WO2024114360A1 - Use of dihydroquercetin in treating ocular surface diseases - Google Patents

Use of dihydroquercetin in treating ocular surface diseases Download PDF

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WO2024114360A1
WO2024114360A1 PCT/CN2023/131428 CN2023131428W WO2024114360A1 WO 2024114360 A1 WO2024114360 A1 WO 2024114360A1 CN 2023131428 W CN2023131428 W CN 2023131428W WO 2024114360 A1 WO2024114360 A1 WO 2024114360A1
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dihydroquercetin
group
ocular surface
cells
corneal
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PCT/CN2023/131428
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French (fr)
Chinese (zh)
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张东蕾
袁振丽
安元龙
王敏楠
龚琪
何向东
何伟
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辽宁何氏医学院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention relates to the technical field of pharmaceuticals, and in particular to application of dihydroquercetin in treating ocular surface diseases.
  • Oxidative damage is widely present in diseases, especially playing an important role in aging and inflammation-related diseases.
  • the free radicals produced by oxidation can directly act on tissue cell membranes and destroy them. Free radicals can also enter cells through channels on the cell membranes, causing damage to proteins and DNA in cells.
  • Oxygen free radicals participate in the metabolism of arachidonic acid, which is an important process in the inflammatory response.
  • the lipid peroxides produced are chemokines, which can aggravate the inflammatory response.
  • oxidation products can also induce the production of chemokines different from arachidonic acid, inactivate protease inhibitors, increase collagenase, etc., and thus destroy connective tissue.
  • Common eye diseases caused by oxidative damage include dry eye and corneal alkali burns.
  • Dry eye is a common disease in ophthalmology. It mainly refers to a disease caused by insufficient tears or excessive tear evaporation, which is caused by multiple factors such as tear stability and ocular surface inflammation.
  • researchers at home and abroad have conducted in-depth research on the pathogenesis of dry eye, and believe that the occurrence and development of the disease is related to the body's immune inflammatory response, cell apoptosis and sex hormone levels.
  • T cell-mediated immune inflammatory response is the most critical factor in the onset of dry eye.
  • the decrease in tear secretion or increase in evaporation leads to high osmotic pressure in tears, which is also a key factor in causing the vicious cycle of dry eye symptoms.
  • the high osmotic pressure of tears can cause morphological changes such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, and trigger an inflammatory cascade reaction, further causing the death of corneal epithelial cells.
  • morphological changes such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, and trigger an inflammatory cascade reaction, further causing the death of corneal epithelial cells.
  • the content of mucin and lipids in tears will be reduced, thereby aggravating the instability of the tear film and promoting this vicious cycle.
  • the disease causes varying degrees of dry eyes, foreign body sensation, photophobia and pain. In severe cases, corneal damage occurs, and the patient's visual function is threatened. Therefore, clinical treatment research on dry eye is imminent.
  • Corneal alkali burns are a common type of ocular trauma in clinical practice, with a very high blindness rate.
  • cell apoptosis and activation of inflammatory response are important factors causing corneal damage; in the late stage of alkali burns, corneal neovascularization will seriously affect corneal transparency and visual acuity.
  • choroidal neovascularization CNV of the fundus
  • the formation of CNV is a complex pathological process, which is regulated by multiple factors.
  • the growth factor network formed by many cytokines precisely regulates the formation of CNV.
  • Vascular endothelial growth factor (VEGF) is the strongest angiogenic factor discovered so far.
  • VEGF vascular endothelial growth factor
  • glucocorticoids include Cushing's syndrome, infection, gastrointestinal reaction, edema, glucose metabolism disorder, electrolyte imbalance, abnormal nervous system excitement, etc.
  • anti-VEGF drugs include conjunctival congestion, eye pain, foreign body sensation, corneal abrasion, corneal edema, increased intraocular pressure, floating black shadows, intraocular infection, retinal detachment or vitreous hemorrhage, etc.
  • the technical problem to be solved by the present invention is to provide a use of dihydroquercetin in treating ocular surface diseases.
  • the dihydroquercetin has a protective effect on H 2 O 2- induced oxidative stress damage of corneal epithelial cells (HCE).
  • the present invention provides the use of dihydroquercetin in preparing a medicine for preventing, alleviating and/or treating ocular surface diseases.
  • Dihydroquercetin also known as taxifolin, exists in many plants, and its content is higher in larch, especially Douglas fir. Dihydroquercetin was first extracted and separated from the leaves of the conifer Chamaecyparisobtusa by Japanese scholar Fukui. In recent years, dihydroquercetin has also been found in many fruits, such as grapes, oranges and grapefruits. Studies have shown that dihydroquercetin contains more phenolic hydroxyl groups, has multiple biological activities, and can inhibit or activate multiple enzymes, thereby producing different physiological effects. The structural formula of dihydroquercetin is as follows:
  • the present invention applies dihydroquercetin to the prevention, alleviation and/or treatment of ocular surface diseases for the first time.
  • the ocular surface disease is an ocular surface disease caused by corneal epithelial cell damage and/or apoptosis.
  • the corneal epithelial damage is oxidative damage.
  • the oxidative damage is damage caused by hydrogen peroxide.
  • the dihydroquercetin of the present invention has a protective effect on oxidative stress damage of corneal epithelial cells (HCE) induced by H 2 O 2 .
  • dihydroquercetin has a proliferation effect on HCE cells and can increase the relative cell viability of HCE cells after oxidative damage.
  • the present invention establishes a H2O2 - induced corneal epithelial cell (HCE) damage model, determines the H2O2 modeling concentration, then performs modeling at this concentration to cause oxidative damage to HCE cells, and finally acts on HCE cells with different concentrations of dihydroquercetin. It is found that a certain concentration of dihydroquercetin has a proliferation effect on HCE cells, while too high a concentration will produce certain toxicity to HCE cells.
  • HCE corneal epithelial cell
  • the modeling concentration is specifically 300 ⁇ M.
  • the concentration of dihydroquercetin having a proliferation effect on HCE cells is 25-200 ⁇ M, and the concentration of dihydroquercetin that can produce a certain degree of toxicity to HCE cells is 600 ⁇ M.
  • the ocular surface disease of the present invention is dry eye or a disease related to corneal neovascularization.
  • the present invention also provides a dihydroquercetin ophthalmic preparation, comprising dihydroquercetin and a solvent.
  • the mass concentration of the dihydroquercetin is 0.01% to 0.5%; more preferably, the mass concentration of the dihydroquercetin is 0.03% to 0.3%; further preferably, the mass concentration of the dihydroquercetin is 0.03% or 0.3%.
  • the preparation is in the form of eye drops, eye ointment, periocular and intraocular injection, eye gel or liposome.
  • the present invention provides the use of dihydroquercetin in the preparation of a drug for preventing, alleviating and/or treating ocular surface diseases.
  • the present invention applies dihydroquercetin to the prevention, alleviating and/or treating ocular surface diseases for the first time.
  • the dihydroquercetin has a good protective effect on diseases related to oxidative damage of corneal epithelial cells (HCE).
  • HCE corneal epithelial cells
  • the dihydroquercetin can promote the proliferation of HCE cells and improve cell viability, and can also increase the tear secretion and tear film stability of HCE cells to significantly improve ocular surface diseases.
  • Figure 1 is a graph showing the results of using the MTS method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and a combination of 300 ⁇ M hydrogen peroxide and different concentrations of dihydroquercetin (C) after acting on corneal epithelial cells for 24 hours, wherein P represents a significant level, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, ## represents P ⁇ 0.01, and ### represents P ⁇ 0.001;
  • FIG2 is a diagram showing the results of tear secretion testing of live mice in animal experiments
  • FIG3 is a diagram showing the tear film breakup time test results of live mice in animal experiments.
  • FIG4 is a diagram of the corneal morphology of mice in each experimental group after corneal fluorescein sodium staining
  • FIG5 is a graph showing the scoring results of corneal epithelial damage in mice examined by corneal sodium fluorescein staining
  • FIG6 is a morphological diagram of new blood vessels on the cornea of New Zealand rabbits in each experimental group detected by corneal slit lamp;
  • FIG. 7 is a graph showing the results of the detection of corneal neovascularization areas in New Zealand rabbits in each experimental group, where **** represents P ⁇ 0.0001.
  • dihydroquercetin provided by the present invention in treating ocular surface diseases is described in detail below in conjunction with examples.
  • Human corneal epithelial cells were purchased from the ATCC cell bank in the United States.
  • the cell culture medium used was high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100 ⁇ g/mL gentamicin.
  • the cells were cultured in an incubator containing 5% carbon dioxide at 37°C.
  • the cells were plated in a 96-well plate and the cells were used in the logarithmic growth phase.
  • the cells were plated at a density of 1.5 ⁇ 10 5 cells/mL, 100 ⁇ L per well, and 6 duplicate wells per group.
  • the cells were grouped into normal cell group, hydrogen peroxide model group, and dihydroquercetin intervention groups at different concentrations.
  • the detection method was the MTS kit method (represented by Promega), and the absorbance was detected by a microplate reader at 490 nm.
  • Survival rate survival rate of each group / survival rate of normal cell group ⁇ 100%
  • Human corneal epithelial cells were purchased from the ATCC cell bank in the United States.
  • the cell culture medium used high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100 ⁇ g/mL gentamicin.
  • the cells were cultured in an incubator containing 5% carbon dioxide at 37°C.
  • the cells were plated in a 96-well plate. Cells in the logarithmic growth phase were plated at a density of 1.5 ⁇ 10 5 /mL, 100 ⁇ L per well, and 6 wells per group. After 24 hours, the cells were treated with H 2 O 2 modeling or dihydroquercetin administration according to the purpose of the experiment.
  • Color development 20 ⁇ L of MTS solution was added to each well and incubated for 2 to 4 hours.
  • Colorimetry Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, record the results, and draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.
