Application of AMG-487 in preparation of medicine for treating retinopathy of premature infant

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to application of AMG-487 in preparation of a medicine for treating premature infant retinopathy, and the medicine is used for treating the premature infant retinopathy by blocking combination of CXCL10 or / and CXCR3 in the ROP high oxygen period. Through research, the inventor finds that expression of CXCL10 in a blood sample of a patient with ROP is remarkably increased, TFR, HIF-1alpha, STAT3 and PTGS2 in a ferroptosis pathway have remarkable correlation with the CXCL10, AMG-487 can inhibit combination of CXCL10 / CXCR3 so as to reduce expression of TFR, HIF-1alpha, STAT3 and PTGS2 in the ferroptosis pathway, the effect of preventing and / or treating the retinopathy (ROP) of premature infants is achieved, ROP can be effectively treated, and the application prospect is wide. The compound is expected to become a novel medicine for treating ROP diseases and has important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular, relates to the application of AMG-487 in the preparation of a drug for treating retinopathy of prematurity. Background Art

[0002] Retinopathy of prematurity (ROP) is a blinding eye disease in children caused by abnormal development of retinal blood vessels in premature infants. According to statistics, there are 50,000 to 100,000 new ROP patients worldwide each year. With the advancement of medicine and the improvement of the survival rate of premature infants, the proportion of ROP patients is on the rise. It is foreseeable that in the context of my country's encouragement of multiple births, the increase in ROP patients will be more obvious, which will seriously affect the happiness of newborn families and cause heavy economic and social burdens.

[0003] Laser photocoagulation is a traditional method for clinical treatment of ROP, but the effect is very limited and there is a risk of causing the patient's visual field to narrow. VEGF antibodies (ranibizumab, conbercept, aflibercept, etc.) have been successfully used in ophthalmology for fundus neovascularization diseases such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). Studies have shown that intravitreal injection of VEGF antibodies also has a certain therapeutic effect on ROP. However, it should be noted that the challenges faced by VEGF antibodies in the treatment of ROP far exceed those of adult diseases such as AMD and DR: the physiological blood vessels of adults have developed well, and VEGF antibodies will only interfere with the newly formed pathological blood vessels; the eyes of premature infants are not yet mature, and VEGF antibodies not only interfere with pathological new blood vessels, but also hinder the normal development of physiological blood vessels. Therefore, the use of existing VEGF antibodies to treat ROP often leads to a decrease in permanent retinal vascular density in newborns, which in turn causes permanent visual impairment to patients. Caffeine is used clinically to treat apnea in premature infants, and studies have shown that ROP symptoms are also alleviated in apnea patients using caffeine.

[0004] Intravitreal injection is the most direct and effective route of administration for the treatment of retinal diseases. It can reduce systemic exposure and allow the drug to accumulate at the lesion site as much as possible. This method of administration is somewhat traumatic, so the frequency of administration must be minimized during clinical application, that is, sustained-release dosage forms must be used first. Caffeine is a hydrophilic drug with a solubility of 21.6 mg / mL. The problem with being too hydrophilic is that caffeine will be quickly dissolved and metabolized by body fluids when it enters the human body, so it must be administered frequently. Traditional drug sustained-release systems are often only effective for hydrophobic drugs, while sustained-release of hydrophilic drugs is a global problem.

[0005] Therefore, the clinical application of antibody drugs is greatly limited, and there is still a need to develop alternative, effective, and conveniently administered drugs to treat ROP. The problem of sustained release of caffeine has not been well solved.

[0006] AMG-487 is an effective and selective targeted blocker of the chemokine receptor CXCR3, which can inhibit CXCL10 / CXCR3 and thus block the inflammatory cycle to improve inflammatory symptoms. Currently, AMG-487 has been widely used in different animal models to inhibit the effects of chemokines and cytokines.

[0007] The inventors have found that AMG-487 has a significant effect on the treatment of retinopathy of prematurity (ROP), thereby providing a new drug for the treatment of retinopathy of prematurity. Summary of the invention

[0008] The purpose of the present invention is to provide the use of AMG-487 in the preparation of a drug for treating retinopathy of prematurity.

[0009] To achieve the purpose of the present invention, the present invention adopts the following technical solution:

[0010] Application of AMG-487 in the preparation of drugs for treating retinopathy of prematurity.

[0011] Furthermore, the drug treats retinopathy of prematurity by blocking the binding of CXCL10 and / or CXCR3 during the hyperoxic period of ROP.

[0012] Furthermore, the drug treats retinopathy of prematurity by inhibiting the ferroptosis pathway during the hyperoxic period of ROP and blocking the binding of CXCL10 and / or CXCR3.

[0013] Furthermore, the drug treats retinopathy of prematurity by blocking the binding of CXCL10 and / or CXCR3 in the ferroptosis pathway during the hyperoxic period of ROP, thereby reducing the expression of TFR, HIF-1α, STAT3, and PTGS2.

