Application of Vimentin in preparation of medicine for preventing and treating retinal ischemia reperfusion injury

Abstract

The invention relates to the technical field of development of retinal ischemia reperfusion injury medicines, discloses application of Vimentin in preparation of medicines for preventing and treating retinal ischemia reperfusion injury, and discloses a safer and more effective medicine for treating retinal ischemia reperfusion injury. According to the invention, the nerve protection characteristic and good biocompatibility of Vimentin are utilized, so that the side effect of the medicine is reduced, and the treatment safety is improved. In addition, the Vimentin is proved to have a good effect in the aspects of reducing oxidative stress injury and improving mitochondrial functions, and a new therapeutic target is provided for treating the retinal ischemia reperfusion injury. Meanwhile, the Vimentin disclosed by the invention can be used as a supplement or replacement of an existing treatment method, provides a new treatment choice for patients, and is particularly suitable for patients with poor response to traditional treatment medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the use of Vimentin in the preparation of drugs for preventing and treating retinal ischemia-reperfusion injury or retinal ganglion cell apoptosis caused by it. Background Technology

[0002] Retinal ischemia-reperfusion injury (IR) is a complex pathophysiological event that occurs when the blood supply to the retina is interrupted and then restored, followed by oxidative stress, inflammatory responses, and the death of retinal ganglion cells (RGCs), leading to alterations in the retinal microenvironment. Currently, there are almost no effective treatments to reverse this condition. Clarifying the specific mechanisms of IR damage and developing comprehensive, multi-target therapeutic strategies is urgently needed, and this invention proposes a novel solution for this purpose. Therefore, reducing or delaying RGC apoptosis in IR damage and finding new drugs to prevent and treat IR damage have significant clinical implications. Summary of the Invention

[0003] Vimentin (Vim) (>sp|P20152|VIME_MOUSE Vimentin OS=Musmusculus OX=10090 GN=Vim PE=1 SV=3) is a type III intermediate filament protein encoded by the VIM gene, playing a crucial role in fundamental cellular functions such as cell migration, proliferation, and division. Studies on neurological diseases such as spinal cord injury, stroke, and glioma have revealed that Vimentin is a multifunctional protein that regulates axonal regeneration, myelination, apoptosis, and neuroinflammation, and may be related to neural development and injury repair.

[0004] Intracellular vimentin is a key component of the cytoskeleton. Under certain physiological or pathological conditions, vimentin filaments depolymerize, thereby increasing their solubility. Unlike typical intracellular vimentin filaments, extracellular vimentin exists primarily as oligomers, and studies have confirmed its involvement in inflammatory responses and tissue repair processes. Therefore, when cells face pathological states such as injury, inflammation, or tumors, vimentin not only functions intracellularly but can also be secreted extracellularly or released extracellularly via exosomes to exert its effects.

[0005] Therefore, the main objective of this invention is to propose the application of Vimentin in the preparation of drugs for preventing and treating retinal ischemia-reperfusion injury or its induced RGC apoptosis, aiming to provide a new prevention and treatment strategy to improve the therapeutic effect of retinal ischemia-reperfusion injury and related diseases. This mainly includes the following aspects:

[0006] Application of Vimentin in the preparation of drugs for the prevention and treatment of retinal ischemia-reperfusion injury.

[0007] Application of Vimentin in the preparation of drugs for preventing and treating apoptosis of retinal ganglion cells.

[0008] Application of Vimentin and its derivatives, fragments, or analogs in the preparation of drugs for the prevention and treatment of retinal ischemia-reperfusion injury.

[0009] Application of Vimentin and its derivatives, fragments, or analogs in the preparation of drugs for preventing and treating apoptosis of retinal ganglion cells.

[0010] The drug also includes pharmaceutically acceptable carriers, excipients, or diluents; the dosage forms of the drug include: injections, eye drops, ointments, implants, sustained-release formulations, or nanoformulations.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] 1. Safety and Efficacy: This invention provides a safer and more effective drug for treating retinal ischemia-reperfusion injury. By utilizing the neuroprotective properties and good biocompatibility of Vimentin, the side effects of the drug can be significantly reduced, the safety of treatment can be improved, and patients can have a more reliable treatment option.

