Use of an antibacterial peptide in the preparation of a preparation for treating acetaminophen-induced acute liver injury and / or liver failure

Abstract

The application discloses application of antibacterial peptides in preparation of a preparation for treating acetaminophen-induced acute liver injury and / or liver failure, and belongs to the field of biomedical technology. The application verifies the key role of the antibacterial peptides in promoting the liver repair process after the acetaminophen-induced acute liver injury and / or liver failure from multiple angles by using Camp gene knockout mice and exogenous injection of LL-37, CRAMP and CRAMP(1-39) synthetic peptides, and reveals that the mechanism of LL-37, CRAMP and CRAMP(1-39) proteins participating in the liver repair is related to promoting inflammation clearance and hepatocyte proliferation. The antibacterial peptides LL-37, CRAMP and CRAMP(1-39) have a good application prospect in treating the acetaminophen-induced acute liver injury and / or liver failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the use of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) in the preparation of therapeutic agents for acetaminophen-induced acute liver injury and / or liver failure. Background Technology

[0002] Acetaminophen (APAP) is a widely used over-the-counter antipyretic and analgesic in clinical practice. It is generally safe and effective when taken within the recommended dosage range (≤4g / day). However, overdose can lead to acute liver injury, which can progress to acute liver failure and death in severe cases. Unintentional abuse is one of the main causes of APAP overdose. Patients may take large doses of APAP for rapid pain relief, or may be unaware that many cold medicines contain APAP and take multiple cold medicines simultaneously, leading to APAP poisoning. In many European and American countries, APAP is also a drug of choice for suicide. In Western countries, APAP-induced acute liver injury is a leading cause of acute liver failure. In my country, APAP-induced liver failure is second only to hepatitis B virus-related causes, and APAP liver poisoning has become a serious public health problem that cannot be ignored globally. N-acetylcysteine ​​(NAC) is currently the only clinically available rescue drug for treating APAP liver poisoning. However, its most significant drawback is its narrow therapeutic window. Its efficacy is significantly reduced in patients who seek medical attention more than 8 hours after APAP ingestion. Furthermore, administering NAC to patients who seek medical attention later, or administering it for extended periods, may even lead to toxic side effects. Unfortunately, most APAP liver poisoning patients miss the optimal treatment window due to delayed medical attention. Once acute liver failure occurs, liver transplantation becomes their only treatment option. However, the shortage of donor organs, post-transplant rejection, high surgical costs, and long-term immunosuppressive therapy cause immense suffering for patients and impose a heavy emotional and economic burden on their families and society. Therefore, given the widespread use of APAP and the limitations of its treatment for APAP liver poisoning, finding new drugs that can effectively treat patients who seek medical attention later (with liver poisoning exceeding 8 hours) and thus prevent or even reverse liver failure has significant clinical and social implications.

[0003] Mouse models are widely recognized as the most suitable animal models for studying APAP hepatotoxicity. Studies using mouse models have shown that the process of APAP-induced toxic liver injury can be divided into two phases: the acute injury phase (0-24 hours after APAP ingestion) and the liver repair phase (24-72 hours after APAP ingestion). Under normal circumstances, most APAP (approximately 90%) is metabolized by glucuronyltransferase and sulfotransferase, binding to glucuronic acid and sulfides in the liver, and is then metabolized by the kidneys to form non-toxic products that are excreted in the urine. A small amount of APAP (approximately 5%-9%) is metabolized by cytochrome P450 enzymes (mainly Cyp2e1) to form the toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI), which is rapidly detoxified by binding to glutathione (GSH). When APAP is overdosed, the aforementioned metabolic pathways become saturated, GSH is depleted, and NAPQI, which is not bound to GSH, can bind to cellular proteins, especially mitochondrial proteins, thereby triggering an intracellular signaling cascade, leading to oxidative stress and mitochondrial dysfunction, ultimately causing hepatocyte necrosis. The antidote NAC targets the early metabolic process of APAP in the liver, acting as a precursor to GSH synthesis to counteract the toxic effects of NAPQI and protect the liver. APAP-induced liver injury is often accompanied by compensatory liver regeneration, which plays a crucial role in preventing damage progression and promoting liver repair. Moderate doses of APAP (300 mg / kg) can cause significant liver damage in mice, but spontaneous liver repair can follow, while high doses of APAP (600 mg / kg) can lead to persistent liver damage, ultimately causing liver failure and even death. Studies have shown that during the acute injury phase, there is no significant difference in the degree of liver damage caused by these two doses of APAP. However, moderate-dose APAP liver poisoning can initiate a spontaneous liver regeneration response, while high-dose APAP liver poisoning cannot. Therefore, liver regeneration and repair are crucial for the final outcome of liver disease. Previous studies have largely focused on elucidating the pathogenesis of acute liver injury following APAP overdose, attempting to screen for effective intervention targets to mitigate the severity of acute liver injury. However, given the importance of liver regeneration and repair for the prognosis of APAP liver poisoning, identifying effective intervention targets to promote regeneration and repair after liver injury to avoid or reverse liver failure may be a more effective and feasible treatment strategy for patients who have missed the NAC treatment window and are seeking medical attention later in life.

[0004] Antimicrobial peptides are peptides encoded by the genes of organisms that possess antimicrobial activity. Cathelicidin is one of the endogenous antimicrobial peptide families, characterized by a highly conserved N-terminal cathelin-like precursor sequence and a variable C-terminal sequence corresponding to the mature antimicrobial peptide. Under normal circumstances, cathelicidin is stored intracellularly as an inactive protein precursor. When stimulated by external factors, the inactive precursor protein is released extracellularly, where it undergoes hydrolysis by proteases to remove the conserved N-terminal fragment, becoming the active mature peptide and thus exerting its biological function. Currently, only one member of the cathelicidin family has been found in humans and mice; their active forms are named LL-37 and cathelicidin-related antimicrobial peptide (CRAMP), respectively, and they are similar in structure, function, and tissue distribution. CRAMP(1-39) is an extended form of CRAMP, with its sequence extending five amino acids from the N-terminus of the CRAMP sequence. Antimicrobial peptides are an important component of the body's innate immune system. In addition to their antimicrobial activity, they also participate in many important physiological processes such as immune regulation, angiogenesis, and wound healing. Studies have found that LL-37 is significantly elevated in the serum of patients with chronic liver diseases (including alcoholic liver disease, hepatitis C, and non-alcoholic fatty liver disease) (Svenja Wertenbruch et al., Digestion 91:707-317, 2015). Animal studies have shown that CRAMP plays a protective role in chronic liver injury (Fengyuan Li et al., J Pathol 252(4):371-383, 2020). The pathogenesis and treatment of the above-mentioned chronic liver diseases are significantly different from those of acute toxic liver injury caused by APAP. Currently, there are no reports of LL-37, CRAMP, or CRAMP(1-39) participating in APAP-induced acute liver injury and / or liver failure. Summary of the Invention

