A ferroptosis inhibitor and its application in radiation pneumonitis drugs

Abstract

The present invention discloses a ferroptosis inhibitor and its application in radiation-induced pneumonia drugs, and relates to the field of biomedicine technology. The specific preparation method comprises: adding a ferroptosis inhibitor and an auxiliary agent to a solvent to dissolve to obtain a ferroptosis inhibitor composition; wherein the ferroptosis inhibitor is isoorientin; the auxiliary agent is a glucosamine derivative; and the solvent is a serum-free DMEM culture medium. The ferroptosis inhibitor composition prepared by the method of the present invention can effectively reduce the increase in the levels of reactive oxygen species, ferrous ions, and malondialdehyde in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 caused by irradiation, restore the enrichment of cell mitochondrial membrane potential, and inhibit the expression of various inflammatory factors in irradiated cells, thereby significantly improving the progression of radiation-induced pneumonia and providing a new medication strategy for the research of radiation-induced pneumonia drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a ferroptosis inhibitor and its application in radiation-induced pneumonia drugs. Background Art

[0002] Ferroptosis is a novel iron-dependent cell death mechanism. Unlike apoptosis, necrosis, and autophagy, ferroptosis is driven by lipid peroxidation. Intracellular iron accumulation and oxidation of polyunsaturated fatty acids by reactive oxygen species are key hallmarks of ferroptosis. Unrestricted lipid peroxidation is a hallmark of ferroptosis, typically characterized by the peroxidation of phospholipids bearing polyunsaturated fatty acid chains, which triggers the accumulation of lipid-reactive oxygen species and leads to cell death. Driven by iron ions, arachidonic acid lipoxygenase is activated and plays a key role in this oxidation process. Within the cell, acyl-CoA synthetase, long-chain family member 4, catalyzes the conjugation of free arachidonic acid or adrenic acid with CoA, generating AA / AdA-CoA derivatives. These derivatives are then esterified by lysophosphatidylcholine acyltransferase 3 to form AA / AdA-phosphatidylethanolamine, which is then oxidized by arachidonic acid 15-lipoxygenase to form lipid peroxides, thereby promoting ferroptosis. A wealth of evidence suggests that ferroptosis may be a key factor in the tumor-killing effects mediated by radiotherapy. Radiotherapy leads to the accumulation of intracellular lipid peroxidation markers malondialdehyde and 4-hydroxyquinal, and upregulates the expression of prostaglandin endoperoxide synthase 2. Post-irradiation cells display distinct morphological features of ferroptosis, namely mitochondrial atrophy and increased membrane density. With increasing radiotherapy dose, the level of intracellular lipid peroxidation and the extent of ferroptosis further increase. Mechanistically, radiotherapy may induce ferroptosis in multiple ways. First, radiotherapy degrades intracellular water, generating excessive reactive oxygen species (ROS), including a large number of lipid-ROS. These ROS promote lipid peroxidation, thereby inducing ferroptosis. Second, radiotherapy upregulates the expression of acyl-CoA synthetase, long-chain fatty acid family member 4, promoting the oxidation of unsaturated fatty acids and catalyzing the formation of lipid peroxides. Radiotherapy also depletes intracellular glutathione, inhibiting the clearance of lipid peroxides mediated by the membrane lipid repair enzyme glutathione peroxidase GPX4, accelerating the process of ferroptosis.

[0003] Radiation therapy is a treatment modality that can improve survival rates for cancer patients. Effective treatment eradicates tumor cells with minimal damage to healthy cells. However, radiation therapy for cancer patients who meet radiotherapy criteria may result in normal cell inflammation. The first case of radiation pneumonitis was reported in 1898 in a patient with tuberculosis. Today, most patients with breast or other thoracic malignancies, lymphomas, and metastatic lung disease are expected to undergo radiation therapy and are at risk for radiation pneumonitis. Radiation pneumonitis is characterized by dyspnea, dry cough, hypoxemia, and low-grade fever. An estimated 10–30% of patients receiving radiation therapy for thoracic malignancies develop radiation pneumonitis, with a dose-dependent incidence. Currently, glucocorticoids are the first-line treatment for radiation pneumonitis. However, their use is associated with significant side effects such as leukocytosis and immunosuppression. The exact incidence of radiation pneumonitis remains unknown and depends on tumor location, radiographic changes, and the presence of clinical symptoms. Previous studies have found that the lungs of patients with radiation pneumonitis exhibit pathological changes consistent with ferroptosis. Therefore, the molecular mechanisms of ferroptosis inhibition and the development of drugs that can treat radiation pneumonitis remain unresolved. Summary of the Invention

[0004] The purpose of the present invention is to provide a ferroptosis inhibitor and its application in radiation pneumonia drugs, to explore the inhibitory molecular mechanism of ferroptosis inhibitors and to prepare radiation pneumonia drugs, thereby providing new ideas and new approaches for the treatment of radiation pneumonia.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions:

[0006] The invention discloses a ferroptosis inhibitor composition, comprising: a ferroptosis inhibitor, an adjuvant and a solvent; the ferroptosis inhibitor is isoorientin, and the dosage ratio of the ferroptosis inhibitor to the solvent is 10-30 μm:1 ml.