[0013] 2. Diversity of Treatment Options: Vimentin of this invention can complement or replace existing treatment methods, providing patients with new treatment options. Especially for patients who do not respond well to traditional drugs, the application of Vimentin is expected to bring better treatment results and broaden treatment pathways.

[0014] 3. Preparation Process and Cost-Effectiveness: The Vimentin preparation process of this invention is relatively mature, with high extraction efficiency and purity, and is easy to implement for large-scale production and application. This not only helps reduce treatment costs but also makes related treatments more affordable for more patients, demonstrating significant economic advantages and social benefits.

[0015] 4. Experimental Validation and Innovation: The experimental results of this invention demonstrate that Vimentin has significant effects in reducing oxidative stress damage and improving mitochondrial function. This provides new ideas and methods for developing novel drugs to treat retinal ischemia-reperfusion injury, and promotes research progress in related fields.

[0016] 5. Novel Therapeutic Target: The experiments in this invention also confirmed that Vimentin can significantly inhibit apoptosis in retinal ganglion cells. This regulatory mechanism helps protect cells from oxidative stress damage while maintaining normal mitochondrial function and alleviating ischemia-reperfusion injury. This provides a new therapeutic target for treating eye diseases, with significant clinical implications and application potential.

[0017] 6. Synergistic effect through multiple mechanisms: Vimentin exerts its effects through multiple mechanisms, including inhibiting apoptosis signaling pathways, enhancing antioxidant capacity, and maintaining mitochondrial membrane potential. This synergistic effect through multiple mechanisms provides a comprehensive therapeutic strategy for preventing and treating retinal ischemia-reperfusion injury, and can more effectively address complex pathological processes.

[0018] 7. Potential for Combination Therapy: Vimentin of this invention can also be used in combination with other drugs to enhance therapeutic effects. Such combination therapy offers greater flexibility and choice in clinical treatment, and is expected to further improve treatment success rates and the quality of patient recovery.

[0019] 8. Wide Applicability in Clinical Practice: Vimentin of this invention is not only applicable to the treatment of retinal ischemia-reperfusion injury and related diseases, but may also have potential therapeutic value for other diseases related to oxidative stress and mitochondrial dysfunction. This provides new ideas and methods for the treatment of more related diseases and has broad clinical application prospects.

[0020] In summary, this invention, through the various properties of Vimentin, provides a novel treatment strategy for preventing and treating retinal ischemia-reperfusion injury. It not only has significant clinical application value but also broad market prospects, offering new options and hope for the treatment of related diseases.

[0021] The present invention will now be described in detail with reference to the accompanying drawings and embodiments, without limiting the scope of protection of the present invention. Attached Figure Description

[0022] Figure 1 : Schematic diagram of IR modeling (in vivo);

[0023] Figure 2 Representative ERG waveforms (A) and quantitative statistics of the amplitudes of the a and b waves of the corresponding ERG in the three groups of mice at a stimulation intensity of 3.0 cd.s / m² in Example 1.

[0024] Figure 3 Example 1: Schematic diagram of the optomotor response detection device (A) and quantitative statistics of the optomotor response index (OMR) of each group of mice (B).

[0025] Figure 4 Example 1: RGC counts (A) and corresponding RGC survival rates (B) of each group of mice in whole retinal patch.

[0026] Figure 5 Representative ERG waveforms (A) and quantitative statistics of the amplitudes of the a and b waves of the corresponding ERG in the three groups of mice at a stimulation intensity of 3.0 cd.s / m² in Example 2.

[0027] Figure 6 Quantitative statistical analysis of the Optical Motion Response Index (OMR) of mice in each group in Example 2;

[0028] Figure 7 Example 2: RGC counts (A) and corresponding RGC survival rates (B) of each group of mice in whole retinal patch.

