Construction method of rheumatoid arthritis photodynamic therapy animal model

Abstract

The invention discloses a construction method of a rheumatoid arthritis photodynamic therapy animal model, and relates to the technical field of rheumatoid arthritis photodynamic therapy. The invention aims to facilitate optimization of the treatment effect on rheumatoid arthritis. Comprising the following steps: S11, selecting a sample; s12, performing experiment evaluation; s13, performing immunohistochemical examination; s14, carrying out histological analysis; s15, carrying out cell culture; s16, carrying out Cur-PDT treatment; s17, performing si-RNA (Ribonucleic Acid) transfection; and after S18: siNrf2 + PDT combined treatment, determining the fibroblast-like synovial cell tumor-like proliferation (invasion, migration, scratch and cloning) capability. According to the invention, the effect of the photosensitizer (curcumin) under the illumination of a proper wavelength can be observed through the animal experiment model, so that the specific mechanism of photochemical reaction is revealed, the interaction between the photosensitizer and light is known, and meanwhile, the research on how PDT affects the immune system, including the infiltration of inflammatory cells, is facilitated. And activating, migrating and ferroptosis of rheumatoid arthritis fibroblast-like synovial cells.

Description

Technical Field

[0001] The invention relates to the technical field of photodynamic therapy for rheumatoid arthritis, and in particular to a method for constructing an animal model for photodynamic therapy for rheumatoid arthritis. Background Art

[0002] In the prior art, rheumatoid arthritis is an autoimmune disease, which is mainly manifested by joint pain, swelling and stiffness. Therefore, the treatment of rheumatoid arthritis is in the stage of research and experimentation. Animals need to be used for the experiment. Photodynamic therapy has been introduced in the current treatment process. In the treatment of rheumatoid arthritis, photodynamic therapy can reduce symptoms such as inflammation and pain by selectively destroying the synovial tissue of the joints. Therefore, constructing an animal model and performing PDT treatment can provide new ideas and methods for the treatment of the disease.

[0003] However, there are still some deficiencies in the construction of animal models for photodynamic therapy. For example, the preparation method of animal models is relatively complicated, and specific inducers or gene editing technologies are needed to simulate human pathological and physiological processes. Animal models also have certain instability. Therefore, how to ensure that the establishment of animal models makes the entire establishment process more systematic and comprehensive, and how to ensure the safety of photodynamic therapy in the process of human pathological treatment has become an important goal.

[0004] In summary, this application proposes a method for constructing an animal model of photodynamic therapy for rheumatoid arthritis. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for constructing an animal model of photodynamic therapy for rheumatoid arthritis.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for constructing an animal model of rheumatoid arthritis photodynamic therapy comprises the following steps:

[0008] Sample selection: 8-week-old healthy SPF male Sprague-Dawley (SD) rats were selected, a total of 25 rats, 5 rats per cage;

[0009] Among 25 SD rats, 5 were randomly selected as the normal control group (Control), and the remaining 20 rats were used to establish the collagen-induced arthritis (CIA) model;

[0010] Bovine type II collagen was mixed with incomplete Freund's adjuvant and fully emulsified on ice to prepare type II collagen emulsion;

[0011] After anesthesia with amobarbital, the rats were injected with the prepared type II collagen emulsion at multiple points on the back and tail base under sterile conditions, which was regarded as the day 0 of the primary immunization;

[0012] On the 7th day after modeling, the mice were given a booster immunization with the same dose of bovine type II collagen mixed with complete Freund's adjuvant; the control group was treated with an equal volume of normal saline.

[0013] On the 14th day after modeling, the 20 CIA rats with successfully established models were randomly divided into 4 groups: model group (CIA), blue-violet light group (Light), curcumin group (Cur), and photodynamic therapy group (PDT);

[0014] Starting from the 15th day after the initial immunization, rats in each group were treated accordingly, with daily treatment for 28 days;

[0015] Arthritis scores were performed every 7 days after the second immunization;

[0016] On the 42nd day after modeling, all rats were euthanized with 5% amobarbital, and ankle joints and blood samples were collected from the rats.

[0017] Preferably, body weight measurement, paw volume monitoring and joint scoring are performed on the 14th day after the initial immunization, and body weight measurement, paw volume monitoring and arthritis scoring are performed every 7 days. The arthritis scoring criteria are as follows: 0 point: no redness or swelling of the ankle joint and paw; 1 point: mild swelling or redness of the ankle joint and paw; 2 points: moderate swelling or mild redness of the ankle joint and paw; 3 points: redness and swelling of the paw below the ankle joint; 4 points: severe redness, swelling and deformation of the ankle and paw joints.

[0018] Further: The resected ankle joint tissue was fixed, decalcified, paraffin-embedded, sliced ​​and stained, observed and images were acquired through a microscope, and the degree of fibrosis was quantitatively analyzed using ImageJ software.

[0019] Furthermore: Hematoxylin-eosin (H&E) staining was used to observe the inflammatory cell infiltration in the synovial tissue, the destruction of bone and cartilage, and the synovial hyperplasia, so as to provide a pathological score for the joint section, and the score was determined based on previous literature reports.

[0020] As a preferred solution of the present invention: 1.2×10^5MH7A cells / well are inoculated in DMEM culture medium containing 10% FBS, and after the cells adhere to the wall, the cells are stimulated with recombinant human interferon TNF-α for 24 hours to induce an inflammatory environment.

[0021] As a further embodiment of the present invention, curcumin dissolved in DMSO was treated with cells at a final concentration of 15 μg / ml for 24 hours, and the parameters were 460 nm wavelength and 21 J / cm2 The cells were irradiated with blue-violet light of high energy intensity and the lysate was finally harvested.

[0022] As a further scheme of the present invention: si-Nrf2 and negative control group small interfering RNA were transfected using Lipofectamine3000 reagent. After 6 hours of transfection, the culture medium was replaced with fresh culture medium without penicillin-streptomycin. Subsequent experiments were carried out according to the above-mentioned cell culture method, and the transfection efficiency was evaluated by measuring the fluorescence intensity and Western blot technology.

[0023] On the basis of the previous protocol: commercial ELISA kits were used to measure the levels of Nrf2, SLC7A11, and GPX4 in serum samples and the levels of IL-1β and IL-6 in cell culture supernatants.

[0024] On the basis of the above scheme: cell viability was assessed using the cell counting kit-8 (CCK-8), MH7A cells were seeded in a 96-well plate at a density of 4×10^3 cells / well, and incubated in an incubator at 5% CO2 and 37°C. After the cells adhered to the wall, they were treated with different concentrations of curcumin solution for 24 hours, 48 ​​hours and 72 hours. DMEM solution and CCK-8 reagent were mixed at a ratio of 1:9, 100 μl of the mixture was added to each well, and incubated for another 1 hour. The optical density (OD) was measured at 450 nm using a microplate reader, and the optical density (OD) of the cells under different light intensities was tested using the same method.

