Application of MCP1 inhibitor in preparation of medicine for treating high myopia related inflammation

Abstract

The invention relates to application of an MCP1 inhibitor in preparation of a medicine for treating high myopia complicated with inflammatory complication diseases. The invention also provides application of the MCP1 as a biomarker in preparation of a kit for diagnosing high myopia complicated with anxiety. The reagent for detecting the MCP1 protein content is applied to preparation of a kit for diagnosing high myopia complicated with anxiety. According to the invention, through specific inhibition of MCP1 effect, the composition can effectively alleviate high myopia in-vivo inflammatory response and reduce eye inflammatory cell infiltration, thereby alleviating local lesion injury of eyes. By protecting the immune barrier function, the invention can reduce the infiltration of mononuclear cells to the central nervous system, thereby relieving the emotional symptoms, and hopefully improving the life quality of patients. The pharmaceutical preparation provided by the invention has the advantages of strong pertinence, obvious curative effect, small side effect and the like, provides a new thought and method for treating complications related to high myopia inflammation, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an MCP1 inhibitor in the preparation of a drug for treating high myopia-related inflammation. Background Art

[0002] High myopia is one of the leading causes of blindness worldwide and is defined as an axial length (AL) ≥ 26 mm. It not only leads to multiple serious complications of the eye, such as macular degeneration, cataracts, and optic neuropathy, but can also cause emotional deficits, especially anxiety symptoms, which seriously affect visual health and quality of life.

[0003] Studies have shown that the occurrence and development of many complications of high myopia are closely related to its special inflammatory environment, especially the increase of monocyte chemoattractant protein-1 (MCP1). The level of MCP1 in the aqueous humor of patients with high myopia is elevated and is related to the stage of myopic maculopathy. However, there is currently no effective intervention drug for high myopia-related inflammation.

[0004] MCP1 inhibitors are agents that can inhibit the biological activity of MCP1 by specifically binding to MCP1 or its receptor active site, interfering with its receptor binding and downstream signal transduction pathways. MCP1 is a small molecule protein belonging to the chemokine family, and its gene is located on human chromosome 17q11.2-q12. It is mainly produced by a variety of cells such as monocytes, macrophages, endothelial cells, and fibroblasts when stimulated by inflammation. MCP1 is a monomeric protein with a typical chemokine structure, with two pairs of disulfide bonds formed by four cysteine ​​residues. However, there are currently no reports on the relationship between MCP1 inhibitors and inflammation associated with high myopia. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the purpose of the present invention is to provide an application of an MCP1 inhibitor in the preparation of a drug for treating high myopia-related inflammation. The drug reduces high myopia-related inflammatory reactions by inhibiting the expression of MCP1 or its binding to receptors, thereby delaying or preventing the development of complications.

[0006] In a first aspect, the present invention provides the use of an MCP1 inhibitor in the preparation of a drug for treating high myopia combined with inflammatory complications.

[0007] As a preferred example, the disease refers to high myopia complicated by anxiety.

[0008] As another preferred example, the MCP1 inhibitor is used to: (1) inhibit the secretion of peripheral chemokine MCP1; (2) inhibit the secretion of peripheral inflammatory factors MIP-1β, RANTES, TNF-α, IFN-γ and IL-6; (3) inhibit the infiltration of peripheral immune cells; (4) reduce the infiltration of monocytes / macrophages in the eyes and brain; (5) protect the blood-eye barrier / blood-brain barrier.

[0009] In a second aspect, the present invention provides the use of MCP1 as a biomarker in the preparation of a diagnostic kit for high myopia and anxiety.

[0010] As a preferred example, the sample to be detected is blood.

[0011] In a third aspect, the present invention provides the use of a reagent for detecting the content of MCP1 protein in the preparation of a diagnostic kit for high myopia complicated with anxiety.

[0012] As a preferred example, the sample to be detected is blood.