[0005] The primary objective of this invention is to provide the application of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) in the preparation of therapeutic agents for APAP-induced acute liver injury and / or liver failure, in order to compensate for the shortcomings of existing NAC treatments and provide new treatment options for patients who have missed the optimal treatment window, especially those who have developed liver failure.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The use of antimicrobial peptides in the preparation of therapeutic agents for APAP-induced acute liver injury and / or liver failure, wherein the antimicrobial peptides include at least one of LL-37, CRAMP, and CRAMP(1-39);

[0008] The sequence of the antimicrobial peptide LL-37 is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; see SEQ NO. 1;

[0009] The sequence of the antimicrobial peptide CRAMP is GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ; see SEQ NO.2;

[0010] The sequence of the antimicrobial peptide CRAMP(1-39) is ISRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ, see SEQ NO.3.

[0011] The antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) are mature peptide forms with biological activity in vivo. CRAMP(1-39) is an extended form of CRAMP, whose sequence is based on the CRAMP sequence extended to the N-terminus by 5 amino acids.

[0012] At least one of the antimicrobial peptides may be used in combination with NAC to prepare a therapeutic agent for APAP-induced acute liver injury and / or liver failure.

[0013] Furthermore, NAC is used to prepare formulations for early use in APAP-induced acute liver injury and / or liver failure, and at least one of the antimicrobial peptides is used to prepare formulations for later use in APAP-induced acute liver injury and / or liver failure.

[0014] Furthermore, NAC is used to prepare formulations for use in the later stages of APAP-induced acute liver failure, and at least one of LL-37 and CRAMP (1-39) is used to prepare formulations for use in the later stages of APAP-induced acute liver failure.

[0015] The application described,

[0016] Early APAP-induced acute liver injury and / or liver failure specifically refers to the period within 6 hours after APAP poisoning.

[0017] APAP-induced acute liver injury and / or late-stage liver failure specifically refers to 6–72 hours after APAP poisoning.

[0018] A second object of the present invention is to provide the use of antimicrobial peptides in the preparation of preparations that promote liver regeneration and repair after APAP-induced acute liver injury and / or liver failure, said antimicrobial peptides including at least one of LL-37, CRAMP and CRAMP(1-39).

[0019] A third objective of this invention is to provide the use of antimicrobial peptides in the preparation of formulations that reduce the necrotic area of ​​the liver or improve the survival rate of acute liver failure following APAP-induced acute liver injury and / or liver failure, wherein the antimicrobial peptides include at least one of LL-37, CRAMP, and CRAMP(1-39).

[0020] A fourth objective of this invention is to provide the use of the antimicrobial peptides LL-37 and / or CRAMP in the preparation of agents to improve liver inflammation following APAP-induced acute liver injury.

[0021] A fifth objective of this invention is to provide the use of the antimicrobial peptides LL-37 and / or CRAMP in the preparation of formulations that promote the phagocytic function of hepatic neutrophils after APAP-induced acute liver injury.

[0022] The sixth objective of this invention is to provide the use of the antimicrobial peptide CRAMP in the preparation of an agent that promotes the proliferation of mouse hepatocytes after APAP-induced acute liver injury.

[0023] This invention promotes liver regeneration and repair after APAP-induced acute liver injury and / or liver failure. The antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) can reduce the necrotic area of ​​the liver, and their efficacy is superior to that of NAC injected alone at the same time.

[0024] This invention promotes liver regeneration and repair after APAP-induced acute liver injury and / or liver failure. The combination of antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) with NAC can further reduce the necrotic area of ​​the liver.

[0025] This invention promotes liver regeneration and repair after APAP-induced acute liver injury and / or liver failure. The combination therapy of antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) with NAC can improve the survival rate of acute liver failure.

[0026] This invention evaluates the promoting effects of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) on liver regeneration and repair through a series of in vitro and in vivo experiments, and further explores their possible mechanisms, providing a theoretical basis and experimental evidence for the development of drugs to treat APAP-induced acute liver injury and / or liver failure.

[0027] Animal experiments showed that CRAMP protein expression was significantly elevated during both the acute injury and repair phases of liver after APAP liver poisoning, with the elevated CRAMP mainly originating from neutrophils infiltrating the liver and a small portion from hepatocytes. Compared with control mice, Camp gene knockout (CampKO) mice showed no significant changes in liver injury during the acute injury phase, but exhibited significantly increased liver necrosis area, higher serum alanine aminotransferase (ALT) levels, and fewer proliferating hepatocytes during the liver repair phase, suggesting that Camp gene knockout can delay liver repair. Exogenous supplementation with CRAMP synthetic peptides 23 hours after liver injury significantly reversed the aforementioned liver damage in Camp KO mice, demonstrating that CRAMP can specifically promote liver repair after liver injury. Injection of antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 23 hours after moderate-dose APAP (300 mg / kg) liver poisoning significantly promoted the repair of damaged liver. Compared with NAC, a classic treatment for APAP-induced liver toxicity, the combination of the three antimicrobial peptides mentioned above with NAC further reduced the area of ​​liver necrosis following acute liver injury. In mice with acute liver failure induced by high-dose APAP (500 mg / kg), the combination of the three antimicrobial peptides with NAC significantly reduced the area of ​​liver necrosis and improved the survival rate of mice with liver failure compared with early treatment with NAC alone. Compared with later treatment with NAC alone, the combination of LL-37 and CRAMP (1-39) with NAC significantly reduced the area of ​​liver necrosis after liver failure and significantly improved the survival rate of mice with liver failure.

