Application of Cus3 in preparation of medicine for preventing and / or treating retina degenerative diseases

Abstract

The invention provides application of Cus3 in preparation of drugs for preventing and / or treating retina degenerative diseases, and particularly belongs to the technical field of drugs for retinopathy. The invention provides application of Cus3 in preparation of a medicine for preventing and / or treating retina degenerative diseases. The invention finds that the compound Cus3 has a potential treatment effect on retinal injury, and a new strategy can be provided for research and development of drugs for treating retinal degenerative diseases. Specifically, the Cus3 can significantly improve the phenotype of the ommatidium of the visually impaired eye, improve the light sensitivity of the visually impaired eye, repair the retinal cell layer structure of the visually impaired eye, and realize the treatment of the retinal degenerative disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of retinal disease drugs, and particularly relates to the use of Cus 3 in preparing drugs for preventing and / or treating retinal degenerative diseases. Background Art

[0002] Retinal degenerative diseases, such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are among the leading causes of irreversible vision loss worldwide. Currently, clinical treatment options are extremely limited, only delaying disease progression in some patients but failing to effectively reverse retinal damage. Therefore, the development of novel drugs is urgently needed to provide new strategies for the research and development of drugs for retinal degenerative diseases. Summary of the Invention

[0003] The present invention aims to provide the use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal degenerative diseases. The present invention discovers that the compound Cus 3 has therapeutic effects on retinal degenerative diseases, providing a new strategy for the development of drugs for retinal degenerative diseases.

[0004] The present invention provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal degenerative diseases. The structural formula of Cus 3 is shown in Formula I:

[0005]

[0006] The present invention also provides the use of Cus 3 in the preparation of a drug having a visual protection effect. The structural formula of the Cus 3 is shown in Formula I:

[0007]

[0008] The present invention also provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating visual impairment. The structural formula of the Cus 3 is shown in Formula I:

[0009]

[0010] The present invention also provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal damage. The structural formula of the Cus 3 is shown in Formula I:

[0011]

[0012] The present invention also provides the use of Cus 3 in the preparation of a drug for improving the morphology of eyes with visual impairment. The structural formula of Cus 3 is shown in Formula I:

[0013]

[0014] Preferably, improving the morphology of the visually impaired eye comprises increasing the horizontal axis distance and / or the vertical axis distance of the visually impaired eye to a normal level.

[0015] The present invention also provides the use of Cus 3 in the preparation of a drug for improving the sensitivity of visually impaired eyes to light. The structural formula of the Cus 3 is shown in Formula I:

[0016]

[0017] The present invention also provides the use of Cus 3 in the preparation of a drug for repairing the retinal cell layer structure of visually damaged eyes. The structural formula of the Cus 3 is shown in Formula I:

[0018]

[0019] Preferably, repairing the structure of the retinal cell layer of the visually damaged eye includes increasing the thickness of the retinal cell layer of the visually damaged eye.

[0020] Preferably, the retinal cell layer includes any one or more of a pigment epithelial cell layer, an inner nuclear layer, an outer nuclear layer, and a ganglion cell layer.

[0021] The present invention provides the use of Cus 3 in the preparation of drugs for preventing and / or treating retinal degenerative diseases. The present invention uses a zebrafish retinal damage model induced by myclobutanil and finds that the compound Cus3 has a potential therapeutic effect on retinal damage, which can provide a new strategy for the development of drugs for retinal degenerative diseases. Specifically, Cus 3 can significantly improve the small eye phenotype of visually damaged eyes, increase the sensitivity of visually damaged eyes to light, repair the retinal cell layer structure of visually damaged eyes, and achieve the treatment of retinal degenerative diseases. The experimental results show that Cus 3 intervention can significantly improve the small eye phenotype of visually damaged zebrafish, and restore the transverse and longitudinal axis diameters of the eyeball to normal levels. At the behavioral level, Cus 3 significantly improved the swimming distance and movement rate of zebrafish in the monochromatic light stimulation escape experiment, restoring them to normal levels. Histological analysis further confirmed that Cus 3 can effectively repair the retinal cell layer structure of visually damaged zebrafish. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a graph showing the effect of the monomer Cus 3 provided by the present invention on the eye morphology of visually impaired zebrafish; wherein, A: a typical image of the eye; B: a graph showing the body length and eye data results;

