Application of L-type calcium channel blocker as ferroptosis inhibitor in medicine preparation

Abstract

The invention discloses application of an L-type calcium channel blocker as a ferroptosis inhibitor in preparation of drugs, and belongs to the technical field of biological medicines. In order to solve the problem that an effective acute pancreatitis and organ injury treatment means is lacked in the prior art, the invention provides application of an L-type calcium channel blocker as a ferroptosis inhibitor in preparation of a medicine for preventing / treating acute pancreatitis and organ injury, and the L-type calcium channel blocker is Benidipine, Xinidipine or Manidipine; proved by detection such as western blot and the like, the L-type calcium channel blocker can inhibit activation of ferroptosis key protein and generation of intracellular lipid active oxygen, is high in safety and provides a new means for disease treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of L-type calcium channel blockers as inhibitors of ferroptosis in the preparation of drugs. Background Technology

[0002] Ferroptosis is a novel form of programmed cell death characterized by abnormal intracellular iron metabolism leading to lipid peroxidation, which in turn causes cell membrane rupture and cell death. Unlike other types of cell death (such as apoptosis, necrosis, and autophagy), ferroptosis depends on intracellular iron levels and lipid peroxidation, typically accompanied by a decrease in glutathione (GSH) levels and the accumulation of lipid peroxides. Ferroptosis is closely linked to various diseases, including neurodegenerative diseases, cardiovascular diseases, and cancer.

[0003] Acute pancreatitis (AP) is a local and systemic inflammatory response of the pancreas caused by enzyme activation due to various etiologies. Its core pathophysiology involves the premature and abnormal activation of trypsinogen in acinar cells, triggering a chain of digestive enzyme reactions that lead to pancreatic autodigestion, tissue damage, and microcirculatory disturbances. Pathological classifications include interstitial edematous pancreatitis and necrotizing pancreatitis. Clinical manifestations are characterized by acute upper abdominal pain, nausea, and vomiting. Severe cases may be complicated by systemic inflammatory response syndrome, organ failure, and local pancreatic complications. Diagnosis is based on typical clinical manifestations, significantly elevated serum amylase / lipase levels, and imaging evidence. Although the diagnosis and treatment of AP are becoming increasingly standardized, improving the trend of AP becoming more severe remains a challenge in critical care medicine.

[0004] Ferroprelation, a novel iron-dependent programmed cell death mechanism, is closely related to the development and progression of pancreatitis. Its core mechanism involves the accumulation of lipid peroxides in pancreatic acinar cells, leading to the inactivation of the antioxidant defense system. In acute pancreatitis, various pathogenic factors induce ferroptosis in pancreatic acinar cells; this process exacerbates oxidative damage and inflammatory necrosis, releasing large amounts of damage-related molecules, further activating immune cells, amplifying the systemic inflammatory response, and potentially driving the disease towards severe illness. Studies have shown that in experimental pancreatitis models, the use of ferroptosis inhibitors can significantly reduce pancreatic tissue damage and inflammation; this confirms that ferroptosis is a key step in the pathological process of pancreatitis, and targeted inhibition of ferroptosis has become a highly promising new therapeutic strategy.

[0005] Calcium plays a crucial role in regulating cell death, including apoptosis, necroptosis, and pyroptosis. Elevated cytoplasmic calcium levels are observed before cell lysis in ferroptosis. (The text abruptly shifts to a seemingly unrelated topic: "Calcium during ferroptosis...") 2+Flow-induced activation of ESCRT-III-dependent membrane repair mechanisms for membrane repair may delay cell death dynamics by counteracting membrane damage, suggesting an anti-ferroptosis function of calcium. Furthermore, mitochondrial calcium... 2+ Calcium levels are considered to directly affect the cell's response to ferroptosis. Calcium ions mainly enter the cell through calcium channel proteins on the cell membrane; calcium channel proteins include various forms such as L, P / Q, N, and R, which are expressed in different types of cells and are responsible for the uptake of calcium ions by the cell.

[0006] Ferric death in pancreatic acinar cells exacerbates tissue necrosis and inflammatory responses. By using specific inhibitors (such as iron chelators and Liproxstatin-1), the core process of lipid peroxidation can be directly blocked, effectively protecting acinar cells and reducing local and systemic damage to the pancreas. This therapy targets key pathogenic mechanisms and demonstrates good clinical translational potential. The function and molecular regulatory mechanism of L-type calcium channel blockers in acute pancreatitis (AP) are still unknown, and no existing technology has reported the related biological effects of L-type calcium channel blockers in AP.