[0025] On the basis of the above scheme, the following steps are also included: using lactate dehydrogenase assay kit to judge the integrity of cell membrane; using AM-PI kit to evaluate the ratio of live cells to dead cells; using EDU kit to evaluate the proliferation ability of cells; using JC-1 dye to detect mitochondrial membrane potential; using transmission electron microscopy to observe the morphological structure of cell membrane, organelles and nucleus of each group of cells after treatment; analyzing lipid reactive oxygen species (ROS); determining GSH, MDA, SOD levels and iron content; analyzing the relative expression level of target genes through qRT-PCR experiments; based on qRT-PCR experiments, using ImageJ software to quantitatively analyze protein blot bands; performing immunofluorescence experiments; performing crystal violet staining, and observing and acquiring images through a fluorescent inverted microscope.

[0026] The beneficial effects of the present invention are:

[0027] 1. A method for constructing an animal model of photodynamic therapy for rheumatoid arthritis. The animal model can be used to observe the effects of different photosensitizers under different wavelengths of light, thereby revealing the specific mechanism of photochemical reactions and understanding the interaction between photosensitizers and light. It is also convenient for studying how PDT affects the immune system, including the activation, migration and apoptosis of inflammatory cells.

[0028] 2. A method for constructing an animal model for photodynamic therapy of rheumatoid arthritis. The animal model can provide important reference data for clinical trials and accelerate the development of new photosensitizers.

[0029] 3. A method for constructing an animal model of photodynamic therapy for rheumatoid arthritis, by adjusting factors such as photosensitizer dosage, light intensity and time, to find the best treatment conditions, so as to facilitate the determination of the best diagnostic and treatment parameters during treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the experimental results of the method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed by the present invention, in which CUR-PDT inhibits the progression of arthritis in CIA rats;

[0031] Figure 2 It is a schematic diagram of experimental results after CUR-PDT treatment of a method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed by the present invention;

[0032] Figure 3 It is a schematic diagram of the experimental results of the CUR-PDT reducing the viability, proliferation and migration ability of MH7A cells in a method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed by the present invention;

[0033] Figure 4 It is a schematic diagram of the experimental results that CUR-PDT can effectively induce ferroptosis in MH7A cells according to a method for constructing an animal model of photodynamic therapy for rheumatoid arthritis proposed in the present invention;

[0034] Figure 5 It is a schematic diagram of the experimental results of the Nrf2-xCT-GPX4 pathway participating in CUR-PDT promoting the ferroptosis process of MH7A cells in a method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed in the present invention;

[0035] Figure 6 It is a schematic diagram of the experimental results of the method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed by the present invention, in which the combined application of down-regulating Nrf2 and CUR-PDT aggravates the ferroptosis of MH7A cells;

[0036] Figure 7It is a schematic diagram of the experimental results of the method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed in the present invention, in which the combined action of si-Nrf2 and CUR-PDT accelerates the ferroptosis process of MH7A cells through the Nrf2-xCT-GPX4 pathway;

[0037] Figure 8 This is a schematic diagram of the experimental results of the method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed by the present invention, in which si-Nrf2 and CUR-PDT acted in combination on the Nrf2-xCT-GPX4 pathway to significantly inhibit the proliferation, invasion and migration ability of MH7A cells;

[0038] Fig. 9 It is a structural schematic diagram of a CUR-PDT-induced RA-MH7A ferroptosis pathway model of a method for constructing a photodynamic therapy animal model of rheumatoid arthritis proposed in the present invention. DETAILED DESCRIPTION

[0039] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0040] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0041] Embodiment 1:

[0042] A method for constructing an animal model of rheumatoid arthritis photodynamic therapy comprises the following steps:

[0043] S11: Sample selection

[0044] 8-week-old healthy SPF male Sprague-Dawley (SD) rats were selected, with a total of 25 rats, 5 rats in each cage. At the beginning of the first study, the animal weight was (270±2) g;

[0045] Among 25 SD rats, 5 were randomly selected as normal control group (Control, n=5), and the remaining 20 rats were used to establish collagen-induced arthritis (CIA) model. Bovine type II collagen and incomplete Freund's adjuvant were mixed in equal volumes and fully emulsified on ice to prepare type II collagen emulsion;

[0046] After anesthesia with 2% amobarbital (50 mg / kg), the prepared type II collagen emulsion (0.2 ml / rat) was injected at multiple points on the back and tail base of the rats under sterile conditions, which was regarded as the day 0 of the primary immunization;

[0047] On the 7th day after modeling, an equal volume of bovine type II collagen and complete Freund's adjuvant were mixed and then boosted with the same dose according to the above method; the control group was injected with an equal volume of normal saline for treatment;

[0048] On the 14th day after modeling, the 20 CIA rats with successfully established models were randomly divided into 4 groups (n=5): model group (CIA, n=5), blue-violet light group (Light, n=5), curcumin group (Cur, n=5), photodynamic therapy group (PDT, n=5);

[0049] Starting from the 15th day after the initial immunization, the blue-violet light group was given 300 J / cm 2 The curcumin group was given 0.1 ml of 15 mM curcumin solution in the bilateral popliteal fossa; the photodynamic therapy group (PDT) was injected with 0.1 ml of 15 mM curcumin solution in the bilateral popliteal fossa and 300 J / cm 2 of blue-violet light;

[0050] The rats in the control group and CIA group were injected with 0.1 ml of normal saline in the bilateral popliteal fossa, and the treatment was continued daily for 28 days. Arthritis scores were performed every 7 days after the second immunization. On the 42nd day after modeling (the 28th day of treatment), all rats in each group were euthanized with 5% amobarbital, and the ankle joints of the rats were collected. Blood samples were collected from the inferior vena cava of the rats and centrifuged at 3000×g, 4°C for 20 minutes to collect serum, which was stored at -80°C;

[0051] S12: Experimental Evaluation

[0052] On the 14th day after the initial immunization, body weight measurement, paw volume monitoring and joint scoring were performed. Thereafter, body weight measurement, paw volume monitoring and arthritis scoring were performed every 7 days. The arthritis scoring criteria were as follows: 0 point: no redness or swelling of the ankle joint and paw; 1 point: mild swelling or redness of the ankle joint and paw; 2 points: moderate swelling or mild redness of the ankle joint and paw; 3 points: redness and swelling of the paw below the ankle joint; 4 points: severe redness, swelling and deformation of the ankle joint and paw;

[0053] S13: Immunohistochemistry

[0054] On the 42nd day after the first immunization, the ankle joints were removed from the sacrificed rats, and the tissues were fixed in 4% paraformaldehyde for 2 days, then decalcified in 10% disodium ethylenediaminetetraacetic acid (EDTA) decalcification solution for 1 month, embedded in paraffin, and pathologically sectioned into 4 μm sections, dewaxed in xylene. In addition, the sections were treated with citric acid antigen retrieval solution, and then 3% hydrogen peroxide was used to block endogenous peroxidase activity. After blocking with normal goat serum blocking solution, the sections were incubated with primary antibodies at 4°C overnight. Subsequently, after incubation with secondary antibodies, the sections were labeled with a DAB detection kit, cell nuclei were stained with hematoxylin, and images were acquired using a fluorescent inverted microscope;

