Application of curcumin in preparation of medicine for treating retinopathy of premature infants

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to application of curcumin in preparation of a medicine for treating premature infant retinopathy, and the medicine is used for treating premature infant retinopathy by reducing the expression level of CXCL10 or / and CXCR3 in the OIR hyperxia period. Through research, the inventor finds that the 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, curcumin can inhibit the expression of TFR, HIF-1alpha, STAT3 and PTGS2 in the ferroptosis pathway so as to reduce the expression of CXCL10, the effect of preventing and / or treating the retinopathy (ROP) of premature infants is achieved, ROP can be effectively treated, and the curative effect is good. The compound is expected to become a novel medicine for treating ROP diseases and has important clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to the application of curcumin in preparing a medicine 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] Compared with macromolecular biological drugs, small molecule drugs have the advantages of high stability, easy administration, low cost, etc. For example, CN113288889A discloses the use of probenecid in the prevention and treatment of retinopathy of prematurity (ROP). However, the efficacy of the drug is still not ideal.

[0007] Curcumin is an active ingredient extracted from turmeric, and its molecular formula is C 21 H 20 O6, chemical name is 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione, molecular weight is: 368.380, structural formula is as follows:

[0008]

[0009] Curcumin has been found to have anti-inflammatory, anti-tumor and anti-oxidative stress effects in various studies, but its pharmacological properties need to be further explored, which restricts its use in the treatment of diseases. The curcumin nanocrystal injection prepared at this stage can be infused intravenously and distributed to various parts of the body with the blood, exerting anti-inflammatory and anti-oxidative stress effects in the body.

[0010] The inventors have found that curcumin has a significant effect on treating retinopathy of prematurity (ROP), thereby providing a new drug for the treatment of retinopathy of prematurity. Summary of the invention

[0011] The purpose of the present invention is to provide application of curcumin in preparing a medicine for treating retinopathy of prematurity.

[0012] In order to achieve the above purpose, the present invention adopts the following technical solutions

[0013] The invention discloses an application of curcumin in the preparation of a drug for treating retinopathy of prematurity (ROP), wherein the drug treats retinopathy of prematurity by downregulating the expression level of CXCL10 and / or CXCR3 during the hyperoxic period of OIR.

[0014] Furthermore, the drug treats retinopathy of prematurity by inhibiting the ferroptosis pathway during the hyperoxic period of OIR to downregulate the expression level of CXCL10 and / or CXCR3.

[0015] Furthermore, the drug treats retinopathy of prematurity by downregulating the expression level of CXCL10 and / or CXCR3 during the hyperoxic period of OIR and inhibiting the expression of TFR, HIF-1α, STAT3, and PTGS2 in the ferroptosis pathway.

[0016] 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. Curcumin can inhibit the expression of CXCL10 and thereby reduce 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).

[0017] Furthermore, the retinopathy of prematurity is oxygen-induced retinal neovascularization (OIR).

[0018] Through further research, the inventors found that curcumin can play a protective role on retinal blood vessels during the hyperoxic period of OIR, protect vascular proliferation, reduce vascular occlusion, alleviate vascular damage, inhibit ferroptosis, and effectively improve the progression of OIR.

[0019] The present invention also provides the use of curcumin in preparing a drug having at least one of the following functions 1)-5):

[0020] 1) Inhibit ferroptosis during the hyperoxic phase of OIR;

[0021] 2) Protect retinal blood vessels during the hyperoxic phase of OIR;

[0022] 3) Reduce intracellular ROS accumulation;

[0023] 4) Reduce oxidative stress levels;

[0024] 5) Improve the migration ability of endothelial cells.

[0025] Furthermore, the inhibition of ferroptosis during the hyperoxic period of OIR refers to at least one of the following 1)-4):

[0026] 1) Inhibit the expression of CXCL10 / CXCR3;

[0027] 2) Inhibit the expression of HIF-1α, PTGS2, and STAT3;

[0028] 3) Reduce the expression of MDA and TFRmRNA;

[0029] 4) Increase the expression of GSH and FTH mRNA.

[0030] Furthermore, the drug is used during the hyperoxic period of OIR.

[0031] Furthermore, the drug is administered by intraperitoneal injection and nasal inhalation.

[0032] Furthermore, the drug is prepared into a liquid reagent with a concentration of 200 mM by 0.9% NaCl solution.

[0033] Furthermore, the intraperitoneal dosage of the drug is 30ul / g / mouse, and the nasal dosage is 10ul / mouse.

