Application of Reelin protein in the preparation of drugs for treating retinal damage

Abstract

The present invention provides a use of a Reelin protein in the preparation of a drug for treating retinal damage, relating to the technical field of protein drugs. The Reelin protein is used in the preparation of a drug for treating retinal damage to regulate the process of retinal ischemia-reperfusion injury by improving retinal tissue morphological abnormalities, retinal optic nerve cell death, retinal dysfunction, and retinal cell apoptosis after retinal damage. The use of Reelin protein in the preparation of a drug for treating retinal damage provides a basis for a new drug for treating retinal ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the technical field of protein drugs, and in particular to the application of Reelin protein in the preparation of drugs for treating retinal damage. Background Art

[0002] Ischemia-reperfusion (IR) injury is a pathological condition associated with high morbidity, disability, and mortality. Besides causing serious consequences in vital organs such as the brain, heart, and kidneys, it is also implicated in a variety of retinal diseases. In the diagnosis and treatment of retinal IR injury, the primary manifestation is the further exacerbation of tissue damage after restoration of retinal blood flow. In the absence of effective treatment, retinal IR injury leads to gradual neuronal loss, altered retinal morphology and function, and ultimately, vision loss.

[0003] Retinal IR damage reduces the oxygen and nutrient supply to retinal cells, ultimately triggering oxidative stress, inflammation, immune responses, and calcium overload, leading to neuronal cell death. Neuronal death can occur through apoptosis, ferroptosis, and autophagy. Apoptosis is considered one of the primary mechanisms of retinal damage after IR, primarily through the mitochondrial-mediated intrinsic apoptosis pathway. Therefore, inhibiting the expression of apoptotic proteins involved in this pathway could help prevent and treat retinal IR damage.

[0004] In humans and mice, the Reln gene encodes an extracellular macromolecular matrix glycoprotein, Reelin, which plays an important role in the mammalian central nervous system. However, it is not clear how Reelin plays a role in retinal IR injury and whether it can be used to treat retinal IR injury.

[0005] Therefore, the present invention proposes the use of an exogenous recombinant protein Reelin in the preparation of a drug for treating retinal ischemia-reperfusion injury to solve the above problems. Summary of the Invention

[0006] The present invention provides an application of Reelin protein in the preparation of a drug for treating retinal damage, so as to solve the problems of how Reelin protein plays a role in retinal IR damage and whether it can be used to treat retinal IR damage.

[0007] In a first aspect of the embodiments of the present invention, a use of Reelin protein in the preparation of a drug for treating retinal damage is proposed.

[0008] In an optional embodiment of the present invention, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving abnormal retinal tissue morphology after retinal injury.

[0009] In an optional embodiment of the present invention, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving retinal optic nerve cell death after retinal injury.

[0010] In an optional embodiment of the present invention, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving retinal dysfunction after retinal injury.

[0011] In an optional embodiment of the present invention, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving retinal cell apoptosis after retinal injury.

[0012] In an optional embodiment of the present invention, the Reelin protein regulates the retinal ischemia-reperfusion injury process through the Dab1-Pi3k / Akt pathway.

[0013] In an optional embodiment of the present invention, the Reelin protein activates the Pi3k / Akt pathway to inhibit retinal cell apoptosis after injury by causing phosphorylation of the intracellular Dab1 protein.

[0014] In a second aspect of an embodiment of the present invention, a drug for treating retinal damage is provided, wherein the active ingredient of the drug is Reelin protein.

[0015] In an optional embodiment of the present invention, the drug further includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier at least includes physiological saline or PBS.

[0016] In an optional embodiment of the present invention, the dosage range of the drug is 300 ng / ul to 500 ng / ul.

[0017] The present invention has the following advantages: An embodiment of the present invention provides an application of Reelin protein in the preparation of a drug for treating retinal damage. The Reelin protein is applied to the preparation of a drug for treating retinal damage, thereby regulating the retinal ischemia-reperfusion injury process by improving abnormal retinal tissue morphology, retinal optic nerve cell death, retinal dysfunction, and retinal cell apoptosis after retinal damage. The Reelin protein activates the Pi3k / Akt pathway to inhibit retinal cell apoptosis after damage by causing phosphorylation of the intracellular Dab1 protein. The use of Reelin protein in the preparation of a drug for treating retinal damage provides a basis for a new drug for treating retinal ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0019] Figure 1 Schematic diagram of a mouse retinal IR injury model provided by an embodiment of the present invention;

[0020] Figure 2 This is a rendering of a mouse model with localized retinal Reln gene knockdown provided by an embodiment of the present invention;

[0021] Figure 3 This is a graph showing the expression results of the Reln gene in a retinal IR injury mouse model provided in an embodiment of the present invention;

[0022] Figure 4 This is a diagram showing the results of retinal tissue damage repair regulated by the Reln gene provided by an embodiment of the present invention;

[0023] Figure 5 This is a result diagram of a retinal IR injury-induced cell apoptosis promoted by the Reln gene through the Dab1-Pi3k / Akt pathway provided by an embodiment of the present invention;