[0028] Mechanistic studies showed that CampKO mice exhibited increased inflammatory cell infiltration in the liver during the repair phase, while LL-37 injection 23 hours after APAP liver poisoning significantly reduced this infiltration. These results suggest that antimicrobial peptides promote liver repair by accelerating the clearance of inflammation in damaged liver tissue. Further research revealed that Camp gene knockout significantly weakened the phagocytic function of neutrophils infiltrating the liver, while LL-37 dose-dependently enhanced the phagocytic function of human neutrophils. These results suggest that the role of antimicrobial peptides in promoting inflammation clearance may be related to enhancing the phagocytosis of necrotic cell debris by neutrophils. Furthermore, antimicrobial peptides can directly promote hepatocyte proliferation. These results demonstrate that antimicrobial peptides can participate in promoting liver repair after APAP liver injury by accelerating inflammation clearance and hepatocyte regeneration.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] This invention utilizes Camp gene knockout mice and exogenous injection of LL-37, CRAMP, and CRAMP(1-39) synthetic peptides to verify from multiple perspectives the key role of antimicrobial peptides in promoting liver repair after acute toxic liver injury (APAP). It demonstrates the effectiveness of injecting LL-37, CRAMP, or CRAMP(1-39) proteins in promoting liver repair in the later stages of APAP acute liver injury, and reveals that the mechanism by which antimicrobial peptides participate in liver repair is related to promoting inflammation clearance and hepatocyte proliferation.

[0031] This invention discloses for the first time the novel use of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) in the treatment of APAP-induced liver injury and / or liver failure. Antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) significantly prolong the therapeutic time window for APAP-induced liver toxicity, effectively compensating for the narrow therapeutic time window of existing NAC treatments, and providing a new treatment strategy for patients who have missed the optimal treatment window, or even those who have already developed acute liver failure.

[0032] This invention suggests that a better treatment strategy may be to use NAC in combination with antimicrobial peptides LL-37, CRAMP, or CRAMP (1-39) to treat patients with APAP liver toxicity who present late in clinical practice.

[0033] The antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) in this invention have small molecular weights, are simple to synthesize, and are endogenous proteins. They can be safely developed and utilized as drugs and have potential and good application prospects in the treatment of acute liver injury. Attached Figure Description

[0034] Figure 1 This study investigated the expression levels of Pro-CRAMP (CRAMP precursor protein) and CRAMP protein in the liver of wild-type (WT) mice after APAP overdose. (A) Western blotting was used to detect the protein expression of Pro-CRAMP and CRAMP in the liver at different time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h) after APAP overdose. (B) Immunofluorescence staining was used to detect the protein expression of CRAMP in the liver at 0 h, 6 h, and 24 h after APAP overdose. The CampKO+APAP group served as the negative control group. The APAP dose was 300 mg / kg.

[0035] Figure 2This study investigated the cellular origin of CRAMP in the liver following APAP overdose. (A) Western blotting was used to detect the protein expression of Pro-CRAMP in hepatocytes and non-parenchymal cells (NPCs). (B) Immunofluorescence co-localization of CRAMP with the neutrophil marker Ly6G was performed. (C) Western blotting was used to detect the protein expression of Pro-CRAMP in the liver of mice after administration of the neutrophil scavenger α-Ly6G. The APAP dose was 300 mg / kg, and the α-Ly6G dose was 100 μg / mouse.

[0036] Figure 3 To investigate the effect of Camp gene knockout on liver injury following APAP overdose. (A) H&E staining results of liver tissue from WT and CampKO mice at 12 hours (acute injury phase) and 48 hours (regeneration and repair phase) after APAP (300 mg / kg) overdose; (B) Serum ALT levels from WT and CampKO mice at 12 hours and 48 hours after APAP (300 mg / kg) overdose; (C) Immunohistochemical staining results of Ki67 (hepatocyte proliferation marker) in hepatocytes from WT and CampKO mice at 48 hours after APAP (300 mg / kg) overdose; (D) Western blotting detection of Pro-CRAMP protein expression levels in the liver after poisoning with different doses of APAP (300 mg / kg and 500 mg / kg); (E) Survival curves of acute liver failure induced by high-dose APAP (500 mg / kg) in WT and CampKO mice.

[0037] Figure 4 The effect of exogenous supplementation with the synthetic peptide CRAMP on liver damage following APAP overdose. (A) H&E staining results of liver tissue in CampKO mice 23 hours after exogenous CRAMP supplementation and 48 hours after APAP overdose; (B) Serum ALT levels in CampKO mice 23 hours after exogenous CRAMP supplementation and 48 hours after APAP overdose; (C) Ki67 immunohistochemical staining results of hepatocytes in CampKO mice 23 hours after exogenous CRAMP supplementation and 48 hours after APAP overdose. The APAP dose was 300 mg / kg, and the CRAMP dose was 4 mg / kg.

[0038] Figure 5The effects of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) on the treatment of APAP-induced acute liver injury were investigated. (A) H&E staining results of liver tissue in WT mice 48 hours after exogenous administration of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) 23 hours after APAP overdose; (B) H&E staining results of liver tissue in WT mice 48 hours after APAP overdose, after administration of NAC 1 hour or 23 hours after APAP overdose, or after administration of antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 23 hours after APAP overdose, or in combination with NAC 1 hour after APAP overdose. The APAP dose was 300 mg / kg, the NAC dose was 500 mg / kg, and the doses of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) were all 4 mg / kg.

[0039] Figure 6 The role of antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) in the treatment of APAP-induced acute liver failure. (A) Liver tissue H&E staining results 72 hours after APAP overdose, treated with NAC 1 hour or 6 hours after APAP overdose; (B) Liver tissue H&E staining results 72 hours after APAP overdose, treated with NAC 1 hour after APAP overdose, combined with treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 6 hours and 23 hours after APAP overdose; (C) Liver tissue H&E staining results 72 hours after APAP overdose, treated with NAC 6 hours after APAP overdose, combined with treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 6 hours and 23 hours after APAP overdose; (D) Survival curves of mice treated with NAC 1 hour or 6 hours after APAP overdose, or treated with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 6 hours and 23 hours after APAP overdose, or treated without any treatment after APAP overdose. The dosage of APAP was 500 mg / kg, the dosage of NAC was 500 mg / kg, and the dosage of antimicrobial peptides LL-37, CRAMP and CRAMP(1-39) was 4 mg / kg.

