Application of compound for regulating pH value in ferroptosis related preparation

Abstract

The invention provides application of a compound for regulating the pH value in ferroptosis related preparations. Experimental results show that in the ferroptosis process, the extracellular pH value is in positive correlation with the ferroptosis death rate, ferroptosis can be effectively delayed by reducing the extracellular pH value, and ferroptosis can be effectively promoted by improving the extracellular pH value. The regulation and control of the pH value can be realized by adding / reducing the pH value regulating compounds such as hydrochloric acid, bicarbonate radical ions, NaH2PO4, sodium hydroxide and the like. In addition, the low pH value can delay ferroptosis of the cells and has high lipid peroxidation, which indicates that the low pH value affects the ferroptosis execution stage after the lipid peroxidation is increased. Therefore, the compound for regulating the pH value has basic and clinical application prospects in the application of preventing and treating ferroptosis and developing ferroptosis drugs, and new small molecules, methods and directions are provided for research and treatment of ferroptosis-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of compounds for regulating pH in ferroptosis-related preparations. Background Art

[0002] Programmed cell death has important physiological and pathological effects and is a research hotspot in the field of biomedicine. In May 2012, researchers proposed a new type of programmed cell death and named it ferroptosis. Ferroptosis is an iron-dependent programmed cell death that is different from apoptosis, necroptosis, and pyroptosis. The typical characteristics of ferroptosis are the accumulation of intracellular reactive oxygen species (ROS), the increase in the level of lipid peroxidation (Lipid ROS) in the cell membrane system, the shrinkage of mitochondria, and the reduction or disappearance of mitochondrial cristae. It is generally believed that intracellular iron mainly promotes the production of reactive oxygen species and lipid peroxidation through the Fenton reaction, which then induces ferroptosis. Recent studies have shown that ferroptosis is closely related to the occurrence and treatment of many diseases such as cancer, neurodegenerative diseases, kidney damage, organ ischemia-reperfusion injury, survival rate after stem cell transplantation, diabetes, and heart disease. Inducing or inhibiting ferroptosis provides a feasible strategy for the treatment of the above diseases. How to intervene in the occurrence and development of related diseases by regulating ferroptosis has become the focus of current basic research and clinical treatment.

[0003] The pH value (Potential of hydrogen) refers to the strength of the acidity or alkalinity of an aqueous solution, and is represented by the symbol pH. Under standard thermodynamic conditions, an aqueous solution with a pH of 7 is neutral, pH < 7 represents acidity, and pH > 7 represents alkalinity. Cell culture medium (such as DMEM) is required in the process of cell culture. Carbon dioxide (CO2) connected to the incubator will dissolve in the culture medium, react with water to form carbonic acid, and then be in equilibrium with the bicarbonate ions in the ordinary culture medium, using the CO2 bicarbonate reaction to interact and buffer / balance its pH value. As cells grow and metabolize, more CO2 lactic acid and other metabolites will be produced, and the pH value of the culture medium will drop in the later stage. Adding sodium bicarbonate to the culture medium can maintain the pH value in the optimal range of 7.2 to 7.4.

[0004] Bicarbonate ion (HCO3-) is the conjugate base of carbonic acid and the conjugate acid of carbonate ion (CO32-). In aqueous solution, bicarbonate can be ionized to form carbonate ion and hydrogen ion (H+), and can also be hydrolyzed to form hydroxide ion (OH-) and carbonic acid (H2CO3). However, because the degree of hydrolysis (producing OH-) of bicarbonate ion is greater than its ionization (producing H +) level, so the pH of pure water solution containing bicarbonate ions will be weakly alkaline. Sodium bicarbonate added to the culture medium can interact with the 5% CO2 supplied in the incubator to maintain the pH value in the range of 7.2 to 7.4. In addition, bicarbonate ions can enter and exit cells through transport proteins or ion channel proteins on the surface of the cell plasma membrane, thereby regulating cell function.

[0005] The main buffer system for regulating pH in animals and most cell culture media is sodium bicarbonate (NaHCO3) and CO2. The combination of the two maintains the pH value of the intracellular and extracellular environment in an appropriate range. Bicarbonate ion is the third most abundant ion in most cell culture media (after chloride ion and sodium ion), and its concentration is positively correlated with the pH value of the culture medium.

[0006] Adding hydrochloric acid or weak acid (such as sodium dihydrogen phosphate NaH2PO4) to the culture medium or the extracellular environment can cause the pH value of the culture medium to decrease. Adding sodium hydroxide (NaOH) to the culture medium or the extracellular environment can cause the pH value of the culture medium to increase.

[0007] So far, there is no report on the relationship between pH-regulating compounds and ferroptosis in cancer cells. Therefore, discovering new pH-regulating compounds will help provide new ideas and methods for the treatment of ferroptosis-related diseases. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an application of a compound for regulating pH in ferroptosis-related preparations.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] One way to promote cell ferroptosis is by regulating the pH of the extracellular environment.