[0007] Furthermore, ferroptosis is closely linked to various diseases such as neurodegenerative diseases and damage to multiple organs including the heart, liver, and kidneys. By using ferroptosis-specific inhibitors, the core process of lipid peroxidation in these diseases can be directly blocked, effectively protecting cells and reducing local and systemic organ damage. This therapy targets key pathogenic links and shows good potential for clinical translation. The function and molecular regulatory mechanisms of L-type calcium channel blockers in these organ damage diseases are still unknown, and currently no existing technology reports the relevant biological effects of L-type calcium channel blockers in ferroptosis-related organ damage. Summary of the Invention

[0008] This invention addresses the lack of effective treatments for acute pancreatitis or organ damage in the prior art by providing the application of L-type calcium channel blockers as inhibitors of ferroptosis in drug preparation.

[0009] One of the objectives of this invention is to provide the application of L-type calcium channel blockers as inhibitors of ferroptosis in the preparation of drugs.

[0010] In a preferred embodiment of the present invention, the drug is a drug for the prevention / treatment of acute pancreatitis or organ damage.

[0011] In a preferred embodiment of the present invention, the L-type calcium channel blocker can disrupt the activation of ferroptosis key regulatory protein kinase PKCβ and the lipid droplet transfer of the ACSL4-PKCβ-ALOX15 complex.

[0012] In a preferred embodiment of the present invention, the L-type calcium channel blocker is Benidipine, Xinidipine, or Manidipine.

[0013] In a preferred embodiment of the present invention, the benidipine has the molecular formula C2. 28 H 31 N3O, with a molecular weight of 505.56 and CAS number 105979-17-7, has the following structural formula: .

[0014] In a preferred embodiment of the present invention, the Xinidipine has the molecular formula C 27 H 28 N₂O₇, with a molecular weight of 492.520 and CAS number 132203-70-4, has the following structural formula: .

[0015] In a preferred embodiment of the present invention, the Manidipine has the molecular formula C0. 35 H 38 N4O6, with a molecular weight of 610.70 and CAS number 89226-50-6, has the following structural formula: .

[0016] In a preferred embodiment of the invention, the concentration of Benidipine applied at the cellular level is 50 µM.

[0017] In a preferred embodiment of the present invention, the concentration of Xinidipine applied at the cellular level is 20 µM.

[0018] In a preferred embodiment of the invention, the concentration of Manidipine applied at the cellular level is 50 µM.

[0019] Compared with the prior art, the beneficial effects of the present invention are: the present invention is the first to discover that L-type calcium channel blockers can be used as ferroptosis inhibitors in the preparation of drugs for the prevention / treatment of acute pancreatitis and organ damage, wherein the L-type calcium channel blockers are Benidipine, Xinidipine or Manidipine; the detection by Western blotting and other methods confirms that it inhibits the activation of key ferroptosis proteins and inhibits the production of intracellular lipid reactive oxygen species.

[0020] This invention also provides L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) that can inhibit the interaction between the key ferroptosis enzymes ACSL4 and ALOX15 and inhibit phosphorylation at multiple sites of ACSL4 and ALOX15. ACSL4 (long-chain acyl-CoA synthase 4) is a key enzyme in lipid metabolism, responsible for catalyzing the activation of long-chain polyunsaturated fatty acids. It plays an irreplaceable role in ferroptosis by specifically driving lipid peroxidation through promoting the incorporation of these fatty acids into membrane phospholipids. ALOX15 is a lipoxygenase that specifically catalyzes the conversion of arachidonic acid to 15-peroxyeicosatetraenoic acid; it is highly expressed in macrophages and plays a crucial role in pathophysiological processes such as ferroptosis, inflammatory responses, and atherosclerosis by regulating lipid peroxidation.

[0021] The present invention also provides that L-type calcium channel blockers (Benidipine, Xinidipine or Manidipine) at cellular application concentrations of 50 µM, 20 µM and 50 µM can effectively inhibit ferroptosis, inhibit the increase of reactive oxygen species, inhibit the activation of the key regulatory protein kinase PKCβ of ferroptosis and the lipid droplet transfer of the PKCβ complex, and do not produce toxic effects on cells.