[0024] Figure 6 This is a diagram showing the results of Reelin protein promoting the repair of retinal tissue damage provided by an embodiment of the present invention;

[0025] Figure 7 This is a diagram showing the results of Reelin protein inhibiting cell apoptosis through the Dab1-Pi3k / Akt pathway after retinal IR damage, as provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0027] Ischemia-reperfusion (IR) injury is a pathological process with high morbidity, disability, and mortality. Its essence is tissue damage caused by inflammatory factors, inflammatory cells, and oxygen free radicals, in addition to blood flow reperfusion in ischemic tissue. IR injury in vital organs such as the brain, heart, and kidneys can lead to serious consequences, including stroke, myocardial infarction, and acute kidney injury. IR injury is also implicated in a variety of retinal diseases, such as retinal vascular occlusion, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Retinal IR injury is a common pathological process in clinical ophthalmology, characterized by further aggravation of tissue damage after restoration of retinal blood flow. Due to the lack of effective treatment, retinal IR injury leads to gradual neuronal loss, altered retinal morphology and function, and ultimately vision loss. Therefore, the prevention and treatment of IR injury has long been a research hotspot in ophthalmology. Exploring the mechanisms of retinal IR injury and developing effective prevention and treatment methods will provide new insights into its prevention and treatment.

[0028] Retinal IR damage leads to reduced oxygen and nutrient supply to retinal cells, ultimately causing oxidative stress, inflammation, immune responses, and calcium overload in the tissue, leading to neuronal cell death. Neuronal death can be triggered by various mechanisms, including apoptosis, ferroptosis, and autophagy. Apoptosis is considered one of the major mechanisms of retinal damage after IR injury, primarily through the mitochondrial-mediated intrinsic apoptosis pathway. Therefore, inhibiting the expression of apoptotic proteins involved in this pathway may help prevent and treat retinal IR injury.

[0029] In humans and mice, the Reln gene encodes Reelin, an extracellular macromolecular matrix glycoprotein. This glycoprotein plays an important role in the mammalian central nervous system. During embryonic development, it regulates radial neuronal migration and the maturation of dendrites and spines; in adulthood, it maintains synaptic plasticity, regulates neural signaling, and modulates synaptogenesis. Recent studies have shown that Reelin signaling in endothelial cells and neurons facilitates communication between blood vessels, glial cells, and neurons, and that this integration is crucial for the integrity of the vascular barrier in the mature retina. As an extension of the central nervous system, the role of Reelin in the visual system has primarily been reported in the retina. In the adult retina, Reelin expression is primarily localized in RGCs (retinal ganglion cells) and RBCs (red blood cell life span). Studies have shown that the Reeln signaling pathway is involved in the maturation of excitatory and inhibitory synaptic mechanisms, which influence the development and fine-tuning of retinal neural networks during postnatal development. Related studies have shown that Reelin protein in the central nervous system can promote neuronal cell survival by inhibiting apoptosis and play a protective role in cerebral ischemia-reperfusion injury. However, how the Reeln gene and Reelin protein play a role in retinal IR injury in mice and the related mechanism of action remain unknown.

[0030] Therefore, the present invention proposes the use of an exogenous recombinant protein Reelin in the preparation of a drug for treating retinal ischemia-reperfusion injury to solve the above problems.

[0031] In a first aspect of an embodiment of the present invention, a method for preparing a drug for treating retinal damage is proposed. Reelin protein is a highly conserved extracellular matrix glycoprotein encoded by the Reln gene and is an important regulator of neuronal migration and synaptogenesis. The Reln signaling pathway regulates the targeted projection of retinal ganglion cell (RGC) dendrites to central neurons. Exogenous recombinant protein reelin is an extracellular matrix protein that can be purchased commercially.

[0032] Based on the application proposed in the first aspect above, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving the abnormal morphology of retinal tissue after retinal injury, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving the death of retinal optic nerve cells after retinal injury, the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving retinal dysfunction after retinal injury, and the Reelin protein regulates the retinal ischemia-reperfusion injury process by improving retinal cell apoptosis after retinal injury. Among them, ischemia-reperfusion injury is tissue damage caused by ischemia and is the main cause of fatal diseases, such as myocardial infarction and stroke caused by coronary artery sclerosis. In the process of rescue and treatment of ischemic diseases, the main factor causing damage to tissues is not ischemia itself, but after the blood supply is restored, the excessive free radicals attack the cells in the tissue that has regained blood supply. This damage is called "tissue ischemia-reperfusion injury."

[0033] Based on the application proposed in the first aspect above, the Reelin protein regulates the retinal ischemia-reperfusion injury process through the Dab1-Pi3k / Akt pathway. Specifically, the Reelin protein causes phosphorylation of the intracellular Dab1 protein to activate the Pi3k / Akt pathway to inhibit retinal cell apoptosis after injury.

