Application of sennoside A in drugs for preventing and / or treating retinal light damage

Abstract

The present invention provides the use of sennoside A in drugs for preventing and / or treating retinal light damage, belonging to the technical field of ophthalmic drug manufacturing. The present invention used a mouse model of retinal light damage as an experimental subject to verify the efficacy of sennoside A. The results showed that sennoside A can improve the pathological morphology of retinal outer nuclear layer and inner nuclear layer cells, reduce retinal cell apoptosis, reduce oxidative stress levels and inflammatory responses in the body, and enhance antioxidant capacity. Therefore, sennoside A has a good protective effect against retinal light damage. Therefore, the present invention provides the use of sennoside A in drugs for preventing and / or treating retinal light damage and / or products for alleviating visual fatigue.

Description

Technical Field

[0001] The present invention belongs to the technical field of ophthalmic drug manufacturing, and particularly relates to the application of sennoside A in drugs for preventing and / or treating retinal light damage. Background Art

[0002] Sennoside A (SA), the primary active anthraquinone component of rhubarb, is widely used in China and other Asian countries as a stimulant laxative, weight loss drug, or dietary supplement. SA also exhibits antimicrobial properties, inhibits enzyme activity, inhibits HIV-1 replication, restores gut microbiota balance, improves glucose metabolism in obese mice, and ameliorates hepatic steatosis in mice with non-alcoholic fatty liver disease. Recent studies have reported that SA is also active against various tumors and cancers, such as pancreatic and liver cancer. Currently, there are no reports on the use of SA for repairing retinal light damage. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a new use of sennoside A, namely, use of sennoside A in the preparation of a drug for preventing and / or treating retinal light damage.

[0004] The present invention provides use of sennoside A in preparing a medicine for preventing and / or treating retinal light damage.

[0005] Preferably, the disease causing retinal light damage includes at least one of the following: myopia, retinal detachment, retinal vascular disease, macular degeneration, eye fatigue and blurred vision.

[0006] Preferably, the retinal light damage disease includes at least one of the following pathological changes: changes in the morphology of cells in the outer nuclear layer and inner nuclear layer of the retina, apoptosis of retinal cells, increased oxidative stress levels in the body, enhanced inflammatory response in the body and decreased antioxidant capacity.

[0007] Preferably, the retinal light damage is caused by acute / chronic exposure to natural light, ultraviolet light or blue light.

[0008] Preferably, the illumination time is more than 20 h.

[0009] Preferably, the dosage form of the drug includes at least one of the following: tablets, powders, oral liquids, capsules, and granules.

[0010] Preferably, the drug includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include at least one of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, flavoring agents, sweeteners, nutrients and flavor enhancers.

[0011] Preferably, the concentration of sennoside A in the drug is not less than 2.24 μM.

[0012] The present invention provides the use of sennoside A in preparing an ALDH2 enzyme agonist.

[0013] Preferably, the concentration of sennoside A in the ALDH2 enzyme agonist is not less than 2.24 μM.

