Use of hard delphinine for the preparation of a medicament for the treatment of lung injury

Abstract

The application discloses application of hard delphinine in preparation of a drug for treating lung injury. The application first proposes and verifies that hard delphinine can inhibit lipid peroxidation of cells, and then inhibit apoptosis and ferroptosis, and finally realize effective relief of acute lung injury, especially acute lung injury secondary to sepsis, thereby providing a new direction for lung injury treatment in clinic.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, and in particular to the application of delphinidin in the preparation of drugs for treating lung injury. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. As one of the most common and serious complications of sepsis, sepsis-associated acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are critical illnesses with high morbidity and mortality rates in intensive care units. The mortality rate of ALI / ARDS exceeds 30%, and it is often accompanied by multiple organ failure, significantly increasing the difficulty of treatment and the risk of poor prognosis. Its pathogenesis is extremely complex, involving multiple molecular and cellular mechanisms such as endothelial dysfunction, immune dysregulation, epigenetic alterations, and oxidative stress. Currently, the clinical treatment of ALI / ARDS mainly relies on a comprehensive approach including anti-infection, fluid resuscitation, respiratory support, and immunomodulation. However, to date, there are still no specific drugs that can effectively reverse or interrupt the intrinsic process of lung injury; supportive care remains the primary approach.

[0003] Delsoline (DEL) is a compound found in *Lysimachia christinae* (a type of herb). Delphinium anthriscifolium Hance The main alkaloid of ) belongs to C 19 Diterpenoid alkaloids. In traditional medicine, *Deltametes aromatica* is used to dispel wind and dampness, promote blood circulation, and relieve pain, and is commonly used for rheumatism, hemiplegia, indigestion, and cough. Modern pharmacological studies have shown that DEL has effects similar to curare and ganglion blocking, and can be used to relieve muscle tension or overexertion. However, there are currently no reports on the use of DEL in the treatment of diseases such as lung injury. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the application of delphinidin in the preparation of drugs for the treatment of related diseases by inhibiting lipid peroxidation in cells, thereby inhibiting apoptosis and ferroptosis.

[0005] Technical solution: The application of the physalisine described in this invention in the preparation of drugs for treating lung injury.

[0006] Preferably, the CAS number of the delphinidin is 509-18-2.

[0007] Preferably, the lung injury is acute lung injury; more preferably, the acute lung injury is acute lung injury caused by sepsis.

[0008] Preferably, the application is in the preparation of a drug that inhibits lipid peroxidation.

[0009] Preferably, the application is in the preparation of drugs that inhibit apoptosis and / or ferroptosis.

[0010] Preferably, the drug contains delphinidin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0011] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, transdermal patches, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that delphinidin can inhibit lipid peroxidation of cells, thereby inhibiting apoptosis and ferroptosis, and ultimately achieve effective relief of acute lung injury, especially acute lung injury secondary to sepsis, providing a new direction for the clinical treatment of lung injury. Attached Figure Description

[0014] Figure 1 This is a schematic diagram of the experimental procedure for the treatment of an acute lung injury model using delphinidin. Figure 2 A graph showing the statistical results of the wet-to-dry ratio of the lungs in a mouse model of acute lung injury after treatment with delphinidin. Figure 3 Representative H&E staining images of lung tissue from mice with acute lung injury model after treatment with delphinidin; Figure 4 A graph showing the statistical results of lung tissue damage scores in a mouse model of acute lung injury after treatment with delphinidin. Figure 5 Representative Masson staining images of lung tissue from mice with acute lung injury model after delphinidin treatment; Figure 6 Representative images of TUNEL staining in lung tissue of mice with acute lung injury model after delphinidin treatment; Figure 7 Figure 1 shows the results of measuring cytokine levels in lung tissue of a mouse model of acute lung injury after treatment with delphinidin. Figure 8These are representative immunohistochemical staining images of iNOS and TNFα in lung tissue of mice with acute lung injury model after treatment with delphinidin. Figure 9 Figure 1 shows the results of lipid peroxidation-related indicators in lung tissue of mice with acute lung injury model after treatment with delphinidin. Figure 10 Figure 1 shows the results of GPX4 level measurement in lung tissue of mice with acute lung injury model after treatment with delphinidin. Figure 11 The graph shows the statistical results of the relative viability of normal MLE12 cells after treatment with delphinidin. Figure 12 A graph showing the statistical results of the relative cell viability of MLE12 cells, an acute lung injury model, after treatment with delphinidin. Figure 13 Figure 1 shows the flow cytometry results of apoptosis level in MLE12 cells, an acute lung injury model after treatment with delphinidin. Figure 14 Figure 1 shows the results of cytokine level measurement in MLE12 cells, an acute lung injury model, after treatment with delphinidin. Figure 15 Figure 1 shows the ROS level flow cytometry analysis results of MLE12 cells, an acute lung injury model after treatment with delphinidin. Figure 16 Representative images of ROS staining in MLE12 cells, an acute lung injury model after treatment with delphinidin. Figure 17 Figure 1 shows the flow cytometry results of lipid peroxidation levels in MLE12 cells, an acute lung injury model, after treatment with delphinidin. Figure 18 Figure 1 shows the results of lipid peroxidation-related index measurements in MLE12 cells, an acute lung injury model, after treatment with delphinidin. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: In vivo efficacy verification of delsoline (DEL) in the treatment of lung injury 1. Establishment and drug treatment of animal models of acute lung injury (ALI) Eight-week-old female C57BL / 6 mice weighing 20-25 g were purchased from the Experimental Animal Center of Nantong University and randomly divided into three groups: sham group, acute lung injury group (ALI group), and acute lung injury treatment group (ALI+DEL group), with n=12 in each group.

