Application of extracellular trapping net in prevention and inhibition of myopia development

Abstract

The invention discloses application of an extracellular trapping net in prevention and inhibition of myopia development, and belongs to the field of biological medicine. It is found that release increase of the extracellular trapping net has a close relationship with myopia, and after release of the extracellular trapping net is inhibited by knocking out a key release gene Padi4 of the extracellular trapping net, the experimental myopia degree is obviously reduced. Therefore, the extracellular trapping net can be used as a new target for preventing and controlling myopia, and myopia can be prevented and controlled by adjusting the release of the extracellular trapping net aiming at the situation that the release of the extracellular trapping net is increased in a myopia-prone individual, a myopic individual or an individual with myopia tendency.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of extracellular traps in preventing and inhibiting the development of myopia. Background Art

[0002] Myopia is the main cause of visual impairment in children and also an important cause of vision decline in the global population. According to the statistical data of the National Health Commission, the overall myopia rate of children and adolescents in China has reached 53.6% in 2020, and the retinal lesions caused by pathological myopia have also become the leading cause of irreversible blindness in China. However, existing myopia prevention and treatment measures, such as wearing glasses (frame glasses and contact lenses), corneal laser correction, drugs (such as atropine, etc.), and posterior scleral reinforcement surgery, etc., can mostly only treat the symptoms and cannot effectively prevent the occurrence and development of myopia fundamentally. Moreover, low-concentration atropine has side effects such as pupil dilation. Therefore, effective and side-effect-free myopia prevention and control drugs are technical problems that urgently need to be solved in this field.

[0003] The sclera is the final effector of myopia formation. Myopia is essentially a mismatch between the refractive power of the eye and the axial length of the eye, resulting in the imaging focus falling in front of the retina. Research has pointed out that this mismatch is mainly caused by the increase in axial length. Abnormal external visual stimuli reach the retina through the eye refractive system and cause scleral remodeling through the "retina-choroid-sclera" pathway, resulting in a decrease in scleral strength and thickness, and then causing an increase in axial length and myopia formation.

[0004] Extracellular traps (ETs) are reticular structures with an aperture of about 200 nm containing various proteins composed of all or part of nuclear DNA / mitochondrial DNA. And the understanding of the function of ETs has expanded from targeting and clearing pathogens to promoting tissue fibrosis, participating in autoimmune diseases, and damaging host cells, etc. In recent years, researchers have also observed the generation of ETs in macrophages, monocytes, mast cells, etc. Extracellular traps (ETs) play an important role in extracellular matrix remodeling. Researchers have observed the presence of ETs in specimens of diseases such as pulmonary fibrosis in patients; in in vitro experiments, ETs can activate fibroblasts derived from organs such as the lung, skin, blood vessels, heart, etc. to transdifferentiate into myofibroblasts. However, whether ETs play a role in myopic scleral remodeling has not been reported.

[0005] Lactoferrin is an iron-binding protein that is present in very high levels in colostrum and is also present in small amounts in tears, saliva, mucus secretions, and secondary granules of neutrophils. After inflammatory stimulation of cells such as mucosal epithelium and neutrophils, these cells can synthesize lactoferrin. Lactoferrin can inhibit bacterial growth, increase the permeability of the bacterial cell wall, inhibit viral infection, block cell-virus fusion, activate the host defense system, etc. However, whether lactoferrin can inhibit myopia progression has not been reported. Summary of the Invention

[0006] The present invention discovers that there is a close connection between increased extracellular trap release and myopia. Therefore, the first object of the present invention is to provide an extracellular trap that can be used as a biomarker for the diagnosis of myopia, the assessment of the degree of myopia, and the monitoring and evaluation of the therapeutic effect of myopia treatment. The present invention discovers that inhibiting extracellular trap release has the effect of preventing and improving myopia and inhibiting myopia progression. Therefore, the second object of the present invention is to provide the use of an extracellular trap release inhibitor in preventing and / or improving myopia, and in slowing down, delaying, or inhibiting progression. The third object of the present invention is to provide a pharmaceutical composition, which includes an extracellular trap release inhibitor, especially lactoferrin, and the pharmaceutical composition can prevent and / or improve myopia, and slow down, delay, or inhibit progression.