[0034] In a second aspect, an embodiment of the present invention proposes a drug for treating retinal damage, wherein the active ingredient of the drug is Reelin protein, and the drug also includes a pharmaceutically acceptable carrier such as physiological saline or PBS. The above-mentioned drug can be administered by vitreous cavity injection, and the dosage range of the administration is 300ng / ul to 500ng / ul, which is obtained through experiments.

[0035] The embodiment of the present invention provides an application of Reelin protein in the preparation of a drug for treating retinal damage. Reelin protein is applied to the preparation of a drug for treating retinal damage, and regulates the retinal ischemia-reperfusion injury process by improving abnormal retinal tissue morphology, retinal optic nerve cell death, retinal dysfunction and retinal cell apoptosis after retinal damage. The Reelin protein activates the Pi3k / Akt pathway to inhibit retinal cell apoptosis after damage by causing phosphorylation of the intracellular Dab1 protein. The use of Reelin protein in the preparation of a drug for treating retinal damage can provide a basis for a new drug for treating retinal ischemia-reperfusion injury.

[0036] In the central nervous system, Reelin promotes neuronal survival by inhibiting apoptosis and plays a protective role against cerebral ischemia-reperfusion injury. However, how the Reelin gene plays a role in retinal IR injury in mice and its related mechanisms of action remain unknown. This study first established a mouse model and verified the mechanism of action of the Reeln gene in mouse retinal IR injury.

[0037] Example 1: Construction and grouping of mouse models

[0038] A: Construction of retinal IR injury model

[0039] Mice were selected as the construction object of the retinal IR injury model. The mice used were 6-8 weeks old, healthy C57BL / 6J wild-type male mice weighing 16-22g (purchased from Beijing Sibeifu Experimental Animal Technology Co., Ltd.). A 33-gauge needle containing a balanced salt solution was inserted into the anterior chamber to maintain intraocular pressure (IOP) at 110mmHg for 60 minutes. A sham-operated group as a control was performed without increasing intraocular pressure. After 60 minutes, the IOP was restored to normal by carefully removing the needle. In order to prevent bacterial infection, tobramycin eye ointment (Alcon, USA) was used to treat it. 3d and 7 days after IR injury, mice were over-anesthetized and their eyeballs were removed.

[0040] The specific operation of fluorescent staining of retinal flat mounts is as follows: 7 days after I / R injury, the eyeballs are collected as described above, the posterior eyeball muscles and connective tissues and other eye accessory tissues are removed, and the eyes are fixed in 4% paraformaldehyde fixative for 3 hours. Under a stereomicroscope, a 15° ophthalmic surgical puncture knife is used to make a puncture at the corneoscleral limbus. Micro spring scissors are used to cut along the corneoscleral limbus from the puncture, and the cornea, iris, lens, and vitreous are removed. The eye cup is cut radially and evenly with the optic nerve as the center to divide it into 4 parts. The sclera is placed on a glass slide with the sclera facing up, and the optic nerve and the transition between the sclera and the optic nerve are cut off to completely separate the retina from the uvea. The residual pigment tissue on the sclera, uvea, and retina is removed. The separated retina is rinsed in 0.1% PBST (washing buffer) and dehydrated in a 50% and 100% methanol gradient. Goat serum is blocked at room temperature for 1 hour, and RBPMS Polyclonal After incubation with antibody (15187-1-AP, Proteintech) at 4°C in the dark for 24 h, the retinas were rinsed with 0.1% PBST and incubated with secondary antibody (Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody) (A-11011, Thermo Fisher Scientific) at room temperature in the dark for 1 h. The retinas were mounted on slides and mounted with anti-fluorescence quenching mounting medium containing DAPI (ab104139; Abcam). Images were captured and analyzed using a high-content imaging system (Operetta CLS, PerkinElmer, UK).

[0041] Optical coherence tomography of the retina is performed as follows:

[0042] Mice were immobilized and anesthetized (with an intraperitoneal injection of 15 mL / kg 1% sodium pentobarbital + 20 μl 10% sulfanilamide) and then placed on a lifting platform. Tropicamide compound eye drops were used to dilate the pupils. Oxybuprocaine hydrochloride eye drops were used for ocular surface anesthesia. Carbomer eye drops (Dr. Gerhard Mann, Chem.-Pharm. Fabrik GmbH, Germany) were then applied to the cornea to be measured. An ophthalmic ultramicroscopy system (OPTOPROBE, UK) was used to adjust the light source focus and focus the lens on the retina for optical coherence tomography. Total retinal thickness (from the inner limiting membrane to the retinal pigment epithelium) was then analyzed using software from OptoProbe Research Ltd. (version 2.0). Measurements were taken at four randomly selected points within a range of 200 to 300 μm from the optic disc, and the average was calculated.

