Application of icariin in preparation of medicine for preventing and treating diabetic cataract

Abstract

The invention provides an application of icariin in preparation of a medicine for preventing and treating diabetic cataract, the icariin can relieve oxidative stress and inhibit apoptosis, and the icariin has a potential treatment value for the diabetic cataract. The icariin can obviously improve the cataract grading of diabetic cataract rats and reduce the pathological score.

Description

[0001] This case is a divisional application of application number 202411270083.6, entitled "Application of Icariin in the Preparation of Drugs for Preventing and Treating Cataract", the parent application of which has the application number 202411270083.6. TECHNICAL FIELD

[0002] The present application belongs to the technical field of medicine, and relates to a new use of Icariin, in particular to the application of Icariin in the preparation of drugs for preventing and treating diabetic cataract. BACKGROUND

[0003] Cataract is the opacity of the lens of the eye and is the leading cause of blindness worldwide. Although the overall prevalence of cataract has been declining, it is estimated that more than 10 million people worldwide are blind due to cataract, and more than 35 million people have severely impaired vision. Diabetes is a systemic disease that can affect multiple organs and can induce multiple complications, and can also cause long-term damage to the eye. Diabetic cataract is one of the most common ocular complications of diabetes and has become a major cause of low vision and increased blindness in patients with diabetes. Compared with non-diabetic patients, diabetic patients have an earlier onset and faster maturation. Cataract surgery is the main treatment for diabetic cataract, but there are many postoperative complications and poor prognosis. It is estimated that if the development of cataract is delayed for about 10 years, about 50% of cataract surgery is unnecessary. The formation of diabetic cataract involves multiple mechanisms.

[0004] Icariin (ICA) is a flavonoid compound extracted from the leaves and stems of Epimedium, which has various pharmacological effects and biological functions such as anti-inflammatory and antioxidant effects in vivo and in vitro. Our previous studies found that ICA can alleviate uveitis by reducing oxidative stress and regulating microglial M1 / M2 phenotype polarization. In addition, some scholars found that ICA can improve diabetic retinopathy by promoting the growth of retinal ganglion cell (RGC) neurites. However, it is not yet clear whether the protective effect of Icariin on diabetic cataract is through antioxidant stress or anti-apoptosis.

[0005] At present, there is no effective treatment drug for diabetic cataract, and the mainstream treatment method is mainly surgery, but there are many postoperative complications and adverse reactions. Therefore, it is urgent to develop a new effective drug for preventing and treating diabetic cataract. SUMMARY

[0006] In order to solve the problems in the prior art, the application provides application of icariin in preparation of a medicine for preventing and treating diabetic cataract, aims to explore the protective effect of icariin on diabetic cataract through IGFBP3, and explore the possible mechanism, so as to provide a theoretical basis for developing a new, economical, safe and effective natural biological agent for preventing and treating diabetic cataract.

[0007] The technical problem of the application is solved by the following technical scheme:

[0008] The application aims to provide application of icariin in preparation of a medicine for preventing and treating diabetic cataract.

[0009] Further, the icariin is applied to preparation of a medicine for inhibiting high-glucose-induced oxidative stress and apoptosis.

[0010] Further, the icariin reduces the production of ROS under high-glucose stimulation and reduces oxidative stress in SRA01 / 04 cells.

[0011] Further, the icariin improves high-glucose-induced cell apoptosis in human lens epithelial cells.

[0012] Further, the icariin relieves oxidative stress and apoptosis by activating the PI3K / AKT pathway.

[0013] Further, the icariin activates the PI3K / AKT pathway through expression of IGFBP3.

[0014] Further, the icariin and a pharmaceutically acceptable carrier form a clinically acceptable preparation.

[0015] Further, the preparation is one of an oral preparation, an injection preparation and a buccal preparation.

[0016] Further, the icariin has an effective dose of 10-50 mg / kg / day.