[0014] The present invention provides the use of sennoside A in the preparation of a drug for preventing and / or treating retinal light damage. The present invention first used 40 compounds obtained through molecular docking virtual screening as test subjects to conduct a test on the ALDH2 target response strength. The results showed that sennoside A (C3) exhibited the strongest ALDH2 enzyme activity activation level, with the cell fluorescence intensity 10 times higher than that of the blank control group. At the same time, the present invention also used a light-damaged mouse model as a subject to verify the efficacy of sennoside A. The results showed that compared with the model group, both low-dose and high-dose sennoside A could significantly improve the morphological changes of the retinal outer nuclear layer and increase the thickness of the outer nuclear layer and inner nuclear layer. The protective effect of sennoside A on the retinal outer nuclear layer was comparable to that of the positive drug lutein. TUNEL fluorescence staining results showed that both low-dose and high-dose sennoside A could significantly improve retinal cell apoptosis caused by light, and the protective effect of low-dose sennoside A on retinal cell apoptosis was comparable to that of the positive drug lutein. Plasma MDA level measurement results showed that high-dose sennoside A could significantly reduce the level of lipid peroxidation in the body, indicating that sennoside A It has a protective effect on cellular oxidative stress damage; the results of plasma interleukin-6 assays showed that high doses of sennoside A can significantly reduce the level of interleukin-6 (IL-6) in the body, indicating that sennoside A has an improving effect on light-induced cellular inflammatory responses; the results of plasma total antioxidant capacity assays showed that high doses of sennoside A can significantly improve the body's antioxidant capacity, indicating that sennoside A can improve the total antioxidant capacity of cells. Based on the above verification results, it can be seen that the sennoside A has a good effect on improving retinal light damage and can be used for the prevention and / or treatment of diseases related to retinal light damage. At the same time, it broadens the medical application field of sennoside A and improves the medicinal value of sennoside A. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of drug screening based on ALDH2 fluorescence response; A is the virtual docking model diagram of sennoside A and ALDH2; B is the relative quantitative diagram of the fluorescence intensity of the increase in ALDH2 enzyme activity induced by natural drugs;

[0016] Figure 2 Confocal fluorescence imaging of HepG2 cells treated with sennoside A; A is a representative image, scale bar = 60 μm; B is the quantitative curve of fluorescence intensity of HepG2 cells;

[0017] Figure 3H&E staining of mouse retina; A is H&E staining of mouse retinal tissue and thickness of retinal outer nuclear layer and inner nuclear layer, scale bars: 500 μm and 20 μm; B and C are quantitative images of retinal outer nuclear layer and inner nuclear layer thickness, number of mice n = 5-6; Note: ## P <0.01, ### P <0.001;

[0018] Figure 4 Figure 2: TUNEL staining of mouse retinal apoptotic cells. A is a TUNEL fluorescence staining image of mouse retina. DAPI blue fluorescence represents cell nuclei, and TUNEL red fluorescence represents apoptotic cells. Scale bars: 500 μm and 200 μm. B is a quantitative image of the fluorescence intensity of retinal apoptotic cells. Number of mice: n = 5-6. Note: ## P <0.01, ### P <0.001;

[0019] Figure 5 Figure 2 is the malondialdehyde content in mouse plasma, number of mice n = 5; Note: # P <0.05,## P <0.01, ns indicates no significant difference compared with the model group;

[0020] Figure 6 is the plasma IL-6 level of mice, number of mice n = 6-7; Note: # P <0.05,### P <0.001;

[0021] Figure 7 is the total antioxidant capacity (T-AOC) of mouse plasma, number of mice n = 5; Note: # P <0.05; the total antioxidant capacity of mice in the high-dose sennoside A group showed a trend of enhancement compared with the model group. P The value is 0.054. DETAILED DESCRIPTION

[0022] The present invention provides use of sennoside A in preparing a medicine for preventing and / or treating retinal light damage.

[0023] In the present invention, the molecular formula of the sennoside A is: C 42 H 38 O 20 , molecular weight: 862.74, structural formula is shown in Formula I: Formula I.

[0024] In the embodiment of the present invention, the sennoside A is purchased from MCE, product number: HY-N0365. Usage: Sennoside A is dissolved in DMSO.

[0025] In the present invention, the diseases caused by retinal light damage preferably include at least one of the following: myopia, retinal detachment, retinal vascular disease, macular degeneration, eye fatigue, and blurred vision. Myopia, retinal detachment, and retinal vascular disease caused by retinal light damage refer to prolonged exposure to high-intensity blue light or ultraviolet light, which can cause photochemical reactions in retinal cells, generate free radicals, and subsequently damage retinal cells. This damage can affect retinal function and may lead to decreased vision and the development of myopia. Macular degeneration refers to long-term exposure to strong light, especially blue light, which can damage cells in the macula, causing macular degeneration. This disease typically manifests as blurred central vision, distortion, or the appearance of dark spots. Retinal detachment refers to retinal light damage that may weaken the adhesion between retinal layers, thereby increasing the risk of retinal detachment. Retinal detachment is a serious disease that can lead to permanent blindness if left untreated. Retinal vascular disease refers to retinal light damage that affects retinal blood vessels, causing damage to the blood vessel walls, thereby causing retinal vascular disease. Retinal light damage is caused by acute or chronic exposure to natural light, ultraviolet light, or blue light, such as natural light or light emitted by electronic devices. The illumination time is preferably more than 20 h, and can be 22 to 30 h, or even 26 h.