[0007] The objects of the present invention are achieved by the following technical solutions:

[0008] In the first aspect of the present invention, the present invention provides the use of an extracellular trap as a biomarker in at least one of the following:

[0009] a1) Use in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of myopia;

[0010] a2) Use in the preparation and / or screening of products for evaluating the degree of myopia;

[0011] a3) Use in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic effect of myopia treatment.

[0012] The products include but are not limited to reagents, kits, chips, test strips, membrane strips, or detection platforms.

[0013] The myopia treatment described in the present invention includes but is not limited to drug treatment, surgical treatment, and instrument correction.

[0014] In the second aspect of the present invention, the present invention provides the use of a substance for detecting extracellular traps in at least one of the following:

[0015] b1) Use in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of myopia;

[0016] b2) Use in the preparation and / or screening of products for evaluating the degree of myopia;

[0017] b3) Use in the preparation and / or screening of products for evaluating or assisting in evaluating the therapeutic effect of myopia treatment.

[0018] The products include but are not limited to reagents, reagent kits, chips, test strips, membrane strips or detection platforms.

[0019] In a specific embodiment of the present invention, the substance for detecting extracellular trap nets is a substance for detecting the key protein citH3 and / or the marker dsDNA in the test sample, and the substance for detecting citH3 and / or the marker dsDNA includes any reagent required for detecting the expression level of citH3 protein or the expression level of the marker dsDNA gene by RT-PCR method, RT-qPCR method, biochip detection method, DNA blotting method, in situ hybridization method, enzyme-linked immunosorbent assay, immunoblotting method, immunohistochemistry method, immunofluorescence method, fluorescent dye method, ultraviolet photometry method, spectrophotometry method, microtitration method.

[0020] In some specific embodiments of the present invention, the substance for detecting citH3 is a reagent for detecting the expression level of citH3 protein by immunofluorescence staining.

[0021] In some specific embodiments of the present invention, the substance for detecting citH3 is a reagent for detecting the expression level of citH3 protein by immunoblotting.

[0022] In some specific embodiments of the present invention, the substance for detecting the marker dsDNA is a reagent for detecting the expression level of the marker dsDNA gene by fluorescent dye method.

[0023] The test sample includes but is not limited to plasma, aqueous humor or sclera.

[0024] In the third aspect of the present invention, the present invention provides an application of extracellular trap nets as a target in at least one of the following:

[0025] c1) Use in the preparation and / or screening of drugs for preventing myopia;

[0026] c2) Use in the preparation and / or screening of drugs for treating or improving myopia;

[0027] c3) Use in the preparation and / or screening of drugs for slowing down, delaying or inhibiting the progression of myopia.

[0028] In the fourth aspect of the present invention, the present invention provides an application of an extracellular trap net release inhibitor in at least one of the following:

[0029] d1) Use in the preparation and / or screening of drugs for preventing myopia;

[0030] d2) Use in the preparation and / or screening of drugs for treating or improving myopia;

[0031] d3) Use in the preparation and / or screening of drugs for slowing down, delaying or inhibiting the progression of myopia.

[0032] The extracellular trap release inhibitor includes substances that inhibit the release of extracellular traps or reduce the activity of extracellular traps, or substances that inhibit the expression of key genes of extracellular traps. The substances include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

[0033] In some specific embodiments of the present invention, the extracellular trap release inhibitor is a gene editing vector that inhibits the expression of the key gene Padi4 of extracellular traps.

[0034] In some specific embodiments of the present invention, the extracellular trap release inhibitor is lactoferrin.

[0035] In some specific embodiments of the present invention, the extracellular trap release inhibitor is a drug with the effect of inhibiting the release of extracellular traps. The drugs include dornase alfa inhalant, danirixin, budesonide, Cevimeline, PHP-303, lonodelestat, CHF6333, alvelestat, disulfiram, NucleoCapture device.

[0036] Cevimeline is a neutrophil elastase inhibitor and has been approved for marketing in China.

[0037] PHP-303 was developed by pH Phanma Company in South Korea and has completed Phase I clinical trials.

[0038] Lonodelestat was developed by Santhera Pharmaceuticals Company in Switzerland and has completed Phase I clinical trials.

[0039] CHF6333 was developed by Chiesi Farmaceutici S.p.A in Italy and has completed Phase I clinical trials.

[0040] Alvelestat is a small molecule NE oral inhibitor developed by Mereo BioPharma Company in the UK and has completed Phase II clinical trials in April 2022.

