Application of YY1 in inhibition of lung epithelial cell ferroptosis and removal of intracellular mycobacteria through nitrogen killing

Abstract

The invention discloses application of YY1 in inhibition of pulmonary epithelial cell ferroptosis and removal of intracellular mycobacteria through nitrogen killing, belongs to the field of development and application of anti-tuberculosis drug targets, and aims to clarify a target capable of inhibiting the pulmonary epithelial cell ferroptosis and killing the intracellular mycobacteria. Research finds that YY1 inhibits ferroptosis in mycobacterium marinum infected pulmonary epithelial cells, increases GSH expression quantity, promotes cell survival rate and reduces MDA expression; overexpression of YY1 promotes iNOS expression, generated NO is induced to kill intracellular bacteria, and intracellular bacteria removal is facilitated; yY1 is in targeted combination with-655 to-1018bp segments of an iNOS promoter, so that the effect of enhancing iNOS expression to promote sterilization is realized.

Description

Technical Field

[0001] The present invention belongs to the field of development and application of anti-tuberculosis drug targets, and specifically relates to the application of YY1 in inhibiting ferroptosis of lung epithelial cells and removing intracellular mycobacteria through nitrogen killing. Background Art

[0002] The TB pathogen Mycobacterium tuberculosis has a unique survival strategy that allows it to evade the host's innate immune response, persist in the body without causing obvious symptoms, and spread by exploiting the host's inflammatory response. Infection with Mycobacterium tuberculosis can induce different forms of host cell death, thereby affecting the pathogen's survival within the cell.

[0003] Mycobacterium tuberculosis infection is highly correlated with iron dependence. Many studies have shown that sufficient iron sources not only promote the proliferation of Mycobacterium tuberculosis in the body, but may also trigger oxidative stress and apoptosis signals in macrophages, thereby weakening the host's anti-tuberculosis immune response. In addition, increased iron content has been considered an independent factor in the increased risk of patients with active pulmonary tuberculosis, further emphasizing the importance of iron metabolism regulation in the pathogenesis of tuberculosis.

[0004] Ferroptosis is a programmed cell death characterized by excess iron and lipid peroxidation, and it has been shown to be associated with a variety of diseases. In tumor research, ferroptosis can inhibit the proliferation and metastasis of tumor cells by regulating iron metabolism and lipid peroxidation. In the study of neurodegenerative diseases, by regulating the ferroptosis process, the death of neurons can be slowed down, thereby improving patient prognosis. However, the role of ferroptosis in pathogen infection in vivo is still unclear. Microbial infection involves complex interactions between the host and pathogens. In-depth exploration of the mechanism of ferroptosis will help provide new perspectives for the treatment of related diseases.

[0005] The transcription factor YinYang 1 (YY1) is a member of the zinc finger protein class and is an important transcription factor involved in a variety of biological functions. YY1 is overexpressed in a range of cancers and is both an activator and an inhibitor, which can promote tumor growth or inhibit tumors. YY1 induces drug resistance by regulating cell death patterns in cancer development and avoids tumor cell death. YY1 has been closely related to cancer since its discovery, but research on its interaction with pathogens is relatively limited. We studied the expression and regulation of YY1 in ferroptosis caused by mycobacterium infection, hoping to discover new strategies for YY1 to regulate mycobacterium intracellular survival during mycobacterium infection by regulating the relevant mechanisms of ferroptosis. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an application of YY1 in inhibiting ferroptosis of lung epithelial cells and removing intracellular mycobacteria through nitrogen killing. The present invention firstly finds that YY1 inhibits ferroptosis in lung epithelial cells infected with marine mycobacterium, increases the expression of glutathione (glutathione, GSH), reduces the expression of lipid peroxidation product malondialdehyde (malondialdehyde, MDA), and improves cell survival rate. At the same time, YY1 promotes the increase of nitric oxide synthase (Inducible nitric oxide synthase, iNOS) expression, and induces the production of nitric oxide (Nitric Oxide, NO) to kill intracellular bacteria and reduce the content of intracellular bacteria. YY1 has a binding site with the upstream -655 to -1018bp segment of the iNOS promoter, thereby regulating the expression of iNOS.