[0026] In an embodiment of the present invention, the experimental subject, a mouse model of retinal light damage, is induced by light exposure of 8,000 to 10,000 Lux. The light exposure time is preferably 22 to 26 hours. In a specific embodiment of the present invention, the treatment condition can be 9,000 Lux of light exposure for 24 hours. The mouse model of retinal light damage preferably includes at least one of the following pathological changes: changes in morphological cells in the retinal outer nuclear layer and inner nuclear layer, retinal cell apoptosis, increased levels of oxidative stress in the body, enhanced inflammatory response, and decreased antioxidant capacity. Changes in morphological cells in the retinal outer nuclear layer and inner nuclear layer refer to morphological changes such as loosening and reduced thickness of the outer nuclear layer and inner nuclear layer of the mouse retina after light damage treatment. Retinal cell apoptosis refers to an increase in the number of red fluorescent apoptotic cells in the mouse retinal cells after light treatment, as measured by TUNEL fluorescence staining. Increased levels of oxidative stress in the body refer to the degree of cell membrane lipid peroxidation, as reflected by plasma malondialdehyde in the model mice, which is used as an indicator of lipid peroxidation. Compared with the control group, plasma malondialdehyde levels were significantly increased after light treatment, indicating increased lipid peroxidation in the model mice. An enhanced inflammatory response in the body is reflected by IL-6 levels. Light-treated mice showed an increase in IL-6-mediated inflammatory responses. Reduced antioxidant capacity refers to the total antioxidant capacity of various antioxidants and antioxidant enzymes. This capacity is used to evaluate the antioxidant capacity of bioactive substances to protect cells from oxidative stress caused by reactive oxygen free radicals. Light-treated mice showed a significant decrease in total antioxidant capacity.

[0027] In the present invention, the sennoside A was set up in a low-dose group (100 mg / kg) and a high-dose group (200 mg / kg) to verify its efficacy on the retinal light-damaged mouse model. The results showed that H&E histopathological results showed that both low-dose and high-dose sennoside A under light conditions could well improve the morphological changes of the retinal outer nuclear layer, increase the thickness of the outer nuclear layer and the inner nuclear layer, and the protective effect of sennoside A on the retinal outer nuclear layer was comparable to that of the positive drug lutein; TUNEL staining results of retinal apoptotic cells showed that both low-dose and high-dose sennoside A could well improve retinal cell apoptosis caused by light, and the protective effect of low-dose sennoside A on retinal cell apoptosis was comparable to that of the positive drug lutein. The results of plasma MDA level determination showed that high doses of sennoside A could significantly reduce the level of lipid peroxidation in the body, indicating that sennoside A has a protective effect on cellular oxidative stress damage; the results of plasma interleukin-6 determination showed that high doses of sennoside A could significantly reduce the level of IL-6 in the body, indicating that sennoside A has an improving effect on the cellular inflammatory response caused by light; the results of plasma total antioxidant capacity (T-AOC) level determination showed that high doses of sennoside A could significantly improve the antioxidant capacity in the body, indicating that sennoside A can improve the total antioxidant capacity of cells

[0028] In the present invention, ALDH2 is an aldehyde dehydrogenase that plays a crucial role in the catabolism of exogenous and endogenous toxic aldehydes, such as those associated with oxidative stress-induced lipid peroxidation. Studies have shown that increased ALDH2 expression can ameliorate oxidative stress and inflammatory damage in the retina, protecting its structure and function. To clarify the mechanism by which sennoside A prevents and treats retinal light damage, the present invention investigated the effect of sennoside A on ALDH2 enzyme activity. The results demonstrated that sennoside A is highly responsive to the ALDH2 target and protects against retinal light damage by upregulating ALDH2 enzyme activity. Therefore, the present invention provides the use of sennoside A in the preparation of a medicament for treating retinal light damage.

