Hepu Pearl Polypeptide Nacrein-LP20 for Treating Cataract and Its Application

Abstract

The present invention discloses a Hepu pearl polypeptide Nacrein-LP20 for treating cataracts and its application, belonging to the technical field of biomedicine. Its amino acid sequence is shown in SEQ ID NO.1. The active pearl polypeptide Nacrein-LP20 provided by the present invention has strong ability to scavenge DPPH free radicals and hydroxyl free radicals, and has certain application value in antioxidant aspect. The active pearl polypeptide Nacrein-LP20 provided by the present invention has a significant protective effect on oxidative stress of human lens cells, can improve the cataract lesions of selenium-induced rats, and has certain application value in cataract treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a Heping pearl polypeptide Nacrein-LP20 for treating cataract and its application. Background Art

[0002] The pathogenesis of cataract is not yet fully understood, and there is still a lack of effective therapeutic drugs clinically. Currently, surgical treatment is the only cure. Generally, mature cataracts can be surgically treated, but mild cataracts are not recommended for surgery, and various complications may still occur after surgical treatment, including postoperative intraocular inflammation, tearing of the lens capsule, retinal detachment, etc. These factors limit the availability and accessibility of cataract surgery globally. Therefore, exploring the pathogenesis of cataract and finding economical and effective drugs for the prevention and treatment of cataract are of great significance. Summary of the Invention

[0003] The purpose of the present invention is to provide a Heping pearl polypeptide Nacrein-LP20 for treating cataract and its application to solve the problems existing in the above-mentioned prior art.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] One of the technical solutions of the present invention is the application of Heping pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating cataract, and the amino acid sequence of the Heping pearl Nacrein-LP20 polypeptide is as shown in SEQ ID NO.1.

[0006] Another technical solution of the present invention is a drug for treating cataract, which includes the Heping pearl Nacrein-LP20 polypeptide.

[0007] A third technical solution of the present invention is the application of Heping pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating oxidative stress of lens cells.

[0008] Based on the above technical solutions, the present invention has the following technical effects:

[0009] The present invention provides a Heping pearl active peptide Nacrein-LP20 and its application. The Heping pearl active polypeptide Nacrein-LP20 provided by the present invention has strong ability to scavenge DPPH free radicals and hydroxyl free radicals, and has certain application value in antioxidant aspects. The Heping pearl active polypeptide Nacrein-LP20 provided by the present invention has a significant protective effect on oxidative stress of human lens cells, can improve the cataract lesions of selenium-induced rats, and has certain application value in the treatment of cataract. Description of the Drawings

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0011] Figure 1 It is the structure diagram, high performance liquid chromatography and mass spectrometry analysis diagram of Nacrein-LP20 polypeptide from Hepu pearl. Among them, A is the structure diagram of Nacrein-LP20 polypeptide from Hepu pearl; B is the high performance liquid chromatography analysis diagram; C is the mass spectrometry analysis diagram.

[0012] Figure 2 It is the hemolytic activity of Nacrein-LP20 polypeptide from Hepu pearl. Among them, 1 is the PBS treatment group; 2 is the 1% TritonX-100 treatment group; 3-12 are the Nacrein-LP20 polypeptide treatment groups with concentrations of 1280-2.5 μg / mL.

[0013] Figure 3 It is the antioxidant activity of Nacrein-LP20 polypeptide from Hepu pearl. Among them, A is the standard curve of the standard product Trolox; B is the total antioxidant capacity of Nacrein-LP20 polypeptide from Hepu pearl; C is the ability of Nacrein-LP20 polypeptide to scavenge hydroxyl radicals; D is the ability of Nacrein-LP20 polypeptide to scavenge DPPH radicals.

[0014] Figure 4 It is the toxicity of Nacrein-LP20 polypeptide and H2O2 to human lens epithelial cells SRA01 / 04. Among them, A is the evaluation of the effect on cell viability after treating human lens epithelial cells with different concentrations of Nacrein-LP20 for 24 h; B is the evaluation of the effect on cell viability after treating human lens epithelial cells with different concentrations of H2O2 for 24 h; C is the evaluation of the effect on cell viability after treating human lens epithelial cells with different concentrations of H2O2 for 48 h. * indicates P < 0.05 compared with the control group (Nacrein-LP concentration is 0 μg / mL or H2O2 concentration is 0 μM).

