Construction method of mixed rat xerophthalmia animal model

Abstract

The invention discloses a construction method of a mixed type rat xerophthalmia animal model. The xerophthalmia animal model is obtained by intragastric administration of flutamide and eye drop of benzalkonium chloride. The invention aims to establish a mixed xerophthalmia animal model through a combined modeling mode of combining flutamide intragastric administration with benzalkonium chloride solution eye dropping, and provides a new idea and possibility for researching the pathogenesis of xerophthalmia and finding a new treatment method and medicine to prevent or delay the course of xerophthalmia.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a method for constructing a mixed rat dry eye animal model. Background Art

[0002] Dry eye affects about 20-30% of the world's population and is one of the most common eye diseases in the world. It not only causes eye discomfort and even vision damage to patients, but also has serious adverse effects on vision-related quality of life. Since the exact physiological and pathological mechanisms of dry eye have not yet been fully elucidated, it is of great significance to establish an animal model of dry eye to study the pathogenesis of dry eye for the treatment of dry eye.

[0003] Ophthalmological associations at home and abroad agree that dry eye is a multifactorial disease, and tear film instability is one of the core causes of dry eye. The pathogenesis of dry eye is complex, including age, sex hormone levels, meibomian gland dysfunction, external environmental factors, autoimmune diseases, etc. It can be mainly divided into three categories: insufficient tear secretion dry eye, excessive evaporation dry eye, and mixed dry eye caused by the above two reasons. At present, the construction of animal models of dry eye includes inducing single or mixed dry eye through drugs, surgery or environmental factors, simulating some influencing factors in the course of dry eye, and most patients with dry eye in clinical practice are mixed dry eye.

[0004] Androgens in sex hormones have receptors expressed in many places in the eye, including the meibomian glands, cornea, conjunctiva and retina, etc. They can regulate the lipid secretion of the lacrimal glands and meibomian glands, and affect the composition and stability of the tear film. Aging, autoimmune diseases, menopause and the use of related anti-androgen drugs can cause androgen deficiency and lead to the occurrence of dry eye. Flutamide is an androgen receptor antagonist that can simulate dry eye caused by sex hormone imbalance in the body; benzalkonium chloride (BZK) is a quaternary ammonium cation that is widely used as a preservative to preserve ophthalmic preparations, which can destroy the lipid phase of the tear film, thereby affecting the stability of the tear film. The dry eye model used in the present invention is closely related to the clinical dry eye detection indicators: tear film stability, meibomian glands, tear secretion, etc., so we speculate that it can better simulate most pathological environments in dry eye. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a mixed rat dry eye animal model.

[0006] The method for constructing a mixed dry eye disease animal model of the present invention comprises the steps of gavaging animals with flutamide and dropping benzalkonium chloride into the eyes to obtain the dry eye disease animal model.

[0007] Preferably, the animal can be various experimental animals, such as rats.

[0008] Preferably, the model is established by intragastric administration of 13 mg / kg body weight of flutamide and eye drops of 0.2% benzalkonium chloride once a day.

[0009] More preferably, the modeling time is 35 days.

[0010] The present invention also provides application of the above construction method in the field of dry eye model construction.

[0011] Preferably, it is a rat dry eye model.

[0012] The present invention provides application of the dry eye model obtained above in the study of the pathogenesis of dry eye.

[0013] The present invention also provides the use of the dry eye model obtained above in finding a new dry eye treatment method.

[0014] The present invention also provides the use of the dry eye model obtained above in screening drugs for preventing, treating or delaying dry eye.

[0015] The present invention aims to establish a mixed dry eye animal model by a combined modeling method of flutamide oral gavage combined with benzalkonium chloride solution eye drops, so as to provide new ideas and possibilities for studying the pathogenesis of dry eye and finding new treatment methods and drugs to prevent, treat or delay the course of dry eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a method for establishing a mixed dry eye rat animal model;

[0017] Figure 2 is the tear secretion of mixed dry eye rats;

[0018] Figure 3 are the representative images and statistical results of PAS and AB staining;

[0019] Figure 4 These are representative images and statistical results of the corneal sodium fluorescein staining test;

[0020] Figure 5 These are representative images and statistical results of corneal HE staining;