[0055] S14: Histological analysis

[0056] After dewaxing the ankle sections with xylene, Masson trichrome (MT) staining was used to evaluate the fibrosis of the synovial tissue of the rat ankle joints. The collagen fiber area ratio of the three regions of each rat was calculated by ImageJ software to quantify the degree of synovial fibrosis. In addition, hematoxylin-eosin (H&E) staining was used to observe the inflammatory cell infiltration, bone and cartilage destruction, and synovial hyperplasia in the synovial tissue to provide a pathological score for the joint sections. The score was determined according to previous reports, as previously reported. The severity of joint arthritis was graded from 0 to 4 according to the intensity of lining hyperplasia, mononuclear cell infiltration, and pannus formation (0 = normal ankle, 1 = normal synovium with occasional mononuclear cells, 2 = definite arthritis with a few layers of flat to rounded synovial lining cells and scattered mononuclear cells and dense infiltration of mononuclear cells, 3 = marked synovial hyperplasia with three or more layers of loosely arranged lining cells and dense infiltration of mononuclear cells, 4 = severe synovitis with pannus and erosion of articular cartilage and subchondral bone);

[0057] S15: Cell culture

[0058] The cells were cultured at 37°C in an environment of 5% CO2 in DMEM high glucose medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin;

[0059] S16: Cur-PDT treatment

[0060] A systematic PDT experiment was conducted. First, 1.2×10^5MH7A cells / well were inoculated in DMEM medium containing 10% FBS. After the cells attached to the wall, the cells were stimulated with recombinant human interferon TNF-α for 24 hours to induce an inflammatory environment. Then, curcumin dissolved in DMSO was treated with a final concentration of 15μg / ml for 24 hours, and the parameters were 460nm wavelength and 21J / cm 2 Irradiate with blue-violet light of high energy intensity, and finally harvest the lysate;

[0061] S17: si-RNA transfection

[0062] MH7A cells were seeded in 6-well plates at a density of 1.2×10^5, and si-Nrf2 and negative control group small interfering RNA were transfected using Lipofectamine3000 reagent. After 6 hours of transfection, the culture medium was replaced with fresh medium without penicillin-streptomycin, and subsequent experiments were performed according to the above cell culture method. The transfection efficiency was evaluated by measuring the fluorescence intensity and Western blot technology;

[0063] S18: Determination of tumor-like proliferation (invasion, migration, scratching and cloning) ability of fibroblast-like synoviocytes after siNrf2+PDT combined treatment

[0064] The cells were divided into a control group, a siNrf2 treatment group, a PDT treatment group, and a siNrf2+PDT combined treatment group. MTT reagent was added to the cells in each group. After incubation for a period of time, the absorbance value was measured to reflect the proliferation ability of the cells.

[0065] Matrigel was spread on the upper layer of the Transwell chamber, and each group of cells was seeded on the lower layer. After incubation for a period of time, the number of cells that passed through the Matrigel was stained and counted;

[0066] A scratch was made on the culture dish, and each group of cells was inoculated into the scratch. After incubation for a period of time, the degree of healing of the scratch was observed and measured;

[0067] Each group of cells was inoculated into soft agar medium, and after incubation for a period of time, the number and size of the colonies formed were observed and counted;

[0068] S19: Cell Counting Kit-8 (CCK-8) to assess cell viability

[0069] MH7A cells were seeded in a 96-well plate at a density of 4 × 10^3 cells / well and incubated in a 5% CO2 and 37°C incubator. After the cells adhered to the wall, they were treated with different concentrations of curcumin solution (0, 10, 15, 20, 25, 30, 40, 50 μM) for 24, 48 and 72 hours. Subsequently, DMEM solution and CCK-8 reagent were mixed at a ratio of 1:9, 100 μl of the mixture was added to each well, and incubated for another 1 hour. The optical density (OD) was measured at 450 nm using an enzyme reader. The same method was used to test the effects of different light intensities (18, 21, 24, 27, 30, 33 J / cm 2 ) The optical density (OD) of the cells;

[0070] S20: Lactate dehydrogenase release

[0071] Use lactate dehydrogenase assay kit to judge the integrity of cell membrane. Inoculate cells at a density of 4×10^4 cells / well in a 96-well plate, and set the maximum release well of the cells. After treating the cells as above, centrifuge the cell culture plate at 400g for 5 minutes in a multi-well plate centrifuge. Remove the supernatant as much as possible, add 150ul PBS and 10ul LDH release reagent mixture to each well, add 160ul of 2.5% TritonX-100 to the maximum release well of the cells, shake and mix appropriately, and then continue to incubate in a microincubator for 1 hour, then centrifuge the cell culture plate at 400g for 5 minutes in a multi-well plate centrifuge, take 120μl of the supernatant from each well, add it to the corresponding well of a new 96-well plate, and then use an enzyme reader to measure the sample at 490nm, and calculate the ratio level of the experimental group to the control group to determine the LDH level;

[0072] S21: Assessing the ratio of live cells to dead cells using the AM-PI kit

[0073] MH7A cells were seeded in a confocal culture dish at a density of 2×10^4 cells / well. After the cells were treated according to the above method, AM, PI and detection buffer were mixed at a ratio of 1:1:1000 according to the instructions, and added to each well in equal amounts, and incubated in a 37°C incubator for 30 minutes, and then observed and acquired images using a fluorescent inverted microscope;

[0074] S22: Assessing cell proliferation using EdU kit

[0075] MH7A cells were seeded into confocal culture dishes at a density of 2×10^4 cells / well. After the cells were treated as described above, EdU assay was performed according to the manufacturer's instructions. Subsequently, the nuclei were stained with Hoechst33342 for 10 min at room temperature, and the cells were counted under a fluorescence microscope.

[0076] S23: Using JC-1 dye to detect mitochondrial membrane potential

[0077] JC-1 dye (200×) and JC-1 buffer were fully mixed at a ratio of 1:200 and added evenly to each well. Incubate at 37°C for 30 min to stain the mitochondrial membrane. Subsequently, wash twice with JC-1 buffer and add anti-fluorescence quencher. Finally, observe and acquire images by fluorescence inverted microscopy, and evaluate the relative ΔΨm level by the red / green fluorescence ratio of JC-1 dye in mitochondria;

[0078] S24: Use transmission electron microscopy to observe the morphological structure of cell membrane, organelles and nucleus of each group of cells after treatment

[0079] After treatment, cells were collected and fixed with 2.5% glutaraldehyde, and TEM imaging was performed by Servicebio (Wuhan, China);

[0080] S25: Analysis of lipid reactive oxygen species (ROS)

[0081] The cells were seeded in 6-well plates at a density of 1.2×10^5 cells / well, and then stained with 10μM ROS-sensitive probe DCFH-DA for 25min at 37°C. The images were observed and acquired by fluorescence microscopy, and the data were processed using ImageJ. The cells were collected and analyzed by flow cytometry, and the data were processed using FlowJo software.

[0082] S26: cells were seeded in 6-well plates at a density of 1.2×10^5 cells / well;

[0083] After treating the cells as described above, the cell lysates of each group were collected to measure the levels of GSH, MDA, SOD, and iron content using the corresponding kits. The experiments were performed according to the manufacturer's instructions;

[0084] S27: MH7A cells were seeded in 6-well plates at 1×10^6 cells / well.

