Method for verifying effect of ferroptosis in PVR

Abstract

The invention discloses a method for verifying the effect of ferroptosis in PVR. The method comprises the following steps: S1, cell experiment; s2, cell culture; s3, verifying the interaction between ferroptosis and EMT from multiple aspects by adopting a kit, a WB experiment, a CCK8 method and a scratch experiment; and S4, comparing a plurality of experiment results. According to the method, the influence mechanism of ferroptosis in the MeCP2-induced RPE cell EMT can be quickly verified, the effect of ferroptosis in the fibrosis process is determined, and the ferroptosis becomes a new target for treating PVR, so that a new theoretical basis is provided for effective treatment or prevention of clinical PVR.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for verifying the effect of ferroptosis in PVR. Background Art

[0002] Currently, there is no effective treatment for proliferative vitreoretinopathy (PVR) except surgery, and PVR is prone to recurrence after surgery. Clinical treatment and prevention strategies are not yet mature. PVR is characterized by intraretinal fibrosis and the formation of contractile epiretinal or subretinal membranes (ERMs). Retinal pigment epithelial (RPE) cells are the main cellular components in the PVR membrane and play a key role in the pathogenesis of PVR. At present, the pathogenesis of PVR has not been fully clarified. Mature RPE cells are in a mitotic quiescent state under physiological conditions. Once the retina is torn or detached, RPE cells undergo type II epithelial-mesenchymal transition (EMT), which is also an early change in the formation of PVR. EMT refers to the biological process in which epithelial cells are transformed into cells with a mesenchymal phenotype through a specific program. It plays an important role in embryonic development, chronic inflammation, tissue reconstruction, cancer metastasis and various fibrotic diseases. Its main characteristics include decreased expression of cell adhesion molecules (such as E-cadherin) and increased expression of α-smooth muscle actin (α-SMA). Through EMT, epithelial cells lose epithelial phenotypes such as cell polarity and connection with the basement membrane, and acquire mesenchymal phenotypes such as higher migration and invasion, anti-apoptosis and ability to degrade extracellular matrix. According to current research, EMT can be divided into three types, each of which involves different biological processes. PVR mainly involves type 2 EMT, which usually occurs in chronic organ damage and inflammation, and participates in tissue trauma and fibrotic scar formation. Although EMT is essentially a process of body repair, wound healing may lead to dysfunction and lesions of fibrous tissue, causing varying degrees of damage to surrounding tissues and organs, depending on the location and degree of recovery. For the eye, this process does more harm than good. When the retina is damaged, it stimulates RPE cell activation. Under the action of various inflammatory mediators, a variety of cytokines are released, such as interleukin (IL), transforming growth factor (TGF), fibroblast growth factor (FGF), etc. Under the action of various stimulating factors, RPE transforms into fibroblast-like cells, migrates to the retinal surface and vitreous body, forms a contractile membrane, namely PVR membrane, and causes retinal detachment. The epithelial-mesenchymal transition (EMT) of RPE cells is considered to be an early change in the onset of PVR.

[0003] Gene expression is regulated by epigenetics. DNA methylation is one of the most characteristic epigenetic marks in the human genome and plays a key role in human gene transcription regulation and other biological processes. Under the catalysis of DNA methyltransferase, a methyl group is added to the 5' position of cytosine, that is, the cytosine of the CpG dinucleotide is methylated to 5-methylated cytosine - this is the most common form of DNA methylation. Among the many reversible histone tail modifications, DNA methylation is a more stable epigenetic change. Biological processes such as wound healing and fibrosis, DNA repair, cell cycle regulation, inflammation / stress response, apoptosis and tumorigenesis are all related to changes in DNA methylation status. Our previous work found that in RPE cells, DNA methylation is a key link in regulating EMT.