[0040] Figure 7To investigate the effect of Camp gene knockout on liver inflammation following APAP overdose. (A) Immunohistochemical staining results of exogenously administered CRAMP synthetic peptide in CampKO mice 23 hours after APAP overdose, and CRAMP immunohistochemical staining results 24 hours after APAP overdose. The CampKO+APAP group served as the negative control group; (B) Flow cytometry analysis of the number of neutrophils infiltrating the liver in WT and CampKO mice 24 and 48 hours after APAP overdose; (C) Flow cytometry analysis of the number of macrophages infiltrating the liver in WT and CampKO mice 24 and 48 hours after APAP overdose. The APAP dose was 300 mg / kg, and the CRAMP dose was 4 mg / kg.

[0041] Figure 8 The effect of exogenous administration of the antimicrobial peptide LL-37 on liver inflammation following APAP overdose was investigated. (A) 23 hours after APAP overdose, WT mice were given exogenous antimicrobial peptide LL-37, and the number of neutrophils infiltrating the liver was detected by flow cytometry 48 hours after APAP overdose. (B) 23 hours after APAP overdose, WT mice were given exogenous antimicrobial peptide LL-37, and the number of macrophages infiltrating the liver was detected by flow cytometry 48 hours after APAP overdose. The APAP dose was 300 mg / kg, and the LL-37 dose was 4 mg / kg.

[0042] Figure 9 The effects of antimicrobial peptides LL-37 and CRAMP on the phagocytic function of hepatic neutrophils were investigated. (A) Flow cytometry was used to detect the phagocytic function of hepatic neutrophils in WT and CampKO mice 24 hours after APAP (300 mg / kg) overdose. (B) Flow cytometry was used to detect the effects of different concentrations of LL-37 (0, 25, 50, 100 μg / ml) on the phagocytic function of human peripheral blood neutrophils.

[0043] Figure 10 The effects of different concentrations of CRAMP (0, 0.1, 1, 2.5, 5, 10 μg / ml) on the proliferation of mouse primary hepatocytes under EGF stimulation were investigated. (A) Western blotting was used to detect the protein expression of Cyclin D1 in mouse primary hepatocytes; (B) Ki67 immunofluorescence staining was used to detect the proliferation of mouse primary hepatocytes. The EGF concentration was 30 ng / ml, and the Control group was the control group without EGF and CRAMP. Detailed Implementation

[0044] The specific embodiments of the present invention will be described in further detail below with reference to the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0045] The following is a detailed experimental procedure for this invention.

[0046] 1. Main reagents and instruments

[0047] APAP and type IV collagenase were purchased from Sigma-Aldrich; synthetic peptides LL-37, CRAMP, and CRAMP(1-39) were purchased from Shanghai Kaijing Biotechnology Co., Ltd.; neutrophil scavenger α-Ly6G and its control were purchased from BioXCell; NAC was purchased from MCE; EGF was purchased from Sigma-Aldrich; CRAMP antibody was purchased from Innovagen; CD11b and Ki67 antibodies were purchased from Abcam; Cyclin D1 antibody was purchased from Cell Signaling Technology; β-actin antibody was purchased from Sigma-Aldrich; Ly6G antibody was purchased from Biolegend; DAPI was purchased from Beyotime Biotechnology Co., Ltd.; flow cytometry antibodies were all purchased from Biolegend; fluorescent microspheres were purchased from Shanghai Yiyuan Biotechnology Co., Ltd.; Tris-Tricine-SDS-PAGE gel preparation kit was purchased from Solarbio Science & Technology Co., Ltd.; Polymorphprep separation buffer was purchased from Axis-Shield.

[0048] Centrifuges were purchased from Eppendorf, Germany; electrophoresis apparatus and gel imaging system were purchased from Bio-Rad Laboratories, USA; dehydrator, embedding machine and microtome were purchased from Leica, Germany; inverted fluorescence microscope was purchased from Olympus, Japan; laser confocal microscope was purchased from Zeiss, Germany; CO2 incubator was purchased from Thermo Fisher Scientific, USA; flow cytometer was purchased from BD Laboratories, USA.

[0049] 2. Animals and their raising

[0050] Experimental animals were selected from male C57BL / 6J mice aged 8-12 weeks and weighing 20-25g, including WT mice (purchased from Hunan Slack Jingda Laboratory Animal Co., Ltd.) and CampKO mice (purchased from Jackson Laboratory, USA). The Camp KO mice and the control WT mice were littermates bred from Camp KO heterozygotes. All experimental mice were housed in the SPF-grade Medical Laboratory Animal Center of Xiangya Second Hospital, Central South University. The housing temperature was maintained at 22-24℃, humidity at 40-70%, with alternating light and dark lighting for 12 hours, and free access to water and food.

[0051] 3. Construction of an APAP acute liver injury model

[0052] Mice were fasted overnight for 15-16 hours the day before APAP injection, but allowed free access to water. APAP powder was dissolved in 55°C physiological saline to prepare a fresh APAP solution (prepared immediately before use), with a final drug concentration of 25 mg / ml. The solution was continuously heated until completely dissolved. An APAP acute liver injury or acute liver failure model was established by intraperitoneal injection at a dose of 300 mg / kg or 500 mg / kg (injection volume of 12 μl / g or 20 μl / g). The solution temperature was lowered to 37°C before intraperitoneal injection. Mice were returned to food 20 minutes after APAP injection.

[0053] 4. Extraction of mouse primary hepatocytes and liver NPCs

[0054] Primary mouse hepatocytes were isolated using a reverse collagenase perfusion method. Mice were anesthetized with 1% sodium pentobarbital, and the abdomen was cut open to expose the inferior vena cava and portal vein. After inserting an indwelling intravenous catheter into the inferior vena cava, the portal vein was quickly severed. The liver was perfused with calcium- and magnesium-free HBSS buffer (containing EDTA). After the liver turned white, in situ digestion was performed with 0.05% type IV collagenase buffer. During collagenase perfusion, the portal vein was first clamped with forceps, and after the liver swelled, the clamping was paused for 30 seconds before releasing the forceps. This process was repeated several times, for a total perfusion of approximately 10 mL, while maintaining the temperature at around 37°C. After perfusion, the liver was quickly removed and transferred to pre-cooled HBSS buffer containing EDTA. The gallbladder was removed, and the liver was then transferred to a culture dish containing 10% FBS in DMEM medium. The liver capsule was gently torn open with forceps, and the liver pedicle was held and gently shaken to fully release the hepatocytes. The cell suspension was collected and filtered through a 200-mesh cell sieve. After centrifugation at 4℃ and 50g for 3 minutes, the precipitate is hepatocytes. If culture is required, resuspend the cell pellet in DMEM medium containing 10% FBS, seed it in a 24-well plate, and change the medium after 2-4 hours.

