CN115737781A - New application and medicine of lycium barbarum glycopeptide - Google Patents
New application and medicine of lycium barbarum glycopeptide Download PDFInfo
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- CN115737781A CN115737781A CN202210591023.9A CN202210591023A CN115737781A CN 115737781 A CN115737781 A CN 115737781A CN 202210591023 A CN202210591023 A CN 202210591023A CN 115737781 A CN115737781 A CN 115737781A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The application discloses application of lycium barbarum glycopeptide in preparation of a medicine for preventing and treating tear film injury and/or corneal epithelial cell injury. The present application also provides a medicament. Experiments prove that the Lycium Barbarum Glycopeptide (LBGP) has the effect of repairing the damage of the tear film and the corneal epithelium, and has the effects of promoting the repair of the corneal epithelium, increasing the tear secretion and the tear MUC5AC content, promoting the recovery of the corneal sensory nerve function, regenerating nerve fibers and inhibiting inflammation as an eye drop treatment-induced mouse tear film and corneal epithelium damage model.
Description
Technical Field
The application relates to the technical field of medicines, in particular to a new application and a medicine of lycium barbarum glycopeptide.
Background
The tear film is the interface between the ocular surface epithelium and the environment, and its primary function is to maintain optimal extracellular structure, pH, oxygen and carbon dioxide levels, and in addition, to provide nutrients to the cornea, playing an important role in protecting the ocular surface, lubricating cleanliness, and maintaining visual stability.
The corneal epithelium is located in the most superficial layer of the cornea and consists of 4-6 layers of non-keratinizing squamous epithelial cells. The corneal epithelium is a physiological barrier that plays a critical role in protecting the eye, and is the first line of defense against external damage behind the tear film.
Damage to the tear film and corneal epithelium can cause the patient to develop the following symptoms: (1) ocular irritation symptoms: the eye drops are easy to be seen in fatigue, dry and astringent, photophobia and lacrimation, red eyes, itchy eyes, stabbing pain of eyes, foreign body sensation, thick secretion and sensitive to external stimulation, but some patients may have slight subjective symptoms due to abnormal function of corneal nerves. (2) visual function-related symptoms: blurred vision, fluctuating or declining vision. These discomfort symptoms can reduce the quality of life of the patient and, in severe cases, can cause visual impairment.
At present, eye drops for treating tear film and corneal epithelium injury mainly comprise artificial tears, growth factor eye drops, glucocorticoid, immunosuppressant, non-steroidal anti-inflammatory drugs and the like, and have the problems of single action or obvious toxic and side effects on the surface of eyes.
Therefore, the development of an eye drop solution for treating tear film and corneal epithelial damage is a technical problem to be solved by those skilled in the art.
Disclosure of Invention
In order to solve the technical problems, the invention provides an application of lycium barbarum glycopeptide in preparing a medicament for preventing and treating tear film damage and/or corneal epithelial cell damage; a second object of the present invention is to provide a medicament; experiments prove that the Lycium Barbarum Glycopeptide (LBGP) has the effect of repairing damages of a tear film and a corneal epithelium, and has the effects of promoting the repair of the corneal epithelium, increasing the content of lacrimal secretion and lacrimal MUC5AC, promoting the recovery of the function of a corneal sensory nerve, regenerating nerve fibers and inhibiting inflammation as an eye drop treatment-induced mouse tear film and corneal epithelium damage model.
The technical scheme provided by the invention is as follows:
application of lycium barbarum glycopeptide in preparing medicine for preventing and treating tear film injury and/or corneal epithelial cell injury.
Preferably, the controlling comprises: improving corneal epithelial cell activity, improving corneal turbidity, improving corneal epithelial integrity, increasing tear secretion, prolonging tear film rupture time, increasing tear MUC5AC content, promoting corneal sensory nerve function recovery, promoting corneal nerve fiber regeneration, reducing mRNA expression level of corneal inflammation factor IL-1 beta, and reducing mRNA expression level of corneal inflammation factor TNF-alpha.
Preferably, the lycium barbarum glycopeptide is administered by eye drop administration.