[0029] Figure 8 Representative images of flow cytometry apoptosis in each group in Example 3 (A) and statistics of the percentage of apoptotic cells in each group (B);

[0030] Figure 9 Example 3: Representative images of TUNEL staining in each group of cells under a 40x objective lens taken using a Zeiss fluorescence microscope (A) and the corresponding statistics of the percentage of TUNEL-positive cells in each group (B).

[0031] Figure 10 : Representative JC-1 fluorescence staining images (A) and corresponding quantitative results of fluorescence intensity of JC-1 ratio (polymer / monomer) in each unmodeled group in Example 4 (B);

[0032] Figure 11 : Representative JC-1 fluorescence staining images (A) and corresponding quantitative results of fluorescence intensity of JC-1 ratio (polymer / monomer) in each group after modeling in Example 4 (B);

[0033] Figure 12 : A representative graph of ROS levels in each group detected by flow cytometry in Example 4 and quantitative statistics of the average fluorescence intensity of DCF in each group (B). Detailed Implementation

[0034] Example 1: Improvement of retinal ischemia-reperfusion injury in mice by intravitreal injection of adenovirus vector overexpressing Vimentin.

[0035] I. Experimental Materials

[0036] C57BL / 6J mice were purchased from Hunan Slack Jingda Company (Animal Qualification Certificate No.: NO.430727251101394286).

[0037] The Vimentin-overexpressing adenovirus vector was constructed by Wuhan Shumi Brain Science Technology Co., Ltd.

[0038] II. Experimental Methods

[0039] Eighteen 5-week-old male C57BL / 6J mice were selected and housed in an SPF-grade standard environment with standard feed and free access to water. The animals were randomly divided into three groups: a normal control group (WT group), a control virus + IR model group (AAV-EGFP+IR group), and a Vimentin overexpression virus + IR model group (AAV-Vimentin+IR group / AAV-Vim+IR group), with 6 mice in each group. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 μL / g). After pupil dilation with compound tropicamide eye drops, 1 μL of control / Vimentin overexpression virus was injected intravitreally into the model group mice using a microsyringe. Levofloxacin eye ointment was applied. After the mice regained consciousness, they were fed routinely and their condition was observed.

[0040] Three weeks after viral injection, mice in the AAV-EGFP+IR and AAV-Vimentin+IR groups were modeled. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital after weighing. Antibiotic eye drops and oxybuprocaine hydrochloride eye drops were administered sequentially. The pressure control device was assembled and its airtightness was checked, maintaining a pressure of 120 mmHg. The mouse's head position was adjusted to keep its eyeballs horizontal. A 34G insulin needle was inserted with the bevel facing down at the midpoint of the line connecting the pupil and the limbus. When 1 / 3 to 1 / 2 of the needle bevel entered the anterior chamber, the pressure control device was turned on to allow gas to enter the anterior chamber. The needle was rotated 180° with the bevel facing up and continued to be inserted until the entire needle bevel entered the anterior chamber. The needle and connecting tubing were then securely fixed. The device pressure was readjusted to 120 mmHg and maintained for 60 minutes. The pressure was slowly reduced in the last 5 minutes. At the end of the countdown, the needle was removed, and levofloxacin eye ointment was applied. After the mice regained consciousness, they were fed normally and their condition was observed (for modeling methods, see: Hu T, Meng S, Liu C, et al. LCN2deficiency mitigates the neuroinflammatory damage following acute...). glaucoma.Theranostics. 2025;15(7):2967-2990. Published 2025 Feb 10. doi:10.7150 / thno.104752) (see Figure 1 ).

[0041] Studies have shown that IR damage leads to visual function impairment in mice (decreased optomotor response index, decreased amplitude of a or b waves in Flash ERG detection) and RGC death. Therefore, we observed this using the following indicators. Seven days after modeling, mice in each group underwent optomotor response testing while awake and Flash ERG visual function testing after anesthesia and pupil dilation. After the tests, the anesthetized mice were perfused with PBS (phosphate buffered solution) and 4% paraformaldehyde, and the eyeballs were collected and fixed in 4% paraformaldehyde. Retinal slides were then prepared for RGC counting.