[0014] Through a large number of studies, the inventors found that the expression of CXCL10 in the blood samples of ROP patients was significantly increased, and TFR, HIF-1α, STAT3, and PTGS2 in the ferroptosis pathway were significantly correlated with CXCL10. AMG-487 can block the CXCL10 / CXCR3 binding and thus inhibit the expression of TFR, HIF-1α, STAT3, and PTGS2 in the ferroptosis pathway, thereby achieving the effect of preventing and / or treating retinopathy of prematurity (ROP).

[0015] The present invention also provides the use of AMG-487 in the preparation of a drug having at least one of the following functions 1)-4):

[0016] 1) Inhibit ferroptosis during the hyperoxic phase of ROP;

[0017] 2) Reduce intracellular ROS accumulation;

[0018] 3) Improve cell proliferation ability;

[0019] 4) Improve cell migration ability.

[0020] Furthermore, the inhibition of ferroptosis during the hyperoxic period of ROP refers to at least one of the following 1)-4):

[0021] 1) Blocking the binding of CXCL10 / CXCR3;

[0022] 2) Inhibit the expression of HIF-1α, PTGS2, and STAT3;

[0023] 3) Reduce the expression of MDA;

[0024] 4) Increase the expression of GSH.

[0025] Furthermore, the drug is used under a state of cellular oxidative stress (i.e., simulating the hyperoxia period of ROP patients).

[0026] Furthermore, the drug is administered by adding AMG-487 to the cell culture medium and incubating the cells normally in a cell culture incubator.

[0027] Furthermore, the dosage of the drug is 2.5 μM.

[0028] Furthermore, the drug is administered once and incubated for 12 hours.

[0029] The present invention overcomes the shortcomings of anti-VEGF antibody drugs in the prior art in clinical application, discovers and proves the application of AMG-487 in the preparation of drugs for treating retinopathy of prematurity (ROP), can exert excellent therapeutic effects in the treatment of ROP disease, can effectively treat ROP, and is expected to become a new drug for the treatment of ROP disease, with important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The difference in gene expression between normal (N) and ROP patients (R);

[0031] Figure 2 The difference in gene expression between blood samples of ROP patients (RB) on day 12-17 and those of the same patients (RA) on day 4-6 after oxygen therapy;

[0032] Figure 3 The results of PPI interaction analysis of differentially expressed genes between ROP and normal groups and differentially expressed genes between RA and RB groups are shown;

[0033] Figure 4 This is the KEGG pathway result diagram;

[0034] Figure 5 Figure 1 is a KEGG pathway enrichment diagram of overlapping differentially expressed genes; Figure A is a Venn diagram of the ROP and normal groups, RA and RB groups, and overlapping differentially expressed gene enrichment pathways and ferroptosis enrichment pathways; Figure B is a KEGG pathway enrichment analysis result diagram, showing all ferroptosis-related factor enrichment pathways, among which ferroptosis, amebiasis, and transcriptional dysregulation in cancer are the overlapping expression pathways in Figure A;

[0035] Figure 6 The GEO database compares the expression of various factors in the lung tissues of normal mice and mice subjected to hyperoxia stimulation, and selects differentially expressed factors related to ferroptosis, and performs a correlation analysis with CXCL10;

[0036] Figure 7 This is the expression result diagram of CXCL10, TFR, HIF-1α, STAT3 and PTGS2 in the lung tissue of hyperoxic mice in the GEO database;

[0037] Figure 8 This is the result graph of exploring the concentration of H2O2 drugs;

[0038] Fig. 9 The results of differential expression of CXCL10, TFR, HIF-1α, STAT3 and PTGS2 in NC and H2O2 groups are shown in the figure;

[0039] Fig.10 This is the expression change result of each factor at different H2O2 stimulation time;

[0040] Fig.11 This is the expression result diagram of each factor in the 9-hour H2O2 stimulation group;

[0041] Fig.12 This is the expression result diagram of each factor in the 12-hour H2O2 stimulation group;

[0042] Fig.13 The graphs are the results of drug concentration exploration; among them: Figure A is the graph of cell viability changes after stimulation with different concentrations of Fer-1; Figure B is the graph of cell viability changes after stimulation with different concentrations of Erastin;

[0043] Fig.14 This is the result diagram of AMG-487 drug concentration exploration;

[0044] Fig.15 The results of MDA expression in each group are shown;

[0045] Fig.16 The results of GSH expression in each group are shown;

[0046] Fig.17The results of TFRmRNA expression in each group are shown;

[0047] Fig.18 The expression results of CXCL10, CXCR3, HIF-1α, STAT3, and PTGS2 in each group are shown in the figure;

[0048] Fig.19 The fluorescence results of ROS in each group of cells, among which: Figure A is the fluorescence results of ROS in NC, Fer-1, Erastin, and E+C group cells, and Figure B is the quantitative statistics of the average fluorescence brightness of ROS in cells;

[0049] Fig. 20 The fluorescence results of live and dead cells in each group, green fluorescence: live cells; red fluorescence: dead cells;

[0050] Fig.21 Quantitative graph of the average fluorescence brightness of living cells in each group of cells;

[0051] Fig. 22 It is a quantitative comparison diagram of the cell proliferation ability of each group;

[0052] Fig.23 The changes in the number of migrated cells in each group;

[0053] Fig.24 Quantitative analysis of the changes in the number of cells that migrated in different groups;

[0054] Fig.25 Cell migration area diagrams at 0 h and 9 h in each group;

[0055] Fig.26 This is a quantitative analysis result diagram of the ratio of the migration area to the original area of ​​each group. DETAILED DESCRIPTION

[0056] The following are specific embodiments of the present invention, and the embodiments are intended to further describe the present invention rather than to limit the present invention.

