Hypertonic dry eye cell model-based ferroptosis inhibition research method

Abstract

The invention discloses a ferroptosis inhibition research method based on a hypertonic dry eye cell model, and relates to the technical field of xerophthalmia. Comprising the following steps: S1, establishing a hypertonic dry eye cell model, and selecting human corneal epithelial cells to perform hypertonic treatment in a hypertonic culture medium; s2, detecting a ferroptosis marker; performing GPX4 protein expression detection; s2.1, detecting the lipid peroxidation level; s2.2, detecting the iron ion level of the sample; s2.3, observing the mitochondrial morphology of the sample; s3, carrying out an autoserum intervention experiment, and treating the culture media of different groups by using autoserum; s4, data statistics and analysis; by detecting expression of proteins such as a ferroptosis marker GPX4 and the like, it is determined that autoserum can inhibit ferroptosis and improve corneal epithelial cell survival by enhancing GPX4 expression and reducing lipid peroxidation and iron ion level, a scientific basis is provided for further optimization and accurate application, a treatment strategy for xerophthalmia is improved, and the application has a wide application prospect. And a new direction is opened up for mechanism research and personalized treatment of the xerophthalmia in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of dry eye diseases, and in particular to a research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model. Background Art

[0002] Dry Eye Disease (DED) is a chronic ocular surface disease caused by multiple factors, and its main features include tear film instability, corneal epithelial damage, and chronic inflammation, which affect visual quality and quality of life. According to the report of TFOSDEWS II (International Dry Eye WorkGroup), dry eye can be divided into aqueous-deficient dry eye (ADDE) and evaporative dry eye (EDE). In the pathological process of both, corneal epithelial cell damage and cell death are involved. In recent years, studies have found that ferroptosis may play an important role in the development of dry eye disease. Especially in a hyperosmotic environment, corneal epithelial cells show increased lipid peroxidation, elevated iron ion levels, and decreased expression of GPX4 (glutathione peroxidase 4), suggesting the occurrence of ferroptosis. However, the existing treatment options mainly focus on tear replacement, immunosuppression, and anti-inflammatory treatment, and fail to intervene from the perspective of ferroptosis;

[0003] Autologous Serum Eye Drops (ASED) have been widely used in the clinical treatment of severe dry eye because they are rich in growth factors (EGF, TGF-β, NGF), anti-inflammatory factors (albumin, lysozyme), and antioxidants (vitamin A, antioxidant enzymes). Current studies have shown that autologous serum can promote corneal epithelial repair, reduce inflammatory responses, and improve tear film stability, but its mechanism of action is still mainly limited to anti-inflammation and growth factor supplementation, and does not involve the regulation of ferroptosis. Although there have been studies exploring antioxidant methods for treating dry eye, such as vitamin E, N-acetylcysteine (NAC), and iron chelator (Deferoxamine, DFO), these regimens lack targeting and do not combine the application of autologous serum eye drops. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies existing in the prior art and propose a research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model.

[0005] In order to achieve the above purpose, the present invention adopts the following technical scheme:

[0006] A research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model, comprising the following steps:

[0007] S1: Establish a hyperosmotic dry eye cell model. Select human corneal epithelial cells and perform hyperosmotic treatment in a hyperosmotic culture medium.

[0008] S3: Carry out an autologous serum intervention experiment. Use autologous serum to treat the culture media of different groups.

[0009] S2: Detect ferroptosis markers; detect the expression of GPX4 protein.

[0010] S2.1: Detect the level of lipid peroxidation.

[0011] S2.2: Detect the iron ion level in the sample.

[0012] S2.3: Observe the mitochondrial morphology of the sample.

[0013] S3: Conduct an autologous serum intervention experiment and use autologous serum to treat the culture media of different groups.

[0014] S4: Data statistics and analysis.

[0015] Preferably: In step S1, the osmotic pressure of the hyperosmotic culture medium is 500 mosm / L, which is achieved by adding NaCl, and a normal osmotic pressure group is set as a negative control.

[0016] Furthermore: In step S2, the other proteins related to ferroptosis include Xc-, ACSL4, and FTH1.

[0017] Furthermore: In step S2, detect the expression of GPX4 protein by Western blot, and detect the intracellular ROS level using a fluorescent probe, detect the MDA content by the thiobarbituric acid method, and measure the total glutathione and oxidized glutathione levels using a glutathione detection kit.

[0018] As a preferred embodiment of the present invention: In step S2.1, use an iron ion-specific fluorescent probe to detect the intracellular free iron concentration, and observe the mitochondrial morphology by transmission electron microscopy.