  • HCE human corneal epithelial cell
  • HCE cells were subcultured, and the plate was dripped at 1.5 ⁇ 10 5 cell/mL, 100 ⁇ L/well. HCE cells were treated with different concentrations of H 2 O 2 (100 ⁇ M, 200 ⁇ M, 300 ⁇ m, 400 ⁇ M, 500 ⁇ M, 600 ⁇ M, 700 ⁇ M, 800 ⁇ M, 900 ⁇ M) for 24h, 6 replicates per group to ensure the stability and accuracy of the experimental data, and the degree of damage to cells by H 2 O 2 was observed. Color development: 20 ⁇ L of MTS solution was added to each well, and the incubation continued for 2-4h. Colorimetry: 490nm wavelength was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the results were recorded.
  • H 2 O 2 100 ⁇ M, 200 ⁇ M, 300 ⁇ m, 400 ⁇ M, 500 ⁇ M, 600 ⁇ M, 700 ⁇ M, 800 ⁇ M, 900 ⁇ M
  • the cell growth curve was drawn with different concentrations of H 2 O 2 as the horizontal axis and the absorbance value as the vertical axis.
  • the H 2 O 2 concentration when the cell survival rate was about 50% detected by MTT method was used as the modeling concentration of the oxidative damage model.
  • Survival rate survival rate of each group / survival rate of normal cell group ⁇ 100%
  • Figure 1 is a graph showing the results of using MTT method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and 300 ⁇ M hydrogen peroxide and different concentrations of dihydroquercetin (C) acting on corneal epithelial cells for 24 hours, where P represents significant level, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, ## represents P ⁇ 0.01, and ### represents P ⁇ 0.001.
  • the H 2 O 2 concentration is 300 ⁇ M
  • the cell survival rate is 49%, so 300 ⁇ M H 2 O 2 is determined as the modeling concentration of the oxidative damage model in subsequent experiments.
  • the experiment was divided into 8 groups: normal control group, H 2 O 2 model group, and different concentrations of dihydroquercetin (0.1-200 ⁇ M) + H 2 O 2 intervention groups.
  • HCE cells were modeled with 300 ⁇ M H 2 O 2 , and different concentrations of drugs within the safe range were applied to HCE cells after modeling.
  • the cell survival rate among the groups was detected by MTS method, and the protective effect of dihydroquercetin on the antioxidant damage of HCE cells was detected.
  • the cell survival rate of the H2O2 modeling group was lower than that of the control group (P ⁇ 0.001), and that of the 25-200 ⁇ M dihydroquercetin group was higher than that of the H2O2 modeling group (P ⁇ 0.01), indicating that H2O2 can induce oxidative stress damage in HCE cells, and dihydroquercetin has a protective effect on H2O2 - induced oxidative stress damage in HCE cells.
  • mice Thirty mice were selected from 50 healthy female C57/BL6 mice aged 8 weeks, and randomly divided into 6 groups with 5 mice in each group, which were recorded as blank control group (N), model group (M), positive drug group (CsA), low concentration group of Chinese herbal medicine compound (SL), medium concentration group of Chinese herbal medicine compound (SM), and high concentration group of Chinese herbal medicine compound (SH).
  • N blank control group
  • M model group
  • CsA positive drug group
  • SL low concentration group of Chinese herbal medicine compound
  • SM medium concentration group of Chinese herbal medicine compound
  • SH high concentration group of Chinese herbal medicine compound
  • mice were raised in the S-rated PF laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, 5 mice were kept in each cage (length 40cm ⁇ width 20cm ⁇ height 20cm), for a total of 6 cages.
  • the bedding was corn cob bedding. This experiment used sterilized complete nutritious feed. (Purchased from Liaoning Changsheng Biological Co., Ltd.).
  • the drinking water was purified water, which was prepared by the HT-RO1000 water purification system of the Animal Center. The water quality is tested by a dedicated person every year. During the experiment, the mice were free to drink water. The mice were fasted the night before the dissection, but they were not forbidden to drink water.
  • the temperature and humidity of the laboratory are automatically controlled by the air conditioning unit at 20°C-25°C and 40%-70%, and the temperature and humidity changes are recorded daily.
  • the lighting system automatically controls the light cycle changes in the animal laboratory, and the light and dark are evenly distributed for 24 hours.
  • the noise in the animal laboratory is controlled to no more than 60 decibels.
  • This experiment was approved and supervised by the Institutional Animal Care Committee (IACUC) and strictly complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well".
  • IACUC Institutional Animal Care Committee
  • an intelligent dry environment control system combined with scopolamine injection was used to induce dry eye models in mice.
  • a two-step dehumidification method was used to control the humidity of the model environment.
  • an adjustable temperature industrial dehumidifier was used to reduce the humidity in the room and adjust the humidity to 40% ⁇ 5%.
  • an intelligent drying box (dehumidification range RH10%-80%) was used to further reduce the humidity of the mouse breeding environment to 15% ⁇ 3%.
  • a noiseless, adjustable speed (wind speed range 0-5m/s) fan was placed in the intelligent drying box. The fan was placed 20cm away from the mouse cage, and the fan was set at the same vertical height as the mouse.
  • mice in the model control group (M), positive drug group (CsA), low-concentration dihydroquercetin group (E1), high-concentration dihydroquercetin group (E2), and solvent control group were placed in the dry environment (humidity 15% ⁇ 3%; wind speed 2.1 ⁇ 0.2 m/s; temperature 21-23°C), and the mice in the blank control group were raised in a normal environment (humidity 60%-80%; temperature 21-23°C).
  • mice in the other groups were subcutaneously injected with 0.5 mg/0.2 mL scopolamine solution 3 times a day (9:00 am; 12:00 noon; 3:00 pm), and the model establishment time lasted for 2 weeks.
  • the drug was administered through the eyes, and the dosage was 5 ⁇ L in the conjunctival sac of each eye twice a day (9:00 am; 3:00 pm).
  • Each drug group started drug administration the day before modeling and continued for 15 days.
  • the drug administration method was ocular administration, and all experimental mice were administered the day before modeling.
  • the blank control group and the model control group were not administered;
  • the positive drug group was administered with 0.05% cyclosporine A eye drops 5 ⁇ L/time, which was dripped into the conjunctival sac;
  • the low-concentration administration group was administered with 0.03% dihydroquercetin eye drops 5 ⁇ L/time, and the high-concentration administration group was administered with 0.3% dihydroquercetin eye drops 5 ⁇ L/time.
  • Each group of mice was administered twice/day (9:00 am and 3:00 pm).
  • mice were given an ophthalmic examination (including visual defects, eyeball abnormalities, and corneal damage) to ensure that there were no abnormalities in the experimental mice.
  • the body weight was measured once a week during the adaptation period and the experiment.
  • the experimental mice were observed before and after dosing every day to record their health status and whether they had abnormal physical signs.
  • the observation of the mouse condition included: appearance (eyes, ears, mouth, nose, vulva, fur, excrement, limb activity, and mental state), vomiting, activity, diet, gait, dying, death, etc.
  • mice were fixed under non-anesthesia, the lower eyelids of the mice were clamped with soft-tipped forceps, and then one end of the phenol red cotton thread was folded and placed in the lower conjunctival sac, and the lower eyelids of the mice were relaxed to close naturally.
  • the phenol red cotton thread was fixed, the phenol red cotton thread was taken out after 1 minute of timing, and the phenol red cotton thread was placed on a ruler paper, and the length of the red part was calculated and recorded.
  • the data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 2 is a graph showing the results of tear secretion in live mice in animal experiments.
  • N is a blank control group
  • M is a model control group
  • R is a solvent control group
  • Y is a positive drug group (mass concentration is 0.05% cyclosporine A)
  • E1 is a low-concentration dihydroquercetin group
  • E2 is a high-concentration dihydroquercetin group.
  • P represents a significant difference
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001.
  • the length of the phenol red cotton thread (mm) was counted. The longer the phenol red cotton thread, the more tear secretion, and vice versa.
  • the tear secretion of animals in each group was normal before modeling, and there was no significant difference between the groups (P>0.05).
  • the tear secretion of the positive drug group (CsA), the low concentration of dihydroquercetin group (E1), and the high concentration of dihydroquercetin group (E2) increased significantly relative to the model control group (M), and the differences were statistically significant (P ⁇ 0.001). There was no significant change in tear secretion between the solvent control group and the model control group.
  • the above results show that after dihydroquercetin treatment, the tear secretion of dry eye model mice has increased to a certain extent.
  • mice were fixed in a non-anesthetized state, and sodium fluorescein solution was dripped into the mouse conjunctival sac. After the mouse eyelids were passively closed several times, the eyelids were opened and the time when the first tear film rupture black spot was observed under the cobalt blue light of the slit lamp was recorded, which was the tear film rupture time. The data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 3 is a graph showing the tear film breakup time test results of live mice in animal experiments.
  • N is the blank control group
  • M is the model control group
  • R is the solvent control group
  • Y is the positive drug group (mass concentration is 0.05% cyclosporine A)
  • E1 is the low-concentration dihydroquercetin group
  • E2 is the high-concentration dihydroquercetin group.
  • P represents significant difference, * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the corneal morphology of each group of experimental mice was observed before and 14 days after modeling, and the corneas of both eyes were stained with sodium fluorescein solution, and a slit lamp was used for observation and photography. After fixing the mice, 5 ⁇ L of 2% sodium fluorescein saline solution was dripped into the conjunctival sac of the mice. After 1 minute, the eyes were gently rinsed with 2 mL of saline, and the excess solution was absorbed with a cotton swab. The mouse cornea stained with sodium fluorescein was irradiated with cobalt blue light, and the slit lamp was used for observation, photography and recording. If there is green fluorescence residue on the surface of the cornea, it means that the corneal epithelium is damaged. The sodium fluorescein residue on the corneal surface was scored and recorded. The data were statistically analyzed using GraphPad Prism 8.0 software
  • Figure 4 is a corneal morphology of mice in each experimental group after corneal fluorescein sodium staining.
  • the corneas of mice in the blank control group were intact and smooth, without sodium fluorescein attachment, indicating that the corneal epithelium of mice in the blank control group was intact and undamaged.
  • the model group and each drug-treated group had varying degrees of sodium fluorescein attachment, indicating that the corneal epithelium of mice in the model control group and each drug-treated group had varying degrees of damage.
  • the positive drug group and the dihydroquercetin-treated group also had dense punctate fluorescent attachment, but compared with the model group, there was a significant reduction.