[0042] III. Experimental Results

[0043] 1. Flash ERG showed that IR (irradiation) induced visual impairment in mice, and overexpression of Vimentin effectively improved visual function in mice with IR damage:

[0044] Figure 2 A shows the representative ERG waveforms of the three groups of mice at a stimulation intensity of 3.0 cd.s / m². It can be seen that IR significantly reduced the amplitude of the a and b waves in the mice, while overexpression of Vimentin could reduce the degree of visual impairment.

[0045] Figure 2 B represents the quantitative statistics of the a-wave and b-wave amplitudes of the ERG in each group of mice, using one-way ANOVA. P < 0.05 P < 0.01 P < 0.0001; Scotopic a / b-Wave Amp: Dark-adapted a / b wave amplitude.

[0046] Note: Flash ERG is an important technique for assessing visual function. By emitting standardized, full-field flashes of light into the eyes of mice, a series of potential responses are induced in the retina. These responses are recorded by highly sensitive electrodes and processed using specialized analysis software. The alpha wave primarily reflects the function of photoreceptors; a decrease in amplitude or a prolongation of latency suggests photoreceptor damage or dysfunction. The beta wave primarily reflects the activity of neurons in the inner retinal layer; changes in its amplitude and latency can be used to assess the normality of retinal neural function.

[0047] 2. Optokinetic response showed that IR caused severe visual impairment in mice, and overexpression of Vimentin could significantly salvage the visual impairment:

[0048] Figure 3 A is a schematic diagram of the visual-motor response detection device;

[0049] Figure 3B represents the quantitative statistics of the Optomotor Response Index (OMR) of mice in each group, using an unpaired t-test; it can be seen that IR leads to visual impairment in mice, while overexpression of Vimentin can salvage some of the damaged vision; P < 0.05 P < 0.01; Optomotor Acuity (0.1 / 0.2 Cycles Per Degree): Optomotor response visual acuity (0.1 or 0.2 cycles / degree).

[0050] Note: Visuomotor response testing is a method for assessing an animal's visual function by observing its head movement responses to visual stimuli. Its principle is based on the animal's response to moving visual patterns (such as...) Figure 3 The natural reaction of animals to the moving patterns (shown in Figure A) is a head-following movement, known as the optomotor response, when they observe these patterns. By varying parameters such as the speed, direction, and contrast of the moving patterns, an animal's visual sensitivity and visual tracking ability can be systematically assessed. The optomotor response index is a quantitative indicator measuring the intensity of the optomotor response, typically calculated by measuring the frequency and amplitude of the animal's head-following movements. When an animal has normal vision, its optomotor response index is high, characterized by a strong response to visual stimuli and stable head-following movements. Conversely, if an animal's vision is impaired, its optomotor response index decreases, manifesting as a weakened response to visual stimuli or increased instability in head movements.

[0051] 3. Whole retinal patch RGC counting showed a significant increase in RGC loss after IR injury, and overexpression of Vimentin could reduce RGC loss:

[0052] Figure 4 A shows representative images of three groups of mouse retinal patches taken under a 40x objective lens of a Zeiss fluorescence microscope (top image) and a representative image of a whole retinal patch taken under a 20x objective lens (bottom image). It can be seen that IR leads to a significant reduction in the number of mouse RGCs, while overexpression of Vimentin can reduce the loss of RGCs; the image shows RGC cell bodies labeled with Tuj1 antibody.

[0053] Figure 4 B represents the statistics of RGC survival rate for each group, using an unpaired t-test; RGC survival rate = (mean number of RGCs in the sample / mean number of RGCs in the WT group) × 100% (mean ± standard error, n = 6); % of RGC Survival: RGC survival rate (%).

[0054] Example 2: Improvement of retinal ischemia-reperfusion injury in mice by intravitreal injection of recombinant human Vimentin protein.

[0055] I. Experimental Materials

[0056] C57BL / 6J mice were purchased from Hunan Slack Jingda Company (Animal Qualification Certificate No.: NO.430727251101394286).

[0057] Recombinant human Vimentin protein was purchased from Changsha Anjixing Biotechnology Co., Ltd. (Catalog No.: 10028-H08B-100).