[0013] In the present invention, the safe and effective dose of the MCP1 inhibitor is 25 mg / kg, to ensure that the MCP1 inhibitor has the effect of inhibiting the chemokine MCP1 in vivo without causing toxic side effects. The safe and effective dose refers to: the dose is sufficient to significantly improve the condition without causing serious side effects.

[0014] The administration route of the MCP1 inhibitor is peripheral administration. The administration mode of the MCP1 inhibitor is minimally invasive administration. The administration mode of the MCP1 inhibitor includes but is not limited to intraperitoneal injection. The injection cycle of the agent of the present invention includes but is not limited to injection for four weeks, with injection twice a week; it can also be a single injection or continuous administration. Those skilled in the art can monitor the individual's condition during the entire treatment process and adjust the injection cycle of the injection agent of the present invention accordingly.

[0015] The advantages of the present invention are: by specifically inhibiting the action of MCP1, the present invention can effectively reduce the inflammatory response in the body of high myopia, reduce the infiltration of inflammatory cells in the eye, and thus reduce the damage to local eye lesions. By protecting the immune barrier function, the present invention can reduce the infiltration of monocytes into the central nervous system, thereby alleviating emotional symptoms and is expected to improve the quality of life of patients. The pharmaceutical preparation provided by the present invention has the advantages of strong pertinence, significant efficacy, and few side effects, and provides a new idea and method for the treatment of inflammatory complications related to high myopia, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a correlation diagram between anxiety scores and peripheral blood MCP1 in patients with high myopia in Example 1 of the present invention;

[0017] Figure 2 This is a graph showing a correlation between increased anxiety-like behavior and peripheral blood MCP1 in the high myopia mouse model in Example 2 of the present invention;

[0018] Figure 3 The figure shows the increase of MCP1 expression in the high myopia mouse model in Example 3 of the present invention (left) and the further increase of inflammatory infiltration of mononuclear macrophages into the eyes and brain (right);

[0019] Figure 4 This is a diagram showing that the inflammatory infiltration of mononuclear macrophages in the high myopia mouse model in Example 4 of the present invention aggravates the destruction of the immune barrier of the eye (left) and brain (right);

[0020] Figure 5 Graph showing the levels of MCP1 and related pro-inflammatory factors in the peripheral blood of highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention (Experimental is the MCP1 inhibitor group);

[0021] Figure 6 This is a graph showing the proportion of mononuclear macrophages in the peripheral blood of highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention;

[0022] Figure 7 This is a result diagram of the change in local MCP1 expression level in highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention;

[0023] Figure 8 This is a result diagram of the changes in local mononuclear macrophage inflammatory infiltration in highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention;

[0024] Fig. 9 This is a result diagram of the changes in tight junction protein expression in the local immune barrier of highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention;

[0025] Fig.10 This is a result diagram of the behavioral phenotype of highly myopic mice treated with MCP1 inhibitor or saline in Example 5 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0027] Example 1

[0028] This example evaluates the correlation between anxiety level and peripheral blood MCP1 level in patients with high myopia, and the steps are as follows:

[0029] 1. Grouping

[0030] Data from 104 high myopia patients and 106 emmetropia controls were collected. There were no significant differences between the two groups in terms of age, gender, body mass index, smoking, hypertension, drinking, occupational type, and education level. High myopia was defined as binocular axial length (AL) equal to or greater than 26 mm, and emmetropia controls were defined as binocular AL between 22 mm and 24.5 mm.

[0031] 2. Sample Collection

[0032] (1) Anxiety assessment questionnaire: The Hamilton Anxiety Inventory (HAMA) was used to assess anxiety levels. The questionnaire was completed by professionally trained physicians. The HAMA questionnaire has 14 sub-items, including psychological anxiety (anxious mood, tension, fear, depressed mood, behavioral performance during interviews, insomnia, cognitive symptoms) and physiological anxiety.