[0004] Methyl CpG binding protein 2 (MeCP2) is a group of sequence-specific DNA binding proteins located in region 8 (Xq28) of band 2 on the long arm of chromosome X, which can bind DNA in a methylation-specific manner. MeCP2 is a multifunctional protein that is not only involved in transcriptional silencing, but also in transcriptional activation, chromatin compaction, and RNA splicing regulation. As a reader of methylation, MeCP2 is highly expressed in fibrotic and transformed RPE cells. MeCP2 can regulate chromatin structure and promote RPE epithelial-mesenchymal transition and RPE fibrosis.

[0005] Ferroptosis is an iron-dependent programmed cell death that is associated with a variety of metabolic disorders. It is different from apoptosis, necrosis, autophagy and other programmed cell deaths, and is characterized by elevated lipid peroxides and reactive oxygen species (ROS) and decreased levels of glutathione (GSH) and glutathione peroxidase 4 (GPX4).

[0006] In the past few years, the interaction between ferroptosis and EMT has been confirmed, that is, EMT increases the susceptibility of cells to ferroptosis, and ferroptosis in turn affects the process of EMT. However, the underlying mechanism between ferroptosis and EMT has not been clarified. Recent studies have confirmed the relationship between ferroptosis and retinal diseases such as retinal ischemia-reperfusion injury, age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, retinoblastoma, etc., but the relationship between ferroptosis and PVR has not been reported. Ferroptosis is mainly caused by an imbalance between oxidative stress and antioxidant response, driven by the accumulation of iron-dependent lipid peroxides. Multiple organelles including mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes are involved in the regulation of ferroptosis, which is manifested by reduced mitochondrial volume, increased mitochondrial membrane density, reduced or disappeared mitochondrial cristae, increased ROS in the cytoplasm and rupture of the mitochondrial outer membrane. This is due to the increase in intracellular iron concentration and the increase in ROS levels caused by lipid peroxidation caused by the consumption of the antioxidant glutathione. Current studies on the mechanism of ferroptosis have identified two cellular components, GPX 4 and system xc-. As the fourth member of the selenium-containing GPX family, GPX4 is an antioxidant enzyme that can inhibit lipid peroxidation and oxidative stress-related cell death and is an important regulator of ferroptosis. When GPX4 is reduced or inactivated, mitochondrial damage occurs. Recent studies have demonstrated that GPX-4 plays an important role in oxidative stress in human RPE cells. When ferroptosis occurs, GPX-4 deficiency is accompanied by cytotoxicity and cell death of RPE cells. It has been determined that wound healing is significantly delayed after GPX-4 knockout, while GPX-4 overexpression strongly protects the retina from oxidative damage-induced retinal degeneration and inhibits the occurrence of ferroptosis.

[0007] MeCP2, as an epigenetic modifier, participates in the regulation of chromatin structure and promotes epithelial-mesenchymal transition and retinal fibrosis in human RPE cells. Ferroptosis may affect MeCP2-induced EMT in RPE cells, but the relationship between MeCP2 and ferroptosis is not yet clear. Therefore, we need to study the mechanism of ferroptosis in RPE cell EMT, clarify the role of ferroptosis in the occurrence of PVR, and elucidate its role in the fibrosis process. Summary of the invention

[0008] In order to solve the above problems, the present invention proposes a method for verifying the role of ferroptosis in PVR.

[0009] The method for verifying the effect of ferroptosis in PVR of the present invention comprises the following steps:

[0010] S1. Cell experiments;

[0011] S2. Cell culture;

[0012] S3. The interaction between ferroptosis and EMT was verified from multiple aspects using kits, microplate reader, WB assay, CCK8 method and scratch assay;

[0013] S4. Comparison of multiple experimental results.

[0014] The cell experiment is as follows: the well-grown RPE cells of the 5th generation are prepared into a single cell suspension, about 20 microliters of the above cell suspension is added to a 159μl Ep tube with a pipette gun, an equal amount of trypan blue is mixed, and then a small amount of the mixture is aspirated and added to a cell counting plate. According to the cell counting method, the cells are counted under an upright microscope, and about 10,000, 8,000, and 5,000 cells are implanted in the wells selected in the 96-well plate for culture. It is found that the cell growth density of each well is moderate at a seeding density of 5,000 cells in 24 hours, so "5000" is selected as the standard seed plate in the CCK8 test using a 96-well plate, and then the number of cells in each well of the 6-well plate is determined by the same method.