[0021] Figure 6 are representative images and statistical results of corneal TUNEL staining;

[0022] Figure 7 It is the level of inflammatory factors in corneal and conjunctival tissue. DETAILED DESCRIPTION

[0023] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0024] Embodiment 1:

[0025] 1. Experimental Methods

[0026] 1. Experimental instruments

[0027] Table 1 Experimental instruments

[0028]

[0029]

[0030] 2. Experimental reagents and test drugs

[0031] Table 2 Experimental reagents and test drugs

[0032]

[0033]

[0034] 3. Solution configuration:

[0035] Ultrapure water (containing 0.5% sodium carboxymethyl cellulose powder):

[0036] Use a measuring cylinder to measure 900 mL of ultrapure water and place it in a 1 L beaker. Add 5.0 g of sodium carboxymethyl cellulose powder and stir until completely dissolved. Pour into a 1000 mL volumetric flask and add ultrapure water to make up to 1000 mL.

[0037] Flutamide suspension:

[0038] Accurately weigh 3.25 g of flutamide powder, add appropriate volume of ultrapure water (containing 0.5% sodium carboxymethylcellulose powder) to make up to 1000 mL, and obtain 3.25 mg / mL flutamide suspension. Store in a -80°C refrigerator and take out before use.

[0039] 0.2% Benzalkonium chloride solution:

[0040] Take 1 mL of 10% benzalkonium chloride standard solution in a 50 mL centrifuge tube, add 49 mL of normal saline to obtain 50 mL of 0.2% benzalkonium chloride solution, store in a 4°C refrigerator, and prepare two portions in total.

[0041] 1% fluorescein sodium solution:

[0042] Dissolve 100 mg of fluorescein sodium powder in 10 mL of normal saline and shake to dissolve to obtain 10 mL of 1% fluorescein sodium solution. Dispense 1.5 mL into each tube and store at -80°C.

[0043] Yanyanming extract solution:

[0044] Zhanyanming extract (Guangzhou Baiyunshan Zhongyi Pharmaceutical, CA1007), the extraction process is shown in the 2020 edition of the "Chinese Pharmacopoeia" Part 1 Zhanyanming tablet preparation method, the crude drug content is 2.48g / g;

[0045] Weigh 82.46 g of the Zhanyanming extract into a 500 mL volumetric flask, add ultrapure water to the scale line, and obtain 500 mL of 164.92 mg / mL Zhanyanming extract mother solution. After mixing, divide into 15 mL per tube and store in a -80°C refrigerator. Take out before use.

[0046] Low-dose group: Take 2 mL of the mother solution of the Zhanyanming extract and add 6 mL of ultrapure water to dilute to 41.23 mg / mL;

[0047] Medium-dose group: Take 4 mL of the mother solution of the Zhanyanming extract and add 4 mL of ultrapure water to dilute to 82.46 mg / mL;

[0048] High-dose group: The mother solution of the Zhanyanming extract was used directly without dilution.

[0049] 4. Establishment of dry eye animal model:

[0050] The rats were examined by slit lamp, and rats without obvious visible damage or lesions in the eyes were included in the study. After one week of adaptive feeding, in order to induce a mixed dry eye model in rats, a combined modeling scheme of oral flutamide suspension (13 mg / kg, about 1.2 mL / rat) combined with 0.2% benzalkonium chloride solution eye drops (one drop / time / day) was used once a day for 35 consecutive days, and distilled water gavage (1.2 mL / rat) and normal saline eye drops (one drop / time / day) were used as controls, and the interval between drug administration and modeling gavage was four hours.

