Application of PGAM5 inhibitor in preparation of medicine for acute pancreatitis
By inhibiting ferroptosis using the PGAM5 inhibitor LFHP-1c, the problem of tissue damage caused by oxidative stress in acute pancreatitis was solved, achieving an effective treatment for acute pancreatitis.
Patent Information
- Application Number
- CN202511552908.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-02-27
AI Technical Summary
Current technologies lack specific interventions for acute pancreatitis, the role of ferroptosis in acute pancreatitis is not fully understood, leading to difficulty in controlling the progression to severe illness, and severe tissue damage caused by oxidative stress.
The PGAM5 inhibitor LFHP-1c was used to treat acute pancreatitis by inhibiting PGAM5 expression, promoting the expression of the death-negative regulatory gene FSP1, and inhibiting the production of reactive oxygen species.
It effectively inhibits ferroptosis, reduces the production of reactive oxygen species, lowers the expression of inflammatory factors, significantly improves the condition of acute pancreatitis, and provides a new treatment approach.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a PGAM5 inhibitor in the preparation of drugs for acute pancreatitis. Background Technology
[0002] Acute pancreatitis (AP) is an acute digestive system disease caused by the abnormal activation of pancreatic enzymes, leading to pancreatic tissue self-digestion and necrosis. Its progression to severe illness can result in multiple organ failure and even death, with approximately 20%-30% of cases progressing to severe acute pancreatitis (SAP), characterized by high mortality and recurrence rates. The global incidence of AP is increasing annually. Its pathogenesis is multifaceted and complex, but clinical treatment remains primarily supportive, lacking specific interventions targeting the pathological mechanisms. Although AP diagnosis and treatment are becoming increasingly standardized, mitigating the trend towards severe AP remains a challenge in critical care medicine. The term "ferroptosis," coined in 2012, describes a regulated form of cell death induced by the ferroptosis inducer Erastin. This cell death inhibits cysteine uptake, relies on reactive oxygen species (ROS)-induced iron-mediated lipid peroxidation, and is catalyzed by the Fenton reaction and lipoxygenase-initiated iron, leading to polyunsaturated fatty acids becoming the primary targets of membrane lipid peroxidation. Many serious and common human degenerative diseases, such as Parkinson's disease and Huntington's disease, as well as some acute injury conditions, such as stroke, cerebral hemorrhage, traumatic brain injury, and ischemia-reperfusion injury, may be associated with ferroptosis.
[0003] Numerous studies have shown that ferroptosis is a crucial link between the initial damage and the subsequent severe inflammatory response in acute pancreatitis. The initiation phase: Ferroptosis occurs in pancreatic acinar cells. In the early stages of acute pancreatitis, various pathogenic factors (such as bile reflux and alcohol metabolites) lead to the production of large amounts of reactive oxygen species within pancreatic acinar cells, triggering oxidative stress. These factors deplete intracellular glutathione and inhibit GPX4 activity, causing the cell's anti-ferroptosis defense system to collapse. Pancreatic cells themselves contain abundant polyunsaturated fatty acids, providing "raw materials" for lipid peroxidation. Simultaneously, acute pancreatitis induces the high expression of ACSL4, a key enzyme in lipid peroxidation. Under the combined effect of these conditions, massive ferroptosis occurs in pancreatic acinar cells. This is a violent death process that releases large amounts of intracellular contents (including digestive enzymes that are not yet fully activated). The amplification phase: Ferroptosis triggers and exacerbates the inflammatory "storm." After ferroptotic cells rupture, the released intracellular substances (such as digestive enzymes and damage-associated molecular patterns (DAMPs)) act as strong "alarm signals."
[0004] These signals are recognized by immune cells (such as macrophages), activating key inflammatory pathways such as the NLRP3 inflammasome. The activated immune cells release large amounts of inflammatory factors such as interleukin-1β and tumor necrosis factor-α, triggering a systemic "inflammatory storm" that leads to severe damage to the pancreas and distant organs.