[0043] The retinal IR injury model was constructed. Figure 1 , Figure 1A schematic diagram of a mouse retinal IR injury model provided in an embodiment of the present invention is shown. Figure 1 Part A in the middle is a representative image of the anterior chamber of the mouse model before and after IR injury with a 33G needle injection. Figure 1 Part B in the middle is a fundus photograph showing the retina damaged by IR. It can be seen that after IR injury, the retinal blood vessels of the mouse are intermittently supplied with blood, the peripheral retina atrophies, and the choroid is visible. Figure 1 Part C in the middle shows a representative fluorescent staining image of the retinal flat mount before and after IR injury, showing the distribution of RBPMS (RNA binding protein with multiple splicing, a marker that can be used for RGC quantification in various RGC degeneration models) positive RGCs (see orange marks in the figure), and the number of RGCs is significantly reduced. Figure 1 Part D in the middle is an OCT (Optical Coherence Tomography) scan of the retina after IR injury. It can be seen that the retina of the mouse is significantly thinned after IR injury (Ctrl in the figure is the control group).

[0044] B: Construction of a retinal Reln gene knockdown mouse model

[0045] Adeno-associated virus (AAV-2 / 9-shRNA-Reln, HANBIO, China) was used to locally knock down the expression of the Reln gene in the mouse retina. The mice used were healthy C57BL / 6J wild-type male / female mice aged 6-8 weeks and weighing 16-22 g (purchased from Beijing Sibeifu Laboratory Animal Technology Co., Ltd.). Three weeks before the start of the model, adeno-associated virus (AAV) solution loaded with specific targets (2 μL, 1.0×10 12 vg / ml) was injected into the vitreous to establish a mouse model with localized retinal knockdown of the Reln gene. The AAV virus carries enhanced green fluorescent protein (EGFP), and the siRNA targeting the Reln gene is CCAGGATACATGATGCAATTT.

[0046] See Figure 2 , Figure 2 This is a diagram showing the effect of a retinal local Reln gene knockdown mouse model provided by an embodiment of the present invention. Figure 2 Part A in the middle shows the retinal infection effect of the mouse model 3 weeks after AAV was passed through the vitreous cavity. The green part in the figure is the EGFP carried by AAV, and the scale bar is 40 microns. Figure 2Part B shows the expression results of Reln mRNA. NC is the blank control group, AAV-EGFP is the AAV virus-transfected enhanced green fluorescent protein-tagged group, and AAV-Reln is the adeno-associated virus-mediated local knockdown of the Reln genome in the mouse retina. As can be seen, the expression of the Reln gene in the AAV-Reln group is significantly lower than that in the NC and AAV-EGFP groups. Therefore, the adeno-associated virus AAV-2 / 9-shRNA-Reln can locally knock down the Reln gene expression in mice, successfully establishing a mouse model with localized retinal Reln gene knockdown.

[0047] The specific operation of intravitreal injection is as follows:

[0048] After anesthesia, mice were anesthetized and pupils were dilated for 5 minutes with compound tropicamide eye drops (Santen Pharmaceutical Co., Ltd., Japan). A 34G microsyringe (Hamilton, Switzerland) was used to collect an exact 2 μl of exogenous recombinant mouse Reelin protein, PBS, AAV-shReln, and AAV-EGFP. The eyeball was then slowly punctured from the superior temporal quadrant (approximately 1 mm from the corneal scleral margin), with the needle tip directed toward the optic disc to avoid puncturing the lens. As the dilated pupil clearly indicated, the needle tip was positioned within the vitreous cavity. The drug was slowly introduced until all the drug was infused, and the needle tip was withdrawn to complete the injection.

[0049] C: Experimental group

[0050] The experimental mice were divided into two groups: the Reelin protein group and the Reln gene knockdown group. Among them, the mice in the Reelin protein group were randomly injected with 2ul of exogenous recombinant mouse Reelin protein (300ng / μL, R&D Systems, USA) and 2ul of PBS into the vitreous cavity 24h before modeling. The mice were divided into the Reelin group and the PBS group, and the retinal IR injury model was established. After the model was established, the mice were divided into the Reelin-IR group and the PBS-IR group. The mice in the Reln gene knockdown group were randomly injected with 2ul of AAV-shReln and AAV-EGFP into the vitreous cavity 3 weeks before modeling. The mice were divided into the AAV-shReln group and the AAV-EGFP group, and the retinal IR injury model was established. After the model was established, the mice were divided into the AAV-shReln-IR group and the AAV-EGFP-IR group.

[0051] Example 2: Expression of Reln gene after retinal IR injury

[0052] Using Reln-CreERT2 mTmG mice, a transgenic mouse that permanently expresses enhanced green fluorescent protein (EGFP) in Reln-positive cells and their progeny after tamoxifen induction, we investigated Reln gene expression in the retina during homeostasis and after IR injury. Frozen sections of eyeball samples from these mice and confocal microscopy revealed that EGFP signals from Reln-positive cells were expressed in the inner granular layer, inner plexiform layer, and ganglion cell layer of the retina.

[0053] Immunofluorescence staining was performed as follows: Mouse eyeballs were fixed overnight in 4% paraformaldehyde, dehydrated with graded sucrose solutions, embedded in OCT embedding medium, and snap-frozen in liquid nitrogen. Retinal cryosections, 5 μm thick, were cut through the optic nerve plane and permeabilized with 0.3% Triton X-100 (solarbio, China). After blocking with goat serum for 1 hour, the sections were incubated with the primary antibody overnight. The primary antibody was Reelin Polyclonal Antibody (1:500, Thermo Fisher Scientific, USA), followed by incubation with Alexa Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific, MA, USA) for 2 hours at room temperature. Slides were mounted with anti-fading mounting medium containing DAPI (ab104139; abcam, UK). Images were captured using an Olympus VS200 virtual slide scanning microscope (Olympus, Japan).