[0007] Preferably, the adjuvant is a glucosamine derivative, which is prepared by reacting salicylic acid, N-hydroxysuccinimide, and 4-methoxybenzoyl chloride. Using glucosamine derivatives to prepare ferroptosis inhibitor compositions can further effectively restore the irradiation-induced decrease in viability of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12. It can also reduce elevated levels of malondialdehyde, a marker of ferroptosis associated with radiation pneumonitis, thereby alleviating the pathological progression of radiation pneumonitis.

[0008] Preferably, the dosage ratio of the auxiliary agent to the solvent is 3-30 mg:1 ml.

[0009] Preferably, the solvent is serum-free DMEM medium.

[0010] Preferably, the method for preparing the glucosamine derivative comprises:

[0011] Add active salicylic acid ester and triethylamine to tetrahydrofuran, lower the temperature and add 4-methoxybenzoyl chloride to react, then increase the temperature to continue the reaction, and after the reaction is completed, rotary evaporation is performed to obtain an intermediate; D-glucosamine is mixed with an aqueous solution of N,N-dimethylformamide, and then triethylamine is added dropwise with stirring, and then the intermediate is added to react, and after the reaction is completed, rotary evaporation is performed to obtain a glucosamine derivative.

[0012] More preferably, the mass ratio of D-glucosamine to the intermediate is 1:2.3-2.8

[0013] More preferably, the reaction temperature for adding 4-methoxybenzoyl chloride is -25---15° C., and the reaction time is 1-3 h.

[0014] More preferably, the heating temperature is 20-30°C, and the heating reaction time is 10-15 hours.

[0015] More preferably, the reaction temperature for adding the intermediate is -2-5°C, and the reaction time is 6-10 h.

[0016] The present invention discloses use of a ferroptosis inhibitor composition in preparing a radiation-induced pneumonia drug. The ferroptosis inhibitor composition comprises the above-mentioned ferroptosis inhibitor composition.

[0017] The present invention discloses a method for preparing a ferroptosis inhibitor composition, comprising:

[0018] The ferroptosis inhibitor and the auxiliary agent are added into a solvent and dissolved to obtain a ferroptosis inhibitor composition.

[0019] Preferably, the ferroptosis inhibitor is isoorientin.

[0020] Preferably, the dosage ratio of the ferroptosis inhibitor to the solvent is 10-30 μM:1 ml.

[0021] Preferably, the auxiliary agent is a glucosamine derivative.

[0022] Preferably, the dosage ratio of the auxiliary agent to the solvent is 3-30 mg:1 ml.

[0023] Preferably, the solvent is serum-free DMEM medium.

[0024] The present invention discloses a method for preparing an active ester of salicylic acid, which is specifically as follows:

[0025] Salicylic acid and N-hydroxysuccinimide were dissolved in tetrahydrofuran, and N,N'-dicyclohexylcarbodiimide solution was added dropwise at 0°C for 1-3 hours. The mixture was then heated to 20-30°C and reacted for 20-30 hours. After the reaction, the filtrate was collected and rotary evaporated to obtain active salicylic acid ester.

[0026] Preferably, the mass ratio of salicylic acid to N-hydroxysuccinimide is 1:0.2-0.5.

[0027] Preferably, the usage ratio of salicylic acid to tetrahydrofuran is 1 g:30-45 ml.

[0028] Preferably, the N,N'-dicyclohexylcarbodiimide solution consists of N,N'-dicyclohexylcarbodiimide and tetrahydrofuran.

[0029] Preferably, the ratio of N,N'-dicyclohexylcarbodiimide to tetrahydrofuran is 1 g: 3-4 ml.

[0030] Preferably, the mass ratio of salicylic acid to N,N'-dicyclohexylcarbodiimide solution is 1:3-4.

[0031] The present invention discloses a method for preparing a glucosamine derivative, which specifically comprises:

[0032] Add salicylic acid active ester and triethylamine to tetrahydrofuran at -2-5°C, then lower the temperature to -25--15°C, add 4-methoxybenzoyl chloride, and react for 1-3 hours. Then raise the temperature to 20-30°C and react for 10-15 hours. After completion of the reaction, evaporate the mixture to obtain the intermediate. Mix D-glucosamine with an aqueous solution of N,N-dimethylformamide at -2-5°C, then dropwise add triethylamine and stir for 8-15 minutes. Then, add the intermediate and react for 6-10 hours. After completion of the reaction, evaporate the mixture to obtain the glucosamine derivative.

[0033] Preferably, the mass ratio of the active salicylic acid ester to the triethylamine is 1:0.2-0.5.

[0034] Preferably, the ratio of active salicylic acid ester to tetrahydrofuran is 1g:10-15.

[0035] Preferably, the mass ratio of the active salicylic acid ester to the 4-methoxybenzoyl chloride is 1:0.2-0.5.

[0036] Preferably, the N,N-dimethylformamide aqueous solution consists of N,N-dimethylformamide and water.