[0058] II. Experimental Methods

[0059] Eighteen 8-week-old male C57BL / 6J mice were selected and housed in an SPF-grade standard environment with standard feed and free access to water. The animals were randomly divided into three groups: a normal control group (WT group), a PBS+IR model group (PBS+IR group), and a Vimentin protein+IR model group (Vimentin pr.+IR group), with six mice in each group. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (10 μL / g). Modeling was performed in the PBS+IR and Vimentin+IR groups (specific method as in Example 1). On the second day after modeling, the mice were anesthetized and their pupils dilated. 1 μL of PBS / Vimentin protein was injected intravitreally into each mouse in the modeling group using a microsyringe, and levofloxacin eye ointment was applied. After the mice regained consciousness, they were fed normally and their condition was observed. On the fourth day after modeling, 1 μL of PBS / Vimentin protein was injected intravitreally again. On the 7th day after modeling, mice in each group underwent optomotor response testing while awake and Flash ERG visual function testing after anesthesia and pupil dilation. After the test, mice under anesthesia were perfused with PBS and 4% paraformaldehyde, and the eyeballs were collected and fixed in 4% paraformaldehyde. Retinal slides were then prepared for RGC counting.

[0060] III. Experimental Results

[0061] 1. Flash ERG showed that IR-induced visual impairment in mice, and that injection of Vimentin protein effectively improved visual function in mice with IR-induced visual impairment.

[0062] Figure 5 A shows the representative ERG waveforms of the three groups of mice at a stimulation intensity of 3.0 cd.s / m². It can be seen that IR significantly reduced the amplitude of the a and b waves in the mice, while Vimentin protein can alleviate the degree of visual impairment.

[0063] Figure 5 B represents the quantitative statistics of the a-wave and b-wave amplitudes of the ERG in each group of mice, using one-way ANOVA. P < 0.05 P < 0.001 P < 0.0001.

[0064] 2. Optokinetic response showed that IR caused severe visual impairment in mice, and injection of Vimentin protein could significantly salvage the visual impairment:

[0065] Figure 6 To quantitatively analyze the visual-motor response index (OMR) of mice in each group, an unpaired t-test was used; it was found that IR leads to visual impairment in mice, while the use of Vimentin protein can salvage some of the damaged vision. P < 0.05 P < 0.01; Optomotor Acuity (0.1 / 0.2 Cycles Per Degree): Optomotor response visual acuity (0.1 or 0.2 cycles / degree).

[0066] 3. Whole retinal patch RGC counting showed a significant increase in RGC loss following IR damage; injection of Vimentin protein reduced RGC loss.

[0067] Figure 7 A shows three representative images of mouse retinal patches taken under a 40x objective lens of a Zeiss fluorescence microscope (top image) and a representative image of a whole retinal patch taken under a 20x objective lens (bottom image). It can be seen that IR leads to a significant reduction in the number of RGCs in mice, while the use of Vimentin protein can reduce the loss of RGCs.

[0068] Figure 7 B represents the statistics of RGC survival rate for each group, using an unpaired t-test; RGC survival rate = (mean number of RGCs in the sample / mean number of RGCs in the WT group) × 100% (mean ± standard error, n = 6); % of RGC Survival: RGC survival rate (%).

[0069] Example 3: Reducing apoptosis in cultured R28 cells (rat retinal progenitor cell line) after IR injury by transfecting with Vimentin overexpression plasmid.

[0070] I. Experimental Materials

[0071] The Lip3000 transfection kit was purchased from Thermo Fisher Scientific (catalog number: L3000015).

[0072] Vimentin overexpression plasmid was constructed by Hesheng Beiyin (Qingdao) Technology Co., Ltd.;

[0073] Antimycin A was purchased from Shanghai Maokang Biotechnology Co., Ltd. (Catalog No.: MS0070-10MG);

[0074] The calcium ion carrier was purchased from Aladdin Company (product number: C137844).

[0075] TUNEL staining kit was purchased from Novizan (catalog number: A113-03).