[0051] 5. Treatment with Zhanyanming extract:

[0052] The rats were randomly divided into the following 6 groups, with 7 rats in each group:

[0053] (1) Blank group: intragastric administration of distilled water (1.2 mL / mouse);

[0054] (2) Dry eye model group: intragastric administration of distilled water (1.2 mL / mouse);

[0055] (3) Low-dose group of Zhanyanming (164.92 mg / kg, 1x clinical dose): The diluted low-dose group of Zhanyanming extract solution (41.23 mg / mL) was injected into rats according to their body weight, about 1.2 mL per rat;

[0056] (4) Medium-dose group of Zhanyanming (329.84 mg / kg, 2x clinical dose): The diluted medium-dose group of Zhanyanming extract solution (82.46 mg / mL) was injected into rats according to their body weight, about 1.2 mL per rat;

[0057] (5) High-dose group of Zhanyanming (659.68 mg / kg, 4x clinical dose): The high-dose group of Zhanyanming extract solution (164.92 mg / mL) was infused into rats according to their body weight, about 1.2 mL / rat;

[0058] (6) Positive control group: distilled water was gavaged (1.2 mL / mouse) and 0.05% cyclosporine A was dripped into the eye (one drop / time / day).

[0059] 6. Tear secretion test:

[0060] The tear secretion of rats was measured by tear phenol red cotton thread test on days 0, 7, 14, 21, 28 and 35 after induction. The phenol red cotton thread was cut into 3 cm lengths and carefully placed in the lower eyelid of the rat, and kept still for 30 seconds. Due to the siphoning effect of tears, the phenol red cotton thread turned from red to yellow, and the length of the yellow part of the cotton thread was measured and recorded as a reference indicator of tear secretion capacity.

[0061] 7. Corneal permeability test:

[0062] On days 0, 7, 14, 21, 28, and 35 after induction, rats were subjected to corneal sodium fluorescein staining test to evaluate the permeability of their corneal epithelial barrier. 5 μL of 1% sodium fluorescein solution was dropped into the surface of the rat eyeball, and after closing the eye for 30 seconds, the corneal area was photographed with a slit lamp microscope. According to the area of ​​sodium fluorescein positive area, the lesions were quantitatively scored using the "12-point method".

[0063] 8. Histological examination:

[0064] On the 35th day after model induction, the rats were killed by overdose anesthesia, and the eyeballs and surrounding accessory eye tissues were collected, fixed with 4% FPA solution for 24 hours, dehydrated with gradient ethanol, transparent with xylene, embedded in wax, and cut into 5μm paraffin slices. After dewaxing, the sections were stained with hematoxylin for 5 minutes, rinsed with running tap water, and counterstained with eosin for 30 seconds. The corneal area was photographed with an inverted microscope and the corneal thickness was quantified using ImageJ software.

[0065] The morphology and number of conjunctival goblet cells were evaluated by Alcian Blue (AB) and Periodic acid-Schiff (PAS) staining. For AB staining, the sections were stained with 1% Alcian blue solution in 3% acetic acid solution for 15 minutes and then rinsed with running tap water. For PAS staining, the sections were stained with 0.5% periodic acid for 5 minutes, rinsed with distilled water, and then stained with Schiff reagent for 15 minutes, rinsed with running tap water, and finally counterstained with hematoxylin, and rinsed with running tap water to turn blue. The goblet cells in the conjunctival sac area were photographed with an inverted microscope, and AB or PAS positive cells appeared as blue and purple cells, respectively. The number of positive cells per millimeter of conjunctival sac length represents the number of goblet cells.

[0066] 9. Cell apoptosis detection:

[0067] The one-step TUNEL apoptosis detection kit was used to detect apoptotic cells in corneal sections (day 35). After dewaxing, 5 μm paraffin sections were permeabilized with 20 μg / mL proteinase K solution at 37°C for 30 minutes. After washing the slides with PBS, they were stained with TUNEL working solution for 30 minutes at 37°C in the dark, and the nuclei were counterstained with DAPI. The corneal area was photographed using a confocal laser scanning microscope. The TUNEL / DAPI double-positive apoptotic cells in the corneal view (235 mm × 235 mm) were counted and quantified using ImageJ software.

[0068] 10. Detection of inflammatory factors:

[0069] The levels of cytokines (IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-18, IL-33, CXCL1, KC, CCL2, MCP-1, GM-CSF, IFN-γ, and TNF-α) were measured using the bead-based LEGENDplex multifactorial assay kit.