[0005] Phosphoglycerate mutase 5 (PGAM5) is a mitochondrial serine / threonine phosphatase. PGAM5 is normally located in the inner mitochondrial membrane. When mitochondrial function is impaired, PGAM5 recruits DRP1 and dephosphorylates it at Ser637, activating DRP1's GTPase activity and promoting mitochondrial division. PGAM5 can be cleaved and released into the cytoplasm via the PARKIN pathway, activating the Wnt signaling pathway and regulating mitochondrial homeostasis. PGAM5 also regulates antioxidant responses by forming a tertiary complex with KEAP1 and NRF2. PGAM5 has multiple functions and can act as a signaling center to sense mitochondrial stress, regulate mitochondrial dynamics, and enhance antioxidant responses.
[0006] Oxidative stress is a major factor contributing to pancreatic damage in acute pancreatitis (AP). In the early stages of AP, the accumulation of reactive oxygen species (ROS) in pancreatic acinar cells and immune cells exacerbates tissue damage. Therefore, targeting oxidative stress is a potential approach to alleviating AP. Multiple studies have confirmed that inhibiting oxidative stress by providing exogenous antioxidants or activating endogenous antioxidant mechanisms can protect against AP. The function and molecular regulatory mechanism of PGAM5 in AP remain unknown, and no existing technology has reported the relevant biological effects of the PGAM5 inhibitor LFHP-1c in AP. Summary of the Invention
[0007] To overcome the shortcomings of the prior art, the present invention aims to provide an application of a PGAM5 inhibitor in the preparation of drugs for acute pancreatitis, specifically including the following:
[0008] The primary objective of this invention is:
[0009] This invention provides the application of a PGAM5 inhibitor in the preparation of drugs for acute pancreatitis. The PGAM5 inhibitor is LFHP-1c, and the molecular formula of LFHP-1c is C2. 55 H 64 N6O4, structural formula as follows:
[0010]
[0011] Furthermore, the acute pancreatitis drug works by inhibiting PGAM5 expression.
[0012] Furthermore, the excipients of the drug include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbent carriers, and lubricants;
[0013] Furthermore, the drug is introduced into the body tissues through oral administration, injection, spraying, nasal drops, eye drops, penetration, absorption, and physical or chemical mediated methods; or it is introduced into the body after being mixed with or encapsulated by other substances.
[0014] The second objective of this invention is to provide the application of LFHP-1c as a reactive oxygen species inhibitor.
[0015] The third objective of this invention is to provide the application of LFHP-1c in the gene FSP1, which promotes negative regulation of death.
[0016] Beneficial effects
[0017] This invention is the first to discover that LFHP-1c can be used to prepare a ferroptosis inhibitor. Western blotting and real-time quantitative PCR confirmed that LFHP-1c can promote the expression of anti-ferroptosis-related genes, inhibit the production of intracellular lipid reactive oxygen species, and exhibits high safety. In vivo experiments showed that it effectively treated arginine-induced acute pancreatitis in mice. Therefore, it can be used as a ferroptosis inhibitor, providing a new therapeutic approach to cellular ferroptosis and offering novel treatment options for the prevention and treatment of acute pancreatitis. Attached Figure Description
[0018] Figure 1 This is a schematic diagram illustrating the effect of LFHP-1c on ferroptosis observed under a fluorescence microscope in this invention.
[0019] Figure 2 This is a schematic diagram illustrating the effect of flow cytometry LFHP-1c on ferroptosis in this invention.
[0020] Figure 3 This is a schematic diagram illustrating the effect of LFHP-1c inhibitor on Lipid ROS in cells using flow cytometry, as described in this invention.
[0021] Figure 4 This is a schematic diagram illustrating the expression of FSP1, a key gene inhibiting ferroptosis, in real-time quantitative PCR analysis of this invention after treatment with LFHP-1c.
[0022] Figure 5 This invention utilizes Western blot analysis to identify FSP1, a key gene in inhibiting ferroptosis.
[0023] A schematic diagram of the expression after LFHP-1c treatment.
[0024] Figure 6This is a schematic diagram illustrating the biochemical analysis of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) levels in mouse blood during this invention.
[0025] Figure 7 This invention provides a real-time quantitative PCR analysis of pancreatic tissue in mice treated with LFHP-1c.