[0033] (2) Blood samples: Blood samples were collected for subsequent analysis. Whole blood was collected in 5 ml EDTA anticoagulant tubes at 9-11 am. The whole blood was centrifuged at 1500 g for 10 minutes at 4°C to separate the plasma, which was then divided into centrifuge tubes and stored at -80°C for later use.

[0034] 3. Anxiety level test: Anxiety is scored using the HAMA questionnaire. Each sub-item is scored from 0 to 4, indicating the presence and severity of anxiety-related symptoms. The total score is 0 to 56. The anxiety level is graded according to the total score, including no anxiety (0 to 6 points), mild to moderate anxiety (7 to 13 points), and severe anxiety (≥14 points). The higher the score, the higher the anxiety level.

[0035] 4. Analysis of MCP1 Levels in Blood Samples Using Suspension Cytokine Array

[0036] (1) Reagent selection: Use the Bio-Plex Pro Human Cytokine Screening Panel kit (Product No.: 12007283) provided by Bio-Rad Laboratories (USA). This kit can accurately and quantitatively detect 48 human cytokines including MCP1.

[0037] (2) Performing experimental procedures: Processing human blood samples according to the steps in the kit instructions, and using the kit to detect inflammatory cytokines.

[0038] (3) Data analysis: The test results were analyzed using BioPlex Manager 6.0 software provided by Bio-Rad Laboratories to obtain the specific levels of inflammatory cytokines in human blood. Pearson correlation analysis was used to evaluate the correlation between anxiety level and plasma MCP1 concentration variables.

[0039] Test results such as Figure 1 shown.

[0040] Depend on Figure 1 It can be seen that the anxiety level of patients with high myopia is positively correlated with the peripheral blood MCP1 level. As the MCP1 level increases, the anxiety score gradually increases, with a correlation coefficient r of 0.350 and a P value of 0.0003. The above results indicate that MCP1 may play a key role in the complications of high myopia.

[0041] Example 2

[0042] This example conducts animal experiments to verify the findings in patients with high myopia, and the steps are as follows:

[0043] 1. Establishment of a high myopia mouse model: A -10D lens was attached to one eye of the mouse for 4 weeks to establish a high myopia model group, and a sham operation control group was worn with a 0D plano lens. Through MRI and refractive power testing, the axial length of the lens group increased significantly and myopic refractive changes occurred, successfully establishing a high myopia mouse model.

[0044] 2. Anxiety behavior assessment: Anxiety-like behavior was assessed using the open field test (OFT). In the OFT test, mice were placed individually in an open field of 40*40*25 cubic centimeters. Each time the mice were placed, they were kept facing the same corner and allowed to move freely in the open field. The movement trajectory of the mice was recorded using an automatic tracking system (EthoVision XT12, Noldus Technologies, USA) for a total of ten minutes. The center of the open field is defined as an area of ​​20*20 square centimeters in the center. The time each mouse stayed in the center of the open field, the number of times it entered the center of the open field, and the total distance moved were assessed. In the absence of significant differences in the total distance moved, the shorter the time the mouse stayed in the center of the open field and the fewer times it entered, the more obvious the anxiety behavior.

[0045] 3. Detection of blood MCP1 expression using pro-inflammatory marker array

[0046] (1) Reagent selection: MSD U-PLEX Proinflammatory Biomarker Kit (MSD, No. K15069L-1). This kit can accurately and quantitatively detect six factors including MCP1 in mouse plasma.

[0047] (2) Experimental operation: Blood samples were processed and tested according to the instructions. Blood was collected from mice by intraperitoneal injection of 0.1 ml of 10% chloral hydrate, and blood was collected from behind the eyeball under anesthesia. The blood was collected in a 1.5 ml EDTA anticoagulant centrifuge tube and centrifuged at 3000 g for 20 minutes at 4°C to separate the plasma, which was then divided into centrifuge tubes and stored at -80°C for later use.