[0076] II. Experimental Methods

[0077] R28 cells were seeded at a density of 400,000 cells / well in 6-well plates and divided into four groups, named Control group, Vimentin group, Control+IR group, and Vimentin+IR group. The culture medium consisted of 90% low-glucose DMEM medium + 10% FBS + 1% penicillin antibiotics. The cells were incubated at 37°C for 24 hours until they adhered to the plates. After 24 hours, the transfection reagent and Vimentin / control plasmid complex were prepared according to the Lip3000 transfection kit instructions and added to the corresponding cell culture plates. Twenty-four hours later, an in vitro cell IR model was established. The culture medium was changed to a solution containing 10 μM antimycin A and 2 μM calcium ion carrier prepared with Hanks' Balanced Salt Solution (HBSS) for 2 hours to simulate ischemia. Then, the medium was changed back to normal and cultured for another 2 hours to simulate reperfusion (for modeling methods, see: Zhang R, Feng Y, Lu J, Ge Y, Li H. lncRNA Ttc3-209 Promotes the Apoptosis of Retinal Ganglion Cells in Retinal Ischemia Reperfusion Injury by Targeting the miR-484 / Wnt8a Axis. Invest Ophthalmol Vis Sci. 2021;62(3):13. doi:10.1167 / iovs.62.3.13). After modeling, flow cytometry and TUNEL staining were performed to detect cell apoptosis.

[0078] Note:

[0079] R28 cells are an immortalized rat retinal progenitor cell line with non-tumorigenic, multi-lineage differentiation potential and a high degree of similarity to the in vivo retinal environment. They express a variety of retinal and neuronal markers and are widely used in vitro to study retinal cell differentiation, neuroprotection, cytotoxicity, and photostimulation response.

[0080] Antimycin A is a mitochondrial respiratory chain inhibitor that inhibits aerobic respiration by blocking complex III in the mitochondrial electron transport chain, leading to reduced intracellular ATP production and thus mimicking cellular energy metabolism disorders under ischemic conditions. Calcium ion carriers, on the other hand, increase intracellular calcium ion concentration, mimicking intracellular calcium overload during reperfusion. Calcium overload activates various calcium-dependent enzymes, such as proteases and phosphatases, triggering a series of intracellular oxidative stress responses and cell damage. By using antimycin A and calcium ion carriers in combination, key pathological mechanisms of ischemic injury can be effectively simulated in in vitro cell culture.

[0081] 1. Flow cytometry detection of apoptosis (Annexin V / PI staining) experimental method

[0082] After modeling, discard the old culture medium, wash once with PBS, digest the cells with 0.25% trypsin without EDTA, centrifuge and resuspend, and adjust the cell density to approximately 1×10⁻⁶. 6 / ml, take 100 µl of cell suspension and add 5 µl of Annexin V-FITC / Annexin V-APC, incubate at room temperature in the dark for 15 minutes, then add 10 µl of PI staining solution and incubate in the dark for 5 minutes, and finally analyze by flow cytometry. Annexin V labels early apoptotic cells (PS eversion), and PI labels late apoptotic or necrotic cells (cell membrane damage). The fluorescence signal distinguishes live cells, early apoptotic cells, and late apoptotic / necrotic cells.

[0083] 2. TUNEL staining method for detecting cell apoptosis

[0084] After modeling, cell samples were first fixed, then treated with protease to increase permeability, followed by the addition of TUNEL reaction solution, in which terminal deoxynucleotidyl transferase (TdT) binds fluorescently labeled dUTPs to the 3'-OH at the DNA break ends. After incubation, fluorescence signals were observed using a fluorescence microscope; positive signals indicated cell apoptosis.

[0085] III. Experimental Results

[0086] 1. Flow cytometry analysis of apoptosis revealed that intraepithelial neoplasia (IR) induced increased apoptosis in R28 cells, while overexpression of Vimentin alleviated IR-induced apoptosis.

[0087] Figure 8 A shows representative images of apoptosis in each group via flow cytometry, where Q1 represents mechanically damaged cells, Q2 represents late-stage apoptotic / necrotic cells, Q3 represents early-stage apoptotic cells, and Q4 represents live cells.