[0015] Figure 5To investigate the protective effect of Nacrein-LP20 from Haliotis cumingii on oxidative damage of human lens epithelial cells induced by H2O2. Among them, A shows the effect on cell viability after treating human lens epithelial cells with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 24 h; B shows the effect on cell viability after treating human lens epithelial cells with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 48 h. * indicates P < 0.05 compared with the control group. # indicates P < 0.05 compared with the H2O2 group.

[0016] Figure 6 To detect the change of ROS level in human lens epithelial cells by flow cytometry. Among them, A shows the intracellular ROS level after treating human lens epithelial cells with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 48 h by flow cytometry; B shows the statistical chart of the average fluorescence intensity in cells of different groups. * indicates P < 0.05 compared with the control group. # indicates P < 0.05 compared with the H2O2 group.

[0017] Figure 7 To detect the change of apoptosis level in human lens epithelial cells by flow cytometry. Among them, A shows the apoptosis level after treating human lens epithelial cells with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 48 h by flow cytometry; B shows the statistical chart of the apoptosis rate of cells in different groups. * indicates P < 0.05 compared with the control group. # indicates P < 0.05 compared with the H2O2 group.

[0018] Figure 8 To observe the morphology of human lens epithelial cells by scanning electron microscopy.

[0019] Figure 9 To detect the contents of GSH-Px (A), SOD (B) and MDA (C) in human lens epithelial cells by biochemical assay. * indicates P < 0.05 compared with the control group; # indicates P < 0.05 compared with the H2O2 group.

[0020] Figure 10 To investigate the therapeutic effect of Nacrein-LP20 polypeptide from Haliotis cumingii on selenium-induced cataract in rats. A shows the change of the lens observed by fundus microscopy in the selenium-induced cataract rat model; B shows the therapeutic effect of the drug on selenium-induced cataract in rats. Detailed implementation manners

[0021] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0025] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0026] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified, and the reagents or raw materials used are all purchased from commercial channels or have been made public unless otherwise specified.

[0027] An embodiment of the present invention provides the application of the Hepu pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating cataracts, and the amino acid sequence of the Hepu pearl Nacrein-LP20 polypeptide is as shown in SEQ ID NO.1.

[0028] An embodiment of the present invention also provides a drug for treating cataracts, comprising the Hepu pearl Nacrein-LP20 polypeptide.

[0029] In some specific embodiments, it further comprises a pharmaceutically acceptable excipient.

[0030] In some specific embodiments, the drug is in a liquid dosage form.

[0031] In some specific embodiments, the cataract is a selenium-induced cataract lesion.

[0032] The embodiments of the present invention also provide the use of Hepu Pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating oxidative stress in lens cells.

[0033] Most cataracts develop after birth. While some non-modifiable and modifiable risk factors can accelerate cataract formation, aging and oxidative stress are the primary causes. A growing body of research indicates that Traditional Chinese Medicine (TCM) holds a unique position in the global anti-aging field, demonstrating its potential to slow and treat aging and age-related diseases. The Daoist Canon, dating from the Kaiyuan period of the Tang Dynasty, refers to "Dendrobium, Tianshan Snow Lotus, Three-ounce Ginseng, 120-year-old Polygonum multiflorum, Sixty-year-old Poria cocos, Wild Ganoderma Lucidum, Sea Pearl, Cordyceps, and Cistanche deserticola" as the Nine Immortal Herbs of China. Among them, seabed pearls, also known as Hepu pearls, are produced in abundance in Beihai, Guangxi Zhuang Autonomous Region, and are particularly famous. They have a long history of medicinal use and were first recorded in the "Compendium of Materia Medica" during the Southern and Northern Dynasties: "If you suffer from eye pain, take real pearls and coptis chinensis and put them in it. After a long time, the juice will come out. Take it and look at the eyes, and the pain will be cured." The 2020 edition of the "Pharmacopoeia of the People's Republic of China" also records the functions and main indications of pearls: "Calming the mind and calming the nerves, improving eyesight and removing cataracts, detoxifying and regenerating tissues, moisturizing the skin and removing spots. It is used for palpitations, insomnia, convulsions, epilepsy, red eyes, cataracts, sores that do not heal, and skin spots." This shows that pearls have unique effects on eye diseases, but the specific therapeutic effect and mechanism of action for cataracts are still unclear. Therefore, the present invention explores the biological activity and antioxidant damage of human lens epithelial cells from the active polypeptide Nacrein-LP20 excavated from Hepu pearls, and clarifies its mechanism of action, as well as its effect on improving cataracts in rats, providing a solid scientific basis for the further development of safer and more effective pearl-based cataract treatment drugs.

