Application of fatty acid synthase inhibitors in the prevention and treatment of drug-induced liver injury

Abstract

The present invention provides the use of fatty acid synthase inhibitors for the prevention and treatment of drug-induced liver injury (DILI), relating to the field of biotechnology. This invention demonstrates that increased fatty acid synthesis mediated by FASN regulates macrophage inflammatory responses and migratory chemotactic behavior. Targeting macrophage fatty acid synthesis can alleviate acute liver injury and inflammatory responses. This discovery represents the first discovery of new targets and new efficacy for fatty acid synthase inhibitors, potentially resolving the current lack of specific treatments for acute liver injury.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the use of a fatty acid synthase inhibitor in preventing and treating drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) is the most common adverse drug reaction and a significant obstacle to the development and marketing of new drugs. Acetaminophen (APAP) is the most widely used analgesic and antipyretic drug worldwide. As an over-the-counter medication, APAP is readily available in various dosage forms and is typically used alone or in combination with other medications to treat fever caused by the common cold and influenza, as well as to relieve mild to moderate pain. In my country, most combination cold medications contain APAP. Although APAP is safe and effective at therapeutic doses, improper use and overdose can easily lead to acute liver injury (ALI), even acute liver failure (ALF), and death. When other risk factors are present, APAP can cause hepatotoxicity and liver damage even within the therapeutic range. APAP-induced acute liver injury is currently the most common cause of ALF, accounting for 40-45% of all causes.

[0003] Despite years of extensive research on APAP hepatotoxicity, its precise mechanism remains unclear. Oxidative stress and mitochondrial dysfunction are currently considered key events in APAP hepatotoxicity. While most APAP is converted into non-toxic glucuronide and sulfate metabolites that are excreted in the urine, approximately 5-10% is oxidized by cytochrome P450 2E1 (CYP2E1) and other enzymes to form the highly active and toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). Normally, NAPQI covalently binds to the thiol group of GSH. However, when GSH is depleted, NAPQI binds to mitochondrial proteins, forming protein adducts that induce mitochondrial oxidative stress and generate large amounts of ROS. ROS further activate c-jun N-terminal kinase (JNK), leading to increased mitochondrial permeability and dysfunction, triggering cell apoptosis and necrosis. Sterile inflammation is another important pathogenesis of APAP-induced ALI. ALI is often the result of the combined effects of liver cells, immune cells, and the inflammatory mediators they secrete. Necrotic hepatocytes release a large amount of mediators, which activate immune cells, exacerbating the inflammatory response and liver tissue necrosis. Furthermore, autophagy, endoplasmic reticulum stress, and microcirculatory dysfunction are also important pathogenic mechanisms of ALI.

[0004] However, currently, there are very limited drugs available for the prevention and treatment of APAP-induced DILI. N-acetylcysteine ​​(NAC), a prodrug for glutathione (GSH), is the only clinically recommended antidote, but it is only effective when administered early in the course of toxicity. Although it can mitigate APAP-induced hepatotoxicity, some patients taking the recommended dose of NAC experience adverse reactions or still develop ALI. Nanoparticle-based drug delivery systems reduce hepatobiliary clearance of drugs by conjugating specific ligands that bind to receptors on the surface of hepatocytes. The asialoglycoprotein receptor (ASGPR) is a specific receptor on the surface of hepatocytes with a high affinity for galactose, lactose, and glucose. Recently, researchers synthesized a polymer called D4-Gal that selectively targets and binds to the ASGPR in hepatocytes of healthy mice and a mouse model of ALI. Furthermore, a D4-Gal conjugate of NAC, Gal-D-NAC, was synthesized and used to treat APAP-ALI in a mouse model. Results showed that Gal-D-NAC reduced cellular oxidative stress, reduced the area of ​​hepatocyte necrosis, and improved the survival rate of ALI mice. However, this type of nanomedicine is still in the animal testing stage, and its clinical efficacy needs further verification. Therefore, how to prevent and treat ALI induced by drugs such as APAP remains a key issue. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the deficiencies in the prior art, the present invention provides an application of a fatty acid synthase inhibitor in the prevention and treatment of drug-induced liver injury, solving the technical problem that currently there are very limited drugs for the prevention and treatment of drug-induced acute liver injury.

[0007] (2) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] On the one hand, the present invention provides a use of a fatty acid synthase inhibitor in the preparation of a medicament for preventing and / or treating drug-induced liver injury, wherein the fatty acid synthase inhibitor inhibits fatty acid synthesis by inhibiting the activity of the fatty acid synthase gene (FASN) to prevent and / or treat drug-induced liver injury.