A medicament for the prevention and treatment of tear film damage and/or corneal epithelial cell damage comprises an effective amount of a glycopeptide of Lycium barbarum.
Preferably, the dosage form of the medicament is eye drops.
Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.
Lycium Barbarum Glycopeptide (LBGP), the main active ingredient of Lycium barbarum, is a water-soluble glycoprotein with a sugar content of 70% and molecular weights of 72kDa and 88kDa, respectively, as measured by sugar and protein standards, and has a sugar composition Ara: gal: glc = 2.5. The invention discloses a preparation method of raw material LBGP powder (CN 107021995B-lycium barbarum glycopeptide and a preparation method and application thereof). The invention removes partial impurities by a temperature-rising flocculation method, replaces the step of using a large amount of ethanol for precipitation in the traditional extraction method, obviously improves the uronic acid content of the obtained lycium barbarum glycopeptide, and has obvious immunocompetence. The existing research shows that LBGP has the effects of regulating the immune function of an organism, resisting oxidation, inflammation, aging, tumor, blood sugar and blood fat and the like, but the effect of LBGP on tear film and corneal epithelium is not researched.
The invention utilizes the characteristic that benzalkonium chloride (BAK) has toxic injury effect on the tear film and the corneal epithelium to prepare a cell model of corneal epithelium injury and an animal model of tear film and corneal epithelium injury. Firstly, carrying out in-vitro cell experiments, and primarily exploring the protective effect of LBGP on corneal epithelial injury; and performing in-vivo experiments, and using the LBGP eye drops to treat the mouse model with the damaged tear film and the damaged corneal epithelium, thereby providing important reference for the LBGP serving as the eye drops to be applied to clinically treating the diseases of the damaged tear film and the damaged corneal epithelium.
Specifically, the tear film is on the surface of the corneal epithelium, which can be divided into three layers: a lipid layer on the surface, an aqueous liquid layer in the middle and a mucin layer at the bottom. The lipid layer is produced by the meibomian glands and its primary function is to reduce the surface tension of the tear film; the aqueous layer is produced by the lacrimal and accessory lacrimal glands, which act as a smooth surface with refraction enhancing light, while lubricating the ocular surface to prevent conjunctival corneal desiccation; the mucin layer is produced by conjunctival goblet cells and ocular surface epithelial cells, forming a gel-like structure that allows the aqueous layer to re-diffuse after each blink. Tear film damage in this study was mainly due to the toxic effects of BAK itself on tear film due to: first, BAK is a cationic molecular compound that can electrostatically aggregate with glycogenase anionic polysaccharide molecules, can penetrate the lipid membrane, alter the structure and properties of the lipid membrane, and cause instability of the tear film; second, BAK can also produce toxicity by inducing ocular surface inflammation; third, BAK has a toxic effect on conjunctival goblet cells, while the mucin layer of the tear film is mainly produced by conjunctival goblet cells. Fourth, the tear film has a certain protective effect on the corneal epithelium, and the damage of the tear film can cause the corneal epithelial cells to be more easily damaged, and the damage of the corneal epithelium can further damage the tear film.
Meanwhile, BAK has damage effect on corneal epithelial cells due to the reasons that BAK can promote inflammatory cell and inflammatory factor infiltration such as TNF-alpha and the like, promote corneal epithelial K10 expression to cause corneal epithelial squamous epithelization, and KI-67 expression is down-regulated, so that corneal epithelial proliferation is inhibited, corneal epithelial apoptosis is promoted, cells have toxicity effect and the like. Therefore, the application uses benzalkonium chloride for molding, and can simultaneously research the effects of the lycium barbarum glycopeptide on tear film damage and corneal epithelial cell damage.
The experimental results are as follows: (1) In vitro experiments show that LBGP has the function of improving the cell activity of injured corneal epithelial cells. (2) In vivo tests show that after being treated by 5mg/mL LBGP eye drops, the corneal turbidity and the corneal epithelial integrity of a model mouse are improved, the lacrimal secretion amount is increased, the lacrimal film rupture time is prolonged, and the lacrimal MUC5AC content is increased; LBGP promotes corneal sensory nerve functional recovery and corneal nerve fiber regeneration in the modeled mice. The qPCR detection shows that LBGP reduces the mRNA expression quantity of the cornea inflammatory factors IL-1 beta and TNF-alpha of the modeling mice. The RNA-seq results showed that, in the results of the KEGG enrichment analysis, the first 20 most significantly different signaling pathways comprise multiple immune-related pathways: cytokine-cytokine receptor interactions, cell adhesion molecules, th17 cell differentiation, chemokine signaling pathway, IL-17 signaling pathway.