[0022] This invention validates the findings at the protein level using Western blotting and subcellular observation using laser confocal microscopy. Results show that L-type calcium channel blockers inhibit ferroptosis by disrupting the activation of the key regulatory protein kinase PKC and the lipid droplet transfer of the PKC complex. Lipid peroxidation probe labeling and fluorescence microscopy and flow cytometry analysis revealed that the addition of Erastin or RSL3 increased intracellular lipid peroxidation levels, which were reversed by the addition of L-type calcium channel blockers. In vivo experiments showed that Benidipine, Xinidipine, and Manidipine effectively treated arginine-induced acute pancreatitis in mice. Therefore, the L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) provided by this invention can be used as ferroptosis inhibitors, offering a new treatment for ferroptosis-related diseases and providing novel treatment options for the prevention and treatment of acute pancreatitis. Attached Figure Description

[0023] Figure 1 To observe the effect of L-type calcium channel blockers on ferroptosis in HT1080 cells using fluorescence microscopy; Figure 2 To observe the effect of L-type calcium channel blockers on ferroptosis in MDA-MB-231 cells using fluorescence microscopy; Figure 3To detect the effect of L-type calcium channel blockers on ferroptosis in HT1080 cells by flow cytometry; A represents RSL3 treatment, B represents Erastin treatment; Beni represents the L-type calcium channel blocker Benidipine, Xini represents the L-type calcium channel blocker Xinidipine, and Mani represents the L-type calcium channel blocker Manidipine. Figure 4 To detect the effect of L-type calcium channel blockers on ferroptosis in MDA-MB-231 cells by flow cytometry; A represents RSL3 treatment, and B represents Erastin treatment; Figure 5 To detect the effect of L-type calcium channel blockers on Lipid ROS in HT1080 cells by flow cytometry; A represents RSL3 treatment, and B represents Erastin treatment; Figure 6 To detect the effect of L-type calcium channel blockers on Lipid ROS in MDA-MB-231 cells by flow cytometry; A represents RSL3 treatment, and B represents Erastin treatment; Figure 7 To detect the effect of L-type calcium channel blockers on the activity of PKCβ, a key regulatory protein kinase of ferroptosis, using Western blotting. Figure 8 To observe the lipid droplet transfer of the ferroptosis-regulating complex PKCβ after treatment with an L-type calcium channel blocker using laser confocal microscopy; Figure 9 To determine serum amylase AMS activity using an enzymatic method; Figure 10 To conduct a biochemical analysis of the lactate dehydrogenase (LDH) content in mouse serum; Figure 11 To analyze the expression of relevant validation markers in mouse pancreatic tissue after treatment with L-type calcium channel blockers using real-time quantitative PCR; A represents mil-1b expression; B represents Tnf-a expression. Figure 12 HE staining was used to detect the degree of pancreatic inflammatory infiltration in mice after treatment with L-type calcium channel blockers. Figure 13 To analyze the phosphorylation of 4-HNE, ACSL4, ALOX15, and ACSL4 and ALOX15 at multiple sites after treatment with L-type calcium channel blockers following pancreatic inflammation using Western blotting. Detailed Implementation

[0024] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0026] Example 1: In this embodiment, ferroptosis inducers Erastin and RSL3 were used to induce ferroptosis in human fibrosarcoma cells HT1080 and human breast cancer cells MDA-MB-231, and the effects of L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) on ferroptosis were investigated.

[0027] 1. Cell Culture Human fibrosarcoma cells HT1080 and human breast cancer cells MDA-MB-231 were cultured in DMEM high glucose medium (purchased from Procell, catalog number PM150210), which also contained 10% fetal bovine serum, 1% penicillin (100 U / mL) and streptomycin (100 U / mL), in an incubator at 37°C and 5% CO2 saturated humidity.

[0028] 2. Effects of L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) on ferroptosis detected by fluorescence microscopy. The HT1080 cells and MDA-MB-231 cells obtained above were cultured at a rate of 1.2 × 10⁻⁶. 5Cells were seeded at a density of 100 cells / well in 12-well plates and cultured overnight at 37°C. Erastin (15 μM / 30 μM) or RSL3 (0.2 μM / 0.4 μM), as well as Benidipine (50 μM), Xinidipine (20 μM), and Manidipine (50 μM) were added to HT1080 cells and MDA-MB-231 cells, respectively, and the cells were stimulated for 18 h. PI (1 mg / mL) dye was added to the cells at a ratio of 1:1000, and the cells were incubated at 37°C for 5 min. Cell ferroptosis was observed under a fluorescence microscope.