  • the corneal epithelial injury scores of the mice in each experimental group after corneal fluorescein sodium staining are shown in Figure 5, where N is the blank control group; M is the model control group; R is the solvent control group; Y is the positive drug group (mass concentration is 0.05% cyclosporine A); E1 is the low-concentration dihydroquercetin group; and E2 is the high-concentration dihydroquercetin group.
  • * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the corneal epithelium of mice in the model group and each drug-treated group was damaged to varying degrees.
  • the positive drug group and the dihydroquercetin-treated group also showed corneal epithelial cell damage, but compared with the model group, it was significantly reduced (P ⁇ 0.001). This indicates that the positive drug group and the dihydroquercetin-treated groups at various concentrations reduced the degree of damage to the corneal epithelium of mice and had a certain protective effect on the cornea.
  • N blank control group
  • M model control group
  • LUV positive drug group
  • E1 dihydroquercetin low concentration group
  • E2 dihydroquercetin high concentration group
  • R solvent control group
  • Feeding conditions The experimental animals were raised in the general rabbit laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, one rabbit was kept in each cage, for a total of 6 cages. The animals were raised in stainless steel hanging cages, and they were allowed to eat and drink freely. No animals of other species were kept in the same room.
  • the temperature and humidity of the animal room are automatically controlled, the temperature is controlled at 20°C-25°C, and the humidity is controlled at 40%-70%.
  • Artificial lighting, the light cycle is automatically controlled, 12 hours light, 12 hours dark, and the noise is below 60dB.
  • Animal Welfare Animal use complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well” (2006, Ministry of Science and Technology). The animal use plan is approved by the Institutional Animal Care Committee (IACUC), and the experimental process is subject to its supervision. During the experiment, it may be necessary to humanely kill the animals to alleviate the animals' suffering or pain. The treatment of animals killed for humane reasons is the same as that of animals that died in the experiment. Surviving animals are euthanized at the end of the experiment. Animal carcasses are entrusted to professional institutions for disposal.
  • IACUC Institutional Animal Care Committee
  • the chemical injury method was used to establish a rabbit corneal neovascularization model.
  • 1% sodium pentobarbital was injected into the ear vein for general anesthesia, and proparacaine eye drops were used for ocular surface anesthesia.
  • a 6mm diameter filter paper was soaked in 1mol/L NaOH solution for 1 minute, and then the soaked filter paper was applied to the center of the cornea. After 30 seconds, the filter paper was removed, and then the ocular surface and conjunctival sac were rinsed with saline for 1 minute.
  • levofloxacin was added to the modeled eye to prevent postoperative infection.
  • the administration method was ocular administration, and all animals in the drug administration groups began to be administered the day before modeling. No drug was given to the blank control group and the model control group; the positive drug group was given levofloxacin eye drops 50 ⁇ L/time, which were dropped into the conjunctival sac; the low-concentration drug administration group was given 0.03% dihydroquercetin eye drops 50 ⁇ L/time, and the high-concentration drug administration group was given 0.3% dihydroquercetin eye drops 50 ⁇ L/time. Each group of New Zealand rabbits was given 2 times/day (9:00 am and 3:00 pm).
  • the ocular surface inflammation and neovascularization area of the animals in each experimental group were detected 14 days after administration. Photos were taken and the neovascularization area was calculated. The data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 6 is a morphological diagram of corneal neovascularization detected by corneal slit lamp in New Zealand rabbits of each experimental group 14 days after administration.
  • the black circle area is the area of corneal neovascularization.
  • the cornea of the blank control group is transparent and has no neovascularization; a large number of dendritic neovascularization appeared in the cornea of the model group and the solvent control group, expanding along the corneal limbus toward the central area of the cornea, which was significantly different from the blank control group; only a small amount of neovascularization appeared on the cornea of the positive drug control group and the high and low concentrations of dihydroquercetin administration groups, which was significantly improved compared with the model group.
  • N is the blank control group
  • M is the model control group
  • R is the solvent control group
  • Y is the positive drug group (levofloxacin)
  • E1 is the low concentration of dihydroquercetin group
  • E2 is the high concentration of dihydroquercetin group.
  • ** represents P ⁇ 0.01
  • **** represents P ⁇ 0.0001.
  • the area of corneal neovascularization in the model group and solvent control group was significantly different from that in the blank control group (P ⁇ 0.0001); the area of corneal neovascularization in the positive drug control group and the high and low concentrations of dihydroquercetin groups was smaller, which was significantly improved compared with the model group (P ⁇ 0.01), indicating that dihydroquercetin can inhibit the appearance of neovascularization and reduce the degree of corneal alkali burns.
  • dihydroquercetin plays an outstanding role in the treatment of ocular surface diseases.
  • Dihydroquercetin at a concentration of 50-200 ⁇ M promotes the proliferation of HCE cells
  • dihydroquercetin at a concentration of 25-200 ⁇ M protects HCE cells from oxidative stress damage induced by H2O2 .
  • the tear secretion of dry eye mice increased to a certain extent, and the stability of the tear film of mice was improved.
  • dihydroquercetin can reduce the degree of damage to the corneal epithelium of mice, has a certain protective effect on the cornea, and can inhibit the appearance of new blood vessels and reduce the degree of corneal alkali burns.

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Abstract

Disclosed is use of dihydroquercetin in treating ocular surface diseases, pertaining to the technical field of pharmacy. The use of dihydroquercetin in treating ocular surface diseases of the present invention is specifically use of dihydroquercetin in preparing a medicament for preventing, alleviating and/or treating ocular surface diseases. The present invention applies dihydroquercetin to the prevention, alleviation and/or treatment of ocular surface diseases for the first time. The dihydroquercetin serving as an antioxidant has a good protective effect against diseases related to oxidative damage in human corneal epithelial (HCE) cells. Moreover, in a certain concentration range, the dihydroquercetin can promote the proliferation and improve the vitality of the HCE cells, and can also increase the tear secretion amount and tear film stability of the HCE cells, resulting in significant amelioration of the ocular surface diseases.

Description

二氢槲皮素在治疗眼表疾病中的应用Application of dihydroquercetin in the treatment of ocular surface diseases
本申请要求于2022年12月02日提交中国专利局、申请号为202211536112.X、发明名称为“二氢槲皮素在治疗眼表疾病中的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application filed with the China Patent Office on December 2, 2022, with application number 202211536112.X and invention name “Application of dihydroquercetin in the treatment of ocular surface diseases”, the entire contents of which are incorporated by reference into this application.
技术领域Technical Field
本发明涉及药学技术领域，尤其涉及二氢槲皮素在治疗眼表疾病中的应用。The invention relates to the technical field of pharmaceuticals, and in particular to application of dihydroquercetin in treating ocular surface diseases.
背景技术Background technique
氧化损伤在疾病中广泛存在，尤其是在衰老、炎症相关疾病中起到重要作用。氧化作用产生的自由基可直接作用于组织细胞膜，破坏细胞膜，并且自由基可通过细胞膜上的通道进入细胞内，对细胞内的蛋白、DNA产生损伤；氧自由基参与花生四烯酸的代谢为炎症反应中的重要过程，其产生的脂质过氧化物为趋化因子，会加剧炎症反应。另外，氧化产物还可以诱导不同于花生四烯酸的趋化因子产生，使蛋白水解酶抑制剂失活，使胶原酶等增加，从而破坏结缔组织。常见的因氧化损伤引起的眼部疾病有干眼症和角膜碱烧伤。Oxidative damage is widely present in diseases, especially playing an important role in aging and inflammation-related diseases. The free radicals produced by oxidation can directly act on tissue cell membranes and destroy them. Free radicals can also enter cells through channels on the cell membranes, causing damage to proteins and DNA in cells. Oxygen free radicals participate in the metabolism of arachidonic acid, which is an important process in the inflammatory response. The lipid peroxides produced are chemokines, which can aggravate the inflammatory response. In addition, oxidation products can also induce the production of chemokines different from arachidonic acid, inactivate protease inhibitors, increase collagenase, etc., and thus destroy connective tissue. Common eye diseases caused by oxidative damage include dry eye and corneal alkali burns.
干眼症是眼科中常见的疾病，主要是指泪液不足或泪液过度蒸发所致的一种由泪液稳定性和眼表炎症性等多种因素引起的疾病。近年来国内外学者对干眼症发病机制做了深入研究，认为该病发生发展与机体免疫炎症反应、细胞凋亡以及性激素水平有关，其中T细胞介导的免疫炎症反应是干眼发病中最为关键的因素。另外，泪液分泌量减少或蒸发量增加导致泪液形成高渗透压，也是引起干眼症状恶性循环的关键因素，泪液的高渗透压会导致结膜上皮细胞凋亡、功能性杯状细胞数量减少等形态学上的改变并引发炎症级联反应，进一步引起角膜上皮细胞死亡，这些功能性细胞缺失后，会减少泪液中粘蛋白和脂质含量，进而加剧泪膜的不稳定性，推动这一恶性循环。该疾病导致不同程度的眼干涩、异物感、畏光感和疼痛感，严重的出现角膜损伤，患者视觉功能受到威胁，因此关于干眼症的临床治疗研究迫在眉睫。Dry eye is a common disease in ophthalmology. It mainly refers to a disease caused by insufficient tears or excessive tear evaporation, which is caused by multiple factors such as tear stability and ocular surface inflammation. In recent years, scholars at home and abroad have conducted in-depth research on the pathogenesis of dry eye, and believe that the occurrence and development of the disease is related to the body's immune inflammatory response, cell apoptosis and sex hormone levels. Among them, T cell-mediated immune inflammatory response is the most critical factor in the onset of dry eye. In addition, the decrease in tear secretion or increase in evaporation leads to high osmotic pressure in tears, which is also a key factor in causing the vicious cycle of dry eye symptoms. The high osmotic pressure of tears can cause morphological changes such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, and trigger an inflammatory cascade reaction, further causing the death of corneal epithelial cells. After the loss of these functional cells, the content of mucin and lipids in tears will be reduced, thereby aggravating the instability of the tear film and promoting this vicious cycle. The disease causes varying degrees of dry eyes, foreign body sensation, photophobia and pain. In severe cases, corneal damage occurs, and the patient's visual function is threatened. Therefore, clinical treatment research on dry eye is imminent.