[0054] See Figure 3 , Figure 3 This is a graph showing the expression of the Reln gene in a mouse model of retinal IR injury provided by an embodiment of the present invention (Ctrl and Control are both control groups). Figure 3 Part A in the middle shows the results of Reln-CreERT2 mTmG mice expressing Reln-positive cells and their progeny at different time points after retinal IR injury. Figure 3 Part B in the middle is a representative image of Reelin immunofluorescence staining at different time points after IR injury in the retina of wild-type mice. Figure 3 Parts C and D in the middle are the statistical results of the percentage of Reln-positive cells and Reelin immunofluorescence intensity in the retina. Figure 3Panel E shows the expression of Reln mRNA after retinal IR injury. As shown, Reln expression initially decreases and then increases after retinal IR injury, but remains below the level in the normal control group. Scale bar, 40 μm. Data are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0055] According to the fluorescence images of frozen sections of Reln-CreERT2 mTmG mice at different time points after IR injury, the EGFP fluorescence signal in the 24h group after IR injury began to gradually weaken, and this trend reached the lowest point 7 days after IR injury. After that, the EGFP fluorescence signal began to gradually increase until 28 days after injury, but it was still lower than the control group level (see Figure 3 Part A and Part C, P < 0.05). The results of Reelin immunofluorescence staining in wild-type mice were completely consistent with the above results (see Figure 3 In addition, the inventors used qRT-PCR to determine the mRNA expression of the Reln gene in retinal IR injury. The results showed that the Reln gene level decreased significantly 24 hours and 3 days after IR injury, and the retinal Reln gene level gradually increased 7 days, 14 days and 28 days after IR injury, but was still lower than the control group level (see Figure 3 The above results indicate that the level of Reln gene decreases rapidly in the short term after retinal IR injury, suggesting that Reln gene may participate in the damage regulation process and play an important role in IR injury.

[0056] Example 3: Reln gene regulates retinal tissue damage and repair

[0057] In order to verify the regulatory role of the Reln gene in the IR injury process, it is first necessary to construct Reln gene knockdown mice. However, due to the full mutation of the Reln gene in mice (reeler mice), they usually die 1-2 weeks after birth, and their body tissue morphology is changed, including cerebellar hypoplasia, structural disorder of the cerebral cortex and hippocampal neuron layer. Therefore, this example chooses to successfully establish a retinal Reln gene knockdown mouse model by intravitreal injection of AAV 3 weeks before modeling. Seven days after IR injury modeling, the retina was stained with HE, and the thickness of the inner retina was measured and counted.

[0058] The specific procedure was as follows: 7 days after I / R injury, eyeballs were collected as described above and immediately immersed in 4% PFA (polyfluoroalkoxy, soluble polytetrafluoroethylene) overnight and embedded in paraffin. Five-micrometer-thick sections cut through the optic nerve were stained with hematoxylin and eosin (H&E) to observe structural changes in the retina. To quantify retinal damage, the thickness of the inner retinal layer (extending from the inner limiting membrane to the inner edge of the outer plexiform layer) was measured at four randomly selected points in each eye within a range of 200 to 300 micrometers from the optic disc, and the data were averaged. Images were captured using an optical microscope (Olympus).

[0059] The results can be found in Figure 4 , Figure 4 This is a diagram showing the results of Reln gene regulation of retinal tissue damage repair provided in this example. Figure 4 Part A in the middle is the result of HE staining retinal tissue pathology. Figure 4 Part B in the middle is a representative OCT image of retinal tissue changes 7 days after IR injury. Figure 4 Part C in the middle is a representative fluorescence staining result of the retinal flat mount 7 days after IR injury. Figure 4 Part D in the middle is a representative oscillatory potential waveform of visual function assessed by FERG 7 days after IR injury. Figure 4 Part E in the middle is a quantitative image of the inner retinal layer thickness (n=6). Figure 4 Part F in the middle is the quantitative result of total retinal thickness (n=6). Figure 4 Part G in the middle shows the number of RBPMS-positive RGCs (n=4). Figure 4 Part H in the middle shows the quantitative results of a-wave and b-wave amplitudes (n=6), with scale bars of 20 μm, 100 μm, or 1 mm, respectively. Data are expressed as mean ± standard deviation, *P<0.05, ***P<0.001.