[0041] The NucleoCapture device can highly selectively remove neutrophil extracellular traps from human blood during plasma exchange.

[0042] In a fifth aspect of the present invention, the present invention provides a pharmaceutical composition comprising an effective amount of the extracellular trap release inhibitor described in the fourth aspect of the present invention.

[0043] The extracellular trap release inhibitor includes substances that inhibit extracellular trap release or extracellular trap activity, or substances that inhibit the expression of key genes of extracellular traps. The substances include, but are not limited to, one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

[0044] In some specific embodiments of the present invention, the extracellular trap release inhibitor is a gene editing vector that inhibits the expression of the key extracellular trap gene Padi4.

[0045] In some specific embodiments of the present invention, the extracellular trap release inhibitor is lactoferrin.

[0046] In some specific embodiments of the present invention, the extracellular trap release inhibitor is a drug having the effect of inhibiting extracellular trap release, and the drugs include alfadeoxyribonuclease inhalant, danirixin, budesonide, cevimeline, PHP-303, lonodelestat, CHF6333, alvelestat, disulfiram, NucleoCapture device.

[0047] Furthermore, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0048] As used in the present invention, "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammalian subject being treated therewith. Preferably, "pharmaceutically acceptable" means approved by a federal regulatory agency or a national government or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans. The pharmaceutically acceptable excipients of the present invention can include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a particular target dosage form.

[0049] The pharmaceutical composition of the present invention can be used for the treatment of diseases and also for in vitro cell culture experiments. When used for the treatment of diseases, the pharmaceutical composition is usually in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with excipients that constitute one or more accessory components.

[0050] The treatment of the disease refers to administering a therapeutically effective amount of the extracellular trap net release inhibitor or pharmaceutical composition of the present invention to a subject in need thereof.

[0051] Furthermore, the treatment of the disease further includes co-administering the extracellular trap net release inhibitor or pharmaceutical composition of the present invention with other conventional medicaments or conventional treatment methods in the art.

[0052] The contribution of the technical solution provided by the present invention to the art lies in:

[0053] Through experiments such as immunoblotting and immunofluorescence, the inventors found that the release amount of macrophage extracellular trap nets in the sclera of the experimental eyes of experimental myopia mice was significantly increased, and there was a significant difference compared with the control eyes. By knocking out the key gene Padi4 for extracellular trap net release through transgenic technology to inhibit extracellular trap nets, the inventors found that the degree of experimental myopia in mice was significantly reduced. Therefore, the increase in extracellular trap net release is closely related to myopia, and extracellular trap net release may be a key mechanism for myopia progression. Therefore, scleral extracellular trap nets can be used as a brand-new myopia prevention and control target to prevent and inhibit myopia progression. For individuals prone to myopia, those already myopic, or those with a tendency to be myopic who show an increase in scleral extracellular trap net release, the purpose of preventing and controlling myopia can be achieved by regulating the release of scleral extracellular trap nets.

[0054] In addition, the inventors also found that the extracellular trap net release level in the plasma of experimental myopia mice was significantly increased, indicating that extracellular trap nets in the plasma can be used as biomarkers for myopia diagnosis and auxiliary diagnosis. In clinical trials, the inventors found that the release amount of extracellular trap nets in the plasma and aqueous humor of myopic patients was significantly higher than that of the normal population, and as the degree of myopia increased, the release amount of extracellular trap nets in the plasma and aqueous humor increased, showing a positive correlation between the two. Therefore, the extracellular trap net release levels in the plasma and aqueous humor can be used as biomarkers for the diagnosis and auxiliary diagnosis of myopia, and the degree of myopia can be evaluated by detecting the extracellular trap net levels in the plasma and aqueous humor, and the myopia treatment level can be monitored.

[0055] The content of lactoferrin in colostrum is very high, and it is also present in small amounts in tears, saliva, mucus secretions, and secondary granules of neutrophils. However, whether lactoferrin can inhibit myopia progression has not been reported in the prior art. The inventors intraperitoneally injected lactoferrin into experimental myopia mice and found that the degree of myopia in the mice was significantly reduced. The present invention confirms that lactoferrin can inhibit extracellular trap net release and has the efficacy of improving myopia and inhibiting myopia progression. Description of the Drawings

[0056] Figure 1(A) Immunofluorescence staining images of the sclera of both eyes of experimental myopia model mice and one eye of control mice; (B) Immunoblotting images of citH3 protein in the sclera of both eyes of experimental myopia model mice and one eye of control mice; (C) Statistical chart of the relative expression level of citH3 protein.