角膜碱烧伤是临床上常见的眼外伤类型，致盲率极高。在碱烧伤早期，细胞凋亡及炎症反应激活是造成角膜损伤的重要因素；在碱烧伤后期，角膜新生血管会严重影响角膜透明度及视力水平。但是，眼底的脉络膜新生血管(CNV) 的形成是一个复杂的病理过程，受到多种因素的调节，许多细胞因子形成的生长因子网络精确地调控着CNV的形成。血管内皮生长因子(VEGF)是目前发现最强的促血管形成因子，VEGF的过度表达与CNV的发生发展密切相关。目前，抑制新生血管发生机制的药物种类繁多，但很多药物的不良反应在治疗过程中被逐步证实，比如糖皮质激素常见的副作用有库欣综合征、感染、消化道反应、水肿、糖代谢紊乱、电解质失衡、神经系统兴奋异常等，抗VEGF药物常见的副作用有结膜充血、眼痛、异物感角膜擦伤、角膜水肿眼压升高、黑影飘动、眼内感染、视网膜脱离或玻璃体出血等。Corneal alkali burns are a common type of ocular trauma in clinical practice, with a very high blindness rate. In the early stage of alkali burns, cell apoptosis and activation of inflammatory response are important factors causing corneal damage; in the late stage of alkali burns, corneal neovascularization will seriously affect corneal transparency and visual acuity. However, choroidal neovascularization (CNV) of the fundus The formation of CNV is a complex pathological process, which is regulated by multiple factors. The growth factor network formed by many cytokines precisely regulates the formation of CNV. Vascular endothelial growth factor (VEGF) is the strongest angiogenic factor discovered so far. The overexpression of VEGF is closely related to the occurrence and development of CNV. At present, there are many kinds of drugs that inhibit the mechanism of neovascularization, but the adverse reactions of many drugs have been gradually confirmed during the treatment process. For example, the common side effects of glucocorticoids include Cushing's syndrome, infection, gastrointestinal reaction, edema, glucose metabolism disorder, electrolyte imbalance, abnormal nervous system excitement, etc. The common side effects of anti-VEGF drugs include conjunctival congestion, eye pain, foreign body sensation, corneal abrasion, corneal edema, increased intraocular pressure, floating black shadows, intraocular infection, retinal detachment or vitreous hemorrhage, etc.
因此，研究开发出新型的对眼部氧化损伤相关疾病有保护作用的抗氧化剂至关重要。Therefore, it is crucial to research and develop new antioxidants that have protective effects on diseases related to ocular oxidative damage.
发明内容Summary of the invention
有鉴于此，本发明要解决的技术问题在于提供二氢槲皮素在治疗眼表疾病中的应用。所述二氢槲皮素对H2O2诱导的角膜上皮细胞(HCE)的氧化应激损伤具有保护作用。In view of this, the technical problem to be solved by the present invention is to provide a use of dihydroquercetin in treating ocular surface diseases. The dihydroquercetin has a protective effect on H 2 O 2- induced oxidative stress damage of corneal epithelial cells (HCE).
为达到以上目的，本发明采用的技术方案如下：In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:
本发明提供了二氢槲皮素在制备用于预防、减轻和/或治疗眼表疾病的药物中的应用。The present invention provides the use of dihydroquercetin in preparing a medicine for preventing, alleviating and/or treating ocular surface diseases.
二氢槲皮素(dihydroquercetin，DHQ)，别名花旗松素(taxifolin)。它存在于多种植物中，在落叶松中含量较高，特别是花旗松。二氢槲皮素最早由日本学者Fukui从针叶植物Chamaecyparisobtusa叶中提取分离。近年来，在很多水果中也发现了二氢槲皮素的存在，如葡萄、橘子和西柚等。研究表明，二氢槲皮素含有较多的酚羟基，具有多种生物学活性，能够抑制或激活多种酶，从而产生不同的生理效应。二氢槲皮素的结构式如下：
Dihydroquercetin (DHQ), also known as taxifolin, exists in many plants, and its content is higher in larch, especially Douglas fir. Dihydroquercetin was first extracted and separated from the leaves of the conifer Chamaecyparisobtusa by Japanese scholar Fukui. In recent years, dihydroquercetin has also been found in many fruits, such as grapes, oranges and grapefruits. Studies have shown that dihydroquercetin contains more phenolic hydroxyl groups, has multiple biological activities, and can inhibit or activate multiple enzymes, thereby producing different physiological effects. The structural formula of dihydroquercetin is as follows:
本发明首次将二氢槲皮素应用于预防、减轻和/或治疗眼表疾病。The present invention applies dihydroquercetin to the prevention, alleviation and/or treatment of ocular surface diseases for the first time.
优选的，所述眼表疾病为由角膜上皮细胞损伤和/或凋亡引发的眼表疾病。Preferably, the ocular surface disease is an ocular surface disease caused by corneal epithelial cell damage and/or apoptosis.
优选的，所述角膜上皮损伤为氧化损伤。Preferably, the corneal epithelial damage is oxidative damage.
优选的，所述氧化损伤为由过氧化氢导致的损伤。Preferably, the oxidative damage is damage caused by hydrogen peroxide.
本发明所述的二氢槲皮素对H2O2诱导的角膜上皮细胞(HCE)的氧化应激损伤具有保护作用。The dihydroquercetin of the present invention has a protective effect on oxidative stress damage of corneal epithelial cells (HCE) induced by H 2 O 2 .
上述保护作用具体为：在一定浓度范围内，二氢槲皮素对HCE细胞具有增殖作用，能提高氧化损伤后的HCE细胞的相对细胞活力。The above protective effect is specifically as follows: within a certain concentration range, dihydroquercetin has a proliferation effect on HCE cells and can increase the relative cell viability of HCE cells after oxidative damage.
本发明通过建立H2O2诱导角膜上皮细胞(HCE)损伤的模型，确定了H2O2造模浓度，然后在该浓度下进行造模引起HCE细胞的氧化损伤，最后将不同浓度的二氢槲皮素分别作用于HCE细胞，发现一定浓度的二氢槲皮素对HCE细胞具有增殖作用，而浓度过高则会对HCE细胞产生一定毒性。The present invention establishes a H2O2 - induced corneal epithelial cell (HCE) damage model, determines the H2O2 modeling concentration, then performs modeling at this concentration to cause oxidative damage to HCE cells, and finally acts on HCE cells with different concentrations of dihydroquercetin. It is found that a certain concentration of dihydroquercetin has a proliferation effect on HCE cells, while too high a concentration will produce certain toxicity to HCE cells.
上述造模浓度具体为300μM。在本发明的一些具体实施例中，对HCE细胞具有增殖作用的二氢槲皮素的浓度为25-200μM，对HCE细胞能产生一定毒性的二氢槲皮素的浓度为600μM。The modeling concentration is specifically 300 μM. In some specific embodiments of the present invention, the concentration of dihydroquercetin having a proliferation effect on HCE cells is 25-200 μM, and the concentration of dihydroquercetin that can produce a certain degree of toxicity to HCE cells is 600 μM.
本发明优选的，所述眼表疾病为干眼症或角膜新生血管相关疾病。Preferably, the ocular surface disease of the present invention is dry eye or a disease related to corneal neovascularization.
试验结果表明，通过二氢槲皮素治疗后，HCE细胞的泪液分泌量和泪膜稳定性会有一定的增加，干眼症会得到明显改善。The experimental results show that after treatment with dihydroquercetin, the tear secretion and tear film stability of HCE cells will increase to a certain extent, and dry eye syndrome will be significantly improved.
本发明还提供了一种二氢槲皮素眼用制剂，包括二氢槲皮素和溶剂。The present invention also provides a dihydroquercetin ophthalmic preparation, comprising dihydroquercetin and a solvent.
优选的，所述二氢槲皮素的质量浓度为0.01％～0.5％；更优选的，所述二氢槲皮素的质量浓度为0.03％～0.3％；进一步优选的，所述二氢槲皮素的质量浓度为0.03％或0.3％。 Preferably, the mass concentration of the dihydroquercetin is 0.01% to 0.5%; more preferably, the mass concentration of the dihydroquercetin is 0.03% to 0.3%; further preferably, the mass concentration of the dihydroquercetin is 0.03% or 0.3%.
本发明优选的，所述制剂的剂型为滴眼液、眼膏、眼周及眼内注射液、眼用凝胶或脂质体。Preferably, the preparation is in the form of eye drops, eye ointment, periocular and intraocular injection, eye gel or liposome.
与现有技术相比，本发明提供了二氢槲皮素在制备用于预防、减轻和/或治疗眼表疾病的药物中的应用。本发明首次将二氢槲皮素应用于预防、减轻和/或治疗眼表疾病。所述二氢槲皮素作为抗氧化剂对角膜上皮细胞(HCE)氧化损伤的相关疾病具有很好的保护作用。并且，在一定浓度范围内，所述二氢槲皮素可以促进HCE细胞的增殖提高细胞活力，也可以增加HCE细胞的泪液分泌量和泪膜稳定性使眼表疾病得到明显改善。Compared with the prior art, the present invention provides the use of dihydroquercetin in the preparation of a drug for preventing, alleviating and/or treating ocular surface diseases. The present invention applies dihydroquercetin to the prevention, alleviating and/or treating ocular surface diseases for the first time. As an antioxidant, the dihydroquercetin has a good protective effect on diseases related to oxidative damage of corneal epithelial cells (HCE). Moreover, within a certain concentration range, the dihydroquercetin can promote the proliferation of HCE cells and improve cell viability, and can also increase the tear secretion and tear film stability of HCE cells to significantly improve ocular surface diseases.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是采用MTS法分别检测不同浓度二氢槲皮素(A)、不同浓度H2O2(B)以及300μM过氧化氢与不同浓度二氢槲皮素组合(C)作用于角膜上皮细胞24小时后的结果图，其中，P代表显著水平，**代表P<0.01，***代表P<0.001，##代表P<0.01，###代表P<0.001；Figure 1 is a graph showing the results of using the MTS method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and a combination of 300 μM hydrogen peroxide and different concentrations of dihydroquercetin (C) after acting on corneal epithelial cells for 24 hours, wherein P represents a significant level, ** represents P<0.01, *** represents P<0.001, ## represents P<0.01, and ### represents P<0.001;
图2是动物实验活体小鼠泪液分泌量检测结果图；FIG2 is a diagram showing the results of tear secretion testing of live mice in animal experiments;
图3是动物实验活体小鼠泪膜破裂时间检测结果图；FIG3 is a diagram showing the tear film breakup time test results of live mice in animal experiments;
图4是各实验组小鼠经角膜荧光素钠染色后的角膜形态图；FIG4 is a diagram of the corneal morphology of mice in each experimental group after corneal fluorescein sodium staining;
图5是角膜荧光素钠染色检查小鼠角膜上皮损伤的评分结果图；FIG5 is a graph showing the scoring results of corneal epithelial damage in mice examined by corneal sodium fluorescein staining;
图6是角膜裂隙灯检测各实验组新西兰兔角膜上新生血管的形态图；FIG6 is a morphological diagram of new blood vessels on the cornea of New Zealand rabbits in each experimental group detected by corneal slit lamp;
图7是各实验组新西兰兔角膜新生血管面积检测结果图，其中****代表P<0.0001。FIG. 7 is a graph showing the results of the detection of corneal neovascularization areas in New Zealand rabbits in each experimental group, where **** represents P<0.0001.