[0017] The carrier includes conventional diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the field of pharmacy, and if necessary, flavoring agents, sweetening agents and the like can also be added. The medicine of the application can be prepared into an oral preparation, an injection preparation and a buccal preparation, and the oral preparation can be prepared into tablets, powders, granules, capsules and various forms, and the medicines of the above-mentioned dosage forms can be prepared according to the conventional method in the field of pharmacy.

[0018] It should be noted that the IGFBP3 in the present application refers to insulin-like growth factor (IGF) binding protein 3, which is the most abundant member of the IGFBP family. One of the main functions of IGFBP3 is to bind to IGF1 and prevent IGF1 from binding to its receptor (IGF1R), thereby inhibiting the activation of the IGF1 cell survival pathway. ROS (Reactive oxygen species) refers to active oxygen, which is a kind of molecule produced by the body during oxidative stress.

[0019] Compared with the prior art, the beneficial technical effects of the present application are:

[0020] The medicine prepared from icariin can not only reduce the damage caused by oxidative stress, but also has a positive therapeutic effect on the adverse effects of apoptosis. After icariin treatment, the cataract classification of diabetic cataract rats is improved, and the pathological score is reduced.

[0021] The use of the present application promotes the PI3K / AKT pathway by binding ICA to IGFBP3, and ultimately inhibits high glucose-induced oxidative stress and apoptosis. IGFBP3 mediates high glucose-induced apoptosis of proximal tubular epithelial cells by increasing oxidative stress, and Diallyl Trisulfide inhibits high glucose-induced apoptosis of cardiomyocytes by targeting ROS-mediated IGFBP3 activation. Icariin can effectively protect the development of diabetic cataract, and icariin as an effective ingredient has important practical significance and social value in the preparation of drugs for preventing and treating diabetic cataract.

[0022] The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, the contents of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, the contents of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described.

[0024] Figure 2 The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, the contents of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described.

[0025] Figure 3 The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, the contents of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described.

[0026] Figure 4 The above description is only a summary of the technical solutions of the present application, in order to more clearly understand the technical means of the present application, the contents of the specification can be implemented, and in order to make the above and other purposes, characteristics and advantages of the present application more obvious and easy to understand, the following specific embodiments of the present application are described.

[0027] Figure 5Figure of surface plasmon resonance (SPR) in Example 5 of the present application.

[0028] Figure 6 Results of rat protein immunoblotting experiment in Example 6 of the present application. DETAILED DESCRIPTION

[0029] The technical solutions of the present application are further described in detail below in combination with the drawings and specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of the present application intended to be protected.

[0030] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or prepared by existing methods.

[0031] In the present application, "prevention" means preventing the occurrence of diabetic cataract or inhibiting the progression of diabetic cataract, and in particular means preventing the lens inside the eyeball from becoming cloudy or hardened, so that the lens remains transparent, and the vision is not deteriorated. "Treatment" means alleviating cataract caused by diabetes, the lens inside the eyeball returns to normal level, i.e. returns to a transparent state, or reduces the scope and degree of cataract.

[0032] In the examples of the present application, unless otherwise specified, icariin is purchased from source leaves (item number B21576, HPLC≥98%); rats are male SD rats, weighing about 200 grams, 8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; cells are SRA01 / 04 human lens epithelial cells, purchased from Shanghai Sunny Biotechnology Co., Ltd.

[0033] Icariin is a flavonoid compound extracted from the leaves and stems of Epimedium, which has various pharmacological effects and biological functions such as anti-inflammatory, antioxidant, and anti-diabetic in vivo and in vitro.

[0034] The structural formula of icariin in the present application is:

[0035]

[0036] Sample construction:

[0037] Animal experiment samples:

[0038] Forty-five rats were equally divided into three groups, 15 rats in each group. The first group was the control group: rats were fasted for 16 h, and injected intraperitoneally with 0.1 mol / L citrate-phosphate buffer, pH 4.5. The second group was the diabetes group: rats were fasted for 16 h, and injected intraperitoneally with 65 mg / kg of freshly prepared streptozotocin (STZ) purchased from Sigma Chemical Co, St Louis, MO, USA, and gavaged with normal saline for 12 weeks. The third group was the drug treatment group: rats were fasted for 16 h, and injected intraperitoneally with 65 mg / kg of freshly prepared streptozotocin, and gavaged with icariin at a daily dose of 20 mg / kg for 12 weeks.

