Hepu pearl polypeptide Nacrein-LP20 for treating cataract and application of Hepu pearl polypeptide Nacrein-LP20

Abstract

The invention discloses a Hepu pearl polypeptide Nacrein-LP20 for treating cataract and application of the Hepu pearl polypeptide Nacrein-LP20, and belongs to the technical field of biological medicine. The amino acid sequence of the gene is as shown in SEQ ID NO. 1. The active polypeptide Nacrein-LP20 provided by the invention has strong capability of removing DPPH free radicals and hydroxyl free radicals, and has a certain application value in the aspect of oxidation resistance. The active polypeptide Nacrein-LP20 provided by the invention has a remarkable protective effect on oxidative stress of human lens cells, can improve the cataract lesion of selenium rats, and has a certain application value in the aspect of cataract treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a Hepu pearl polypeptide Nacrein-LP20 for treating cataracts and an application thereof. Background Art

[0002] Cataract is a disease in which the lens in the eye becomes cloudy, causing visual impairment. It is the leading cause of blindness worldwide and the second leading cause of moderate to severe visual impairment (MSVI) worldwide. According to statistics, 17.01 million people worldwide will be blinded by cataracts in 2020, and 84.48 million people will suffer from MSVI due to cataracts. Many factors can cause lens opacity, such as aging, genetics, local nutritional disorders, immune and metabolic disorders, radiation, long-term use of glucocorticoids, trauma, etc. Therefore, according to the cause of the disease, cataracts can be divided into congenital cataracts, senile cataracts, metabolic cataracts, and traumatic cataracts. The most common of these is senile cataracts, especially as the global population ages, its prevalence is gradually increasing, and it has become one of the global public health issues. Therefore, the treatment of senile cataracts is the most urgent issue in the field of cataract treatment. However, its pathogenesis is not yet fully understood, and there is still a lack of effective therapeutic drugs in clinical practice. Currently, surgical treatment is the only cure. Generally, mature cataracts can be treated surgically, but mild cataracts are not recommended. Moreover, a variety of complications may still occur after surgical treatment, including postoperative intraocular inflammation, tearing of the lens capsule, retinal detachment, etc., as well as the lack of surgical facilities in poor and developing countries. These factors limit the availability and accessibility of cataract surgery worldwide. Therefore, it is of great significance to explore the pathogenesis of cataracts and find economical and effective cataract prevention and treatment drugs. Summary of the invention

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

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

[0005] One of the technical solutions of the present invention is the use of 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 shown in SEQ ID NO.1.

[0006] The second technical solution of the present invention is a drug for treating cataracts, comprising the Hepu Pearl Nacrein-LP20 polypeptide.

[0007] The third technical solution of the present invention is the use of Hepu Pearl Nacrein-LP20 polypeptide in the preparation of drugs for treating oxidative stress of lens cells.

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

[0009] The present invention provides a Hepu pearl active peptide Nacrein-LP20 and its application. The Hepu pearl active peptide Nacrein-LP20 provided by the present invention has a strong ability to scavenge DPPH free radicals and hydroxyl free radicals, and has certain application value in anti-oxidation. The Hepu pearl active peptide Nacrein-LP20 provided by the present invention has a significant protective effect on oxidative stress of human lens cells, can improve selenium-induced cataract lesions in rats, and has certain application value in the treatment of cataracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0011] Figure 1 The following are the structure of Hepu pearl Nacrein-LP20 polypeptide and the high performance liquid chromatography and mass spectrometry analysis diagrams. A is the structure diagram of Hepu pearl Nacrein-LP20 polypeptide; B is the high performance liquid chromatography analysis diagram; C is the mass spectrometry analysis diagram.

[0012] Figure 2 The hemolytic activity of Hepu Pearl Nacrein-LP20 polypeptide. 1 is the PBS treatment group; 2 is the 1% TritonX-100 treatment group; 3-12 are the 1280-2.5μg / mL Nacrein-LP20 polypeptide treatment groups.