[0060] It can be seen that the thickness of the inner layer of the retina of mice was significantly thinner after injury, and the thickness of the inner layer of the retina of Reln gene knockdown mice was further reduced after injury compared with the negative control group (see Figure 4 Similarly, the inventors used OCT to evaluate the dynamic in vivo retinal tissue, and the results showed that the thickness of the retinal layer of Reln knockdown mice was further reduced after injury (see Figure 4 The results of immunofluorescence staining of mouse retina showed that the RGC density in the AAV-shReln-IR group was significantly lower than that in the AAV-EGFP-IR group, and the RGC count in the whole retina was also significantly reduced (see Figure 4Parts C and G, P < 0.05). The retinal thickness and RGC number of the undamaged AAV-shReln group were slightly lower than those of the AAV-EGFP group, but there was no statistical difference. The inventors then evaluated visual function using flash electroretinogram (FERG). The results showed that compared with the AAV-EGFP-IR group, the retinal a-wave or b-wave amplitude of the AAV-shReln-IR group was significantly reduced. In addition, the retinal a-wave or b-wave amplitude of the undamaged AAV-shReln group was also lower than that of the AAV-EGFP group, and there was a statistical difference (see Figure 4 (D and H, P < 0.05). AAV-shReln-mediated local knockdown of the Reln gene exacerbated the symptoms of IR-injured mice, primarily manifested by thinning of the retina, decreased number of RGCs, and retinal dysfunction. These findings suggest that the Reln gene regulates retinal homeostasis and injury repair, and knockdown of the retinal Reln gene further exacerbated retinal IR damage.

[0061] Example 4: Reln gene knockdown promotes cell apoptosis through the Dab1-Pi3k / Akt pathway

[0062] As described in Examples 2 and 3, Reln gene knockdown promoted the death of mouse retinal RGCs after IR injury. To elucidate the mechanism of action of Reln gene in mouse retinal IR injury, the effect of local knockdown of Reln gene on cell apoptosis was evaluated by TUNEL staining.

[0063] The specific procedures were as follows: 3 days after I / R injury, eyeballs were harvested as described above, and retinal cryosections were stained using a TUNEL kit (C1090, Beyotime, Jiangsu, China). Cells were fixed with 4% PFA for 30 minutes. After washing with PBS for 20 minutes, the membranes were permeabilized with 0.5% Triton X-100 at room temperature for 5 minutes. TUNEL detection solution (TDT enzyme + fluorescent labeling solution) was added to the samples and incubated at 37°C in the dark for 60 minutes. Slides were mounted with anti-fluorescence quenching mounting medium containing DAPI. Images were captured using an Olympus VS200 virtual slide scanning microscope (Olympus).

[0064] The results refer to Figure 5 , Figure 5 This is a graph showing the results of the Reln gene promoting post-IR cell apoptosis through the Dab1-Pi3k / Akt pathway after retinal IR injury provided by an embodiment of the present invention. Figure 5 Part A in the middle is a representative TUNEL staining image of the frozen section of the retina of AAV-shReln mice 3 days after IR injury. Figure 5Part B in the middle is the quantitative result of TUNEL-positive cells (n=6). Figure 5 Part C in the middle is a representative blot showing the effects of local knockdown of Reln on retinal Dab1, p-Dab1, Pi3k, Akt, p-Akt, Bcl-2, Bax, and cleaved caspase-3 3 days after IR injury. Figure 5 Middle D shows the quantitative analysis of the effects of local knockdown of Reln on retinal protein levels of Dab1, p-Dab1, Pi3k, Akt, p-Akt, Bcl-2, Bax, and cleaved caspase-3 3 days after IR injury. Scale bar, 40 μm. Data are expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

[0065] In the frozen sections of the retina of the non-IR-injured group, TUNEL-positive cells were basically not observed. However, 7 days after IR-injured, TUNEL-positive cells increased significantly in the retina of mice, and the number of TUNEL-positive cells increased significantly after local knockdown of the Reln gene (see Figure 5 (Parts A and B, P < 0.05), indicating that Reln gene knockdown promotes cell apoptosis after IR injury. To further explore its mechanism of action, Western blot experiments were performed on the samples based on the results of single-cell transcriptome analysis.

[0066] The specific procedure was as follows: 3 days after I / R injury, eyeballs were harvested as described above, and retinas were isolated under a surgical microscope. Retinal proteins were thoroughly lysed using RIPA (Radio Immunoprecipitation Assay Lysis Buffer) containing protease inhibitors, and total retinal protein was extracted from the lysed samples. Protein concentration was determined using a BCA protein assay kit (Promega, Madison, WI, USA). Protein samples were separated by electrophoresis on a 10% polyacrylamide gel, and proteins were transferred to a nitrocellulose membrane using a semi-dry blotting system. After blocking with 5% skim milk for 0.5 h at room temperature, the membranes were incubated with the following primary antibodies: anti-phospho-Dab1 (ab78200; Abcam), anti-Dab1 (ab111684; Abcam), anti-Pi3k (ab302958; Abcam), anti-phospho-Akt (4060S; Cell Signaling Technology, Massachusetts, USA), anti-pan-Akt (ab8805; Abcam), anti-Bcl-2 (ab182858; Abcam), anti-Bax (ab32503; Abcam), anti-cleaved caspase-3 (9661S; Cell Signaling Technology), and anti-β-actin (66009-1, Proteintech, Rosemont, IL, USA) overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 2 h at room temperature. Proteomic analysis was performed using ChemiDoc. TM Protein bands were photographed using the MP Imaging System (Bio-Rad) and analyzed using ImageJ software (https: / / imagej.nih.gov / ij / ).