[0085] After treatment according to the above method, total RNA of MH7A cells was extracted using Trizol reagent, and then reverse transcribed into cDNA using a reverse transcription kit. qRT-PCR experiments were performed using TBGreenPCR master mix. Total RNA was extracted using RNAzol reagent and reverse transcribed into cDNA. According to the manufacturer's instructions, RT-qPCR analysis was performed using the SYBRSelectMasterMix kit. The relative expression level of the target gene was normalized with GAPDH as an internal reference and analyzed using the 2-ΔΔCt method. The primers of the genes were synthesized by Qingke and are listed as follows:

[0086] GAPDH(Forward:5'-GGAGTCCACTGGCGTCTTCA-3'Reverse:5'-GTCATGAGTCCTTCCACGATACC-3');

[0087] Nrf2(Forward: 5'-GAGCGGCTTTGTCTTTGGATTTA-3'Reverse: 5'-CTCATACTCTTTCCGTCGCTGAC-3');

[0088] xCT(Forward: 5'-CCAGGTTCGGGACATAGAGAATC-3'Reverse: 5'-CCCAGTAGAACCAGAATCAGACA-3');

[0089] GPX4(Forward: 5'-GTAAACTACACTCAGCTCGTCGA-3'Reverse: 5'-TTGATCTCTTCGTTACTCCCTGG-3');

[0090] S28: Cultivation of MH7A cells in six-well plates

[0091] After treatment as described above, the cells were lysed with RIPA lysis buffer containing 1% protease inhibitor and 1% phosphatase inhibitor, and the protein concentration was determined using a BCA protein quantification kit (Mishu, Xi'an, China). Subsequently, the protein samples were heat denatured and electrophoresed using a 12% SDS-PAGE gel, and the proteins were subsequently transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk powder at 37°C, and after blocking for 1 hour, the membranes were incubated with a variety of antibodies at 4°C overnight, including Nrf2, xCT, GPX4, GAPDH, and the membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at 37°C for 1 hour. The protein bands were visualized using ECL chemiluminescent solution and detected by ChemiDoc Imaging System. The protein blot bands were quantitatively analyzed using ImageJ software.

[0092] S29: According to the above cell culture method, the MH7A cells after treatment in each group were counted to 2×10^4 cells / well and inoculated into confocal culture dishes.

[0093] After 24 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, permeabilized with 0.3% Triton-100 for 15 minutes, and then blocked with immunofluorescence fast blocking solution for 30 minutes at room temperature. After that, the cells were incubated with various antibodies at 4°C overnight, including Nrf2, xCT and GPX4, and then incubated with fluorescent secondary antibody goat anti-rabbit antibody at room temperature for 1 hour. After that, the cells were incubated with DAPI at room temperature for 10 minutes, and then fluorescence detection was performed using a fluorescence microscope.

[0094] S30: After culturing cells as described above, MH7A cells were seeded in a 12-well plate at a density of 800 cells per well.

[0095] After the cells adhered, the culture medium was changed every 72 h. After 10 days of culture, the cells were fixed with 4% paraformaldehyde and washed with phosphate buffered saline (PBS), then stained with crystal violet and observed and images were obtained using a fluorescent inverted microscope.

[0096] MH7A cells were seeded in 6-well plates at 1×10^5 cells per well, and the cells were cultured to 90% confluence as described above. The cell layer was gently scratched with a 200μL pipette tip to form a wound, and the cell debris was washed away with ice-cold sterile PBS. Subsequently, the cells were cultured in serum-free medium. At 0, 24, and 48 hours after scratching, wound healing was imaged using an inverted microscope (50x magnification), and the wound area was analyzed using ImageJ software. The results were expressed as the migration index, that is, the migration distance of the treated MH7A cells relative to the control group;

[0097] S31: Transwell chamber migration assay was used to detect the migration ability of cells after Nrf2 knockdown

[0098] 500 μl of DMEM medium containing 10% serum was placed in the bottom chamber; in the upper chamber, MH7A cells (final concentration of 5×10^4 cells / ml) were suspended in serum-free medium, and the chamber was incubated at 37°C for 24 hours, and the non-migrated cells in the upper chamber were carefully removed with a cotton swab, and then the cells on the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with crystal violet solution, and then the total number of migrating cells was counted in 5 randomly selected fields of view of each insert using an optical microscope (magnification ×200);

[0099] S32: Cell invasion assay using Transwell inserts

[0100] First, the upper surface of Transwell was pre-coated with Matrigel (1.25 mg / ml, 20 μl / well) at 37°C for 30 min. 500 μl of DMEM medium containing 10% serum was added to the bottom chamber. MH7A cells (5×10^4 cells / well) were added to the upper chamber and incubated in DMEM medium without serum. After incubation for 48 hours at 37°C and 5% CO2, non-invasive cells on the upper membrane surface were gently wiped with a cotton swab. Next, the cells were fixed and stained with crystal violet solution. Finally, the cells on the lower surface were counted at 200x magnification under a fluorescent inverted microscope to quantify the invasive ability of the cells, and the average number of invasive cells was calculated in five random areas.

[0101] S33: Data are expressed as mean ± standard deviation (SD)

[0102] All statistical analyses were performed using GraphPad Prism 9.0. One-way and two-way ANOVA were used to analyze the data, where P < 0.05 was considered statistically significant. Each experiment was repeated three times.

[0103] Example 2

[0104] A method for constructing an animal model of photodynamic therapy for rheumatoid arthritis. In order to judge the therapeutic effect of the therapy, this embodiment conducts different experiments and draws the following conclusions after the experiments:

[0105] (1) CUR-PDT inhibits the progression of arthritis in CIA rats

[0106] In SPF, a CIA rat model was established to evaluate the effects of CUR-PDT on RA. Figure 1 As shown in A, compared with the control group, significant swelling of the left hind paw was observed in both the model group and the treatment group on day 28. However, on day 35, the edema of the left hind paw in the treatment group was reduced, and the reduction in swelling in the CUR-PDT group was the most significant ( Figure 1 B and F). Meanwhile, during the whole experiment, the arthritis scores and left hind limb toe swelling of CIA group rats did not improve, while PDT intervention significantly improved these symptoms, but there was no significant difference in body weight between the groups ( Figure 1 E). In addition, the histopathological examination of the ankle joints of rats by H&E staining showed that the CIA group had obvious pathological features, including inflammatory cell infiltration and abnormal synovial hyperplasia, while the PDT group significantly reduced the number of synovial tissue infiltrating cells and significantly alleviated inflammation ( Figure 1 C), reduced the histopathological scores of CIA rats ( Figure 1 G). In addition, the collagen fibers of each group were quantitatively analyzed by Masson staining, and it was found that the deposition of collagen fibers in the synovial tissue of the CIA group was more severe than that of the healthy control group (Control); while the proportion of collagen fibers in the synovial tissue of the PDT group was significantly lower than that of the Light group and the CUR group; however, there was no significant difference between the Light group and the CIA group, and between the CUR group and the CIA group ( Figure 1 D and I), these findings indicate that the blue-violet light effect alone and the curcumin effect alone did not achieve the purpose of alleviating RA, but after CUR-PDT intervention, the synovial inflammation and hyperplasia of CIA rats can be significantly improved, and the fibrosis of synovial tissue can be alleviated. The above results indicate that CUR-PDT has further research value in the process of alleviating RA;