[0048] (3) Data reading: MESO QuickPlex SQ 120 reader was used. Pearson correlation analysis was used to evaluate the correlation between anxiety level and plasma MCP1 concentration.

[0049] Test results such as Figure 2 shown.

[0050] Depend on Figure 2 It can be seen that the anxiety-like behavior (staying time in the center of the open field) exhibited by the high myopia mouse model is closely related to the peripheral blood MCP1 level. The higher the blood MCP1, the more anxious behavior, the correlation coefficient r is 0.876, and the P value is 0.0221. These findings are consistent with the research results of patients with high myopia.

[0051] Example 3

[0052] This example was conducted to further explore the role of elevated MCP1 in anxiety associated with high myopia, and the steps were as follows:

[0053] 1. Detection of MCP1 expression in mouse eye tissue by immunofluorescence staining:

[0054] (1) Mouse eye tissue sampling: According to the high myopia modeling method in Example 2, high myopia mice and control mice were obtained. After the mice were killed, the mouse eyeballs were removed with scissors, immersed in FAS eyeball fixative, and routinely fixed and embedded to make frozen sections. The section thickness was 10 μm, and the section direction was the sagittal plane (the largest section included the cornea, lens, iris and retina).

[0055] (2) Immunofluorescence staining

[0056] 1) Tissue fixation: Tissue sections were fixed in 4% paraformaldehyde.

[0057] 2) Permeabilization: PBS containing 0.5% Triton X-100 was used for permeabilization.

[0058] 3) Blocking treatment: Use 3% bovine serum albumin for blocking.

[0059] 4) Antibody incubation: The following antibodies were used for fluorescently labeled probe treatment and incubated overnight at 4°C: MCP1 (Abcam, catalog number ab25124, 1:100).

[0060] 5) Secondary antibody incubation: Incubation was performed using a secondary antibody (Invitrogen, catalog number A-11012, 1:1000).

[0061] 6) Microscopic examination: Examination was performed using a confocal microscope (TCS SP5, Leica Microsystems, Germany).

[0062] 2. Quantification of monocyte levels in blood using flow cytometry

[0063] (1) Sample collection and processing: The mice were intraperitoneally injected with 0.1 ml of 10% chloral hydrate, and the brain was removed by cardiac perfusion under anesthesia. The brain tissue was minced with scissors, and a digestion solution containing 0.02 mg / ml collagenase IV, 5% fetal bovine serum, and DMEM culture medium was added. The cells were incubated in a 37°C water bath for 2 hours, shaken every 30 minutes, and then the digestion was terminated with a culture solution containing 10% fetal bovine serum, filtered through a 70 μm cell sieve, and washed twice with PBS to obtain a single cell suspension. After the mice were killed, the eyeballs were removed, and the uveal tissue was carefully separated with scissors and forceps. The fresh uveal tissue was digested with a digestion solution containing 0.02 mg / ml collagenase IV, 5% fetal bovine serum, and DMEM culture medium, and then digested on a shaker at 37°C at 70 rpm for 2 hours. The digestion was terminated with a culture solution containing 10% fetal bovine serum, filtered through a 70 μm cell sieve, and washed twice with PBS to obtain a single cell suspension.

[0064] (2) Fc receptor blocking: The single cell suspension obtained above was blocked before staining using CD16 / CD32 (BD Biosciences, No. 553142).

[0065] (3) Cell staining:

[0066] Antibodies used for mouse cells (uveal tract): anti-CD45 (BD Biosciences, No. 550994, dilution ratio 1:200), anti-CD11b (BD Biosciences, No. 552850, dilution ratio 1:200), anti-Ly6C (BD Biosciences, No. 560592, dilution ratio 1:200), and anti-F4 / 80 (BD Biosciences, No. 565411, dilution ratio 1:200).

[0067] Mouse brain cells (marking intravascular immune cells): Labeled with anti-CD45 (BD Pharmingen, No. 557659, dilution ratio 1:200), anti-CD11b (BD Pharmingen, No. 562605, dilution ratio 1:200), anti-Ly6C (BD Pharmingen, No. 560595, dilution ratio 1:200) and anti-TMEM119 (Thermo, No. 25-6119-82, dilution ratio 1:200).

