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

Abstract

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

Description

Technical Field

[0001] The invention belongs to the field of medical technology, relates to a new use of icariin, and specifically relates to the use of icariin in preparing a drug for preventing and treating cataracts. Background Art

[0002] Cataracts are clouding of the lens of the eye and are the leading cause of blindness worldwide. Although the overall prevalence of cataracts has been decreasing, it is estimated that more than 10 million people worldwide are still blind due to cataracts and more than 35 million people are severely visually impaired. Cataract surgery is the main treatment for cataracts, but there are many postoperative complications and poor prognosis. It is estimated that approximately 50% of cataract surgeries would be unnecessary if the development of cataracts was delayed by about 10 years. There are multiple mechanisms involved in the formation of cataracts.

[0003] Icariin (ICA) is a flavonoid compound extracted from the leaves and stems of Epimedium, which has multiple pharmacological and biological functions such as anti-inflammatory and antioxidant effects in vivo and in vitro. Our previous studies have found that ICA can relieve uveitis by alleviating oxidative stress and regulating the polarization of microglia M1 / M2 phenotype. In addition, some scholars have found that ICA can improve retinopathy by promoting the growth of ganglion cell (RGC) neurites. However, it is still unclear whether the protective effect of icariin on cataracts works through anti-oxidative stress or anti-apoptosis.

[0004] Currently, there is no effective drug for cataracts. The mainstream treatment is surgery, but there are many complications and adverse reactions after surgery. Therefore, it is urgent to develop a new and effective drug for the prevention and treatment of cataracts. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides an application of icariin in the preparation of a drug for preventing and treating cataracts, aiming to explore the protective effect of icariin on cataracts through IGFBP3 and its possible mechanism of action, so as to provide a theoretical basis for developing a new, economical, safe and effective natural biological preparation for preventing and treating cataracts.

[0006] The present invention solves the technical problem by adopting the following technical solutions:

[0007] The purpose of the present invention is to provide an application of icariin in the preparation of a drug for preventing and treating cataracts.

[0008] Furthermore, the icariin is used in the preparation of a drug for inhibiting high glucose-induced oxidative stress and apoptosis.

[0009] Furthermore, the icariin reduces the generation of ROS under high glucose stimulation in SRA01 / 04 cells, thereby alleviating oxidative stress.

[0010] Furthermore, the icariin improves high glucose-induced cell apoptosis in human lens epithelial cells.

[0011] Furthermore, the icariin relieves anti-oxidative stress and anti-apoptosis by activating the PI3K / AKT pathway.

[0012] Furthermore, the icariin activates the PI3K / AKT pathway through the expression of IGFBP3.

[0013] Furthermore, icariin and a pharmaceutically acceptable carrier form a clinically acceptable preparation.

[0014] Furthermore, the preparation is one of an oral preparation, an injection preparation, and a buccal preparation.

[0015] Furthermore, the effective dose of icariin is 10 to 50 mg / kg / day.

[0016] The carrier includes conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field, and flavoring agents, sweeteners, etc. may be added when necessary. The drug of the present invention can be made into oral preparations, injection preparations, and buccal preparations. The oral preparations can be made into tablets, powders, granules, capsules, etc., and the drugs in the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0017] It should be noted that IGFBP3 in the present invention 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 reactive oxygen species, a class of molecules produced when the body is under oxidative stress.

[0018] Compared with the prior art, the beneficial technical effects of the present invention are:

[0019] The medicine prepared by the icariin of the present invention can not only reduce the damage caused by oxidative stress, but also has a positive therapeutic effect on the adverse effects of cell apoptosis. After being treated with icariin, the cataract grade of cataract rats is improved and the pathological score is reduced.

[0020] The use of the present invention promotes the PI3K / AKT pathway through the combination of ICA and IGFBP3, and finally inhibits high-sugar-induced oxidative stress and apoptosis. IGFBP3 increases high-sugar-induced apoptosis and ROS-mediated oxidative stress. The icariin of the present invention can effectively protect the occurrence and development of cataracts. As an effective ingredient, icariin has important practical significance and social value in the preparation of drugs for the prevention and treatment of cataracts.

[0021] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The grading of rat cataracts and example photos in Example 1 of the present invention are shown.

[0023] Figure 2 This is a diagram of the histopathological analysis of the rat eyeball in Example 2 of the present invention.

[0024] Figure 3 The expression of ROS in lens epithelial cells under different stimuli in Example 3 of the present invention.