[0007] To solve the above technical problems, the embodiments of the present invention provide the following technical solutions:

[0008] Application of transcription factor YY1 in the preparation of ferroptosis inhibitors.

[0009] Application of transcription factor YY1 as a ferroptosis inhibitor in the preparation of anti-Mycobacterium tuberculosis drugs.

[0010] Preferably, the transcription factor YY1 inhibits ferroptosis of lung epithelial cells.

[0011] Preferably, transcription factor YY1 inhibits ferroptosis of lung epithelial cells, increases glutathione expression, and reduces the expression of lipid peroxidation product malondialdehyde.

[0012] Preferably, the transcription factor YY1 promotes the expression of nitric oxide synthase and induces the production of nitric oxide.

[0013] The beneficial effects of the above technical solution of the present invention are as follows:

[0014] In the present invention, studies have shown that YY1 inhibits ferroptosis in lung epithelial cells infected with Mycobacterium marinum, increases GSH expression, promotes cell survival rate, and reduces MDA expression; YY1 expression promotes iNOS expression, and the induced NO kills intracellular bacteria, which helps to eliminate intracellular bacteria; YY1 targets and binds to the -655 to -1018bp segment of the iNOS promoter, thereby enhancing iNOS expression and promoting bactericidal effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is an analysis diagram of the experimental results of YY1 inhibiting ferroptosis caused by Mycobacterium marinum infection of lung epithelial cells in Example 1, wherein: Figure 1 A is a schematic diagram of flow cytometry detection of cell ferroptosis after Mycobacterium marinum infected wild-type lung epithelial cell lines and YY1 knockdown A549 cell lines; Figure 1 B is a quantitative statistical graph of cell ferroptosis detected by flow cytometry after Mycobacterium marinum infected wild-type lung epithelial cell lines and YY1 knockdown A549 cell lines; Figure 1 C is the result of the detection of glutathione (GSH) release after Mycobacterium marinum infected wild-type lung epithelial cells and YY1 knockdown A549 and H1299 cell lines;

[0016] Figure 1 D is the result of detecting malondialdehyde (MDA) release after Mycobacterium marinum infected wild-type lung epithelial cells and YY1 knockdown A549 and H1299 cell lines;

[0017] Figure 2 This is a result analysis diagram of the experiment of overexpressing YY1 to kill intracellular bacteria in Example 2, wherein: Figure 2 A is a schematic diagram of the intracellular bacterial load (CFU) after Mycobacterium marinum infected H1299 cells overexpressing YY1; Figure 2 B is a quantitative statistical graph of intracellular bacterial load (CFU) after Mycobacterium marinum infected H1299 cells overexpressing YY1; Figure 2 C is a flow cytometry diagram of cells overexpressing YY1 after using iNOS inhibitor 1400W; Figure 2 D is a statistical graph showing the quantification of intracellular bacterial content in cells overexpressing YY1 using the iNOS inhibitor 1400W;

[0018] Figure 3 This is an analysis diagram of the experimental results of YY1 in Example 3 that affects sterilization by regulating the iNOS specific region promoter, wherein: Figure 3 A is a schematic diagram of the construction of the iNOS promoter plasmid, P0 is the dual-luciferase empty plasmid, P1 is the promoter sequence without predicted binding sites, P2 is the promoter sequence containing five predicted sites, and P3 is the full-length promoter sequence; Figure 3 B is the quantitative statistical graph of dual luciferase assay. DETAILED DESCRIPTION

[0019] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0020] The embodiments of the present invention provide the application of YY1 in inhibiting ferroptosis of lung epithelial cells and eliminating intracellular mycobacteria through nitrogen killing, and the application of transcription factor YY1 as a ferroptosis inhibitor in the preparation of anti-Mycobacterium tuberculosis drugs, which is specifically manifested in that transcription factor YY1 inhibits ferroptosis of lung epithelial cells, increases the expression of glutathione, reduces the expression of lipid peroxidation product malondialdehyde, and improves cell survival rate. At the same time, transcription factor YY1 promotes the expression of nitric oxide synthase, induces the production of nitric oxide to kill intracellular bacteria, and reduces the content of intracellular bacteria.