[0029] like Figure 1-2 As shown, HT1080 and MDA-MB-231 cells were co-treated with 40 μM Benidipine, Xinidipine, and Manidipine along with 15 μM / 30 μM Erastin or 0.2 μM / 0.4 μM RSL3. After 18 hours, ferroptosis was observed under an inverted fluorescence microscope after PI staining. Compared with the control group, the addition of the L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) provided in this invention inhibited ferroptosis induced by Erastin or RSL3.

[0030] 3. Flow cytometry to determine the degree of cell death The HT1080 cells and MDA-MB-231 cells obtained above were cultured at a rate of 1.2 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 12-well plates and cultured overnight at 37°C. Erastin (15 μM / 30 μM) or RSL3 (0.2 μM / 0.4 μM), as well as Benidipine (50 μM), Xinidipine (20 μM), and Manidipine (50 μM) were added to HT1080 cells and MDA-MB-231 cells, respectively, to stimulate the cells for 18 h. PI (1 mg / mL) dye was added to the cells at a ratio of 1:1000, and the cells were incubated at 37°C for 5 min. Cells were collected and flow cytometry was used to detect ferroptosis.

[0031] like Figure 3-4As shown, HT1080 and MDA-MB-231 cells were co-treated with 40 μM Benidipine, Xinidipine, and Manidipine along with 15 μM / 30 μM Erastin or 0.2 μM / 0.4 μM RSL3. After 18 hours, ferroptosis was detected by flow cytometry after PI staining. Compared with the control group, ferroptosis induced by Erastin or RSL3 was significantly inhibited after the addition of the L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) provided in this invention.

[0032] 4. Flow cytometry analysis of intracellular lipid peroxide (lipid ROS) changes The HT1080 cells and MDA-MB-231 cells obtained above were cultured at a rate of 1.2 × 10⁻⁶. 5 Cells were seeded at a density of 15 μM / well in 12-well plates and cultured overnight at 37°C. Cells were then stimulated for 10 h with either 15 μM / 30 μM or RSL3 (0.2 μM / 0.4 μM) and Benidipine (50 μM), Xinidipine (20 μM), or Manidipine (50 μM), respectively. Finally, BODIPY was added to the cells at a 1:1000 ratio. TM Cells were incubated at 581 / 591 C11 (5 mM) at 37°C for 30 min, and then collected. The production of lipid ROS in the cells was detected by flow cytometry.

[0033] like Figure 5-6 As shown, 40 μM Benidipine, Xinidipine, and Manidipine were added to cells induced by 15 μM / 30 μM Erastin or 0.2 μM / 0.4 μM RSL3. After 10 hours, BODIPY C11 was added to the cells, and the accumulation of Lipid ROS in the cells was detected by flow cytometry. Compared with the control group, the Lipid ROS content was significantly reduced after adding the L-type calcium channel blockers (Benidipine, Xinidipine, or Manidipine) provided in this invention.

[0034] 5. Western blot assay Sample preparation: Take 80 μL of lysed cells (MDA-MB-231 cells or HT1080 cells) or tissue (pancreatic tissue) into a 1.5 mL centrifuge tube, add 20 μL of 5×Sample buffer, and boil for 10 min; Electrophoresis: Centrifuge at 12000 rpm for 5 min, and take the supernatant for 12% SDS-PAGE electrophoresis; Transfer: After electrophoresis, remove the gel, cut one NC membrane and six filter papers according to the gel size, with the order of gel > NC membrane > filter paper. Arrange the three layers in the following order from bottom to top: 3 layers of filter paper, NC membrane, gel, and 3 layers of filter paper. Cover with the transfer cap and transfer for 70 min; Blocking: After transfer, remove the NC membrane and immerse it in 5% skim milk blocking solution. Incubate at room temperature in a shaker for 2 minutes. h; Primary antibody: Add the corresponding primary antibody to 5 mL of blocking buffer at a ratio of (1:1000), place the membrane in the NC membrane, incubate overnight at 4℃, and wash the membrane 4 times with TBST for 10 min each time the next day; Secondary antibody: Add the corresponding horseradish peroxidase-labeled secondary antibody to 5 mL of TBST at a ratio of (1:5000), place the membrane in the NC membrane, incubate at 37℃ for 50 min in a shaker, and wash the membrane 3 times with TBST for 10 min each time; Imaging: Remove the NC membrane, add an appropriate amount of ECL (solution A:solution B = 1:1), and take pictures using a multicolor fluorescence imager.