[0010] In one embodiment, the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

[0011] In one embodiment, the fatty acid synthase inhibitor is a macrophage fatty acid synthase inhibitor.

[0012] In one embodiment, the macrophage fatty acid synthase inhibitor is an intrahepatic macrophage fatty acid synthase inhibitor.

[0013] In one embodiment, the fatty acid synthase inhibitor comprises orlistat or a pharmaceutically acceptable salt thereof.

[0014] In one embodiment, orlistat or a pharmaceutically acceptable salt thereof is used as the main active ingredient.

[0015] In one embodiment, orlistat or a pharmaceutically acceptable salt thereof as the main active ingredient means that orlistat or a pharmaceutically acceptable salt thereof accounts for more than 10% of the active ingredient; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0016] In a second aspect, the present invention provides a pharmaceutical composition for treating drug-induced liver injury, wherein the drug for treating drug-induced liver injury comprises a fatty acid synthase inhibitor.

[0017] In one embodiment, the fatty acid synthase inhibitor comprises orlistat or a pharmaceutically acceptable salt thereof.

[0018] In one embodiment, the drug for treating drug-induced liver injury further comprises pharmaceutically acceptable excipients.

[0019] In one aspect, the present invention provides a pharmaceutical preparation for treating drug-induced liver injury, comprising the above-mentioned pharmaceutical composition.

[0020] In one embodiment, the pharmaceutical formulation comprises a solid dosage form, a semisolid dosage form, a liquid dosage form, or a gaseous dosage form.

[0021] In one embodiment, the solid dosage form includes powders, pills, tablets, and capsules; the semisolid dosage form includes ointments, suppositories, and gels; the liquid dosage form includes lotions, mixtures, solutions, and injections; and the gaseous dosage form includes aerosols and sprays.

[0022] In one aspect, the present invention provides use of the above-mentioned pharmaceutical composition or pharmaceutical preparation in the preparation of a drug for preventing and / or treating drug-induced liver injury.

[0023] In one aspect, the fatty acid synthase inhibitor comprises orlistat or a pharmaceutically acceptable salt thereof.

[0024] In one embodiment, the above-mentioned orlistat or a pharmaceutically acceptable salt thereof is used as the main active ingredient in a pharmaceutical composition or pharmaceutical preparation.

[0025] In one embodiment, orlistat or a pharmaceutically acceptable salt thereof as the main active ingredient means that orlistat or a pharmaceutically acceptable salt thereof accounts for more than 10% of the active ingredient; preferably, more than 20%; preferably, more than 30%; preferably, more than 40%; preferably, more than 50%; preferably, more than 60%; preferably, more than 65%; preferably, more than 70%; preferably, more than 75%; preferably, more than 80%; preferably, more than 85%; preferably, more than 90%; preferably, more than 95%.

[0026] (3) Beneficial effects

[0027] The present invention provides a fatty acid synthase inhibitor for use in preventing and treating drug-induced acute liver injury. Compared with the prior art, it has the following beneficial effects:

[0028] This study, for the first time, explores the mechanism of macrophage involvement in the development and progression of APAP-induced ALI from the perspective of fatty acid metabolism. In vitro and in vivo experiments confirm that targeting macrophage fatty acid synthase (FASN) can alleviate ALI, revealing a novel mechanism of DILI pathogenesis that has not been reported to date. This approach addresses the current lack of effective treatments for DILI and provides a new target and therapeutic approach for its clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The fatty acid synthesis of liver macrophages in mice with acetaminophen-induced acute liver injury (ALI) model increased; Figure 1 A: Mouse serum ALT / AST; Figure 1 B: H&E staining of mouse liver tissue; Figure 1 C: IBA1 marked liver macrophages, Bodipy493 / 503 stained neutral lipids, and immunofluorescence analysis of neutral lipid levels in mouse liver macrophages; Figure 1 D: RAW264.7 macrophages were stimulated with ACM for 24 h, stained with Bodipy493 / 503, and the intracellular neutral lipid levels were observed under a confocal microscope; Figure 1E: Liver macrophages of DILI model mice were obtained by collagenase perfusion, and the expression level of Fasn mRNA in cells was analyzed by qPCR; Figure 1 FG: RAW264.7 macrophages were stimulated with ACM for 12 h, and the expression level of FASN in the cells was analyzed by qPCR and Western blotting.