[0068] (4) Flow cytometry

[0069] Fluorescence activated cell sorting data were obtained using a BD FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo (V10, Tree Star, Inc). Figure 3 shown.

[0070] Depend on Figure 3 It can be seen that (left figure) immunofluorescence staining showed that compared with the control group, the local MCP1 expression in the retina of highly myopic mice was significantly upregulated; (right figure) flow cytometric analysis showed that compared with the control group, the proportion of mononuclear macrophages in the uveal tract and brain of highly myopic mice was significantly increased, that is, the infiltration of mononuclear macrophages was significantly increased, revealing the potential mechanism of MCP1-mediated inflammatory infiltration.

[0071] Example 4

[0072] This example investigates the effect of mononuclear macrophage infiltration on further destruction of the blood-eye barrier (BOB) and blood-brain barrier (BBB) ​​in highly myopic mice, and the steps are as follows:

[0073] 1. Detection of blood-eye barrier (BOB):

[0074] (1) Mouse eye tissue sampling and protein group extraction: According to the high myopia modeling method in Example 2, high myopia mice and control mice were obtained. After the mice were killed, the mouse eyeballs were removed with scissors, and the retinal tissue was carefully separated. The conventional RIPA lysis method, ultrasound and BCA kit were used for protein extraction and quantification.

[0075] (2) Western Blot quantitative detection of BOB tight junction protein Occludin expression level

[0076] 1) Protein separation by SDS-PAGE electrophoresis and conventional membrane transfer

[0077] 2) Blocking: The membrane was blocked with 10% skimmed milk powder solution for 2 hours.

[0078] 3) Primary antibody incubation: The following antibodies were used to bind to the target protein and incubated overnight at 4°C: Occludin (Proteintech, catalog number PA5-85256, 1:1000) and internal reference β-actin (Sigma Chemical, catalog number A5441, 1:5000). All antibodies were diluted with Beyotime antibody diluent.

[0079] 5) Secondary antibody incubation: After washing the membrane three times with TBST, incubate with secondary antibody (CST, catalog number 7074 and 7076, 1:1000).

[0080] 6) Band development: After washing the membrane three times with TBST, the membrane was developed using Beyotime enhanced chemiluminescence reagent (Catalog No.: P0018FS). The bands were photographed and the expression level of the target protein relative to the internal reference protein was analyzed using ImageJ. 2. Detection of the blood-brain barrier (BBB):

[0081] (1) Brain tissue sampling and slicing: The high myopia mouse model was established according to the method in Example 2 to obtain high myopia mice and control mice. The mice were intraperitoneally injected with 0.1 ml of 10% chloral hydrate, and the brain was removed by cardiac perfusion under anesthesia. The brain tissue was immediately fixed in 10 times the volume of paraformaldehyde fixative, dehydrated, embedded and frozen. The slicing position was the basolateral amygdala (BLA) section, and the slice thickness was 70 μm.

[0082] (2) The expression level of BBB tight junction protein occludin was detected by immunofluorescence.

[0083] 1) Tissue fixation: Tissue sections were fixed in 4% paraformaldehyde.

[0084] 2) Permeabilization: PBS containing 0.5% Triton X-100 was used for permeabilization.

[0085] 3) Blocking treatment: Use 3% bovine serum albumin for blocking.

[0086] 4) Antibody incubation: The following antibodies were used for fluorescently labeled probe treatment and incubated overnight at 4°C: Occludin (Proteintech, catalog number: 27260-1-AP, 1:500).

[0087] 5) Secondary antibody incubation: Incubation was performed using a secondary antibody (Invitrogen, catalog number A-11012, 1:1000).

[0088] 6) Microscopic examination: Examination was performed using a confocal microscope (TCS SP5, Leica Microsystems, Germany).