[0035] like Figure 7 As shown, 40 μM Benidipine or Xinidipine was added to wild-type HT1080 and B16-F10 cells. After 9 hours, the phosphorylation level of PKC protein was detected. Compared with the control group, the addition of Benidipine or Xinidipine could effectively inhibit the phosphorylation level of Erastin or RSL3-induced PKC protein.

[0036] 6. Observation of lipid droplet transfer of the ferroptosis-regulating complex PKCβ after treatment with Benidipine, Xinidipine and Manidipine using laser confocal microscopy. HT1080 cells stably expressing PKCβ-GFP were cultured in 6-well plates containing glass slides. After 24 hours, erastin was added alone, or erastin was co-cultured with Benidipine, Xinidipine, or Manidipine for 8 hours. After incubation with Nile Red (200 μM) for 30 minutes, the culture medium was aspirated, and the cells were washed 5 times with PBS buffer and 2 times with HBSS buffer. The cells were then fixed to the glass slides and the lipid droplet transfer of PKCβ after treatment with Benidipine, Xinidipine, and Manidipine was observed and photographed using a laser confocal microscope.

[0037] like Figure 8 As shown, 40 μM Benidipine or Xinidipine was added to HT1080 cells. After 9 hours, the subcellular localization of the PKC complex was detected. Compared with the control group, the addition of Benidipine or Xinidipine could block the lipid droplet transfer of the PKC complex induced by Erastin treatment.

[0038] Example 2: In this embodiment, C57 mice (purchased from Liaoning Changsheng Biotechnology Co., Ltd.) were treated with a single injection of arginine to explore the application of benidipine, xinidipine, and manidipine in the treatment of acute pancreatitis.

[0039] 1. Biochemical detection of serum amylase (AMS) levels in mice with acute pancreatitis after treatment with benidipine, xinidipine, and manidipine. Mouse models were prepared as follows: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) once, followed by injection of benidipine, xinidipine, and manidipine (5 mg / kg) the next day. Blood samples were collected from the eyes of mice in each treatment group on the third day for biochemical analysis.

[0040] like Figure 9 As shown, the amylase AMS content in mice decreased significantly after treatment with Benidipine, Xinidipine, and Manidipine provided by this invention.

[0041] 2. Biochemical detection of serum lactate dehydrogenase (LDH) levels in mice with acute pancreatitis after treatment with Benidipine, Xinidipine, and Manidipine. Mouse models were prepared as follows: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) once, followed by injection of Benidipine, Xinidipine, and Manidipine (5 mg / kg) the next day. Blood samples were collected from the eyes of mice in each treatment group on the third day for biochemical analysis.

[0042] like Figure 10 As shown, the serum lactate dehydrogenase (LDH) levels in mice were significantly reduced after treatment with Benidipine, Xinidipine, and Manidipine provided by this invention.

[0043] 3. Real-time quantitative PCR detection of the expression of relevant inflammatory factors in pancreatic tissue of mice treated with benidipine, xinidipine, and manidipine. Preparation of the required mouse model: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) once, followed by injection of benidipine, xinidipine, and manidipine (5 mg / kg) the next day. On the third day, RNA was extracted from the pancreatic tissue of the mice in the above different treatment groups and detected by real-time quantitative PCR.

[0044] (1) RNA extraction RNA was obtained using the TRizol RNA extraction kit (Thermo Fisher Scientific catalog number: 15596026). The specific steps are as follows: Aspirate all culture medium. Add 1 mL Trizol to each well of a 6-well plate and 0.5 mL Trizol to each well of a 12-well plate. Shake 3-5 times, then pipette 2-3 times to ensure complete lysis. Transfer the lysate to a centrifuge tube. Incubate at room temperature for 5 minutes to allow for complete lysis. Add 0.2 mL chloroform per mL of Trizol, vortex for 15 seconds, and incubate at room temperature for 2-3 minutes. Centrifuge at 12000 g, 4°C for 15 minutes. Then, transfer the colorless upper aqueous phase containing total RNA to a new centrifuge tube. 0.5-0.55 mL of Trizol per mL of Trizol is recommended. Add 0.5 mL of isopropanol to each mL of the initial Trizol, invert to mix, and precipitate at room temperature for 10 minutes (microRNA extraction requires overnight precipitation at -70°C). Centrifuge at 12000 g, 4°C for 10 minutes; RNA precipitate will be visible at the bottom of the tube; discard the supernatant. Add 1 mL of 75% ethanol (prepared with DEPC water) to each mL of the initial Trizol, vortex to mix, and centrifuge at 7500 g, 4°C for 5 minutes; discard the supernatant. Centrifuge at >5000 rpm, 4°C for 1 second, and aspirate all liquid. After the RNA has slightly dried, dissolve it in 20 μL of DEPC water and store at -70°C. Note: Do not allow the RNA to dry excessively, otherwise it will be extremely difficult to dissolve, and the measured A... 260 / 280 The value will be lower than 1.6.