[0031] Figure 2 It is FASN-mediated fatty acid synthesis that regulates hepatic macrophage inflammation and infiltration in ALI; Figure 2 A: qPCR analysis of the expression levels of inflammatory factors in liver macrophages of ALI model mice; Figure 2 B: Construction of myeloid conditional Fasn knockout mice (FASN △Mye ), bone marrow-derived macrophages (BMDM) were isolated and stimulated with ACM for 12 h, and the expression levels of inflammatory factors in cells were analyzed by qPCR; Figure 2 C: IBA1 marks monocyte-derived macrophages, CLEC4F marks liver-resident macrophages, and immunofluorescence analysis of mouse liver macrophage composition; Figure 2 D: Transwell chamber assay to analyze the effect of ACM stimulation on the migration ability of RAW264.7 macrophages; Figure 2 E: siRNA silenced FASN expression, and Transwell chamber assay was used to analyze the effect of ACM stimulation on the migration of RAW264.7 macrophages; Figure 2 F: Transwell chamber assay to analyze the effect of ACM stimulation on Fasn △Mye Effects on BMDM migration ability.

[0032] Figure 3 The effect of myeloid conditional knockout of Fasn on liver injury and inflammation in ALI model mice; Figure 3 A: Mouse serum ALT / AST; Figure 3 B: H&E staining of mouse liver tissue; Figure 3 C: TUNEL staining of frozen sections of liver tissue and immunofluorescence analysis of mouse liver cell apoptosis; Figure 3 D: qPCR analysis of the mRNA expression levels of inflammatory factors IL-1β and TNF-α in liver tissue; Figure 3 E: Immunofluorescence analysis of liver macrophage subsets, IBA1 + CLEC4F + Hepatic resident Kupffer cells, IBA1 + CLEC4F - Monocyte-derived macrophages.

[0033] Figure 4 Orlistat intervention can alleviate liver damage and inflammation in ALI model mice; Figure 4A: Mouse serum ALT / AST; Figure 4 B: H&E staining of mouse liver tissue; Figure 4 C: TUNEL staining of frozen sections of liver tissue and immunofluorescence analysis of mouse liver cell apoptosis; Figure 4 D: Immunofluorescence analysis of liver macrophage subsets, IBA1 + CLEC4F + Liver Kupffer cells, IBA1 + CLEC4F - Monocyte-derived macrophages; Figure 4 E: qPCR analysis of the mRNA expression levels of inflammatory factors IL-1β and TNF-α in liver tissue; Figure 4 F: 600 mg / k to draw a survival curve to observe the survival status of mice; Figure 4 G: C57 / B6J male mice were given 10 mg / kg orlistat by gavage and fasted for 12 h. APAP (300 mg / kg) was injected intraperitoneally to establish the ALF model. The same dose of orlistat was administered every 24 h. Serum was collected 72 h later for ALT / AST measurement. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0035] The present application provides an application of a fatty acid synthase inhibitor in preventing and treating acetaminophen-induced drug-induced liver injury, thereby solving the technical problem of very limited drugs currently available for preventing and treating APAP-induced ALI.

[0036] The technical solution in the embodiments of the present application is to solve the above technical problems, and the overall idea is as follows:

[0037] Currently, there are very limited drugs available for the prevention and treatment of APAP-induced ALI. N-acetylcysteine ​​(NAC), a prodrug of glutathione (GSH), is the only clinically recommended antidote, but it is only effective when administered early in the course of toxicity. Although it can mitigate APAP-induced hepatotoxicity, some patients taking the recommended dose of NAC experience adverse reactions or still develop ALI. Nanoparticle-based drug delivery systems reduce hepatobiliary clearance of drugs by conjugating specific ligands that bind to receptors on the surface of hepatocytes. The asialoglycoprotein receptor (ASGPR) is a specific receptor on the surface of hepatocytes with a high affinity for galactose, lactose, and glucose. Recently, researchers synthesized a polymer called D4-Gal that selectively targets and binds to the ASGPR in hepatocytes of healthy mice and a mouse model of ALI. Furthermore, a D4-Gal conjugate of NAC, Gal-D-NAC, was synthesized and used to treat APAP-ALI in a mouse model. Results showed that Gal-D-NAC can reduce cellular oxidative stress, shrink the area of ​​liver cell necrosis, and improve the survival rate of ALI mice. However, this type of nanomedicine is still in the animal testing stage, and its clinical effectiveness needs further verification.