[0017] Using mice in the ALI and ALI+DEL groups, sepsis was induced by cecal ligation and puncture (CLP) to establish an animal model of acute lung injury. Specifically, mice were anesthetized by intraperitoneal injection of 50 mg / kg sodium pentobarbital. A 1-2 cm incision was made along the midline of the abdomen to expose the cecum. The cecum was then ligated 1 cm from the tip with 3-0 silk suture and punctured once with an 18-gauge needle. After squeezing out a small amount of feces, the cecum was returned to the abdominal cavity, and the abdomen was sutured in layers to complete the cecal ligation and puncture. In the sham group, the cecum was only exposed after anesthesia and then directly sutured without ligation or puncture.

[0018] All mice were administered medication immediately after surgery. Mice in the ALI+DEL group received an intraperitoneal injection of 20 mg / kg of delphinidin (purchased from MedChemExpress LLC., catalog number HY-N0789), while mice in the ALI and sham groups received an equal volume of physiological saline intraperitoneally. Mice were euthanized 24 hours later, and lung tissue was collected for subsequent experiments. The experimental procedure is as follows: Figure 1 As shown.

[0019] 2. Lung wet-to-dry ratio measurement The lung tissue collected from mice was weighed and recorded as wet weight. It was then dried in a constant-temperature oven at 80°C until constant weight, and the dry weight of the lung tissue was obtained again. The wet-to-dry weight ratio of the lung tissue for each group of mice was calculated using the following formula: Lung wet-to-dry weight ratio = Lung tissue wet weight / Lung tissue dry weight.

[0020] The results are as follows Figure 2 As shown, the lung wet-to-dry weight ratio of mice in the sham group was 3.9, that of mice in the ALI group was 8.3, and that of mice in the ALI+DEL group after DEL treatment decreased to 5.1. The wet / dry weight ratio of the lung is a direct indicator of pulmonary edema and a sensitive indicator of the severity of lung injury. This result indicates that DEL can effectively alleviate lung injury symptoms, relieve pulmonary edema, and achieve ALI treatment.

[0021] 3. Lung tissue pathological analysis 3.1 H&E staining The mouse lung tissues collected were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 4 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0022] The results are as follows Figure 3As shown, the alveolar structures of mice in the Sham group were intact without inflammatory infiltration, while the lungs of mice in the ALI group showed severe damage, including alveolar wall thickening, edema, and accumulation of a large number of inflammatory cells. These pathological changes were significantly reduced under DEL treatment, indicating that DEL can be used as a potential natural product for the treatment of ALI.

[0023] The degree of lung injury was further evaluated based on the H&E staining results of lung tissue. Inflammatory cell infiltration, edema, and damage in lung tissue were observed under low magnification to assess the distribution of lung injury; alveolar structures were observed under high magnification. Six high-power fields were used to assess and score the degree of lung injury. The specific scoring criteria were: 0 points, no injury; 1 point, <25% of the injury area; 2 points, 25%–50% of the injury area; 3 points, 50%–75% of the injury area; 4 points, >75% of the injury area.

[0024] The results are as follows Figure 4 As shown, the lung injury score of mice in the sham group was 0.86, the lung injury score of mice in the ALI group was 8.96, and after DEL treatment, the lung injury score decreased to 2.21, indicating that DEL can effectively alleviate ALI and effectively restore lung function.