Therefore, LBGP has the function of improving the activity of damaged corneal epithelial cells, and has the effects of promoting corneal epithelial repair, increasing tear secretion and tear MUC5AC content, promoting corneal sensory nerve function recovery, nerve fiber regeneration and inhibiting inflammation as eye drops for treating induced mouse tear film and corneal epithelial damage models.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without creative efforts.
Figure 1 shows the effect of different concentrations of LBGP on the cellular activity of normal HCECs, # P <0.01, compared to the 0mg/mL LBGP group (i.e. cells cultured in corneal epithelial cell culture medium without LBGP addition).
FIG. 2 shows CCK-8 measures the cellular activity of HCECs; a P<0.05, compared to the BAK-/LBGP-group; b P<0.01, compared to the BAK-/LBGP-group; c P<0.05, compared to the BAK +/LBGP-group; d P<0.01, compared to the BAK +/LBGP-group; ns, differences are not statistically significant.
FIG. 3 shows cell viability assays for HCECs using dead and live staining; wherein, (A) Live/Dead cell staining; (B) dead cell ratio: * P <0.05, P <0.01, P <0.001. (scale unit: 100 μm)
Figure 4 shows corneal haze and corneal epithelial integrity in mice after treatment; wherein, (A) white light and cobalt blue light map of mouse cornea; (B) corneal opacity score; (C) scoring of sodium fluorescein staining. * P<0.05, compared to Control group; ns, differences are not statistically significant, compared to Control group; a P<0.001, compared to Control group; b P<0.05, compared to Control group; c P<0.001, compared to the Vehicle group; d P<0.05, compared to the Hycoan group.
FIG. 5 shows the change in tear secretion and tear film break-up time of mice after treatment; wherein (A) tearLiquid secretion amount: a P<0.001, compared to Control group; b P<0.001, compared to the Vehicle group; c P>0.05, compared to the Hycoan group. (B) tear break up time: a P<0.001, compared to Control group; b P<0.01, compared to Control group; c P<0.001, compared to the Vehicle group; d P>0.05, compared to the Hycoan group.
Figure 6 shows the change in tear MUC5AC content in mice following treatment; wherein, a P<0.001, compared to Control group; b P<0.01, compared to Control group; c P>0.05, compared to Control group; d P<0.01, compared to the Vehicle group; e P<0.05, compared to the Hycoan group.
Figure 7 shows the change in corneal sensitivity of mice after treatment; wherein, a P<0.001, compared to Control group; b P<0.001, compared to the Vehicle group; c P<0.001, compared to the Hycoan group.
FIG. 8 shows immunofluorescence staining of Anti- β III Tubulin Antibody in mouse corneal nerves after treatment; wherein, (A, B) is a Vehicle group; (C, D) LBGP group. (E) statistical map of nerve density: * P <0.05. (scale unit: 2000. Mu.m in A and C, and 100. Mu.m in B and D.)
FIG. 9 shows the mRNA expression changes of mouse corneal inflammatory factors IL-1. Beta. And TNF-. Alpha.after treatment; wherein (A) the relative expression level of IL-1 beta mRNA: a P<0.001, compared to Control group; b P<0.05, compared to Control group; c P>0.05, compared to Control group; d P<0.05, compared to the Vehicle group; e P<0.001, compared to the Vehicle group; f P<0.05, compared to the Hycoan group. (B) relative expression amount of TNF-. Alpha.mRNA: a P<0.05, compared to Control group; b P>0.05, compared to Control group; c P>0.05, compared to the Vehicle group; d P<0.01, compared to the Vehicle group; e P<0.05, compared to the Hycoan group.