[0067] The results showed that there was no significant change in the expression level of AKT protein between the AAV-shReln-IR group and the AAV-EGFP-IR group. However, the expression of phosphorylation-related proteins p-Akt and Pi3k in the AAV-shReln-IR group was significantly lower than that in the AAV-EGFP-IR group. In addition, the expression of apoptosis-related protein Caspase-3 and the ratio of Bax / Bcl2 were significantly higher in the AAV-EGFP-IR group (refer to Figure 5(C and D, P < 0.05). Furthermore, there was no significant difference in Dab1 protein expression between the AAV-shReln-IR and AAV-EGFP-IR groups, but p-Dab1 protein expression was significantly lower in the AAV-shReln-IR group than in the AAV-EGFP-IR group. These results suggest that knockdown of the Reln gene reduces Dab1 phosphorylation after retinal IR injury and inhibits activation of the Pi3k-Akt pathway, thereby promoting retinal cell apoptosis after injury.

[0068] From the results of Examples 2 to 4, it can be seen that after local knockdown of the Reln gene, the retinal thickness of mice becomes thinner, the number of RGC cells decreases, and retinal dysfunction occurs, leading to further aggravation of retinal IR damage. In addition, knockdown of the Reln gene leads to reduced phosphorylation of the Dab1 protein after retinal IR damage, and can inhibit the activation of the Pi3k-Akt pathway, thereby promoting retinal cell apoptosis after damage. In order to further confirm the potential therapeutic role of Reelin protein in IR damage, Reelin protein was injected into the IR-injured mouse model to explore its damage and repair effect on retinal tissue.

[0069] Example 5: Reelin protein promotes retinal tissue damage repair

[0070] In order to determine the potential therapeutic effect of Reelin protein in IR injury, exogenous recombinant Reelin protein was administered into the vitreous cavity 24 h before modeling, and PBS was injected into the vitreous cavity as a control group. The specific operation is referred to the operation in Example 1 and will not be repeated here. Then, the retina of mice 7 days after IR injury was used and HE staining was performed to measure and count the thickness of the inner retinal layer. The results are referred to Figure 6 , Figure 6 This is a graph showing the results of Reelin protein promoting retinal tissue damage repair provided by an embodiment of the present invention. Figure 6 Part A in the middle is the result of HE staining retinal tissue pathology examination. Figure 6 Part B is a representative OCT image of retinal tissue showing the changes 7 days after IR injury. Figure 6 Part C in the middle is the distribution of RBPMS-positive RGCs (orange) shown by epifluorescence staining of retinal flat mounts 7 days after IR injury. Figure 6 Part D in the middle is a representative oscillatory potential waveform result of visual function assessed by FERG 7 days after IR injury. Figure 5 Part E in the middle is the quantitative results of retinal thickness (n=6). Figure 6 Part F in the middle is the quantitative result of total retinal thickness (n=6). Figure 6 Part G in the middle is the number of RBPMS-positive RGCs (n=4). Figure 6The middle H part shows the quantitative results of a-wave and b-wave amplitude (n=6). Scale bars are 20 μm, 100 μm, or 1 mm. Data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001.

[0071] It can be seen that the thickness of the inner retina of mice in the IR injury group was significantly lower than that of the uninjured group. The thickness of the inner retina of mice was significantly reduced 7 days after IR injury. Moreover, this IR-induced thinning of the inner retina was significantly improved in mice given exogenous reelin protein (see Figure 6 Part A and Part E, P < 0.05). The inventors then used OCT to perform a dynamic assessment of retinal tissue in vivo, and the results showed that Reelin protein also has a protective effect on the thickness of the entire retina after injury (see Figure 6 Parts B and F in the middle, P < 0.05). Immunofluorescence staining of mouse retinas revealed that the number of RGCs in the mouse retina was significantly reduced 7 days after IR injury compared with the uninjured group. Reelin protein supplementation has a significant protective effect on RGCs after injury, as evidenced by a significant increase in the RGC count in the Reelin-IR group compared with the PBS-IR group (see Figure 6 In addition, visual function was assessed using FERG (flash electroretinogram, a set of potential changes recorded at the corneal end when retinal neurons are stimulated by light. Its waveform is the result of the joint action of multiple retinal neurons). The results showed that the amplitude of the retinal a-wave and b-wave in mice was significantly decreased after IR injury, while the amplitude of the retinal a-wave and b-wave in mice in the Reelin-IR group was significantly increased compared with the PBS-IR group (see Figure 6 (D and H, P < 0.05). Furthermore, the b-wave amplitude in the Reelin group without IR injury was slightly increased compared to the PBS group, but the difference was not statistically significant. In summary, appropriate supplementation of Reelin protein can promote retinal tissue repair, as evidenced by mitigating retinal thinning, RGC reduction, and retinal dysfunction after IR injury. Therefore, Reelin protein may serve as a new target for injury repair intervention in the treatment of retinal diseases.