[0057] Experimental Example 1: Analysis of gene expression differences in ROP patients using the GEO database

[0058] Test method:

[0059] (1) In the GEO database, we compared the gene expression differences between normal (N) and ROP patients (R) and the significantly differentially expressed genes in the blood samples of ROP patients (RB) and patients (RA) treated with oxygen for 12-17 days. Then, we performed PPI protein interaction analysis on the two groups of significantly different factors.

[0060] In the GEO database, information about ROP patients was searched (GSE32472). Patient inclusion criteria: (1) premature infants with a gestational age of less than 32 weeks; (2) birth weight less than 1500 g. Children with or without ROP were divided into two groups: ROP and N. Blood samples of ROP children were divided according to age. The blood samples of the same ROP patient on days 4-6 after birth were defined as the RA group, and the blood samples on days 12-17 after birth were defined as the RB group. To find differentially expressed variables, the screening criteria of P value 0.05 and FC absolute value >1 were used. PPI network The data of the STRING database (a search engine for interacting genes) were imported into the Cytoscape software, and an interaction network was created.

[0061] (2) The differentially expressed genes in the R vs. N group and the RB vs. RA group were compared, and the KEGG pathway enrichment analysis was performed on the genes with overlapping differential expression. Subsequently, the Wayne coincidence analysis was performed on the overlapping differentially expressed gene enrichment pathways and the ferroptosis enrichment pathways.

[0062] The Blast program was used to compare the gene sequences with the KEGG database, and then the pathway and GO annotation information of all genes were extracted. KEGG enrichment analysis of differentially expressed factors was performed through the Metascape online analysis website (https: / / metascape.org / gp / index.html# / main / step1). Overlap analysis with ferroptosis-related factors was performed, referring to the ferroptosis-significant gene library (http: / / www.zhounan.org / ferrdb / current / ).

[0063] (3) The lung tissues of normal and hyperoxia-stimulated C57 mice were compared using the GEO database (GSE51039), and differentially expressed factors were selected based on the condition of P < 0.05. The overlap analysis with ferroptosis-related factors was performed, and the Pearson rank correlation test was used to discover factors significantly correlated with CXCL10, and the factors were ranked according to the correlation coefficient.

[0064] (4) H2O2 cell model to verify the expression of various factors

[0065] HRMECs were selected to simulate the condition of retinal endothelial cells in patients. H2O2 was used to construct a cell model to simulate retinal vascular damage in vitro caused by oxidative stress. MTT was used to detect changes in cell viability after stimulation with different concentrations of H2O2. PCR was used to observe changes in the expression of CXCL10, CXCR3, HIF-1α, PTGS2, and STAT3.

[0066] Human retinal microvascular endothelial cells (HRMECs) were grown in complete medium in a 37°C, 5% CO2 cell culture incubator. DMEM complete medium contains 89% DMEM, 10% fetal bovine serum, 1% 10000U / ml penicillin and 10000g / ml streptomycin. Cells were revived from liquid nitrogen in advance and cultured to the third generation for daily experiments.

[0067] MTT assay for cell viability

[0068] Cell viability was determined using MTT following the manufacturer's instructions.

[0069] ① Digest the cells from the T25 flask and evenly seed them in a 96-well plate.

[0070] ② After the cells adhered overnight, the supernatant was discarded and drug stimulation was added according to different groups.

[0071] ③ After reaching the specified stimulation time, discard the supernatant and wash three times with 1×PBS.

[0072] ④ Add 10ul MTT (5mg / ml) and 100ul DMEM to each well of the cell culture plate. After culturing at 37°C for 4 hours, discard the supernatant.

[0073] ⑤ Add 150ul dimethyl sulfoxide to each well and wait for 15 minutes at room temperature.

[0074] ⑥ After shaking for 10 seconds using an enzyme-linked immunosorbent assay, measure the A value at a wavelength of 490 nm.

[0075] ⑦ Final result = actual measured value - blank well measured value. Repeat the experiment 3 times using a 96-well plate, with at least 3 wells in each group. Real-time quantitative PCR was used to detect the expression of mRNA of each factor

[0076] 1. RNA Extraction

[0077] ① After stimulation, the cells in each group were digested with trypsin, collected into 15 ml centrifuge tubes and neutralized by adding 2 ml of complete culture medium.

[0078] ②After washing with PBS for 3 times, add 500ul Lysis Buffer and mix thoroughly by repeatedly blowing with the pipette tip.

[0079] ③ Add 500ul of anhydrous ethanol to each lysate, carefully blow with the pipette tip to mix thoroughly, then add to the centrifuge column, centrifuge at 7000rpm for 1min, discard the waste liquid, and centrifuge at 12000rpm for 1min.