[0019] As a further embodiment of the present invention: In step S2.1, the fluorescent probe is DCFH-DA, which is used to detect the intracellular ROS level; the detection method for MDA content is the thiobarbituric acid (TBA) method; the measurement of GSH and GSSG levels uses a glutathione detection kit.

[0020] As a still further embodiment of the present invention: In step S2.2, the iron ion-specific fluorescent probe is FerroOrange.

[0021] Based on the foregoing embodiments: In step S3, the autologous serum is collected from the venous blood of healthy volunteers, stored after centrifugation and filtration sterilization, and diluted to a concentration of 20% before use.

[0022] Based on the above-mentioned solution: In step S3, the treatment of the positive control group in the autologous serum intervention experiment includes adding the ferroptosis inhibitor Ferrostatin-1 to the hypertonic culture medium.

[0023] Based on the above-mentioned solution: In step S3, during the centrifugation of venous blood in the autologous serum intervention experiment, the centrifugal force is 2000×g and the centrifugation time is 10 minutes.

[0024] The beneficial effects of the present invention are as follows:

[0025] 1. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model. By detecting the expression of ferroptosis markers such as the protein GPX4, it is determined that autologous serum can inhibit ferroptosis, improve the survival of corneal epithelial cells, and provide a scientific basis for further optimization and precise application, enhance the treatment strategy for dry eye, and open up a new direction for the mechanism research and personalized treatment of dry eye in the future by enhancing the expression of GPX4, reducing lipid peroxidation and iron ion levels.

[0026] 2. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model. Through experiments, it is determined that autologous serum treatment can significantly reduce the intracellular ROS level, alleviate oxidative stress, and reduce the accumulation of free radicals, thereby effectively inhibiting the process of ferroptosis; this effect is of great significance for protecting corneal epithelial cells from oxidative damage.

[0027] 3. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model. Through experiments, it is determined that autologous serum treatment can significantly reduce the MDA content and increase the total glutathione (GSH) / oxidized glutathione (GSSG) ratio, indicating that autologous serum can regulate the antioxidant system, reduce the occurrence of lipid peroxidation, and further inhibit ferroptosis.

[0028] 4. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model. Through experiments, it is determined that autologous serum treatment can significantly reduce the intracellular iron ion concentration, indicating that autologous serum may inhibit the occurrence of ferroptosis by regulating the intracellular iron homeostasis or promoting the excretion of iron ions and reducing the accumulation of iron ions. Description of the Drawings

[0029] Figure 1 is a schematic flowchart of a research method for inhibiting ferroptosis based on a hypertonic dry eye cell model proposed by the present invention;

[0030] Figure 2 is a schematic diagram showing the increased lipid peroxidation level caused by hyperosmotic pressure and the occurrence of ferroptosis in HCE-T cells in a research method for inhibiting ferroptosis based on a hypertonic dry eye cell model proposed by the present invention;

[0031] Figure 3It is a schematic diagram showing that in a research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model proposed by the present invention, AS alleviates ferroptosis of HCE-T cells caused by hyperosmotic stress;

[0032] Figure 4 It is a schematic diagram showing that in a research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model proposed by the present invention, Fer-1 and AS improve the abnormal iron homeostasis of HCE-T cells under hyperosmotic stress and inhibit ferroptosis through the Xc- / GPX4 pathway. Specific embodiments

[0033] The technical solutions of this patent will be further described in detail below in conjunction with specific embodiments.

[0034] The embodiments of this patent are described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the drawings are exemplary and are only used to explain this patent and should not be construed as a limitation to this patent.

[0035] Example 1:

[0036] A research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model, as Figures 1-4 shown, includes the following steps:

[0037] S1: Establishment of a hyperosmotic dry eye cell model; The selected cell type is: human corneal epithelial cells (HCE-T) are cultured in DMEM / F12 medium containing 10% fetal bovine serum (37 °C, 5% CO 2 ) until an appropriate cell density is reached; First, the cell viability of corneal epithelial cells at different osmotic pressures was measured by CCK8, and finally 90 mM NaCl was selected as the modeling stimulation concentration; Subsequently, the cells were exposed to a hyperosmotic medium (osmotic pressure 500 mosm / L, by adding NaCl) for 24 hours for hyperosmotic treatment to simulate the pathological environment of dry eye disease;

[0038] And a normal osmotic pressure group (310 mosm / L) control group was set as a negative control;

[0039] S2: Detection of ferroptosis markers; Detection of GPX4 protein expression; Cell proteins were collected by using Western blot, and anti-GPX4 primary antibody and HRP-labeled secondary antibody were used to detect the protein expression level by chemiluminescence method; Preferably, the anti-GPX4 primary antibody is Abcam#ab125066, diluted 1:1000;

[0040] The proteins related to ferroptosis, such as Xc-, ACSL4, and FTH1, were detected by Western blot, and then immunofluorescence analysis was performed.