[0013] Figure 3 It is the antioxidant activity of Hepu Pearl Nacrein-LP20 polypeptide. Among them, A is the standard curve of standard substance Trolox; B is the total antioxidant capacity of Hepu Pearl Nacrein-LP20 polypeptide; C is the hydroxyl radical scavenging ability of Nacrein-LP20 polypeptide; D is the DPPH free radical scavenging ability of Nacrein-LP20 polypeptide.

[0014] Figure 4The toxicity of Nacrein-LP20 peptide and H2O2 on human lens epithelial cells SRA01 / 04. A is the effect of different concentrations of Nacrein-LP20 on human lens epithelial cells after 24 hours of treatment, and the effect of different concentrations of H2O2 on cell viability is evaluated; B is the effect of different concentrations of H2O2 on human lens epithelial cells after 24 hours of treatment, and the effect of different concentrations of H2O2 on cell viability is evaluated after 48 hours of treatment. * indicates that compared with the control group (Nacrein-LP concentration is 0μg / mL or H2O2 concentration is 0μM), P<0.05.

[0015] Figure 5 The protective effect of Hepu Pearl Nacrein-LP20 on H2O2-induced oxidative damage in human lens epithelial cells. A is the effect of H2O2 alone or combined with different concentrations of Nacrein-LP20 on human lens epithelial cells after 24 hours of treatment, and B is the effect of H2O2 alone or combined with different concentrations of Nacrein-LP20 on human lens epithelial cells after 48 hours of treatment, and the effect of H2O2 alone or combined with different concentrations of Nacrein-LP20 on cell viability. * indicates P<0.05 compared with the control group. # indicates P<0.05 compared with the H2O2 group.

[0016] Figure 6 The figure shows the changes of ROS levels in human lens epithelial cells measured by flow cytometry. A shows the intracellular ROS levels of human lens epithelial cells treated with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 48 hours by flow cytometry; B shows the statistical graph of the intracellular mean fluorescence intensity of different groups. * indicates P<0.05 compared with the control group. # indicates P<0.05 compared with the H2O2 group.

[0017] Figure 7 The changes in the apoptosis level of human lens epithelial cells were measured by flow cytometry. A is the apoptosis level of human lens epithelial cells treated with H2O2 alone or in combination with different concentrations of Nacrein-LP20 for 48 hours by flow cytometry; B is the statistical chart of cell apoptosis rate in different groups. * indicates P<0.05 compared with the control group. # indicates P<0.05 compared with the H2O2 group.

[0018] Figure 8 The morphology of human lens epithelial cells was observed under scanning electron microscopy.

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

[0020] Fig.10 The therapeutic effect of Hepu Pearl Nacrein-LP20 peptide on selenium-induced cataract in rats. A is the changes of lens observed by fundus ophthalmoscopy in rat selenium-induced cataract model; B is the therapeutic effect of the drug on selenium-induced cataract in rats. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

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

[0027] The embodiment of the present invention provides the use of Hepu Pearl Nacrein-LP20 polypeptide in the preparation of a drug for treating cataracts. The amino acid sequence of the Hepu Pearl Nacrein-LP20 polypeptide is shown in SEQ ID NO.1.

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

[0029] In some specific embodiments, a pharmaceutically acceptable excipient is also included.

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

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

[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 of lens cells.