[0037] Preferably, the usage ratio of N,N-dimethylformamide to water is 1:0.2-0.7.

[0038] Preferably, the usage ratio of D-glucosamine to N,N-dimethylformamide aqueous solution is 1 g:10-15 ml.

[0039] Preferably, the mass ratio of D-glucosamine to triethylamine is 1:0.3-0.5.

[0040] Preferably, the mass ratio of D-glucosamine to the intermediate is 1:2.3-2.8.

[0041] More preferably, in the process of preparing the ferroptosis inhibitor composition of the present invention, in addition to using glucosamine derivatives, shikonin derivatives can also be used. The shikonin derivatives and glucosamine derivatives are used synergistically to exert a synergistic effect, further restore the reduced cell viability of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 caused by irradiation, increase the level of malondialdehyde, and effectively relieve radiation pneumonia as a ferroptosis inhibitor composition.

[0042] Preferably, the usage ratio of the glucosamine derivative to the shikonin derivative is 1:0.3-1.

[0043] The present invention discloses a method for preparing a shikonin derivative, which specifically comprises:

[0044] Fumaric acid, N',N'-dimethylcarbodiimide and 4-dimethylaminopyridine are added to dichloromethane, and then shikonin is added, and the reaction is carried out at -2-5°C for 10-15 hours. After the reaction is completed, the mixture is placed at -15--25°C for separation to obtain a filtrate, and the filtrate is evaporated and concentrated to obtain a concentrated product, which is then separated by thin layer chromatography to obtain a shikonin derivative.

[0045] Preferably, the molar ratio of fumaric acid to N',N'-dimethylcarbodiimide is 1:1.6-2.

[0046] Preferably, the molar ratio of fumaric acid to 4-dimethylaminopyridine is 1:0.1-0.5.

[0047] Preferably, the usage ratio of fumaric acid to dichloromethane is 1 mol: 60-90 ml.

[0048] Preferably, the molar ratio of fumaric acid to shikonin is 1:0.7-1.

[0049] Preferably, the volume ratio of ethyl acetate to petroleum ether is 1:5-10.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The present invention provides a ferroptosis inhibitor and its use in radiation-induced pneumonitis treatment. The ferroptosis inhibitor and an adjuvant are added to a solvent and dissolved to obtain a ferroptosis inhibitor composition; the ferroptosis inhibitor is isoorientin; the adjuvant is a glucosamine derivative; and the solvent is serum-free DMEM medium. The ferroptosis inhibitor composition prepared by the present method effectively reduces irradiation-induced increases in reactive oxygen species, ferrous ions, and malondialdehyde levels in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12, restores mitochondrial membrane potential enrichment, and inhibits the expression of various inflammatory factors in irradiated cells, thereby significantly improving the progression of radiation-induced pneumonitis. This provides a new medication strategy for radiation-induced pneumonitis treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0053] Figure 1 is the infrared spectrum of glucosamine derivatives;

[0054] Figure 2 Heat map of differentially expressed genes in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 after 6 hours of irradiation;

[0055] Figure 3 Heat map of differentially expressed genes in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 after 7 days of irradiation;

[0056] Figure 4 Cluster analysis of differential gene functions before and after irradiation;

[0057] Figure 5 This is a Venn diagram of the cross-genes between differentially expressed genes before and after irradiation and ferroptosis-related genes;

[0058] Figure 6 This is the result diagram of gene enrichment after irradiation;

[0059] Figure 7 is the logarithm of the ratio of gene expression levels before and after irradiation;

[0060] Figure 8 This is a diagram of cell viability detection of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0061] Figure 9This is a diagram of the detection of cellular reactive oxygen species in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0062] Figure 10 The mean fluorescence intensity of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 was measured;

[0063] Figure 11 This is a graph showing the measurement of ferrous ion levels in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0064] Figure 12 The figure shows the determination of glutathione levels in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0065] Figure 13 This is a graph showing the measurement of malondialdehyde levels in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0066] Figure 14 This is a diagram of the mitochondrial membrane potential detection of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0067] Figure 15 Figure 2 shows the cell viability assay of different groups of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0068] Figure 16 Figure 2 shows the detection of reactive oxygen species in different groups of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0069] Figure 17 The graph shows the measurement of ferrous ion levels in different groups of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0070] Figure 18 The figure shows the malondialdehyde level measurement in different groups of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0071] Figure 19 This is a graph showing the mitochondrial membrane potential detection of different groups of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0072] Figure 20 This is a graph showing the mRNA expression levels of related inflammatory factors in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12;

[0073] Figure 21 Heatmap of gene expression differences between different cell types and groups;

[0074] Figure 22 Graph showing the effects of different ferroptosis inhibitor combinations on cell viability;

[0075] Figure 23 This figure shows the effects of different ferroptosis inhibitor combinations on malondialdehyde levels. DETAILED DESCRIPTION

[0076] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0077] The following first describes the concepts involved in this application with reference to the accompanying drawings. It should be noted that the following description of each concept is intended only to make the content of this application easier to understand and does not limit the scope of protection of this application. At the same time, the embodiments and features in the embodiments of this application can be combined with each other unless there is a conflict. The following detailed description of this application will be made with reference to the accompanying drawings and in conjunction with the embodiments.