[0011] The pH value is adjusted to be greater than 7.

[0012] The pH adjustment is achieved by adding alkaline compounds.

[0013] One method to inhibit cell ferroptosis is to regulate the pH of the extracellular environment.

[0014] The pH value is adjusted to be less than or equal to 7.

[0015] The pH adjustment is achieved by adding an acidic compound.

[0016] An inhibitor of ferroptosis comprising an acidic compound.

[0017] A ferroptosis promoter comprising a basic compound.

[0018] A tumor therapeutic drug comprises a ferroptosis inducer and an alkaline compound.

[0019] The ferroptosis inducing agent is at least one of RSL3, ML210 and Erastin.

[0020] The tumor is preferably a fibrosarcoma.

[0021] A drug for treating / preventing tissue ischemia-reperfusion and / or neurological diseases, comprising a compound that makes a solution acidic.

[0022] The tissue ischemia-reperfusion is cerebral tissue ischemia-reperfusion.

[0023] The neurological disease is a brain tissue related disease.

[0024] A culture medium that promotes cell ferroptosis is obtained by adding alkaline compounds to the culture medium.

[0025] A culture medium for inhibiting cell ferroptosis is obtained by adding acidic compounds to the culture medium or replacing some components.

[0026] The culture medium for inhibiting cell ferroptosis is obtained by replacing NaHCO3 in the culture medium with sodium gluconate.

[0027] The alkaline compound is a compound that can increase the pH value; preferably at least one of NaOH or NaHCO3.

[0028] The acidic compound is a compound that can lower the pH value; preferably at least one of HCl or NaH2PO4.

[0029] The culture medium is preferably DMEM culture medium.

[0030] The cells are human fibrosarcoma HT-1080 cells.

[0031] Ferroptosis is a cell death mode caused by iron-dependent lipid oxidation damage.

[0032] Compared with the prior art, the present invention has the following advantages and effects:

[0033] 1. The difference between the present invention and other ferroptosis patents: The relationship between the pH-regulating compound in this patent and ferroptosis is not reported. There are many types of pH-regulating compounds with wide flexibility.

[0034] 2. Another difference between the present invention and other ferroptosis patents is that the bicarbonate ions in this patent exist in large quantities under the physiological environment of organisms, and there is no report on their relationship with ferroptosis. The regulation of bicarbonate ions provides a new choice for the basis and application of ferroptosis regulating preparations, and has broad application prospects.

[0035] 3. Another difference between the present invention and other ferroptosis patents is that under acidic pH or no bicarbonate ions or high concentration of disodium hydrogen phosphate, although ferroptosis can be significantly delayed, the level of cellular lipid peroxidation is still maintained at a high level. At present, ferroptosis inhibitors mainly inhibit ferroptosis by reducing the level of intracellular lipid peroxidation. This patent achieves a significant delay in ferroptosis without reducing the level of lipid peroxidation, indicating that this regulation method is different from most existing ferroptosis inhibition technologies.

[0036] 4. The effect of pH regulation on ferroptosis found in the present invention is of great significance in the treatment and drug development of ferroptosis-related diseases, and has broad application prospects in the preparation of drugs for the treatment / prevention of tissue ischemia-reperfusion and / or neurological diseases and tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a graph showing the death rate caused by ferroptosis observed at 6 h under DMEM culture conditions with an acidic pH gradient (adjusted using hydrochloric acid); the ordinate shows the cell death rate.

[0038] Figure 2 This is a graph showing the results of cellular lipid peroxidation levels detected by laser confocal microscopy under DMEM culture conditions with the pH adjusted to 5.5 (adjusted using hydrochloric acid).

[0039] Figure 3 The results of the cell lipid peroxidation level detected by flow cytometry at 2h, 4h, and 6h under DMEM culture conditions with pH adjusted to 5.5 (adjusted with hydrochloric acid); the ordinate is the number of cells, and the abscissa is the fluorescence intensity of the lipid peroxidation dye C11-BODIPY oxidation state. D refers to DMEM culture medium. R refers to the ferroptosis inducer RSL3.

[0040] Figure 4 No bicarbonate ions (no HCO3 - The osmotic difference caused by the cell death rate was observed at 9 h under DMEM culture conditions (the osmotic difference caused by the cell death rate was supplemented by sodium gluconate SG); the vertical axis shows the cell death rate.

[0041] Figure 5This is the result of laser confocal microscopy detection of cellular lipid peroxidation levels under DMEM culture conditions without bicarbonate ions (the osmotic difference caused by the absence of NaHCO3 is compensated by sodium gluconate SG). No N2+SG refers to the DMEM condition in which SG replaces bicarbonate ions.