[0055] Collect the supernatant after centrifugation, centrifuge at 50g for 3 minutes, discard the precipitate, and further remove hepatocytes. Centrifuge the supernatant at 2500rpm for 5 minutes, and the precipitate is NPCs.

[0056] 5. H&E staining of liver tissue

[0057] Mouse liver tissue samples were taken in approximately 5mm × 5mm sizes (preferably from the same location for each mouse). The liver tissue was fixed with 4% paraformaldehyde for 24 hours, then dehydrated with a gradient of ethanol (concentration increasing), cleared with xylene, and finally embedded in paraffin. Sections were serially prepared using a microtome to a thickness of 5μm, laid up, and dried in a 45℃ oven. The main steps of H&E staining were as follows: sections were baked in a 60℃ oven for approximately 30 minutes, then sequentially immersed in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 2 minutes, and 70% ethanol for 2 minutes, followed by rinsing with tap water. Sections were then stained with hematoxylin for 3-5 minutes, and rinsed with tap water to remove excess stain. Differentiation was performed with 1% hydrochloric acid alcohol for 30 seconds, followed by rinsing with tap water, followed by blue staining for 5 seconds, and rinsing with running water. Sections were then stained with eosin for 5 minutes. Sections were sequentially immersed in 95% ethanol for 5 minutes, then anhydrous ethanol I for 5 minutes, then anhydrous ethanol II for 5 minutes, then xylene I for 5 minutes, and finally xylene II for 5 minutes to dehydrate and clear. The sections were then mounted with neutral resin. The sections were observed and photographed using an Olympus inverted microscope (IX71) (magnification: 40x). ImageJ image analysis software was used to calculate the percentage of necrotic area and assess the degree of necrosis.

[0058] 6. Serum ALT measurement

[0059] Mice were anesthetized with 1% sodium pentobarbital, and their blood was collected. The blood was left to stand overnight at 4°C, centrifuged at 3000g for 10 minutes, and the supernatant serum was collected. The serum was diluted 7 times with deionized water and sent to the Biochemistry Laboratory of Xiangya No. 2 Hospital of Central South University. The serum ALT level was detected by a fully automated biochemistry analyzer (HITACHI 7600).

[0060] 7. Immunohistochemical staining of liver tissue

[0061] The procedures for tissue sampling, fixation, dehydration, paraffin embedding, and sectioning are the same as for H&E staining. The main steps for immunohistochemical staining are as follows: Tissue sections are placed in a retrieval chamber filled with citrate antigen retrieval buffer (pH 6.0) and microwaved for antigen retrieval. Heat on medium heat for 8 minutes until boiling, then turn off the heat and incubate for 8 minutes, followed by 7 minutes on medium-low heat. After natural cooling, slides are placed in PBS (pH 7.4) and washed three times on a destaining shaker, 5 minutes each time. Sections are then placed in 3% hydrogen peroxide solution and incubated at room temperature in the dark for 25 minutes. Sections are then placed in PBS and washed three times on a destaining shaker, 5 minutes each time. 3% BSA is added to the histochemistry zone to evenly cover the tissue, and the section is blocked at room temperature for 30 minutes. The blocking solution is gently removed, and primary antibody is added to the section. The section is then incubated overnight at 4°C in a humidified chamber. The next day, the section is placed in PBS and washed three times on a destaining shaker, 5 minutes each time. After slightly drying the section, the corresponding species secondary antibody is added to the zone to cover the tissue, and the section is incubated at room temperature for 50 minutes. The slides were placed in PBS and washed three times on a destaining shaker for 5 minutes each time. After slightly drying the slides, freshly prepared DAB chromogenic solution was added to the circle. The chromogenic time was controlled under a microscope; a positive result was brownish-yellow. The slides were then rinsed with tap water to stop the chromogenic process. The cell nuclei were counterstained with hematoxylin for 3 minutes, washed with tap water, differentiated with hematoxylin differentiation solution for a few seconds, rinsed with tap water, and then restained with hematoxylin blue solution and rinsed with running water. The slides were then sequentially immersed in 95% ethanol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to dehydrate and clear. The slides were then mounted with neutral resin.

[0062] The method for counting Ki67-positive hepatocytes is as follows: Observe and photograph using an Olympus inverted microscope (IX71) (magnification: 200x). Select 7-8 high-power fields under a light microscope to identify Ki67-positive hepatocytes (the nuclei of hepatocytes are large and round, stained brown). Count them using Image-ProPlus image software. The ratio of Ki67-positive hepatocyte nuclei to the total number of hepatocyte nuclei is taken as the hepatocyte proliferation rate.

[0063] 8. Immunofluorescence staining

[0064] Liver tissue samples from mice were taken in approximately 5mm × 5mm sizes (preferably from the same location for each mouse). Liver tissue was embedded in OCT, frozen into blocks at -20°C, and serially sectioned using a cryostat to a thickness of 5μm. Frozen sections were then air-dried at room temperature for 15 minutes, fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed three times (5 minutes each time) in PBS on a destaining shaker. The tissue to be stained was circled with a histochemical pen, permeabilized with 0.2% Triton X-100 (prepared in PBS) at room temperature for 20 minutes, and then blocked with 3% BSA at room temperature for 1 hour. The blocking solution was gently removed, and primary antibody was added to the sections. The sections were then incubated overnight at 4°C in a humidified chamber (if using immunofluorescence co-staining, incubate with one antibody overnight, wash three times with PBS, and then incubate with the other antibody overnight). The next day, the sections were washed three times (5 minutes each time) in PBS on a destaining shaker. After slightly drying the slides, add the corresponding species' secondary antibody to the circle to cover the tissue, and incubate at room temperature in the dark for 1 hour (if co-staining with immunofluorescence, the two secondary antibodies can be mixed). Place the slides in PBS and wash three times on a destaining shaker, 5 minutes each time. Stain the cell nuclei with DAPI for 5 minutes, then place the slides in PBS and wash once on a destaining shaker. Add anti-fluorescence quenching agent and mount. Observe under a fluorescence microscope or confocal microscope.