[0021] Next, the technical scheme of the present application is further described in detail in conjunction with specific preparation examples and examples. The following preparation examples and examples are only exemplary illustrations and explanations of the present application, and should not be construed as limiting the scope of protection of the present application. Those skilled in the art make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned invention content, which still belong to the scope of protection of the present application. Unless otherwise stated, the raw materials and reagents used in the following preparation examples and examples are all commercially available products, or can be prepared or formulated by known methods or reagent instructions.

[0022] Example 1: YY1 inhibits ferroptosis caused by Mycobacterium marinum infection of lung epithelial cells

[0023] 1. Experimental purpose and principle

[0024] The purpose of the experiment is to detect the ferroptosis phenotype of lung epithelial cells A549 and H1299 with knockdown of YY1 after infection with mycobacteria. The experimental principle is to detect cell death with or without ferroptosis inhibitors, and to detect the release of GSH and MDA to translate the occurrence of ferroptosis.

[0025] 2. Preparation of experimental reagents

[0026] Preparation of Fer-1: 5 mg of powder was dissolved in 1.9059 mL of DMSO to prepare a 10 mM Fer-1 solution (Ferrostatin-1 (Fer-1) from Shanghai Yien Chemical Technology Co., Ltd., product number R022075);

[0027] Dead / live dye preparation: After the FVS700 powder is equilibrated to room temperature, add 310 μL of cell culture grade dimethyl sulfoxide (DMSO) and mix thoroughly until the powder is completely dissolved. The FVS700 solution is divided into 10 μL portions and frozen to avoid repeated freezing and thawing (using BD Biosciences' FixableViability Stain 700 (FVS-700), catalog number 564997);

[0028] Preparation of PBS containing 1% BSA: weigh 1 g of bovine serum albumin (BSA) and add it to 100 mL of PBS and mix thoroughly;

[0029] GSH standard solvent application solution: before use, take 1mL of standard solvent stock solution and add it to 9mL of double distilled water and mix well. Prepare and use it immediately; (Use the reduced glutathione (GSH) determination kit (microplate method) of Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., item number A006-2-1)

[0030] 20μmol / L GSH standard: Add 10mL of GSH solvent application solution to the standard powder (3.07mg) and mix thoroughly to obtain a 1mmol / L GSH standard solution; add 0.2mL of the 1mmol / L GSH standard solution to 9.8mL of GSH solvent application solution and mix thoroughly for later use;

[0031] MDA working solution: prepare the working solution according to the ratio of reagent 1:2:3=0.2:3:1 mentioned in the reagent manual, and use it immediately (using the cell malondialdehyde (MDA) determination kit (microplate method) of Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., product number A003-4-1).

[0032] 3. Experimental Procedure

[0033] 1. Cell ferroptosis detection:

[0034] 1) Cell subplating and bacterial infection:

[0035] Trypsinize the cells, resuspend them in complete medium and count them at 1 × 10 5 After the cells were completely attached to the wall, Fer-1 (working concentration 10 μM) was added, and an equal volume of DMSO was added to the other groups. After 1 hour, Mycobacterium marinum was added to infect the cells at an MOI of 4 and cultured at 32°C for 12 hours.

[0036] 2) Sample collection:

[0037] The culture supernatant was removed and the cells were rinsed three times with PBS (to remove extracellular bacteria); the cells were digested with EDTA-free trypsin, 300 μL PBS was added to blow off and resuspend the cells, and the cell suspension was transferred to a 1.5 mL centrifuge tube; the cells were centrifuged at 500 g for 5 min at 4°C and the supernatant was discarded; the cells were washed twice with PBS containing 1% BSA.