[0042] Figure 6 This is the result of the flow cytometer-detected lipid peroxidation level under DMEM culture conditions without bicarbonate ions (the osmotic difference caused by the absence of NaHCO3 is supplemented by sodium gluconate SG); the ordinate is the number of cells, and the abscissa is the fluorescence intensity of the oxidation state of the lipid peroxidation dye C11-BODIPY. -N2 refers to the absence of bicarbonate ions, and D refers to DMEM culture medium. R refers to the ferroptosis inducer RSL3.

[0043] Figure 7 This is a graph showing the death rate caused by ferroptosis at 6h and 9h measured by lactate dehydrogenase (LDH) cytotoxicity assay under acidic pH (adjusted with 100mM NaH2PO4) DMEM culture conditions; the ordinate shows the OD value of the LDH assay result. The higher the OD value, the higher the mortality rate. P refers to NaH2PO4, and D refers to DMEM culture medium.

[0044] Figure 8 This is the result of laser confocal microscopy detection of cellular lipid peroxidation levels under DMEM culture conditions with acidic pH (adjusted with 100mM NaH2PO4). The white light image shows the color of phenol red under each condition: the redder the phenol red, the more alkaline the pH; the yellower the phenol red, the more acidic the pH. D refers to DMEM culture medium. R refers to the ferroptosis inducer RSL3. 100P refers to DMEM culture medium supplemented with 100mM NaH2PO4. -N2+SG refers to the DMEM condition in which SG replaces bicarbonate ions.

[0045] Fig. 9 This is the result of the flow cytometer detection of cell lipid peroxidation levels at 2h, 4h, and 6h under DMEM culture conditions with acidic pH (adjusted with 100mM NaH2PO4); the ordinate is the number of cells, and the abscissa is the fluorescence intensity of the oxidation state of the lipid peroxidation dye C11-BODIPY. D refers to DMEM culture medium, and R refers to the ferroptosis inducer RSL3. 100P refers to DMEM culture medium supplemented with 100mM NaH2PO4.

[0046] Fig.10This is a graph showing the death rate caused by ferroptosis under the condition of phosphate buffer solution PBS with an acidic pH gradient (adjusted with hydrochloric acid) at 6 hours; the vertical axis shows the cell death rate. The PBS in the figure refers to the purchased PBS buffer solution (pH ~ 7.2).

[0047] Fig.11 This is the result of the cell lipid peroxidation level detected by flow cytometry at 1h and 4h under the condition of acidic pH (adjusted by hydrochloric acid) phosphate buffer solution PBS culture condition; the vertical axis is the number of cells, and the horizontal axis is the fluorescence intensity of the lipid peroxidation dye C11-BODIPY oxidation state. R refers to the ferroptosis inducer RSL3.

[0048] Fig.12 This is a graph showing the death rate caused by ferroptosis observed at 3 h under DMEM culture conditions with an alkaline pH gradient (adjusted using sodium hydroxide); the ordinate shows the cell death rate. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0050] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0051] Experimental reagents:

[0052] Fetal bovine serum: ExCell;

[0053] 0.25% pancreatin: Biotime;

[0054] Double antibody (penicillin-streptomycin mixture) 100x: Biotime Company;

[0055] PI (Propidium iodide, dead cell dye): Bio-Time;

[0056] Hoechst (total cell count dye): Bio-Time;

[0057] SYTOX: KeyGen Biotech;

[0058] Hydrochloric acid: Guangzhou Chemical Reagent Factory;

[0059] Sodium hydroxide: Guangzhou Chemical Reagent Factory;

[0060] Sodium phosphate monobasic (NaH2PO4): Vetec;

[0061] Sodium bicarbonate (NaHCO3): Sigma;

[0062] Sodium gluconate (SG): Sigma;

[0063] PBS buffer: Biosharp;

[0064] DMEM (Dulbecco's Modified Eagle Medium) high glucose medium: Gibco;

[0065] DMEM high-glucose basal medium (customized with lack of nutrients, such as no NaHCO3): Shann Biotechnology Co., Ltd.