[0034] Furthermore, the drug is administered once every 2 days during the hyperoxic period.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] (1) The present invention overcomes the shortcomings of the prior art anti-VEGF antibody drugs in clinical application, discovers and proves the application of curcumin in the preparation of drugs for treating retinopathy of prematurity (ROP), can play an excellent therapeutic effect in treating ROP, can effectively treat ROP, and is expected to become a new drug for treating ROP, with important clinical application value;

[0037] (2) The curcumin of the present invention is used in the treatment of retinopathy of prematurity (ROP), and the administration method is intraperitoneal injection and nasal inhalation, which improves patient compliance and avoids the side effects caused by intravitreal injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the result diagram of exploring the concentration of curcumin drug;

[0039] Figure 2 The graphs are the expression levels of MDA and GSH in each group; Figure A is the expression level of MDA, and Figure B is the expression level of GSH;

[0040] Figure 3 The expression result diagram of each factor in each group;

[0041] Figure 4 The expression result diagram of each factor in each group;

[0042] Figure 5 Figure 1 is the expression graph of CXCL10 and CXCR3 proteins in each group; Figure A is the expression graph of CXCL10 protein, and Figure B is the expression graph of CXCR3 protein;

[0043] Figure 6 The fluorescence results of CXCL10, CXCR3 and TFR in each group; Figure A is the statistical diagram of the average fluorescence intensity data of each group, ***P<0.001, **P<0.01, *P<0.05; Figure B is the fluorescence results of CXCL10, CXCR3 and TFR in NC, H2O2 and H+C groups;

[0044] Figure 7Figure 2 is the fluorescence results of ROS in each group of cells; Figure A is the fluorescence results of ROS in NC, H2O2, H+C(2μm), H+C(4μm), C(2μm)C and (4μm) groups of cells; Figure B is the quantitative statistics of the average fluorescence brightness of ROS in cells;

[0045] Figure 8 The fluorescence images of live and dead cells in each group; Figure A is the fluorescence results of live and dead cells in the H2O2 and H+C groups, green fluorescence: live cells; red fluorescence: dead cells; Figure B is the quantitative image of the average fluorescence brightness of live cells in the cells;

[0046] Fig. 9 This is a quantitative comparison of the cell proliferation ability of each group;

[0047] Fig.10 Figure 1 is a graph of cell migration in each group; Figure A shows the changes in the number of migrated cells in the H2O2 and H+C groups, and Figure B is a quantitative analysis of the changes in the number of migrated cells in different groups;

[0048] Fig.11 is the migration area map for each group;

[0049] Fig.12 This is the result of retinal flat mount;

[0050] Fig.13 The results of retinal flat mounts of P12 OIR mice treated with different concentrations of curcumin; Figure A is a retinal flat mount of OIR mice treated with 100mM, 200mM, and 400mM curcumin at P12; Figure B is the percentage of avascular area / whole retina of each group of mice;

[0051] Fig.14 The following are statistical diagrams of retinal flat-mount blood vessel areas; Figure A shows the blood vessel-free area of ​​each group; Figure B shows the new blood vessel area of ​​each group;

[0052] Fig.15 Figure 1 is a graph of filopodia; Figure A is a statistical graph of the number of filopodia; Figure B is a statistical graph of the length of filopodia;

[0053] Fig.16 For P12R, P12R+C P7-12 , P17R and P17R+C P7-12 Figure 3: Vascular filopodia in the avascular area of ​​the retina in group A;

[0054] Fig.17 This is a statistical diagram of the number of tip cells in the avascular area of ​​the retina;

[0055] Fig.18 For P12R, P12R+C P7-12 Figure 1. Tip cell map of the avascular zone of the group retina;

[0056] Fig.19 For P12NC, P12R and P12R+C P7-12 Immunofluorescence images of retinal astrocytes in group 2;

[0057] Fig. 20 The figure is the statistical diagram of astrocyte density in the avascular area of ​​each group of mice during the hyperoxia period;

[0058] Fig.21 For P17NC, P17R, P17R+C P7-12 and P17R+C P12-17 Immunofluorescence images of retinal astrocytes in the avascular area of ​​group 2;

[0059] Fig. 22 For P17NC, P17R, P17R+C P7-12 and P17R+C P12-17 Immunofluorescence images of retinal astrocytes in the neovascular area of ​​group 2;

[0060] Fig.23 The figures are the statistical graphs of astrocyte density in the avascular area and neovascular area of ​​each group of mice during the hypoxia period; Figure A is the statistical graph of astrocyte density in the avascular area; Figure B is the statistical graph of astrocyte density in the neovascular area;

[0061] Fig.24 Figure 1 is the weight graph of mice measured at different times;

[0062] Fig.25 Figure 1 is the expression result diagram of MDA and GSH; Figure A is the expression result diagram of MDA; Figure B is the expression result diagram of GSH;

[0063] Fig.26 The results of TFR mRNA and FTH mRNA expression in the retina of mice in the P12NC and P12R groups are shown in the figure;

[0064] Fig. 27 For P12R and P12R+C P7-12 The results of TFR mRNA and FTH mRNA expression in the retina of mice in group A;

[0065] Fig.28 It is a quantitative statistical graph of fluorescence results; among them, Figure A is the fluorescence result graph of TFR; Figure B is the fluorescence result graph of FTH;

[0066] Fig.29 The immunofluorescence results of TFR and FTH in the retina of mice in each group;