[0078] Example 1:

[0079] Preparation of the ferroptosis inhibitor composition: isoorientin is added to serum-free DMEM medium to obtain the ferroptosis inhibitor composition, wherein the dosage ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml.

[0080] Example 2:

[0081] Preparation of active salicylic acid ester: Salicylic acid and N-hydroxysuccinimide were dissolved in tetrahydrofuran to obtain a mixture. N,N'-dicyclohexylcarbodiimide solution was added dropwise to the mixture at 0°C for 2 h, then the temperature was raised to 25°C for 25 h. After completion of the reaction, the filtrate was collected and rotary evaporated to obtain active salicylic acid ester. The mass ratio of salicylic acid to N-hydroxysuccinimide was 1:0.31, the mass ratio of salicylic acid to tetrahydrofuran was 1 g:36.71 ml, the N,N'-dicyclohexylcarbodiimide solution was composed of N,N'-dicyclohexylcarbodiimide and tetrahydrofuran in a ratio of 1 g:3.23 ml, and the mass ratio of salicylic acid to N,N'-dicyclohexylcarbodiimide solution was 1:3.25.

[0082] Preparation of glucosamine derivatives: Add salicylic acid active ester and triethylamine to tetrahydrofuran at 0°C, then lower the temperature to -20°C, add 4-methoxybenzoyl chloride, and allow to react for 2 hours. Then, raise the temperature to 25°C and allow to react for 12 hours. After completion of the reaction, rotary evaporation is performed to obtain the intermediate. D-glucosamine is mixed with an aqueous solution of N,N-dimethylformamide at 0°C, followed by the dropwise addition of triethylamine and stirring for 10 minutes. The intermediate is then added and allowed to react for 7 hours. After completion of the reaction, rotary evaporation is performed to obtain the glucosamine derivative. The mass ratio of salicylic acid active ester to triethylamine is 1:0.4, the mass ratio of salicylic acid active ester to tetrahydrofuran is 1 g:11.87 ml, the mass ratio of salicylic acid active ester to 4-methoxybenzoyl chloride is 1:0.36, the N,N-dimethylformamide aqueous solution is composed of N,N-dimethylformamide and water, the mass ratio of N,N-dimethylformamide to water is 1:0.5, the mass ratio of D-glucosamine to N,N-dimethylformamide aqueous solution is 1 g:13.91 ml, the mass ratio of D-glucosamine to triethylamine is 1:0.47, and the mass ratio of D-glucosamine to the intermediate is 1:2.58.

[0083] Preparation of a ferroptosis inhibitor composition: Isoorientin and a glucosamine derivative are added to a serum-free DMEM medium to obtain a ferroptosis inhibitor composition. The ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml, and the ratio of the glucosamine derivative to serum-free DMEM medium is 8 mg:1 L.

[0084] Example 3:

[0085] The preparation of salicylic acid active ester is the same as in Example 2.

[0086] The preparation of glucosamine derivatives is the same as in Example 2.

[0087] Preparation of ferroptosis inhibitor composition: The preparation of the ferroptosis inhibitor composition in this example is compared with that in Example 2, except that the usage ratio of the glucosamine derivative to the serum-free DMEM culture medium is 15 mg:1 L, and other conditions and parameters are the same as in Example 2.

[0088] Example 4:

[0089] The preparation of salicylic acid active ester is the same as in Example 2.

[0090] The preparation of glucosamine derivatives is the same as in Example 2.

[0091] Preparation of ferroptosis inhibitor composition: The preparation of the ferroptosis inhibitor composition in this example is compared with that in Example 2, except that the usage ratio of the glucosamine derivative to the serum-free DMEM culture medium is 5 mg:1 L, and other conditions and parameters are the same as in Example 2.

[0092] Example 5:

[0093] The preparation of salicylic acid active ester is the same as in Example 2.

[0094] The preparation of glucosamine derivatives is the same as in Example 2.

[0095] Preparation of Shikonin Derivatives: Fumaric acid, N',N'-dimethylcarbodiimide, and 4-dimethylaminopyridine are added to dichloromethane to obtain a mixture, followed by the addition of Shikonin, and the mixture is reacted at 0°C for 12 hours. After completion of the reaction, the mixture is separated at -20°C to obtain a filtrate, which is evaporated and concentrated to obtain a concentrated product. Finally, the Shikonin Derivatives are separated by thin-layer chromatography. The molar ratio of fumaric acid to N',N'-dimethylcarbodiimide is 1:1.725, the molar ratio of fumaric acid to 4-dimethylaminopyridine is 1:0.22, the molar ratio of fumaric acid to dichloromethane is 1 mol:80 ml, the molar ratio of fumaric acid to shikonin is 1:0.875, and the volume ratio of ethyl acetate to petroleum ether is 1:7.