[0024] Figure 2 The graphs of the effects of Cus 3 on the behavior of visually impaired zebrafish provided by the present invention; wherein, A: graph of movement distance and speed results; B: graph of movement trajectory;

[0025] Figure 3 Figure 3 shows the effect of the monomer Cus 3 provided by the present invention on the morphology and thickness of the retina in visually impaired zebrafish; A: H&E staining of eye sections; B: Thickness of the pigment epithelium, outer nuclear layer, inner nuclear layer, and ganglion cell layer; C: Thickness of the retina and lens. DETAILED DESCRIPTION

[0026] The present invention provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal degenerative diseases. The structural formula of Cus 3 is shown in Formula I:

[0027]

[0028] Cus 3 monomer is a known natural product isolated from Convolvulaceae plants, and its molecular formula is C 37 H 66 O 18 . The embodiment of the present invention uses pyraclostrobin to construct a zebrafish retinal degenerative disease model. The experimental results show that the zebrafish in the model group exhibited a significant small eye-like phenotype, and this phenotypic change is highly consistent with the characteristics of retinal degenerative diseases. It has been verified by the present invention that Cus 3 intervention can significantly improve the small eye phenotype of visually damaged zebrafish, and restore the transverse and longitudinal axis diameters of the eyeball to normal levels. At the behavioral level, Cus 3 significantly improved the swimming distance and movement rate of zebrafish in the monochromatic light stimulation escape experiment, restoring them to normal levels. Histological analysis further confirmed that Cus 3 can effectively repair the retinal cell layer structure of visually damaged zebrafish.

[0029] The present invention also provides the use of Cus 3 in the preparation of a drug having a visual protection effect. The structural formula of the Cus 3 is shown in Formula I:

[0030]

[0031] The examples of the present invention demonstrate the protective effect of Cus 3 on eye vision from multiple aspects of morphology, behavior and histology.

[0032] The present invention also provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating visual impairment. The structural formula of the Cus 3 is shown in Formula I:

[0033]

[0034] The present invention demonstrates that Cus 3 has an ameliorative effect on visual impairment, specifically by improving the small eye phenotype in visually impaired zebrafish, restoring the eyeball's horizontal and vertical axis diameters to normal levels; and by increasing the swimming distance and movement rate in a monochromatic light escape experiment, restoring them to normal levels. Furthermore, it repairs the retinal cell layer structure in visually impaired zebrafish.

[0035] The present invention also provides the use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal damage. The structural formula of the Cus 3 is shown in Formula I:

[0036]

[0037] The experimental results showed that after Cus 3 intervention, the thickness of the pigment epithelium, inner nuclear layer, outer nuclear layer, and ganglion cell layer increased significantly and returned to normal levels. Cus 3 can restore the thickness and structure of the retina.

[0038] The present invention also provides the use of Cus 3 in the preparation of a drug for improving the morphology of eyes with visual impairment. The structural formula of Cus 3 is shown in Formula I:

[0039]

[0040] In a specific embodiment, improving the morphology of the visually impaired eye includes increasing the horizontal and / or vertical axis distances of the visually impaired eye to normal levels. Experimental results showed that after Cus 3 intervention, the zebrafish eye size was significantly improved, with the horizontal axis diameter restored to (313.73±12.98) μm and the vertical axis diameter restored to (254.36±12.54) μm, which were significantly different from the model group and essentially reached normal physiological levels.

[0041] The present invention also provides the use of Cus 3 in the preparation of a drug for improving the sensitivity of visually impaired eyes to light. The structural formula of the Cus 3 is shown in Formula I:

[0042]

[0043] The experimental results showed that after Cus 3 intervention, the swimming distance and speed of zebrafish increased significantly compared with the model group. The swimming distance and speed in the red, green and blue light stimulation experiments returned to normal levels. Cus 3 intervention increased the sensitivity of visually damaged zebrafish to light.