[0067] Fig.30Figure 1 is the expression results of CXCL10, CXCR3, and HIF-1α mRNA and protein in the retina of mice in the P12NC group and the P12R group; Figure 2 is the expression results of CXCL10, CXCR3, and HIF-1α mRNA in the retina of mice in the P12R group, the P12R+C group, and the P12R+C group. P7-12 Figure A shows the expression results of CXCL10, CXCR3 and HIF-1α mRNA in the retina of mice in each group; Figure C shows the expression results of CXCL10 protein in the retina of mice in each group; Figure D shows the expression results of CXCR3 protein in the retina of mice in each group;

[0068] Fig.31 The immunofluorescence results of CXCL10 / CXCR3 in the retina of mice in each group are shown;

[0069] Fig.32 The results of HIF-1α immunofluorescence in the retina of mice in each group;

[0070] Fig.33 This is a quantitative statistical diagram of the CXCL10 fluorescence results in the retina of each group of mice;

[0071] Fig.34 This is a quantitative statistical diagram of the CXCR3 fluorescence results in the retina of each group of mice;

[0072] Fig.35 This is a quantitative statistical graph of the HIF-1α fluorescence results in the retina of each group of mice. DETAILED DESCRIPTION

[0073] 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.

[0074] Experimental Example 1: Using HRMECs to construct a cell model to evaluate the effect of curcumin on the ferroptosis pathway

[0075] Test method:

[0076] HRMECs were used to construct NC, H2O2, H+C (H2O2+curcumin), Erastin (ferroptosis inducer) and E+C (Erastin+curcumin) models.

[0077] 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:

[0078]

[0079]

[0080] (1) Using MTT to explore the IC of curcumin 50 ;

[0081] Cell viability was determined using MTT following the manufacturer's instructions.

[0082] ① Digest the cells from the T25 flask and evenly seed them in a 96-well plate.

[0083] ② After the cells adhered overnight, the supernatant was discarded and drug stimulation was added according to different groups.

[0084] ③ After reaching the specified stimulation time, discard the supernatant and wash three times with 1×PBS.

[0085] 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.

[0086] ⑤ Add 150ul dimethyl sulfoxide to each well and wait for 15 minutes at room temperature.

[0087] ⑥ After shaking for 10 seconds using an enzyme-linked immunosorbent assay, measure the A value at a wavelength of 490 nm.

[0088] 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) Use MDA and GSH to evaluate changes in ferroptosis;

[0089] Determination of MDA

[0090] 1. Determine protein concentration using BCA.

[0091] ①According to the experimental groups, cells were collected for total protein extraction.

[0092] ② Preparation of diluted albumin standard: Standard ratio description

[0093] 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

[0094] ③ Prepare BCA working solution according to the instructions, and add sample to BCA working solution (1:20) into each well.

[0095] ④ After shaking for 30 seconds, let it stand at 37°C for 30 minutes and record the absorbance using an enzyme-labeled instrument.

[0096] 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.

[0097] 2. MDA Assay Kit for Assessing the Effects of Lipid Peroxidation

[0098] 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)

[0099]

[0100]

[0101] 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).

[0102] ③Take the supernatant and measure the absorbance of each test tube at 532nm using an ELISA reader.

[0103] 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).

[0104] Determination of GSH

[0105] 1. Measure protein concentration using BCA assay as above.

[0106] 2. Use the GSH detection kit to quantitatively detect the expression of reduced glutathione.

[0107] 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.

[0108] Add reagents as follows, and set up blank wells, standard wells, and measurement wells.

[0109] 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

[0110] ② Mix and let stand for 5 minutes, and measure the absorbance of each well at 405 nm using an ELISA reader.

[0111] 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)

[0112] (3) PCR was used to observe the expression changes of TFR, CXCL10, CXCR3, HIF-1α, PTGS2, and STAT3;

[0113] Real-time quantitative PCR detection of mRNA expression of each factor

[0114] 1. RNA Extraction

[0115] 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.

[0116] ②After washing with PBS for 3 times, add 500ul Lysis Buffer and mix thoroughly by repeatedly blowing with the pipette tip.

[0117] 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.

[0118] 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.

[0119] ⑤ Transfer the centrifugal column to a 1.5 ml EP tube and let it dry on the operating table for 2 minutes.

[0120] ⑥Add 30ul Elution Buffer, leave at room temperature for 2min, then centrifuge at 12000rpm for 1min.

[0121] ⑦ Collect RNA and determine the RNA concentration using a spectrophotometer.

[0122] 2. Synthesis of cDNA

[0123] 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.

[0124] ② Add Nuclease-free water to 16ul according to the RNA volume, then add 4ul MasterMix

[0125] 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.

[0126] 3.RT-PCR

[0127] ① Design primer sequence:

[0128] Table 1. Primer sequences

[0129]

[0130] 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.

[0131] 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.

[0132] 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).

[0133] (4) Elisa assay was used to observe the changes in CXCL10 and CXCR3 protein expression in the cell supernatants of the NC, H2O2, and H+C groups; immunofluorescence was used to observe the fluorescence intensity of CXCL10, CXCR3, and TFR in the N, H2O2, and H+C groups;

[0134] ELISA

[0135] ① The concentrations of CXCL10 and CXCR3 in the samples were determined by enzyme-linked immunosorbent assay (ELISA) kits. After stimulation of each group of cells, the cells were centrifuged to obtain the supernatant. Each standard was prepared according to the following ratios.