[0080] ④ Add 500ul Wash Water to the centrifuge column, centrifuge at 12000rpm for 1min, discard the waste liquid, and centrifuge again at 12000rpm for 1min.

[0081] ⑤ Transfer the centrifugal column to a 1.5 ml EP tube and let it dry on the operating table for 2 minutes.

[0082] ⑥Add 30ul Elution Buffer, leave at room temperature for 2min, then centrifuge at 12000rpm for 1min.

[0083] ⑦ Collect RNA and determine the RNA concentration using a spectrophotometer.

[0084] 2. Synthesis of cDNA

[0085] ① The total cDNA system is 20ul. Calculate the volume corresponding to 1ug RNA based on the RNA concentration, add it to an enzyme-free 0.2ml PCR tube, and place it on ice.

[0086] ② Add Nuclease-free water to 16ul according to the volume of RNA, and then add 4ul MasterMix.

[0087] ③ After thorough mixing, incubate at 37°C for 15 min, incubate at 60°C for 10 min, and heat at 95°C for 3 min to obtain cDNA.

[0088] 3.RT-PCR

[0089] ① Design primer sequence:

[0090] Table 1. Primer sequences

[0091]

[0092] ② In the amplification reaction, add 4ul cDNA, 4ul DEPC water, 2ul equal ratio mixture of upstream and downstream primers, and 10ul qPCR MasterMix in sequence, and the final volume is 20ul;

[0093] ③ Add the mixed solution to a 96-well plate in sequence, operate on ice throughout the process, with 3 replicate wells in each group, and repeat the experiment 3 times;

[0094] ④ After 3 min at 95°C, 15 s at 95°C, 1 min at 60°C, and repeating this sequence for 40 cycles, the temperature was then lowered to 4°C at 72°C for 5 min. The relative expression level of the cathepsin gene was determined using the comparative threshold cycle (2-ΔΔCt).

[0095] Test results:

[0096] (1) GEO database showed significant differential expression of CXCL10 in ROP patients ( Figure 1 and Figure 2), and after PPI protein interaction analysis of the significant difference factors between the two groups, it was found that CXCL10 was differentially expressed in both groups and played a major hub role ( Figure 3 ). Therefore, the inventors believe that CXCL10 plays an important role in the early progression of ROP. KEGG pathway shows that CXCL10 exerts its effect by binding to CXCR3 receptor ( Figure 4 ).

[0097] (2) Comparison of differentially expressed genes between the R and N groups, and between the RB and RA groups revealed 588 overlapping differentially expressed genes ( Figure 5 A). KEGG pathway enrichment analysis was performed on genes with overlapping differential expression, and it was found that the differential factors were enriched in the ferroptosis pathway ( Figure 5 A), which is consistent with vascular occlusion caused by ferroptosis during the hyperoxic phase of ROP. Subsequently, pathway enrichment was performed for all factors related to ferroptosis, among which three pathways of transcriptional dysregulation in ferroptosis, amebiasis, and cancer were expressed in both groups ( Figure 5 B).

[0098] (3) Comparing the expression of various factors in the lung tissues of normal mice and mice receiving hyperoxia stimulation in the GEO database, the differentially expressed factors related to ferroptosis were selected and the correlation analysis was performed with CXCL10. It was found that TFR, HIF-1α, STAT3, PTGS2 in the ferroptosis pathway were significantly correlated with CXCL10 (correlation coefficients: 0.829, 0.829, 0.886, 0.771; P = 0.042, 0.042, 0.019, 0.072) ( Figure 6 ). At the same time, the expression of CXCL10, TFR, HIF-1α, STAT3 and PTGS2 in the lung tissues of hyperoxic mice was increased (P=0.009, 0.033, <0.001, 0.001, <0.001) ( Figure 7 ) This is consistent with expectations, and the inventors speculate that CXCL10 and ferroptosis play a role in the hyperoxic phase of ROP.

[0099] (4) According to the MTT results, there was no significant difference in cell activity between the normal group (N) and the control group (NC) (adding the same amount of DMSO) after 12 hours of treatment. 100 and 200 μ m H2O2 significantly reduced cell viability (P < 0.001), and 400 μ m H2O2 greatly increased cytotoxicity (P < 0.001) ( Figure 8 The ideal concentration for HRMECs injury is 400 μM.

[0100] The gene expression levels in the H2O2 group were CXCL10: 2.19±0.42, CXCR3: 0.90±0.47, TFR: 2.38±0.32, HIF-1α: 1.74±0.40, STAT3: 1.36±0.08 and PTGS2: 2.28±0.30, respectively. Compared with the control group (NC), CXCL10, TFR, HIF-1α, STAT3 and PTGS2 were significantly different (F=9.65, 10.04, 12.71, 4.11, 0.28, 12.51, P=0.039, 0.695, 0.017, 0.034, 0.003, 0.019, respectively) ( Fig. 9 ). HIF-1α has the effect of inhibiting ferroptosis, but its expression was significantly increased in the H2O2 group, so further research was conducted.