[0057] Figure 2 (A) Padi4 - / - and the trend chart of the change in axial length of the eyes after experimental myopia induction in wild-type mice; (B) Padi4 - / - and the trend chart of the change in diopter after experimental myopia induction in wild-type mice; (C) Immunoblotting images of COL1A1, α-SMA, and citH3 proteins in the sclera of Padi4 - / - and wild-type mice after experimental myopia induction; (D) Statistical chart of the relative expression level of citH3 protein.

[0058] Figure 3 (A) Trend chart of the effect of lactoferrin on the axial length of experimental myopia model mice; (B) Trend chart of the effect of lactoferrin on the diopter of experimental myopia model mice; (C) Immunoblotting images of COL1A1, α-SMA, and citH3 proteins in the sclera of experimental myopia model mice after administration of lactoferrin; (D) Statistical chart of the relative expression levels of COL1A1, α-SMA, and citH3 proteins.

[0059] Figure 4 Statistical chart of the content of extracellular trap marker dsDNA in the plasma of myopic mice.

[0060] Figure 5 Statistical chart of the content of extracellular trap marker dsDNA in the plasma of myopic patients.

[0061] Figure 6 Statistical chart of the content of extracellular trap marker dsDNA in the aqueous humor of myopic patients. Detailed implementation manners

[0062] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0063] The present invention has verified the following conclusions through experiments:

[0064] Conclusion 1: Extracellular traps (ETs) are highly expressed in the sclera of myopic mice

[0065] Purpose of the experiment: To verify the relationship between the release of extracellular traps in the sclera and myopia.

[0066] Experimental method: The experimental animals were 4-week-old C57 / BL6 mice. In the experimental group, the right eyes of the mice were subjected to lens-induced myopia (LIM) modeling with a -25D lens. After 4 weeks of induction, the animals were anesthetized and sacrificed. The right eyes were LIM-T eyes, and the left eyes were self-control LIM-F eyes. The scleras of both eyes were taken for immunoblotting and immunofluorescence observation. The control group of mice was fed under normal conditions and sacrificed after 4 weeks. The right sclera (natural control eye) was taken as the NC eye for immunoblotting and immunofluorescence observation.

[0067] Experimental results: The eyes of the mice in the experimental group induced with LIM using a -25D lens were LIM-T eyes; the other eye without induction was the LIM-F eye; the right eyes of the mice without lens induction in both eyes were NC eyes. The results of immunofluorescence staining of the scleras of LIM-T eyes, LIM-F eyes, and NC eyes are as Figure 1 shown in A. As can be seen from the figure, after 4 weeks of optical defocus, compared with LIM-F eyes and NC eyes, the expression level of the key protein citH3 of extracellular trap nets in the sclera of the myopic eyes (LIM-T eyes) of mice increased significantly (as shown by the white arrows), indicating that the release of extracellular trap nets in the sclera of myopic eyes was significantly enhanced. The results of immunoblotting experiments and relative protein expression levels of citH3 protein in the scleras of LIM-T eyes, LIM-F eyes, and NC eyes are as Figure 1 shown in B and Figure 1 C. As can be seen from the figure, compared with the natural control eyes and the contralateral control eyes, the expression level of citH3 protein in the sclera of LIM eyes increased significantly, indicating that the increase in the expression level of scleral citH3 protein (release of extracellular trap nets in the sclera) was closely related to myopia. The extracellular trap nets in the sclera can be used as biomarkers for the diagnosis or auxiliary diagnosis of myopia, or the degree of myopia can be evaluated by detecting the release level of extracellular trap nets in the sclera.

[0068] Conclusion 2: Knocking out the key gene Padi4 of extracellular trap nets can inhibit myopia

[0069] Experimental purpose: To verify the relationship between extracellular trap nets and myopia at the gene level.