[0033] Most cataract cases occur after birth. Although some unchangeable and changeable risk factors can accelerate the formation of cataracts, aging and oxidative stress are the main causes. More and more studies have shown that traditional Chinese medicine is unique in the world's anti-aging field and has a certain delaying and therapeutic effect on aging and aging-related diseases. In the Daoist Canon during the Kaiyuan period of the Tang Dynasty in China, "Dendrobium, Tianshan Snow Lotus, Three-liang Ginseng, 120-year-old Polygonum Multiflorum, Sixty-year-old Poria, Deep Mountain Wild Ganoderma, Seabed Pearls, Cordyceps Sinensis, and Cistanche" were collectively called the Nine Great Immortal Herbs of China. The seabed pearls, also known as Hepu pearls, are produced in abundance in Beihai, Guangxi, 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 and be focused on your eyes, and most of them will be cured." The 2020 edition of the "Pharmacopoeia of the People's Republic of China" also records the functions and indications of pearls: "Calming the mind and calming the nerves, improving eyesight and removing cataracts, detoxifying and regenerating tissue, moisturizing the skin and removing freckles. 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 of the active polypeptide Nacrein-LP20 excavated from Hepu pearls, and clarifies its mechanism of action, as well as its improvement effect on cataracts in rats, to provide a solid scientific basis for the further development of safer and more effective pearl-based cataract treatment drugs.

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

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

[0036] Example 1

[0037] Preparation of Hepu Pearl Nacrein-LP20 Peptide

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

[0039] The structure of Hepu pearl Nacrein-LP20 peptide and its HPLC and MS analysis Figure 1 shown.

[0040] Example 2

[0041] Hemolytic activity test of Hepu Pearl Nacrien-LP20 peptide

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

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

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

[0045] Example 3

[0046] Antioxidant activity test of Hepu pearl Nacrein-LP20 peptide

[0047] The total antioxidant capacity of Hepu Pearl Nacrein-LP20 polypeptide was detected using the Biyuntian Total Antioxidant Capacity Test Kit (ABTS Rapid Method) (Product No.: S0121). The hydroxyl radical test kit (A018-1-1) and DPPH free radical scavenging capacity kit (A153-1-1) of Nanjing Jiancheng Bioengineering Institute were used to detect the scavenging hydroxyl and DPPH free radicals of Hepu Pearl Nacrein-LP20 polypeptide, and the positive control was set to the same concentration of vitamin C.

[0048] The results are as follows Figure 3 As shown, the standard curve equation of standard Trolox is y=1.2593x-0.0868, 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 Hepu Pearl Nacrein-LP20 polypeptide is 0.25mg / mL, its total antioxidant capacity is equivalent to 1.7861mM Trolox. In addition, Hepu Pearl Nacrein-LP20 polypeptide has a significant ability to scavenge hydroxyl free radicals and DPPH free radicals, which is almost equivalent to vitamin C. Its IC 50 The concentrations of Nacrein-LP20 peptide from Hepu Pearl were 151.36 μg / mL and 1.39 μg / mL, respectively, indicating that the Nacrein-LP20 peptide from Hepu Pearl has strong antioxidant activity in vitro.

[0049] Example 4

[0050] CCK-8 assay

[0051] SRA01 / 04 human lens epithelial cells (iCell-h408, Cybio (Shanghai) Biotechnology Co., Ltd.) (STR identified) were cultured in DMEM (low glucose) complete medium (containing 10% FBS, 1% penicillin-streptomycin) (KGM31600S-500, Jiangsu KeyGen Biotechnology Co., Ltd.) in a 37°C 5% CO2 incubator (BPN-80CW, Shanghai Yiheng Scientific Instrument Co., Ltd.). SRA01 / 04 human lens epithelial cells were seeded into 96-well plates, with 1×10 per well. 4cells, and the Hepu Pearl Nacrein-LP20 polypeptide was dissolved in DMEM (low sugar) complete medium to prepare a 1 mg / mL stock solution. The 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, respectively. The 96-well plate cells to be tested were replaced with the same culture medium, 100 μL per well, and 10 μL CCK8 reagent (KGA317, Jiangsu Keygen Biotechnology Co., Ltd.) was added to each well, and the cells were placed in an incubator and incubated for 4 h. The absorbance of each well was detected at a wavelength of 450 nm by an enzyme reader (SuPerMax3100, Shanghai Shanpu Biotechnology Co., Ltd.). Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 peptide (160, 320, 640 μg / mL) for 24 h and 48 h. CCK8 was used to detect cell survival rate.