[0096] Preparation of a ferroptosis inhibitor composition: Isoorientin, a glucosamine derivative, and a shikonin derivative are added to a serum-free DMEM medium to obtain a ferroptosis inhibitor composition. The ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml, the ratio of the glucosamine derivative to serum-free DMEM medium is 8 mg:1 L, and the ratio of the glucosamine derivative to the shikonin derivative is 1:0.5.

[0097] Example 6:

[0098] The preparation of salicylic acid active ester is the same as in Example 2.

[0099] The preparation of glucosamine derivatives is the same as in Example 2.

[0100] The preparation of shikonin derivatives is the same as in Example 5.

[0101] Preparation of ferroptosis inhibitor composition: The preparation of the ferroptosis inhibitor composition in this example is compared with that in Example 5, except that the usage ratio of the glucosamine derivative to the shikonin derivative is 1:0.9, and other conditions and parameters are the same as in Example 5.

[0102] The ferroptosis inhibitor composition prepared by the present invention was evaluated for safety in an acute toxicity experiment in mice. The mice were randomly divided into a normal control group and a ferroptosis inhibitor composition group. After fasting for approximately 16 hours, the mice were weighed and administered 10 ml / kg of the ferroptosis inhibitor composition three times daily via intraperitoneal injection. The control group received an equal volume of deionized water. The rats were observed for 14 consecutive days for appearance, behavior, respiration, food and water intake, defecation, and mortality. After the experiment, no rats died in any group, and no obvious toxic reactions were observed. This indicates that the ferroptosis inhibitor composition prepared by the present invention is practically non-toxic.

[0103] Comparative Example 1:

[0104] The preparation of shikonin derivatives is the same as in Example 5.

[0105] Preparation of ferroptosis inhibitor composition: The preparation of the ferroptosis inhibitor composition in this example is compared with that in Example 5, except that no glucosamine derivative is used, and other conditions and parameters are the same as in Example 5.

[0106] Experimental Example 1:

[0107] The glucosamine derivatives were characterized by infrared spectroscopy analysis of the glucosamine derivatives in Example 1, with a scanning range of 500-4000 cm -1 The results are as follows Figure 1 As shown, 720cm -1 and 1590cm -1 The absorption peaks of aromatic ring CH and C=C are 1050-1150cm -1 is the absorption peak of CO, 1550 cm -1 is the absorption peak of CN, 1640 cm -1 is the absorption peak of NH, 1705cm -1 is the absorption peak of C=O, 3200-3600cm -1 is the absorption peak of OH.

[0108] Experimental Example 2:

[0109] The correlation analysis between ferroptosis and radiation pneumonia was conducted by using human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12. The normal cells, namely human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12, were irradiated with 3.5Gy and 2Gy, respectively, using a small animal x-irradiator to generate irradiated cells. The radiation pneumonia dataset GSE242840 was analyzed using the R package, the transcriptome data was differentially analyzed using the DESeq2 package, and differential gene enrichment was performed using the clusterProfiler and enrichplot packages. The results are shown in Figure 2. Figure 2-7As shown, 28 of the differentially expressed genes are ferroptosis-related genes, and the expression of ferroptosis-related genes is significantly increased after radiation exposure. These fully demonstrate that ferroptosis is closely related to radiation pneumonia.

[0110] Experimental Example 3:

[0111] Experimental determination of the correlation between ferroptosis and radiation pneumonia: First, an in vitro cell model of radiation pneumonia was constructed, using human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12. Using a small animal X-ray irradiator, normal cells, namely human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12, were irradiated with 3.5Gy and 2Gy, respectively, to obtain irradiated cells. Ferroptosis markers were then detected:

[0112] For cell viability testing, normal cells and irradiated cells were inoculated in DMEM medium containing 10% serum and 1% penicillin-streptomycin and cultured at 37°C in an atmosphere containing 5% CO2 for 24 hours. The normal and irradiated cells were then collected and cultured in serum-free DMEM medium. Cell viability was measured using the CCK-8 kit at different time points. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2 × 10 5 The ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1. The CCK-8 kit was purchased from Novozyme Biotechnology Co., Ltd. Figure 8 As shown, the cell activity assay results of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent, and the growth rate of the irradiated cells was lower than that of normal cells, that is, the cell activity of the irradiated cells was significantly lower than that of normal cells as time went by.

[0113] To detect the level of cellular reactive oxygen species, normal cells and irradiated cells were inoculated in DMEM medium containing 10% serum and 1% penicillin-streptomycin and cultured at 37°C in an atmosphere containing 5% CO2 for 24 hours. The cells were then collected and serum-free DMEM medium was added for a further 48 hours of culture. After the culture was completed, the cells were washed three times with PBS, digested with trypsin for 2 minutes, and then digested with complete DMEM medium. The normal cells and irradiated cells were collected and the reactive oxygen species level was measured using an active oxygen kit at an excitation wavelength of 495 nm and an emission wavelength of 530 nm. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5The ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1, the volume ratio of trypsin to serum-free DMEM medium was 1:10, and the active oxygen species test kit was purchased from Novozyme Biotechnology Co., Ltd. Figure 9 and 10 As shown, Figure 9 This is a flow cytometric analysis of lipid-reactive oxygen species in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12. After irradiation, the cell flow cytometric curves shifted to the right, and the level of reactive oxygen species increased significantly. Figure 10 The mean fluorescence intensity of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 is measured. The mean fluorescence intensity values ​​of the irradiated cells are significantly higher than those of normal cells, indicating that the irradiated cells have higher reactive oxygen species production.