[0136] 48ng / L Standard No. 5 150ul of original standard is added to 150ul of standard diluent 24ng / L Standard No. 4 150ul of standard No. 5 was added to 150ul of standard diluent 12ng / L Standard No. 3 150ul of standard No. 4 was added to 150ul of standard diluent 6ng / L Standard No. 2 150ul of standard No. 3 was added to 150ul of standard diluent 3ng / L Standard No. 1 150ul of standard No. 2 was added to 150ul of standard diluent

[0137] ② Grind the mouse retina and centrifuge to obtain the supernatant.

[0138] ③ Set up blank wells, standard wells and sample wells to be tested respectively. Add 50ul of standard to the standard wells, and add 10ul of sample and 40ul of sample diluent to the sample wells to be tested. Try to add the liquid directly to the bottom of the enzyme-labeled plate to avoid liquid residue on the tube wall. The microplate has purified antibodies pre-coated with target proteins. After sealing the plate with a sealing film, incubate in an incubator for 30 minutes.

[0139] ④Discard the liquid, spin dry, add 100ul washing solution / well, let it stand for 30s and then discard, repeat washing 5 times.

[0140] ⑤Except for the blank wells, add 50ul of enzyme-labeled reagent to other wells. Seal the plate with a sealing film and incubate for 30 minutes, then wash 5 times.

[0141] ⑥Add 50ul / well of color developer A and 50ul / well of color developer B in sequence, mix well and incubate in the dark for 10 minutes.

[0142] ⑦ Add 50ul / well of stop solution to stop the reaction.

[0143] ⑧The blank wells were adjusted to zero and the absorbance of each well at 450 nm was detected using an ELISA reader.

[0144] ⑨Read the CXCL10 and CXCR3 protein concentrations from the standard curve for each sample.

[0145] Immunofluorescence

[0146] ① Plant the cells in a glass-bottomed culture dish. After each group completes the corresponding stimulation, wash them three times with 1×PBS.

[0147] ② Add 4% paraformaldehyde 500ul / dish, fix for 20min, then wash with 1×PBS 500ul / dish, 5min / time, for 3 times.

[0148] ③ Add 0.1% Triton-X 500ul / dish and let stand for 10 min to improve cell permeability. Then wash with 1×PBS 500ul / dish, 5 min / time, for 3 times.

[0149] ④ Add 500ul / dish of blocking solution, including PBST (PBS+Tween-20) and goat serum, and block for 1 hour.

[0150] ⑤Discard the blocking solution, add the diluted primary antibody, and incubate at 4°C overnight (shaking incubation). The dilution ratio of CXCL10, CXCR3, and TFR is 1:250. The antibody diluent includes 90% PBS, 10% goat serum, 0.1% Tween-20, 0.02252g / ml glycine, and 0.01g / ml bovine serum albumin.

[0151] ⑥Discard the primary antibody, soak in PBST and wash for 10 minutes, repeat 3 times.

[0152] ⑦ Add goat anti-rabbit IgG antibody (1:1000) and / or goat anti-mouse IgG antibody (1:1000) at room temperature and let stand for 1 hour.

[0153] ⑧Discard the secondary antibody, soak in PBST and wash for 10 minutes, repeat three times.

[0154] ⑨Add DAPI for counterstaining and take pictures with a fluorescence microscope. Use image software to analyze the fluorescence intensity.

[0155] (5) ROS test of cellular oxidative stress status;

[0156] 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.

[0157] ② After the cells adhered overnight, the supernatant was discarded and drug stimulation was added according to different groups.

[0158] ③ After reaching the specified stimulation time, discard the supernatant and wash three times with 1×PBS.

[0159] 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.

[0160] ⑤Discard the supernatant, wash three times with serum-free medium, and then observe under a fluorescence microscope.

[0161] 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.

[0162] (6) Live-death cell assay and MTT test for cell viability and proliferation;

[0163] Live and dead cell staining

[0164] 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.

[0165] 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.

[0166] 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.

[0167] ③ After centrifugation, resuspend the cells in 1×AssayBuffer to make the cell count of the cell suspension 1×10 5～6 Pieces / ml.

[0168] ④ Add 2ul Calcein-AM for every 1ml of cells and incubate at 37℃ in the dark for 25min.

[0169] ⑤ Then add 5ul of PI to the above cell suspension and stain for 5 minutes at room temperature in the dark.

[0170] 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.

[0171] MTT assay for cell viability

[0172] Same as above.

[0173] (7) Transwell and scratch assays were used to examine cell migration ability.

[0174] Transwell cell migration assay

[0175] 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.

[0176] ②Digest the cells in the T25 culture flask, neutralize and centrifuge, and then add serum-free culture medium to form a cell suspension.

[0177] 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.

[0178] ④ After removing the upper chamber, wash it with PBS and add 800ul 4% paraformaldehyde to the lower chamber for fixation for 30 minutes.

[0179] 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.

[0180] ⑥After staining, gently wipe the surface of the chamber with a cotton swab to remove excess non-migrated cells.