[0089] Test results such as Figure 4 shown.

[0090] Depend on Figure 4 It can be seen that (left figure) Western Blot results showed that compared with the control group, the ocular infiltration of mononuclear macrophages in highly myopic mice significantly promoted the downregulation of the expression of BOB tight junction protein occludin, which aggravated the destruction of the blood-eye barrier; (right figure) Immunofluorescence staining analysis showed that compared with the control group, the encephalitis infiltration in highly myopic mice significantly promoted the downregulation of the expression of BBB tight junction protein Occludin, which aggravated the destruction of the blood-brain barrier.

[0091] Example 5

[0092] This example involves exploring the effect of using MCP1 inhibitor (purchased from Abmart Pharmaceutical Technology (Shanghai, China) Co., Ltd.) on inflammatory infiltration and immune barrier destruction in high myopia and the intervention effect on complications. The steps are as follows:

[0093] 1. Application of MCP1 inhibitors to intervene in high myopia mice: During the construction of a high myopia model in C57BL / 6J mice, the experimental group (Experimental) was intraperitoneally injected with MCP1 inhibitors. Taking a 20g mouse as an example, each mouse was injected with 50μg, and the injection concentration was 0.5mg / ml. The control group (Control) was intraperitoneally injected with an equal amount of normal saline, twice a week for 4 consecutive weeks.

[0094] 2. Use pro-inflammatory marker array to detect blood MCP1 expression.

[0095] (1) Reagent selection: MSD U-PLEX Proinflammatory Biomarker Kit (MSD, No. K15069L-1). This kit can accurately and quantitatively detect the concentrations of proinflammatory factors such as MCP1, interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α).

[0096] (2) Experimental procedures: Blood was collected from mice by intraperitoneal injection of 0.1 ml of 10% chloral hydrate. Blood was collected from behind the eyeball under anesthesia and collected in a 1.5 ml EDTA anticoagulant centrifuge tube. The blood was centrifuged at 3000 g for 20 min at 4°C to separate the plasma, which was then dispensed into centrifuge tubes and stored at -80°C for later use.

[0097] (3) Data reading: MESO QuickPlex SQ 120 reader was used.

[0098] 3. The proportion of monocytes and macrophages in the blood of the two groups of mice was detected by flow cytometry.

[0099] (1) Sample collection and processing: Mice were intraperitoneally injected with 0.1 ml of 10% chloral hydrate, and blood was collected from behind the eyeball under anesthesia. 4 ml of red blood cell lysis buffer was added to every 100 ul of fresh blood in an EDTA anticoagulation centrifuge tube to lyse the red blood cells and remove the interference of red blood cells. The samples were washed with PBS three times for subsequent staining.

[0100] (2) Fc receptor blocking: The single cell suspension obtained above was blocked before staining using CD16 / CD32 (BD Biosciences, No. 553142).

[0101] (3) Cell staining:

[0102] Antibodies used for mouse cells (blood): anti-CD45 (BD Biosciences, No. 550994, dilution ratio 1:200), anti-CD11b (BD Biosciences, No. 552850, dilution ratio 1:200), anti-Ly6C (BD Biosciences, No. 560592, dilution ratio 1:200), and anti-F4 / 80 (BD Biosciences, No. 565411, dilution ratio 1:200).

[0103] (4) Flow cytometry

[0104] Fluorescence activated cell sorting data were obtained using a BD FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo (V10, Tree Star, Inc). 4. Detection of local MCP1 expression in the eye by immunofluorescence staining

[0105] (1) Mouse eye tissue sampling: After the mice were killed, the eyeballs were removed with scissors, immersed in FAS eyeball fixative, and routinely fixed and embedded to make frozen sections. The section thickness was 10 μm, and the section direction was the sagittal plane (the largest section included the cornea, lens, iris, and retina).