[0029] In the present invention, the dosage form of the drug includes at least one of the following: tablets, powders, oral liquids, capsules, and granules. The drug includes pharmaceutically acceptable excipients; such pharmaceutically acceptable excipients include at least one of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, flavoring agents, sweeteners, nutrients, and flavor enhancers. The concentration of sennoside A in the drug or product is preferably no less than 2.24 μM, and preferably 10 to 50 μM.

[0030] In an embodiment of the present invention, the dosage of sennoside A for mice is preferably 100-200 mg / kg. The dosage of sennoside A for humans is preferably 10.99-21.98 mg / kg.

[0031] In view of the fact that sennoside A can upregulate the activity of ALDH2 enzyme in cells, the present invention provides the use of sennoside A in the preparation of ALDH2 enzyme agonists.

[0032] In the present invention, the half effective concentration (EC 50 ) is 2.24 μM. The concentration of sennoside A in the ALDH2 enzyme agonist is preferably not less than 10 mM, preferably 12 to 20 mM, and can be 15 to 18 mM. The ALDH2 enzyme agonist can be used to upregulate ALDH2 enzyme activity in HepG2 cell lines.

[0033] The application of sennoside A provided by the present invention in the prevention and / or treatment of retinal light damage is described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] Screening of ALDH2-responsive drugs based on HepG2 cells

[0036] 1. Experimental Methods:

[0037] 1.1 Cell culture and passaging

[0038] HepG2 cell lines were purchased from the National Authenticated Cell Culture Bank of the Chinese Academy of Sciences. Cells were cultured in complete medium (Dulbecco's modified Eagle's medium + 10% fetal bovine serum + 1% antibiotics) at 37°C in a 5% CO2 incubator. When cells reached 80% confluence, they were carefully harvested and passaged to ensure exponential cell growth. In all experiments, cells remained mycoplasma-free at passage number <25.

[0039] 1.2 Drug screening based on ALDH2 response

[0040] Molecular docking and virtual screening identified 40 compounds with promising binding to the ALDH2 enzyme. HepG2 cells were incubated with each of these compounds. Drug solutions were provided by MCE at a concentration of 10 mM. Cells were treated overnight with 10 μM of drug. A 0.5 mM azidoacetaldehyde (AAN3) was used as a substrate for the ALDH2 enzyme reaction and incubated for 8 hours. Cells were then fixed with formaldehyde at 4°C, stained with rhodamine (RHO), and photographed using a confocal fluorescence microscope. Images were analyzed using ImageJ software, and fluorescence intensity was quantified by densitometry. Cell fluorescence intensity was positively correlated with ALDH2 activity.

[0041] 1.3 Quantitative determination of the response intensity of sennoside A and ALDH2 target

[0042] HepG2 cells were treated with various concentrations of sennoside A overnight, followed by incubation with 0.5 mM AAN3 for 8 hours. Cells were fixed with methanol at 4°C and stained with RHO. Images were taken using a confocal fluorescence microscope, analyzed using ImageJ software, and fluorescence intensity was quantified by densitometry. Quantitative analysis of HepG2 cell fluorescence intensity was performed using Nonlin fitting to analyze the quantitation effect of sennoside A. Signal intensities were normalized to those of the blank control.