[0181] ⑦ After drying, cut off the bottom of the chamber with a sterile sharp knife and seal it with neutral gum.

[0182] 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.

[0183] Cell scratch assay

[0184] ① On the back of the 24-hole plate, use a ruler to draw a vertical line through the center of the plate;

[0185] ② For cells in the logarithmic growth phase, trypsinize them into single cell suspension and centrifuge them at 2*10 5 The cells were seeded at a density of 100 cells / well in a 24-well plate and placed in a cell incubator overnight.

[0186] ③After the cells adhered, each group was stimulated and the culture medium was discarded.

[0187] 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.

[0188] ⑤Wash the cells 3 times with PBS, remove the scratched cells, and add serum-free culture medium.

[0189] 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.

[0190] ⑦ Analyze the results using Image J software. Cell migration rate = (initial scratch area - scratch area at time t) / initial scratch area.

[0191] Test results:

[0192] (1) Cell viability was tested at different curcumin doses and the curcumin IC 50 =43.35μm( Figure 1 ). Since curcumin plays a role in protecting blood vessels as a drug, 2 μM was selected as the commonly used concentration.

[0193] (2) Compared with the NC group, the expression level of MDA in the H2O2 group was 0.90±0.06, which was significantly increased (P=0.011); compared with the H2O2 group, the expression level of MDA in the H+C group was 0.57±0.06, which was significantly decreased (P=0.003). Compared with the control group, the expression level of GSH in the H2O2 group was 167.14±27.31, which was significantly decreased (P=0.019); compared with the H2O2 group, the expression level of GSH in the H+C group was 208.54±8.21, which was significantly increased (P=0.046). In the H2O2 group, the expression of MDA increased and the expression of GSH decreased, indicating that oxidative stress caused aggravated ferroptosis, while the H+C group reversed this result, indicating that curcumin can effectively inhibit ferroptosis ( Figure 2 ).

[0194] (3) Compared with the H2O2 group, the expression of each factor in the H+C group decreased, and the difference was statistically significant ( Figure 3 ). CXCL10: 0.56±0.06, CXCR3: 0.55±0.11, TFR: 0.50±0.18, HIF-1α: 0.62±0.07, STAT3: 0.80±0.10, PTGS2: 0.78±0.13 (P=0, 0.016, 0.009, 0.001, 0.027, 0.046). The decrease in TFR mRNA expression indicates that curcumin effectively alleviates the progression of ferroptosis. The expression of CXCL10 and CXCR3 mRNA decreased. Combined with the results in the previous section, the inventors believe that curcumin reduces ferroptosis by inhibiting CXCL10 / CXCR3. The decrease in HIF-1α, STAT3, and PTGS2 mRNA expression indicates that curcumin can also regulate the expression of HIF-1α, STAT3, and PTGS2 by inhibiting CXCL10 / CXCR3.

[0195] Compared with group E, the expression of each factor in group E+C decreased, and the difference was statistically significant ( Figure 4 ). CXCL10: 0.64±0.09, CXCR3: 0.76±0.06, TFR: 0.64±0.10, HIF-1α: 0.51±0.06, STAT3: 0.61±0.06, PTGS2: 0.45±0.08 (P=0.02, 0.009, 0.027, 0, 0.008, 0.006). In the environment of simply aggravated ferroptosis, curcumin can still improve ferroptosis and inhibit the expression of CXCL10, CXCR3, TFR, HIF-1α, STAT3, and PTGS2.

[0196] (4) Compared with the control group (NC), the expression of CXCL10 protein in the H2O2 group was 159.43±4.05ng / L, which was significantly increased (P=0.018); compared with the H2O2 group, the expression of CXCL10 in the H+C group was 128.92±9.41ng / L, which was significantly decreased (P<0.001). Compared with the control group (NC), the expression of CXCR3 in the H2O2 group was 2.65±0.13ng / L, which was significantly increased (P<0.001); compared with the H2O2 group, the expression of CXCR3 in the H+C group was 1.95±0.35ng / L, which was significantly decreased (P=0.002). It was verified again at the protein level that H2O2 aggravated the expression of CXCL10 and CXCR3, while curcumin could effectively reduce their expression ( Figure 5 ). The immunofluorescence results supported the above results. Compared with the control group (NC), the average fluorescence brightness of CXCL10, CXCR3, and TFR in the H2O2 group was significantly increased (P<0.001, <0.001, <0.001); compared with the H2O2 group, the average fluorescence brightness of CXCL10, CXCR3, and TFR in the H+C group was significantly decreased (P<0.001, <0.001, <0.001) ( Figure 6 This is consistent with the above results, in which curcumin inhibited ferroptosis by inhibiting CXCL10 / CXCR3.