Figure 10 shows KEGG enrichment analysis of LBGP group DEGs most significant 20 signaling pathways compared to the Vehicle group.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
1. Test materials
Test drugs: lycium Barbarum Glycopeptide (LBGP) powder is provided by Ningxia Riben Lycium Barbarum Biotech GmbH.
Test cells: human Corneal Epithelial Cells (HCECs) were proffered by professor Wu Kaili, the Zhongshan university center of ophthalmology, zhongshan.
Test animals: SPF grade 6-8 week old C57BL/6 female mice were used and purchased from Guangdong provincial centers for medical laboratory animals. All experimental mice were in good health and were housed in a good environment: grain and moisture are sufficient, the change law is that day and night (the illumination time is 8-20: 00 every day), the temperature is 22 ℃ +/-2 ℃, the humidity is 60% +/-10%, the noise is less than 60dB, and the ventilation is good. All procedures related to animal experiments were in compliance with The animal use regulations established by The American society for Research in Vision and Ophthalology, ARVO, approved by The Experimental animal ethical Committee of river-south university for animal ethical review (approved code IACUC-20191202-06).
2. Test method
2.1 preparation of the drug
(1) Normal corneal epithelial cell culture medium preparation
The formula is as follows:
| reagent | Dosage of | Manufacturer of the product |
| DMEM/F-12(Gibco TM ) | 44mL | Thermofisher,USA |
| Fetal Bovine Serum(FBS) | 5mL | Invitrogen,USA |
| Penicillin/Streptomycin(P/S) | 500μL | Thermofisher,USA |
| Insulin-Transferrin-Selenium(ITS) | 500μL | Thermofisher,USA |
| Human Epidermal Growth Factor(hEGF) | 10ng/mL | Peprotech,USA |
The above liquid was added to a 50mL sterile centrifuge tube and mixed well, filtered through a 0.22 μm sterile microporous membrane, and stored at 4 ℃.
(2) Human corneal epithelial cell medium (LBGP medium) supplemented with Lycium Barbarum Glycopeptide (LBGP): weighing required LBGP powder, dissolving the LBGP powder in a prepared human corneal epithelial cell culture medium to prepare a stock solution with the final concentration of 10mg/mL, adjusting the pH to 7.0, performing suction filtration through a 0.22 mu m sterile microporous filter membrane, storing at 4 ℃, and adding the LBGP powder into the human corneal epithelial cell culture medium in proportion to dilute the LBGP powder to the target concentration when in use.
(3) Lycium barbarum glycopeptide eye drops (LBGP eye drops): weighing the required LBGP powder, dissolving the LBGP powder in sterile physiological saline, adjusting the pH value to be 6.0-8.0, and filtering the LBGP powder by using a 0.22 mu m sterile microporous membrane. Stored at 4 ℃.
2.2 cell model building and group design
An in vitro corneal epithelial cell injury model was constructed by treating Human Corneal Epithelial Cells (HCECs) with 0.002% benzalkonium chloride (BAK) for 5 min. The specific operation is as follows: treating cells with BAK for 5min, removing BAK by suction, washing residual BAK solution with PBS, and adding corneal epithelial culture medium containing no Lycium barbarum glycopeptide or corneal epithelial culture medium containing Lycium barbarum glycopeptide.
Then, the optimum LBGP concentration in the culture medium of the human corneal epithelial cells is screened out by utilizing a CCK-8 Kit (Cell Counting Kit-8 Cell Counting Kit). The specific operation is as follows: (1) After cell counting, the cells were seeded in a 96-well plate at 6000 cells/well, and the cells were cultured in an incubator for 12 hours. (2) Old corneal epithelial cell culture medium is aspirated, LBGP culture medium of corresponding different concentrations is added, and the cells are placed in the incubator again for 24 hours. (3) LBGP medium was aspirated off, and the corneal epithelial cell medium without LBGP was replaced with fresh LBGP, and 10. Mu.L of CCK-8 solution was added to each well. (4) incubate the plates in an incubator for 2 hours. And (5) measuring the absorbance of each hole at 450nm by using a microplate reader.