具体实施方式Detailed ways
为了进一步说明本发明，下面结合实施例对本发明提供的二氢槲皮素在治疗眼表疾病中的应用进行详细描述。To further illustrate the present invention, the application of dihydroquercetin provided by the present invention in treating ocular surface diseases is described in detail below in conjunction with examples.
人角膜上皮细胞购自美国ATCC细胞库，细胞培养基采用高糖DMEM培养基辅以10％(V/V)胎牛血清、100U/mL青霉素、100μg/mL庆大霉素。于含5％二氧化碳，37℃培养箱中培养。细胞铺于96孔板中，用对数生长期细胞， 以1.5×105个/mL的密度，每孔100μL铺板，每组6个副孔。细胞分组均为正常细胞组、过氧化氢模型组、各浓度二氢槲皮素干预组。检测方法为MTS试剂盒法(代表Promega)，490nm处酶标仪检测吸光度。
存活率＝各组存活率/正常细胞组存活率×100％Human corneal epithelial cells were purchased from the ATCC cell bank in the United States. The cell culture medium used was high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100μg/mL gentamicin. The cells were cultured in an incubator containing 5% carbon dioxide at 37°C. The cells were plated in a 96-well plate and the cells were used in the logarithmic growth phase. The cells were plated at a density of 1.5×10 5 cells/mL, 100 μL per well, and 6 duplicate wells per group. The cells were grouped into normal cell group, hydrogen peroxide model group, and dihydroquercetin intervention groups at different concentrations. The detection method was the MTS kit method (represented by Promega), and the absorbance was detected by a microplate reader at 490 nm.
Survival rate = survival rate of each group / survival rate of normal cell group × 100%
实验例1Experimental Example 1
(一)MTS检测方法(I) MTS detection method
人角膜上皮细胞购自美国ATCC细胞库，细胞培养基采用高糖DMEM培养基辅以10％(V/V)胎牛血清、100U/mL青霉素、100μg/mL庆大霉素。于含5％二氧化碳，37℃培养箱中培养。细胞铺于96孔板中，用对数生长期细胞，以1.5×105个/mL的密度，每孔100μL铺板，每组6个副孔。24h后根据实验目的对细胞进行H2O2建模或二氢槲皮素给药处理。呈色：每孔加MTS溶液20μL，继续孵育2～4h。比色：选择490nm波长，在酶联免疫监测仪上测定各孔光吸收值，记录结果，以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线。Human corneal epithelial cells were purchased from the ATCC cell bank in the United States. The cell culture medium used high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100μg/mL gentamicin. The cells were cultured in an incubator containing 5% carbon dioxide at 37°C. The cells were plated in a 96-well plate. Cells in the logarithmic growth phase were plated at a density of 1.5×10 5 /mL, 100μL per well, and 6 wells per group. After 24 hours, the cells were treated with H 2 O 2 modeling or dihydroquercetin administration according to the purpose of the experiment. Color development: 20μL of MTS solution was added to each well and incubated for 2 to 4 hours. Colorimetry: Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, record the results, and draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.
(二)H2O2诱导人角膜上皮细胞(HCE)损伤模型的建立(II) Establishment of H2O2 - induced human corneal epithelial cell (HCE) injury model
HCE细胞传代培养，按照1.5×105cell/mL滴板，100μL/孔。将不同浓度的H2O2(100μM，200μM，300Μm，400μM，500μM，600μM，700μM，800μM，900μM)分别作用HCE细胞24h，每组6复孔以保证实验数据的稳定性和准确性，观察H2O2对细胞的损伤程度。呈色：每孔加MTS溶液20μL，继续孵育2-4h。比色：选择490nm波长，在酶联免疫监测仪上测定各孔光吸收值，记录结果，以不同浓度H2O2为横坐标，吸光值为纵坐标绘制细胞生长曲线。MTT法检测细胞存活率在50％左右时的H2O2浓度做为氧化损伤模型的造模浓度。存活率＝各组存活率/正常细胞组存活率×100％HCE cells were subcultured, and the plate was dripped at 1.5×10 5 cell/mL, 100μL/well. HCE cells were treated with different concentrations of H 2 O 2 (100μM, 200μM, 300μm, 400μM, 500μM, 600μM, 700μM, 800μM, 900μM) for 24h, 6 replicates per group to ensure the stability and accuracy of the experimental data, and the degree of damage to cells by H 2 O 2 was observed. Color development: 20μL of MTS solution was added to each well, and the incubation continued for 2-4h. Colorimetry: 490nm wavelength was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the results were recorded. The cell growth curve was drawn with different concentrations of H 2 O 2 as the horizontal axis and the absorbance value as the vertical axis. The H 2 O 2 concentration when the cell survival rate was about 50% detected by MTT method was used as the modeling concentration of the oxidative damage model. Survival rate = survival rate of each group / survival rate of normal cell group × 100%
图1是采用MTT法分别检测不同浓度二氢槲皮素(A)、不同浓度H2O2(B)以及300μM过氧化氢与不同浓度二氢槲皮素组合(C)作用于角膜上皮细胞24小时后的结果图，其中，P代表显著水平，**代表P<0.01，***代表P<0.001，##代表P<0.01，###代表P<0.001。如图1中的B图所示，当H2O2浓度在300μM时细胞存活率为49％，所以300μM的H2O2确定为后续实验的氧化损伤模型的造模浓度。Figure 1 is a graph showing the results of using MTT method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and 300 μM hydrogen peroxide and different concentrations of dihydroquercetin (C) acting on corneal epithelial cells for 24 hours, where P represents significant level, ** represents P<0.01, *** represents P<0.001, ## represents P<0.01, and ### represents P<0.001. As shown in Figure B of Figure 1, when the H 2 O 2 concentration is 300 μM, the cell survival rate is 49%, so 300 μM H 2 O 2 is determined as the modeling concentration of the oxidative damage model in subsequent experiments.
(三)二氢槲皮素对HCE细胞的增殖及毒性作用 (III) Proliferation and toxicity of dihydroquercetin on HCE cells
不同浓度的二氢槲皮素(0.01-600μM)分别作用于HCE细胞，每组6复孔以保证实验数据的稳定性和准确性，观察药物对细胞的增殖作用。Different concentrations of dihydroquercetin (0.01-600 μM) were applied to HCE cells, with 6 replicate wells in each group to ensure the stability and accuracy of the experimental data, and the effect of the drug on cell proliferation was observed.
如图1中的A图所示，不同浓度的二氢槲皮素处理HCE细胞24h后，浓度为50μM、100μM、200μM实验组HCE细胞的细胞存活率(cell viability)与对照组相比有显著性差异，明显高于对照组，这说明50-200μM浓度的二氢槲皮素对HCE细胞有促增值作用。当二氢槲皮素浓度升高到600μM时，HCE细胞存活率(cell viability)与对照组相比细胞存活率显著降低，这说明600μM浓度的二氢槲皮素对HCE细胞有毒性。As shown in Figure 1A, after HCE cells were treated with different concentrations of dihydroquercetin for 24 hours, the cell viability of HCE cells in the experimental groups with concentrations of 50μM, 100μM, and 200μM was significantly different from that in the control group, and was significantly higher than that in the control group, indicating that dihydroquercetin at a concentration of 50-200μM has a proliferative effect on HCE cells. When the concentration of dihydroquercetin increased to 600μM, the cell viability of HCE cells was significantly reduced compared with that in the control group, indicating that dihydroquercetin at a concentration of 600μM is toxic to HCE cells.
(四)二氢槲皮素对HCE细胞的抗氧化损伤作用(IV) Antioxidant effect of dihydroquercetin on HCE cells
实验分8组：正常对照组，H2O2模型组，不同浓度二氢槲皮素(0.1-200μM)+H2O2干预组。利用300μM的H2O2对HCE细胞进行造模，造模后安全范围内不同浓度药物作用于HCE细胞，通过MTS方法检测各组间细胞存活率，检测二氢槲皮素对HCE细胞的抗氧化损伤保护作用情况。The experiment was divided into 8 groups: normal control group, H 2 O 2 model group, and different concentrations of dihydroquercetin (0.1-200μM) + H 2 O 2 intervention groups. HCE cells were modeled with 300μM H 2 O 2 , and different concentrations of drugs within the safe range were applied to HCE cells after modeling. The cell survival rate among the groups was detected by MTS method, and the protective effect of dihydroquercetin on the antioxidant damage of HCE cells was detected.
如图1中的C图所示，各实验组的细胞存活率中H2O2建模组低于对照组(P＜0.001)，25-200μM的二氢槲皮素组高于H2O2建模组(P＜0.01)，这说明H2O2能够诱导HCE细胞氧化应激损伤，而二氢槲皮素对H2O2诱导HCE细胞氧化应激损伤的具有保护作用。As shown in Figure 1C, among the experimental groups, the cell survival rate of the H2O2 modeling group was lower than that of the control group (P＜0.001), and that of the 25-200μM dihydroquercetin group was higher than that of the H2O2 modeling group (P＜0.01), indicating that H2O2 can induce oxidative stress damage in HCE cells, and dihydroquercetin has a protective effect on H2O2 - induced oxidative stress damage in HCE cells.