[0088] Figure 8 B is the correct answer. Figure 8A. Statistical analysis of the percentage of apoptotic cells in each group: Apoptotic cell rate (%) = Q2 percentage of apoptotic cells + Q3 percentage of apoptotic cells. Comparisons were based on one-way ANOVA, n=6. P < 0.0001 P < 0.05.

[0089] 2. TUNEL staining showed that IR induced increased apoptosis in R28 cells, while overexpression of Vimentin reduced apoptosis.

[0090] Figure 9 A shows representative images of TUNEL staining in each group of cells under a 40x objective lens taken using a Zeiss fluorescence microscope. During apoptosis, when DNA double-strand breaks or single-strand breaks occur, the 3'-OH end of the DNA can be labeled with fluorescein or biotin by terminal deoxynucleotidyl transferase (TdT). The nuclear regions of cells that are TUNEL positive and DAPI-labeled are apoptotic cells as observed under a microscope.

[0091] Figure 9 B is the correct answer. Figure 9 A. Statistical analysis of the percentage of TUNEL-positive cells in each group. TUNEL-positive cell rate (%) = Number of TUNEL-positive and DAPI-labeled cell nuclei / Total number of DAPI-labeled cell nuclei. Comparisons were based on one-way ANOVA, n=6. P < 0.0001.

[0092] Example 4: Reduction of oxidative stress damage caused by IR injury in in vitro cultured R28 cells and improvement of mitochondrial function after transfection with Vimentin overexpression plasmid.

[0093] I. Experimental Materials

[0094] The Lip3000 transfection kit was purchased from Thermo Fisher Scientific (catalog number: L3000015).

[0095] Vimentin overexpression plasmid was constructed by Hesheng Beiyin (Qingdao) Technology Co., Ltd.;

[0096] Antimycin A was purchased from Shanghai Maokang Biotechnology Co., Ltd. (Catalog No.: MS0070-10MG);

[0097] The calcium ion carrier was purchased from Aladdin Company (product number: C137844).

[0098] The mitochondrial membrane potential detection kit (JC-1) was purchased from Beyotime International Co., Ltd. (catalog number: C2006).

[0099] The reactive oxygen species detection kit was purchased from Solarbio (catalog number: CA1410-100T).

[0100] II. Experimental Methods

[0101] R28 cells were seeded in 6-well plates at a density of 400,000 cells / well and divided into four groups: Control, Vimentin, Control+IR, and Vimentin+IR. The culture medium consisted of 90% low-glucose DMEM, 10% FBS, and 1% antibiotics. Cells were incubated at 37°C for 24 hours until adherence. After 24 hours, transfection reagents and the Vimentin / control plasmid complex were prepared according to the Lip3000 transfection kit instructions and added to the corresponding cell culture plates. An IR model was established after 24 hours by changing the culture medium to a solution containing 10 μM antimycin A and 2 μM calcium ionomer in HBSS for 2 hours to simulate ischemia, followed by a 2-hour reperfusion period in normal culture medium (see Example 3 for detailed IR modeling method). Mitochondrial membrane potential and intracellular reactive oxygen species levels were measured after modeling.

[0102] 1. Experimental method for detecting mitochondrial membrane potential using the JC-1 kit

[0103] After modeling, discard the old culture medium, wash once with PBS, digest the cells with 0.25% trypsin without EDTA, centrifuge and resuspend, and adjust the cell density to 1×10⁻⁶. 6 The cell suspension was then mixed with JC-1 dye working solution at a ratio of approximately 1:1000, and incubated at room temperature in the dark for 15-30 minutes. After incubation, the cells were washed twice with PBS to remove unbound dye. Finally, the fluorescence intensity of the cells was observed using a Leica fluorescence microscope. JC-1 aggregates in mitochondria with high membrane potential to form red fluorescent J-mers, while in mitochondria with low or lost membrane potential, it exists as monomers emitting green fluorescence. The change in the red-green fluorescence ratio can be used to assess the state of mitochondrial membrane potential, thereby determining mitochondrial function and apoptosis status.