Then, cells are grouped for subsequent detection, and the specific groups are as follows: (1) pseudo model group (BAK-/LBGP-): when the model group is induced by adding BAK, the group is added with PBS for false treatment, and then is cultured by using a normal corneal epithelial cell culture medium; (2) drug group for pseudotype (BAK-/LBGP +): when BAK induction is added to the model group, PBS pseudo-treatment is added to the group, and then the model non-drug group (BAK +/LBGP-): after BAK is used for model induction, normal corneal epithelial cell culture medium is used for subsequent culture; (4) model drug group (BAK +/LBGP +): after model induction using BAK, culture was subsequently performed using medium supplemented with LBGP at the optimum concentration.
In addition, the normal Control group (Control) was cultured under the same conditions using a normal corneal epithelial cell medium, and was used for the assay.
2.3 establishment and group design of animal models
Mice were dropped into both eyes with 0.2% bak eye drops, 5 μ L per eye each time, 2 times per day (8 AM and 8PM daily) for 14 consecutive days, inducing a mouse model of tear film and corneal epithelial damage.
The 30 successfully molded experimental mice were randomly divided into 10 groups of solvent control group (Vehicle), positive control group (Hycosan) and LBGP eye drop group, and the subsequent experiments were performed. Wherein, the solvent control group uses sterile normal saline; the positive control group used artificial tear sea water (Hycosan, preservative free; ursubapharmam, saarbr ü cken, germany) without preservative component; the LBGP eye drops concentration is 5mg/mL.
The corresponding eye drops were used 3 times per day (8 am,2PM and 8PM per day), 5 μ L per eye per time, for 10 days, according to the group. In addition, the normal Control group (Control) was also 10 healthy mice, which were normally bred in the same environment without any treatment such as molding and medication for detection.
2.4 statistical analysis
Statistical analysis of the data was performed using the software IBM SPSS Statistics 26.0, the comparison between the groups of the population was performed using One-Way analysis of variance (One-Way ANOVA), the comparison between the two groups was performed using the two-sample T-Test (Independent-Samples T Test), the results were expressed as mean ± standard deviation (mean ± standard deviation), the differences were considered statistically significant when P <0.05, significant when P <0.01, and significant when P <0.001. Statistical plots were drawn using the software GraphPad Prism 8.
3. Test results
3.1 LBGP increases cellular activity of BAK-treated HCECs
First, in order to evaluate the effect of Lycium Barbarum Glycopeptide (LBGP) itself on Human Corneal Epithelial Cells (HCECs), the activity of cells in each group was measured by CCK-8 assay after culturing the cells in LBGP media (0, 0.1, 0.5, 1, 2.5, 5, 7.5, 10 mg/mL) at various concentrations for 24 hours. The CCK-8 detection is specifically carried out as follows: (1) After cell counting, the cells were seeded in a 96-well plate at 6000 cells/well, and the cells were cultured in an incubator for 12 hours. (2) Old corneal epithelial cell culture medium was aspirated, the cells were treated with 0.002% BAK solution for 5min, the control group or the sham-model group was replaced with PBS, and after the BAK solution treatment was completed, each well was washed twice with 150. Mu.L of PBS. Then, fresh corneal epithelial cell culture medium with or without LBGP was added, and the cells were cultured in an incubator for 24 hours. (3) Old medium was aspirated off, and fresh corneal epithelial cell medium without LBGP was replaced, and 10. Mu.L of CCK-8 solution was added to each well. (4) incubate the plates in an incubator for 2 hours. And (5) measuring the absorbance of each hole at 450nm by using a microplate reader.
The results show that 0-1mg/mL LBGP had no effect on the cellular activity of HCECs, 2.5mg/mL LBGP increased the cellular activity, but too high a concentration of LBGP (5-10 mg/mL) caused a significant decrease in cellular activity instead (P <0.01, see FIG. 1). Based on the above experimental results, LBGP media at concentrations of 0.1, 0.5 and 1mg/mL were selected for the next CCK-8 assay.