[0101] By screening the database and analyzing the results of stimulating Huvecs with H2O2, it can be found that the relative difference of TFR increases significantly with time; and as the expression of TFRmRNA increases, the expression of HIF-1α first decreases significantly, and then gradually increases in 8-16 hours. Through the HIF-1 signaling pathway, it can be found that HIF-1 is the hub of multiple pathways. It is considered that when TFR increases, negative feedback inhibits HIF-1α expression, and as TFR increases, the expression of other HIF-1 pathways such as VEGF increases to resist the damage to cells caused by excessive TFR expression ( Fig.10 ). For this reason, this study compared the expression of different factors after 9h and 16h H2O2 stimulation: Compared with the control group (NC), the expression of CXCL10, CXCR3, and TFR in the 9-hour H2O2 stimulation group was significantly increased (P=0.005, 0.049, 0.038), and the expression of HIF-1α was significantly decreased (P=0.012) ( Fig.11 ); Compared with the control group (NC), the expression of CXCL10, TFR, and HIF-1α in the 12-hour H2O2 stimulation group was significantly increased (P=0.035, <0.001, 0.044), and the expression of CXCR3 was not significantly different but accompanied by a downward trend ( Fig.12 ). Therefore, the inventors believe that the expression change of HIF-1α is related to the expression degree or time of TFR. CXCR3 expression is closely related to ferroptosis, but when the stimulation time is long, CXCR3 expression may gradually decrease due to TFR negative feedback inhibition. Considering the consistency of the conclusion with the animal model, the present invention chooses to stimulate for 12h to construct the relevant model.

[0102] Experimental Example 2: Using HRMECs to construct a cell model to evaluate the effect of AMG-487 on the ferroptosis pathway

[0103] Test method:

[0104] HRMECs were used to construct Fer-1 (ferroptosis inhibitor), Erastin (ferroptosis inducer) and E+A (Erastin+AMG-487) models.

[0105] Human retinal microvascular endothelial cells (HRMECs) were grown in complete culture medium in a 37°C, 5% CO2 cell culture incubator. DMEM complete culture medium contains 89% DMEM, 10% fetal bovine serum, 1% 10000U / ml penicillin and 10000g / ml streptomycin. The cells were revived from liquid nitrogen in advance and cultured to the third generation for daily experiments. The cells were stimulated and grouped as follows:

[0106] Group Reagents Stimulation time Stimulating concentration Normal group (N) none none none Control group (NC) DMSO 12h Reference experimental group Fer-1 group Fer-1 12h 20μm Erastin Group Erastin 12h 5μm AMG-487 group AMG-487 12h 0.5μm E+A group Erastin+AMG-487 12h E(5μm)+A(2.5μm)

[0107] (1) MTT was used to detect the changes in cell viability after stimulation with different concentrations of Fer-1, Erastin and AMG-487

[0108] HRMECs cells were stimulated with 5, 10, 20, 40, and 80 μM Fer-1; 1.25, 2.5, 5, 10, and 20 μM Erastin; and 0.125, 0.25, 0.5, 1, and 2 μM AMG-487 for 12 h, and then cell viability was determined using MTT according to the manufacturer's instructions.

[0109] ① Digest the cells from the T25 flask and evenly seed them in a 96-well plate.

[0110] ② After the cells adhered overnight, the supernatant was discarded and drug stimulation was added according to different groups.

[0111] ③ After reaching the specified stimulation time, discard the supernatant and wash three times with 1×PBS.

[0112] ④ Add 10ul MTT (5mg / ml) and 100ul DMEM to each well of the cell culture plate. After culturing at 37°C for 4 hours, discard the supernatant.

[0113] ⑤ Add 150ul dimethyl sulfoxide to each well and wait for 15 minutes at room temperature.

[0114] ⑥ After shaking for 10 seconds using an enzyme-linked immunosorbent assay, measure the A value at a wavelength of 490 nm.

[0115] ⑦ Final result = actual measured value - blank well measured value. Repeat the experiment three times using a 96-well plate, with at least 3 wells in each group. (2) Evaluation of ferroptosis changes using MDA and GSH

[0116] Determination of MDA

[0117] 1. Determine protein concentration using BCA.

[0118] ①According to the experimental groups, cells were collected for total protein extraction.

[0119] ② Preparation of diluted albumin standard: Standard ratio description

[0120] Group Diluent volume (ul) BSA volume and composition (ul) BSA final concentration (ug / ml) A 0 300 original solution 2000 B 125 375 original solution 1500 C 325 325 original solution 1000 D 175 175B group diluent 750 E 325 325C group dilution 500 F 325 325E group dilution 250 G 325 325F group diluent 125 H 400 100G group dilution 25 I 400 0 0

[0121] ③ Prepare BCA working solution according to the instructions, and add sample to BCA working solution (1:20) into each well.

[0122] ④ After shaking for 30 seconds, let it stand at 37°C for 30 minutes and record the absorbance using an enzyme-labeled instrument.

[0123] ⑤ Draw a standard curve based on the relationship between the absorbance value and the concentration of each standard. Calculate the protein concentration according to the standard curve formula.