[0197] (5) Fluorescence staining was used to detect intracellular ROS. Compared with the control group (NC), the average fluorescence brightness of the H2O2 group was significantly different (P<0.001). Compared with the H2O2 group, the average fluorescence brightness of the H+C (2μm) and H+C (4μm) groups decreased significantly (P<0.001, <0.001). There was no significant change in the average fluorescence brightness of the C (2μm) and C (4μm) groups that only applied curcumin ( Figure 7). This indicates that the C (2μm) and C (4μm) groups of curcumin do not aggravate the accumulation of ROS, while the H2O2 group has significant accumulation of intracellular ROS. Curcumin can effectively reduce the accumulation of intracellular ROS caused by H2O2. The increase in drug concentration has a positive correlation with the reduction of intracellular ROS. Therefore, curcumin has a dose-dependent effect on inhibiting the accumulation of ROS.

[0198] (6) Live-dead cell staining was used to observe the protective effect of curcumin on cells. Compared with the H2O2 group, the average fluorescence brightness of dead cells in the H+C group was significantly decreased (P<0.001) ( Figure 8 ). This indicates that curcumin can alleviate cell death caused by H2O2. 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 H2O2 group decreased significantly (P<0.001), and the difference was statistically significant. Compared with the H2O2 group, the number of cells in the H+C (2μm) and H+C (4μm) groups increased significantly (P=0.004, <0.001) ( Fig. 9 ). There was no significant change in the number of cells in the C (2μm) and C (4μm) groups that were treated with curcumin alone. This indicates that oxidative stress caused by H2O2 significantly inhibits cell proliferation, and curcumin can protect cells from damage caused by oxidative stress. And as the drug concentration increases, the number of cells increases. Therefore, curcumin has a dose-dependent effect on cell proliferation.

[0199] (7) Transwell cell migration assay can determine the vertical migration ability of retinal endothelial cells. Curcumin effectively improved the migration ability of cells in the H2O2 group. Compared with the H2O2 group, the number of cells passing through the small holes in the H+C group was 98.33±4.16, which was significantly increased (P<0.001) ( Fig.10 ). This indicates that curcumin can improve the migration ability of endothelial cells. The scratch test showed that the cell migration area was compared at 0 hours and 15 hours. Compared with the control group (NC), the cell migration in the H2O2 group was significantly decreased (P<0.001); adding different concentrations of curcumin can improve the cell migration ability of the H2O2 group (P=0.003, 0.001) ( Fig.11 ). Curcumin protects cell migration ability.

[0200] Experimental Example 2: Evaluation of the effect of curcumin on retinal blood vessels in premature infants during hyperoxia using the OIR mouse model

[0201] Test method:

[0202] (1) An OIR mouse model was constructed to effectively simulate the changes in the retinal state of premature infants after inhaling hyperoxia.

[0203] Construction of OIR mouse model

[0204] ① In order to effectively simulate the changes in the retinal state of premature infants after inhaling hyperoxia, an oxygen-induced retinal ischemia model was constructed, and the effects of hyperoxia on retinal blood vessels were observed through in vivo experiments.

[0205] ② About 40 newborn mice were mixed with their 8 lactating mother mice (C57BL / 6J mice were purchased from Beijing Huafukang Company) under normal conditions to ensure that the mother mice and mice were fully familiar with each other so that the lactating mother mice could effectively feed the mice in the later stage of modeling.

[0206] ③ 40 newborn mice were randomly divided into a normal group and a model group, with 20 mice in each group. On the 7th day after birth (P7), the mice in the model group were placed in a hyperoxia chamber together with 4 lactating mother mice, and the oxygen concentration in the chamber was ensured to reach 75%, the temperature in the chamber was appropriate, and there was enough water and mouse food. Pay attention to check whether the hyperoxia chamber is airtight.

[0207] ④ On the 12th day after birth, the mice were collected or returned to a normal environment (21% O2). During this period, the mother mice were replaced with the normal group every 2 days to prevent the mother mice from failing to feed the mice or dying due to long-term inhalation of high oxygen.

[0208] ⑤ The mice that were sampled directly on the 12th day were marked as the P12R group. The mice were in the hyperoxic period from P7 to P12. Note that the sampling of mice should be rapid and accurate to reduce exposure to normoxic air and prevent deviations in experimental results.

[0209] ⑥ After being transferred to a normal environment on the 12th day, the mice survived in a normoxic environment until the 17th day. The mice were sampled on the 17th day and marked as the P17R group. The mice were in a hypoxic period from P12 to P17. Pathological neovascularization reached its maximum at P17. The main culture process of the P17R group mice: they experienced hyperoxia from 7 to 12 days after birth and switched to normoxia from 12 to 17 days.

[0210] ⑦ The control group mice were fed under normal conditions until the 12th day after birth and then directly sampled, and were labeled as P12NC. The control group mice were fed under normal conditions until the 17th day after birth and then directly sampled, and were labeled as P17NC.

[0211] (2) Curcumin was prepared into a liquid reagent with a concentration of 200 mM using 0.9% NaCl solution according to the instructions.

[0212] Randomly divide mice into groups:

[0213] Normal group (N): The mice were not treated with any treatment and were kept under normal oxygen conditions.