[0043] 2. Experimental results:

[0044] 2.1 Drug screening experiments based on ALDH2 response

[0045] First, we used molecular docking to virtually screen compounds that interact with ALDH2. The docking prediction results showed that sennoside A strongly interacts with the ALDH2 allosteric site ( Figure 1In Figure A, the ALDH2 docking pocket is ribbon-shaped, and sennoside A is skeleton-shaped. Forty compounds from A2-3A2 (see Table 1) were subsequently screened using cell incubation experiments. Confocal fluorescence detection and analysis revealed that sennoside A (C3) exhibited the strongest ALDH2 enzyme activation, with cell fluorescence intensity 10 times higher than that of the blank control group ( Figure 1 This result is consistent with the docking prediction.

[0046] Table 1 Information of 40 compounds of A2-3A2

[0047]

[0048] 2.2 Determination of the median effective concentration of sennoside A in response to ALDH2 target

[0049] After HepG2 cells were treated with different concentrations of sennoside A, they were photographed and analyzed using a confocal fluorescence microscope. The signal intensity was normalized to that of the blank group, and the quantitative effect analysis was performed using Nonlin fitting.

[0050] Imaging results showed that sennoside A significantly upregulated the enzyme activity of ALDH2 ( Figure 2 A), and the half effective concentration of sennoside A (EC 50 ) was 2.24 μM ( Figure 2 Middle B).

[0051] Example 2

[0052] Drug administration experiment in mouse light damage model

[0053] 1. Experimental Methods

[0054] 1.1 Experimental Animals

[0055] Eight-week-old BALB / c male mice were purchased from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd. Before the experiment, all mice were housed in an animal room with a 12:12 h light / dark cycle for 1 week and provided with normal chow and drinking water daily. All experimental procedures adhered to the Guidelines for the Care and Use of Animals of the Institute of Animal Science, Zhejiang University.

[0056] 1.2 Construction of mouse light damage model and drug treatment

[0057] A homemade light box (108 cm long, 50 cm wide, and 72 cm high) was used, with a light intensity of 8,500-10,000 lux as measured by a light meter. Mice underwent ocular examination, and those with normal ocular abnormalities were randomly divided into a control group (12 mice), a model group (10 mice), a lutein group (100 mg / kg, 10 mice), a low-dose sennoside A group (100 mg / kg, 10 mice), and a high-dose sennoside A group (200 mg / kg, 10 mice). Lutein and sennoside A were suspended in 5% sodium carboxymethylcellulose (CMC-Na) solution and administered orally once daily. Mice in the control and model groups received an equal volume of 5% CMC-Na for 10 consecutive days. Ten days later, mice in the model, lutein, low-dose, and high-dose sennoside A groups were kept in darkness for 36 hours. Atropine ophthalmic gel was then applied to both eyes to induce pupil dilation. The mice were then exposed to 8,000 to 10,000 Lux light for 24 hours. Mice in the control group were housed in a normal environment. After the light exposure period, eyeballs were removed from each group for blood collection and tissue analysis for subsequent experiments.

[0058] 1.3 Experimental instruments are shown in Table 2.

[0059] Table 2 Instrument source description

[0060]

[0061] 1.4 Experimental consumables and reagents are shown in Table 3.

[0062] Table 3 Sources of experimental consumables and reagents

[0063]

[0064] 1.5 Histopathological examination

[0065] Eyeball tissues of 5-6 mice were collected from each group for hematoxylin-eosin staining (H&E) and TUNEL fluorescence staining of apoptotic cells, and the pathological changes of mouse eyeball tissues were observed under an optical microscope (provided by Wuhan Sevier Biotechnology Co., Ltd.).

[0066] 1.6 Determination of MDA levels in mouse plasma

[0067] Blood was collected from mice in anticoagulant tubes and centrifuged at 3000 rpm for 10 min at 4°C. The upper plasma layer was collected and the MDA content was determined according to the method provided by the kit manufacturer.

[0068] 1.7 Statistical Analysis

[0069] Prism8 software was used for statistical analysis and plotting. All data are expressed as mean ± standard error (SEM ± ). The differences between the two groups were analyzed using independent sample t-tests, and the others were analyzed using one-way anova. P <0.05 indicated that the difference was statistically significant.