[0039] Cell experiment samples:

[0040] Control group: the control group cells in the animal experiment samples were added with DMSO;

[0041] Diabetes group: the diabetes group cells in the animal experiment samples were added with DMSO and D-glucose at a concentration of 50 mM;

[0042] Drug treatment group: the drug treatment group cells in the animal experiment samples were added with DMSO, D-glucose at a concentration of 50 mM, and icariin at a concentration of 15 μM.

[0043] Example 1: Cataract score

[0044] After the rats were modeled for 12 weeks, the eyes of the rats were evaluated using a digital slit lamp after mydriasis (mydriasis used the drug tropicamide phenylephrine eye drops, purchased from Santen Pharmaceutical Co., Ltd., Osaka, Japan). The study was conducted by a trained observer who was unaware of the treatment.

[0045] The severity of cataract was graded in the form of a 5-grade scoring system as follows:

[0046] 0 grade: transparent normal lens;

[0047] 1 grade: peripheral capsular vacuoles;

[0048] 2 grade: peripheral vacuoles plus cortical opacities;

[0049] 3 grade: diffuse central opacity;

[0050] 4 grade: mature cataract.

[0051] After the rats were scored for cataract, statistical analysis was performed.

[0052] Referring to the accompanying drawings, Figure 1 , the cataract grading of rats in each group of animal experiment samples and example photographs are shown. Figure 1A is a representative photo of the eyeballs of animals in each sample group. It can be seen that the eyeballs of the control rats are normal, transparent lens, no cataract; the eyeballs of the diabetic group are basically cloudy white, cataract modeling is successful; the eyeballs of the drug treatment group have obvious vesicles in the periphery, and the lens turbidity is basically improved. It can be seen that the cataract condition after drug treatment has improved significantly and has basically approached the normal level. Figure 1 B is the cataract grading of each group of rats, Figure 1 C is a statistical chart of the cataract grading of each group of rats, combined Figure 1 A can be seen that most of the eyes of the control rats are at level 0, and only one is at level 1; most of the eyes of the diabetic rats are at level 4; most of the eyes of the drug treatment group are at level 1 or 2, and the severity of cataract has improved significantly. It can be seen that icariin has obvious therapeutic effect on diabetic cataract.

[0053] Example 2 Histopathological analysis

[0054] After 12 weeks of modeling, the eyeball tissue samples of the three groups of SD rats were fixed in 10% formalin for 24 hours, and then embedded in paraffin. Then the tissue was stained with hematoxylin and eosin (H&E), and examined using an optical microscope.

[0055] Referring to the attached Figure 2 , it can be seen that the anterior epithelium of the lens of the control group is cubic, the lens fibers are arranged in concentric circles, arranged in order, and lack organelles. The lens capsule thickness and epithelial cell density of the diabetic group decreased, and the differentiated lens fibers, vacuoles and homogeneous area decreased. The drug treatment group was treated with icariin, and these results were improved, the lens capsule thickness and epithelial cells improved to some extent, and the lens fiber differentiation was not obvious. It can be seen that icariin has obvious therapeutic effect on diabetic cataract in histopathology.

[0056] Example 3 Detection of reactive oxygen species

[0057] The logarithmic growth phase SRA01 / 04 cells of each animal experiment group were inoculated in a 12-well culture plate at 1×10 5 cells / well, and the experiment was performed when the cell confluence reached 60%. Different drugs were added according to the grouping (three replicate wells were set for each group).

[0058] Control group: the control group cells in the animal experiment sample were added with DMSO;

[0059] Diabetic group: the cells in the animal experiment sample of the diabetic group were added with DMSO and D-glucose with a concentration of 50mM;

[0060] Drug treatment group: the cells in the drug treatment group of the animal experiment sample were added with DMSO, D-glucose with a concentration of 50 mM and 15 μM of icariin.

[0061] The volume of the added drug was the same in each group.