[0070] Experimental method: The experimental animals were divided into two groups. The experimental group of mice were 4-week-old Padi4 - / - KO C57 / BL6 mice, and the control group of mice were 4-week-old wild-type C57 / BL6 mice. A single-eye optical defocus experimental myopia model was established for both groups of mice using a -25D lens. The diopter was measured with an infrared eccentric photorefractor and the axial length of the eye was measured with OCT before the experiment, 2 weeks after LIM induction, and 4 weeks after LIM induction. The release of extracellular trap nets in the sclera was analyzed by immunoblotting.

[0071] Test results: The eyes of the experimental group mice induced by LIM were Padi4 - / - LIM eyes, and the eyes not induced by LIM on the other side were Padi4 - / - NC eyes. The eyes of the control group mice induced by LIM were WT LIM eyes, and the eyes not induced by LIM on the other side were WT NC eyes. The changing trends of the diopter and axial length of each eye at 0, 2, and 4 weeks after LIM induction were as Figure 2 shown in Figure 2 Figure B and Figure 2 the results of the Western blot experiments of COL1A1, α-SMA, and citH3 proteins and the relative protein expression levels in the sclera of each eye were as Figure 2 shown in Figure C and

[0072] Figure D Figure 2 - / - According to the diopter change trend graph shown in - / - Figure B, as the LIM induction time increased, the diopters of WT NC eyes and Padi4 - / - NC eyes both showed a slow increasing trend, and the increasing trend of Padi4 - / - NC eyes was more obvious, indicating that knocking out the key gene of extracellular trap network (inhibiting the release of extracellular trap network) had the effect of increasing the diopter level of normal eyes and could prevent myopia. The diopters of WT LIM eyes and Padi4 - / - LIM eyes induced by LIM both showed a decreasing trend. From the decreasing trend, it could be seen that the diopter of WT LIM eyes dropped suddenly from week 0 and until week 4, the diopter of WT LIM eyes had approached 0D. In contrast, the decreasing trend of the diopter of Padi4 - / - LIM eyes was significantly slower, indicating that knocking out the key gene of extracellular trap network (inhibiting the release of extracellular trap network) had the effect of delaying the change of diopter to negative, could improve myopia, and could inhibit the development of optically defocused myopia. - / - According to the axial length change trend graph shown in - / - Figure A, as the induction time increased, the axial lengths of WT natural control eyes (WT NC eyes) and Padi4 - / - natural control eyes (Padi4 - / -The upward trend of the eye axis length in LIM eyes slowed down significantly, indicating that knocking out the key gene of extracellular trap nets (inhibiting the release of extracellular trap nets) has the effect of delaying the elongation of the eye axis, can inhibit myopia, and can inhibit the development of optically defocused myopia.

[0074] According to Figure 2 C and Figure 2 As shown in C and D are the results of immunoblotting experiments of COL1A1, α-SMA, and citH3 in the sclera of each eye. It can be seen that compared with wild-type mice, the expression level of citH3 protein in the sclera of the experimental group of mice with Padi4 gene knockout decreased significantly, indicating that Padi4 - / - The degree of extracellular trap net release in the sclera of the Padi4 KO group was less than that of the wild-type group, indicating that knocking out the key gene Padi4 can inhibit the release of extracellular trap nets in the sclera; compared with wild-type mice, the expression level of COL1A1 protein in the sclera of the experimental group of mice with Padi4 gene knockout increased significantly, and the expression of α-SMA protein decreased significantly, and the myopic manifestation of the mouse sclera was inhibited, indicating that knocking out the key gene Padi4 can inhibit myopic scleral remodeling.

[0075] In this experiment, Padi4 - / - The refractive myopia degree and the degree of eye axis elongation of LIM eyes were less than those of WT LIM eyes, and there was a statistically significant difference compared with WT LIM eyes, and Padi4 - / - The release of extracellular trap nets in the sclera of mice was significantly reduced. It shows that knocking out the key gene Padi4 of extracellular trap nets - / - can inhibit the release of extracellular trap nets, has the effect of delaying the change of refractive power to negative and the elongation of the eye axis, can inhibit the formation of optically defocused myopia in mice, and slow down the development of optically defocused myopia in mice.

[0076] Conclusion 3: Lactoferrin inhibits the release of extracellular trap nets and thus inhibits myopia

[0077] Purpose of the experiment: To verify that lactoferrin has the effect of inhibiting extracellular trap nets, and thus lactoferrin has the effect of inhibiting the progression of myopia.