[0025] Figure 4 This is the effect of icariin in Example 4 of the present invention on high glucose-induced lens epithelial cell apoptosis.

[0026] Figure 5 This is a surface plasmon resonance (SPR) graph in Example 5 of the present invention.

[0027] Figure 6 The results of the rat protein immunoblotting experiment in Example 6 of the present invention are shown. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection that the present invention is intended to protect.

[0029] In addition, unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0030] In the present invention, "prevention" means preventing the occurrence of cataracts or inhibiting the progression of cataracts, and in particular means preventing the lens in the eye from becoming cloudy or hardened, keeping the lens transparent, and preventing vision from deteriorating. "Treatment" means alleviating cataracts, restoring the lens in the eye to normal levels, i.e., returning to a transparent state, or reducing the scope and degree of cataracts.

[0031] In the embodiments of the present invention, if not otherwise specified, icariin was purchased from source leaves (item number B21576, HPLC ≥ 98%); the rats were male SD rats, weighing about 200 grams, 8 weeks old, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; the cells were SRA01 / 04 human lens epithelial cells, purchased from Shanghai Xinyu Biotechnology Co., Ltd.

[0032] Icariin is a flavonoid compound extracted from the leaves and stems of Epimedium, which has multiple pharmacological effects and biological functions such as anti-inflammatory and antioxidant effects both in vivo and in vitro.

[0033] The structural formula of icariin in the present invention is:

[0034]

[0035] Sample build:

[0036] Animal experiment samples:

[0037] 45 rats were divided into three groups, 15 in each group. The first group was the normal control group: the rats were fasted for 16 hours and intraperitoneally injected with 0.1 mol / L citrate-phosphate buffer, pH 4.5. The second group was the cataract group: the rats were fasted for 16 hours, intraperitoneally injected with 65 mg / kg 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: the rats were fasted for 16 hours, intraperitoneally injected with 65 mg / kg freshly prepared streptozotocin, and gavaged with icariin at a daily dose of 20 mg / kg for 12 weeks.

[0038] Cell experiment samples:

[0039] Ordinary control group: Add DMSO to the control group cells in the animal experimental samples;

[0040] Cataract group: DMSO and 50 mM D-glucose were added to the cataract group cells in the animal experimental samples;

[0041] Drug treatment group: DMSO, 50 mM D-glucose and 15 μM icariin were added to the drug treatment group cells in the animal experimental samples.

[0042] Example 1 Cataract Scoring

[0043] Twelve weeks after modeling, the eyes of the rats were evaluated using a digital slit lamp after mydriasis (mydriasis was performed using the drug tropicamide phenylephrine eye drops, purchased from Santen Pharmaceutical Co., Ltd., Osaka, Japan). The study was performed by a trained observer who was blinded to the treatment.

[0044] The severity of cataracts is graded on a 5-point scoring system as follows:

[0045] Grade 0: transparent normal lens;

[0046] Level 1 peripheral vesicles;

[0047] Grade 2: peripheral vesicles plus cortical opacities;

[0048] grade 3 diffuse central opacity;

[0049] Grade 4 mature cataract.

[0050] The rats were scored for cataract and then statistically analyzed.

[0051] See attached Figure 1 , shows the grading of cataracts in rats of each group of animal experimental samples and example photos. Figure 1 A is a representative photo of the eyeballs of the animals in each sample group. It can be seen that the eyeballs of the rats in the ordinary control group are normal, with transparent lenses and no cataracts; the eyeballs of the cataract group are almost all turbid white, and the cataract model is successfully established; there are obvious vesicles on the periphery of the eyeballs in the drug treatment group, and the opacity of the lens has been basically improved. It can be seen that the cataract condition has been significantly improved after drug treatment and is basically close to the normal level. Figure 1 B is the cataract grading of rats in each group. Figure 1 C is a statistical chart of cataract grading in rats in each group. Figure 1 A It can be seen that most of the eyes of the rats in the ordinary control group were at level 0, and only one was at level 1; most of the eyes of the rats in the cataract group were at level 4; most of the eyes of the rats in the drug treatment group were at level 1 or 2, and the severity of cataracts was significantly improved. It can be seen that icariin has a significant therapeutic effect on cataracts.

[0052] Example 2 Histopathological Analysis

[0053] Twelve weeks after rat modeling, eyeball tissue samples of the three groups of SD rats were fixed in 10% formalin for 24 hours and then embedded in paraffin. The tissues were then stained with hematoxylin and eosin (H&E) and examined using an optical microscope.