[0070] 20 mg of corneal and conjunctival tissue (day 35) was placed in 0.5 mL of pre-cooled PBS solution containing a protease inhibitor cocktail, homogenized until there were no obvious particles, and centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was collected for total protein quantification (BCA method), and 25 μL of each supernatant was transferred for multifactor kit detection. The multifactor analysis was performed according to the manufacturer's instructions. 25 μL of microbead mixture and 25 μL of detection buffer were added to the filter plate, 25 μL of the sample solution to be tested was added, the plate was sealed and incubated in the dark for 2 hours with shaking. After thorough washing, 25 μL of detection antibody was added, and the plate was incubated in a closed state with shaking for 1 hour, and then 25 μL of SA-PE was added and incubated for another 30 minutes. After thorough washing, 150 μL of washing buffer was added, and the analysis was performed using CyExpert 2.4.0.28. The quantification of the detection factors was expressed as mean fluorescence intensity (MFI), calculated according to the standard curve, and normalized to the total protein concentration in ng per mg of protein.

[0071] 2. Experimental Results

[0072] This study used male Sprague-Dawley rats weighing 300-350g, and induced a mixed dry eye model by oral administration of flutamide (13mg / kg) once a day combined with 0.2% benzalkonium chloride solution eye drops for 35 consecutive days. The sedative group was treated with 1x clinical dose (165mg / kg), 2x clinical dose (330mg / kg) and 4x clinical dose (660mg / kg) by oral administration, while the positive control group was treated with 0.05% cyclosporine A eye drops once a day as a positive control. Figure 1 shown.

[0073] Tear secretion and corneal barrier permeability were detected on days 0, 7, 14, 21, 28, and 35. The tear phenol red cotton thread was placed at 1 / 3 of the lower eyelid of the rat, and the length of the tear-soaked cotton thread was recorded after keeping it still for 30 seconds to directly characterize the total tear secretion of the rat. On day 0, there was no significant difference in the tear secretion of the rats in the model group compared with the rats in the blank group. In the later stage of modeling, the total tear secretion of the rats in the model group gradually decreased, especially on days 28 and 35, the tear secretion of the rats in the model group decreased significantly, such as Figure 2 shown.

[0074] The mucus layer is the innermost structure of the three layers of the tear film and is mainly produced by goblet cells located in the conjunctiva. Goblet cells rich in mucin play a vital role in eye health. In the goblet cells of the conjunctival sac, PAS staining makes acidic mucin appear purple, and AB staining makes neutral mucin appear blue. Compared with the blank group, the number of goblet cells in the model group decreased significantly, and the cell morphology changed, shrinking or disappearing, such as Figure 3This suggests that in this mixed dry eye animal model, the normal secretion function of goblet cells in the conjunctival area is impaired and the tear film structure may be destroyed.

[0075] In addition to the decrease in the total amount of tear secretion and the decrease in the number of goblet cells, dry eye often causes increased permeability and even shedding of the corneal and conjunctival epithelium. The corneal permeability is evaluated by the sodium fluorescein staining test, and the "12-point method" is used to quantify the severity of corneal damage after taking pictures under a slit lamp microscope. Before the modeling began, almost all rats had no corneal epithelium damage, and the sodium fluorescein-positive spots were negligible. However, after the modeling, the corneal epithelium of the rats will increase in permeability, and punctate or even sheet-like positive sodium fluorescein staining areas will appear. Figure 4 As shown in the figure, in the first two weeks, the corneal fluorescein sodium staining scores of the rats in the model group were all greater than 6.0, and reached a peak on the 14th day with a staining score of 7.0, which was significantly different from the blank group. This means that in this mixed dry eye animal model, the corneal permeability of rats increased and the corneal damage was significant. This observation may also indicate that compared with other days, the 14th day is the best time point to evaluate the changes in corneal permeability in this mixed dry eye model.

[0076] In the later stage of the experiment, the corneal damage of the model group rats was alleviated, which may be due to the self-repair ability of the cornea, which means that the model may simulate early mild dry eye. In order to further study the reasons, H&E staining and TUNEL staining were used to reveal the changes in corneal structure and morphology after drug treatment.

[0077] like Figure 5 As shown in the representative pictures of HE staining, the corneal tissue of the model group is thinner than that of the blank group, which indicates that the tissue morphology and structure have changed. Through ImageJ statistics, the thickness of the corneal tissue of the model group is significantly lower than that of the blank group. The statistical analysis results are shown in Figure 5 Shown right.