To investigate whether LBGP has a repairing effect on BAK-induced HCECs damage, an in vitro corneal damage model induced by BAK was cultured in the LBGP medium of the above three concentrations, respectively. The results are shown in FIG. 2, and it can be seen from FIG. 2 that the cell activities of the sham-model group (BAK-/LBGP-) and the sham-model group (BAK-/LBGP +) were not significantly different from the cell activity of the normal culture group (Control). The cellular activity of model non-drug group (BAK +/LBGP-) decreased to (46.64% + -8.87%) (P < 0.01), and the cellular activity of BAK-treated HCECs was increased by three different concentrations of 0.1, 0.5 and 1mg/mL of LBGP in the model drug group, wherein the cellular activities of 0.1mg/mL and 1mg/mL of LBGP were increased to (75.05% + -10.36%) and (80.37% + -10.10%), respectively, which were statistically different (P < 0.05) compared to the model non-drug group (BAK +/LBGP-) and increased to (82.07% + -7.45%) compared to the model non-drug group (BAK +/LBGP-) (P < 0.01). Based on the above experimental results, the subsequent experiments will use LBGP medium with a concentration of 0.5mg/mL as the drug group for model.
In order to further verify that LBGP has an effect of improving the cell activity of HCECs treated by BAK, the cell activity is detected by carrying out cell death and activity staining on a pseudomodel group (BAK-/LBGP-), a model non-drug group (BAK +/LBGP-), and a model drug group (BAK +/LBGP +). The experimental results are shown in figure 3. As can be seen from fig. 3, the cell death rates of the cells in the pseudo model group (BAK-/LBGP-) group, the model non-drug group (BAK +/LBGP-) group and the model drug group (BAK +/LBGP +) were (5.64% ± 1.51%), (27.75% ± 2.15%) and (13.49% ± 3.05%), respectively, and the differences between the three groups were statistically significant two by two, P <0.05 (, P <0.01 (, P <0.001 (, x). Wherein, the cell death rate of HCECs (P < 0.001) is obviously increased by the model drug-free group (BAK +/LBGP-), while the cell death rate of the model drug-free group (BAK +/LBGP +) is obviously lower than that of the model drug-free group (BAK +/LBGP-) (P < 0.01). It was further demonstrated that 0.5mg/mL LBGP increased the cellular activity of BAK treated HCECs.
3.2 Effect of LBGP on modeling improvement of corneal haze and corneal epithelial integrity in mice
To investigate the effect of LBGP on modeling corneal haze and corneal epithelial integrity in mice, observations were made on day 9 of instillation therapy and the results are shown in figure 4.
The results showed that corneal opacity scores were (0.30. + -. 0.48), (1.10. + -. 0.57), (0.50. + -. 0.53) and (0.40. + -. 0.52) for the Control group, the Vehicle group, the Hycosan group and the LBGP group, respectively (FIG. 4B). The corneal opacity of mice in the LBGP group was elevated compared to that in the Vehicle group, and the difference was statistically significant (P < 0.05). The corneal opacity scores of the LBGP group and the Hycosan group were statistically insignificant compared to the Control group (P > 0.05).
The fluorescein sodium staining scores of the Control group, the Vehicle group, the Hycosan group and the LBGP group were (1.60. + -. 0.70), (6.20. + -. 1.40), (3.60. + -. 0.97) and (2.90. + -. 1.45), respectively (FIG. 4C). The staining of the mice in LBGP group with sodium corneal fluorescein was significantly reduced compared with that in the Vehicle group, and the difference has significant statistical significance (P < 0.001); compared with the Hycosan group, the staining is less, the epithelial repair effect is better, and the difference has statistical significance (P < 0.05).
Thus, it is demonstrated that LBGP can improve corneal haze and corneal epithelial integrity in modeled mice.
3.3 LBGP increases the amount of tear secretion in a model mouse and prolongs the tear film rupture time
In order to investigate the influence of LBGP on the amount of tear secretion and tear film break-up time of the modeled mice, tear secretion tests and tear film break-up time tests were performed on day 7 after the modeled mice were treated with the corresponding eye drops of each group, and the results are shown in fig. 5.