[0066] LDH detection kit: Biotime Company;

[0067] BODIPY TM 581 / 591 C11 (lipid peroxidation tag): Invitrogen;

[0068] Experimental equipment:

[0069] HT-1080 cells: Shanghai Cell Bank, Chinese Academy of Sciences;

[0070] 96-well plate: Nest Biotechnology;

[0071] Six-well plate: Thermo Fisher Scientific;

[0072] 30ml disposable dosing syringe: Shanghai Kangdeli Company;

[0073] 0.22μm filter membrane: Millipore;

[0074] 50 ml centrifuge tube: Thermo Fisher Scientific;

[0075] 15 ml centrifuge tube: Thermo Fisher Scientific;

[0076] 1.5 ml centrifuge tube: Thermo Fisher Scientific;

[0077] Four-partition confocal dish: Shanghai Jingan Company;

[0078] pH meter: Mettler-Toledo;

[0079] Electronic balance: Ohaus;

[0080] Electric constant temperature water tank: Shanghai Boyuan Company;

[0081] CO2 incubator: ESCO;

[0082] Ordinary low-temperature refrigerator: Haier Company;

[0083] -80℃ ultra-low temperature freezer: Thermo Fisher Scientific;

[0084] Ordinary optical microscope: Olympus;

[0085] Ultrapure water instrument: MILLIPORE;

[0086] Biological safety cabinet: ESCO;

[0087] FV3000 laser confocal microscope: Olympus;

[0088] Multifunctional microplate reader: BioTek

[0089] CytoFLEX flow cytometer: Beckman;

[0090] Cytation 5 Cell Imaging Multi-function Microplate Detection System: Agilent.

[0091] Preparation of main reagents

[0092] Prepare DMEM complete medium for HT-1080 cell culture: store fetal bovine serum in a -80°C refrigerator for future use. Prepare 50 mL / tube of DMEM complete medium containing 10% serum and 1% double antibody at a final concentration, and place in a 4°C refrigerator for future use.

[0093] Prepare 10 mg / ml PI dye solution: add 20 mg of packaged PI powder to 2 mL of sterile water and place in a 4°C refrigerator for later use.

[0094] Prepare 10 mg / ml Hoechst staining solution: add 20 mg of packaged Hoechst powder to 2 mL of sterile water and place in a 4°C refrigerator until ready for use.

[0095] Example 1 Human fibrosarcoma HT-1080 cell culture

[0096] HT-1080 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% double antibody in a 37°C, 5% CO2, saturated humidity incubator. The medium was changed every 2-3 days for passage. Cells in the logarithmic growth phase were selected for the experiment.

[0097] Example 2 Ferroptosis mortality experiment under DMEM culture conditions with acidic pH gradient (adjusted with hydrochloric acid)

[0098] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 96-well plates, and 100 μL of cell suspension was added to each well;

[0099] (2) DMEM culture media with different pH values ​​(pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 6.0, and pH 7.0) were prepared (the pH was adjusted by adding hydrochloric acid and using a pH meter), and a DMEM culture medium without pH adjustment was set as a control (pH of approximately 7.5);

[0100] Each of the above groups was set up as follows: Control group (no drug treatment), RSL3 (2 μM final concentration) group, RSL3 (2 μM final concentration) + Fer-1 (4 μM final concentration) group. Three replicate wells were set up for each of the above treatment groups. 100 μL of the corresponding culture medium was added to each replicate well;

[0101] (3) After 6 h, add 10 μL of PI / Hoechst staining solution to each well and stain for 15-30 min;

[0102] (4) Use the Cytation 5 cell imaging microplate detection system to image and calculate the cell viability.

[0103] The results are as follows Figure 1 As shown in the figure, as the pH decreased (between pH 5.5 and 7.0), the ferroptosis mortality rate gradually decreased, suggesting that acidic pH can delay RSL3-induced ferroptosis.

[0104] Example 3 Laser confocal microscopy test of lipid peroxidation during ferroptosis under DMEM culture conditions with pH adjusted to 5.5 (adjusted with hydrochloric acid)

[0105] (1) Dilute the HT-1080 in the logarithmic growth phase obtained in Example 1 to 1×10 5 / mL were inoculated into four-grid confocal dishes, and 500μL of cell suspension was added to each well;

[0106] (2) After the cells in the four-partition confocal dish adhered to the wall, the culture medium was aspirated and the cells were washed 2-3 times with normal culture medium DMEM. The culture medium was changed and the cells were divided into the following groups: DMEM group (DMEM group), DMEM+RSL3 group (RSL3 group), DMEM with pH 5.5 group (pH 5.5 group), and DMEM with pH 5.5+RSL3 group (pH5.5+R group).

[0107] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator and incubated for 2 h;

[0108] (3) The cells in each group were imaged using the FITC channel of a laser confocal microscope (receiving light wavelength: 485-565 nm), and the images were processed using ImageJ.

[0109] The experimental results are as follows Figure 2 As shown in the figure, when the pH was adjusted to an acidic state of 5.5, ferroptosis was significantly delayed, and the lipid peroxidation level of the cells remained high, suggesting that the way acidic pH regulates ferroptosis is different from general ferroptosis inhibition conditions.