[0015] The cell culture refers to the culture of ARPE19 cells, and the specific steps are: maintaining the cells in DMEM + 10% serum, placing them at 37°C and 5% CO 2 The cells were cultured in a tissue culture incubator and observed under a microscope. When the growth reached 90%, the cells were passaged.

[0016] The kit is used in S3 to verify the interaction between ferroptosis and EMT, specifically referring to the use of a kit method to explore the effect of recombinant human MeCP2 protein treatment on the content of ferroptosis markers in RPE cells, including the following steps:

[0017] (1) Cell treatment: Well-growing RPE cells were seeded in 6-well plates and divided into a control group, a MeCP2 100 ng / mL treatment group, an Erastin 10 uM treatment group, and a MeCP2 protein and ferroptosis inducer Erastin co-treatment group. When the cell fusion rate reached 90%, the cells were treated with recombinant human MeCP2 protein, ferroptosis inducer Erastin, and MeCP2 protein and ferroptosis inducer Erastin for 24 h.

[0018] (2) Microplate reader or flow cytometry: Add reagents to the treated cells according to the instructions and use a microplate reader or flow cytometry to measure changes in ferroptosis-related indicators using the corresponding wavelength;

[0019] (3) Content measurement: After treating the cells according to the instructions, the OD value was detected by a microplate reader at the corresponding wavelength, and the expression levels of MDA and GSH were calculated using the corresponding formula; the treated cells were treated with a positive control and a negative control using a ROS kit according to the instructions, and the changes in ROS in the cells were analyzed using FlowJo 7.6.2 software (Tree Star Inc., Ashland, OR, USA).

[0020] The WB experiment in S3 verifies the interaction between ferroptosis and EMT, specifically, the WB experiment is used to explore the effect of recombinant human MeCP2 protein treatment on the expression of ferroptosis-related factors GPX4, GCLM, and Nrf2 in RPE cells; and the effect of inducing ferroptosis in cells on the expression of EMT-related factors such as α-SMA, E-cadherin, and FN, including the following steps:

[0021] (1) Cell processing;

[0022] (2) extracting protein;

[0023] (3) Protein denaturation;

[0024] (4) Gel preparation, sample loading and electrophoresis;

[0025] (5) Transfer membrane;

[0026] (6) Closed;

[0027] (7) Immune response;

[0028] (8) Chemiluminescence test.

[0029] In S3, CCK8 is used to explore and verify the interaction between ferroptosis and EMT, specifically, CCK8 is used to explore the effect of ferroptosis inducers on RPE cell proliferation, including the following steps:

[0030] (1) Cell processing;

[0031] (2) CCK8 experiment: ARPE-19 cells were cultured in 10% DMEM in a culture dish. When they grew to 90%, they were seeded in a 96-well plate with about 5,000 cells per well. A blank control with only PBS was kept for 24 hours. Then, different dilutions of ferroptosis inducers were added. After 6 replicate wells were designed, different concentrations of ferroptosis inducers were added to the 96-well plate. The plates were cultured at 37°C and 5% CO2 for 24 hours. 10 μl of CCK-8 solution was added to each well of the plate and incubated for 1 hour. The absorbance at 450 nm was then measured using a microplate reader. Three biological replicates were performed (note the effects of cell number, treatment time, and CCK8 incubation time on the results).

[0032] The scratch experiment in S3 is to explore and verify the role of ferroptosis in MeCP2-induced EMT. Specifically, the scratch experiment is used to explore the effect of ferroptosis inducers on RPE cell migration, including the following steps:

[0033] (1) ARPE-19 cells were cultured in a culture dish with 10% DMEM. When they grew to 90%, they were seeded in a six-well plate, including the control group, MeCP2 group, Erastin group, and MeCP2+Erastin group, with 2 ml per well;

[0034] (2) After 24 hours, a vertical line was drawn in the center of each well with a 1000 ul pipette tip. After washing three times with PBS, 2 ml of 1% DMEM medium was added, and MeCP2 and Erastin were added respectively, where the concentration of MeCP2 was 100 ng / ml and the concentration of Erastin was 10 uM. Photographs were taken and recorded.