The results showed that tear secretion amounts of the Control group, the Vehicle group, the Hycosan group and the LBGP group were (5.75. + -. 0.35 mm), (1.68. + -. 0.37 mm), (2.80. + -. 0.54 mm) and (3.34. + -. 0.73 mm), respectively (FIG. 5A). The lacrimal secretion amount of the mice in the LBGP group is obviously increased compared with that in the Vehicle group, and the difference has obvious statistical significance (P < 0.001); the difference was not statistically significant compared to the Hycosan group (P > 0.05).
Tear film rupture times were (8.90. + -. 1.37 s), (4.20. + -. 0.92 s), (6.70. + -. 1.16 s) and (6.50. + -. 1.35 s) for the Control group, the Vehicle group, the Hycosan group and the LBGP group, respectively (FIG. 5B). Tear film break up time was significantly increased in LBGP group mice compared to the Vehicle group, with differences of significant statistical significance (P < 0.001); the difference was not statistically significant compared to the Hycosan group (P > 0.05).
The results show that LBGP increased tear secretion in the modeled mice, prolonged tear film break-up time, significantly improved compared to the Vehicle group, and no statistical difference compared to the Hycosan group.
3.4 Modeling increased tear MUC5AC content in mice by LBGP
To investigate the effect of LBGP on the content of MUC5AC in the modeled mice, the modeled mice were treated with the corresponding eye drops for each group, and tears were collected on day 8 for ELISA detection of MUC5AC (mucin 5 AC) content. The results are shown in FIG. 6.
The results showed that the tear MUC5AC content of the Control group, the Vehicle group, the Hycosan group and the LBGP group were (59.90. + -. 5.32 ng/mL), (35.85. + -. 1.95 ng/mL), (45.03. + -. 3.57 ng/mL) and (59.15. + -. 6.80 ng/mL), respectively. The lachrymal MUC5AC content of the LBGP group was significantly increased compared to the Vehicle group, the difference having significant statistical significance (P < 0.01); compared with the Hycosan group, the content of the gene is increased, and the difference has statistical significance (P < 0.05); the difference was not statistically significant compared to Control group (P > 0.05).
The results show that the lachrymal MUC5AC content of the LBGP group was significantly increased compared to the Vehicle group, and was restored to a level substantially consistent with that of the Control group.
3.5 LBGP promotes corneal sensory nerve function recovery in modeled mice
To assess whether LBGP had an effect on the corneal sensory nerve function of the modeled mice, corneal sensitivity assays were performed on day 9 of instillation therapy. The results are shown in FIG. 7.
Determination method of corneal sensitivity: it must be done in the mouse unaesthetized and all manipulations must be done by the same blinded investigator to reduce bias.
Corneal sensitivity measurements were performed using a Cochet-Bonnet tactile measuring instrument. Before measurement, whether the nylon wire of the tactile measurement instrument is complete is checked to ensure that the instrument is complete. The mouse corneal central region was touched using a preset longest nylon filament length (6.0 cm) as the starting measurement length. If the mouse does not have effective blink reflex, the length of the longest nylon wire which is shortened by 0.5cm in sequence and is measured continuously until the mouse just can induce the effective blink reflex is considered as a threshold value of the sensitivity and is recorded as the cornea sensitivity. The measurement was repeated 3 times per mouse, and the average value was taken as the measurement result.
The manufacturer of the Cochet-Bonnet tactile meter: luneau SAS, france.
The results showed that the corneal sensitivities of the Control group, the Vehicle group, the Hycosan group and the LBGP group were (5.70. + -. 0.35 cm), (2.40. + -. 0.39 cm), (3.45. + -. 0.50 cm) and (4.65. + -. 0.24 cm), respectively. The cornea sensitivity of the LBGP group is obviously improved compared with that of the Vehicle group, and the difference has significant statistical significance (P is less than 0.001); the differences were still statistically significant (P < 0.001) compared to the increased sensitivity of the Hycosan group.
The results show that LBGP promotes the recovery of corneal sensory nerve function in the modeled mice.