[0114] To measure ferrous iron levels, normal and irradiated cells were inoculated in DMEM medium containing 10% serum and 1% penicillin-streptomycin and cultured at 37°C in an atmosphere containing 5% CO₂ for 24 hours. The cells were then harvested and incubated in serum-free DMEM medium for an additional 48 hours. After incubation, the cells were washed three times with PBS, digested with trypsin for 2 minutes, and then terminated with complete DMEM medium. The normal and irradiated cells were harvested and ferrous iron levels were measured using a ferrous iron detection kit at an excitation wavelength of 542 nm and an emission wavelength of 575 nm. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2 × 10 5 The ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1, the volume ratio of trypsin to serum-free DMEM medium was 1:10, and the ferrous ion detection kit was purchased from Novozyme Biotechnology Co., Ltd. Figure 11 As shown in the figure, the results of ferrous ion level determination in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. After irradiation, the cell flow cytometry curve shifted to the right and the ferrous ion level increased significantly.

[0115] To test glutathione levels, normal cells and irradiated cells were inoculated in DMEM medium containing 10% serum and 1% penicillin-streptomycin and cultured at 37°C in an atmosphere containing 5% CO2 for 24 hours. The cells were then collected and cultured in serum-free DMEM medium for another 48 hours. After the culture, the cells were washed three times with PBS, digested with trypsin for 2 minutes, and then digested with complete DMEM medium. The cells were then washed three times with PBS again, and the normal cells and irradiated cells were collected and disrupted. The supernatant after disruption was taken and the glutathione content was measured using a glutathione kit. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1, the volume ratio of trypsin to serum-free DMEM medium was 1:10, and glutathione was purchased from Novozyme Biotechnology Co., Ltd. Figure 12 As shown in the figure, the results of glutathione level determination in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent, and the glutathione levels of cells were significantly decreased after irradiation.

[0116] For the detection of malondialdehyde levels, normal cells and irradiated cells were inoculated in DMEM medium containing 10% serum and 1% penicillin-streptomycin, and cultured at 37°C in an atmosphere containing 5% CO2 for 24 hours. The cells were then collected and serum-free DMEM medium was added to continue culturing for 48 hours. After the culture, the cells were washed three times with PBS, lysed with RIPA buffer at 0°C for 30 minutes, and the lysate was collected and added with malondialdehyde working solution to react at 100°C for 2 hours. After the reaction, the supernatant was collected and the absorbance was measured at 532nm to calculate the malondialdehyde content. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×10 5 The volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1, the volume ratio of trypsin to serum-free DMEM medium was 1:10, the malondialdehyde working solution consisted of TBA working solution, TBA storage solution and antioxidant, and the volume ratio of TBA working solution, TBA storage solution and antioxidant was 1:0.33:0.02, and the volume ratio of lysis buffer to malondialdehyde working solution was 1:2. Figure 13As shown in the figure, the results of malondialdehyde level determination in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent, and the malondialdehyde levels of cells increased significantly after irradiation.

[0117] To measure mitochondrial membrane potential, normal and irradiated cells were inoculated in DMEM medium supplemented with 10% serum and 1% penicillin-streptomycin and incubated at 37°C in an atmosphere containing 5% CO₂ for 24 hours. The cells were then harvested and incubated in serum-free DMEM medium for an additional 48 hours. After incubation, the cells were washed three times with PBS and fixed in paraformaldehyde for 15 minutes. After fixation, changes in mitochondrial membrane potential in normal and irradiated cells were measured using a mitochondrial membrane potential assay. Observation and photography were performed using an excitation wavelength of 490 nm and an emission wavelength of 530 nm. The ratio of normal cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×105 cells / ml, the ratio of irradiated cells to DMEM medium containing 10% serum and 1% penicillin-streptomycin was 2×105 cells / ml, and the volume ratio of DMEM medium containing 10% serum and 1% penicillin-streptomycin to serum-free DMEM medium was 1:1. The mitochondrial membrane potential kit was purchased from Beyotime Biotechnology Co., Ltd. Figure 14 As shown in the figure, the results of mitochondrial membrane potential measurement of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. After irradiation, the green light of the cells was significantly enhanced and the red light was weakened. The mitochondrial membrane potential of the cells was mainly enriched outside the mitochondria, that is, the cells may be in the early stage of apoptosis.

[0118] The above results indicate that radioactive irradiation can induce ferroptosis of lung epithelial cells.