[0041] The Western blot results showed that compared with the normal osmotic pressure group, the expression of GPX4 protein in corneal epithelial cells of the hypertonic treatment group was significantly decreased (P<0.05), indicating that the intracellular antioxidant capacity was impaired. The decrease in the expression of the antioxidant enzyme GPX4 related to ferroptosis provided a basis for the occurrence of ferroptosis.

[0042] S2.1: Detection of lipid peroxidation level;

[0043] First, ROS detection was carried out. The DCFH-DA fluorescent probe (10 μM, incubated at 37 °C for 30 minutes) was used, and the intracellular ROS level was quantitatively analyzed by fluorescence microscopy; the excitation wavelength was 488 nm, and the emission wavelength was 525 nm.

[0044] Then, the thiobarbituric acid (TBA) method was used to detect the content of malondialdehyde (MDA) in the samples.

[0045] Finally, the levels of total glutathione (GSH) and oxidized glutathione (GSSG) were determined by colorimetry using a glutathione detection kit.

[0046] After detecting the intracellular ROS level using the DCFH-DA probe, fluorescence microscopy observation showed that the intracellular ROS in the hypertonic treatment group was significantly increased, indicating that oxidative stress was activated in the hypertonic environment, providing a catalytic condition for ferroptosis.

[0047] The measurement results of MDA content showed that the MDA content in cells of the hypertonic group was significantly increased (P<0.05), further confirming the occurrence of lipid peroxidation, which was consistent with the typical characteristics of ferroptosis.

[0048] S2.2: Detection of iron ion level in samples; A specific fluorescent probe for iron ions was used, and the intracellular free iron concentration was detected by fluorescence microscopy. The preferred probe was FerroOrange, 1 μM, incubated at 37 °C for 30 minutes, the excitation wavelength was 540 nm, and the emission wavelength was 590 nm.

[0049] In the detection, it was detected that the intracellular free iron ion concentration in the hypertonic group cells was significantly increased (P<0.05), indicating that the accumulation of iron might be an important mechanism of ferroptosis.

[0050] S2.3: Observation of mitochondrial morphology in samples; After cell fixation, the changes in mitochondrial cristae structure, such as mitochondrial shrinkage, increased membrane density, and reduced cristae, were observed by transmission electron microscopy, and MitoTracker Red staining was performed.

[0051] The observation results showed that typical ferroptosis morphological changes occurred in the mitochondria of cells in the hypertonic treatment group, including mitochondrial volume reduction, increased membrane density, and decreased cristae; in addition, MitoTracker Red staining showed that obvious changes occurred in mitochondrial morphology, indicating impaired mitochondrial function in cells.

[0052] S3: Autologous serum intervention experiment; First, prepare autologous serum. Collect samples from the venous blood of healthy volunteers, let them stand for 30 minutes and then centrifuge, collect the supernatant serum, then filter and sterilize it using a 0.22 μm filter membrane, aliquot and store it at -80 °C; dilute the autologous serum to a concentration of 20% before use; preferably, the centrifugal force is 2000 × g and the centrifugation time is 10 minutes.

[0053] Subsequently, use the processed autologous serum to treat the culture media of different groups.

[0054] The samples to be treated include: normal control group (310 mosm / L culture medium), hypertonic model group (450 mosm / L culture medium), hypertonic + autologous serum group (450 mosm / L culture medium + 20% autologous serum), and positive control group (hypertonic culture medium + 1 μM Ferrostatin-1, a ferroptosis inhibitor); the hypertonic culture medium and autologous serum in these groups are co-treated for 24 hours.

[0055] S4: Data statistics and analysis; Repeat the above experiment at least 3 times, and the data are expressed as mean ± standard deviation.

[0056] Subsequently, perform statistical processing on the experimental data, and use GraphPad Prism software for one-way analysis of variance (ANOVA) or t-test; when comparing multiple groups, use Tukey's post hoc test; a P value less than 0.05 is considered statistically significant.

[0057] The experimental results in this method showed that a typical ferroptosis process occurred in corneal epithelial cells under hypertonic conditions, which was caused by oxidative stress and iron ion accumulation.