[0078] Experimental method: The experimental animals were 4-week-old wild-type C57 / BL6 mice, and a -25D lens was used to establish an experimental myopia model of monocular optical defocus in the right eye of the mice. The experimental animals were randomly divided into 2 groups: the drug group (lactoferrin 20 μg / g) and the solvent control group (saline). The drug group was intraperitoneally injected with lactoferrin 20 μg / g body weight dissolved in saline. The injection was carried out at 9 am every day for 4 consecutive weeks, and the solvent control group was given saline. The refractive power was measured with an infrared eccentric photorefractor (EIR), and the eye axis length was measured with OCT before the experiment, 2 weeks after drug administration, and 4 weeks after drug administration. The release of extracellular trap nets in the sclera was analyzed by immunoblotting.

[0079] Test results: The eyes of the lactoferrin (Lf) drug group receiving LIM induction were denoted as "Lf+LIM", and the other eye was denoted as "Lf+NC". The eyes of the solvent control group (normal saline, NS) receiving LIM induction were denoted as "NS+LIM", and the other eye was denoted as "NS+NC". The changing trends of the diopter and axial length of each eye at 0, 2, and 4 weeks after intraperitoneal injection of lactoferrin were as shown in Figure 3 B and Figure 3 A. The results of the immunoblotting experiments of COL1A1, α-SMA, and citH3 proteins and the relative protein expression levels in the sclera of each eye were as shown in Figure 3 C and Figure 3 D.

[0080] From Figure 3 the changing trend of the diopter shown in B, it can be seen that with the increase of the LIM induction time, the diopter of the NS+LIM eye showed a significant downward trend. By the 4th week, the diopter of the NS+LIM eye was close to 0D. Under the same induction conditions, the downward trend of the diopter of the Lf+LIM eye intervened with lactoferrin became significantly slower, indicating that lactoferrin has the effect of inhibiting and delaying the change of the diopter to negative, can improve myopia, and inhibit the development of optical defocus myopia.

[0081] Figure 3 The same conclusion was shown in the effect of lactoferrin on the axial length shown in A. With the increase of the LIM induction time, the axial length of the NS+LIM eye showed a significant upward trend. In contrast, the upward trend of the Lf+LIM eye became significantly slower, indicating that lactoferrin has the effect of delaying the elongation of the axial length, can improve myopia, and inhibit the development of optical defocus myopia.

[0082] Figure 3 C and Figure 3 D are the results of the immunoblotting experiments of COL1A1, α-SMA, and citH3 proteins in the sclera of each eye. It can be seen from the figure that compared with the mice given normal saline, the expression level of citH3 protein in the sclera of the mice intervened with lactoferrin decreased significantly, indicating that the degree of extracellular trap release in the sclera of the lactoferrin intervention group was less than that of the wild-type group, indicating that lactoferrin can inhibit the release of extracellular traps in the sclera; compared with the mice given normal saline, the expression level of COL1A1 protein in the sclera of the mice intervened with lactoferrin increased significantly, and the expression of α-SMA protein decreased significantly, and the myopic manifestation of the mouse sclera was inhibited, indicating that lactoferrin can inhibit myopic scleral remodeling.

[0083] The data of this experiment prove that the degree of refractive myopia and the degree of axial elongation of the LIM eyes intervened by lactoferrin are both less than those of the normal saline control group, showing statistical significance compared with the control group. Moreover, the release of extracellular trap nets in the sclera of the lactoferrin intervention group of mice is significantly reduced. The above results indicate that lactoferrin can inhibit the release of extracellular trap nets in the sclera, delay the change of refractive power to negative and the elongation of the eye axis, thereby inhibiting the formation or slowing down the development of optical defocus myopia in mice.

[0084] Conclusion 4: The content of extracellular trap net marker dsDNA in the plasma of myopic mice increases

[0085] Purpose of the experiment: To verify the relationship between the release of plasma extracellular trap nets and myopia.

[0086] Experimental method: The experimental animals were 4-week-old wild-type C57 / BL6 mice. The experimental animals were randomly divided into 2 groups: the optical defocus group (LIM group) and the control group (NC group). The right eyes of the mice in the LIM group were fitted with -25D lenses for LIM modeling; the mice in the NC group were not fitted with defocus lenses. After 4 weeks of modeling, the blood of the two groups of mice was collected by submandibular blood collection. After extracting the plasma, the content of dsDNA in the plasma was detected by fluorescence quantitative detection method.