[0044] The present invention also provides the use of Cus 3 in the preparation of a drug for repairing the retinal cell layer structure of visually damaged eyes. The structural formula of the Cus 3 is shown in Formula I:

[0045]

[0046] In a specific embodiment, the repairing of the retinal cell layer structure of the visually damaged eye includes increasing the thickness of the retinal cell layer of the visually damaged eye. In a specific embodiment, the retinal cell layer includes any one or more of the pigment epithelium, inner nuclear layer, outer nuclear layer and ganglion cell layer.

[0047] To further illustrate the present invention, the application of Cus 3 provided by the present invention in the preparation of a medicament for preventing and / or treating retinal degenerative diseases is described in detail below in conjunction with examples, but these examples should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] 1. Experimental Materials

[0050] 1.1 Zebrafish

[0051] Wild-type TU strain male and female zebrafish, 6 to 8 months old, were purchased from the National Zebrafish Resource Center and cultured in a recirculating aquaculture system. The zebrafish culture medium was maintained at a pH of 7.0 to 7.2 and a temperature of 28 ± 0.5°C. The system was maintained under a 14-h light / 10-h dark cycle. Freshly hatched brine shrimp were fed 2 to 3 times daily.

[0052] Zebrafish culture medium: Prepared by dissolving 17.5 g of sodium chloride, 0.25 g of calcium chloride, 0.25 g of potassium chloride, and 0.25 g of sodium bicarbonate in 5 L of pure water.

[0053] 1.2 Compound Information

[0054] Cus 3 monomer is a known natural product isolated from Convolvulaceae plants, and its molecular formula is C 37 H 66 O 18 , the structural formula is shown in Formula I.

[0055]

[0056] 2. Experimental Methods

[0057] 2.1 Zebrafish Embryo Collection

[0058] After feeding for 1.5 hours in the evening, place adult male and female fish in a 1:1 ratio in a spawning tank, block them with a baffle, and place them in a breeding tank. Add zebrafish culture medium, maintaining a shallow layer of water just above the zebrafish. The next morning, within 60 minutes of the onset of light, collect embryos, select normal embryos, and place them in zebrafish culture medium at 26-28°C.

[0059] 2.2 Modeling and grouping

[0060] Healthy embryos at 0.5 hpf (hours post-fertilization) were plated into 6-well plates, 30 per well, and 5 mL of zebrafish culture medium was added. Each group consisted of 6 wells, with one plate representing one group. Between 0.5 hpf and 72 hpf, visual impairment models were established using zebrafish culture medium supplemented with 0.01% DMSO as a blank control group and zebrafish culture medium supplemented with 0.5 mg / L mepanipyrim (Mep, stock solution prepared with DMSO to 5000 mg / L) as a model group (refer to the following reference for modeling methods: Shen C, Cai Y, Li J, et al. Mepanipyrim induces visual developmental toxicity and vision-guided behavioral alteration in zebrafish larvae [J]. Journal of Environmental Sciences, 2023, 124:76-88). At 72 hpf, the blank control and model groups were given zebrafish culture medium, the treatment group was given zebrafish culture medium containing 80 μg / mL Cus 3, and the positive control group was given zebrafish culture medium containing 10 μg / mL lutein. Cultures were continued until 7 dpf (day post-fertilization), with the corresponding culture medium replaced daily.

[0061] 2.3 Observation of zebrafish eye morphology

[0062] At 7 dpf, 24 juveniles (4 / well) were randomly selected from 6 wells in each group, anesthetized with MS-222, fixed with 8% sodium carboxymethylcellulose, and placed in the lateral position for observation under a stereomicroscope. Pictures of their bodies and eyes were taken, and the body length, eye horizontal (X-axis), and vertical (Y-axis) data were obtained using DanioScope software.

[0063] 2.4 Behavioral Analysis

[0064] At 156 hpf, 24 zebrafish were randomly selected from each group and placed in a 24-well plate, with one zebrafish and 1 mL of zebrafish culture medium placed in each well. The zebrafish were then placed in an incubator and allowed to acclimate for 12 hours. At 168 hpf, the zebrafish in each group were observed in darkness, monochromatic red light, blue light, and green light for 5 minutes. Parameters such as total distance moved, average speed, and movement trajectory were recorded within the 5-minute period.