[0065] For immunofluorescence staining of primary mouse hepatocytes, the slides in which the cells have already grown were washed with PBS in a cell culture plate, the cells were fixed with 4% paraformaldehyde, and the remaining steps were the same as for tissue immunofluorescence staining.

[0066] 9. Western blotting analysis

[0067] Weigh 20 mg of liver tissue or extracted primary mouse hepatocytes and NPCs, add lysis buffer containing protease inhibitors, grind the liver tissue using a tissue homogenizer, centrifuge at 12000 rpm for 10 minutes, and collect the supernatant for Western blotting analysis. Determine the protein concentration in the supernatant using a BCA protein kit. Dilute and quantify the protein using lysis buffer and loading buffer, and denature the protein by heating at 100°C for 10 minutes. Immediately after heating, cool on ice. Separate the denatured protein by SDS-PAGE at a loading rate of 30 μg, transfer to a PVDF membrane, block with 5% BSA for 60 minutes at room temperature, and incubate overnight with the first antibody at 4°C. The next day, wash the membrane three times with TBST for 5 minutes each time, then incubate with diluted secondary antibody of the corresponding species at room temperature for 60 minutes, followed by three more washes with TBST for 5 minutes each time. Develop the blot using chemiluminescence immunoassay with an ECL kit, and measure the signal intensity on the blot using ImageJ software. β-actin was used as an internal control protein. For the detection of CRAMP small molecule proteins, the procedure was performed according to the Tris-Tricine-SDS-PAGE gel preparation kit.

[0068] 10. Flow cytometry

[0069] NPCs were extracted from mouse livers. After lysing the red blood cells with erythrocyte lysis buffer, the cells were resuspended in PBS containing 1% FBS. 10 cells were then collected. 6 Cell surface staining was performed. Zombie staining was performed to determine cell viability; cells were incubated at room temperature for 20 minutes, followed by a single wash with PBS containing 1% FBS. CD16 / 32 blocking and non-specific staining were performed, followed by incubation on ice for 10 minutes. Antibodies corresponding to the cell surface molecules were added at a 1:100 ratio, mixed, and incubated at 4°C in the dark for 30 minutes. Cells were washed once with PBS containing 1% FBS, centrifuged, and resuspended in 200 μl of PBS for flow cytometry analysis (FACS Canto II). If flow cytometry analysis could not be performed immediately, an equal volume of 4% paraformaldehyde was added for fixation.

[0070] 11. Extraction of human peripheral blood neutrophils

[0071] 10 ml of peripheral blood was collected from healthy adults using EDTA anticoagulant tubes. 5 ml of peripheral blood was added to 5 ml of Polymorphprep separation buffer. The cells were centrifuged at 500 g for 35 minutes at room temperature (without acceleration or deceleration). After centrifugation, two ring-shaped milky-white cell layers were observed: an upper layer of mononuclear cells and a lower layer of neutrophils. The upper mononuclear cell layer was aspirated, and the neutrophils were collected. The cells were washed once with PBS, lysed with erythrocyte lysis buffer, and resuspended in RP1640 containing 2% FBS. The cells were then centrifuged at 4.5 × 10⁻⁶. 5 10 cells / well were seeded in a 48-well plate for further processing.

[0072] 12. Statistical Analysis

[0073] All values ​​are expressed as mean ± standard error (SEM). All analyses were performed using GraphPad Prism 7 software. Statistical analysis was performed using either the two independent samples t-test or one-way ANOVA. P < 0.05 was considered statistically significant.

[0074] Example 1: The expression level of Pro-CRAMP / CRAMP protein in the liver was significantly increased after APAP overdose.

[0075] An APAP-induced acute liver injury model was established in WT mice. For example... Figure 1 As shown in Figure A, the protein expression of Pro-CRAMP and CRAMP in the liver significantly increased after APAP injection, peaking at 24 hours, then gradually declining, returning to baseline levels at 72 hours. This is consistent with the Western blot results. Figure 1As shown in Figure B, the CRAMP immunofluorescence staining results revealed that CRAMP signal gradually increased over time at 6 and 24 hours after APAP overdose. These results suggest that APAP overdose can induce increased expression of CRAMP protein in the liver.

[0076] Example 2: CRAMP is mainly expressed in neutrophils infiltrating the liver.

[0077] like Figure 2 As shown in Figure A, hepatocytes and neutrophils were isolated 24 hours after APAP injection in WT mice. Western blot results showed that CRAMP was mainly expressed by NPCs, with a small portion expressed by hepatocytes. Previous studies have indicated that neutrophils are the main source of CRAMP, and the trend of hepatic CRAMP protein expression over time is consistent with that of neutrophils. Figure 2 As shown in Figure B, immunofluorescence colocalization results of WT mice 24 hours after APAP injection showed that CRAMP signaling in the liver was mainly colocalized with infiltrating neutrophils. Figure 2 As shown in Figure C, neutrophils in WT mice were cleared using the neutrophil neutralizing antibody α-Ly6G. Twenty-four hours after APAP injection, Western blotting was used to detect the protein expression of Pro-CRAMP in the liver. The results showed that the elevated Pro-CRAMP protein expression level was significantly suppressed. These results indicate that the elevated CRAMP levels after APAP overdose mainly originate from neutrophils infiltrating the liver, followed by hepatocytes.

[0078] Example 3: Camp gene knockout significantly delayed liver repair after acute liver injury with acute liver injury (APAP).

[0079] APAP-induced acute liver injury models were established in WT and Camp KO mice. Figure 3 As shown in Figure A, H&E staining results indicated that 12 hours after APAP injection, the area of ​​liver necrosis was comparable in Camp KO and WT mice, while 48 hours after APAP injection, the area of ​​liver necrosis was larger in Camp KO mice compared to WT mice. This is consistent with the histological results, as shown in Figure A. Figure 3 As shown in Figure B, serum ALT results indicated that 12 hours after APAP injection, Camp KO and WT mice had similar serum ALT levels, while 48 hours after APAP injection, Camp KO mice had higher serum ALT levels compared to WT mice. Figure 3 As shown in Figure C, Ki67 staining was used to detect hepatocyte proliferation 48 hours after APAP injection. The results showed that the number of proliferating hepatocytes in Camp KO mice was significantly reduced. These results indicate that Camp gene knockout specifically inhibits the liver repair process after APAP acute liver injury.