[0070] 2. Experimental Results

[0071] 2.1 H&E histopathological evaluation

[0072] The retina is the most critical part of the eye, and its hierarchical structure includes the retinal ganglion cell layer, the inner nuclear layer, the outer nuclear layer, and the retinal pigment epithelium. The outer nuclear layer of the retina is mainly composed of photoreceptor cells such as rods and cones, which can receive light stimulation and convert light signals into electrical signals. The inner nuclear layer of the retina contains different types of neuronal cells that can convert electrical signals into nerve impulses. It can be seen that the cells of the inner / outer nuclear layer of the retina play an important role in the reception and transmission of visual signals. According to the results of retinal H&E staining, light damage caused morphological changes such as loosening and reduced thickness of the outer nuclear layer and inner nuclear layer of the retina of the model group mice, indicating that light caused damage to retinal cells ( Figure 3 Middle A). However, both low-dose and high-dose sennoside A administration under light conditions can significantly improve the morphological changes of the outer nuclear layer of the retina and increase the thickness of the outer nuclear layer and the inner nuclear layer ( Figure 3 BC), and the protective effect of sennoside A on the outer nuclear layer of the retina is comparable to that of the positive drug lutein, indicating that sennoside A has a good effect in improving retinal light damage.

[0073] 2.2 TUNEL staining of retinal apoptotic cells

[0074] According to the TUNEL fluorescence staining results of retinal apoptotic cells, light induced a large number of apoptosis in the retinal cells of the model group mice (red fluorescence, Figure 4 A), and both low and high doses of sennoside A can effectively improve retinal cell apoptosis caused by light, and the protective effect of high doses of sennoside A on retinal cell apoptosis is comparable to that of the positive drug lutein ( Figure 4 A and B).

[0075] 2.3 Determination of plasma MDA levels

[0076] Plasma malondialdehyde (MDA) is one of the main products of cell membrane lipid peroxidation. Its content can be used as an indicator of lipid peroxidation, reflecting the degree of cell membrane lipid peroxidation. In this experiment, it was found that light exposure caused an increase in lipid peroxidation levels in the model mouse group, while high doses of sennoside A could significantly reduce lipid peroxidation levels in the body, indicating that sennoside A has a protective effect on cell oxidative stress damage ( Figure 5 ).

[0077] 2.4 Plasma interleukin-6 (IL-6) level measurement

[0078] Interleukins are a type of cytokine produced by and acting on a variety of cells, and have important regulatory effects. They play an important role in transmitting information, activating and regulating immune cells, and mediating inflammatory responses. In this experiment, it was found that light exposure can lead to an increase in the plasma IL-6 level in the model group mice, indicating that light exposure leads to an increase in IL-6-mediated inflammatory response in the body, while high doses of sennoside A can significantly reduce the level of IL-6 in the body, indicating that sennoside A has an improving effect on the cellular inflammatory response caused by light ( Figure 6 ).

[0079] 2.5 Determination of plasma total antioxidant capacity (T-AOC) level

[0080] Plasma total antioxidant capacity (T-AOC) refers to the total antioxidant level composed of various antioxidant substances and antioxidant enzymes. In order to protect cells from oxidative stress damage caused by reactive oxygen free radicals, total antioxidant capacity can be used to evaluate the antioxidant capacity of bioactive substances. This experiment showed that light exposure caused a decrease in the total antioxidant capacity level of the model mouse group, while high doses of sennoside A showed a trend of increasing the antioxidant capacity in the body. This indicates that sennoside A can increase the total antioxidant capacity of cells ( Figure 7 ).

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of sennoside A in the preparation of a medicament for preventing and / or treating retinal light damage.

2. The application according to claim 1, characterized in that The dosage form of the drug is selected from at least one of the following: tablets, powders, oral liquids, capsules and granules.

3. The application according to claim 1, characterized in that The drug includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include at least one of the following: a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavor enhancer.

4. The use according to any one of claims 1 to 3, characterized in that The concentration of sennoside A in the drug is not less than 2.24 μM.

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