[0058] It was shown in step S2 that under hypertonic conditions, in corneal epithelial cells treated with the addition of autologous serum, the expression of GPX4 protein increased significantly (P < 0.05). The results of Western blot and immunofluorescence analysis showed that the expression level of GPX4 in the treatment group cells recovered to a level close to that of the normal control group; autologous serum may promote the synthesis of GPX4 by providing additional growth factors or antioxidant components, thereby enhancing the antioxidant capacity of cells and inhibiting the occurrence of ferroptosis.

[0059] In step S2.1, it was shown that compared with the hyperosmotic model group, the intracellular ROS level in cells treated with autologous serum was significantly decreased (P<0.05); this indicates that autologous serum can effectively reduce oxidative stress, decrease the accumulation of free radicals, and thus inhibit the process of ferroptosis;

[0060] Meanwhile, the measurement results of MDA content and the ratio of total glutathione (GSH) / oxidized glutathione (GSSG) showed that the level of lipid peroxidation in the autologous serum treatment group was significantly lower than that in the hyperosmotic model group (P<0.05); autologous serum can reduce the occurrence of lipid peroxidation by regulating the antioxidant system and further inhibit ferroptosis;

[0061] In step S2.2, the iron ion concentration in cells treated with autologous serum was significantly lower than that in the untreated hyperosmotic model group (P<0.05); this indicates that autologous serum may reduce iron ion accumulation by regulating intracellular iron homeostasis or promoting the excretion of iron ions, thereby inhibiting ferroptosis;

[0062] In step S2.3, the mitochondrial morphology in the autologous serum treatment group was significantly improved, the mitochondrial volume returned to normal, and the cristae structure was restored to a certain extent; transmission electron microscopy (TEM) also showed that autologous serum treatment could relieve the structural damage of mitochondria and restore mitochondrial function;

[0063] And compared with the positive control group: the effect of autologous serum was similar to that of the positive control group (Ferrostatin-1, a ferroptosis inhibitor). Cells in the Ferrostatin-1 treatment group also showed significantly decreased ROS and iron ion levels, and increased GPX4 expression.

[0064] In this method, autologous serum eye drops inhibit ferroptosis of corneal epithelial cells by enhancing GPX4 expression, reducing lipid peroxidation and iron ion levels, thereby realizing a new treatment mechanism for dry eye;

[0065] The new application strategy of autologous serum eye drops proposed in the present invention is applicable to patients with severe dry eye, hyperosmolarity-induced dry eye and poor existing treatment effects, and can optimize the eye drop frequency, personalized treatment plan and combination drug strategy, optimizing the use of autologous serum eye drops in inhibiting ferroptosis for the treatment of dry eye, the new mechanism of action and its application in precision treatment, providing an important scientific basis for the mechanism research and clinical transformation of dry eye.

[0066] Example 2:

[0067] A research method for inhibiting ferroptosis based on a hyperosmotic dry eye cell model, as Figures 1-4As shown in the figure, in order to further compare and study the effects of the ferroptosis inhibitor Ferrostatin-1 (Fer-1) and autologous serum (AS) in inhibiting ferroptosis, a hyperosmotic dry eye cell model obtained in step S1 of Example 1 was used, and the samples were divided into a control group, a hyperosmotic injury group, a Fer-1 treatment group, and an AS treatment group;

[0068] Subsequently, cells in the Fer-1 group and the combined group were added to a medium containing 10 μM Fer-1 6 hours in advance, and the other groups were used with an equal volume of DMSO (<0.1%) as a solvent control; 20% autologous serum was added to the AS group and the combined group at the same time; then the cells were cultured for another 24 hours;

[0069] Subsequently, CCK-8 assay was performed. 10 μL of CCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 2 hours. Then, the absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability was calculated;

[0070] Furthermore, immunoblot analysis (Western Blot) was used to detect ferroptosis markers. Cells were lysed with RIPA lysis buffer, and protein extraction was quantified by the BCA method for GPX4, ACSL4, and FTH1;

[0071] The test results showed that the cell viability of HCE-T at different osmotic pressures was measured by CCK-8. Different concentrations of sodium chloride (70, 80, 90, 100 μM) were added to the serum-free medium and cultured for 24 hours. It was found that the cell viability gradually decreased with the increase of osmotic pressure; the changes in the contents of ROS and Fe2+ in HCE-T were observed using H2DCFDA and FerroOrange fluorescent probes, and it was found that hyperosmolarity increased the contents of ROS and Fe2+ in HCET in a concentration-dependent manner; immunoblot analysis showed that in a hyperosmotic environment, the expression of Xc- in HCET did not change significantly, but the expression of GPX4 decreased; in addition, transmission electron microscopy showed that the mitochondria of HCE-T showed typical morphological changes of ferroptosis (mitochondrial atrophy, increased membrane density, disappearance of mitochondrial cristae) in a hyperosmotic environment, indicating that the lipid peroxidation level of HCE-T increased under hyperosmotic stimulation and ferroptosis occurred;