[0026] Schematic diagram of the expression of relevant validation indicators such as TNF-α / IL-6 / IL-1β.
[0027] Figure 8 This is a schematic diagram illustrating the degree of inflammatory infiltration of the heart and pancreas in mice after LFHP-1c treatment using HE staining of pancreatic tissue in this invention.
[0028] Figure 9 This is a schematic diagram illustrating the expression of PTGS2, a ferrodeogenesis-related marker, in the pancreas of mice treated with LFHP-1c, using real-time quantitative PCR analysis.
[0029] Figure 10 This is a schematic diagram illustrating the expression of 4-HNE, a marker of iron death in the pancreas of mice treated with LFHP-1c, as analyzed by Western blot analysis in this invention.
[0030] Figure 11 This is a schematic diagram illustrating the expression of MDA, a ferrodeogenesis-related marker, in the pancreas of mice after LFHP-1c treatment, as detected and analyzed by the kit of this invention. Detailed Implementation
[0031] The technical solutions in the embodiments of the present invention will now be described with reference to the accompanying drawings.
[0032] The invention is further described below through embodiments, including the materials used and their specific sources. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials similar to or identical in type and model, or in nature or function to the following tissues, cells, reagents, and instruments can be used in the implementation of the invention. The drug may be supplemented with one or more pharmaceutically acceptable adjuvants, including but not limited to granules, buffers, surfactants, and other known pharmaceutical adjuvants.
[0033] In some specific embodiments, the drug may be formulated into dosage forms including, but not limited to, microinjection formulations and transfection-suitable dosage forms, which may be prepared according to conventional methods in the pharmaceutical field. Unless otherwise specified, the methods in the following examples are conventional methods.
[0034] Main materials:
[0035] Unless otherwise specified, the reagents used in this invention may be any suitable commercially available reagents; cell lines may be obtained commercially.
[0036] Example 1
[0037] In this embodiment, human cardiomyocyte AC16 cells were induced with the ferroptosis inducer Erastin, and the effect of LFHP-1c on the sensitivity of cells to ferroptosis was demonstrated. The specific method is as follows:
[0038] 1. Cell Culture
[0039] Human cardiomyocytes AC16 were cultured in DMEM low-glucose medium containing 10% Ceg serum, 1% penicillin (100 U / mL), and streptomycin (100 U / mL). The cells were cultured in an incubator at 37°C and 5% CO2 saturated humidity.
[0040] 2. Effect of LFHP-1c on ferroptosis detected by fluorescence microscopy
[0041] Culture the cells to be tested at 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 (20 μM) was added to the cells to stimulate them for 18 h. PI (1 mg / ml) dye was added to the cells at a ratio of 1:1000 and incubated at 37°C for 5 min. Cell ferroptosis was observed under a fluorescence microscope.
[0042] The results are as follows Figure 1 As shown, AC16 cells were co-treated with 2 μM LFHP-1c and 20 μM erastin. After 18 hours, ferroptosis was observed under an inverted fluorescence microscope after PI staining. Compared with the control group, the addition of LFHP-1c inhibited erastin-induced ferroptosis.
[0043] 3. Flow cytometry to determine the degree of cell death
[0044] Culture the cells to be tested at 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 (20 μM) was added to the cells to stimulate them for 18 h. PI (1 mg / ml) dye was added to the cells at a ratio of 1:1000 and incubated at 37°C for 5 min. Cells were collected and flow cytometry was used to detect ferroptosis.
[0045] The results are as follows Figure 2 As shown, 2 μM LFHP-1c was added to AC16 cells induced by 20 μM erastin. After 18 hours, ferroptosis was detected by flow cytometry after PI staining. Compared with the control group, erastin-induced ferroptosis was significantly inhibited after the addition of LFHP-1c.
[0046] 4. Flow cytometry analysis of intracellular lipid ROS changes
[0047] Culture the cells to be tested at 1.2 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 12-well plates and incubated overnight at 37°C. Erastin (20 μM) was added to each well to stimulate the cells for 10 h. BODIPY was then added to the cells at a ratio of 1:1000. TM 581 / 591C11 (5mM) was incubated at 37℃ for 30 min; cells were collected and flow cytometry was used to detect lipid ROS production in the cells.