[0025] 3.2 Masson staining The preliminary steps were the same as those for H&E staining. In the staining section, the collagen deposits in the liver tissue were stained using the Beyotime Masson Tricolor Staining Kit (catalog number C0189S). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0026] The results are as follows Figure 5 As shown, compared with the sham group mice, the ALI group mice had a large amount of collagen deposition in their lung tissue and severe damage to the alveolar structure, resulting in impaired lung function; while the mice treated with DEL showed a significant reduction in lung collagen deposition and effective improvement in alveolar structure, further indicating that DEL can effectively alleviate ALI.

[0027] 4. TUNEL staining of lung tissue After freezing and sectioning the mouse lung tissue collected above, it was fixed with 4% paraformaldehyde for 30 min, washed with PBS, and then incubated with 0.5% (v / v) Triton X-100 in PBS at room temperature for 5 min. Subsequently, it was stained with Beyotime's one-step TUNEL cell apoptosis detection kit (red fluorescence) (C1089). Finally, it was mounted with Beyotime's anti-fluorescence quenching mounting solution (containing DAPI) (P0131) and observed and images were acquired by fluorescence microscopy.

[0028] The results are as follows Figure 6As shown, compared with the sham group mice, the red fluorescence of the lung tissue of the ALI group mice was significantly enhanced, indicating that there was more DNA damage in the lung tissue. After DEL treatment, the red fluorescence of the lung tissue of the mice was significantly weakened, which preliminarily indicates that DEL can improve the DNA damage in lung tissue caused by sepsis and has a certain protective effect against apoptosis.

[0029] 5. Measurement of cytokine levels in lung tissue 100 mg of mouse lung tissue was collected and homogenized with 1 mL of pre-cooled physiological saline. The homogenate was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The cytokine levels in the lung tissue were measured using the Ellete mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay kit (catalog number E-EL-M0037) and the Ellete mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit (catalog number E-EL-M0044).

[0030] The results are as follows Figure 7 As shown, the levels of IL-1β in the sham group mice were 106 pg / mg and IL-6 were 127 pg / mg; the levels of IL-1β in the ALI group mice were 683 pg / mg and IL-6 were 877 pg / mg; while the levels of IL-1β in mice treated with DEL decreased to 320 pg / mg and IL-6 decreased to 400 pg / mg, indicating that DEL can reduce lung inflammation and tissue damage.

[0031] 6. Measurement of inducible nitric oxide synthase (iNOS) and TNFα levels in lung tissue The aforementioned 4 μm thick lung tissue sections were antigen-retrieved at 95°C for 20 min using citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited for 10 min with 3% hydrogen peroxide solution, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were then incubated overnight at 4°C with either a 1:500 dilution of iNOS primary antibody (Proteintech, catalog number 22226-1-AP) or a 1:500 dilution of TNFα primary antibody (Proteintech, catalog number 17590-1-AP). After washing, the sections were incubated at room temperature for 1 h with a 1:500 dilution of horseradish peroxidase (HRP) conjugated secondary antibody (Proteintech, catalog number SA00004-2). Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0032] The results are as follows Figure 8As shown, compared with the sham group mice, the protein expression levels of iNOS and TNF-α in the ALI group mice were significantly increased, while DEL treatment could significantly inhibit this trend.

[0033] 7. Measurement of lipid peroxidation-related indicators in lung tissue 100 mg of mouse lung tissue was collected and 1 mL of pre-cooled physiological saline was added to prepare a tissue homogenate. After centrifugation at 12,000 rpm for 15 min, the supernatant was collected. The levels of LPO, 4-HNE and MDA in the supernatant were detected using the Elite Lipid Peroxide (LPO) Colorimetric Assay Kit (Catalog No. E-BC-K176-M), the Elite 4-HNE Enzyme-Linked Immunosorbent Assay Kit (Catalog No. E-EL-0128), and the Elite MDA Enzyme-Linked Immunosorbent Assay Kit (Catalog No. E-EL-0060).

[0034] The results are as follows Figure 9 As shown, experimental data indicate that DEL can significantly inhibit the generation of lipid peroxidation products, including LPO and its stable byproducts MDA and 4-HNE, ultimately reducing oxidative stress in ALI model mice and achieving the goal of treating ALI.