[0034] The Nacrein-LP20 active peptide from Pinctada fucata martensii provided by the present invention has strong in vitro antioxidant activity and almost no hemolytic activity. An oxidative damage model was constructed by treating human lens epithelial cells SRA01 / 04 with H2O2 (400 μM) for 48 h. The cells were grouped for modeling and treated with Nacrein-LP20 polypeptide from Pinctada fucata martensii at different concentrations (160, 320, 640 μg / mL) for 48 h. CCK8 was used to detect the cell proliferation ability, flow cytometry was used to detect the apoptosis and the content of reactive oxygen species (ROS) in each group of cells, biochemical methods were used to detect the content of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) in each group of cells, and scanning electron microscopy was used to observe the morphological changes of cells, so as to explore the therapeutic effect of Nacrein-LP20 from Pinctada fucata martensii on the selenium-induced cataract rat model. The results showed that after the cells were treated with H2O2, the cell proliferation, the content of GSH-Px and SOD decreased, the apoptosis rate, the content of ROS and MDA increased significantly, and the normal cell morphology could not be observed by scanning electron microscopy. After treatment with Nacrein-LP20 polypeptide from Pinctada fucata martensii at different concentrations, the cell proliferation, apoptosis, the content of ROS, and the content of GSH-Px, SOD and MDA were all restored, and the cell morphology also returned to normal. And Nacrein-LP20 from Pinctada fucata martensii has an improving effect on the cataract lesions of rat eyes. It is indicated that the Nacrein-LP20 polypeptide from Pinctada fucata martensii has a certain protective effect on the oxidative damage of SRA01 / 04 cells induced by H2O2 and has an improving effect on selenium-induced cataract in rats.

[0035] IBM SPSS Statistics 19.0, a statistical software, was used for data analysis, and GraphPad Prism8 software was used for statistical mapping. All experiments were repeated 3 times, and the quantitative results were expressed as mean ± standard deviation (X±S). The independent sample t-test was used for comparing the quantitative values between two groups, and one-way analysis of variance was used for comparing the quantitative values among multiple groups. In all tests, P<0.05 was considered to be a significant difference and was statistically significant, which was indicated by "*" or "#".

[0036] Example 1

[0037] Preparation of Nacrein-LP20 polypeptide from Pinctada fucata martensii

[0038] The Hepu pearl Nacrein-LP20 polypeptide (SEQ ID NO.1: 'GENGHKHGCRVKKAKHLSRI') was synthesized by Wuhan Tiande Biotechnology Co., Ltd. (Wuhan, China) using the solid-phase method, with a purity > 95%. Nacrein-LP20 was dissolved in phosphate buffer PBS to prepare different concentrations for subsequent experiments.

[0039] The structure, high-performance liquid chromatography and mass spectrometry analysis of the Hepu pearl Nacrein-LP20 polypeptide are as Figure 1 shown.

[0040] Example 2

[0041] Detection of the hemolytic activity of the Hepu pearl Nacrien-LP20 polypeptide

[0042] The hemolytic activity was detected using 2% porcine red blood cells. Hemolytic activity detection: Nacrein-LP20 polypeptide was diluted to concentrations of 2560 μg / mL, 1280 μg / mL, 640 μg / mL, 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL using PBS according to the serial dilution method. 100 μL of the polypeptide solution was taken and added to a 96-well plate as the experimental group. Another 100 μL of PBS and 1.0% Triton X-100 were used as the negative control and positive control, respectively. 100 μL of 2% red blood cell suspension was added to each well, with 3 replicates in each group, so that the final concentration of the polypeptide was 1280 - 2.5 μg / mL. Incubate in a 37 °C incubator for 3 h, centrifuge at 1500 rpm for 10 min, take 150 μL of the supernatant and transfer it to a new 96-well plate, measure the absorbance at 450 nm, and calculate the hemolysis rate.