[0038] 3) Sample processing:

[0039] Dilute FVS-700 dye at 1:1000, add 100 μL dye to each tube, and stain for 1 hour at room temperature in the dark; centrifuge at 500g for 5 minutes at 4°C, discard the supernatant; wash the cells once with PBS containing 1% BSA, and add 300 μL PBS to resuspend the cells.

[0040] 4) Flow cytometry detection:

[0041] The detection was performed using the APC-700 channel of a Beckman flow cytometer.

[0042] 5) Use Flowjo software to analyze and export the results.

[0043] 2. GSH content determination:

[0044] 1) Cell subplating and bacterial infection:

[0045] Trypsinize the cells, resuspend them in complete medium and count them at 1 × 10 6 After the cells were completely attached to the wall, Mycobacterium marinum was added at a ratio of MOI = 4 and cultured at 32°C for 8 h.

[0046] 2) Sample collection:

[0047] The culture supernatant was removed, the cells were rinsed twice with PBS, and the cells were collected by trypsin digestion; centrifuged at 800g for 10 min at 4°C, the supernatant was discarded, and 300 μL PBS was added for resuspending.

[0048] 3) Sample processing:

[0049] Ultrasonic disruption was performed on ice at 200W for 10s, with an interval of 15s, and repeated 5 times; 100μL of the cell suspension after disruption was taken, 100μL of reagent 1 was added, and the mixture was thoroughly mixed; centrifuged at 3500g for 10min, and the supernatant was taken as the sample to be tested.

[0050] 4) Determination:

[0051] In a 96-well plate, add the reagents in the order shown in the table below, mix thoroughly and let stand for 5 minutes; measure the absorbance at 405 nm on an ELISA reader.

[0052] Blank hole Standard hole Determination hole Reagent 1 (μL) 100 / / 20μmol / LGSH standard / 100 / Supernatant (μL) / / 100 Reagent 2 (μL) 100 100 100 Reagent 3 (μL) 25 25 25 .

[0053] 3. Determination of MDA content

[0054] 1) Cell subplating and bacterial infection:

[0055] The cells were digested with trypsin, resuspended in complete medium and counted, and plated into a 6-well plate at 1×106 cells / well; after the cells were completely attached to the plate, Mycobacterium marinum was added at an MOI of 4 and cultured at 32°C for 8 h.

[0056] 2) Sample collection:

[0057] The cells were rinsed twice with PBS, digested with trypsin, collected, centrifuged at 800 g, 4°C for 10 min, the supernatant was discarded, and 500 μL of reagent V was added for resuspending.

[0058] 3) Sample processing:

[0059] Ultrasonication was performed on ice at 200 W for 10 s, with an interval of 15 s, and repeated 5 times.

[0060] 4) Determination:

[0061] Add 1 mL of working solution to a 1.5 mL centrifuge tube, add 100 μL of anhydrous ethanol to a blank tube, add 100 μL of 10 nmol / mL standard to a standard tube, and add 100 μL of ultrasonic sample to a measuring tube; mix thoroughly with a vortex mixer, boil in a metal bath at 100°C for 3 h; cool in running water for 5 min, and centrifuge at 4000 g for 10 min; take 250 μL of reaction solution from each tube and add it to a 96-well plate, and measure the absorbance at a wavelength of 530 nm on an ELISA reader.

[0062] According to the above experimental process, the results obtained are as follows Figure 1 As shown, Figure 1 A. Figure 1 B is a schematic diagram and quantitative statistical diagram of flow cytometry detection of cell ferroptosis after wild-type lung epithelial cells and A549 cell lines with YY1 knocked down were infected with marine mycobacterium, indicating that after mycobacterium infection, cell death increased after YY1 knocked down. From the results, it can be seen that ferroptosis increased in YY1 knocked down cells, and there was a higher mortality rate after marine mycobacterium infection; Figure 1 C. Figure 1 D is the detection of glutathione (GSH) and malondialdehyde (MDA) release after Mycobacterium marinum infected wild-type lung epithelial cells and YY1 knockdown A549 and H1299 cell lines. The results showed that the GSH content was reduced, the MDA content was increased, and there was a more serious oxidative reaction in the YY1 knockdown cells. The above results also suggest that YY1 can inhibit ferroptosis.