[0110] Example 4 Flow cytometric detection of lipid peroxidation during ferroptosis under DMEM culture conditions with pH adjusted to 5.5 (using hydrochloric acid)

[0111] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 6-well plates, and 100 μL of cell suspension was added to each well;

[0112] (2) DMEM medium with a pH of 5.5 was prepared (the pH was adjusted by adding hydrochloric acid and using a pH meter), and a DMEM medium without pH adjustment was set as a control;

[0113] (3) After the cells in the 6-well plate adhered to the wall, the culture medium was aspirated and the culture medium was replaced and the cells were divided into the following groups: DMEM group (DMEM group; 6 h), DMEM+RSL3 group (D+R group; 2 h), DMEM at pH 5.5 group (D+pH5.5 group; 6 h), and DMEM at pH 5.5+RSL3 group (D+pH5.5+R group; 2 h, 4 h, and 6 h).

[0114] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator for further incubation;

[0115] (4) After incubation, aspirate the culture medium and place it in a 1.5 ml centrifuge tube;

[0116] (5) Add 1× trypsin to each well for digestion. After 1 min, remove the trypsin, rinse the cells with PBS, and centrifuge at 300g for 3 min. Resuspend the cells in the culture medium of step 3 and detect the FITC fluorescence channel on the machine. Use FlowJo 10 for analysis.

[0117] The experimental results are as follows Figure 3As shown in the figure, when the pH was adjusted to an acidic state of 5.5, ferroptosis was significantly delayed, and the lipid peroxidation level of the cells remained high, suggesting that the way acidic pH regulates ferroptosis is different from general ferroptosis inhibition conditions.

[0118] Example 5 Experiment on delaying ferroptosis without NaHCO3 (osmotic difference caused by lack of NaHCO3 is compensated by sodium gluconate SG)

[0119] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL was inoculated into a 96-well plate, and 100 μL of cell suspension was added to each well;

[0120] (2) For DMEM complete medium (containing 44mM NaHCO3) and customized DMEM medium without NaHCO3 (lacking 44mM NaHCO3), the following groups were set up: Control group (no drug treatment), RSL3 (2μM final concentration) group, RSL3 (2μM final concentration) + Fer-1 (4μM final concentration) group. Three replicate wells were set up for each of the above treatment groups. The culture medium of the treatment group was replaced at 0h, and the cell survival was detected at 9h. 100μL of the corresponding culture medium was added to each replicate well (the culture medium contained 5% serum, the same applies below);

[0121] (3) After 9 h, add 10 μL of PI / Hoechst dye solution to each well and stain for 15-30 min;

[0122] (4) Use the Cytation 5 cell imaging microplate detection system to image and calculate the cell viability.

[0123] The results are as follows Figure 4 As shown, removing NaHCO3 from DMEM medium (the osmotic difference caused by the absence of NaHCO3 is replenished by sodium gluconate SG, and the pH is ~6.1) can significantly delay ferroptosis induced by RSL3, a classic inducer of ferroptosis.

[0124] Example 6 Laser confocal microscopy test of lipid peroxidation during ferroptosis under DMEM culture conditions without bicarbonate ions (the osmotic difference caused by the absence of NaHCO3 is compensated by sodium gluconate SG)

[0125] (1) Dilute the HT-1080 in the logarithmic growth phase obtained in Example 1 to 1×10 5 / mL were inoculated into four-grid confocal dishes, and 500μL of cell suspension was added to each well;

[0126] (2) After the cells in the four-partition confocal dish adhered to the wall, the culture medium was aspirated and the cells were washed 2-3 times with basal nutrient-deficient culture medium (DMEM without NaHCO3). The culture medium was changed and the cells were divided into the following groups: DMEM group (D group), DMEM+RSL3 group (D+R group), SG group without NaHCO3 supplementation (-N2+SG group), and SG+RSL3 group without NaHCO3 supplementation (-N2+SG+R group).

[0127] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator and incubated for 2 h;

[0128] (3) The cells in each group were imaged using the FITC channel of a laser confocal microscope (receiving light wavelength: 485-565 nm), and the images were processed using ImageJ.

[0129] The experimental results are as follows Figure 5 As shown in the figure, in the absence of bicarbonate ions (the osmotic difference caused by the absence of NaHCO3 is replenished by sodium gluconate SG), ferroptosis was significantly delayed, and the lipid peroxidation level of the cells was still high, which also suggests that the way bicarbonate ions regulate ferroptosis is different from the general ferroptosis inhibition conditions (inhibiting ferroptosis by inhibiting lipid peroxidation).

[0130] Example 7 No bicarbonate ions (no HCO3 - Flow cytometric detection of lipid peroxidation during ferroptosis under the condition of sodium gluconate (SG)

[0131] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 6-well plates, and 100 μL of cell suspension was added to each well;

[0132] (2) After the cells in the 6-well plate adhered to the wall, the culture medium was aspirated and the medium was replaced and the groups were divided into the following groups: DMEM group (D group; 6 h), DMEM+RSL3 group (D+R group; 2 h), SG group without NaHCO3 supplementation (-N2+SG group; 6 h), and SG+RSL3 group without NaHCO3 supplementation (-N2+SG+R group; 2 h, 4 h, and 6 h).