[0043] Hemolysis rate = [OD of the experimental group 450 - OD of the negative control group 450 / [OD of the positive control group 450 - OD of the negative control group 450 × 100%.

[0044] The results are as Figure 2 shown. Column 1 is the negative control, with no hemolytic activity; column 2 is the positive control, showing obvious hemolysis; columns 3 - 12 are the Hepu pearl Nacrein-LP20 polypeptide groups with final concentrations of 1280 - 2.5 μg / mL, and their hemolytic activity is almost negligible.

[0045] Example 3

[0046] Detection of the antioxidant activity of the Hepu pearl Nacrein-LP20 polypeptide

[0047] The total antioxidant capacity of Nacrein-LP20 polypeptide from Hepu pearls was detected using the Total Antioxidant Capacity Detection Kit (ABTS rapid method) (Product No.: S0121) from Beyotime; the abilities of Nacrein-LP20 polypeptide from Hepu pearls to scavenge hydroxyl radicals and DPPH radicals were detected using the Hydroxyl Radical Test Kit (A018-1-1) and DPPH Radical Scavenging Capacity Kit (A153-1-1) from Nanjing Jiancheng Bioengineering Institute. Vitamin C at the same concentration was set as the positive control.

[0048] The results are as Figure 3 shown. The standard curve equation of the standard product Trolox was y = 1.2593x - 0.0868, with R 2 = 0.9911. Therefore, according to the standard curve equation, the total antioxidant capacity of the sample to be tested can be calculated. When the concentration of Nacrein-LP20 polypeptide from Hepu pearls was 0.25 mg / mL, its total antioxidant capacity was equivalent to 1.7861 mM Trolox. Moreover, Nacrein-LP20 polypeptide from Hepu pearls had significant abilities to scavenge hydroxyl radicals and DPPH radicals, almost equivalent to those of vitamin C, and its IC 50 values were 151.36 μg / mL and 1.39 μg / mL respectively, indicating that Nacrein-LP20 polypeptide from Hepu pearls had strong antioxidant activity in vitro.

[0049] Example 4

[0050] CCK-8 assay

[0051] SRA01 / 04 human lens epithelial cells (iCell-h408, Cyagen Biosciences (Shanghai) Inc.) (STR identified) were cultured in DMEM (low glucose) complete medium (containing 10% FBS, 1% penicillin-streptomycin) (KGM31600S-500, Jiangsu Kaygee Biotechnology Co., Ltd.) in a 37 °C, 5% CO2 incubator (BPN-80CW, Shanghai Yiheng Scientific Instrument Co., Ltd.). The SRA01 / 04 human lens epithelial cells were seeded into 96-well plates at 1×10 4Cells were used. The Nacrein-LP20 polypeptide from Pinctada fucata martensii was dissolved in DMEM (low glucose) complete medium to prepare a stock solution of 1 mg / mL. Cells were treated with different concentrations of Nacrein-LP20 polypeptide (0, 80, 160, 320, 640, 1280 μg / mL) and H2O2 (0, 200, 300, 400, 500, 600, 800, 1000 μmol / L) for 24 h and 48 h. The cells in the 96-well plate to be tested were replaced with the same medium, 100 μL per well, and 10 μL of CCK8 reagent (KGA317, Jiangsu KeyGen Biotech Co., Ltd.) was added to each well. The plate was incubated in an incubator for 4 h, and the absorbance of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader (SuPerMax3100, Shanghai Flash Spectrum Biotechnology Co., Ltd.). Human lens epithelial cells SRA01 / 04 were cultured and treated with H2O2 at a concentration of 400 μM combined with different concentrations of Nacrein-LP20 polypeptide (160, 320, 640 μg / mL) for 24 h and 48 h. CCK8 was used to detect the cell viability.