[0072] Western blot analysis showed that AS could inhibit the increased expression of ACSL4 and FTH1 in HCE-T cells under hyperosmotic conditions and increase the expression of GPX4; the cell lipid peroxidation level was measured using a related kit. After hyperosmotic stimulation, the levels of ROS and MDA in HCE-T increased significantly, while in the hyperosmotic medium containing 20% AS, the levels of ROS and MDA in HCE-T basically returned to normal levels; GSH is an important antioxidant in cells, and GSH in HCE-T decreased significantly under hyperosmotic conditions, and AS could restore it; the effect of AS on the mitochondrial morphology of HCE-T under hyperosmotic conditions was further observed by transmission electron microscopy. Compared with the normal group, the mitochondria of HCE-T under hyperosmotic conditions shrank, the outer mitochondrial membrane ruptured, and the cristae decreased or disappeared, while AS could improve the mitochondrial damage of HCE-T under hyperosmotic conditions, indicating that AS could reduce the lipid peroxidation level of HCE-T under hyperosmotic conditions and significantly affect the expression of ferroptosis-related genes and proteins;

[0073] And the ferroptosis inhibitor Fer-1 was used. By measuring the activity of HCE-T cells with CCK-8, it was found that 10 μM of Fer-1 could enhance its cell activity under hyperosmotic conditions; through the FRO probe test, it was found that Fer-1 and AS could reduce the Fe2+ content in HCE-T under hyperosmotic conditions; Western blot analysis showed that both AS and Fer-1 could inhibit the occurrence of ferroptosis in HCE-T through the Xc- / GPX4 pathway; Fer-1 and AS could improve the Fe2+ disorder in HCET under hyperosmotic conditions and inhibit the occurrence of ferroptosis through the Xc- / GPX4 pathway, indicating that both AS and Fer-1 treatments could improve the cell activity under hyperosmotic conditions.

[0074] As described above, this is a preferred specific embodiment of the present invention. The protection scope of the present invention is not limited thereto. Any person skilled in the art, within the technical scope disclosed by the present invention, in combination with the prior art or common knowledge of the public, any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model, characterized in that: The steps include: S1: Establish a hypertonic dry eye cell model, select human corneal epithelial cells and treat them in a hypertonic medium; S2: Detection of ferroptosis markers; GPX4 protein expression detection; S2.1: Detection of lipid peroxidation level; S2.2: Sample iron ion level detection; S2.3: Observation of sample mitochondrial morphology; S3: Carry out autologous serum intervention experiment and use autologous serum to treat the culture medium of different groups; S4: Data statistics and analysis.

2. A research method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S1, the osmotic pressure of the hypertonic culture medium is 500 mosm / L, which is achieved by adding NaCl, and a normal osmotic pressure group is set as a negative control.

3. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S2, the other proteins associated with ferroptosis include Xc-, ACSL4 and FTH1.

4. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S2, GPX4 protein expression was detected by western blotting, and intracellular ROS levels were detected by fluorescent probes, malondialdehyde content was detected by thiobarbituric acid method, and total glutathione and oxidized glutathione levels were measured by glutathione detection kit.

5. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S2.1, the intracellular free iron concentration was detected using an iron ion-specific fluorescent probe, and the mitochondrial morphology was observed by transmission electron microscopy.

6. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S2.1, the fluorescent probe is DCFH-DA, which is used to detect the intracellular ROS level; the MDA content is detected by the thiobarbituric acid (TBA) method; and the GSH and GSSG levels are determined using a glutathione detection kit.

7. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 1, characterized in that: In step S2.2, the iron ion-specific fluorescent probe is FerroOrange.

8. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 7, characterized in that: In step S3, the autologous serum is collected from venous blood of healthy volunteers, stored after centrifugation and filtration sterilization, and diluted to a concentration of 20% before use.

9. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 8, characterized in that: In step S3, the treatment of the positive control group in the autologous serum intervention experiment includes adding ferroptosis inhibitor Ferrostatin-1 to the hypertonic culture medium.

10. The method for inhibiting ferroptosis based on a hypertonic dry eye cell model according to claim 9, characterized in that: In step S3, the centrifugal force during the centrifugation of venous blood in the autologous serum intervention experiment was 2000×g, and the centrifugation time was 10 minutes.

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