1.1干眼1.1 Dry eyes
1.1.1分组1.1.1 Grouping
50只8周龄C57/BL6的健康雌性小鼠中筛选出30只，随机划分5只小鼠为一组，共划分6组，记录为空白对照组(N)、模型组(M)、阳性药组(CsA)、中药复方低浓度组(SL)、中药复方中浓度组(SM)、中药复方高浓度组(SH)。分组时在小鼠尾部及笼卡进行标识，标识信息包括：实验名称、实验编号、组别、给药剂量、给药时间等。Thirty mice were selected from 50 healthy female C57/BL6 mice aged 8 weeks, and randomly divided into 6 groups with 5 mice in each group, which were recorded as blank control group (N), model group (M), positive drug group (CsA), low concentration group of Chinese herbal medicine compound (SL), medium concentration group of Chinese herbal medicine compound (SM), and high concentration group of Chinese herbal medicine compound (SH). The mice were marked on the tail and cage card when grouping, and the marking information included: experimental name, experimental number, group, dosage, and administration time.
1.1.2饲养1.1.2 Feeding
实验动物饲养于沈阳何氏医学院动物中心S代表PF级实验室中。实验期间每笼(长40cm×宽20cm×高20cm)饲育5只小鼠，共6笼。垫料为玉米芯垫料。本实验采用灭菌全价营养饲料。(采购自辽宁长生生物有限公司)。饮水为纯净水，由动物中心HT-RO1000型净水系统自行制备。每年专人检测水质。实验期间小鼠自由摄取食水，小鼠解剖取材前夜禁食，不禁饮水。动物实 验室温度湿度由空调机组自动控制在温度20℃-25℃，湿度40％-70％，每日记录温湿度变化情况。光照系统自动控制动物实验室内光照周期变化，24小时明暗平均分配。动物实验室噪音控制，不高于60分贝。本实验经机构动物管理委员会(IACUC)批准并接受其监督，严格遵守国家动物福利法规《关于善待动物的指导性意见》。The experimental animals were raised in the S-rated PF laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, 5 mice were kept in each cage (length 40cm×width 20cm×height 20cm), for a total of 6 cages. The bedding was corn cob bedding. This experiment used sterilized complete nutritious feed. (Purchased from Liaoning Changsheng Biological Co., Ltd.). The drinking water was purified water, which was prepared by the HT-RO1000 water purification system of the Animal Center. The water quality is tested by a dedicated person every year. During the experiment, the mice were free to drink water. The mice were fasted the night before the dissection, but they were not forbidden to drink water. Animal experiment The temperature and humidity of the laboratory are automatically controlled by the air conditioning unit at 20℃-25℃ and 40%-70%, and the temperature and humidity changes are recorded daily. The lighting system automatically controls the light cycle changes in the animal laboratory, and the light and dark are evenly distributed for 24 hours. The noise in the animal laboratory is controlled to no more than 60 decibels. This experiment was approved and supervised by the Institutional Animal Care Committee (IACUC) and strictly complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well".
1.2造模方法1.2 Modeling method
本实验采用智能干燥环境控制系统结合东莨菪碱注射诱导建立小鼠干眼模型。造模过程中应用两阶梯除湿法控制模型环境湿度，首先采用可调温工业除湿机将房间湿度降低，将湿度调节到40％±5％，再使用智能干燥箱(除湿范围RH10％-80％)将小鼠饲养环境湿度进一步降低至15％±3％。同时在智能干燥箱内放置无噪音、可调速(风速范围0-5m/s)风扇，放置风扇距离鼠笼20cm，且设置风扇与小鼠同一垂直高度。通过以上措施，可以将各实验环境参数有效控制在实验所需要的环境条件范围内。将模型对照组(M)、阳性药组(CsA)、二氢槲皮素低浓度组(E1)、二氢槲皮素高浓度组(E2)、溶剂对照组小鼠放置于该干燥环境内(湿度15％±3％；风速2.1±0.2m/s；温度21-23℃)饲养，空白对照组小鼠于正常环境中(湿度60％-80％；温度21-23℃)饲养。除空白对照组外，其余各组小鼠每天皮下注射0.5mg/0.2mL东莨菪碱溶液3次(上午9:00；中午12:00；下午3:00)，模型建立时间持续2周。给药采用经眼给药，给药剂量为每只眼结膜囊内滴入5μL每天给药两次(上午9:00；下午3:00)，各给药组均在造模前一天开始给药，连续给药15天。In this experiment, an intelligent dry environment control system combined with scopolamine injection was used to induce dry eye models in mice. During the modeling process, a two-step dehumidification method was used to control the humidity of the model environment. First, an adjustable temperature industrial dehumidifier was used to reduce the humidity in the room and adjust the humidity to 40% ± 5%. Then, an intelligent drying box (dehumidification range RH10%-80%) was used to further reduce the humidity of the mouse breeding environment to 15% ± 3%. At the same time, a noiseless, adjustable speed (wind speed range 0-5m/s) fan was placed in the intelligent drying box. The fan was placed 20cm away from the mouse cage, and the fan was set at the same vertical height as the mouse. Through the above measures, each experimental environmental parameter can be effectively controlled within the range of environmental conditions required for the experiment. The mice in the model control group (M), positive drug group (CsA), low-concentration dihydroquercetin group (E1), high-concentration dihydroquercetin group (E2), and solvent control group were placed in the dry environment (humidity 15% ± 3%; wind speed 2.1 ± 0.2 m/s; temperature 21-23°C), and the mice in the blank control group were raised in a normal environment (humidity 60%-80%; temperature 21-23°C). Except for the blank control group, the mice in the other groups were subcutaneously injected with 0.5 mg/0.2 mL scopolamine solution 3 times a day (9:00 am; 12:00 noon; 3:00 pm), and the model establishment time lasted for 2 weeks. The drug was administered through the eyes, and the dosage was 5 μL in the conjunctival sac of each eye twice a day (9:00 am; 3:00 pm). Each drug group started drug administration the day before modeling and continued for 15 days.
1.3给药方法与剂量1.3 Administration and dosage
给药方式为经眼给药，全部实验小鼠均在造模前一天开始给药。空白对照组、模型对照组不给药；阳性药组采用质量浓度为0.05％的环孢素A滴眼5μL/次，滴入结膜囊内；低浓度给药组采用质量浓度为0.03％二氢槲皮素滴眼5μL/次，高浓度给药组采用质量浓度为0.3％二氢槲皮素滴眼5μL/次，各组小鼠给药均2次/日(上午9：00，下午3：00)。The drug administration method was ocular administration, and all experimental mice were administered the day before modeling. The blank control group and the model control group were not administered; the positive drug group was administered with 0.05% cyclosporine A eye drops 5 μL/time, which was dripped into the conjunctival sac; the low-concentration administration group was administered with 0.03% dihydroquercetin eye drops 5 μL/time, and the high-concentration administration group was administered with 0.3% dihydroquercetin eye drops 5 μL/time. Each group of mice was administered twice/day (9:00 am and 3:00 pm).
1.4模型评估1.4 Model Evaluation
在造模第14天，完成日常造模和给药操作后，检测空白对照组小鼠及模型对照组小鼠角膜上皮损伤情况、泪液分泌情况。统计分析两组指标具有显著性差异时，表示造模成功。 On the 14th day of modeling, after completing the daily modeling and drug administration operations, the corneal epithelial damage and tear secretion of the blank control group and the model control group were detected. When the statistical analysis of the two groups showed significant differences, it indicated that the modeling was successful.
1.5实验检查指标1.5 Experimental inspection indicators
1.5.1一般情况观察1.5.1 General Observations
在适应期(分组前)对所有小鼠进行一次眼科检查(包括视力缺陷情况、眼球异常情况、角膜损伤情况)，确保实验用小鼠无异常。适应期及实验过程中每周称量1次体重，给药期每天在给药前后观察实验小鼠，记录健康状况，是否出现异常体征。小鼠情况观察包括：外观(眼、耳、口、鼻、外阴、皮毛、排泄物、四肢活动以及精神状态)、是否呕吐、活动情况、饮食、步态、濒死、死亡等。During the adaptation period (before grouping), all mice were given an ophthalmic examination (including visual defects, eyeball abnormalities, and corneal damage) to ensure that there were no abnormalities in the experimental mice. The body weight was measured once a week during the adaptation period and the experiment. During the dosing period, the experimental mice were observed before and after dosing every day to record their health status and whether they had abnormal physical signs. The observation of the mouse condition included: appearance (eyes, ears, mouth, nose, vulva, fur, excrement, limb activity, and mental state), vomiting, activity, diet, gait, dying, death, etc.
1.5.2泪液分泌量检测1.5.2 Tear secretion detection
将小鼠在非麻醉的状态下进行固定，将小鼠的下眼睑使用软头镊夹起，然后将酚红棉线折叠一端置入下结膜囊内，放松小鼠下眼睑使之自然闭合，将酚红棉线固定好后，计时1min后取出酚红棉线，将酚红棉线放在标尺纸上，计算变红色部分的长度，记录。对数据使用GraphPad Prism 8.0软件进行统计分析。The mice were fixed under non-anesthesia, the lower eyelids of the mice were clamped with soft-tipped forceps, and then one end of the phenol red cotton thread was folded and placed in the lower conjunctival sac, and the lower eyelids of the mice were relaxed to close naturally. After the phenol red cotton thread was fixed, the phenol red cotton thread was taken out after 1 minute of timing, and the phenol red cotton thread was placed on a ruler paper, and the length of the red part was calculated and recorded. The data were statistically analyzed using GraphPad Prism 8.0 software.