[0214] Control group (NC): mice were raised under normal oxygen conditions, injected intraperitoneally and inhaled saline solution into the nasal cavity at P7, P9, and P11, and samples were collected at P12, and recorded as P12NC. Mice were raised under normal oxygen conditions, injected intraperitoneally and inhaled saline solution into the nasal cavity at P7, P9, and P11, and samples were collected at P17, and recorded as P17NC.

[0215] OIR+Curcumin group (RC): On the basis of hyperoxia from P7 to P12, curcumin (200mM, 30ul / g) was injected intraperitoneally at P7, P9, and P11, and curcumin (200mM, 10ul) was inhaled through the nasal cavity. The mice were sampled at P12 and recorded as P12R+C P7-P12 .

[0216] On the basis of hyperoxia from P7 to P12, curcumin (200mM, 30ul / g) was injected intraperitoneally and curcumin (200mgM, 10ul) was inhaled intranasally at P7, P9, and P11. The mice were raised under normal oxygen conditions from P12 to P17, and the samples were collected at P17. The mice were recorded as P17R+C P7-P12 .

[0217] From P7 to P12, the mice were placed in a hyperoxia chamber as before without drug injection. From P12 to P17, curcumin (200 mM, 30 ul / g) was injected intraperitoneally and inhaled intranasally (200 mgM, 10 ul) daily under normoxic conditions. The samples were collected on P17 and the mice were recorded as P17R+C. P12-P17 .

[0218] (3) The retina of each group of mice was flat-mounted, and then IB4 staining was used to observe the retinal blood vessels and vascular filopodia in the avascular area of ​​the retina in each group of mice. The retinas of each group of mice were immunostained with GFAP (green) and IB4 (red).

[0219] (4) The weight changes of mice were measured at different time points to determine whether curcumin would interfere with the development of mice; the expression changes of MDA and GSH were measured, and the expression levels of FTH, TFR, CXCL10, CXCR3, and HIF-1α were observed by PCR and immunofluorescence, and the changes in the expression levels of CXCL10 and CXCR3 proteins were observed by WB.

[0220] Test results:

[0221] (1) Compared with the control group, the avascular area in the P12R group increased significantly ( Fig.12 ), which indicates that during the period when retinal blood vessels are not fully developed, high oxygen significantly inhibits the normal growth and development of blood vessels.

[0222] (2) After the OIR mice were given curcumin according to the above administration method, the occluded retinal blood vessel area was observed. When the curcumin concentration was 100mM, there was no significant improvement; when the concentration was 200mM, the occluded retinal blood vessel area was significantly reduced (P=0.001); when the concentration was 400mM, the occluded retinal blood vessel area was significantly reduced (P=0.001), while there was no significant difference in the occluded retinal blood vessel area between 200mM and 400mM, and both had significant effects. Therefore, the drug concentration was determined to be 200mM ( Fig.13 ).

[0223] (3) Compared with the P12R group, the P12R+C P7-12 The avascular area of ​​the group was reduced, indicating that curcumin reduced the avascular area during the hyperoxic period of OIR and weakened the damage of hyperoxia to blood vessels (P<0.001)( Fig.12 , Fig.14 Compared with the P17NC group, the number of new blood vessels in the P17R group increased significantly, which was due to the vascular occlusion leading to local vascular ischemia and hypoxia, and then the proliferation of pathological new blood vessels. P7-12 Both the avascular area and the neovascular area were reduced (P=0.002, 0.017) ( Fig.12 , Fig.14 ), which indicates that curcumin effectively protects blood vessels and reduces vascular occlusion during the hyperoxic period, thereby alleviating the proliferation of new blood vessels; while P17R+C P12-17 The area of ​​new blood vessels did not decrease, indicating that curcumin did not inhibit new blood vessels and only had a protective effect on retinal blood vessels during the hyperoxic period. Therefore, curcumin can protect blood vessels during the hyperoxic period from P7 to 12.

[0224] The tip cells of the vascular endothelium play an important role in the proliferation of vascular sprouting. Studying the level of vascular dilation (filamentous pseudopodia) can help understand vascular proliferation. P7-12 The number and length of filopodia were significantly increased in the group (P<0.001, <0.001) ( Fig.15 , Fig.16 ), and the number of tip cells in the central avascular zone also increased significantly ( Fig.17 , Fig.18 ), indicating that curcumin protected vascular proliferation and alleviated vascular occlusion. However, compared with P17R, P17R+C P7-12 Only a trend of improvement was shown, but no significant difference was found. This suggests that curcumin has a significant protective effect on blood vessels mainly during the hyperoxic period of OIR.