[0035] The expression level of glutathione peroxidase 4 (GPX4) in lung tissue was further determined by Western blot. Specifically, 100 mg of mouse lung tissue was taken, ground in liquid nitrogen using a mortar and pestle, and then lysed with 0.1 volume of cyprotease inhibitor mixture (product number P001) and 0.1 volume of cyprotease inhibitor mixture (product number P003) in cyprotease RIPA lysis buffer (product number WB3100). The lysate was collected at 4°C for 30 min, centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant was collected. The supernatant was quantified using the cyprotease BCA protein quantification kit (product number WB6501). Subsequently, cyprotease 5× Loading buffer (product number WB2001) was added in proportion, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, GPX4 primary antibody (purchased from Proteintech, catalog number 30388-1-AP) diluted 1:1000 or GAPDH primary antibody (purchased from Proteintech, catalog number 10494-1-AP) diluted 1:20000 was added and incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, catalog number SA00001-2) diluted 1:2000 was added and incubated at room temperature for 2 h. Finally, the chemiluminescence was developed and images were acquired using the New Semiconductor Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0036] The results are as follows Figure 10As shown, compared with the sham group mice, the GPX4 protein level in the ALI group mice was significantly decreased, which promoted ferroptosis. However, administration of DEL could restore the GPX4 protein expression level, inhibit ferroptosis, and alleviate ALI.

[0037] Example 2: In vitro efficacy verification of delphinidin in the treatment of lung injury 1. Construction of an acute lung injury cell model Mouse lung epithelial cells MLE12 (purchased from Xiamen Aikexin Biotechnology Co., Ltd., catalog number IM-M015) were cultured in a dedicated incubator at 37°C and 5% CO2 using lung epithelial cell culture medium. The lung epithelial cell culture medium was RPMI 1640 medium supplemented with 2% (v / v) fetal bovine serum, 1% (v / v) GlutaMAX (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., PB180419), 1% (v / v) 1 M HEPES, 1% (v / v) ITS (purchased from Beyotime Biotechnology, C0345-10ml), 10 nM hydrocortisone (purchased from MedChemExpress LLC., HY-N0583), 10 nM dimethoxyestradiol (purchased from MedChemExpress LLC., HY-12033), and 1% (v / v) PS (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., PB180120).

[0038] MLE12 cells in the logarithmic growth phase were collected and treated with LPS (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number L8880) at a final concentration of 100 µg / mL for 24 h to construct an acute lung injury cell model.

[0039] 2. Cell viability assay after DEL treatment 2.1 Viability assay of normal MLE12 cells after DEL treatment Normal MLE12 cells were used at a dose of 1×10 3 Cells were seeded at the specified density in 96-well plates and cultured overnight. The original medium was then replaced with fresh lung epithelial cell culture medium containing DEL at final concentrations of 0, 1, 2, 5, 10, 20, 40, 80, 160, 320, or 640 µM for 24 h. After treatment, each well was replaced with 100 μL of RPMI 1640 basal medium containing 10% CCK8 reagent and incubated at 37°C for 2 h. Absorbance was measured at 450 nm using a microplate reader, and relative cell viability was calculated.

[0040] The results are as follows Figure 11As shown, low concentrations of DEL less than 10 µM did not have a significant cytotoxic effect on MLE12 cells; therefore, the final concentration of DEL in subsequent in vitro experiments was determined to be 10 µM.

[0041] 2.2. Viability determination of MLE12 cells after DEL treatment in an acute lung injury cell model Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 1×10⁻⁶. 3 Cells were seeded at a density of 96-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO (DMSO group). MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO (LPS group) or DEL with a final concentration of 10 µM (LPS+DEL group). After 24 h of treatment, each well was replaced with 100 μL of RPMI 1640 basal medium containing 10% CCK8 reagent and incubated at 37°C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative level of cell viability was calculated.

[0042] The results are as follows Figure 12 As shown, compared with the DMSO group, the cell viability of the LPS group decreased to 0.32, while the cell viability could be restored to 0.69 after treatment with 10 µM DEL, which preliminarily indicates that DEL can effectively restore the viability of MLE12 cells, an acute lung injury cell model induced by LPS.

[0043] 3. Determination of apoptosis level after DEL treatment Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO. MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO or a final concentration of 10 µM DEL.

[0044] Cells were collected 24 h after treatment and stained using the Annexin V-FITC / 7AAD apoptosis detection kit (catalog number A213-01). The stained cells were analyzed by flow cytometry to quantify the level of apoptosis.

[0045] The results are as follows Figure 13 As shown, compared with the DMSO group, the LPS group cells had a higher level of apoptosis, while the level of apoptosis decreased significantly after DEL treatment.

[0046] 4. Measurement of cytokine levels after DEL treatment Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO. MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO or a final concentration of 10 µM DEL.