[0119] Experimental Example 4:

[0120] To investigate the effects of isoorientin on ferroptosis and radiation-induced pneumonia, human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were divided into a control group, an irradiated group, and a post-irradiation drug administration group. Ferroptosis markers were detected:

[0121] Cell viability detection. The cell viability detection method in this experimental example is compared with that in Example 3. The difference is that after adding serum-free DMEM medium, DMSO is added to the control group and the irradiation group, and isoorientin is added to the drug-treated group after irradiation. The dosage ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml. Other conditions and parameters are the same as in Example 3. The results are shown in Figure 3. Figure 15 As shown, the results of cell activity assays of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. Compared with the irradiation group, the cell viability of the drug-treated group after irradiation was significantly increased in growth rate and the cell viability was significantly restored.

[0122] Cellular reactive oxygen species level detection. The detection method of cellular reactive oxygen species level in this experimental example is compared with Example 3, except that after adding serum-free DMEM medium, DMSO is added to the control group and the irradiation group, and isoorientin is added to the drug-treated group after irradiation. The dosage ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml. Other conditions and parameters are the same as Example 3. The results are shown in Figure 3. Figure 16 As shown in the figure, the results of the cellular reactive oxygen species level determination of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. Compared with the irradiated group, the cell flow cytometry curves of the irradiated group were shifted to the left, and the reactive oxygen species levels were significantly decreased.

[0123] Ferrous ion level detection: The method for detecting ferrous ion level in this experimental example is compared with that in Example 3, except that after adding serum-free DMEM medium, DMSO was added to the control group and the irradiation group, and isoorientin was added to the drug-treated group after irradiation. The dosage ratio of isoorientin to serum-free DMEM medium was 16 μM:1 ml. Other conditions and parameters were the same as those in Example 3. Figure 17 As shown in the figure, the results of ferrous ion level determination in human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. Compared with the irradiated group, the cell flow cytometry curve of the irradiated group shifted to the left, and the ferrous ion level in the irradiated group was significantly decreased.

[0124] Malondialdehyde level detection. The detection method of malondialdehyde level in this experimental example is compared with Example 3, except that after adding serum-free DMEM medium, DMSO is added to the control group and the irradiation group, and isoorientin is added to the drug-treated group after irradiation. The dosage ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml. Other conditions and parameters are the same as Example 3. The results are shown in Figure 3. Figure 18 As shown in the figure, the results of malondialdehyde level determination in lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. The malondialdehyde level in the drug-treated group after irradiation was significantly decreased compared with the irradiation group.

[0125] Cell mitochondrial membrane potential detection. The detection method of cell mitochondrial membrane potential in this experimental example is compared with Example 3, except that after adding serum-free DMEM medium, DMSO is added to the control group and the irradiation group, and isoorientin is added to the drug-treated group after irradiation. The dosage ratio of isoorientin to serum-free DMEM medium is 16 μM:1 ml. Other conditions and parameters are the same as Example 3. The results are shown in Figure 3. Figure 19 As shown, the malondialdehyde level determination results of lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12 were consistent. Compared with the irradiation group, the red light of the cells in the irradiation group was enhanced, the green light was weakened, and the mitochondrial membrane potential of the cells was enriched and restored to the mitochondria.

[0126] To measure mRNA expression levels of related inflammatory factors, normal and irradiated cells were plated in DMEM medium supplemented with 10% serum and 1% penicillin-streptomycin and cultured at 37°C in a 5% CO2 atmosphere for 24 hours. The cells were then harvested and replaced with serum-free DMEM medium. The control and irradiated groups were then treated with DMSO. The irradiated and treated groups were then treated with isoorientin at a ratio of 16 μM to 1 ml of serum-free DMEM medium. The cells were then cultured for a further 48 hours. Total RNA was then extracted using Freezol reagent, and cDNA was obtained using the HiScript II Q RT SuperMix kit. qRT-PCR was then performed using the ChamQ Universal SYBR qPCR Master Mix kit. The qRT-PCR protocol consisted of an initial denaturation at 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing / extension at 60°C for 30 seconds. The Freezol reagent, HiScript II Q RT SuperMix kit, and ChamQ Universal SYBR qPCR Master Mix kit were purchased from Novozymes Biotechnology Co., Ltd. The volume ratio of Freezol reagent to serum-free DMEM medium was 1:2. Figure 20 and 21 As shown in the results, the mRNA expression of inflammatory factors such as MCP-1, ICAM-1, IL-1α, TNF-α, IL-6, and IP-10 in the irradiated group cells was significantly reduced. At the same time, the post-irradiation drug administration group could inhibit the expression of various inflammatory factors in the irradiated cells.

[0127] Therefore, isoorientin can significantly improve the progression of radiation pneumonitis.

[0128] Experimental Example 5:

[0129] Effects of different ferroptosis inhibitor compositions on cell viability. The cell viability detection method in this experimental example is compared with that in Example 4, except that after collecting cells, the ferroptosis inhibitor composition was added to the irradiated and dosing group, and the volume ratio of DMEM culture medium containing 10% serum and 1% penicillin-streptomycin to the amount of the ferroptosis inhibitor composition used was 1:1. Cell viability was measured after 96 hours of culture. Other conditions and parameters were the same as in Example 4. The ferroptosis inhibitor composition was the ferroptosis inhibitor composition prepared in Examples 1-6 and Comparative Example 1.