[0063] Example 2: Overexpression of YY1 can kill intracellular bacteria

[0064] 1. Experimental purpose and principle

[0065] The purpose of the experiment is to detect the intracellular bacterial content of lung epithelial cells H1299 overexpressing YY1 after infection with mycobacteria. The experimental principle is to detect the intracellular bacterial content in overexpressing YY1 and empty cells by CFU, and to detect the intracellular fluorescent bacteria content in cells overexpressing YY1 after using iNOS inhibitors by flow cytometry to reflect the interaction between YY1 and iNOS.

[0066] 2. Basic information of reagents

[0067] Middle Brook 7H10 agar was obtained from Haibo Biotechnology Co., Ltd., with the catalog number HB6270; iNOS was obtained from Abcam, with the catalog number ab178945; 1400W was obtained from Shanghai Taoshu Biotechnology Co., Ltd., with the catalog number T3491; and hygromycin B was obtained from Shanghai Aladdin Biochemical Technology Co., Ltd., with the catalog number H113147.

[0068] 3. Preparation of experimental reagents

[0069] 0.1% Triton-X100 preparation: add 10 μL Triton-X100 into 10 mL distilled water and mix well.

[0070] 1400W preparation: Dissolve 5 mg of powder in 1.9986 mL of distilled water to prepare a 10 mM 1400W solution.

[0071] IV. Experimental Procedure

[0072] 1. Colony forming unit (CFU)

[0073] 1) Cell splitting, adding inhibitors, and bacterial infection:

[0074] Trypsinize the cells, resuspend them in complete medium and count them at 1 × 10 5 After the cells were completely attached to the wall, Fer-1 (working concentration 10 μM) was added, and an equal volume of DMSO was added to the other groups. After 1 hour, Mycobacterium marinum was added to infect the cells at an MOI of 4 and cultured at 32°C for 12 hours.

[0075] 2) Sample pre-treatment:

[0076] The culture supernatant was discarded and the cells were washed with PBS for 3 times. 300 μL 0.1% Triton-X100 was added to blow off the cells, and the cells were repeatedly blown to rupture the cell membrane and release the intracellular bacteria.

[0077] 3) Dilute and coat the plate:

[0078] The cell suspension was appropriately diluted, evenly spread on 7H10 plates, and counted after incubation at 32°C for 7-10 days.

[0079] 2. Fluorescent intracellular bacteria detection

[0080] 1) Cell splitting, adding inhibitors, and bacterial infection:

[0081] Trypsinize the cells, resuspend them in complete medium and count them at 1 × 10 5 The cells were plated into 24-well plates. After the cells were completely attached to the wall, 1400W (working concentration 15 μM) and Fer-1 (working concentration 10 μM) were added. An equal volume of DMSO was added to the group without Fer-1. After 1 h, Mycobacterium marinum (containing pGiles-pLeft-hisBFP fluorescent plasmid) was added to infect the cells at an MOI of 4 and cultured at 32°C for 12 h.

[0082] 2) Sample pre-treatment:

[0083] The culture supernatant was removed and the cells were rinsed three times with PBS (to remove extracellular bacteria); the cells were digested with EDTA-free trypsin, 300 μL PBS was added to blow off and resuspend the cells, and the cell suspension was transferred to a 1.5 mL centrifuge tube; the cells were centrifuged at 500 g for 5 min at 4°C and the supernatant was discarded; the cells were washed twice with PBS and 300 μL PBS was added to resuspend the cells.