[0124] 2. MDA Assay Kit for Assessing the Effects of Lipid Peroxidation

[0125] ① Set up blank tubes, standard tubes, test tubes and control tubes. After the centrifuge tube is covered, use a needle to poke a small hole in the cover and mix thoroughly. Add each reagent according to the following instructions. a*: Indicates that the amount of sample, standard, anhydrous ethanol and reagent 1 to be added are all the same. (Usually add 0.1-0.2mL)

[0126]

[0127] ② Place it in a 95℃ water bath (or boil it with the lid open) for 40 minutes, take it out and cool it with running water, then centrifuge it at 3500-4000rpm for 10 minutes (the centrifugation time must be extended to below 3000rpm for complete precipitation).

[0128] ③Take the supernatant and measure the absorbance of each test tube at 532nm using an ELISA reader.

[0129] ④MDA content was substituted into the formula: (measured OD value - blank OD value) / (standard OD value - blank OD value)*standard concentration (10nmol / ml) / protein concentration (mgprot / ml).

[0130] Determination of GSH

[0131] 1. Measure protein concentration using BCA assay as above.

[0132] 2. Use the GSH detection kit to quantitatively detect the expression of reduced glutathione.

[0133] ① After collecting cells from each group, the cells were disrupted by ultrasound, and the reagents were added and mixed, followed by centrifugation to obtain the supernatant.

[0134] Add reagents as follows, and set up blank wells, standard wells, and measurement wells.

[0135] Blank hole Standard hole Determination hole Reagent 1 (ul) 100 20μmol / LGSH standard (ul) 100 Supernatant (ul) 100 Reagent 2 (ul) 100 100 100 Reagent 3 (ul) 25 25 25

[0136] ② Mix and let stand for 5 minutes, and measure the absorbance of each well at 405 nm using an ELISA reader.

[0137] ③ GSH was substituted into the formula: (measured OD value - blank OD value) / (standard OD value - blank OD value) * standard concentration (20 μM) * dilution rate / protein concentration (gprot / L)

[0138] (3) PCR was used to observe the expression changes of CXCL10, CXCR3, HIF-1α, PTGS2, and STAT3

[0139] 1. RNA Extraction

[0140] ① After stimulation, the cells in each group were digested with trypsin, collected into 15 ml centrifuge tubes and neutralized by adding 2 ml of complete culture medium.

[0141] ②After washing with PBS for 3 times, add 500ul Lysis Buffer and mix thoroughly by repeatedly blowing with the pipette tip.

[0142] ③ Add 500ul of anhydrous ethanol to each lysate, carefully blow with the pipette tip to mix thoroughly, then add to the centrifuge column, centrifuge at 7000rpm for 1min, discard the waste liquid, and centrifuge at 12000rpm for 1min.

[0143] ④ Add 500ul Wash Water to the centrifuge column, centrifuge at 12000rpm for 1min, discard the waste liquid, and centrifuge again at 12000rpm for 1min.

[0144] ⑤ Transfer the centrifugal column to a 1.5 ml EP tube and let it dry on the operating table for 2 minutes.

[0145] ⑥Add 30ul Elution Buffer, leave at room temperature for 2min, then centrifuge at 12000rpm for 1min.

[0146] ⑦ Collect RNA and determine the RNA concentration using a spectrophotometer.

[0147] 2. Synthesis of cDNA

[0148] ① The total cDNA system is 20ul. Calculate the volume corresponding to 1ug RNA based on the RNA concentration, add it to an enzyme-free 0.2ml PCR tube, and place it on ice.

[0149] ② Add Nuclease-free water to 16ul according to the volume of RNA, and then add 4ul MasterMix.

[0150] ③ After thorough mixing, incubate at 37°C for 15 min, incubate at 60°C for 10 min, and heat at 95°C for 3 min to obtain cDNA.

[0151] 3.RT-PCR

[0152] ① Design primer sequence:

[0153] Table 1. Primer sequences

[0154]

[0155] ② In the amplification reaction, add 4ul cDNA, 4ul DEPC water, 2ul equal ratio mixture of upstream and downstream primers, and 10ul qPCR MasterMix in sequence, and the final volume is 20ul.

[0156] ③ Add the mixed solution to a 96-well plate in sequence. Perform the whole process on ice. There are 3 replicate wells in each group and the experiment is repeated 3 times.

[0157] ④ After 3 min at 95°C, 15 s at 95°C, 1 min at 60°C, and repeating this sequence for 40 cycles, the temperature was then lowered to 4°C at 72°C for 5 min. The relative expression level of the cathepsin gene was determined using the comparative threshold cycle (2-ΔΔCt).

[0158] (4) Using ROS to examine the cellular oxidative stress state

[0159] ① Digest the cells from the T25 flask and culture them at 2*10 5 The cells were evenly plated in a 24-well plate at a density of 100 cells / well.

[0160] ② After the cells adhered overnight, the supernatant was discarded and drug stimulation was added according to different groups.

[0161] ③ After reaching the specified stimulation time, discard the supernatant and wash three times with 1×PBS.

[0162] ④ Add 200ul / well (1:1000 serum-free medium) of diluted DCFH-DA to the test wells. Add an equal amount of DMSO to the blank group and an equal amount of reactive oxygen species positive control reagent to the positive control group. Incubate for 30 minutes.

[0163] ⑤Discard the supernatant, wash three times with serum-free medium, and then observe under a fluorescence microscope.