[0106] (2) Immunofluorescence staining

[0107] 1) Tissue fixation: Tissue sections were fixed in 4% paraformaldehyde.

[0108] 2) Permeabilization: PBS containing 0.5% Triton X-100 was used for permeabilization.

[0109] 3) Blocking treatment: Use 3% bovine serum albumin for blocking.

[0110] 4) Antibody incubation: The following antibodies were used for fluorescently labeled probe treatment and incubated overnight at 4°C: MCP1 (Abcam, catalog number ab25124, 1:100).

[0111] 5) Secondary antibody incubation: Incubation was performed using a secondary antibody (Invitrogen, catalog number A-11012, 1:1000).

[0112] 6) Microscopic examination: Examination was performed using a confocal microscope (TCS SP5, Leica Microsystems, Germany).

[0113] 5. Flow cytometry analysis of the effect of MCP1 inhibitor on inflammatory infiltration of mononuclear macrophages was performed using mouse tissue. (1) Sample collection and processing: After the mice were killed, the eyeballs were removed and the uveal tissue was carefully separated using scissors and forceps. The fresh uveal tissue was digested with a digestion solution containing 0.02 mg / ml collagenase IV and 5% fetal bovine serum and DMEM culture medium, and then digested at 37°C on a shaker at 70 rpm for 2 hours. The digestion was then terminated with a culture medium solution containing 10% fetal bovine serum, filtered through a 70 μm cell sieve, and washed twice with PBS to obtain a single cell suspension.

[0114] (2) Fc receptor blocking: The single cell suspension obtained above was blocked before staining using CD16 / CD32 (BD Biosciences, No. 553142).

[0115] (3) Cell staining:

[0116] Antibodies used for mouse cells (uveal tract): anti-CD45 (BD Biosciences, No. 550994, dilution ratio 1:200), anti-CD11b (BD Biosciences, No. 552850, dilution ratio 1:200), anti-Ly6C (BD Biosciences, No. 560592, dilution ratio 1:200), and anti-F4 / 80 (BD Biosciences, No. 565411, dilution ratio 1:200).

[0117] (4) Flow cytometry

[0118] Fluorescence activated cell sorting data were acquired using a BD FACSCanto II flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo (V10, Tree Star, Inc).

[0119] 6. Use immunofluorescence to evaluate the effects of MCP1 inhibitors on the local immune barrier.

[0120] (1) Mouse eye tissue sampling: After the mice were killed, the eyeballs were removed with scissors, immersed in FAS eyeball fixative, and routinely fixed and embedded to make frozen sections. The section thickness was 10 μm, and the section direction was the sagittal plane (the largest section included the cornea, lens, iris, and retina).

[0121] (2) Immunofluorescence staining

[0122] 1) Tissue fixation: Tissue sections were fixed in 4% paraformaldehyde.

[0123] 2) Permeabilization: PBS containing 0.5% Triton X-100 was used for permeabilization.

[0124] 3) Blocking treatment: Use 3% bovine serum albumin for blocking.

[0125] 4) Antibody incubation: The following antibodies were used for fluorescently labeled probe treatment at 4°C overnight: Cx43 (Santa Cruz, 1:1000).

[0126] 5) Secondary antibody incubation: Incubation was performed using a secondary antibody (Invitrogen, catalog number A-11012, 1:1000).

[0127] 6) Microscopic examination: The images were taken under a confocal microscope (TCS SP5, Leica Microsystems, Germany) at 20x and 40x magnification to observe the iris and ciliary body regions.