[0080] Previous studies have shown that the liver can spontaneously repair itself after moderate APAP poisoning (300 mg / kg), while high APAP poisoning (500 mg / kg) can inhibit liver regeneration and repair. Figure 3 As shown in Figure D, compared to a moderate dose of APAP, a high dose of APAP significantly inhibited the protein expression of Pro-CRAMP, further supporting the involvement of Pro-CRAMP in the liver repair process. Figure 3 As shown in Figure E, high-dose APAP injection induced acute liver failure in mice. Survival rates of WT and Camp KO mice were monitored, and the results showed that the survival rate of Camp KO mice was significantly reduced, further demonstrating that Camp gene knockout can inhibit liver repair and promote death induced by acute liver failure.

[0081] Example 4: Exogenous supplementation with CRAMP synthetic peptides significantly reversed liver damage in Camp KO mice.

[0082] An APAP-induced acute liver injury model was established in WT and Camp KO mice, and exogenous CRAMP was supplemented in Camp KO mice 23 hours after APAP injection. Figure 4 As shown in Figure A, H&E staining results 48 hours after APAP injection showed that exogenous CRAMP supplementation significantly reduced the area of ​​liver necrosis in CampKO mice. This is consistent with the histological results, as... Figure 4 As shown in Figure B, serum ALT levels 48 hours after APAP injection indicated that exogenous CRAMP supplementation significantly reduced serum ALT levels in CampKO mice. Figure 4 As shown in Figure C, Ki67 immunohistochemical staining results 48 hours after APAP injection showed that exogenous CRAMP supplementation significantly promoted hepatocyte proliferation in Camp KO mice. These results indicate that supplementation with CRAMP synthetic peptides can reverse liver damage in Camp KO mice following APAP overdose.

[0083] Example 5: Treatment with antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) significantly promoted liver repair after acute liver injury caused by APAP.

[0084] An APAP-induced acute liver injury model was established in WT mice, such as... Figure 5 As shown in Figure A, WT mice were treated with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) 23 hours after APAP injection. H&E staining results 48 hours after APAP injection showed that treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) significantly reduced the area of ​​liver necrosis. Figure 5As shown in Figure B, H&E staining results 48 hours after APAP injection revealed that NAC treatment 1 hour after APAP injection significantly reduced the area of ​​liver necrosis, while NAC treatment 23 hours after APAP injection failed to inhibit liver necrosis. Early NAC treatment (1 hour after APAP injection) combined with late-stage (23 hours after APAP injection) treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) further reduced the area of ​​liver necrosis. These results indicate that treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) significantly promotes liver repair after APAP acute liver injury, and that early NAC treatment combined with late-stage treatment with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) has a synergistic effect on the treatment of APAP liver injury.

[0085] Example 6: Antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) treat and protect mice against APAP-induced acute liver failure.

[0086] An APAP-induced acute liver failure model was established in WT mice. NAC treatment was administered 1 hour or 6 hours after APAP injection to simulate patients in the early and late stages of clinical treatment, respectively. Figure 6 As shown in Figure A, H&E staining results 72 hours after APAP injection showed that NAC treatment administered 1 hour after APAP injection significantly reduced the area of ​​liver necrosis, while the therapeutic effect of NAC administered 6 hours after injection was significantly reduced. Figure 6 As shown in Figure B, H&E staining results 72 hours after APAP injection showed that, compared with early NAC monotherapy, NAC combined with late-stage (6 and 23 hours after APAP injection) antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) significantly reduced the area of ​​liver necrosis. Figure 6 As shown in Figure C, H&E staining results 72 hours after APAP injection showed that, compared to NAC alone, NAC combined with late-stage antimicrobial peptides LL-37 or CRAMP (1-39) significantly reduced the area of ​​liver necrosis, while there was no significant difference in the area of ​​liver necrosis between NAC and CRAMP. Figure 6As shown in Figure D, APAP overdose reduced mouse survival. Compared to no treatment, early NAC treatment and early NAC combined with antimicrobial peptides LL-37, CRAMP, or CRAMP(1-39) significantly increased WT mouse survival. Later NAC treatment alone did not significantly improve survival, but combined treatment with antimicrobial peptides LL-37 or CRAMP(1-39) significantly increased mouse survival. However, NAC combined with CRAMP did not improve mouse survival. These results indicate that treatment with antimicrobial peptides LL-37, CRAMP, and CRAMP(1-39) can protect mice against APAP-induced acute liver failure, but the effect of antimicrobial peptide CRAMP in treating APAP-induced acute liver failure is weaker than that of LL-37 and CRAMP(1-39).

[0087] Example 7: Camp gene knockout significantly delayed the clearance of liver inflammation after APAP poisoning.

[0088] To elucidate the mechanism by which antimicrobial peptides promote liver repair after liver injury, we first performed immunohistochemical staining for CRAMP. For example... Figure 7 As shown in Figure A, 24 hours after APAP injection in WT mice, immunohistochemical results of CRAMP in the liver showed that after APAP overdose, endogenous CRAMP expression increased and was mainly concentrated in the necrotic area of ​​the liver. Consistent with this, exogenous supplementation of CRAMP synthetic peptides in Camp KO mice 23 hours after APAP injection resulted in exogenous CRAMP entering the liver 1 hour later and being mainly distributed in the necrotic area of ​​the liver, suggesting that CRAMP mainly functions in the necrotic area of ​​the liver.

[0089] Following an APAP overdose, a large number of immune cells infiltrate the necrotic areas of the liver, triggering an inflammatory response. Neutrophils and macrophages are the main cells involved in this inflammatory response. Previous studies have shown that acute inflammation is a key characteristic of the APAP-induced liver injury phase, and timely clearance of inflammation is a prerequisite for initiating liver regeneration and repair. To clarify whether CRAMP-promoted liver repair following APAP injury is related to inflammation clearance, we therefore examined the number of neutrophils and macrophages infiltrating the liver. Figure 7 As shown in Figure B, APAP-induced acute liver injury models were established in WT and Camp KO mice. Flow cytometry analysis at 24 and 48 hours post-APAP injection showed that at 24 hours post-APAP injection, the number of neutrophils in the liver of Camp KO mice was comparable to that of WT mice, while at 48 hours post-APAP injection, the number of neutrophils in the liver of Camp KO mice was significantly higher than that of WT mice. Similarly, as... Figure 7As shown in Figure C, 24 hours after APAP injection, the number of macrophages in the liver of Camp KO mice was comparable to that of WT mice, while 48 hours after APAP injection, the number of macrophages infiltrating the liver of Camp KO mice showed a significant increasing trend. These results indicate that Camp gene knockout does not affect the recruitment of neutrophils and macrophages, but significantly delays the clearance of inflammation during the liver repair phase.