[0047] After 24 h of treatment, cell culture supernatant was collected, and the levels of cytokines in the supernatant were measured using the Elletet mouse interleukin-1β (IL-1β) enzyme-linked immunosorbent assay kit, the Elletet mouse interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit, and the Elletet tumor necrosis factor-α (TNF-α) enzyme-linked immunosorbent assay kit (catalog number E-EL-M3063).

[0048] The results are as follows Figure 14 As shown, compared with the DMSO group, the LPS group showed significantly increased levels of inflammatory cytokines, while DEL treatment significantly downregulated the levels of inflammatory cytokines.

[0049] 5. Measurement of reactive oxygen species (ROS) levels in cells after DEL treatment Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO. MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO or a final concentration of 10 µM DEL.

[0050] Cells were collected 24 h after treatment and stained with a 5 μM DCFH-DA probe (MedChemExpress LLC., catalog number HY-D0940) at 37°C for 30 min. Quantitative analysis was performed by flow cytometry. Another portion of cells from the same batch were stained with the DCFH-DA probe, fixed, and counterstained with Beyotime's antifluorescence quenching mounting medium. Images were then observed and acquired using a fluorescence microscope.

[0051] The results are as follows Figure 15 , 16As shown, DEL can inhibit the production of ROS in LPS-induced ALI cell models, thereby inhibiting ferroptosis and restoring cell viability.

[0052] 6. Measurement of cellular lipid peroxidation levels after DEL treatment Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO. MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO or a final concentration of 10 µM DEL.

[0053] Cells were collected 24 h after treatment, washed with pre-cooled PBS, and stained with BODIPY 581 / 591 C11 probe (purchased from MedChemExpress LLC., catalog number HY-D1301) at 37°C for 30 min. Intracellular lipid peroxidation levels were detected and analyzed by flow cytometry.

[0054] The results are as follows Figure 17 As shown, DEL can effectively inhibit lipid peroxidation levels, and its changing trend is similar to that of ROS.

[0055] 7. Determination of cell lipid peroxidation-related index levels after DEL treatment Normal MLE12 cells or MLE12 cells from an acute lung injury cell model treated with LPS were used at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 6-well plates and cultured overnight. Normal MLE12 cells were replaced with fresh lung epithelial cell culture medium containing 1‰ DMSO. MLE12 cells, an acute lung injury cell model treated with LPS, were treated with fresh lung epithelial cell culture medium containing 1‰ DMSO or a final concentration of 10 µM DEL.

[0056] Cells were collected 24 h after treatment, homogenized with PBS and centrifuged. The supernatant was collected and the levels of LPO, 4-HNE and MDA were detected using the Elite Lipid Peroxide (LPO) Colorimetric Assay Kit, the Elite 4-HNE Enzyme-Linked Immunosorbent Assay Kit and the Elite MDA Enzyme-Linked Immunosorbent Assay Kit, respectively.

[0057] The results are as follows Figure 18 As shown, DEL can significantly reduce the level of lipid peroxidation products in LPS group cells, further verifying that DEL can inhibit ferroptosis in lung epithelial cells.

[0058] The expression level of GPX4 in DEL-treated cells was further determined by Western blot. Specifically, cells were collected 24 h after treatment and lysed with RIPA lysis buffer containing 0.1 volume of cyprotease inhibitor and phosphatase inhibitor mixtures at 4°C for 30 min. After centrifugation at 12,000 rpm at 4°C for 30 min, the supernatant was collected and quantified using a cyprotein BCA protein quantification kit. Subsequently, 5× loading buffer was added and the mixture was boiled in a metal bath for 10 min. SDS-PAGE electrophoresis was then performed. After transfer and blocking, GPX4 primary antibody diluted 1:1000 or GAPDH primary antibody diluted 1:20000 was added and incubated overnight at 4°C. After rinsing, the cells were incubated with secondary antibody diluted 1:2000 at room temperature for 2 h. Finally, the cells were developed and images were acquired using a cyprotein ECL chemiluminescence kit.

[0059] The results were consistent with in vivo experiments in mice, showing that DEL could restore GPX4 protein levels in an LPS-induced acute lung injury cell model, thereby inhibiting ferroptosis.

Claims

1. The application of a physalis alkaloid in the preparation of a drug for treating lung injury.

2. The application according to claim 1, characterized in that, The CAS number for the delphinidin is 509-18-2.

3. The application according to claim 1, characterized in that, The lung injury described is acute lung injury.

4. The application according to claim 3, characterized in that, The acute lung injury mentioned refers to acute lung injury caused by sepsis.

5. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit lipid peroxidation.

6. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit apoptosis and / or ferroptosis.

7. The application according to claim 1, characterized in that, The drug contains delphinidin or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, transdermal patches, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

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