[0107] like Figure 1As shown, CUR-PDT alleviated synovial inflammation and fibrosis in CIA rats. (A) Cultivation process of CIA-induced RA rats in SPF. (B) Swelling degree of ankle joints of rats in each group on day 42 after CIA modeling (day 28 of treatment). (C and D) Ankle joints harvested on day 42 after modeling (day 28 of treatment). Representative images of H&E staining and Mosson trichrome staining of rats in each group are listed (scale bars: 1 mm and 200 μm). (EG) Body weight, paw volume measurement and joint score of rats in each group were performed every 7 days after the second immunization (0 point: no redness and swelling of ankle joints and paws; 1 point: mild swelling or redness of ankle joints and paws; 2 points: moderate swelling or mild redness of ankle joints and paws; 3 points: redness and swelling of paws below ankle joints; 4 points: severe redness, swelling and deformation of ankle and foot joints). (H) Histological changes of ankle joints The severity of arthritis was scored in three sections of each rat (the severity of arthritis was divided into 4 levels according to the intensity of lining hyperplasia, mononuclear cell infiltration and pannus formation: 0 = normal ankle joint, 1 = normal synovium with occasional mononuclear cells, 2 = definite arthritis with several layers of flat to round synovial lining cells and dense infiltration of scattered mononuclear cells, 3 = marked synovial hyperplasia with three or more layers of loosely arranged lining cells and dense infiltration of mononuclear cells, 4 = severe synovitis with pannus and erosion of articular cartilage and subchondral bone,) (I) The proportion of synovial tissue fibrosis was statistically calculated in three sections of each rat, and statistical analysis was performed by one-way analysis of variance, and multiple comparisons were performed using Tukey's test. Data are expressed as SD ± mean, *p<0.05, **p<0.01, ***p<0.001.

[0108] (2) After CUR-PDT treatment, the level of ferroptosis in RA synovial tissue increased

[0109] Since there were significant differences between the PDT group and the CIA group in alleviating RA synovial inflammation and fibrosis, we selected the Control group, CIA group, and PDT group for subsequent experiments. First, the concentrations of Nrf2, xCT, and GPX4 molecules in the collected rat serum were measured by ELISA, and it was found that the expression of these three molecules in the PDT group was significantly reduced compared with the CIA group ( Figure 2 AC), and then the expression of Nrf2, xCT and GPX4 in the synovial tissue of the ankle joint was detected by immunohistochemistry. The results showed that compared with the Control group, the expression of Nrf2, xCT and GPX4 in the synovial tissue of the CIA group increased, while the expression of these three molecules decreased consistently after PDT intervention ( Figure 2 DI), indicating that PDT may regulate ferroptosis in synovial tissue by affecting the Nrf2-xCT-GPX4 signaling pathway, thereby affecting the progression of rheumatoid arthritis;

[0110] like Figure 2 As shown, CUR-PDT inhibited the expression of Nrf2-xCT-GPX4 molecules in CIA rats. (AC) Elisa assay of Nrf2, xCT and GPX4 in rat serum, (DF) immunohistochemical detection of Nrf2, xCT and GPX4 in rat synovium (scale bar: 100 μm), (GI) quantification of the percentage of Nrf2-positive, xCT-positive and GPX4-positive in the synovium of each group, statistical analysis was performed by one-way ANOVA, and multiple comparisons were performed using Tukey's test, data are expressed as SD ± mean, **p < 0.01, ***p < 0.001.

[0111] (3) CUR-PDT reduces the viability, proliferation and migration ability of MH7A cells

[0112] The above results indicate that PDT may reduce the invasion of synovial cells and the infiltration of inflammatory cells by reducing the expression of Nrf2-xCT-GPX4 molecules in the ferroptosis pathway, thereby inhibiting the progression of RA. Therefore, we hypothesized that CUR-PDT could achieve the purpose of treating RA by promoting ferroptosis in RA synovial tissue, and further explored its mechanism of action in in vitro experiments. In order to detect the effect of photodynamic therapy on the proliferation and migration ability of fibroblast-like synoviocytes, the CCK-8 method was first used to detect the appropriate drug concentration of tumor necrosis factor-a (TNF-a) in the MH7A cell culture system. The results showed that 20 ng / ml TNF-a enhanced the viability of MH7A cells ( Figure 3 A), in addition, to ensure the successful induction of the inflammatory environment of rheumatoid arthritis, IL-1β and IL-6 are the main pro-inflammatory factors in RA. We performed Elisa detection on their contents in the collected cell culture fluid. The results showed that the concentrations of IL-1β and IL-6 in the 20ng / ml TNF-a induced group (TNF-aControl) were significantly higher than those in the uninduced group (NormalControl)( Figure 3 BC), indicating that the RA inflammatory environment was successfully established. Next, in this paper, Control was used to replace the TNF-aControl group in the cell culture system. On the basis of the inflammatory environment, CUR drug concentration and light intensity were detected by CCK-8. After testing, 15μg / mlCUR had no significant effect on cell viability after 24h of treatment, while MH7A activity decreased significantly after treatment with 20, 25 and 30μg / mlCUR ( Figure 3 D), therefore, 15 μg / ml CUR was used in subsequent experiments (CUR group). In addition, cell viability was related to the light intensity of blue-violet light. When the light intensity was 29 and 38 J / cm2, it had a significant inhibitory effect on the viability of MH7A, while the light intensity of 21 J / cm2 had no significant effect on its viability ( Figure 3E), therefore, 21 J / cm2 light intensity was used in subsequent experiments (Light group), but when 15 ug / ml CUR was combined with 21 J / cm2 light intensity to take advantage of PDT, MH7A activity was significantly inhibited ( Figure 3 F), based on this culture condition, we conducted a follow-up study and found that there was no significant difference in the release of lactate dehydrogenase (LDH) under the action of blue-violet light (Light) and curcumin (CUR), but after PDT, the release of lactate dehydrogenase increased significantly ( Figure 3 G), indicating that PDT can damage the MH7A cell membrane to a greater extent and induce cell death. In addition, the proliferation ability of MH7A cells was detected by EdU method and plate cloning experiment. The proliferation ability of MH7A cells was significantly reduced after PDT. However, there was no significant difference between the Light group and the Control group, and between the CUR group and the Control group ( Figure 3 HK), and then the migration ability of MH7A cells was detected by wound healing experiment. The results showed that the wound healing rates of Light group and CUR group at 24h and 48h did not show significant difference with those of Control group, while the wound healing rates of PDT group at 24h and 48h were significantly lower than those of Control group, and the migration area did not change significantly ( Figure 3 LM), these results indicate that blue-violet light alone and curcumin alone do not inhibit the proliferation and migration of MH7A cells, while the combined effect of CUR-PDT can play a significant inhibitory role in disease progression. Therefore, the Control group and PDT group were used for subsequent research;

[0113] like Figure 3As shown in the figure, CUR-PDT reduces the viability, proliferation and migration ability of MH7A cells. (A) Different concentrations of TNF-a were added to perform CCK-8 experiment to detect the viability of MH7A. (BC) IL-1β and IL-6 in the cell culture medium were determined by Elisa. (DF) The viability of MH7A under different conditions was detected. (G) The release of lactate dehydrogenase (LDH) in the Control group, Light group, CUR group and PDT group was detected. (H) The proliferation ability of MH7A was detected by EdU method. The cell nucleus was stained with Hoechst33342 (blue). EdU positive Sex cells are in red (scale bar: 20 μm). (I) EdU-positive cells were quantified in three randomly selected microscopic fields, and the number of positive cells was divided by the total number of cells as the EdU-positive rate. (JK) The proliferation of MH7A was detected by plate cloning assay (scale bar: 500 μm). (LM) The migration ability of each group was detected by wound healing assay (scale bar: 500 μm). Statistical analysis was performed by one-way ANOVA, and multiple comparisons were performed using Tukey's test. Data are expressed as SD ± mean, ns, not significant, *p<0.1, **p<0.01, ***p<0.001.