[0133] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator for further incubation;

[0134] (3) After incubation, aspirate the culture medium and place it in a 1.5 ml centrifuge tube;

[0135] (4) Add 1× trypsin to each well for digestion. After 1 minute, remove the trypsin, rinse the cells with PBS, and centrifuge at 300g for 3 minutes. Resuspend the cells in the culture medium of step 3 and detect the FITC fluorescence channel on the machine. Use FlowJo 10 for analysis.

[0136] The experimental results are as follows Figure 6 As shown in the figure, in the absence of bicarbonate ions (the osmotic difference caused by the absence of NaHCO3 is replenished by sodium gluconate SG), ferroptosis was significantly delayed, and the lipid peroxidation level of the cells was still high, suggesting that the way bicarbonate ions regulate ferroptosis is different from the general ferroptosis inhibition conditions (i.e., inhibiting ferroptosis by inhibiting lipid peroxidation).

[0137] Example 8 Ferroptosis mortality experiment under DMEM culture conditions with acidic pH (adjusted using 100 mM NaH2PO4) (lactate dehydrogenase LDH detection)

[0138] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 96-well plates, and 100 μL of cell suspension was added to each well;

[0139] (2) After the cells in the 96-well plate adhered to the wall, the culture medium was removed and the cells were washed once with PBS. The culture medium was changed and the groups were divided as follows:

[0140] a. Cell-free culture medium group (background blank control group): DMEM-9h, DMEM+100mM NaH2PO4-6 h, DMEM+100mM NaH2PO4-9 h (this group is the group without cell plating);

[0141] b. The control groups without drug treatment include cell groups (sample control group): DMEM-9h, DMEM+100mM NaH2PO4-6h, DMEM+100mM NaH2PO4-9 h;

[0142] c. Drug-treated cell groups (drug-treated sample groups): DMEM+RSL3-9 h, DMEM+100mM NaH2PO4+RSL3-6 h, DMEM+100mM NaH2PO4+RSL3-9 h, DMEM+RSL3+Fer-1-9h, DMEM+100mM NaH2PO4+RSL3+Fer-1-6h, DMEM+100mM NaH2PO4+RSL3+Fer-1-9h;

[0143] RSL3 (2 μM final concentration) group, RSL3 (2 μM final concentration) + Fer-1 (4 μM final concentration) group. Each of the above treatment groups was set up with 4 replicate wells. 200 μL of the corresponding culture medium was added to each replicate well;

[0144] (3) After the predetermined time has elapsed, the 96-well plate was centrifuged at 400 g for 5 min using a multi-well plate centrifuge. 120 μL of the supernatant from each well was taken and added to the corresponding wells of a new 96-well plate for sample measurement;

[0145] (4) Prepare the LDH detection working solution according to the instructions of the LDH kit and add 60 μL of LDH detection working solution to each well;

[0146] (5) After mixing, incubate at room temperature in the dark for 30 min (wrap with aluminum foil and place on a horizontal shaker and shake slowly);

[0147] (6) After the incubation, the 96-well plate was placed in a multifunctional microplate reader, and the absorbance was measured at 490 nm, and a dual-wavelength measurement was performed using 600 nm or any wavelength greater than 600 nm as a reference wavelength;

[0148] (7) To calculate the absorbance OD value of each group, the absorbance of the background blank control well must be subtracted.

[0149] The results are as follows Figure 7 As shown in the figure, due to the destruction of the cell membrane structure during cell ferroptosis, proteins in the cytoplasm are released into the culture medium, including lactate dehydrogenase (LDH) with relatively stable enzyme activity. The experimental results also showed that adjusting the extracellular culture environment to an acidic pH by different methods (using 100mM NaH2PO4 for adjustment) can effectively delay RSL3-induced ferroptosis, suggesting that acidic pH (adjusted by 100mM NaH2PO4) can delay ferroptosis.

[0150] Example 9 Laser confocal microscopy detection experiment of lipid peroxidation during ferroptosis under acidic pH (adjusted by 100 mM NaH2PO4) DMEM culture conditions

[0151] (1) Dilute the HT-1080 in the logarithmic growth phase obtained in Example 1 to 1×10 5 / mL were inoculated into four-grid confocal dishes, and 500μL of cell suspension was added to each well;

[0152] (2) After the cells in the four-partition confocal dish adhered to the wall, the culture medium was aspirated and the cells were washed 2-3 times with normal culture medium DMEM. The medium was changed and the cells were divided into the following groups: DMEM group (DMEM group), DMEM+RSL3 group (RSL3 group), DMEM+100mM NaH2PO4 (100P group), and DMEM+100mM NaH2PO4+RSL3 group (100P+R group).