图2是动物实验活体小鼠泪液分泌量检测结果图。如图2所示，N为空白对照组；M为模型对照组；R为溶剂对照组；Y为阳性药组(质量浓度为0.05％环孢素A)；E1为二氢槲皮素低浓度组；E2为二氢槲皮素高浓度组。另外，P代表显著性差异，*代表P<0.05，**代表P<0.01，***代表P<0.001。Figure 2 is a graph showing the results of tear secretion in live mice in animal experiments. As shown in Figure 2, N is a blank control group; M is a model control group; R is a solvent control group; Y is a positive drug group (mass concentration is 0.05% cyclosporine A); E1 is a low-concentration dihydroquercetin group; and E2 is a high-concentration dihydroquercetin group. In addition, P represents a significant difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001.
统计酚红棉线长短(mm)，酚红棉线越长说明泪液分泌量越多，反之泪液分泌量越少。造模前各组动物泪液分泌量均正常，组间无显著性差异(P＞0.05)如图2所示，造模14d后阳性药组(CsA)、二氢槲皮素低浓度组(E1)、二氢槲皮素高浓度组(E2)相对模型对照组(M)泪液分泌量显著增加，差异均具有统计学意义(P＜0.001)，溶剂对照组与模型对照组相比泪液分泌量无显著性变化。上述结果表明经二氢槲皮素治疗后，干眼模型小鼠泪液分泌量有一定增加。The length of the phenol red cotton thread (mm) was counted. The longer the phenol red cotton thread, the more tear secretion, and vice versa. The tear secretion of animals in each group was normal before modeling, and there was no significant difference between the groups (P>0.05). As shown in Figure 2, 14 days after modeling, the tear secretion of the positive drug group (CsA), the low concentration of dihydroquercetin group (E1), and the high concentration of dihydroquercetin group (E2) increased significantly relative to the model control group (M), and the differences were statistically significant (P<0.001). There was no significant change in tear secretion between the solvent control group and the model control group. The above results show that after dihydroquercetin treatment, the tear secretion of dry eye model mice has increased to a certain extent.
1.5.3泪膜破裂时间检测1.5.3 Tear film breakup time detection
将小鼠在非麻醉的状态下进行固定，并使用荧光素钠溶液滴入小鼠结膜囊内，然后被动闭合小鼠眼睑数次后，打开眼睑，在裂隙灯钴蓝光下，记录观察到第一个泪膜破裂黑斑的时间，即为泪膜破裂时间。对数据使用GraphPad Prism 8.0软件进行统计分析。 The mice were fixed in a non-anesthetized state, and sodium fluorescein solution was dripped into the mouse conjunctival sac. After the mouse eyelids were passively closed several times, the eyelids were opened and the time when the first tear film rupture black spot was observed under the cobalt blue light of the slit lamp was recorded, which was the tear film rupture time. The data were statistically analyzed using GraphPad Prism 8.0 software.
图3是动物实验活体小鼠泪膜破裂时间检测结果图。如图3所示，其中N为空白对照组；M为模型对照组；R为溶剂对照组；Y为阳性药组(质量浓度为0.05％环孢素A)；E1为二氢槲皮素低浓度组；E2为二氢槲皮素高浓度组。另外，P代表显著性差异，*代表P<0.05，**代表P<0.01，***代表P<0.001。小鼠造模前检测BUT数值均在正常范围，差异无统计学意义(P>0.05)；经智能干燥环境系统结合药物诱导造模后，空白对照组造模前后检测BUT值差异无统计学意义(P>0.05)；在造模第14天(day 14)后对各组小鼠进行BUT检测。Figure 3 is a graph showing the tear film breakup time test results of live mice in animal experiments. As shown in Figure 3, N is the blank control group; M is the model control group; R is the solvent control group; Y is the positive drug group (mass concentration is 0.05% cyclosporine A); E1 is the low-concentration dihydroquercetin group; E2 is the high-concentration dihydroquercetin group. In addition, P represents significant difference, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. The BUT values of mice before modeling were all within the normal range, and there was no statistically significant difference (P>0.05); after the intelligent drying environment system combined with drug-induced modeling, there was no statistically significant difference in the BUT values of the blank control group before and after modeling (P>0.05); BUT detection was performed on mice in each group after the 14th day of modeling (day 14).
结果如图3所示：与空白对照组比较，其余5组小鼠泪膜破裂时间均有不同程度变短，差异有统计学意义(P<0.05)；与模型对照组比较，二氢槲皮素给药组泪膜破裂时间明显增长，差异有统计学意义(P<0.05)，说明经二氢槲皮素治疗后，小鼠泪膜稳定性得到改善。The results are shown in Figure 3: Compared with the blank control group, the tear film breakup time of the other five groups of mice was shortened to varying degrees, and the difference was statistically significant (P<0.05); compared with the model control group, the tear film breakup time of the dihydroquercetin-treated group was significantly increased, and the difference was statistically significant (P<0.05), indicating that the tear film stability of mice was improved after dihydroquercetin treatment.
1.5.4角膜荧光素钠染色检查角膜上皮损伤1.5.4 Corneal fluorescein sodium staining to examine corneal epithelial damage
分别在开始造模前及造模后14天观察各组实验小鼠角膜形态，并使用荧光素钠溶液染色双眼角膜，并使用裂隙灯进行观察拍照。固定好小鼠后，将5μL的2％荧光素钠生理盐水溶液滴入小鼠结膜囊内，1min后用2mL生理盐水轻轻淋洗眼睛，用棉棒吸去多余溶液，用钴蓝光照射荧光素钠染色的小鼠角膜，用裂隙灯进行观察、拍照并记录。如角膜表面有绿色荧光残留，代表该处角膜上皮有损伤。对角膜表面荧光素钠残留进行评分，记录。对数据使用GraphPad Prism 8.0软件进行统计分析The corneal morphology of each group of experimental mice was observed before and 14 days after modeling, and the corneas of both eyes were stained with sodium fluorescein solution, and a slit lamp was used for observation and photography. After fixing the mice, 5 μL of 2% sodium fluorescein saline solution was dripped into the conjunctival sac of the mice. After 1 minute, the eyes were gently rinsed with 2 mL of saline, and the excess solution was absorbed with a cotton swab. The mouse cornea stained with sodium fluorescein was irradiated with cobalt blue light, and the slit lamp was used for observation, photography and recording. If there is green fluorescence residue on the surface of the cornea, it means that the corneal epithelium is damaged. The sodium fluorescein residue on the corneal surface was scored and recorded. The data were statistically analyzed using GraphPad Prism 8.0 software
图4是各实验组小鼠经角膜荧光素钠染色后的角膜形态图。如图4所示，空白对照组小鼠角膜均完整光滑，无荧光素钠附着，说明空白对照组小鼠角膜上皮完好无损伤。除空白对照组外，模型组与各给药组均有不同程度的荧光素钠附着，说明模型对照组与各给药组小鼠角膜上皮均有不同程度的损伤。在造模第14天，与模型对照组相比，阳性药组和二氢槲皮素给药组也出现了密集的点状荧光附着，但与模型组相比有显著性减少。Figure 4 is a corneal morphology of mice in each experimental group after corneal fluorescein sodium staining. As shown in Figure 4, the corneas of mice in the blank control group were intact and smooth, without sodium fluorescein attachment, indicating that the corneal epithelium of mice in the blank control group was intact and undamaged. Except for the blank control group, the model group and each drug-treated group had varying degrees of sodium fluorescein attachment, indicating that the corneal epithelium of mice in the model control group and each drug-treated group had varying degrees of damage. On the 14th day of modeling, compared with the model control group, the positive drug group and the dihydroquercetin-treated group also had dense punctate fluorescent attachment, but compared with the model group, there was a significant reduction.
角膜荧光素钠染色检查各实验组小鼠角膜上皮损伤评分结果如图5所示，其中，N为空白对照组；M为模型对照组；R为溶剂对照组；Y为阳性药组(质量浓度为0.05％环孢素A)；E1为二氢槲皮素低浓度组；E2为二氢槲皮素高浓度组。另外，*代表P<0.05，**代表P<0.01，***代表P<0.001。由图可知，除 空白对照组外，模型组与各给药组小鼠角膜上皮均有不同程度的损伤。在造模第14天，与模型对照组相比，阳性药组和二氢槲皮素给药组也出现了角膜上皮细胞损伤，但与模型组相比有显著性减少(P<0.001)。说明阳性药组与各浓度二氢槲皮素给药组减轻了小鼠角膜上皮的损伤程度，对角膜有一定的保护作用。The corneal epithelial injury scores of the mice in each experimental group after corneal fluorescein sodium staining are shown in Figure 5, where N is the blank control group; M is the model control group; R is the solvent control group; Y is the positive drug group (mass concentration is 0.05% cyclosporine A); E1 is the low-concentration dihydroquercetin group; and E2 is the high-concentration dihydroquercetin group. In addition, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001. As can be seen from the figure, except for Except for the blank control group, the corneal epithelium of mice in the model group and each drug-treated group was damaged to varying degrees. On the 14th day of modeling, compared with the model control group, the positive drug group and the dihydroquercetin-treated group also showed corneal epithelial cell damage, but compared with the model group, it was significantly reduced (P<0.001). This indicates that the positive drug group and the dihydroquercetin-treated groups at various concentrations reduced the degree of damage to the corneal epithelium of mice and had a certain protective effect on the cornea.
2.1角膜碱烧伤建立角膜新生血管疾病模型2.1 Establishment of corneal neovascularization disease model by corneal alkali burn
2.1.1分组2.1.1 Grouping
50只3月龄的健康新西兰兔，雌雄各半，随机划分6只小鼠为一组，共划分6组，记录为空白对照组(N)、模型对照组(M)、阳性药组(LEV)、二氢槲皮素低浓度组(E1)、二氢槲皮素高浓度组(E2)和溶剂对照组(R)。分组时在兔耳部及笼卡进行标识，标识信息包括：实验名称、实验编号、组别、给药剂量、给药时间等。Fifty healthy New Zealand rabbits aged 3 months, half male and half female, were randomly divided into 6 groups, including blank control group (N), model control group (M), positive drug group (LEV), dihydroquercetin low concentration group (E1), dihydroquercetin high concentration group (E2) and solvent control group (R). The rabbit ears and cage cards were marked when grouping, and the marking information included: experimental name, experimental number, group, dosage, administration time, etc.