[0048] The results are as follows Figure 3 As shown, 2 μM LFHP-1c was added to AC16 cells induced by 20 μM erastin. 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 content of Lipid ROS was significantly reduced after the addition of LFHP-1c.
[0049] 5. Real-time quantitative PCR experiment
[0050] Discard the AC16 cell culture medium, wash twice with PBS, add 1 ml of Trizol extraction buffer to a 1.5 ml EP tube, pipette for 5 minutes, add 0.2 ml of chloroform, shake vigorously for 10 seconds, let stand for 5 minutes, centrifuge at 13500 rpm for 15 minutes at 4°C, transfer the supernatant to another EP tube, add 0.5 ml of isopropanol, mix slowly, let stand for 10 minutes, centrifuge at 13500 rpm for 10 minutes at 4°C, discard the supernatant, add 1 ml of 75% ethanol (prepared with DEPC water) to the precipitate, mix well, centrifuge at 10600 rpm for 5 minutes at 4°C, discard the supernatant, dry at room temperature for 5-10 minutes, dissolve the precipitate in 10 μL of DEPC water. After RNA extraction, use a reverse transcription kit (Takara Code NO. RR047A) to obtain cDNA. For the real-time quantitative PCR experiment, the primers, cDNA, AceQ qPCR SYBR Green Master Mix (2x), and RNase-free ddH2O were thawed on ice. The PCR reaction mixture was prepared in 10 μL on ice and in the dark according to the kit instructions. After adding the mixture to the eight-tube set, the mixture was vortexed and centrifuged briefly.
[0051] As a result, Figure 4 2 μM LFHP-1c was added to wild-type AC16 cells. After 9 hours, the expression level of FSP1 mRNA was detected. Compared with the control group, the expression level of FSP1 mRNA was significantly upregulated after the addition of M1.
[0052] 6. Western blot assay
[0053] Sample preparation: Transfer 80 μL of lysed cell sample to a 1.5 ml centrifuge tube, add 20 μL of... 5×Samplebuffer, boil for 10 min; Electrophoresis: centrifuge at 12000 rpm for 5 min, take the supernatant for 12% SDS-PAGE electrophoresis; Transfer: after electrophoresis, take out the gel, cut 1 NC membrane and 6 filter papers according to the gel size, the size of the three in the order of gel > NC membrane > filter paper, and lay them flat on the transfer apparatus in the order of 3 layers of filter paper, NC membrane, gel, 3 layers of filter paper from bottom to top, cover the transfer lid, transfer for 70 min; Blocking: after the transfer, take out the NC membrane and soak it in 5% skim milk blocking solution, incubate at room temperature for 2 h in a shaker; Primary antibody: add the corresponding primary antibody to 5 ml of blocking solution at a ratio of (1:1000), put in the NC membrane, incubate at 4℃ overnight, the next day, wash the membrane 4 times with TBST, 10 min each time; Secondary antibody: add to 5 ml of blocking solution at a ratio of (1:5000) Add horseradish peroxidase-labeled secondary antibody to TBST, place it in an NC membrane, incubate at 37°C for 50 min in a shaker, wash the membrane 3 times with TBST, 10 min each time; Imaging: remove the NC membrane, add an appropriate amount of ECL (A solution: B solution = 1:1), and take pictures with a multicolor fluorescence imager.
[0054] The results are as follows Figure 5 2 μM LFHP-1c was added to wild-type AC16 cells. After 9 hours, the expression level of FSP1 protein was detected. Compared with the control group, the expression level of FSP1 protein was significantly upregulated after the addition of LFHP-1c.
[0055] Example 2
[0056] In this embodiment, C57 mice were treated with a single injection of arginine, and the application of LFHP-1c in the treatment of acute pancreatitis was investigated. The specific steps are as follows:
[0057] 1. Biochemical detection of LDH and ALT levels in blood cells of mice with acute pancreatitis after LFHP-1c treatment. Preparation of the required mouse model: 7-8 week old C57 mice were injected with LFHP-1c (3 mg / kg) one day before, on the day of, the second day, and the third day. Blood samples were collected from the eyeballs on the third day for biochemical analysis.