[0104] 2. Experimental Method for Detecting Intracellular Reactive Oxygen Species Levels Using a Reactive Oxygen Species Reagent Kit

[0105] After modeling, discard the old culture medium, wash once with PBS, digest the cells with 0.25% trypsin without EDTA, centrifuge and resuspend, and adjust the cell suspension concentration to 1×10⁻⁶. 6The cells were incubated at approximately 10 µM / ml. Next, a reactive oxygen species reagent (DCFH-DA) was added, and the cells were incubated for 15-30 minutes in the dark. After incubation, the cells were washed twice with PBS to remove unbound reagent. Finally, the cell suspension was collected and analyzed by flow cytometry using a 488 nm excitation wavelength, detecting the fluorescence intensity at 525 nm. The fluorescence intensity reflects the intracellular ROS level, thus assessing the oxidative stress state of the cells.

[0106] III. Experimental Results

[0107] 1. Mitochondrial membrane potential (MMP) assays showed that overexpression of Vimentin increased cellular MMP; MMP decreased after IR (instantaneous transduction), and overexpression of Vimentin significantly reversed this change; the results before and after modeling were as follows: Figure 10 and 11 As shown.

[0108] Figure 10 A and Figure 11 A shows representative JC-1 fluorescence staining images for each group. The JC-1 probe emits green fluorescence at low concentrations (low mitochondrial membrane potential) and forms aggregates emitting red fluorescence at high concentrations (high mitochondrial membrane potential). The change in fluorescence color reflects the level of mitochondrial membrane potential. Transfection with a Vimentin overexpression plasmid can increase the mitochondrial membrane potential of cells; IR damage leads to a decrease in mitochondrial membrane potential, while overexpression of Vimentin can reduce the magnitude of MMP reduction caused by IR damage.

[0109] Figure 10 B and Figure 11 B represents the quantitative results of fluorescence intensity at the JC-1 ratio (Aggregates / Monomers). Comparisons were based on an unpaired t-test, n=6; P < 0.0001.

[0110] 2. Flow cytometry analysis of reactive oxygen species (ROS) showed that reactive oxygen species (IR) induced ROS accumulation in R28 cells, while overexpression of Vimentin reduced this increase.

[0111] Figure 12 A represents the ROS levels of each group as detected by flow cytometry. When detecting ROS using flow cytometry, DCFH-DA, as a probe, enters the cell and is hydrolyzed into DCFH by intracellular esterases. DCFH is then oxidized into fluorescent DCF under the action of ROS. The flow cytometer reflects the intracellular ROS level by detecting the fluorescence intensity of intracellular DCF.

[0112] Figure 12 B is the correct answer. Figure 12 A. Quantitative statistical analysis of intracellular ROS levels (mean ROS intensity, measured by flow cytometry using DCF) in each group. Comparisons were based on one-way ANOVA, n=6; P < 0.0001.

Claims

1. Application of Vimentin in the preparation of drugs for the prevention and treatment of retinal ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or diluents; the dosage forms of the drug include: injections, eye drops, ointments, implants, sustained-release formulations, or nanoformulations.

3. Application of Vimentin in the preparation of drugs for preventing and treating apoptosis of retinal ganglion cells.

4. The application according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or diluents; the dosage forms of the drug include: injections, eye drops, ointments, implants, sustained-release formulations, or nanoformulations.

5. Application of Vimentin and its derivatives, fragments, or analogs in the preparation of drugs for the prevention and treatment of retinal ischemia-reperfusion injury.

6. The application according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or diluents; the dosage forms of the drug include: injections, eye drops, ointments, implants, sustained-release formulations, or nanoformulations.

7. Application of Vimentin and its derivatives, fragments, or analogs in the preparation of drugs for preventing and treating apoptosis of retinal ganglion cells.

8. The application according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable carriers, excipients, or diluents; the dosage forms of the drug include: injections, eye drops, ointments, implants, sustained-release formulations, or nanoformulations.

Images