[0084] 3) Flow cytometry detection:

[0085] The detection was performed using the PB450 channel of a Beckman flow cytometer.

[0086] 4) Use Flowjo software to analyze and export the results.

[0087] According to the above experimental process, the results obtained are as follows Figure 2 As shown, Figure 2 A. Figure 2 B is a schematic diagram and quantitative statistical diagram of the intracellular bacterial load (CFU) after H1299 cells overexpressing YY1 were infected with Mycobacterium marinum. It can be seen that the intracellular bacterial content of cells overexpressing YY1 was significantly lower than that of the control group, while the use of ferroptosis inhibitors led to an increase in the intracellular bacterial content, indicating that overexpression of YY1 can enhance the bactericidal ability. From the results, the intracellular bacteria in cells overexpressing YY1 were significantly reduced after infection, indicating that overexpression of YY1 promoted the expression of bactericidal substances, thereby reducing intracellular bacteria while inhibiting ferroptosis. Figure 2 C. Figure 2 D is the iNOS inhibitor 1400W used in cells overexpressing YY1. The flow cytometry diagram and quantitative statistical graph show the intracellular bacterial content. After using 1400W, the intracellular bacteria increased significantly. After using the ferroptosis inhibitor Fer-1, the intracellular bacterial content increased further, indicating that iNOS is the main bactericidal mechanism. The above results all indicate that overexpression of YY1 enhances the bactericidal effect.

[0088] Example 3: YY1 affects bactericidal activity by regulating the specific region promoter of iNOS

[0089] 1. Experimental purpose and principle

[0090] The purpose of the experiment is to analyze whether there is targeted regulation between transcription factor YY1 and iNOS promoter. The experimental principle is to construct a dual-luciferase plasmid containing promoter sequences with different predicted binding sites, and analyze the binding between transcription factor YY1 and iNOS promoter through a dual-luciferase reporter group experiment.

[0091] 2. Basic information of reagents

[0092] The double restriction enzyme vector pMCS-Fluc-SV40-Rluc-IRES-Puro was obtained from Wuhan Miaoling Biotechnology Co., Ltd., with the catalog number P35079; the endonucleases BamHI and XbaI were obtained from New England Biolabs, with the catalog numbers R0136 and R0145, respectively; the endonuclease SpeI was obtained from Beijing Quanshijin Biotechnology Co., Ltd., with the catalog number JS601; the T4 ligase was obtained from New England Biolabs, with the catalog number M0202; and the DH5α competent cells were obtained from Nanjing Novogene Biotech Co., Ltd., with the catalog number C502. The ReporterAssay System uses Promega, catalog number E1910.

[0093] 3. Preparation of experimental reagents

[0094] Preparation of 1× Passive Lysis Buffer (PLB): Add 1 part of 5× Passive Lysis Buffer to 4 parts of distilled water and mix thoroughly. Make sure to prepare it before use.

[0095] Luciferase Assay Reagent II configuration: Resuspend the freeze-dried Luciferase Assay Substrate with 10 mL of Luciferase Assay Buffer II in the kit, mix the substrate and buffer to prepare Luciferase Assay Reagent II (LAR II).

[0096] Stop& Reagent configuration: 1 portion of 50×Stop& Add the substrate to a 50-pack of Stop& Buffer solution should be prepared before use.

[0097] IV. Experimental Procedure

[0098] 1. Construction of dual luciferase plasmid:

[0099] 1) Using the A549 cell genome as a template and the iNOS promoter primers, PCR was performed respectively. After the correct band was verified by electrophoresis, the PCR reaction solution was purified and recovered;

[0100] 2) Double digest the vector pMCS-Fluc-SV40-Rluc-IRES-Puro with BamHI and SpeI at 37°C, and recover the digestion product; Double digest the PCR purification product with BamHI and XbaI at 37°C, and recover the digestion product;

[0101] 3) Use T4 ligase at a molar ratio of vector: fragment = 1:3 and ligate at room temperature for 1 h;

[0102] 4) Take 5 μL of the ligation product and add it to 50 μL of DH5α competent cells, place on ice for 30 min, heat shock at 42°C for 45 s, and place on ice for 2 min; add 700 μL of LB medium without resistance, and resuscitate on a shaker at 37°C at 200 rpm for 1 h; take 100 μL of the resuscitated bacterial solution, evenly spread it on the corresponding resistance plate, and culture it in a constant temperature incubator at 37°C overnight;

[0103] 5) Single clones were selected for sequencing to verify whether the fragments were connected, and the successfully constructed vector was used as a plasmid for later use.