[0164] ⑥ Randomly select areas for photography based on the morphology and intensity of the staining signal, and use ImageJ to calculate the average fluorescence intensity for subsequent analysis.

[0165] (5) Use live-dead cell assay and MTT to test cell viability

[0166] Live and dead cell staining

[0167] Calcein-AM and PI dual dyes are used to perform dual staining of live cells and dead cells, thereby analyzing the levels of live cells and dead cells.

[0168] ① Take out 10× assay buffer from the refrigerator, take an appropriate amount under sterile conditions, and dilute it with deionized water to obtain 1× assay buffer.

[0169] ② After digestion with 0.25% trypsin, the cells were collected after neutralization and washed with 1× assay buffer. Centrifugation was performed twice at 450 g, 5 min / time, to remove trypsin and residual esterase.

[0170] ③ After centrifugation, resuspend the cells in 1×AssayBuffer to make the cell count of the cell suspension 1×10 5～6 Pieces / ml.

[0171] ④ Add 2ul Calcein-AM for every 1ml of cells and incubate at 37℃ in the dark for 25min.

[0172] ⑤ Then add 5ul of PI to the above cell suspension and stain for 5 minutes at room temperature in the dark.

[0173] ⑥450g, 5min. Centrifuge to remove the staining solution, resuspend the cells in PBS, drop them on a glass slide, observe under a fluorescence microscope, and analyze the fluorescence intensity using ImagJ.

[0174] MTT assay for cell viability

[0175] Same as above.

[0176] (6) Transwell and scratch assays to test cell migration ability

[0177] Transwell cell migration assay

[0178] ① Add 100ul serum-free culture medium to the upper chamber of Transwell in advance and incubate in the cell incubator for 30 minutes for humidification and preheating.

[0179] ②Digest the cells in the T25 culture flask, neutralize and centrifuge, and then add serum-free culture medium to form a cell suspension.

[0180] ③Take out the Transwell chamber, add 750ul of culture medium containing 20% ​​serum to the lower chamber, and add 200ul of cell suspension to the upper chamber. Then, place in a cell culture incubator and incubate for 16h.

[0181] ④ After removing the upper chamber, wash it with PBS and add 800ul 4% paraformaldehyde to the lower chamber for fixation for 30 minutes.

[0182] ⑤ After taking out the upper chamber, wash it with PBS and add 800ul crystal violet to the lower chamber for staining for 30 minutes.

[0183] ⑥After staining, gently wipe the surface of the chamber with a cotton swab to remove excess non-migrated cells.

[0184] ⑦ After drying, cut off the bottom of the chamber with a sterile sharp knife and seal it with neutral gum.

[0185] ⑧The experiment was performed three times, with three compound wells in each group. Images were taken with a microscope, and the field of view was randomly selected to count the number of cells visible under the microscope, i.e., the number of migrated cells. The data were recorded and saved for further statistical analysis.

[0186] Cell scratch assay

[0187] ① On the back of the 24-hole plate, use a ruler to draw a vertical line through the center of the plate;

[0188] ② For cells in the logarithmic growth phase, trypsinize them into a single-cell suspension, seed the cells at a density of 2*105 cells / well in a 24-well plate, and place them in a cell incubator overnight.

[0189] ③After the cells adhered, each group was stimulated and the culture medium was discarded.

[0190] ④ Use a 200ul gun tip to align with the ruler and scratch perpendicular to the horizontal line. Keep the gun tip vertical and try to keep the scratch thickness consistent in each well.

[0191] ⑤Wash the cells 3 times with PBS, remove the scratched cells, and add serum-free culture medium.

[0192] ⑥ Erase the horizontal marker line on the back of the orifice plate and take photos at a fixed point every half an hour from 0 to 15 hours.

[0193] ⑦ Analyze the results using Image J software. Cell migration rate = (initial scratch area - scratch area at time t) / initial scratch area.

[0194] Test results:

[0195] (1) Fer-1 and Erastin were set in concentration gradients to test cell viability. Based on the MTT results, 20 μM and 5 μM were selected as the ideal induction concentrations ( Fig.13 AB). AMG-487 is a blocker of CXCL10 and CXCR3, so an AMG-487 group and a H2O2 group mixed with AMG-487 were constructed to block the binding of CXCL10 and CXCR3, and then the changes in ferroptosis were evaluated ( Fig.14 ).

[0196] (2) Compared with the control group (NC), the expression of MDA in the AMG-487 group and the fer-1 group decreased (P=0.30, 0.006), and the expression of GSH increased (P<0.001, =0.001), indicating that the combination of fer-1 and blocking CXCL10 and CXCR3 can inhibit ferroptosis; the expression of MDA in the Erastin group increased (P=0.001), and the expression of GSH decreased (P=0.006), indicating that the model was successfully constructed and Erastin effectively induced ferroptosis; and the addition of AMG-487 could effectively reverse the expression of MDA and GSH (P=0.005, 0.03) ( Fig.15 and Fig.16 ), which indicates that blocking the binding of CXCL10 and CXCR3 can effectively block ferroptosis, further proving that CXCL10 and CXCR3 play a role in ferroptosis. Transcriptomics results once again verified the above conclusions. Compared with the control group, TFRmRNA expression in the fer-1 group was significantly decreased (P=0.041), and in the Erastin group, it was significantly increased (P=0.044) and significantly different from the E+A group (P=0.014) ( Fig.17 ).