[0035] If at least three of the experimental results are consistent, it can be proved that ferroptosis plays a key role in the occurrence of PVR.

[0036] In the experiment of S3, the results of WB experiment accounted for the largest proportion of all experiments, followed by the kit experiment, and finally the CCK8 experiment.

[0037] The beneficial effects of the present invention are that CCK8 proves the inhibitory effect of ferroptosis on proliferation, and the scratch experiment proves the inhibitory effect of ferroptosis on migration, and these two experiments verify that ferroptosis inhibits the EMT process; the kit and WB experiments (three indicators of WB GPX4, GCLM, Nrf2) verify the effect of recombinant human protein MeCP2 on ferroptosis; WB (α-SMA, E-cadherin, FN) verifies that ferroptosis regulates EMT; the method of the present invention can quickly verify the influence mechanism of ferroptosis on MeCP2-induced RPE cell EMT, clarify the role of ferroptosis in the fibrosis process, and make it a new target for the treatment of PVR, thereby providing a new theoretical basis for the effective treatment or prevention of clinical PVR. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a diagram showing changes in the GSH content in each group of cells detected using the GSH kit of the present invention.

[0039] Figure 2 This is a diagram showing changes in the MDA content in each group of cells detected using the MDA kit of the present invention.

[0040] Figure 3 The present invention uses a ROS kit to detect changes in the ROS content in each group of cells.

[0041] Figure 4 It is a graph showing the experimental results of the WB experiment of the present invention.

[0042] Figure 5 This is a diagram of the present invention using CCK8 test to detect the effect of Erastin on cell proliferation.

[0043] Figure 6 It is a picture of the scratch test of the present invention.

[0044] Figure 7 It is a graph showing the cell migration rate results of the scratch test of the present invention. DETAILED DESCRIPTION

[0045] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0046] The method for verifying the effect of ferroptosis in PVR of the present invention comprises the following steps:

[0047] S1. Cell experiment: determine the number of cells per well of a 96-well plate or a 6-well plate, i.e., the seeding density.

[0048] Prepare the 5th generation well-grown RPE cells into a single cell suspension, add about 20 microliters of the above cell suspension to a 159μl Ep tube with a pipette gun, mix with an equal amount of trypan blue, then aspirate a small amount of the mixture and add it to a cell counting plate, count the cells under an upright microscope according to the cell counting method, implant about 10,000, 8,000, and 5,000 cells into the wells selected in the 96-well plate for culture, and find that the cell growth density in each well after 24 hours is moderate when the inoculation density is 5,000. Therefore, "5000" is selected as the standard plate in the CCK8 test using a 96-well plate, and then the number of cells in each well of the 6-well plate is determined by the same method.

[0049] S2. Cell culture;

[0050] Cell culture refers to the culture of ARPE-19 cells. The specific steps are as follows: the cells are maintained in DMEM + 10% serum at 37°C and 5% CO 2 The cells were cultured in a tissue culture incubator and observed under a microscope. When the growth reached 90%, the cells were passaged.

[0051] S3. The interaction between ferroptosis and EMT was verified from multiple aspects using kits, microplate reader, WB assay, CCK8 method and scratch assay;

[0052] S301. The kit is used to verify the interaction between ferroptosis and EMT, specifically, the kit method is used to explore the effect of recombinant human MeCP2 protein treatment on RPE cell ferroptosis markers (MDA, GSH) and intracellular ROS content, including the following steps:

[0053] (1) Cell treatment: Well-growing RPE cells were seeded in 6-well plates and divided into a control group, a recombinant human MeCP2 protein treatment group (100 ng / ml), a ferroptosis inducer Erastin (10 uM) group, and a group treated with both MeCP2 protein (100 ng / ml) and ferroptosis inducer Erastin (10 uM). When the cell fusion rate reached 90%, the cells were treated with recombinant human MeCP2 protein, ferroptosis inducer Erastin, and a group treated with both MeCP2 protein and ferroptosis inducer Erastin for 24 h.