[0065] 2.5 Histopathological examination

[0066] At 7 dpf, 12 juvenile fish were randomly selected from each group and fixed with 4% paraformaldehyde (PFA) for at least 24 h before use. The samples were then dehydrated, embedded in paraffin, sectioned, and stained with HE. The specific steps were as follows:

[0067] PFA was aspirated and discarded, replaced with double deionized water (ddH2O), soaked for 5 minutes and discarded, and fresh ddH2O was added. This was repeated three times, and the ddH2O was replaced and allowed to stand for 10 minutes. Fresh ddH2O was replaced again and allowed to stand for 30 minutes. The zebrafish was then soaked in 75% ethanol overnight. The zebrafish was then soaked in 85% ethanol for 1 hour, 95% ethanol for 50 minutes, 100% ethanol for 10 minutes, 100% ethanol for 15 minutes, 100% ethanol / xylene 1:1 for 20 minutes, xylene for 9 minutes, xylene for 5 minutes, paraffin / xylene 1:1 (v / v) for 40 minutes (performed in a 60°C oven), paraffin for 2 hours, and embedded in a paraffin embedding machine. During embedding, the zebrafish should be kept with its back or abdomen facing up and its eyes parallel.

[0068] Paraffin blocks containing eye tissue were cut into 4-6 μm paraffin cross-sections using a paraffin microtome and stained using the hematoxylin & eosin (HE) procedure. The specific staining steps were as follows: 1) Oven-dry the slides at 60°C for 1 hour; 2) Rehydrate. Immediately after removal from the oven, the slides were sequentially treated with xylene for 5 minutes, xylene for 5 minutes, 100% ethanol, 95% ethanol, 80% ethanol, 70% ethanol, 50% ethanol, and ddH2O for 3 minutes each, followed by hematoxylin for 3 seconds, and then rinsed three times with tap water; 3) Dehydrate. The slides were sequentially soaked in 50% ethanol, 70% ethanol, 80% ethanol, and 95% ethanol for 3 minutes, followed by eosin for 45 seconds, followed by 100% ethanol, xylene for 3 minutes, xylene for 5 minutes, and xylene for 5 minutes each; 4) Mounting. Remove the slides from the xylene bath and immediately apply an appropriate amount of neutral resin to the slides using a cotton swab. Cover with a coverslip, removing as many bubbles as possible and ensuring that the tissue surface is free of air bubbles. 5) Allow to dry. Incubate in a fume hood at room temperature overnight.

[0069] Images were taken using a digital camera microscope, and the thickness of different retinal cell layers (n=12) was measured using Image J software.

[0070] 3. Results and Analysis

[0071] 3.1 Effects of Cus 3 on eye morphology in visually impaired zebrafish

[0072] Figure 1 The results of the effect of monomer Cus 3 on the eye morphology of visually impaired zebrafish are shown in Figure 1. A: Typical image of the eye; B: Result image of body length and eye data. Figure 1 As shown in the results, there was no significant difference in the body length of zebrafish in each treatment group, but their eye morphological parameters changed significantly, specifically the significant changes in the transverse and longitudinal diameters of the eyes. Compared with the blank control group, the zebrafish in the model group showed a significant microphthalmia-like phenotype, with the transverse diameter of the eyeball shortened from (319.36±17.81)μm to (303.10±18.07)μm, and the longitudinal diameter decreased from (250.30±13.76)μm to (234.95±13.08)μm. This phenotypic change is highly consistent with the characteristics of retinal degenerative diseases (Zheng N, Liao T, Zhang C, et al. Quantumdots-caused retinal degeneration in zebrafish regulated by ferroptosis and mitophagy inretinal pigment epithelial cells through inhibiting spliceosome[J]. Advanced Science, 2024, 11(46)). After Cus 3 treatment, the zebrafish eye size was significantly improved, with the transverse diameter restored to (313.73±12.98) μm and the longitudinal diameter restored to (254.36±12.54) μm, significantly different from the model group and basically reaching normal physiological levels. In contrast, the positive control lutein group only significantly increased the longitudinal distance of the eye.