[0078] In order to further explore the cause of corneal thinning in rats after combined modeling, the broken DNA in apoptotic cells was marked by TUNEL staining test to examine the apoptosis level of corneal epithelial cells. Representative fluorescent staining pictures are shown in Figure 6 Compared with the blank group, most of the corneal epithelial cells in the model group were marked as TUNEL / DAPI double-positive cells, which indicated that in this mixed dry eye animal model, epithelial cell apoptosis increased significantly and corneal barrier function was severely damaged. The count / view of TUNEL / DAPI double-positive cells was statistically analyzed by ImageJ. The statistical analysis results are shown in Figure 2. Figure 6 Shown right.

[0079] In the development of dry eye, inflammation is one of the core factors. The Biolegend multifactor kit was used to detect the level of inflammatory factors in rat corneal conjunctival tissue. The quantitative analysis of the detected factors was expressed as mean fluorescence intensity (MFI), calculated according to the standard curve, and normalized to the total protein concentration in ng per mg of protein. The experimental results showed that ( Figure 7 ), in this mixed dry eye animal model, the levels of inflammatory factors TNF-α and IL12p70 in the corneal conjunctival tissue of rats were significantly increased, indicating the occurrence of inflammation.

[0080] 3. Advantages of combined modeling with flutamide oral gavage and benzalkonium chloride eye drops

[0081] 1. Advantages of combined modeling compared with flutamide modeling alone

[0082] Sex hormone imbalance is one of the influencing factors of dry eye, and androgen deficiency is one of the high-risk factors for dry eye in clinical practice. After oral administration of flutamide, its active form can compete with the active form of androgen in vivo to bind to androgen receptors, simulating the physiological situation of androgen deficiency. Using flutamide alone for modeling can simulate dry eye caused by sex hormone imbalance, but it has the disadvantages of a longer modeling cycle and a milder severity of some key phenotypes, such as:

[0083] SIt (tear secretion): 4 weeks after modeling, there was no significant difference between the low-concentration group and the control group, and the tear secretion of the medium- and high-concentration groups was significantly lower than that of the control group, but the decrease in tear secretion was less. By the 8th week after modeling, the tear secretion of the medium- and high-concentration groups was reduced to the same level as that of the 4th week after combined modeling. The specific data of tear secretion of rats in each group are shown in the table below. 1 .

[0084] Table 1-3 Tear secretion test of rats in each group at different times ( n=8l / mm)

[0085] Table 1-3 The St of rats at different time points of each group

[0086]

[0087] Note: Compared with the control group # P<0.05; compared with high concentration group Δ P<0.05; compared with the medium concentration group ◆ P<0.05VSControl group # P<0.05; VS Highconcentration group ΔP<0.05;VSMediumconcentrationgroup ◆ P<0.05

[0088] Corneal fluorescein sodium staining: Under the cobalt blue light of the slit lamp, the yellow-green fluorescent area is considered to be a positive area for fluorescein sodium staining, indicating that the corneal barrier is damaged. The corneal fluorescein sodium staining score is positively correlated with corneal barrier damage, such as Figure 3 As shown. Two weeks after drug administration, there was no significant change in the fluorescein sodium staining scores of the low, medium and high concentration groups compared with the control group; four weeks after drug administration, the staining scores of the medium and high concentration groups increased significantly compared with the control group, but the staining scores were lower and the degree of corneal damage was milder. At the eighth week after drug administration, the staining score of the high concentration group was equivalent to that of the second week of combined modeling. The fluorescein sodium staining data of rats in each group are shown in the table below.