[0054] See attached Figure 2, which are the histopathological analysis diagrams of the rat eyeballs of various animal experimental samples, it can be seen that the anterior lens epithelium of the ordinary control group is cubic, the lens fibers are concentrically arranged, neatly arranged, and lack organelles. The lens capsule thickness and epithelial cell density of the cataract group decreased, and the differentiated lens fibers, vacuoles, and homogeneous areas decreased. These results were improved in the drug treatment group through icariin treatment, the lens capsule thickness and epithelial cells were improved to a certain extent, and the lens fiber differentiation was not obvious. It can be seen that in terms of histopathology, icariin has a significant therapeutic effect on cataracts.

[0055] Example 3 Active oxygen detection

[0056] In each animal experimental group, SRA01 / 04 cells in the logarithmic growth phase were seeded in a 12-well culture plate at a density of 1×10 5 Cells / well were inoculated, and the experiment was performed when the cell confluence reached 60%. Different drugs were added for treatment according to the groups (three replicate wells were set for each group).

[0057] Ordinary control group: Add DMSO to the control group cells in the animal experimental samples;

[0058] Cataract group: DMSO and 50 mM D-glucose were added to the cataract group cells in the animal experimental samples;

[0059] Drug treatment group: DMSO, 50 mM D-glucose and 15 μM icariin were added to the drug treatment group cells in the animal experimental samples.

[0060] The volume of drugs added to each group was the same.

[0061] After treatment, the cells were added with 1 μM DCFH-DA probe and then incubated at 37°C in the dark for 30 min. Finally, the fluorescence intensity was observed with an inverted fluorescence microscope to determine the amount of reactive oxygen species.

[0062] See attached Figure 3 , is the expression of ROS in lens epithelial cells under different stimuli. The accumulation of ROS in lens epithelial cells was measured using fluorescently labeled DCFH-DA dye as a probe. It can be seen that compared with the cells in the ordinary control group, the fluorescence intensity of ROS in the cataract group cells increased significantly. However, the cells in the drug treatment group significantly reduced the fluorescence intensity of HG-induced ROS. It can be seen that at the cellular level, icariin can reduce the oxidative stress induced by high glucose in lens epithelial cells.

[0063] Example 4 Detection of cell apoptosis by flow cytometry

[0064] use 488Annexin V / PI apoptosis detection kit was purchased from ABclonal, with the catalog number RK05875. Human lens epithelial cells in the logarithmic growth phase were seeded in a 12-well plate (seeding density was 50%) and cultured overnight in a cell incubator at 37°C and 5% CO2. After 24 hours, different drugs were added according to the grouping for treatment (three replicates were set for each group).

[0065] Ordinary control group: Add DMSO to the control group cells in the animal experimental samples;

[0066] Cataract group: DMSO and 50 mM D-glucose were added to the cataract group cells in the animal experimental samples;

[0067] Drug treatment group: DMSO, 50 mM D-glucose and 15 μM icariin were added to the drug treatment group cells in the animal experimental samples.

[0068] The volume of drugs added to each group was the same.

[0069] Then, the cells of each treatment group were cultured for 16 hours in an incubator at 37°C and 5% CO2. The culture medium was discarded, and the cells were washed twice with pre-cooled PBS. After digesting the cells with trypsin without EDTA, DMEM culture medium containing 10% fetal bovine serum was added to terminate the digestion. The cells were gently blown down and transferred to a 1.5mL EP tube. The cells were collected by centrifugation at 600g for 5 minutes. After collecting the cells, pre-cooled PBS solution was added and washed gently with a pipette. The cells were collected by centrifugation and washed twice in total. 100μL 1×Binding buffer working solution was added to the cell pellet, and the cells were resuspended (the total number of cells was about 1×10 5 cell), add 1.5 μL 488Annexin V and 1.5μL PI, gently mix, and incubate at room temperature in the dark for 15 minutes. After staining and incubation, add 100μL 1×Binding Buffer working solution to each tube, mix well, and use flow cytometry to detect (detect within 1 hour). When 488Annexin V and PI are used together, living cells have only very low intensity background fluorescence, and late apoptotic cells are Q2.