[0062] After treatment, the cells were added with 1 μM DCFH-DA probe and then incubated at 37°C in the dark for 30 minutes. Finally, the fluorescence intensity was observed with an inverted fluorescence microscope to determine the amount of reactive oxygen species.

[0063] See the attached Figure 3 , the expression of ROS in lens epithelial cells under different stimuli. With fluorescently labeled DCFH-DA dye as a probe, the accumulation of ROS in lens epithelial cells was determined. It can be seen that compared with the control group cells, the fluorescence intensity of ROS in the diabetic group cells increased significantly. The fluorescence intensity of ROS induced by high glucose in the cells of the drug treatment group was significantly reduced. It can be seen that at the cellular level, icariin can reduce the oxidative stress induced by high glucose in lens epithelial cells.

[0064] Example 4 Flow cytometry detection of cell apoptosis

[0065] ABflo ® 488 Annexin V / PI cell apoptosis detection kit was used, purchased from ABclonal, product number RK05875. Human lens epithelial cells in the logarithmic growth phase were inoculated in a 12-well plate (inoculation density was 50%), and cultured in a cell incubator at 37°C with 5% CO2 overnight. After 24 hours, different drugs were added according to the grouping (three replicate wells were set in each group).

[0066] Control group: the cells in the control group of the animal experiment sample were added with DMSO;

[0067] Diabetic group: the cells in the diabetic group of the animal experiment sample were added with DMSO and D-glucose with a concentration of 50 mM;

[0068] Drug treatment group: the cells in the drug treatment group of the animal experiment sample were added with DMSO, D-glucose with a concentration of 50 mM and 15 μM of icariin.

[0069] The volume of the added drug was the same in each group.

[0070] Then the cells of each treatment group were continued to be cultured at 37°C in a culture box containing 5% CO2 for 16h. After the culture medium was discarded, the cells were washed twice with pre-cooled PBS, and the cells were digested with trypsin without EDTA, and then the digestion was terminated by adding DMEM medium containing 10% fetal bovine serum, and the cells were blown down gently and transferred to a 1.5mL EP tube, and the cells were collected by centrifugation at 600g for 5min. After the cells were collected, the cells were washed twice by adding pre-cooled PBS solution and centrifuging to collect the cells. 100μL of 1×Binding buffer working solution was added to the cell pellet, and after the cells were resuspended (the total number of cells was about 1×10 5 cells), 1.5μL of ABflo 488 Annexin V and 1.5μL of PI were added, and the mixture was mixed gently, and incubated at room temperature for 15min in the dark. After the staining incubation, 100μL of 1×Binding Buffer working solution was added to each tube, and the mixture was mixed and then detected by flow cytometry (within 1 hour).

[0071] See Figure 5 Figure 4 Figure 5 shows the effect of Icariin on high glucose-induced apoptosis of lens epithelial cells. The apoptosis rate was detected by flow cytometry. Flow cytometry analysis showed that the number of cells in the late apoptosis stage in the control group was very small. The rate of late apoptosis in the diabetic group significantly increased after high glucose stimulation. Compared with the diabetic group, the rate of late apoptosis of lens epithelial cells in the drug treatment group significantly decreased after Icariin stimulation. It can be seen that Icariin has a protective effect on high glucose-induced apoptosis of lens epithelial cells.

[0072] Example 5 Surface Plasmon Resonance

[0073] Biacore T200 (Cytiva) was used to evaluate binding affinity. Specifically, IGFBP3 was diluted to 25 ug / ml with sodium acetate (pH=4.0, 10 mM) and immobilized on channel 2 of a CM5 chip as a control with channel 1 of CM5. The ICA was diluted to the specified concentration with a mobile phase buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20). The compound was injected into the chip for 120 seconds, and the electrophoresis buffer was used as a blank control for 180 seconds.

[0074] See Figure 5 Figure 5For surface plasmon resonance (SPR) plot, used to evaluate the affinity of icariin to IGFBP3. IGFBP3 was immobilized on the surface of Biacore Chip CM5. Then, different concentrations of compounds were prepared and injected to pass through the surface. The equilibrium dissociation constant (KD) value of IGFBP3 to ICA was 130.8 μM. It can be seen that icariin can stably interact with IGFBP3, thereby exerting a series of therapeutic effects.

