CD1 mice, as detailed below:

[0075] Ten-week-old male Balb / c (CD1) mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with 3-5 mice per cage. Housing conditions were controlled (23±2℃, 40%-60% humidity, 12-hour light / dark cycle), and mice had free access to food and water. All experimental procedures were approved by the Laboratory Animal Management and Use Committee of Jinan University (Approval No.: IACUC-20241018-06, Guangzhou, China). Animals were randomly divided into three groups: a control group without blue light exposure, a blue light exposure group treated with PBS, and a blue light exposure group treated with sodium DIDS.

[0076] To induce blue light damage, mice were exposed to blue light for 6 consecutive days. Figure 8 (A) Daily, mice were anesthetized with 1.25% tribromoethanol (0.2 mL / 10 g, intraperitoneal injection; Merck, Germany) and mydriatic with compound tropicamide eye drops. Both eyes were exposed to a blue LED array (450–500 nm, 20,000 Lux) for 1 hour, followed by application of carbomer ophthalmic gel to prevent dry eye. Mice were then placed in a light chamber topped with a blue LED array (10,000 Lux) and lined with aluminum foil for uniform illumination (11 hours of light / 12 hours of darkness daily). Control group mice were maintained under standard indoor lighting conditions.

[0077] For DIDS sodium treatment, mice were anesthetized and mydriatic one day before blue light exposure, and then injected intravitreally with 1.2 μL DIDS sodium (1 mM, dissolved in PBS) or PBS. After six days of blue light exposure, mice underwent optomotor response testing, followed by overnight dark adaptation and electroretinography (ERG). Mice were then sacrificed and tissues were collected. The specific testing methods are as follows:

[0078] Visual response test: Mouse vision was assessed using an optomotor response system. Evaluation was performed by monitoring the mouse's head rotation response to moving gratings. The mouse was placed on a centrally elevated platform surrounded by computer screens displaying vertically rotating sinusoidal gratings. These gratings were programmed using MATLAB with 100% contrast, a movement speed of 12 cycles / second, and spatial frequencies gradually increasing from 0.1 cycles / degree to 0.6 cycles / degree. When the animal could see the gratings, it instinctively tracked them with its head (optomotor reflex). Head movements were recorded video, and the maximum spatial frequency that elicited the optomotor response was manually recorded as an indicator of the mouse's vision.

[0079] Electroretinography (ERG) recording: Mice underwent overnight dark adaptation, followed by anesthesia and mydriasis under dark red light. Corneal electrodes, a reference electrode, and a ground wire were then placed. Mice were placed in the spherical stimulator of the Rowland retinal recording system and received green full-field flash stimulation of increasing intensity (0.01, 0.1, and 3.0 cd·s / m²). Dark-adapted retinal light responses were recorded. The amplitudes of the a and b waves were measured. The best response from both eyes was recorded as a data point for each animal.

[0080] Results analysis: Visuomotor response test results showed that, compared with the untreated control group, the visual acuity of mice in the DIDS sodium salt treatment group with blue light damage tended to improve. Figure 8 (B in the text). Electroretinography analysis further showed that after administration of DIDS sodium salt, wave a was significantly restored at all tested flash intensities, while wave b amplitude was also increased, indicating improved function of photoreceptors and neurons in the inner retinal layer, respectively. Figure 8 (C and D in the text).

[0081] To assess structural changes, retinal sections were immunostained with the anti-opsin antibody Opsin to visualize the outer segments of cone cells, as detailed below:

[0082] Immunofluorescence staining of retinal tissue: To examine retinal structure, after fixation and embedding in glycosylated OCT, the eyeballs were sectioned longitudinally along the optic disc to a thickness of 16 μm. The eyeball sections were then incubated overnight at 4°C with rabbit anti-opsin red / green antibody in blocking solution (0.3% PBST containing 10% donkey serum albumin and Triton X-100). After primary antibody incubation, the sections were incubated with Alexa Fluor® 488-labeled donkey anti-rabbit secondary antibody at room temperature for 2 hours. DAPI staining was performed for 10 minutes, followed by washing three times with PBS for 5 minutes each time. The retinal samples were then washed, mounted, and sealed with coverslips.