[0070] See attached Figure 4, is the effect of icariin on high glucose-induced apoptosis of lens epithelial cells. Flow cytometry was used to detect the cell apoptosis rate. Flow cytometry analysis showed that the number of cells in the late stage of apoptosis in the ordinary control group was very small. The late apoptosis rate in the cataract group increased significantly after high glucose stimulation. Compared with the cataract group, the late apoptosis rate of lens epithelial cells in the drug treatment group after icariin stimulation was significantly reduced. It can be seen that icariin has a protective effect on high glucose-induced apoptosis of lens epithelial cells.

[0071] Example 5 Surface Plasmon Resonance

[0072] Binding affinity was assessed using Biacore T200 (Cytiva). Specifically, IGFBP3 was diluted to 25ug / ml with sodium acetate (pH=4.0, 10mM) and immobilized on channel 2 of a CM5 chip using channel 1 of CM5 as a control. ICA was diluted to the specified concentration using mobile phase buffer (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA, 0.05% P20). The compound was injected into the chip for 120 seconds and dissociated for 180 seconds using electrophoresis buffer as a blank control.

[0073] See attached Figure 5 , is a surface plasmon resonance (SPR) graph used to evaluate the affinity of icariin for 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 for ICA was 130.8μM. It can be seen that icariin can stably interact with IGFBP3, thereby exerting a series of therapeutic effects.

[0074] Example 6 Protein immunoblotting experiment

[0075] Human lens epithelial cells in the logarithmic growth phase were seeded in six-well plates (seeding density was about 50%) and cultured overnight in a cell incubator at 37°C with 5% CO2. After 24 hours, different drugs were added for treatment according to the groups.

[0076] Ordinary control group: Add DMSO to the control group cells in the animal experimental samples;

[0077] Cataract group: DMSO and 50 mM D-glucose were added to the cataract group cells in the animal experimental samples;

[0078] Drug treatment group: DMSO, 50 mM D-glucose and 15 μM icariin were added to the drug treatment group cells in the animal experimental samples.

[0079] The volume of drugs added to each group was the same.

[0080] After the cells of each treatment group were cultured for 16 hours in an incubator at 37°C and 5% CO2, the culture medium was discarded, 1 ml of pre-cooled PBS was added for washing 3 times, and 100 μl of lysis solution containing PMSF was added to each well after the PBS was aspirated (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 minutes (the vortex mixer was shaken for 30 seconds every 5 minutes to ensure complete cell lysis); after sufficient lysis, the cells were centrifuged at 12000g and 4°C for 15 minutes, and the supernatant was transferred to a new 1.5 ml centrifuge tube to obtain the total cell protein product. The protein concentration was determined by the BCA method, and the corresponding volume of 20ug protein was calculated and taken out as the sample volume for protein immunoblotting experiment. Then 5*SDS-PAGE protein loading buffer was added (1μl protein loading buffer was added for every 4ul protein sample), and the protein sample was obtained by metal bath at 98℃ for 10min. The protein sample was used for protein immunoblotting experiment to detect the protein expression levels of IGFBP3 / PI3K / P-PI3K / AKT / P-AKT in human lens epithelial cells in each group.

[0081] See attached Figure 6 , are the results of protein immunoblotting experiments in different groups of SD rats. The expression of proteins related to the IGFBP3 / PI3K / Akt signaling pathway was detected by protein immunoblotting. Compared with the rats in the normal control group, the expression levels of p-PI3K and p-Akt in the cataract group rats were significantly decreased, while the expression levels of PI3K and Akt did not change significantly. After drug treatment, p-PI3K and p-AKT were significantly restored. These results indicate that the therapeutic effect of icariin on cataract is mainly achieved through the IGFBP3 / PI3K / Akt signaling pathway.

[0082] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0083] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Application of icariin in the preparation of drugs for preventing and treating cataracts.

2. The use according to claim 1, characterized in that: The icariin is used in preparing a medicine for inhibiting oxidative stress and apoptosis induced by high sugar.

3. The use according to claim 2, characterized in that: The icariin reduces the generation of ROS under high glucose stimulation in SRA01 / 04 cells, thereby reducing oxidative stress.

4. The use according to claim 2, characterized in that: The icariin improves high glucose-induced cell apoptosis in human lens epithelial cells.

5. The use according to claim 2, characterized in that: The icariin relieves anti-oxidative stress and anti-apoptosis by activating the PI3K / AKT pathway.

6. The use according to claim 5, characterized in that: The icariin activates the PI3K / AKT pathway through the expression of IGFBP3.

7. The use according to any one of claims 1 to 6, characterized in that: Icariin and a pharmaceutically acceptable carrier form a clinically acceptable preparation.

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