[0058] The results are as follows Figure 6 After LFHP-1c treatment, the levels of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) in the blood of mice decreased significantly.
[0059] 2. Real-time quantitative PCR detection of the expression of related inflammatory factors in pancreatic tissue of mice after LFHP-1c treatment. Preparation of the required mouse model: 7-8 week old C57 mice were injected with LFHP-1c (3 mg / kg) one day before, on the day of, the second day, and the third day. On the third day, pancreatic tissue was collected for RNA extraction and real-time quantitative PCR detection.
[0060] The results are as follows Figure 7 After LFHP-1c treatment, the expression levels of related validation indicators such as TNF-α / IL-6 / IL-1β in the pancreatic tissue of mice were significantly reduced.
[0061] 3. HE staining of pancreatic tissue to detect the degree of pancreatic inflammatory infiltration in mice after LFHP-1c treatment.
[0062] To prepare the mouse animal model required for the test: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) one day before, on the day of injection, the second day, and the third day. On the third day, mouse pancreatic tissue was prepared and sections were stained with hematoxylin and eosin (HE).
[0063] As a result, Figure 8 After LFHP-1c treatment, the inflammatory infiltration area of the pancreatic tissue in mice was significantly reduced.
[0064] 4. Real-time quantitative PCR analysis of the expression of PTGS2, a ferroptosis-related marker, in the pancreatic tissue of mice after LFHP-1c treatment.
[0065] Preparation of mouse animal models required for detection: 7-8 week old C57 mice were injected with arginine (3.5 g / kg) one day before, on the day of injection, the second day, and the third day. After the third day, pancreatic tissue was taken to extract RNA for real-time fluorescence quantitative PCR detection.
[0066] As a result, Figure 9 The expression level of PTGS2 mRNA in the pancreatic tissue of mice was significantly reduced after LFHP-1c treatment.
[0067] 5. Western blot analysis of the expression of ferroptosis-related marker 4-HNE in pancreatic tissue of mice after LFHP-1c treatment.
[0068] Preparation of mouse animal models required for detection: 7-8 week old C57 mice were injected with LFHP-1c (3mg / kg) one day before, on the day of, the second day, and the third day. After the third day, pancreatic tissue was taken to extract protein for detection.
[0069] The results are as follows Figure 10The expression level of 4-HNE protein in the pancreatic tissue of mice was significantly reduced after LFHP-1c treatment.
[0070] 6. Detect the expression of MDA, a ferroptosis-related marker, in pancreatic tissue of mice after LFHP-1c treatment. Mouse animal model preparation: 7-8 week old C57 mice were injected with LFHP-1c (3 mg / kg) one day before, on the day of, the second day, and the third day. On the third day after injection, pancreatic tissue was collected for protein extraction and detection.
[0071] The results are as follows Figure 11 The MDA content in the pancreatic tissue of mice was significantly reduced after LFHP-1c treatment.
[0072] The above description of the present invention is only a preferred embodiment of the present invention and is not intended to limit the implementation of the present invention. Those skilled in the art can easily make corresponding modifications or alterations based on the main concept and spirit of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of protection claimed in the claims.
Claims
1. Application of PGAM5 inhibitors in the preparation of ferroptosis inhibitors and drugs for acute pancreatitis.
2. The application according to claim 1, characterized in that, The PGAM5 inhibitor is LFHP-1c, and the molecular formula of LFHP-1c is C. 55 H 64 N6O4, structural formula as follows:
3. The application according to claim 2, characterized in that, The acute pancreatitis drug works by inhibiting PGAM5 expression.
4. The application according to claim 3, characterized in that, The excipients of the drug include one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.
5. The application according to claim 3, characterized in that, The drug is introduced into the body tissues through oral administration, injection, spraying, nasal drops, eye drops, penetration, absorption, and physical or chemical mediated methods; or it is introduced into the body after being mixed with or encapsulated by other substances.
6. Application of LFHP-1c as a reactive oxygen species inhibitor.
7. Application of LFHP-1c in promoting death-negative regulatory gene FSP1.