[0225] The vascular structure is constructed with astrocytes as the skeleton. The surviving retinal astrocytes in the P12NC and P17NC groups were labeled with GFAP. Normal astrocytes are star-shaped, with many long and branched processes extending from the cell body, stretching and filling between the cell body and processes of the nerve cells. In contrast, the number of astrocytes in the avascular zone in the P12R group was significantly reduced (P<0.001), and their shape changed, such as thinning and shortening of the branches. Compared with the P12R group, the P12R+C P7-12 The number and shape of astrocytes in the group were greatly improved, which indicates that curcumin can effectively improve the damage of astrocytes induced by hyperoxia ( Fig.19 , Fig. 20 ). Compared with the P17NC group, the number of astrocytes in the avascular and neovascular areas of the P17R group was significantly reduced, and their shapes were changed (P<0.001, <0.001)( Fig.21 , Fig. 22 , Fig.23 ). Compared with P17R, P17R+C P7-12 The number and shape of astrocytes in the avascular and neovascular areas were effectively improved (P = 0.001, < 0.001), while P17R+C P12-17 There was no significant improvement in the Fig.21 , Fig. 22 , Fig.23 ), which indicates that curcumin effectively protects blood vessels and reduces vascular occlusion during the hyperoxic period, and the effect of alleviating vascular damage is still effective in mice at 17 days. This also shows that curcumin plays a protective role in the hyperoxic period and effectively improves the progression of OIR, while curcumin has no obvious effect during the hypoxic period.

[0226] (4) The weight changes of mice were measured at different time points. There was no significant difference in the weight of mice in the normal group (N) and the control group (NC). The weight of OIR mice was significantly lower than that of the normal group (N) and the control group (NC). P7-12 There were no significant differences in body weight between the groups, suggesting that curcumin did not interfere with mouse development ( Fig.24 ). Compared with the P12N group, the expression of MDA in the P12R group increased (P<0.001) and the expression of GSH decreased (P=0.001) ( Fig.25 ). TFR mRNA expression increased significantly (P = 0.029) and FTH mRNA expression decreased significantly (P = 0.042) ( Fig.26 ), indicating that ferroptosis was aggravated in P12R mice and vascular occlusion was caused by ferroptosis. However, the MDA decreased and GSH expression increased in the P17R group (P = 0.011, 0.005), indicating that ferroptosis caused by increased angiogenesis during hypoxia was weakened ( Fig.25 , Fig.26). Compared with the P12N group, the P12R+C P7-12 In the control group, the expression of MDA and TFR mRNA was significantly decreased (P < 0.001, = 0.022), and the expression of GSH and FTH mRNA was significantly increased (P = 0.039, 0.019), indicating that curcumin can effectively inhibit ferroptosis ( Fig.25 , Fig. 27 ). The fluorescence results are consistent with the PCR trend ( Fig.28 , Fig.29 ). Curcumin inhibits ferroptosis during the hyperoxic phase of OIR.

[0227] Compared with the P12N group, the P12R group showed increased expression of CXCL10, CXCR3, and HIF-1α mRNA and protein (P = 0.014, 0.002, 0.003). P7-12 The group showed a decrease in CXCL10 and CXCR3 mRNA and protein (P = 0.001, < 0.001; = 0.011, 0.041) ( Fig.30 ). This indicates that during the hyperoxia period of OIR, the expression of CXCL10 / CXCR3 increases, and curcumin inhibits its expression. Compared with the P17N group, the expression of CXCL10 / CXCR3 protein in the P17R group decreased, but there was no significant difference. The inventors believe that since the expression of CXCL10 / CXCR3 in the retina increases during P7-P12, and the expression of CXCL10 / CXCR3 decreases during P12-P17, the difference with the normal group is not significant ( Fig.30 CD). Therefore, the research focus of the present invention is that curcumin protects the retina during the hyperoxia period of OIR. The fluorescence results are consistent with the PCR trend ( Figure 31-Figure 35 ), curcumin effectively inhibited the expression of CXCL10 / CXCR3.

Claims

1. The use of curcumin in the preparation of a drug for treating retinopathy of prematurity, wherein: The drug treats retinopathy of prematurity by downregulating the expression level of CXCL10 and / or CXCR3 during the hyperoxic period of OIR.

2. The use according to claim 1, characterized in that: The drug treats retinopathy of prematurity by inhibiting the ferroptosis pathway during the hyperoxic period of OIR and downregulating the expression level of CXCL10 and / or CXCR3.

3. The use according to claim 2, characterized in that: The drug treats retinopathy of prematurity by inhibiting the expression of CXCL10 and / or CXCR3 during the hyperoxic period of OIR, thereby downregulating the expression of TFR, HIF-1α, STAT3, and PTGS2 in the ferroptosis pathway.

4. Use of curcumin in the preparation of a drug having at least one of the following functions 1)-5): 1) Inhibit ferroptosis during the hyperoxic phase of OIR; 2) Protect retinal blood vessels during the hyperoxic phase of OIR; 3) Reduce intracellular ROS accumulation; 4) Reduce oxidative stress levels; 5) Improve the migration ability of endothelial cells.

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

6. The use according to claim 5, characterized in that: The drug is used during the hyperoxic phase of OIR.

7. The use according to claim 6, characterized in that: The administration method of the drug is intraperitoneal injection and nasal inhalation.

8. The use according to claim 7, characterized in that: The drug was prepared into a liquid reagent with a concentration of 200 mM by using 0.9% NaCl solution.

9. The use according to claim 8, characterized in that: The intraperitoneal dosage of the drug is 30ul / g / mouse, and the nasal dosage is 10ul / mouse.

10. The use according to claim 9, characterized in that: The drug is administered once every 2 days during the hyperoxic period.

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