[0087] Experimental results: As Figure 4 shown, the concentration of extracellular trap net marker dsDNA in the plasma of the LIM modeling group of mice was significantly higher than that of the control NC group without LIM modeling. The above results confirm that the release level of extracellular trap nets in the plasma of experimentally myopic mice induced by LIM increases compared with that of the control group of mice. Therefore, there is a close relationship between the release of plasma extracellular trap nets and myopia. Plasma extracellular trap nets can be used as biomarkers for the diagnosis or auxiliary diagnosis of myopia, and the degree of myopia can be evaluated by detecting the release level of plasma extracellular trap nets.

[0088] Conclusion 5: The content of extracellular trap net marker dsDNA in the plasma of myopic people increases

[0089] Purpose of the experiment: To verify the relationship between the release of extracellular trap nets in the plasma of myopic people and myopia.

[0090] Test method: From February 2024 to October 2024, 30 myopic patients were enrolled in the outpatient ophthalmology department of Zhongshan Hospital, Fudan University for this study. The patients were aged 21 - 29 years. Exclusion criteria were: having a history of systemic diseases, including diabetes, hypertension, inflammatory and infectious diseases, or having other diseases other than myopia. All subjects underwent a comprehensive ophthalmic evaluation. The patients were divided into two groups according to the axial length (AL) of the eyes: the AL ≤ 24 mm group and the AL > 26 mm group. There were no statistically significant differences in demographic factors between the groups (both P > 0.05), but there were statistically significant differences in axial length and equivalent spherical diameter (both P < 0.05).

[0091] This study was approved by the Ethics Committee for Human Subjects of Zhongshan Hospital, Fudan University (approval number: B2024 - 392R). The research protocol followed the guidelines set by the Declaration of Helsinki and written informed consent was obtained from all subjects.

[0092] The baseline characteristics of the included patients are as follows in the table:

[0093] Table 1

[0094]

[0095] OD: right eye; OS: left eye. There were no statistically significant differences in age and gender between the two groups of patients (P > 0.05, Fisher's exact test). There were statistically significant differences in AL and SE between the two groups (P < 0.05, unpaired T - test).

[0096] Test results: The test results are as Figure 5 shown. The concentration of the extracellular trap - net biomarker dsDNA in the plasma of patients in the AL > 26 mm group was significantly higher than that in the AL ≤ 24 mm group. Clinically, an axial length AL ≤ 24 mm is generally considered emmetropia; AL > 26 mm is considered high myopia. It shows that the degree of myopia is positively correlated with the level of extracellular trap - net release in plasma. The higher the degree of myopia, the higher the level of extracellular trap - net release in plasma. Therefore, the degree of myopia can be evaluated by detecting the level of extracellular trap - net release in plasma, that is, extracellular trap - net in plasma can be used as a biomarker for evaluating the degree of myopia.

[0097] Conclusion 6: The content of the extracellular trap - net biomarker dsDNA in the aqueous humor of myopic people increases

[0098] Experimental method: From May 2024 to October 2024, 14 patients (25 eyes) who underwent implantable collamer lens (ICL) surgery in Zhongshan Hospital Affiliated to Fudan University were included in this study. The patients were aged 18 - 45 years. The aqueous humor samples obtained during the surgery were used in this study. Exclusion criteria were as follows: participants had a history of systemic diseases, including diabetes, hypertension, inflammation, and infectious diseases; participants had contraindications for ICL surgery. In addition, participants with glaucoma, retinopathy, other ophthalmic diseases, or a history of intraocular or corneal surgery were also excluded. All subjects underwent a comprehensive ophthalmic evaluation. The patients were divided into the AL ≤ 27 mm group and the AL > 27 mm group according to the axial length of the eye. There were no statistically significant differences in demographic factors between the groups (both P > 0.05), but there were statistically significant differences in AL and SE (both P < 0.05).

[0099] This study was approved by the Ethics Committee of Human Subjects of Zhongshan Hospital, Fudan University (Approval No.: B2024 - 392R). The research protocol followed the guidelines established in the Declaration of Helsinki and written informed consent was obtained from all subjects.