[0114] (4) CUR-PDT can effectively induce ferroptosis in MH7A cells

[0115] To explore the effect of curcumin-mediated photodynamic therapy (CUR-PDT) on ferroptosis in MH7A, we used the JC-1 fluorescent probe to detect changes in mitochondrial membrane potential to assess mitochondrial damage. The results of the JC-1 experiment showed that the mitochondrial membrane potential of the Control group was higher, and JC-1 aggregated in the mitochondrial matrix in the form of polymers, showing red fluorescence; while in the PDT group, due to the decrease in mitochondrial membrane potential, JC-1 mostly existed in the form of monomers, showing green fluorescence, and the statistical results showed that the ratio of polymers (red fluorescence) to monomers (green fluorescence) in the mitochondria of the PDT group decreased ( Figure 4 AB). In addition, transmission electron microscopy (TEM) observations showed that the mitochondria in the PDT group showed obvious condensation, reduced mitochondrial volume, mitochondrial cristae rupture and reduced number, increased membrane and intramembrane matrix density, and a clear trend of ferroptosis ( Figure 4 C). Since lipid peroxidation is a major feature of ferroptosis, we then focused on detecting intracellular lipid-related products and reactive oxygen species (ROS). We loaded ROS probes into the control group and the PDT group, and performed fluorescence intensity detection and flow cytometry analysis. The results showed that the PDT group had a significant increase in ROS levels ( Figure 4DG), and further detected the concentrations of lipid peroxide (MDA) and glutathione (GSH) to observe the changes in lipid peroxidation-related characteristics in ferroptosis. The results showed that the level of malondialdehyde in the PDT group was significantly higher than that in the control group ( Figure 4 H), while the glutathione content was lower than that of the control group ( Figure 4 I), superoxide dismutase (SOD) is an important antioxidant enzyme that removes oxygen free radicals, especially superoxide anion free radicals, in the body. The results of its level detection showed that the SOD level in the control group was higher than that in the PDT group ( Figure 4 J). In addition, since ferroptosis is a Fe2+-dependent cell death mode, we also detected the iron content in each group. The results showed that the iron content in the PDT group was significantly higher than that in the control group ( Figure 4 K), therefore, based on the impairment of mitochondrial structure and function, the accumulation of ROS and lipid peroxides, the decrease in glutathione and superoxide dismutase levels, and the increase in iron content, it can be concluded that PDT treatment leads to increased ferroptosis in RA fibroblast-like synoviocytes;

[0116] Figure 4 .CUR-PDT induced ferroptosis in MH7A cells. (AB) The changes in mitochondrial membrane potential in the Control group and PDT group were detected by JC-1 fluorescent probe. Red is polymer and green is monomer (scale bar: 500 μm). The polymer / monomer ratio was used to represent the changes. (C) Transmission electron microscopy (TEM) reflected the damage of cell membrane and organelles (scale bar: 1.0 μm and 200 nm). (D and F) The ROS ratio of each group was detected. ROS was green and the cell nucleus was stained blue with Hoechst (scale bar: 20 μm). (E and G) The ROS level was detected by flow cytometry. (HK) The concentrations of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and Fe2+ were detected. Statistical analysis was performed by one-way ANOVA and multiple comparisons were performed using Tukey's test. Data are expressed as SD±mean, ns, not significant, *p<0.05, **p<0.01, ***p<0.001.

[0117] (5) Nrf2-xCT-GPX4 pathway participates in CUR-PDT-induced ferroptosis in MH7A cells

[0118] The above experiments verified that PDT can effectively reduce the cell activity, proliferation and migration ability of RA-MH7A, and obvious ferroptosis was observed in the PDT group. In order to further explore the molecular changes of the Nrf2-xCT-GPX4 pathway in the process of PDT-induced ferroptosis of MH7A cells, we detected the expression of related indicators. Through real-time quantitative polymerase chain reaction (qRT-PCR) analysis, it was found that 4 hours after PDT treatment, the expression levels of Nrf2, xCT and GPX4 genes in the PDT group were significantly decreased compared with those in the control group ( Figure 5 AC). In addition, 8 hours after PDT treatment, the protein abundance of these three molecules was significantly decreased as detected by Western blot technology ( Figure 5 DG), then we used immunofluorescence to observe the expression and distribution of these three molecules in MH7A. The results showed that the fluorescence intensity of Nrf2, xCT and GPX4 molecules in the PDT group was significantly weaker than that in the control group ( Figure 5 HM), and Nrf2 is widely expressed in cells, mainly concentrated in the nucleus; xCT is mainly distributed in the cytoplasm, and GPX4 is distributed in the cytoplasm, cell membrane and nucleus. In summary, it is confirmed that CUR-PDT-induced ferroptosis of rheumatoid arthritis fibroblast-like synoviocytes is closely related to the significant decrease in the expression of molecules in the Nrf2-xCT-GPX4 pathway;

[0119] Figure 5 Nrf2-xCT-GPX4 pathway is involved in CUR-PDT-induced ferroptosis in MH7A cells. (AC) mRNA expression levels of Nrf2, xCT, and GPX4 in the Control and PDT groups. (DG) Protein expression levels of Nrf2, xCT, and GPX4. (HM) Expression of Nrf2, xCT, and GPX4. Nrf2 was stained red with 594 goat anti-rabbit secondary antibody, xCT and GPX4 were stained green with 488 goat anti-rabbit secondary antibody, and cell nuclei were stained blue with DAPI (scale bar: 20 μm). Statistical analysis was performed by one-way ANOVA and multiple comparisons were performed using Tukey's test. Data are expressed as SD ± mean, ns, not significant, *p < 0.05, **p < 0.01, and ***p < 0.001.