[0038] Excessive APAP induction can induce hepatocyte necrosis, leading to the release of cellular contents such as nuclear DNA fragments, high-mobility group protein B1 (HMGB1), mitochondrial DNA, uric acid, and ATP. These immune-stimulating factors act as damage-associated molecular patterns, transcriptionally activating proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-10) and chemokines (MCP-1, MIP-2, and IL-8), mediating hepatic inflammation and recruiting more peripheral neutrophils and monocytes. However, whether the inflammatory response in the pathophysiology of DILI promotes the progression of liver damage or serves as a cellular defense against toxicity remains controversial. Studies have found that, in addition to potentially promoting the progression of DILI, inflammation also helps clear dead cells and debris and later stimulates liver repair and regeneration. Therefore, blocking inflammation may have a protective effect in the initial stages of DILI but may actually be detrimental to the ultimate process of damage repair. Consequently, the role of pure anti-inflammatory therapy in DILI remains controversial, and inflammation as a therapeutic target for DILI is also controversial.

[0039] Other studies have found that natural medicines such as astaxanthin and urolithin can be used to treat DILI by alleviating cellular damage caused by oxidative stress, activating the Nrf2 signaling pathway, reducing the release of inflammatory factors, and regulating the synthesis, conjugation, and excretion of GSH. The liver is a key site for drug biotransformation in the body and is therefore susceptible to the adverse effects of many compounds, dietary supplements, and herbal remedies. Notably, many of these natural medicines require high doses or repeated administration to achieve the desired therapeutic effect. Furthermore, these drugs are co-administered with high concentrations of dimethyl sulfoxide, which may increase the incidence of adverse drug reactions, lead to off-target or toxic reactions, and further aggravate liver damage. Therefore, further clinical trials are needed to determine the appropriate use of natural medicines for the treatment of DILI.

[0040] Therefore, given the current prevalence of APAP use and the limitations of DILI treatment, in-depth research and clarification of the pathogenesis of APAP hepatotoxicity and the search for new therapeutic targets have become important issues that need to be urgently addressed in DILI research.

[0041] The pathogenesis of DILI remains unclear, and treatment options are limited. Antioxidant, anti-inflammatory, and natural medicine interventions have been unsatisfactory. This study analyzed the regulatory mechanisms of fatty acid synthesis on macrophage innate immunity in DILI from an immunometabolism perspective. In vitro and in vivo experiments confirmed the involvement of increased macrophage fatty acid synthesis in the development of DILI. Furthermore, intervention with the weight loss drug orlistat effectively mitigated DPAP-induced liver damage and inflammation, providing a new therapeutic strategy for the clinical treatment of DILI.

[0042] This application involves ① establishing an ALI mouse model to analyze the expression of fatty acid synthase (FASN) in liver macrophages, lipid levels, infiltration of mononuclear macrophages, and expression of inflammatory factors; ② analyzing the effects of inhibiting FASN activity or specifically knocking out FASN on the inflammatory response and migration chemotaxis of macrophages in vitro; ③ establishing a myeloid cell-specific knockout Fsan mouse (Fasn △Mye ) ALI model, analyzing liver damage and inflammatory response; ④ Intervention with the FASN inhibitor orlistat in ALI model mice, analyzing liver damage, inflammation and survival, confirming for the first time the changes in fatty acid metabolism of liver macrophages in ALI, and revealing a new mechanism by which fatty acid synthesis regulates macrophage inflammation to mediate the occurrence of ALI. It was found that myeloid cell-specific knockout of FASN or orlistat intervention can significantly alleviate ALI symptoms, providing new treatment strategies for clinical practice.

[0043] In order to better understand the above technical solution, the above technical solution will be described in detail below with reference to the accompanying drawings and specific implementation methods.

[0044] Example 1

[0045] 1. It was found that fatty acid synthesis in liver macrophages increased when DILI occurred

[0046] 8-10 week old wild-type C57BL6 / J male mice were fasted for 12 hours and then intraperitoneally injected with APAP (300 mg / kg) to establish an ALI model. Mice were sacrificed 24 hours later, and livers and peripheral blood were collected.

[0047] in, Figure 1 Acetaminophen-induced acute liver injury (ALI) is a model of increased fatty acid synthesis in hepatic macrophages in mice. 8-10-week-old C57 / B6J male mice were intraperitoneally injected with APAP (300 mg / kg) to establish the ALI model. 24 hours after the mice were sacrificed, and serum and liver tissue were collected. AML12 mouse hepatocytes were stimulated with 5 mM APAP. After 24 hours of stimulation, the culture medium was discarded and serum-free medium was added for 12 hours. The culture supernatant (ACM) was collected after centrifugation.