[0054] (2) Microplate reader or flow cytometry: Add reagents to the treated cells according to the instructions and use a microplate reader or flow cytometry to measure changes in ferroptosis-related indicators using the corresponding wavelength;

[0055] (3) Content measurement: After treating the cells according to the instructions, the OD value was detected by a microplate reader at the corresponding wavelength, and the expression levels of MDA and GSH were calculated using the corresponding formula; the treated cells were treated with a positive control and a negative control using a ROS kit according to the instructions, and the changes in ROS in the cells were analyzed using FlowJo 7.6.2 software (Tree Star Inc., Ashland, OR, USA).

[0056] S302. Use WB experiments to verify the interaction between ferroptosis and EMT, specifically using WB experiments to explore the effect of recombinant human MeCP2 protein treatment on the expression of ferroptosis-related factors GPX4, GCLM, and Nrf2 in RPE cells; and the effect of inducing cell ferroptosis on the expression of EMT-related factors such as α-SMA, E-cadherin, and FN, including the following steps:

[0057] (1) Cell treatment: Well-growing RPE cells were seeded in 6-well plates and divided into a control group, a recombinant human MeCP2 protein treatment group (100 ng / ml), a ferroptosis inducer Erastin (10 uM) group, and a group treated with both MeCP2 protein (100 ng / ml) and ferroptosis inducer Erastin (10 uM). Each group was treated for 24 h.

[0058] (2) Protein extraction: Take out the cells from the incubator, wash with PBS three times, add 100 μl of cell lysis buffer, 1 μl of protease inhibitor and 1 μl of protein phosphatase inhibitor to each well, lyse on ice for 30 min, scrape the cells, transfer to a 1.5 ml EP tube, and store in a -80°C refrigerator;

[0059] (3) Protein denaturation: Take out the protein from the -80°C freezer, dissolve it on ice, centrifuge it at 4°C at 12,000 rpm for 30 min, discard the precipitate, carefully transfer the supernatant to a new EP tube, add protein:5*SDS loading buffer = 4:1, mix well, seal it with sealing film, place it in a 98°C water bath for 5 min, and store it at -20°C;

[0060] (4) Glue preparation, sample loading and electrophoresis: prepare the glass plate, clean and dry it, install it after it is free of impurities, add the prepared separation gel to the glass plate, add pure water to flatten it, and after it is completely solidified, pour out the pure water, add the prepared 5% concentrated gel, immediately insert the 10-hole comb, and put it into the electrophoresis tank after it is completely solidified, and add the prepared electrophoresis solution; load 25 μl of protein per well, adjust the constant voltage current to 80V, and change the constant voltage to 100V after the marker strips are pulled apart. Stop the electrophoresis after the marker strips are fully separated;

[0061] (5) Transferring the membrane; cut the PVDF membrane of the corresponding size according to the size of the gel, soak it in methanol for 1 min after the electrophoresis is almost finished, and then transfer the PVDF membrane to the transfer solution; the electrophoresis can be completed only after the protein is fully separated. Install the electrotransfer device in the transfer buffer, firmly clamp the transfer clamp, put it in the electrotransfer tank, connect the positive and negative electrodes, and then put the transfer device in ice water, place multiple ice bags around it to ensure that the temperature of the transfer process is constant. The transfer time is 75 min at 120V voltage;

[0062] (6) Blocking: Open the device, observe that the side of the PVDF membrane in contact with the electrophoresis gel is the front side, cut a small corner and make the corresponding mark, carefully remove the membrane with tweezers, place it in the prepared blocking solution, and block at room temperature;