[0052] The results are as Figures 4 - 5 shown. Compared with the control group, the Nacrein-LP20 polypeptide had no significant effect on the cell viability of each group after treatment ( Figure 4 in A); for the H2O2 treatment model group, when the treatment time was 24 h, compared with the control group, the cell viability of the H2O2 groups with a concentration above 400 μM was significantly reduced; when the treatment time was 48 h, the cell viability of the H2O2 groups with a concentration above 200 μM was significantly reduced ( Figure 4 in B and C). Based on the above results, Nacrein-LP20 polypeptides with concentrations of 160, 320, and 640 μg / mL and H2O2 with a concentration of 400 μM were selected for subsequent experiments. The results of the protective effect of Nacrein-LP20 from Pinctada fucata martensii on H2O2-induced oxidative damage of human lens epithelial cells showed that compared with the control group, the cell viability of the H2O2 group was significantly reduced, and compared with the H2O2 group, the cell viability of the H2O2 + 640 μg / mL Nacrein-LP20 polypeptide group was significantly increased after treatment for 24 h and 48 h ( Figure 5 ).

[0053] Example 5

[0054] Flow cytometry was used to detect the intracellular reactive oxygen species level

[0055] Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 polypeptide (0, 160, 320, and 640 μg / mL) for 48 h. The cells were collected, and the culture medium was discarded. 10 μM DCFH-DA was added to each sample and incubated at 37 °C for 20 min, washed three times with serum-free medium, then washed with PBS, and the supernatant was discarded by centrifugation. After resuspending with 300 μL PBS, the change in the level of reactive oxygen species in the cells was detected by flow cytometry.

[0056] The results are as Figure 6 shown. Compared with the control group, the level of cellular ROS in the H2O2 group was significantly increased. Compared with the H2O2 group, the level of cellular ROS was significantly decreased after treatment with Nacrein-LP20 polypeptide.

[0057] Example 6

[0058] Detection of the apoptosis rate of cells by flow cytometry

[0059] Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 polypeptide (0, 160, 320, and 640 μg / mL) for 48 h, washed by centrifugation at 1500 rpm for 3 min with PBS, and repeated twice. 300 μL of pre-cooled 1× AnnexinV-FITC binding solution was taken to resuspend the cells, and 5 μL of AnnexinV-FITC (AP101-100-kit, MULTI SCIENCES) and 10 μL of PI (AP101-100-kit, MULTI SCIENCES) were added to each well. After gently mixing, it was incubated at room temperature in the dark for 10 min, and the apoptosis of the cells was detected by flow cytometry (NovoCyte2060R, ACEA Biosciences (Hangzhou) Co., Ltd.).

[0060] The results are as Figure 7 shown. Compared with the control group, the apoptosis rate of cells in the H2O2 group was significantly increased. Compared with the H2O2 group, the apoptosis rate of cells was significantly decreased after treatment with Nacrein-LP20 polypeptide.

[0061] Example 7

[0062] Observation by scanning electron microscope

[0063] Human lens epithelial cells SRA01 / 04 were seeded on sterile cover slips and placed in 6-well plates. After adhesion, a control group (normal group), a model group (hydrogen peroxide treatment group), and an experimental group (hydrogen peroxide plus low, medium, and high concentrations of Nacrein-LP20 polypeptide treatment groups) were set up, and incubation was continued for 48 h. The cell slides were gently rinsed with PBS, fixed in electron microscopy fixative at room temperature for 2 h, then placed at 4 °C, rinsed with 0.1 M phosphate buffer (pH 7.4), fixed with 1% osmium tetroxide in the dark at room temperature for 1 - 2 h, dehydrated and dried. Then the slides were adhered to the conductive carbon film double-sided tape and placed on the sample stage of an ion sputter (108Auto, Cressington) for gold spraying, and the morphological changes of the cells were observed under a scanning electron microscope (FEI Quanta250, FEI Company, USA).

[0064] The results were as Figure 8 shown. In the control group, the cell morphology was intact. In the H2O2 group, intact cell morphology could not be observed. After treatment with different concentrations of Nacrein-LP02 polypeptide, the cell morphology recovered to normal.