3.6 LBGP for promoting regeneration of corneal nerve fibers of modeled mice
The recovery of corneal sensory nerve function is closely related to the regeneration of corneal nerve fibers. To explore the effect of LBGP on the regeneration of corneal nerve fibers in modeled mice, mice corneas from both the Vehicle group and LBGP group were stained by whole-sheet immunofluorescence staining using Anti-neuron specific β -III Tubulin antibodies (Anti- β III Tubulin antibodies) to stain corneal nerve fibers on day 10. The results are shown in FIG. 8.
The results show that the corneal nerve density of the LBGP group is obviously improved compared with that of the Vehicle group, and the nerve density of the Vehicle group and the LBGP group is respectively (121.61 +/-16.57 mm/mm) through statistics 2 ) And (285.94. + -. 50.64mm/mm 2 ) The difference between the two being statistically significant (P)<0.05)。
The results show that LBGP promotes corneal nerve fiber regeneration in the modeled mice, consistent with the results of corneal sensory nerve function described above.
3.7 LBGP reduces mRNA expression level of IL-1 beta and TNF-alpha of mouse cornea inflammation factor
In order to study the effect of LBGP on mRNA expression level of corneal inflammatory factor in model mice, the corneal tissue of the model mice was taken on day 10 for qPCR assay after the model mice were treated with the corresponding eye drops of each group, and the results are shown in FIG. 9.
The results show that the relative mRNA expression quantity of corneal IL-1 beta of mice in the LBGP group is obviously reduced compared with that in the Vehicle group, and the difference has significant statistical significance (P < 0.001); compared with the Hycosan group, the expression level is reduced, and the difference has statistical significance (P < 0.05); the difference was not statistically significant (P > 0.05) compared to Control group (fig. 9A). The relative expression quantity of mRNA of corneal TNF-alpha of mice in the LBGP group is obviously reduced compared with that in the Vehicle group, and the difference has obvious statistical significance (P < 0.01); compared with the Hycosan group, the expression level is reduced, and the difference has statistical significance (P < 0.05); the difference was not statistically significant (P > 0.05) compared to Control group (fig. 9B).
Experimental data show that LBGP reduces the mRNA expression level of cornea inflammatory factors IL-1 beta and TNF-alpha of a modeling mouse.
3.8 RNA-seq analysis of LBGP on the cornea of modeled mice
To assess the transcriptomic effect of LBGP on the corneas of the modeled mice, corneal tissues from both the Vehicle group (group V) and LBGP group (group L) were collected for total RNA for transcriptome sequencing 10 days after treatment with eye drops. The experimental results are as follows:
KEGG (Kyoto encyclopedia of genes and genes, kyoto encyclopedia of genes and genomes) enrichment analysis of differentially expressed genes: according to Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the DEGs are enriched by the KEGG, and 20 most significant signal channels with difference are selected (figure 10). Wherein, the immune-related pathways comprise significant differences of metabolism pathways such as Cytokine-Cytokine receptor interaction (Cytokine-Cytokine receptor interaction), cell adhesion molecules (Cell adhesion molecules), th17 Cell differentiation (Th 17 Cell differentiation), chemokine signaling pathway (Chemokine signaling pathway), IL-17 signaling pathway (IL-17 signaling pathway), and the like. In combination with the qPCR results, it can be speculated that LBGP has good anti-inflammatory properties.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. Application of lycium barbarum glycopeptide in preparing medicine for preventing and treating tear film injury and/or corneal epithelial cell injury.
2. Use according to claim 1, characterized in that the control comprises: improving corneal epithelial cell activity, improving corneal turbidity, improving corneal epithelial integrity, increasing tear secretion, prolonging tear film rupture time, increasing tear MUC5AC content, promoting corneal sensory nerve function recovery, promoting corneal nerve fiber regeneration, reducing mRNA expression level of corneal inflammation factor IL-1 beta, and reducing mRNA expression level of corneal inflammation factor TNF-alpha.
3. The use of claim 1, wherein the glycopeptide of lycium barbarum is administered by eye drop administration.
4. A medicament for the prevention and treatment of tear film damage and/or corneal epithelial cell damage comprising an effective amount of a glycopeptide of Lycium barbarum.
5. The pharmaceutical of claim 4, wherein the pharmaceutical is in the form of eye drops.
6. The medicament of claim 4, further comprising a pharmaceutically acceptable excipient.
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