[0063] (7) Immune reaction; Determine the position of the target protein and internal reference according to the molecular weight of the marker, cut the required PVDF membrane at the corresponding position, mark it, and put it into (required primary antibodies - α-SMA, E-cadherin, FN, GPX4, GCLM, Nrf2) respectively, and incubate at 4°C overnight; the next day, wash the membrane 3 times with 1×TBST, each time for 10 minutes. According to the source of the primary antibody, add HRP-labeled anti-rabbit and anti-mouse secondary antibodies diluted 1:3000 with blocking solution, incubate at room temperature for about 1 hour, and wash the PVDF membrane 3 times, each time for 10 minutes;

[0064] (8) Chemiluminescence test: After the PVDF membrane is slightly dried with filter paper, it is placed in a developer, and an appropriate amount of chemiluminescence reagent is evenly added to completely cover the membrane. The image is then exposed and collected for analysis.

[0065] S303. Using CCK8 to explore and verify the interaction between ferroptosis and EMT, specifically using CCK8 to explore the effect of ferroptosis inducers on RPE cell proliferation, including the following steps:

[0066] (1) Cell treatment: Well-growing RPE cells were seeded in 96-well plates and 24-well plates and divided into a control group and an Erastin-treated group. When the RPE cells adhered to the plate, the cells were treated with Erastin 10 μM for 24 h.

[0067] (2) CCK8 experiment: ARPE-19 cells were cultured in 10% DMEM in a culture dish. When they grew to 90%, they were seeded in a 96-well plate. The number of cells in each well was about 5000. A blank control with only PBS was kept. After culturing for 24 hours, different dilutions of ferroptosis inducers were added. The concentrations of ferroptosis inducers were 0, 2, 4, 8, 10, and 20 uM, respectively. After designing 6 replicate wells, different concentrations of ferroptosis inducers were inoculated at 37°C and 5% CO. 2 The cells were cultured in a 96-well plate for 24 hours, and then 10 μl of CCK-8 solution was added to each well of the plate and incubated for 1 hour. The absorbance at 450 nm was measured using a microplate reader. Three biological replicates were performed (note the effects of cell number, treatment time, and CCK8 incubation time on the results).

[0068] S304. A scratch test is used to explore and verify the interaction between ferroptosis and EMT, specifically, a scratch test is used to explore the effect of ferroptosis inducer on RPE cell migration, including the following steps:

[0069] (1) ARPE-19 cells were cultured in a culture dish with 10% DMEM. When they grew to 90%, they were seeded in 6-well plates, including the control group, MeCP2 group, Erastin group, and MeCP2+Erastin group, with 2 ml per well;

[0070] (2) After 24 hours, a vertical line was drawn in the center of each well with a 1000 ul pipette tip. After washing three times with PBS, 2 ml of 1% DMEM medium was added, and MeCP2 and Erastin were added respectively, where the concentration of MeCP2 was 100 ng / ml and the concentration of Erastin was 10 uM. Photographs were taken and recorded.

[0071] S4. Comparison of multiple experimental results.

[0072] If at least three conclusions are consistent in the experimental results, it can be proved that ferroptosis plays a key role in the occurrence of PVR. In S3's experiment, the results of the WB experiment accounted for the largest proportion of all experiments, followed by the kit experiment, and finally the CCK8 experiment.

[0073] S301. The kit method was used to investigate the effects of recombinant human MeCP2 protein treatment on RPE cell ferroptosis markers (MDA, GSH) and intracellular ROS content.

[0074] like Figure 1 As shown, the changes in GSH content in each group of cells were detected by GSH kit. It can be found that recombinant human MeCP2 protein can increase the expression level of ferroptosis marker GSH in ARPE-19 cells, thereby inhibiting the process of ferroptosis. ****P<0.0001, ***P<0.001.

[0075] like Figure 2 As shown, the changes in the MDA content in each group of cells were detected by using an MDA kit. It can be found that recombinant human MeCP2 protein can reduce the expression level of the ferroptosis marker MDA in ARPE-19 cells, thereby inhibiting the process of ferroptosis.