[0153] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator and incubated for 2 h;

[0154] (3) Use the FITC channel of a laser confocal microscope to image each group of cells (receiving light wavelength: 485-565 nm). Take white light photos of the culture dish containing phenol red. Use ImageJ to process the images.

[0155] The experimental results are as follows Figure 8 As shown, when 100 mM NaH2PO4 was added to adjust the acidic conditions of DMEM, ferroptosis was significantly delayed, and the lipid peroxidation level of the cells remained high, suggesting that the way acidic pH (regulated by 100 mM NaH2PO4) regulates ferroptosis is different from general ferroptosis inhibition conditions.

[0156] Example 10 Flow cytometric detection of lipid peroxidation during ferroptosis under DMEM culture conditions with acidic pH (adjusted with 100 mM NaH2PO4)

[0157] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 6-well plates, and 100 μL of cell suspension was added to each well;

[0158] (2) After the cells in the 6-well plate adhered to the wall, the culture medium was aspirated and the medium was replaced and the cells were divided into the following groups: DMEM group (DMEM group; 6 h), DMEM+RSL3 group (D+R group; 2 h), DMEM+100 mM NaH2PO4 group (D+100P group; 6 h), and DMEM+100 mM NaH2PO4+RSL3 group (D+100P+R group; 2 h, 4 h, and 6 h).

[0159] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator for further incubation;

[0160] (3) After incubation, aspirate the culture medium and place it in a 1.5 ml centrifuge tube;

[0161] (4) Add 1× trypsin to each well for digestion. After 1 minute, remove the trypsin, rinse the cells with PBS, and centrifuge at 300g for 3 minutes. Resuspend the cells in the culture medium of step 3 and detect the FITC fluorescence channel on the machine. Use FlowJo 10 for analysis.

[0162] The experimental results are as follows Fig. 9 As shown, when 100 mM NaH2PO4 was added to adjust the acidic conditions of DMEM, ferroptosis was significantly delayed, and the lipid peroxidation level of the cells remained high, which also suggested that the way acidic pH regulates ferroptosis is different from the general ferroptosis inhibition conditions.

[0163] Example 11 Ferroptosis mortality experiment under the culture conditions of phosphate buffer solution PBS with acidic pH gradient (adjusted by hydrochloric acid)

[0164] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 96-well plates, and 100 μL of cell suspension was added to each well;

[0165] (2) PBS with different pH values ​​(pH 5.0, pH 6.0, pH 7.0) was prepared (pH was adjusted by adding hydrochloric acid, sodium hydroxide and using a pH meter), and PBS without pH adjustment was set as a control (pH was about 7.2);

[0166] Each of the above groups was set up as follows: Control group (no drug treatment), RSL3 (2 μM final concentration) group, RSL3 (2 μM final concentration) + Fer-1 (4 μM final concentration) group. Three replicate wells were set up for each of the above treatment groups. 100 μL of the corresponding culture medium was added to each replicate well;

[0167] (3) After 6 h, add 10 μL of PI / Hoechst staining solution to each well and stain for 15-30 min;

[0168] (4) Use the Cytation 5 cell imaging microplate detection system to image and calculate the cell viability.

[0169] The results are as follows Fig.10 As shown, as the pH dropped to around 5-6, the ferroptosis mortality rate decreased, suggesting that acidic pH can delay RSL3-induced ferroptosis, suggesting that this result can be reproduced in different culture environments.

[0170] Example 12 Flow cytometric detection of lipid peroxidation during ferroptosis under PBS culture conditions with an acidic pH gradient (adjusted with hydrochloric acid)

[0171] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 6-well plates, and 100 μL of cell suspension was added to each well;

[0172] (2) PBS with different pH values ​​(pH 6.0, pH 7.0) was prepared (pH was adjusted by adding hydrochloric acid, sodium hydroxide and using a pH meter), and PBS without pH adjustment was set as a control (pH was about 7.2);

[0173] (3) After the cells in the 6-well plate adhered to the wall, the culture medium was aspirated and the culture medium was replaced and the cells were divided into the following groups: PBS group (PBS group; 1 h), PBS+RSL3 group (PBS+R group; 1 h), pH 6.0 PBS group (pH 6 group; 1 h, 4 h), pH 6 PBS+RSL3 group (pH 6+R group; 1 h, 4 h), pH 7.0 PBS group (pH 7 group; 1 h), and pH 7 PBS+RSL3 group (pH 7+R group; 1 h).