[0045] (2) Real-time quantitative PCR Using the RNA extracted above as a template, cDNA was synthesized using a cDNA Synthesis Kit (Cat: RR047A, Takara), and the expression of TNF-α and IL-1β genes was detected using a Universal SYBR qPCR Master Mix Kit (Cat: Q711-03, Vazyme). The primer information used is shown in Table 1.

[0046] The real-time quantitative PCR system consisted of: 10.0 μL of 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of Primer F (10 μM), 0.4 μL of Primer R (10 μM), 1 μL of Template DNA / cDNA, and ddH2O added to a final volume of 20 μL.

[0047] The real-time quantitative PCR program is as follows: 95℃, 30 s; 95℃, 10 s, 60℃, 30 s, 40 cycles; using the instrument's default melting curve acquisition program.

[0048] Table 1

[0049] like Figure 11 As shown, the expression levels of IL-1β and TNF-α in the pancreatic tissue of mice were significantly reduced after treatment with Benidipine, Xinidipine and Manidipine provided by this invention.

[0050] 4. HE staining of pancreatic tissue to detect the degree of pancreatic inflammatory infiltration in mice treated with Benidipine, Xinidipine, and Manidipine. Mouse models were prepared as follows: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) once, followed by injection of Benidipine, Xinidipine, and Manidipine (5 mg / kg) the next day. On the third day, pancreatic tissue sections from the different treatment groups were prepared and stained with HE.

[0051] like Figure 12 As shown, the inflammatory infiltration area of ​​the pancreatic tissue in mice was significantly reduced after treatment with Benidipine, Xinidipine and Manidipine provided by this invention.

[0052] 5. To detect the expression of ferroptosis-related markers 4-HNE, ACSL4, and ALOX15 in pancreatic tissue of mice treated with benidipine, Xinidipine, and Manidipine, and the phosphorylation status of multiple sites of ACSL4 and ALOX15. Mouse animal models were prepared: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) once, followed by injection of benidipine, Xinidipine, and Manidipine (5 mg / kg) the next day. Proteins were extracted from pancreatic tissue of the mice in the different treatment groups on the third day for detection.

[0053] like Figure 13As shown, after treatment with Benidipine, Xinidipine and Manidipine provided by this invention, the expression of ferroptosis-related markers 4-HNE, ACSL4 and ALOX15 in the pancreatic tissue of mice was significantly reduced, and phosphorylation at multiple sites of ACSL4 and ALOX15 was significantly reduced.

[0054] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. Application of L-type calcium channel blockers as inhibitors of ferroptosis in drug preparation.

2. The application according to claim 1, characterized in that, The drug is for the prevention / treatment of acute pancreatitis or organ damage.

3. The application according to claim 1, characterized in that, The L-type calcium channel blocker can disrupt the activation of PKCβ, a key regulatory protein kinase of ferroptosis, and the lipid droplet transfer of the ACSL4-PKCβ-ALOX15 complex.

4. The application according to claim 1, characterized in that, The L-type calcium channel blocker is Benidipine, Xinidipine, or Manidipine.

5. The application according to claim 1, characterized in that, The molecular formula of Benidipine is C. 28 H 31 N3O, with a molecular weight of 505.56 and CAS number 105979-17-7, has the following structural formula: 。 6. The application according to claim 1, characterized in that, The molecular formula of Xinidipine is C 27 H 28 N₂O₇, with a molecular weight of 492.520 and CAS number 132203-70-4, has the following structural formula: 。 7. The application according to claim 1, characterized in that, The molecular formula of the Manidipine is C. 35 H 38 N4O6, with a molecular weight of 610.70 and CAS number 89226-50-6, has the following structural formula: 。 8. The application according to claim 1, characterized in that, The concentration of Benidipine used at the cellular level is 50 µM.

9. The application according to claim 1, characterized in that, The concentration of Xinidipine used at the cellular level is 20 µM.

10. The application according to claim 1, characterized in that, The concentration of Manidipine used at the cellular level is 50 µM.

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