[0052] The results are as follows Figures 4-5 As shown, compared with the control group, the cell survival rate of each group was not significantly affected after treatment with Nacrein-LP20 peptide ( Figure 4 For the H2O2 treated model group, when the cells were treated for 24 hours, the cell survival rate of the H2O2 group with a concentration of more than 400 μM was significantly reduced compared with the control group; when the cells were treated for 48 hours, the cell survival rate of the H2O2 group with a concentration of more than 200 μM was significantly reduced ( Figure 4 B, C). Based on the above results, Nacrein-LP20 peptides 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 Hepu Pearl Nacrein-LP20 on H2O2-induced oxidative damage of human lens epithelial cells showed that compared with the control group, the cell survival rate in the H2O2 group was significantly reduced. Compared with the H2O2 group, the cell survival rate of the H2O2 plus 640 μg / mL Nacrein-LP20 peptide group treated for 24h and 48h was significantly increased ( Figure 5 ).

[0053] Example 5

[0054] Flow cytometry to detect intracellular reactive oxygen species levels

[0055] Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 peptide (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 the cells were incubated at 37°C for 20 min. The cells were washed three times with serum-free culture medium and then with PBS, and the supernatant was discarded after centrifugation. After resuspending in 300 μL PBS, the changes in cellular reactive oxygen levels were detected by flow cytometry.

[0056] The results are as follows Figure 6 As shown, compared with the control group, the cellular ROS level in the H2O2 group was significantly increased, and compared with the H2O2 group, the cellular ROS level after Nacrein-LP20 peptide treatment was significantly decreased.

[0057] Example 6

[0058] Flow cytometry to detect cell apoptosis rate

[0059] Human lens epithelial cells SRA01 / 04 were cultured, treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 peptide (0, 160, 320 and 640 μg / mL) for 48 h, washed with PBS by centrifugation at 1500 rpm for 3 min, 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 Lianke Bio) and 10 μL PI (AP101-100-kit, MULTI SCIENCES Lianke Bio) were added to each well; after slight mixing, incubated at room temperature in the dark for 10 min, and cell apoptosis was detected by flow cytometry (NovoCyte2060R, Aisen Bio (Hangzhou) Co., Ltd.).

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

[0061] Example 7

[0062] Scanning electron microscopy observation

[0063] Human lens epithelial cells SRA01 / 04 were inoculated on sterile coverslips 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 Nacrein-LP20 peptide treatment group) were set up, and the incubation was continued for 48 hours. The cell slides were gently rinsed with PBS, placed in an electron microscope fixative and fixed at room temperature for 2 hours, and then placed at 4°C, rinsed with 0.1M phosphate buffer (pH 7.4), fixed with 1% osmium acid at room temperature in the dark for 1-2 hours, dehydrated and dried, and then the slides were closely attached to the conductive carbon film double-sided tape and placed on the sample stage of an ion sputtering instrument (108Auto, Cressington) for gold spraying, and the cell morphology changes were observed under a scanning electron microscope (FEIQuanta250, FEI Company, USA).

[0064] The results are as follows Figure 8 As shown, the cell morphology was intact in the control group, but no complete cell morphology could be observed in the H2O2 group. After treatment with different concentrations of Nacrein-LP02 polypeptide, the cell morphology returned to normal.

[0065] Example 8

[0066] Biochemical testing

[0067] Human lens epithelial cells SRA01 / 04 were cultured and treated with 400 μM H2O2 combined with different concentrations of Nacrein-LP20 peptide (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, Elaruite), SOD (A001-3-2, Nanjing Jiancheng) and MDA (E-EL-0060c, Elaruite) in the cell pellets were detected using biochemical kits. The samples were processed and measured using a microplate reader (WD-2012B, Beijing Liuyi) according to the instructions of the kit.