[0174] The final concentration of the above RSL3 was 2 μM. C11-BODIPY dye was added to each group at 0 h with a final concentration of 5 μM. After changing the medium, the cells were returned to the incubator for further incubation;

[0175] (4) After incubation, aspirate the culture medium and place it in a 1.5 ml centrifuge tube;

[0176] (5) Add 1× trypsin to each well for digestion. After 1 min, remove the trypsin, rinse the cells with PBS, and centrifuge at 300g for 3 min. Resuspend the cells in the culture medium of step 3 and detect the FITC fluorescence channel on the machine. Use FlowJo 10 for analysis.

[0177] The experimental results are as follows Fig.11 As shown in the figure, when the pH dropped to around pH 6.0, ferroptosis was significantly delayed, and the lipid peroxidation level of the cells remained high, indicating that under PBS culture conditions, the way acidic pH regulates ferroptosis is also different from that of general ferroptosis inhibitors.

[0178] Example 13 Ferroptosis mortality experiment under DMEM culture conditions with alkaline pH gradient (adjusted using sodium hydroxide)

[0179] (1) The HT-1080 cells in the logarithmic growth phase obtained in Example 1 were diluted to 5×10 4 / mL were inoculated in 96-well plates, and 100 μL of cell suspension was added to each well;

[0180] (2) DMEM medium with different pH values ​​(pH 8.0, pH 9.0) was prepared (pH was adjusted by adding sodium hydroxide and using a pH meter), and a DMEM medium without pH adjustment was set as a control (pH ~ 7.4);

[0181] Each of the above groups was set up as follows: Control group (no drug treatment), RSL3 (2 μM final concentration) group, RSL3 (2 μM final concentration) + Fer-1 (4 μM final concentration) group. Three replicate wells were set up for each of the above treatment groups. 100 μL of the corresponding culture medium was added to each replicate well;

[0182] (3) After 3 h, add 10 μL of PI / Hoechst staining solution to each well and stain for 15-30 min;

[0183] (4) Use the Cytation 5 cell imaging microplate detection system to image and calculate the cell viability.

[0184] The results are as follows Fig.12 As shown, at the 3 h time point, the ferroptosis mortality rate gradually increased with the increase of pH, suggesting that alkaline pH can promote RSL3-induced ferroptosis.

[0185] Example 14 Experimental study on the effect of acidic environment on improving brain tissue neuronal cell death in mouse organotypic entorhinal-hippocampal slice culture (OEHSC) oxygen glucose deprivation model (OGD, a classic model of tissue ischemia reperfusion)

[0186] Mouse biopsy tissue slices were obtained from wild-type C55Bl6 mice on the fifth day after birth. The brain tissue slices were cultured in vitro for 9 days before the experiment. The experiment was divided into three groups:

[0187] a. Control+DMEM group: mouse brain tissue slices were treated with DMEM for 24 h;

[0188] b. 3h OGD+DMEM group: Mouse brain tissue slices were first treated with ischemic cerebrospinal fluid for OGD for 3h, and then treated in DMEM medium for 24h (the formula of ischemic artificial cerebrospinal fluid was: 0.3mM CaCl2, 70mM NaCl, 5.25mM NaHCO3, 70mM KCl, 1.25mM NaH2PO4, 2mM MgSO4, 10mM sucrose);

[0189] c. 3h OGD + low pH culture medium group: Mouse brain tissue slices were first treated with ischemic cerebrospinal fluid for OGD for 3h, and then treated in low pH DMEM culture medium for 24h.

[0190] After 24 h of treatment, the cells were stained with 2 μg / mL PI dye at 37°C for 1 h and imaged using a 10x objective lens under a conventional wide-field microscope.

[0191] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A ferroptosis promoter, characterized in that: Includes basic compounds.

2. A ferroptosis inhibitor, characterized in that: Including acidic compounds.

3. A tumor treatment drug, characterized in that: Including the ferroptosis promoter as described in claim 1.

4. A drug for treating / preventing tissue ischemia-reperfusion and / or neurological diseases, characterized in that: Including the ferroptosis inhibitor as described in claim 2.

5. A culture medium for promoting cell ferroptosis, characterized in that: It is obtained by adding alkaline compounds to the culture medium.

6. A culture medium for inhibiting cell ferroptosis, characterized in that: It is obtained by adding acidic compounds to the culture medium or replacing some components.

7. The culture medium according to claim 6, characterized in that: The culture medium is obtained by replacing NaHCO3 in the culture medium with sodium gluconate.

8. The culture medium according to any one of claims 5 to 7, characterized in that: The culture medium is DMEM culture medium.

9. The ferroptosis promoter according to claim 1 or the culture medium according to claim 5, characterized in that: The alkaline compound is at least one of NaOH and NaHCO3.

10. The inhibitor according to claim 2 or the culture medium according to any one of claims 6 to 7, characterized in that: The acidic compound is at least one of HCl or NaH2PO4.

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