[0120] (6) Downregulation of Nrf2 combined with CUR-PDT exacerbates MH7A cell ferroptosis

[0121] In order to explore the relationship between the upstream transcription factor Nrf2 and xCT-GPX4 in the classical ferroptosis pathway, this study, based on the use of CUR-PDT treatment, further knocked down the expression of Nrf2 molecules by siRNA transfection technology to study the effect of CUR-PDT on ferroptosis of RA-MH7A cells. The experiments were divided into five groups: control group (Control), photodynamic therapy group (PDT), negative control group (NC), Nrf2 knockdown group (si-Nrf2) and Nrf2 knockdown combined with photodynamic therapy group (si-Nrf2+PDT). First, through cell live-death staining, it was observed that the number of live cells (green fluorescence) in the Nrf2 knockdown combined with photodynamic therapy group (siNrf2+PDT) was more significantly reduced than that in the photodynamic therapy group (PDT) and the Nrf2 knockdown group (si-Nrf2), while the number of dead cells (red fluorescence) was significantly increased, resulting in a significant decrease in the ratio of live cells to dead cells in this group ( Figure 6 AB). In addition, the CCK-8 method and lactate dehydrogenase (LDH) release experiment further verified that the cell viability of the siNrf2+PDT group was significantly lower than that of the PDT group and the si-Nrf2 group, while the LDH release was significantly higher than that of the latter ( Figure 6 CD), further indicating that the siNrf2+PDT group could significantly promote the death of MH7A cells. Subsequently, more significant mitochondrial condensation and mitochondrial cristae damage were observed in the siNrf2+PDT group than in the other groups, and a significant decrease in mitochondrial membrane potential was detected, which was determined by transmission electron microscopy (TEM) and JC-1 method ( Figure 6 EG), and then, after loading with ROS probe, fluorescence detection and flow cytometry analysis were performed, and the results showed that the ROS level in the siNrf2+PDT group was significantly higher than that in the PDT group and the si-Nrf2 group ( Figure 6 HK), at the same time, other signs of ferroptosis, such as the concentrations of glutathione (GSH) and superoxide dismutase (SOD), malondialdehyde (MDA) and iron content, showed that the concentrations of GSH and SOD in the si-Nrf2+PDT group were significantly lower than those in the other groups, while the concentration of malondialdehyde (MDA) and iron content were significantly higher than those in the other groups ( Figure 6 LO), therefore, it can be concluded that CUR-PDT further promotes ferroptosis of MH7A cells through the knockdown of Nrf2, which provides a basis for the subsequent exploration of the effect of CUR-PDT on the proliferation of MH7A cells after the knockdown of Nrf2;

[0122] Figure 6The combination of downregulation of Nrf2 and CUR-PDT exacerbated ferroptosis in MH7A cells. (AB) Live-dead staining of MH7A cells. Calcein stained live cells green and PI stained dead cells red (scale bar: 500 μm). (C) CCK-8 was used to detect MH7A viability. (D) LDH release was detected. (EF) Changes in mitochondrial membrane potential were detected by JC-1 fluorescent probe. Red is polymer and green is monomer (scale bar: 500 μm). The ratio of polymer / monomer was used to represent the changes. (G) Transmission electron microscopy (TEM) reflected the damage of cell membrane and organelles (scale bar: 1.0 μm and 200nm), (HK) The ROS level of each group was detected by fluorescence inverted microscopy and flow cytometry, ROS was green, and the cell nucleus was stained blue with Hoechst (scale bar: 20μm), (LO) The concentrations of malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and Fe2+ in each group were detected, and statistical analysis was performed by one-way analysis of variance, and multiple comparisons were performed using Tukey's test, and the data are expressed as SD±mean, ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0123] (7) The combined action of si-Nrf2 and CUR-PDT accelerates the ferroptosis process of MH7A cells through the Nrf2-xCT-GPX4 pathway

[0124] The above experimental results showed that after knocking down Nrf2 molecules, CUR-PDT significantly enhanced ferroptosis in RA-MH7A cells. Subsequently, to further explore whether CUR-PDT regulates ferroptosis in MH7A cells by affecting the classical ferroptosis pathway xCT-GPX4 under the condition of Nrf2 knockdown, we detected the changes in the molecular expression of this pathway. The specific methods included qRT-PCR ( Figure 7 AC)、Western blot( Figure 7 DG) and immunofluorescence experiments ( Figure 7 HM) to verify the correlation of Nrf2-xCT-GPX4 pathway. The results showed that after Nrf2 knockdown and CUR-PDT treatment, the expression levels of Nrf2, xCT and GPX4 were significantly decreased compared with those of Nrf2 knockdown group and PDT group ( Figure 7 These results further confirmed that CUR-PDT could regulate the Nrf2-xCT-GPX4 pathway and promote ferroptosis of rheumatoid arthritis fibroblast-like synoviocytes by reducing the expression of molecules in this pathway, thereby reducing the number of MH7A cells and decreasing their cell viability;

[0125] Figure 7.si-Nrf2+CUR-PDT accelerates ferroptosis in MH7A cells through the Nrf2-xCT-GPX4 pathway. (AC) mRNA expression levels of Nrf2, xCT and GPX4 in the Control group, PDT group, NC group, si-Nrf2 group and si-Nrf2+PDT group. (DG) Protein expression levels of Nrf2, xCT and GPX4. (HM) Expression of Nrf2, xCT and GPX4. Nrf2 was stained red by 594 goat anti-rabbit secondary antibody, xCT and GPX4 were stained green by 488 goat anti-rabbit secondary antibody, and cell nuclei were stained blue by DAPI (Scale bar: 20 μm). Statistical analysis was performed by one-way ANOVA and multiple comparisons were performed using Tukey's test. Data are expressed as SD ± mean, ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0126] (8) The combined action of si-Nrf2 and CUR-PDT on the Nrf2-xCT-GPX4 pathway significantly inhibited the proliferation, invasion and migration of MH7A cells

[0127] In addition, to clarify the role of PDT in reducing MH7A cell activity and weakening MH7A proliferation, migration and invasion through the Nrf2-xCT-GPX4 pathway, we performed the following verification: plate cloning and EDU methods were used to detect the proliferation ability of MH7A. The results showed that the colony number and proliferation rate of the si-Nrf2+PDT group were significantly lower than those of the other groups. These results indicate that the PDT group and si-Nrf2 group weakened the proliferation ability of MH7A cells, while the combined effect of Nrf2 knockdown and CUR-PDT had a more significant inhibitory effect on the proliferation ability of MH7A ( Figure 8 AD), the horizontal and vertical migration abilities of MH7A cells were determined by wound healing and Transwell migration assays, respectively. The results of the wound healing experiment showed that the wound healing rates of the PDT group at 24h and 48h were significantly lower than those of the control group, indicating that CUR-PDT could inhibit the horizontal migration ability of MH7A. However, after combined treatment, the wound healing rate was further reduced, and the wound healing area was not significantly reduced, which weakened the horizontal migration ability of cells ( Figure 8 EF), the results of Transwell migration assay showed that the number of vertical migration cells in the PDT group was lower than that in the control group, and after the combined action, the vertical migration rate of MH7A was even more significantly decreased than that in the PDT group and si-Nrf2 group. This result shows that the combined action can inhibit the vertical migration ability of MH7A cells ( Figure 8GH), and then the Transwell invasion assay also revealed that the number of invasive cells in the PDT group and the si-Nrf2 group was significantly lower than that in the control group, indicating that CUR-PDT and Nrf2 knockdown both weakened the invasive ability of MH7A cells. After si-Nrf2+PDT, the number of invasive cells decreased significantly, further weakening the invasive ability of the cells and the infiltration of MH7A cells into the surrounding tissues ( Figure 8 IJ), the above results show that the combined effect of CUR-PDT and Nrf2 knockdown can further inhibit the proliferation, migration, invasion and other tumor-like proliferation characteristics of rheumatoid arthritis fibroblast-like synoviocytes compared with the single effect of CUR-PDT and si-Nrf2, and alleviate the infiltration and damage of the cells to the surrounding tissues. These results not only confirm that CUR-PDT can regulate RA-MH7A ferroptosis through the Nrf2-xCT-GPX4 pathway, but also more intuitively present to us the therapeutic significance of CUR-PDT by inhibiting this pathway. At the same time, targeting Nrf2 will also become an efficient therapeutic strategy for the treatment of RA.