[0104] 2. Dual luciferase assay:

[0105] 1) 293T cells were uniformly transferred into a 24-well plate to a density of about 60%. The dual-luciferase reporter plasmid and transcription factor overexpression plasmid (or empty vector) were transferred into 293T cells at the same time. Each well was configured with 500 ng plasmid + 50 μL buffer + 1 μL transfection reagent. The solution was changed after 6 to 8 hours.

[0106] 2) 24 hours after transfection, remove the culture supernatant and gently rinse twice with PBS to remove the detached cells and residual growth medium. Add 100 μL 1×PLB reagent and shake the 24-well plate on a shaker at room temperature for 15 minutes;

[0107] 3) Transfer the lysate to a 1.5 mL centrifuge tube, centrifuge at 12000 g for 30 seconds to remove cell debris from the sample, and use the clarified lysate for detection;

[0108] 4) Add 100 μL of LAR II and 20 μL of cell lysate to a 1.5 mL centrifuge tube, mix thoroughly by pipetting, and place in a luminometer for measurement. Take out the 1.5 mL centrifuge tube, add 100 μL of Stop & reagents, mix thoroughly, and place the 1.5 mL centrifuge tube in the luminescence detector for re-measurement;

[0109] 5) Export the results and analyze them.

[0110] According to the above experimental process, the results obtained are as follows Figure 3As shown in the results, it can be seen that in the samples containing the overexpressed YY1 plasmid, the relative luciferase content of P0-P3 gradually increased, indicating that YY1 binds to the promoter of iNOS. The luciferase content produced by transfected P3 is about twice that of transfected P2, and the median difference is two predicted sites, indicating that YY1 has a strong binding in the -655 to -1018bp segment of the iNOS promoter. This result also shows that YY1 affects the bactericidal mechanism by regulating the binding of the iNOS specific region promoter (-655 to -1018bp segment).

[0111] In summary, transcription factor YY1 can inhibit ferroptosis of lung epithelial cells, increase the expression of glutathione, reduce the expression of lipid peroxidation product malondialdehyde, and improve cell survival rate. At the same time, transcription factor YY1 promotes the increase of nitric oxide synthase expression, induces the production of nitric oxide to kill intracellular bacteria, and reduces the content of intracellular bacteria. YY1 targets and binds to the -655 to -1018bp segment of the iNOS promoter, enhancing the expression of iNOS and promoting bactericidal effect.

[0112] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of transcription factor YY1 in the preparation of ferroptosis inhibitors.

2. Application of transcription factor YY1 as a ferroptosis inhibitor in the preparation of anti-tuberculosis drugs.

3. Use of the transcription factor YY1 according to claim 2 as a ferroptosis inhibitor in the preparation of anti-tuberculosis drugs, characterized in that: Transcription factor YY1 inhibits ferroptosis in lung epithelial cells.

4. The use of the transcription factor YY1 according to claim 3 as a ferroptosis inhibitor in the preparation of anti-tuberculosis drugs, characterized in that: Transcription factor YY1 inhibits ferroptosis of lung epithelial cells, increases glutathione expression, and reduces the expression of lipid peroxidation product malondialdehyde.

5. Use of the transcription factor YY1 according to claim 3 as a ferroptosis inhibitor in the preparation of anti-tuberculosis drugs, wherein the transcription factor YY1 promotes the expression of nitric oxide synthase and induces the production of nitric oxide.

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