[0065] Example 8

[0066] Biochemical detection

[0067] Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 polypeptide (0, 160, 320, and 640 μg / mL) for 48 h. The cell pellets were collected, and the contents of GSH-PX (E-BC-K096-M, Elabscience), SOD (A001-3-2, Nanjing Jiancheng), and MDA (E-EL-0060c, Elabscience) in the cell pellets were detected using a biochemical kit. The samples were processed according to the kit instructions and measured with an enzyme-labeling instrument (WD-2012B, Beijing Liuyi).

[0068] The results were as Figure 9 shown. Compared with the control group, the content of GSH-Px decreased, the content of SOD decreased significantly, and the content of MDA increased significantly in the H2O2 treatment group. Compared with the H2O2 group, after treating the cells with different doses of Nacrein-LP20 polypeptide, the contents of GSH-Px and SOD in the cells increased significantly, and the content of MDA decreased significantly.

[0069] Example 9

[0070] Improvement effect of Nacrein-LP20 from Pinctada fucata on sodium selenite-induced cataract in rats

[0071] Ten-day-old neonatal rats were divided into 4 groups, with 4 rats in each group, namely the normal control group, the sodium selenite-induced cataract group in rats (model group), the model + Blancophthalmus group (positive group), and the model + Nacrein-LP20 polypeptide group (640 μg / mL).

[0072] The method for constructing the sodium selenite-induced cataract model in rats was as follows: SD male and female rats (10 weeks old) were raised at a temperature of 20 - 26 °C and a humidity of 40 - 70%, fed with the growth and reproduction feed for mice and rats, and given free access to water to allow them to reproduce. Neonatal rats in the model group at 10 days old were subcutaneously injected with 3.46 mg / kg of sodium selenite on the nape of the neck for model establishment, once every other day, for a total of 3 injections. The normal group was injected with an equal volume of physiological saline. Subsequently, the changes in the lens were observed with an ophthalmoscope to determine the successful establishment of the selenium-induced cataract model in SD rats.

[0073] The eyes were instilled with drugs once in the morning and once in the evening every day. The normal control group and the model group were instilled with physiological saline in the morning and evening every day, 0.05 mL each time, for 2 consecutive months; the model + Blancophthalmus group was instilled with Blancophthalmus Bingzhen Quyi Eye Drops in the morning and evening every day, 0.05 mL each time, for 2 consecutive months; the model + Nacrein-LP20 polypeptide group was instilled with a 640 μg / mL Nacrein-LP20 polypeptide solution in the morning and evening every day, 0.05 mL each time, for 2 consecutive months.

[0074] During this period, the body weights of the rats were recorded weekly. After 2 months, photographs were taken using a slit lamp to observe the improvement of cataracts.

[0075] The results were as Figure 10 shown. In the normal control group, the lens was transparent and there was no turbidity; while in the model group, turbidity of the lens was visible. Moreover, compared with the model group, the degree of lens turbidity in the model + Blancophthalmus group and the model + Nacrein-LP20 polypeptide group was significantly reduced, and the symptoms of cataracts were significantly improved. This indicates that pearl Nacrein-LP20 has a certain therapeutic and improving effect on selenium-induced cataracts in SD rats.

[0076] In summary, a derivative peptide segment Nacrein-LP20 was obtained from the nacre matrix protein Nacrein of Pinctada fucata martensii, synthesized using the solid-phase polypeptide synthesis method and identified using liquid chromatography-mass spectrometry (LC–MS). It has significant antioxidant activity in vitro, and its hemolytic activity is almost negligible. After treating human lens cells SRA01 / 04 with H2O2, the cell proliferation, GSH-Px and SOD contents decreased, while the apoptosis rate, ROS and MDA contents increased significantly, and the normal cell morphology could not be observed by scanning electron microscopy. After treatment with different concentrations of Nacrein-LP20 polypeptide from Pinctada fucata martensii, the cell proliferation, apoptosis, ROS content, and GSH-Px, SOD and MDA contents were all restored, and the cell morphology also returned to normal. Moreover, Nacrein-LP20 from Pinctada fucata martensii has an improving effect on cataract lesions in rat eyes.

[0077] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, rather than limiting the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. Use of Heping pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating cataract, characterized in that, The amino acid sequence of the Hepu pearl Nacrein-LP20 polypeptide is shown in SEQ ID NO.1.

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