[0089] Table 1-2 Corneal fluorescein sodium staining scores of rats in each group at different time points ( n=8Score)

[0090] Table1-2Fluorescein staining score of rats at different time points of each group

[0091]

[0092] Note: Compared with the control group # P<0.05; compared with high concentration group △ P<0.05; compared with the medium concentration group ◆ P<0.05VSControl group # P<0.05; VS High concentration group Δ P<0.05; VS Mediumconcentration group ◆ P<0.05

[0093] Apoptosis of corneal epithelial cells: When tears are insufficient and the number and function of goblet cells that secrete mucin are reduced or lost, the aqueous layer and mucin layer of the tear film are damaged, the tear film structure is destroyed and unbalanced, and tear evaporation is further accelerated to form a hypertonic environment and induce inflammation. At the same time, the corneal epithelium loses the nutritional support and barrier protection of the tear film and comes into direct contact with the external environment or foreign bodies on the ocular surface, resulting in increased stimulation to the corneal epithelium, which may lead to damage to the corneal barrier function and epithelial cell damage. After combined modeling, the level of epithelial cell apoptosis increased significantly, which was not detected when using flutamide alone. The experimental results are shown in Figure 6 .

[0094] 2. Advantages of combined modeling over single benzalkonium chloride modeling

[0095] Benzalkonium chloride is a commonly used preservative in ophthalmic preparations. Long-term use can cause serious damage to the ocular surface. It is suitable for modeling short-term severe dry eye, but it has the characteristics and limitations of short duration but severe degree of dry eye. [2] .

[0096] Model maintenance time: After using benzalkonium chloride alone to establish the model, indicators such as tear secretion decreased significantly in the first two weeks of modeling, and recovered in the fourth week of modeling, with no significant difference from the control group. The specific experimental results are shown in the table below 2 The tear secretion index under the combined modeling was maintained for a longer time, and compared with the control group, it decreased significantly in the fourth and fifth weeks after modeling. The specific experimental results are shown in Figure 2 .

[0097] Table 1 Changes in tear secretion parameters of the two groups of mice

[0098]

[0099] Note: Compared with the control group, * P<0.05

[0100] Inflammation: The hyperosmotic environment of the ocular surface caused by insufficient tears can cause inflammation. After the combined modeling, the levels of inflammatory factors TNF-α and IL12p70 in the corneal conjunctival tissue of rats increased significantly, indicating the occurrence of inflammation. Among them, the increase of IL12p70 was not detected in the modeling with flutamide or benzalkonium chloride alone. The experimental results are shown in Figure 7 .

[0101] Serum testosterone concentration: After oral administration of flutamide, its active form in vivo will compete with the active form of androgen to bind to the androgen receptor, causing a compensatory increase in the level of testosterone (the main active form of androgen), simulating the imbalance of sex hormones in the influencing factors of dry eye, which is not detected by modeling with benzalkonium chloride alone. For specific experimental results, please refer to the literature 3 .

[0102] References

[0103] [1] Peng Ling. Experimental study on flutamide-induced dry eye model in rats[D]. Southern Medical University, 2016.

[0104] [2] Du Jing, Li Yong, Gao Jinrong, et al. Expression of interleukin-1 receptor-associated kinase 1 (IRAK1) and NF-κB in the cornea and conjunctiva of mice with benzalkonium chloride-induced dry eye[J]. Advances in Ophthalmology, 2019, 39(03): 223-228.

[0105] [3] Peng Ling, Cai Weihao, Lu Xiaohe, et al. Pathological changes of corneal and lacrimal gland tissues and expression of TNF-α and IL-1α in rats with flutamide-induced dry eye model [J]. Advances in Ophthalmology, 2016, 36(06): 506-511.

Claims

1. A method for constructing a mixed dry eye animal model, characterized in that: The animal model of dry eye was obtained by gavage of flutamide and eye drops of benzalkonium chloride.

2. The construction method according to claim 1, characterized in that: The animals are rats.

3. The construction method according to claim 2, characterized in that: The model was established by intragastric administration of 13 mg / kg body weight of flutamide and eye drops of 0.2% benzalkonium chloride once a day.

4. The construction method according to claim 3, characterized in that: The modeling time is 35 days.

5. Application of any one of the construction methods of claims 1 to 4 in the field of dry eye model construction.

6. The use according to claim 5, characterized in that: The dry eye model is a rat dry eye model.

7. Use of the dry eye model obtained by any construction method of claims 1-4 in the study of the pathogenesis of dry eye.

8. Use of the dry eye model obtained by any construction method of claims 1-4 in finding new treatment methods for dry eye.

9. Use of the dry eye model obtained by any one of the construction methods of claims 1 to 4 in screening drugs for preventing, treating or delaying dry eye.

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