[0130] The results are as follows Figure 22As shown, Example 1, compared with Example 2, shows that the use of glucosamine derivatives to prepare ferroptosis inhibitor compositions can improve the cell viability of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12; Example 2, compared with Example 3, shows that increasing the amount of glucosamine derivatives within a certain range can further improve cell viability; Example 2, compared with Example 4, shows that reducing the amount of glucosamine derivatives within a certain range will reduce cell viability; Example 2, compared with Example 5, shows that on the basis of using glucosamine derivatives, the use of shikonin derivatives can improve cell viability; Example 5, compared with Example 6, shows that increasing the amount of shikonin derivatives within a certain range can improve cell viability; Example 5, compared with Comparative Example 1, shows that shikonin derivatives need to be used together with glucosamine derivatives, and the use of shikonin derivatives alone has no obvious effect on improving cell viability.

[0131] Experimental Example 6:

[0132] Effects of different ferroptosis inhibitor compositions on malondialdehyde levels. The method for detecting malondialdehyde levels in this experimental example is compared with that in Example 4, except that after cell collection, the ferroptosis inhibitor composition was added to the irradiated and dosing group, and the volume ratio of DMEM culture medium containing 10% serum and 1% penicillin-streptomycin to the amount of the ferroptosis inhibitor composition used was 1:1. Other conditions and parameters were the same as in Example 4. The ferroptosis inhibitor composition was the ferroptosis inhibitor composition prepared in Examples 1-6 and Comparative Example 1.

[0133] The results are as follows Figure 23 As shown, Example 1, compared with Example 2, shows that the use of glucosamine derivatives to prepare ferroptosis inhibitor compositions can reduce the malondialdehyde levels of human lung epithelial cells BEAS-2b and mouse lung epithelial cells MLE-12; Example 2, compared with Example 3, shows that increasing the amount of glucosamine derivatives within a certain range can further reduce the malondialdehyde level; Example 2, compared with Example 4, shows that reducing the amount of glucosamine derivatives within a certain range will inhibit the effect of reducing the malondialdehyde level; Example 2, compared with Example 5, shows that on the basis of using glucosamine derivatives, the use of shikonin derivatives can further reduce the malondialdehyde level; Example 5, compared with Example 6, shows that increasing the amount of shikonin derivatives within a certain range can reduce the malondialdehyde level; Example 5, compared with Comparative Example 1, shows that shikonin derivatives need to be used together with glucosamine derivatives, and the use of shikonin derivatives alone has no obvious effect on reducing the malondialdehyde level.

[0134] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and do not impose any form of limitation on the implementation methods of the technology of the present invention. Any person skilled in the art may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0135] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression, there are objectively infinite specific structures. For ordinary technicians in this technical field, without departing from the principles of this application, they can also make several improvements, modifications or changes, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A ferroptosis inhibitor composition comprising: Ferroptosis inhibitor, auxiliary agent and solvent; the ferroptosis inhibitor is isoorientin, and the dosage ratio of the ferroptosis inhibitor to the solvent is 10-30 μm: 1 ml; The auxiliary agent is a glucosamine derivative, which is prepared by reacting salicylic acid, N-hydroxysuccinimide and 4-methoxybenzoyl chloride; the dosage ratio of the auxiliary agent to the solvent is 3-30 mg: 1 ml.

2. A ferroptosis inhibitor composition according to claim 1, characterized in that described The solvent was serum-free DMEM medium.

3. A ferroptosis inhibitor composition according to claim 1, characterized in that The preparation method of the glucosamine derivative comprises: Add active salicylic acid ester and triethylamine to tetrahydrofuran, lower the temperature and add 4-methoxybenzoyl chloride to react, then increase the temperature to continue the reaction, and after the reaction is completed, rotary evaporation is performed to obtain an intermediate; D-glucosamine is mixed with an aqueous solution of N,N-dimethylformamide, and then triethylamine is added dropwise with stirring, and then the intermediate is added to react, and after the reaction is completed, rotary evaporation is performed to obtain a glucosamine derivative.

4. A ferroptosis inhibitor composition according to claim 3, characterized in that The mass ratio of the usage of the D-glucosamine to the intermediate is 1:2.3-2.

8.

5. A ferroptosis inhibitor composition according to claim 3, characterized in that: The reaction temperature of the adding 4-methoxybenzoyl chloride reaction is -25---15° C., and the reaction time is 1-3 hours.

6. A ferroptosis inhibitor composition according to claim 3, characterized in that: The heating temperature is 20-30°C, and the heating reaction time is 10-15 hours.

7. A ferroptosis inhibitor composition according to claim 3, characterized in that: The reaction temperature for adding the intermediate is -2-5°C, and the reaction time is 6-10 hours.

8. Use of a ferroptosis inhibitor composition in the preparation of a drug for treating radiation pneumonia, wherein the ferroptosis inhibitor composition comprises the ferroptosis inhibitor composition according to any one of claims 1 to 7.

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