[0100] The baseline characteristics of the included patients are as follows in the table:

[0101] Table 2

[0102]

[0103] There were no statistical differences between the two groups in terms of age, gender, operative eye, etc. (P > 0.05, Fisher's exact test). There were statistically significant differences in AL and SE between the two groups (P < 0.05, unpaired T - test).

[0104] Experimental results: As Figure 6 shown, the concentration of extracellular trap - like web marker dsDNA in the aqueous humor of patients in the AL > 27 mm group was significantly higher than that in the AL ≤ 27 mm group. Clinically, an AL > 26 mm is generally considered high myopia. For highly myopic eyes with AL > 27 mm, the larger the AL, the higher the release level of extracellular trap - like web in the aqueous humor, indicating that the degree of myopia is positively correlated with the release level of extracellular trap - like web in the aqueous humor. Therefore, the degree of myopia can be evaluated by detecting the release level of extracellular trap - like web in the aqueous humor, as well as monitoring the treatment effect of myopia.

[0105] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of extracellular traps as biomarkers in at least one of the following: a1) Use in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of myopia; a2) Use in the preparation and / or screening of products for assessing the degree of myopia; a3) Application in the preparation and / or screening of products for evaluating or assisting in the evaluation of the therapeutic effect of myopia.

2. Use of a substance for detecting extracellular traps in at least one of the following: b1) Use in the preparation and / or screening of products for diagnosing or assisting in the diagnosis of myopia; b2) Use in the preparation and / or screening of products for assessing the degree of myopia; b3) Application in the preparation and / or screening of products for evaluating or assisting in the evaluation of the therapeutic effect of myopia; The products include reagents, test kits, chips, test strips, membrane strips or detection platforms.

3. The use according to claim 2, characterized in that: The substance for detecting the extracellular trap is selected from the key protein citH3 and / or marker dsDNA of the extracellular trap in the detection sample. The substance for detecting citH3 and / or marker dsDNA includes any reagent required for detecting the expression level of citH3 protein or the expression level of marker dsDNA gene by RT-PCR method, RT-qPCR method, biochip detection method, Southern blotting method, in situ hybridization method, enzyme-linked immunosorbent assay, immunoblotting method, immunohistochemistry method, immunofluorescence method, fluorescent dye method, ultraviolet photometry, spectrophotometry, and microtiter method. The detection sample includes plasma, aqueous humor or sclera.

4. The use according to claim 3, characterized in that: The substance for detecting citH3 is a reagent for detecting the expression of citH3 protein by immunofluorescence staining; or, the substance for detecting citH3 is a reagent for detecting the expression of citH3 protein by immunoblotting; or, the marker dsDNA is a reagent for detecting the expression level of the marker dsDNA gene by fluorescent dye method.

5. Use of the extracellular trap as a target in at least one of the following: c1) Use in the preparation and / or screening of drugs for preventing myopia; c2) Application in the preparation and / or screening of drugs for treating or improving myopia; c3) Use in the preparation and / or screening of drugs for slowing down, delaying or inhibiting the progression of myopia.

6. Use of an extracellular trap release inhibitor in at least one of the following: d1) Use in the preparation and / or screening of drugs for preventing myopia; d2) Application in the preparation and / or screening of drugs for treating or improving myopia; d3) Use in the preparation and / or screening of drugs for slowing down, delaying or inhibiting the progression of myopia; The extracellular trap release inhibitor includes a substance that inhibits the release of extracellular traps or reduces the activity of extracellular traps, or a substance that inhibits the expression of key genes of extracellular traps. The substance includes one or more of nucleic acid molecules, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentiviruses or adeno-associated viruses.

7. The use according to claim 6, characterized in that: The extracellular trap release inhibitor is a gene editing vector that inhibits the expression of the key gene Padi4 of the scleral extracellular trap.

8. The use according to claim 6, characterized in that: The extracellular trap release inhibitor is lactoferrin.

9. The use according to claim 6, characterized in that: The extracellular trap release inhibitor is a drug that has the effect of inhibiting the release of extracellular traps, and the drugs include alpha deoxyribonuclease inhaler, danirexin, budesonide, cypermethrin, PHP-303, lonodelestat, CHF6333, alvelestat, disulfiram, and NucleoCapture equipment.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an effective amount of the extracellular trap release inhibitor according to any one of claims 6 to 9.

11. The pharmaceutical composition according to claim 10, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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