[0128] Figure 8 si-Nrf2+PDT significantly inhibited the proliferation, invasion and migration of MH7A cells through the Nrf2-xCT-GPX4 pathway. (AB) The proliferation ability of MH7A in the Control group, CUR-PDT group, NC group, si-Nrf2 group and si-Nrf2+PDT group was detected by plate cloning assay (scale bar: 500μm). (CD) The proliferation ability of MH7A was detected by EdU. The cell nucleus was stained with Hoechst33342 (blue), and EdU-positive cells were stained red (scale bar: 20μm). EdU-positive cells were quantified in three randomly selected microscopic fields. The EdU positivity rate was calculated as the positive cells divided by the total number of cells. (EF) The horizontal migration ability of MH7A was detected by wound healing assay (scale bar: 500 μm), (GH) the vertical migration ability of MH7A was detected by migration assay (scale bar: 500 μm), (IJ) the invasive ability of MH7A was detected by invasion assay (scale bar: 500 μm). Statistical analysis was performed by one-way ANOVA and multiple comparisons were performed using Tukey's test. Data are expressed as SD ± mean, ns, not significant, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

[0129] (9) Figure 9. CUR-PDT induced RA-MH7A ferroptosis pathway model, such as Fig. 9As shown in the figure, treatment of MH7A with CUR-PDT resulted in a decrease in its proliferation, invasion and migration abilities, which may be related to the accumulation of ROS in MH7A and the decreased expression of the Nrf2-xCT-GPX4 signaling pathway, which triggered MH7A ferroptosis.

[0130] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.

Claims

1. A method for constructing an animal model of rheumatoid arthritis photodynamic therapy, characterized in that: The following steps are involved: Sample selection: 8-week-old healthy SPF male Sprague-Dawley (SD) rats were selected, a total of 25 rats, 5 rats per cage; Among 25 SD rats, 5 were randomly selected as the normal control group (Control), and the remaining 20 rats were used to establish the collagen-induced arthritis (CIA) model; Bovine type II collagen was mixed with incomplete Freund's adjuvant and fully emulsified on ice to prepare type II collagen emulsion; After anesthesia with amobarbital, the rats were injected with the prepared type II collagen emulsion at multiple points on the back and tail base under sterile conditions, which was regarded as the day 0 of the primary immunization; On the 7th day after modeling, the mice were given a booster immunization with the same dose of bovine type II collagen mixed with complete Freund's adjuvant; the control group was treated with an equal volume of normal saline. On the 14th day after modeling, the 20 CIA rats with successfully established models were randomly divided into 4 groups: model group (CIA), blue-violet light group (Light), curcumin group (Cur), and photodynamic therapy group (PDT); Starting from the 15th day after the initial immunization, rats in each group were treated accordingly, with daily treatment for 28 days; Arthritis scores were performed every 7 days after the second immunization; On the 42nd day after modeling, all rats were euthanized with 5% amobarbital, and ankle joints and blood samples were collected from the rats.

2. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 1, characterized in that: On the 14th day after the initial immunization, body weight measurement, paw volume monitoring and joint scoring were performed, and body weight measurement, paw volume monitoring and arthritis scoring were performed every 7 days. The arthritis scoring criteria were as follows: 0 point: no redness or swelling of the ankle joint and paw; 1 point: mild swelling or redness of the ankle joint and paw; 2 points: moderate swelling or mild redness of the ankle joint and paw; 3 points: the claw is red and swollen below the ankle joint; 4 points: Severe redness, swelling and deformation of the ankle and foot joints.

3. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 2, characterized in that: The resected ankle joint tissues were fixed, decalcified, paraffin-embedded, sliced, and stained. The images were obtained by microscopy, and the degree of fibrosis was quantitatively analyzed using ImageJ software.

4. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 3, characterized in that: Hematoxylin-eosin (H&E) staining was used to observe the inflammatory cell infiltration, bone and cartilage destruction, and synovial hyperplasia in the synovial tissue, thereby providing a pathological score for the joint sections. The score was determined based on previous reports in the literature.

5. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 4, characterized in that: 1.2×10^5MH7A cells / well were seeded in DMEM medium containing 10% FBS. After the cells adhered, the cells were stimulated with recombinant human interferon TNF-α for 24 hours to induce an inflammatory environment.

6. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 5, characterized in that: Curcumin dissolved in DMSO was treated with a final concentration of 15 μg / ml for 24 h and the parameters were 460 nm wavelength and 21 J / cm 2 The cells were irradiated with blue-violet light of high energy intensity and the lysate was finally harvested.

7. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 6, characterized in that: The si-Nrf2 and negative control group small interfering RNAs were transfected using Lipofectamine3000 reagent. Six hours after transfection, the culture medium was replaced with fresh culture medium without penicillin-streptomycin. Subsequent experiments were performed according to the above cell culture method, and the transfection efficiency was evaluated by measuring the fluorescence intensity and Western blot technology.

8. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 7, characterized in that: The levels of Nrf2, SLC7A11, and GPX4 in serum samples and the levels of IL-1β and IL-6 in cell culture supernatants were determined using commercial ELISA kits.

9. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 8, characterized in that: Cell viability was assessed using the cell counting kit-8 (CCK-8). MH7A cells were seeded in a 96-well plate at a density of 4×10^3 cells / well and incubated in an incubator at 5% CO2 and 37°C. After the cells adhered to the wall, they were treated with different concentrations of curcumin solution for 24 hours, 48 ​​hours and 72 hours. DMEM solution and CCK-8 reagent were mixed in a ratio of 1:9, 100 μl of the mixture was added to each well, and incubated for another hour. The optical density (OD) was measured at 450 nm using a microplate reader. The optical density (OD) of the cells under different light intensities was tested using the same method.

10. The method for constructing an animal model of rheumatoid arthritis photodynamic therapy according to claim 9, characterized in that: The following steps are also included: A lactate dehydrogenase assay kit was used to determine the integrity of the cell membrane; The ratio of live cells to dead cells was evaluated using the AM-PI kit; the proliferation ability of cells was evaluated using the EDU kit; the mitochondrial membrane potential was detected using JC-1 dye; the morphological structure of the cell membrane, organelles and nucleus of each group of cells after treatment was observed using a transmission electron microscope; lipid reactive oxygen species (ROS) were analyzed; the levels of GSH, MDA, SOD and iron content were determined; the relative expression levels of target genes were analyzed by qRT-PCR experiments; based on the qRT-PCR experiments, the protein blot bands were quantitatively analyzed using ImageJ software; immunofluorescence experiments were performed; crystal violet staining was performed, and images were observed and acquired using a fluorescent inverted microscope.

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