2.1.2饲养2.1.2 Feeding
饲养条件：实验动物饲养于沈阳何氏医学院动物中心普通级兔实验室中。实验期间每笼饲育1只兔，共6笼。采用不锈钢悬挂笼单笼饲养，动物自由摄食饮水，同一房间内未饲养其他种属的动物。Feeding conditions: The experimental animals were raised in the general rabbit laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, one rabbit was kept in each cage, for a total of 6 cages. The animals were raised in stainless steel hanging cages, and they were allowed to eat and drink freely. No animals of other species were kept in the same room.
环境条件：动物房的温湿度自动控制，控制温度20℃-25℃，控制湿度40％-70％。人工光照，光周期自动控制，12小时明，12小时暗，噪音在60dB以下。Environmental conditions: The temperature and humidity of the animal room are automatically controlled, the temperature is controlled at 20℃-25℃, and the humidity is controlled at 40%-70%. Artificial lighting, the light cycle is automatically controlled, 12 hours light, 12 hours dark, and the noise is below 60dB.
动物福利：动物使用遵守国家动物福利法规《关于善待动物的指导性意见》(2006年，科技部)。动物使用方案经机构动物管理委员会(IACUC)批准，试验过程接受其监督。试验过程中，可能需要人道处死动物，以减轻动物的痛苦或疼痛。人道原因处死的动物处理方式同试验中死亡的动物。试验结束存活的动物进行安乐死。动物尸体委托专业机构进行处置。Animal Welfare: Animal use complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well" (2006, Ministry of Science and Technology). The animal use plan is approved by the Institutional Animal Care Committee (IACUC), and the experimental process is subject to its supervision. During the experiment, it may be necessary to humanely kill the animals to alleviate the animals' suffering or pain. The treatment of animals killed for humane reasons is the same as that of animals that died in the experiment. Surviving animals are euthanized at the end of the experiment. Animal carcasses are entrusted to professional institutions for disposal.
2.2造模方法2.2 Modeling method
本实验采用化学损伤法建立兔角膜新生血管模型，使用1％戊巴比妥钠耳缘静脉注射进行全身麻醉，丙美卡因滴眼液进行眼表麻醉，取6mm直径的滤纸片浸泡在1mol/L的NaOH溶液中浸泡1分钟，然后将浸泡后的滤纸片贴敷在角膜中心，30秒后取下滤纸片，然后用生理盐水冲洗眼表及结膜囊1分钟，造模完成后将造模眼滴加左氧氟沙星预防术后感染。 In this experiment, the chemical injury method was used to establish a rabbit corneal neovascularization model. 1% sodium pentobarbital was injected into the ear vein for general anesthesia, and proparacaine eye drops were used for ocular surface anesthesia. A 6mm diameter filter paper was soaked in 1mol/L NaOH solution for 1 minute, and then the soaked filter paper was applied to the center of the cornea. After 30 seconds, the filter paper was removed, and then the ocular surface and conjunctival sac were rinsed with saline for 1 minute. After the modeling was completed, levofloxacin was added to the modeled eye to prevent postoperative infection.
2.3给药方法与剂量2.3 Administration and Dosage
给药方式为经眼给药，全部给药组动物均在造模前一天开始给药。空白对照组、模型对照组不给药；阳性药组采用左氧氟沙星滴眼50μL/次，滴入结膜囊内；低浓度给药组采用质量浓度为0.03％二氢槲皮素滴眼50μL/次，高浓度给药组采用质量浓度为0.3％二氢槲皮素滴眼50μL/次，各组新西兰兔给药均2次/日(上午9：00，下午3：00)。The administration method was ocular administration, and all animals in the drug administration groups began to be administered the day before modeling. No drug was given to the blank control group and the model control group; the positive drug group was given levofloxacin eye drops 50 μL/time, which were dropped into the conjunctival sac; the low-concentration drug administration group was given 0.03% dihydroquercetin eye drops 50 μL/time, and the high-concentration drug administration group was given 0.3% dihydroquercetin eye drops 50 μL/time. Each group of New Zealand rabbits was given 2 times/day (9:00 am and 3:00 pm).
2.4角膜新生血管面积检测2.4 Detection of corneal neovascularization area
各实验组动物在给药后14天进行眼表炎症及新生血管面积检测，拍照记录并计算新生血管面积，对数据使用GraphPad Prism 8.0软件进行统计分析。The ocular surface inflammation and neovascularization area of the animals in each experimental group were detected 14 days after administration. Photos were taken and the neovascularization area was calculated. The data were statistically analyzed using GraphPad Prism 8.0 software.
图6为角膜裂隙灯检测各实验组新西兰兔给药后14天角膜上新生血管的形态图，黑色圆圈区域即为角膜新生血管面积，空白对照组角膜透明无新生血管；模型组和溶剂对照组角膜出现大量树枝状新生血管沿着角膜缘向着角膜中心区域扩张，与空白对照组相比有显著性差异；阳性药对照组及高低浓度二氢槲皮素给药组角膜上仅出现少量的新生血管，与模型组相对比有显著性改善。各实验组的新西兰兔的角膜新生血管面积的检测结果如图7所示，其中，N为空白对照组；M为模型对照组；R为溶剂对照组；Y为阳性药组(左氧氟沙星)；E1为二氢槲皮素低浓度组；E2为二氢槲皮素高浓度组。另外，**代表P<0.01，****代表P<0.0001。模型组和溶剂对照组角膜的新生血管面积与空白对照组相比有显著性差异(P<0.0001)；阳性药对照组及高低浓度二氢槲皮素给药组角膜的新生血管面积较小，与模型组相对比有显著性改善(P<0.01)，说明二氢槲皮素能抑制新生血管的出现，减轻角膜碱烧伤程度。Figure 6 is a morphological diagram of corneal neovascularization detected by corneal slit lamp in New Zealand rabbits of each experimental group 14 days after administration. The black circle area is the area of corneal neovascularization. The cornea of the blank control group is transparent and has no neovascularization; a large number of dendritic neovascularization appeared in the cornea of the model group and the solvent control group, expanding along the corneal limbus toward the central area of the cornea, which was significantly different from the blank control group; only a small amount of neovascularization appeared on the cornea of the positive drug control group and the high and low concentrations of dihydroquercetin administration groups, which was significantly improved compared with the model group. The detection results of the corneal neovascularization area of New Zealand rabbits in each experimental group are shown in Figure 7, where N is the blank control group; M is the model control group; R is the solvent control group; Y is the positive drug group (levofloxacin); E1 is the low concentration of dihydroquercetin group; E2 is the high concentration of dihydroquercetin group. In addition, ** represents P < 0.01, **** represents P < 0.0001. The area of corneal neovascularization in the model group and solvent control group was significantly different from that in the blank control group (P<0.0001); the area of corneal neovascularization in the positive drug control group and the high and low concentrations of dihydroquercetin groups was smaller, which was significantly improved compared with the model group (P<0.01), indicating that dihydroquercetin can inhibit the appearance of neovascularization and reduce the degree of corneal alkali burns.
综上可知，二氢槲皮素应用于治疗眼表疾病中作用突出，50-200μM浓度的二氢槲皮素对HCE细胞有促增值作用、25-200μM的二氢槲皮素对H2O2诱导HCE细胞氧化应激损伤的具有保护作用。经二氢槲皮素治疗后，干眼小鼠泪液分泌量有一定增加，小鼠泪膜稳定性得到改善。并且二氢槲皮素能够减轻小鼠角膜上皮的损伤程度，对角膜有一定的保护作用，同时可以抑制新生血管的出现，减轻角膜碱烧伤程度。In summary, dihydroquercetin plays an outstanding role in the treatment of ocular surface diseases. Dihydroquercetin at a concentration of 50-200μM promotes the proliferation of HCE cells, and dihydroquercetin at a concentration of 25-200μM protects HCE cells from oxidative stress damage induced by H2O2 . After treatment with dihydroquercetin, the tear secretion of dry eye mice increased to a certain extent, and the stability of the tear film of mice was improved. In addition, dihydroquercetin can reduce the degree of damage to the corneal epithelium of mice, has a certain protective effect on the cornea, and can inhibit the appearance of new blood vessels and reduce the degree of corneal alkali burns.
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的 保护范围内。 The above embodiments are only used to help understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of the claims of the present invention. Within the scope of protection.

Claims (8)

  1. 二氢槲皮素在制备用于预防、减轻和/或治疗眼表疾病的药物中的应用。Application of dihydroquercetin in the preparation of medicines for preventing, alleviating and/or treating ocular surface diseases.
  2. 根据权利要求1所述的应用，其特征在于，所述眼表疾病为由角膜上皮细胞损伤和/或凋亡引发的眼表疾病。The use according to claim 1 is characterized in that the ocular surface disease is an ocular surface disease caused by corneal epithelial cell damage and/or apoptosis.
  3. 根据权利要求2所述的应用，其特征在于，所述角膜上皮损伤为氧化损伤。The use according to claim 2 is characterized in that the corneal epithelial damage is oxidative damage.
  4. 根据权利要求3所述的应用，其特征在于，所述氧化损伤为由过氧化氢导致的损伤。The use according to claim 3 is characterized in that the oxidative damage is damage caused by hydrogen peroxide.
  5. 根据权利要求2所述的应用，其特征在于，所述眼表疾病为干眼症或角膜新生血管相关疾病。The use according to claim 2 is characterized in that the ocular surface disease is dry eye or a disease related to corneal neovascularization.
  6. 一种二氢槲皮素眼用制剂，其特征在于，包括二氢槲皮素和溶剂。A dihydroquercetin ophthalmic preparation, characterized by comprising dihydroquercetin and a solvent.
  7. 根据权利要求6所述的二氢槲皮素眼用制剂，其特征在于，所述二氢槲皮素的质量浓度为0.01％～0.5％。The dihydroquercetin ophthalmic preparation according to claim 6, characterized in that the mass concentration of the dihydroquercetin is 0.01% to 0.5%.
  8. 根据权利要求6所述的二氢槲皮素眼用制剂，其特征在于，所述制剂的剂型为滴眼液、眼膏、眼周及眼内注射液、眼用凝胶或脂质体。 The dihydroquercetin ophthalmic preparation according to claim 6, characterized in that the preparation is in the form of eye drops, eye ointment, periocular and intraocular injection, ophthalmic gel or liposomes.
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