[0068] The results are as follows Fig. 9 As shown, compared with the control group, the GSH-Px content in the H2O2 treatment group was decreased, the SOD content was significantly decreased, and the MDA content was significantly increased; compared with the H2O2 group, after the cells were treated with different doses of Nacrein-LP20 peptide, the intracellular GSH-Px and SOD contents were significantly increased, and the MDA content was significantly decreased.

[0069] Example 9

[0070] Improvement of Nacrein-LP20 from Hepu Pearl on Sodium Selenite-induced Cataract in Rats

[0071] The 10-day-old rats were divided into 4 groups, with 4 rats in each group, including the normal control group, the sodium selenite-induced cataract group (model group), the model plus cataract group (positive group), and the model plus Nacrein-LP20 peptide group (640 μg / mL).

[0072] The method for constructing the sodium selenite-induced rat cataract model is as follows: SD male and female rats (10 weeks old) are raised at a temperature of 20-26°C, a humidity of 40-70%, and are fed with rat and mouse growth and breeding feed, and are allowed to drink water freely to breed. The 10-day-old offspring rat model group is given 3.46 mg / kg sodium selenite subcutaneously in the neck and back for modeling, and the injection is given once every other day for a total of 3 injections. The normal group is injected with an equal amount of normal saline, and then the changes in the lens are observed with an ophthalmoscopy to determine that the SD rat selenium cataract model is successfully constructed.

[0073] The drugs were dropped into the eyes once in the morning and evening every day. The normal control group and the model group were dripped with normal saline every morning and evening, 0.05 mL each time, for 2 consecutive months; the model plus Baineting group was dripped with Baineting Bingzhen Quyi eye drops every morning and evening, 0.05 mL each time, for 2 consecutive months; the model plus Nacrein-LP20 polypeptide group was dripped with 640 μg / mL Nacrein-LP20 polypeptide solution every morning and evening, 0.05 mL each time, for 2 consecutive months.

[0074] The body weight of the rats was recorded every week during this period, and photos were taken using a slit lamp after 2 months to observe the improvement of cataracts.

[0075] The results are as follows Fig.10 As shown, the normal control group showed transparent lens without turbidity, while the model group showed turbidity in the lens. Compared with the model group, the model plus Nacrein-LP20 peptide group and the model plus Nacrein-LP20 peptide group showed significantly reduced turbidity in the lens, and significantly improved cataract symptoms. This indicates that Nacrein-LP20 has a certain therapeutic effect on selenium-induced cataract in SD rats.

[0076] In summary, the present invention obtains a derivative peptide segment Nacrein-LP20 from the Hepu pearl matrix protein Nacrein protein, synthesized by solid phase peptide synthesis method and identified by liquid chromatography-mass spectrometry (LC–MS). It has significant antioxidant activity in vitro, and its hemolytic activity is almost negligible. After H2O2 treatment of human lens cells SRA01 / 04, cell proliferation, GSH-Px and SOD content are all decreased, apoptosis rate, ROS and MDA content are significantly increased, and the normal cell morphology cannot be observed by scanning electron microscopy. After being treated with different concentrations of Hepu pearl Nacrein-LP20 polypeptide, cell proliferation, apoptosis, ROS content, GSH-Px, SOD and MDA content are all restored, and the cell morphology is also restored to normal. And Hepu pearl Nacrein-LP20 has an improvement 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, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

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

1.

2. A drug for treating cataracts, characterized in that: It includes the Hepu pearl Nacrein-LP20 polypeptide as described in claim 1.

3. The drug according to claim 3, characterized in that Pharmaceutically acceptable excipients are also included.

4. The drug according to claim 3, characterized in that The drug is in liquid dosage form.

5. The drug according to claim 3, characterized in that The cataract is a selenium cataract lesion.

6. Application of Hepu Pearl Nacrein-LP20 polypeptide in the preparation of drugs for treating oxidative stress in lens cells.

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