[0197] (3) Compared with the control group, the expressions of CXCL10, HIF-1α, STAT3, and PTGS2 in the Erastin group were significantly increased (P=0.026, 0.03, 0.011, and 0.007), and the differences were statistically significant; compared with the Erastin group, the expressions of each factor in the E+A group were significantly decreased (P=0.001, 0.01, 0.001, and 0.046), and the differences were statistically significant ( Fig.18 ). Therefore, CXCL10, HIF-1α, STAT3, and PTGS2 increase with ferroptosis expression and are also affected by the regulation of CXCL10 / CXCR3.

[0198] (4) Fluorescence staining was used to detect intracellular ROS. Intracellular ROS can oxidize non-fluorescent DCFH into green fluorescent DCF. Compared with the control group (NC), the average fluorescence brightness of the Erastin group was significantly different (P<0.001), and the fluorescence intensity of the Fer-1 group decreased but was not statistically significant. Compared with the Erastin group, the average fluorescence brightness of the E+A group decreased significantly (P<0.001) ( Fig.19AB). This suggests that intracellular ROS accumulation occurs after ferroptosis is aggravated, and blocking the CXCL10 / CXCR3 axis will reduce intracellular ROS accumulation.

[0199] (5) Fluorescence staining was used to detect live and dead cells. Compared with the control group (NC), the Fer-1 group, and the Erastin group, the average fluorescence brightness of dead cells was significantly different (P = 0.001, < 0.001). Compared with the Erastin group, the average fluorescence brightness of dead cells in the E+A group was significantly decreased (P < 0.001) ( Fig. 20 and Fig.21 ). This indicates that cell death increases after ferroptosis is aggravated, and blocking the CXCL10 / CXCR3 axis will reduce cell death. The results of the cell viability experiment are consistent with this. The MTT test of cell proliferation ability showed that compared with the control group (NC), the number of cells in the Fer-1 group increased (P=0.04), and the number of cells in the Erastin group decreased (P=0.002). The difference was statistically significant ( Fig. 22 Compared with the Erastin group, the number of cells in the E+A group increased significantly (P=0.004). This indicates that ferroptosis significantly inhibits cell proliferation, and blocking the CXCL10 / CXCR3 axis will improve cell proliferation ability.

[0200] (6) The number of cells passing through the pores in the control group (NC), Fer-1 group, and Erastin group were 234.67±16.26, 250.33±8.50, and 78±5.29, respectively. The number of cells passing through the pores in the Erastin group was significantly decreased (P<0.001) ( Fig.23 and Fig.24 ). This indicates that ferroptosis significantly affects the migration ability of endothelial cells. The scratch test showed that: comparing the cell migration area at 0 hours and 9 hours ( Fig.25 ), compared with the control group (NC), the migration of cells in the Fer-1 group was significantly accelerated (P<0.001), and the migration of cells in the Erastin group was significantly decreased (P=0.002); the addition of AMG-487 could improve the migration ability of cells in the Erastin group (P=0.014) ( Fig.26 ).

Claims

1. Application of AMG-487 in the preparation of drugs for the treatment of retinopathy of prematurity.

2. The use according to claim 1, characterized in that: The drug treats retinopathy of prematurity by blocking the binding of CXCL10 or / and CXCR3 during the hyperoxic period of ROP.

3. The use according to claim 2, characterized in that: The drug treats retinopathy of prematurity by inhibiting the ferroptosis pathway during the hyperoxic period of ROP and blocking the binding of CXCL10 and / or CXCR3.

4. The use according to claim 3, characterized in that: The drug treats retinopathy of prematurity by blocking the binding of CXCL10 and / or CXCR3 during the hyperoxic period of ROP, thereby downregulating the expression of TFR, HIF-1α, STAT3, and PTGS2 in the ferroptosis pathway.

5. Use of AMG-487 in the preparation of a drug having at least one of the following functions 1)-4): 1) Inhibit ferroptosis during the hyperoxic phase of ROP; 2) Reduce intracellular ROS accumulation; 3) Improve cell proliferation ability; 4) Improve cell migration ability.

6. The use according to claim 5, characterized in that: The inhibition of ferroptosis during the hyperoxic period of ROP refers to at least one of the following 1)-4): 1) Blocking CXCL10 / CXCR3 binding; 2) Inhibit the expression of HIF-1α, PTGS2, and STAT3; 3) Reduce the expression of MDA; 4) Increase the expression of GSH.

7. The use according to claim 6, characterized in that: The drug is used under conditions of cellular oxidative stress.

8. The use according to claim 7, characterized in that: The drug is administered by adding AMG-487 to the cell culture medium and incubating the cells normally in a cell culture incubator.

9. The use according to claim 8, characterized in that: The drug was administered at a dose of 2.5 μM.

10. The use according to claim 9, characterized in that: The drug was administered once and incubated for 12 hours.

Images