[0083] Image Acquisition and Processing: For retinal tissue, fluorescence images of the retina were acquired using fluorescence microscopy or confocal microscopy. At least three full-length retinal sections were evaluated for each animal to obtain the average expression pattern of the retina for each animal. Images at distances of 500, 1000, and 1500 μm from the center of the optic disc were selected as samples for the central, mid-peripheral, and peripheral zones, respectively. To measure the thickness of the outer nuclear layer of the retina or the length of the outer segments of opsin-stained cone cells, lines were drawn perpendicular to each retinal layer or along the outer segments of the cone cells, and the line lengths were measured using ImageJ. Three to six images were acquired for each retina of each animal, with an image size of 320 × 320 μm. The values ​​were then averaged to obtain a data point for the animal, which is displayed as a single dot in the results image.

[0084] Quantitative analysis showed that, after blue light damage, DIDS sodium treatment significantly restored the length and number of outer segments of cone cells. Figure 8 E, F, and G in the text). Furthermore, DAPI staining showed that, compared to untreated blue light-exposed mice, DIDS sodium-treated mice exhibited significantly preserved outer nuclear layer thickness in the central, peripheral, and overall retinal regions. Figure 8 (H and I in). The above results indicate that sodium DIDS can enhance the survival ability of photoreceptors after blue light exposure.

[0085] In summary, these in vivo experimental results demonstrate that sodium DIDS treatment effectively reduces blue light-induced retinal damage by protecting visual function and the integrity of photoreceptors.

[0086] The various embodiments in the specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0087] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. 4,4'-Diisothiocyanate-2,2'-stilbenesulfonate disodium salt in the preparation of medicaments for the prevention, delay and / or treatment of blue light-induced retinal damage.

2. The application according to claim 1, characterized in that, The sodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate exerts its therapeutic effect by protecting photoreceptor cells damaged by blue light and blue light-induced retinal damage.

3. The application according to claim 2, characterized in that, The protection of photoreceptor cells from blue light damage can be achieved through one or more of the following methods: 1) Restore the mitochondrial membrane potential of photoreceptor cells; 2) Increases the level of adenosine triphosphate (ATP) in photoreceptor cells; 3) Reduce the accumulation of reactive oxygen species in photoreceptor cells; 4) Inhibits the activation of mitochondrial-dependent apoptosis pathways.

4. The application according to claim 2, characterized in that, The sodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate achieves protection against blue light-induced retinal damage through one or more of the following mechanisms: 1) Protect the visual function of animals exposed to blue light, including improving vision and / or restoring the amplitude of a-waves and / or b-waves in electroretinograms; 2) Protect the retinal structures of animals exposed to blue light, including protecting the length and / or number of outer segments of cone cells, and / or maintaining the thickness of the outer nuclear layer of the retina.

5. The application according to claim 1, characterized in that, The dosage form of the drug is ophthalmologically acceptable, including eye drops, ophthalmic gels, ophthalmic ointments, microemulsions, liposomes, nanoparticles, ophthalmic inserts, or intravitreal injections.

6. The application according to claim 5, characterized in that, The drug can be administered via intravitreal injection, subconjunctival injection, eye drops, or topical application to the eye.

7. A pharmaceutical composition for preventing, delaying, or treating blue light-induced retinal damage, characterized in that, It contains a therapeutically effective amount of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate and one or more ophthalmologically acceptable excipients.

8. A method for protecting photoreceptor cells from blue light damage for non-therapeutic purposes, characterized in that, The method includes the step of contacting photoreceptor cells with an effective concentration of disodium 4,4'-diisothiocyanate-2,2'-stilbenesulfonate.

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