[0073] 3.2 Effects of Cus 3 on the behavior of visually impaired zebrafish

[0074] As a model organism, zebrafish has a well-developed visual system similar to that of humans, and there is a significant neurobiological correlation between its visual system and its behavioral performance. Figure 2 The results of Cus 3 on the behavior of visually damaged zebrafish are shown in Figure 1. A: the result of movement distance and speed; B: the result of movement trajectory. Figure 2As shown in the behavioral test, the zebrafish in the Mep-treated group were more "sluggish" in the dark, and their movement distance and speed were significantly lower than those in the blank control group; when given monochromatic light stimulation, the swimming distance and speed driven by the instinctive avoidance and escape of the zebrafish in the model group were significantly decreased compared with the blank control group, indicating that after Mep treatment, the zebrafish's sensitivity to light decreased. After Cus 3 intervention, the zebrafish's swimming distance and speed were significantly increased compared with the Mep-treated group, among which the swimming distance and speed in the red, green and blue light stimulation experiments returned to normal levels, indicating that Cus3 intervention significantly improved the light sensitivity of visually damaged zebrafish.

[0075] 3.3 Effects of Cus 3 on the retinal cell layers of visually impaired zebrafish

[0076] The retinal structure mainly includes the pigment epithelium (RPE), inner nuclear layer (INL), outer nuclear layer (ONL), ganglion cell layer (GCL), rods, cones, etc. The cell status and number of each cell layer are crucial to maintaining the normal functioning of the visual system. Figure 3 The results of the effect of monomer Cus 3 on the morphology and thickness of the retina of visually impaired zebrafish are shown in Figure 1. A: H&E staining of eye sections; B: Thickness of the pigment epithelium, outer nuclear layer, inner nuclear layer and ganglion cell layer; C: Thickness of the retina and lens. Figure 3 As shown in the figure, compared with the blank control group, the integrity of the retinal cell layers in the Mep group was poor. Statistical analysis revealed a significant decrease in the thickness of the RPE, ONL, INL, GCL, and retinal layers. After Cus 3 treatment, the thickness of these cell layers increased significantly and returned to normal levels. These results indicate that Cus 3 can rescue visual impairment in zebrafish.

[0077] This study systematically evaluated the visual protective effects of the monomeric component Cus 3, based on a zebrafish model of visual impairment. Results demonstrated that Cus 3 intervention significantly improved the small eye phenotype in visually impaired zebrafish, restoring the eyeball's transverse and longitudinal diameters to normal levels. Behavioral studies also demonstrated that Cus 3 significantly increased the zebrafish's swimming distance and movement rate in a monochromatic light escape experiment, restoring them to normal levels. Histological analysis further confirmed that Cus 3 effectively repaired the retinal cell layer structure in visually impaired zebrafish.

[0078] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal degenerative diseases, wherein the structural formula of Cus 3 is shown in Formula I:

2. Use of Cus 3 in the preparation of a drug having a visual protection effect, wherein the structural formula of Cus 3 is shown in Formula I:

3. Use of Cus 3 in the preparation of a medicament for preventing and / or treating visual impairment, wherein the structural formula of Cus 3 is shown in Formula I:

4. Use of Cus 3 in the preparation of a medicament for preventing and / or treating retinal damage, wherein the structural formula of Cus 3 is shown in Formula I:

5. Use of Cus 3 in the preparation of a drug for improving the morphology of eyes with visual impairment, wherein the structural formula of Cus 3 is shown in Formula I:

6. The use according to claim 5, characterized in that The improving the morphology of the visually impaired eye includes increasing the horizontal axis distance and / or the vertical axis distance of the visually impaired eye to a normal level.

7. Use of Cus 3 in the preparation of a drug for increasing the sensitivity of visually impaired eyes to light, wherein the structural formula of Cus 3 is shown in Formula I:

8. Use of Cus 3 in the preparation of a drug for repairing the retinal cell layer structure of visually damaged eyes, wherein the structural formula of Cus 3 is shown in Formula I:

9. The use according to claim 8, characterized in that The repairing of the retinal cell layer structure of the visually damaged eye includes increasing the thickness of the retinal cell layer of the visually damaged eye.

10. The use according to claim 8 or 9, characterized in that: The retinal cell layer includes any one or more of a pigment epithelial cell layer, an inner nuclear layer, an outer nuclear layer, and a ganglion cell layer.

Images