[0128] 7. Use animal behavior to evaluate the effect of MCP1 inhibitors on complications related to high myopia: Confirm the anxiety behavior of mice in the elevated plus maze test (EPM). In the EPM test, mice were placed individually in a 75cm high cross-shaped elevated frame. The elevated frame was composed of two intersecting long strips (one runway was open and the other runway was closed, with 19cm high glass baffles on both sides) forming a cross shape. Each runway was 36cm long and 6cm wide, and was divided into 4 arms, 2 open arms and 2 closed arms. Each time, the mouse was placed at the intersection of the elevated frame (central area) and kept facing the open arm. The mouse was allowed to explore the elevated frame from an early age, and the movement trajectory of the mouse was recorded by an automatic tracking system (EthoVision XT12, Noldus Technologies, USA), for a total of 5 minutes. The time each mouse spent in the open arm and the number of times it entered the open arm were analyzed. The shorter the time the mouse stayed in the open arm and the fewer times it entered, the more obvious the anxiety behavior. The test results are as follows Figure 5-10 shown.

[0129] Figure 5The detection of pro-inflammatory factors shown in the figure shows that compared with the normal saline (control) group, the levels of peripheral blood MCP1 and related pro-inflammatory factors IL-6 and TNF-α in the inhibitor (Experimental) group were significantly reduced. Figure 6 The flow cytometric analysis of mouse blood showed that compared with the saline (control) group, the proportion of CD11b+F4 / 80+ monocytes and macrophages in the peripheral blood of the inhibitor (Experimental) group was significantly reduced. Figure 7 The MCP1 immunofluorescence staining of mouse eyes shown in the figure shows that compared with the saline (control) group, the expression of MCP1 in the retina of the inhibitor (Experimental) group was significantly downregulated. Figure 8 The flow cytometric analysis of mouse uveal tract showed that compared with the saline (control) group, the proportion of intraocular CD11b+Ly6C+ proinflammatory monocytes and CD11b+F4 / 80+ macrophages in the inhibitor (Experimental) group was significantly decreased. Fig. 9 The immunofluorescence staining of the blood-eye barrier of mice shown in the figure shows that compared with the normal saline (control) group, the expression of blood-eye barrier tight junction protein Cx43 in the inhibitor (Experimental) group was significantly increased, that is, the blood-eye barrier was improved. Fig.10 The animal behavior analysis shown in the figure shows that compared with the saline (control) group, the mice in the inhibitor (Experimental) group had an increased time in the open arm and the number of entries in the EPM test, that is, the anxiety behavior was reduced. In the figure, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figure 5-10 It can be seen that inhibiting MCP1 can significantly reduce the peripheral blood MCP1 level and the proportion of monocytes and macrophages in patients with high myopia, and change the inflammatory environment in the body of patients with high myopia; MCP1 inhibitors significantly reduce the expression of local MCP1 in the eye, thereby inhibiting inflammatory infiltration and immune barrier destruction, and ultimately regulating high myopia-related inflammation, alleviating the inflammatory-related complications of high myopia to a certain extent.

[0130] In summary, the MCP1 inhibitor of the present invention inhibits the action of MCP1, inhibits the inflammatory response mediated by MCP1, reduces the inflammatory infiltration and immune barrier destruction in the eyes and brain of high myopia, and thus may improve inflammation-related complications such as anxiety. This provides a new strategy for the treatment of high myopia and has potential clinical application value.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Application of MCP1 inhibitors in the preparation of drugs for the treatment of high myopia combined with inflammatory complications.

2. The use according to claim 1, characterized in that: The disease refers to high myopia complicated by anxiety.

3. The use according to claim 1, characterized in that: The MCP1 inhibitor is used to: (1) inhibit the secretion of peripheral chemokine MCP1; (2) inhibit the secretion of peripheral inflammatory factors MIP-1β, RANTES, TNF-α, IFN-γ and IL-6; (3) inhibit the infiltration of peripheral immune cells; (4) reduce the infiltration of monocytes / macrophages in the eyes and brain; (5) protect the blood-eye barrier / blood-brain barrier.

4. Application of MCP1 as a biomarker in the preparation of a diagnostic kit for high myopia and anxiety.

5. Application of reagents for detecting MCP1 protein content in the preparation of diagnostic kits for high myopia and anxiety.

6. The use according to claim 4 or 5, characterized in that: The sample tested is blood.

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