[0076] like Figure 3 As shown, the changes in the ROS content in each group of cells were detected by using a ROS kit. It can be found that the expression level of ferroptosis marker ROS in ARPE-19 cells did not change significantly after the addition of recombinant human MeCP2 protein.

[0077] S302. WB experiments were used to investigate the effects of recombinant human MeCP2 protein treatment on the expression of EMT-related factors α-SMA, E-cadherin, FN and ferroptosis-related factors GPX4, GCLM, and Nrf2 proteins in RPE cells.

[0078] like Figure 4As shown in the figure, WB experiments were used to explore the effects of recombinant human MeCP2 protein treatment on the expression of ferroptosis-related factors GPX4, GCLM, and Nrf2 in RPE cells; and the effects on the expression of EMT-related factors such as α-SMA, E-cadherin, and FN after inducing cell ferroptosis. It can be found that recombinant human MeCP2 protein increased the expression of ferroptosis markers GPX4, GCLM, and Nrf2 in ARPE-19 cells; ferroptosis inducer Erastin reduced the expression of EMT-related factors α-SMA and FN in ARPE-19 cells, and increased the expression of E-cadherin and Occludin. It can be concluded that recombinant human MeCP2 protein can inhibit the process of ferroptosis in ARPE-19 cells, and ferroptosis inducer Erastin can inhibit the process of EMT and fibrosis formation in ARPE-19 cells.

[0079] S303. CCK8 was used to explore and verify the interaction between ferroptosis and EMT, specifically, CCK8 was used to explore the effect of ferroptosis inducers on RPE cell proliferation.

[0080] like Figure 5 As shown, the effect of Erastin on cell proliferation was detected by CCK8 assay, and the results were presented in a bar graph: After Erastin treatment, ARPE-19 cell proliferation was inhibited in a concentration-dependent manner, ****P<0.0001, ***P<0.001.

[0081] S304. A scratch assay was used to investigate and verify the interaction between ferroptosis and EMT. Specifically, a scratch assay was used to investigate the effect of ferroptosis inducers on RPE cell migration.

[0082] like Figure 6 and Figure 7 As shown, compared with the control group, after the cells were treated with recombinant human MeCP2 protein (100ng / ml) for 24h, the migration rate of ARPE-19 cells increased; after the cells were treated with ferroptosis inducer Erastin (10uM) for 24h, the migration rate of ARPE-19 cells decreased; and after the cells were treated with recombinant human MeCP2 protein (100ng / ml) and ferroptosis inducer Erastin (10uM) for 24h, the migration rate of cells was not significantly different from that of the control group. ****P<0.0001, ***P<0.001, **P<0.01.

[0083] In summary, all experiments in S3 were able to prove that recombinant human MeCP2 protein could inhibit the process of ferroptosis in RPE cells, while ferroptosis inducers inhibited the EMT process and fibrosis formation in RPE cells.

[0084] In these experiments, the experimental results of the kit can more intuitively show that recombinant human MeCP2 protein can increase the expression level of GSH in ARPE-19 cells and reduce the expression level of MDA, thereby inhibiting the process of ferroptosis.

[0085] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0086] Although the above embodiments have been shown and described, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. Changes, modifications, substitutions and variations of the above embodiments by those of ordinary skill in the art are all within the scope of protection of the present invention.

Claims

1. A method for verifying the role of ferroptosis in PVR, characterized in that: The following steps are involved: S1. Cell experiments; S2. Cell culture; S3. The interaction between ferroptosis and EMT was verified from multiple aspects using kits, WB experiments, CCK8 methods and scratch experiments; S4. Comparison of multiple experimental results.

2. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: The cell experiment is as follows: the well-grown RPE cells of the 5th generation are prepared into a single cell suspension, the above cell suspension is added to the Ep tube with a pipette gun, an equal amount of trypan blue is mixed, and then a small amount of the mixture is aspirated and added to the cell counting plate, and the cells are counted under an upright microscope according to the cell counting method, and about 10,000, 8,000, and 5,000 cells are implanted in the wells selected in the 96-well plate in sequence for culture, and the number of cells in each well of the 96-well plate is determined, and then the number of cells in each well of the 6-well plate is determined by the same method.

3. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: The cell culture refers to the culture of ARPE-19 cells, and the specific steps are: maintaining the cells in DMEM+10% serum, culturing in a tissue culture incubator at 37°C and 5% CO2, observing under a microscope, and performing cell passaging when the growth reaches 90%.

4. The method for verifying the role of ferroptosis in PVR according to claim 3, characterized in that: The kit is used in S3 to verify the interaction between ferroptosis and EMT, specifically referring to the use of a kit method to explore the effect of recombinant human MeCP2 protein treatment on the content of ferroptosis markers in RPE cells, including the following steps: (1) Cell treatment: Well-growing RPE cells were seeded in 6-well plates and divided into a control group, a recombinant human MeCP2 protein treatment group, a ferroptosis inducer Erastin group, and a MeCP2 protein and ferroptosis inducer Erastin co-treatment group. Each group of cells was treated for 24 h. (2) Microplate reader or flow cytometry: Add reagents to the treated cells according to the instructions and use a microplate reader or flow cytometry to measure changes in ferroptosis-related indicators using the corresponding wavelength; (3) Content measurement: After treating the cells according to the instructions, the OD value was detected by a microplate reader at the corresponding wavelength, and the expression levels of MDA and GSH were calculated using the corresponding formula; the treated cells were treated with a positive control and a negative control using a ROS kit according to the instructions, and the changes in ROS in the cells were analyzed using FlowJo 7.6.2 software.

5. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: The WB experiment in S3 verifies the interaction between ferroptosis and EMT, specifically, the WB experiment is used to explore the effect of recombinant human MeCP2 protein treatment on the expression of ferroptosis-related factors GPX4, GCLM, and Nrf2 in RPE cells; and the effect of inducing ferroptosis in cells on the expression of EMT-related factors such as α-SMA, E-cadherin, and FN, including the following steps: (1) Cell processing; (2) extracting protein; (3) Protein denaturation; (4) Gel preparation, sample loading and electrophoresis; (5) Transfer membrane; (6) Closed; (7) Immune response; (8) Chemiluminescence test.

6. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: In S3, CCK8 is used to explore and verify the interaction between ferroptosis and EMT, specifically, CCK8 is used to explore the effect of ferroptosis inducers on RPE cell proliferation, including the following steps: (1) Cell processing; (2) CCK8 experiment: ARPE-19 cells were cultured in culture dishes. When they grew to 90%, they were seeded in 96-well plates and cultured for 24 h. Different dilutions of ferroptosis inducers were then added. After 24 h of culture, CCK-8 solution was added to each well of the plate and incubated for 1 h. The absorbance at 450 nm was then measured using a microplate reader. Three biological replicates were performed.

7. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: The scratch experiment in S3 is to explore and verify the role of ferroptosis in MeCP2-induced EMT. Specifically, the scratch experiment is used to explore the effect of ferroptosis inducers on RPE cell migration, including the following steps: (1) ARPE-19 cells were cultured in a culture dish. When they grew to 90%, they were seeded in a six-well plate, divided into a control group, a MeCP2 group, an Erastin group, and a MeCP2+Erastin group, with 2 ml per well; (2) After 24 hours, a vertical line was drawn in the center of each well using a 1000 ul pipette tip. The wells were washed three times with PBS, and then 2 ml of 1% DMEM medium was added. MeCP2 and Erastin were added respectively, and the wells were photographed and recorded.

8. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: If at least three of the experimental results are consistent, it can be proved that ferroptosis plays a key role in the occurrence of PVR.

9. The method for verifying the role of ferroptosis in PVR according to claim 1, characterized in that: In the experiment of S3, the results of WB experiment accounted for the largest proportion of all experiments, followed by the kit experiment, and finally the CCK8 experiment.

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