[0072] Example 6: Reelin protein inhibits cell apoptosis through the Dab1-Pi3k / Akt pathway

[0073] As described in Example 5, Reelin protein inhibited the death of mouse retinal RGCs after IR injury. To clarify the neuroprotective mechanism induced by Reelin protein, the effect of Reelin protein on cell apoptosis was evaluated by TUNEL staining. The results are shown in Figure 7 , Figure 7 This is a graph showing the results of Reelin protein inhibiting cell apoptosis through the Dab1-Pi3k / Akt pathway after retinal IR damage provided by an embodiment of the present invention, wherein: Figure 7 Part A in the middle is a representative TUNEL staining image of retinal frozen sections 3 days after IR injury. Figure 7 Part B is the quantification of TUNEL-positive cells (n=6). Figure 7 Part C in the middle is a representative blot of the effects of Reelin protein treatment on retinal Dab1, p-Dab1, Pi3k, Akt, p-Akt, Bcl-2, Bax, and cleaved caspase-3 3 days after IR injury. Figure 7 Part D in the middle shows the quantitative analysis of the effects of Reelin protein treatment on the levels of Dab1, p-Dab1, Pi3k, Akt, p-Akt, Bcl-2, Bax, and cleaved caspase-3 proteins in the retina 3 days after IR injury. The scale bar is 40 μm. The data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0074] In the frozen sections of the retina of the uninjured group, almost no TUNEL-positive cells were observed. However, 7 days after IR injury, the number of TUNEL-positive cells in the retina of mice increased significantly, while the number of TUNEL-positive cells in the Reelin-IR group decreased significantly (refer to Figure 7 Part A and Part B, P < 0.05), indicating that Reelin protein can effectively inhibit retinal cell apoptosis after injury. To further explore the anti-apoptotic mechanism of Reelin protein, Western Blot experiments were performed on the samples based on the results of single-cell transcriptome analysis. The results showed that there was no significant change in the expression level of AKT protein between the Reelin-IR group and the PBS-IR group, but the expression of phosphorylation-related proteins p-Akt and Pi3K in the Reelin-IR group was significantly higher than that in the PBS-IR group, while the expression of apoptosis-related protein Caspase-3 and the ratio of BAX / BCL2 were significantly lower than those in the PBS-IR group (see Figure 7 (C and D, P < 0.05). Furthermore, the expression levels of Dab1, a protein involved in the classical Reln signaling pathway, remained unchanged between the Reelin-IR and PBS-IR groups. However, the expression of the phosphorylation-related protein p-Dab1 was significantly higher in the Reelin-IR group than in the PBS-IR group. This suggests that supplementation of Reelin protein after retinal IR injury can induce the phosphorylation of Dab1 and exert its biological effects, potentially inhibiting retinal cell apoptosis by activating the Pi3k-Akt pathway.

[0075] The embodiment of the present invention provides an application of Reelin protein in the preparation of a drug for treating retinal damage. Reelin protein is applied to the preparation of a drug for treating retinal damage, and the process of retinal ischemia-reperfusion injury is regulated by improving abnormal morphology of retinal tissue after retinal damage, death of retinal optic nerve cells after retinal damage, retinal dysfunction after retinal damage, and retinal cell apoptosis after retinal damage. The Reelin protein activates the Pi3k-Akt pathway to inhibit apoptosis of retinal cells after damage by causing phosphorylation of Dab1 protein. The use of Reelin protein in the preparation of a drug for treating retinal damage can provide a basis for a new drug for treating retinal ischemia-reperfusion injury.

[0076] The above is a detailed introduction to the use of a Reelin protein provided by the present invention in the preparation of a drug for treating retinal damage. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core concept. At the same time, for those skilled in the art, according to the concept of the present invention, there may be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A use of Reelin protein in the preparation of a drug for treating retinal ischemia-reperfusion injury, wherein the drug further comprises a pharmaceutically acceptable carrier and the dosage range of the drug is 300 ng / ul to 500 ng / ul.

2. The use according to claim 1, characterized in that The Reelin protein regulates the retinal ischemia-reperfusion injury process by improving the abnormal morphology of retinal tissue after retinal injury.

3. The use according to claim 1, characterized in that The Reelin protein regulates the retinal ischemia-reperfusion injury process by improving the death of retinal optic nerve cells after retinal injury.

4. The use according to claim 1, characterized in that The Reelin protein regulates the process of retinal ischemia-reperfusion injury by improving retinal dysfunction after retinal injury.

5. The use according to claim 1, characterized in that The Reelin protein regulates the process of retinal ischemia-reperfusion injury by improving retinal cell apoptosis after retinal injury.

6. The use according to claim 1, characterized in that The Reelin protein regulates the retinal ischemia-reperfusion injury process through the Dab1-Pi3k / Akt pathway.

7. The use according to claim 6, characterized in that The Reelin protein inhibits retinal cell apoptosis after injury by activating the Pi3k / Akt pathway through phosphorylation of the intracellular Dab1 protein.

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