[0075] Example 6 Western Blotting Experiment

[0076] Human lens epithelial cells in logarithmic growth phase were inoculated in a six-well plate (inoculation density was about 50%), and cultured overnight in a cell incubator at 37°C containing 5% CO2. After 24 hours, different drugs were added according to the grouping for treatment.

[0077] Control group: the cells in the control group of the animal experiment sample were added with DMSO;

[0078] Diabetic group: the cells in the diabetic group of the animal experiment sample were added with DMSO and D-glucose with a concentration of 50 mM;

[0079] Drug treatment group: the cells in the drug treatment group of the animal experiment sample were added with DMSO, D-glucose with a concentration of 50 mM and icariin with a concentration of 15 μM.

[0080] The volume of the added drugs in each group was the same.

[0081] After the cells in each treatment group were further cultured at 37°C in a culture box containing 5% CO2 for 16 hours, the culture solution was discarded, 1 ml of pre-cooled PBS was added for washing 3 times, and after the PBS was absorbed, 100 μl of lysis solution containing PMSF was added to each well (10 μl of PMSF was added to each 1 ml of lysis solution). The cells were scraped on one side of the culture plate with a clean cell scraper, and then the cell fragments and lysis solution were moved to a 1.5 ml centrifuge tube with a gun and placed on ice for lysis for 30 min (vortex mixing instrument was shaken for 30 s every 5 min to ensure complete lysis of the cells). After complete lysis, centrifugation was performed at 12000 g and 4°C for 15 min, and the supernatant was transferred to a new 1.5 ml centrifuge tube to obtain the total protein product. The protein concentration was determined by BCA method, and the corresponding volume of 20 μg of protein was calculated and taken as the loading amount for Western blotting experiment. Then 5*SDS-PAGE protein loading buffer was added to the protein sample (1 μl of protein loading buffer was added to each 4 ul of protein sample) and incubated at 98°C for 10 min to obtain the protein sample, which was used for Western blotting experiment to detect the protein expression levels of IGFBP3 / PI3K / P-PI3K / AKT / P-AKT in human lens epithelial cells in each group.

[0082] See the attached Figure 6 The results of Western blotting experiments for different groups of SD rats. The expression of proteins related to the IGFBP3 / PI3K / Akt signaling pathway was detected by Western blotting. Compared with the control group of rats, the expression levels of p-PI3K and p-Akt in the diabetic group of rats decreased significantly, while the expression levels of PI3K and Akt had no significant change. After drug treatment, p-PI3K and p-AKT were significantly restored. These results show that the therapeutic effect of Icaritin on diabetic cataract is mainly achieved through the IGFBP3 / PI3K / Akt signaling pathway.

[0083] The above-mentioned embodiment numbers of the present application are only for description, not representing the advantages and disadvantages of the embodiments.

[0084] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above-mentioned specific embodiments, and the above-mentioned specific embodiments are only illustrative, not restrictive. Those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, which are all within the protection of the present application.

Claims

1. Use of icariin in diabetic cataract for preparing IGFBP3 specific binding agent.

2. Use of icariin in diabetic cataract for preparing PI3K / AKT pathway promoter.

3. Use according to claim 2, characterized in that, 3. Use of icariin in diabetic cataract for preparing high glucose-induced oxidative stress and apoptosis inhibitor.

4. A complex in a high sugar environment in a body, characterized by, The complex is formed by binding of icariin and IGFBP3.

5. The composite of claim 4, wherein, The complex has an equilibrium dissociation constant of 130.8 μM.

6. Use of the complex of claim 4 as a marker for evaluating therapeutic effect of diabetic cataract.

7. A kit, wherein the reagents in the kit comprise icariin and IGFBP3.

8. Use of icariin as a modulator of IGFBP3 function for preparing a medicament for preventing and / or treating diabetic cataract.

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