[0090] Example 8: Antimicrobial peptide LL-37 significantly improved liver inflammation after APAP poisoning.

[0091] An APAP-induced acute liver injury model was established in WT mice. Twenty-three hours after APAP injection, the antimicrobial peptide LL-37 was administered exogenously. Figure 8 As shown in Figures A and 8B, flow cytometry analysis 48 hours after APAP injection revealed that exogenous LL-37 injection significantly reduced the number of neutrophils and macrophages infiltrating the liver. These results indicate that the antimicrobial peptide LL-37 significantly promotes the clearance of inflammation during the liver repair phase following APAP-induced liver injury.

[0092] Example 9: Antimicrobial peptide LL-37 / CRAMP significantly promotes phagocytic function of neutrophils

[0093] CRAMP expression peaks 24 hours after APAP overdose, and the primary event at this time point is known to be neutrophil-mediated clearance of necrotic cell debris. Therefore, to clarify whether the antimicrobial peptide's promotion of inflammation clearance after liver injury is related to enhanced neutrophil phagocytic function, we examined neutrophil phagocytic function both in vivo and in vitro. Figure 9 As shown in Figure A, an APAP-induced acute liver injury model was established in WT and Camp KO mice. Twenty-four hours after APAP injection, 1 μm FITC-labeled fluorescent microspheres were injected via the tail vein. Thirty minutes later, liver neutrophils (NPCs) were extracted for flow cytometry analysis. The results showed that compared to WT mice, Camp KO mice exhibited significantly reduced neutrophil phagocytic capacity. This is consistent with in vivo results, such as... Figure 9 As shown in Figure B, human peripheral blood neutrophils were treated with different concentrations of LL-37 (0, 25, 50, and 100 μg / ml) for 2 hours, followed by the addition of 1 μm FITC-labeled fluorescent microspheres. Flow cytometry analysis was performed 30 minutes later. The results showed that LL-37 enhanced the phagocytic function of neutrophils in a concentration-dependent manner. These results indicate that the antimicrobial peptides LL-37 and CRAMP can significantly promote the phagocytic function of neutrophils.

[0094] Example 10: CRAMP synthetic peptide significantly promotes mouse hepatocyte proliferation

[0095] Primary hepatocytes isolated from mice cannot proliferate without additional cytokine stimulation. Epidemiologically active factor (EGF) is a commonly used cytokine for stimulating hepatocyte proliferation in vitro. Primary hepatocytes from WT mice were extracted and cultured, and then treated with EGF and different concentrations of CRAMP (0.1, 1, 2.5, 5, 10 μg / ml) for 24 hours. Figure 10 As shown in Figure A, the Western blot results indicate that CRAMP treatment significantly increases the protein expression of Cyclin D1. This is consistent with the Western blot results, as... Figure 10 As shown in Figure B, CRAMP treatment increased the number of Ki67-positive hepatocytes. These results indicate that the CRAMP-synthesized peptide can directly promote the proliferation of mouse hepatocytes.

[0096] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Xiangya No. 2 Hospital of Central South University <120> Application of antimicrobial peptides in the preparation of therapeutic agents for acetaminophen-induced acute liver injury and / or liver failure <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 37 <212> PRT <213> Homo sapiens <400> 1 Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> 2 <211> 34 <212> PRT <213> Mouse (Mus musculus) <400> 2 Gly Leu Leu Arg Lys Gly Gly Glu Lys Ile Gly Glu Lys Leu Lys Lys 1 5 10 15 Ile Gly Gln Lys Ile Lys Asn Phe Phe Gln Lys Leu Val Pro Gln Pro 20 25 30 Glu Gln <210> 3 <211> 39 <212> PRT <213> mice (Mus musculus) <400> 3 Ile Ser Arg Leu Ala Gly Leu Leu Arg Lys Gly Gly Glu Lys Ile Gly 1 5 10 15 Glu Lys Leu Lys Lys Ile Gly Gln Lys Ile Lys Asn Phe Phe Gln Lys 20 25 30 Leu Val Pro Gln Pro Glu Gln 35

Claims

1. The use of antimicrobial peptides in the preparation of therapeutic agents for acetaminophen-induced acute liver injury and / or liver failure, wherein the antimicrobial peptides include at least one of LL-37, CRAMP, and CRAMP (1-39); The sequence of the antimicrobial peptide LL-37 is LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; The sequence of the antimicrobial peptide CRAMP is GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ; The sequence of the antimicrobial peptide CRAMP (1-39) is ISRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ.

2. The application according to claim 1, characterized in that, At least one of the antimicrobial peptides may be synergistically used with N-acetylcysteine ​​to prepare a therapeutic agent for acetaminophen-induced acute liver injury and / or liver failure.

3. The application according to claim 2, characterized in that, N-acetylcysteine ​​is used in the early stages of acetaminophen-induced acute liver injury and / or liver failure, and at least one of the antimicrobial peptides is used in the later stages of acetaminophen-induced acute liver injury and / or liver failure.

4. The application according to claim 2, characterized in that, The antimicrobial peptides described herein promote liver regeneration and repair following acetaminophen-induced acute liver injury and / or liver failure.

5. The application according to claim 2, characterized in that, The antimicrobial peptide reduces the necrotic area of ​​the liver or improves the survival rate of acute liver failure after acetaminophen-induced acute liver injury and / or liver failure.

6. The application according to claim 2, characterized in that, The antimicrobial peptides LL-37 and / or CRAMP improve liver inflammation following acetaminophen-induced acute liver injury.

7. The application according to claim 2, characterized in that, The antimicrobial peptides LL-37 and / or CRAMP promote hepatic neutrophil phagocytosis following acetaminophen-induced acute liver injury.

8. The application according to claim 2, characterized in that, The antimicrobial peptide CRAMP promotes hepatocyte proliferation in mice following acetaminophen-induced acute liver injury.

Images