[0048] Figure 1 A is mouse serum ALT / AST; Figure 1 B is H&E staining of mouse liver tissue; Figure 1 C: IBA1 labeled liver macrophages, Bodipy493 / 503 stained neutral lipids, and immunofluorescence analysis of the neutral lipid levels in mouse liver macrophages; Figure 1 D: RAW264.7 macrophages stimulated with ACM for 24 h, stained with Bodipy493 / 503, and the intracellular neutral lipid levels were observed under a confocal microscope; Figure 1 E: Liver macrophages of ALI model mice were obtained by collagenase perfusion, and the expression level of FASN mRNA in cells was analyzed by qPCR; Figure 1 FG: RAW264.7 macrophages were stimulated with ACM for 12 h, and the expression level of FASN in the cells was analyzed by qPCR and Western blotting.

[0049] like Figure 1 As shown in A, the serum ALT / AST levels of model mice were significantly increased. H&E staining results also showed that there were obvious necrotic areas in the liver of ALI model mice ( Figure 1 B) Indicates that the DILI model was successfully established. Bodipy493 / 503 staining of tissue neutral lipids and immunofluorescence observation of IBA1 in liver macrophages of DILI model mice + Lipid deposition in macrophages (e.g. Figure 1 C). APAP (5 mM) was further used to induce liver cell damage in AML12 mice, and the culture supernatant (ACM) was collected and used to stimulate RAW264.7 macrophages. Figure 1As shown in D, ACM stimulation can increase the level of intracellular neutral lipids. FASN is the rate-limiting enzyme in fatty acid synthesis. Collagenase perfusion analysis of liver macrophages in ALI model mice revealed a significant increase in Fasn mRNA levels in macrophages in model mice ( Figure 1 E). Consistent with this, in vitro ACM stimulation can also upregulate FASN expression in RAW264.7 cells ( Figure 1 F, G). These results suggest that FASN-mediated fatty acid synthesis in liver macrophages increases during DILI.

[0050] 2. Revealing that FASN-mediated fatty acid synthesis regulates liver macrophage inflammation and infiltration in ALI model mice

[0051] It is known that FASN-mediated fatty acid synthesis is upregulated in liver macrophages of ALI model mice, but its specific role needs further study.

[0052] Collagen perfusion was used to obtain liver macrophages from ALI model mice, among which, Figure 2 FASN-mediated fatty acid synthesis regulates hepatic macrophage inflammation and infiltration in the ALI mouse model.

[0053] Figure 2 A is qPCR analysis of the expression levels of inflammatory factors in liver macrophages of ALI model mice; Figure 2 B is the construction of myeloid conditional Fasn knockout mice (Fasn △Mye ), bone marrow-derived macrophages (BMDM) were isolated and stimulated with ACM for 12 h, and the expression levels of inflammatory factors in cells were analyzed by qPCR; Figure 2 C is the immunofluorescence analysis of the composition of mouse liver macrophages by IBA1 marking monocyte-derived macrophages and CLEC4F marking liver resident macrophages; Figure 2 D is a Transwell chamber experiment analyzing the effect of ACM stimulation on the migration ability of RAW264.7 macrophages; Figure 2 E: siRNA silenced FASN expression, and Transwell chamber assay was used to analyze the effect of ACM stimulation on the migration of RAW264.7 macrophages; Figure 2 F is the Transwell chamber experiment to analyze the effect of ACM on Fasn △Mye Effects on BMDM migration ability.

[0054] Collagen perfusion was used to obtain liver macrophages from ALI model mice. qPCR results showed that the mRNA levels of inflammatory factors TNFα and IL1β increased significantly ( Figure 2 A). To clarify the effect of fatty acid synthesis on the expression of inflammatory factors in macrophages, we constructed myeloid conditional knockout mice (Fasn △Mye) and isolated BMDM. qPCR analysis of inflammatory factor expression in BMDM after ACM stimulation revealed that Fasn △Mye The mRNA levels of TNFα and IL1β in mouse-derived BMDM were significantly reduced ( Figure 2 B) showed that increased fatty acid synthesis can upregulate the inflammatory response of liver macrophages in DILI model mice. When liver damage or inflammation occurs, a large number of peripheral monocyte-derived macrophages infiltrate the liver and participate in the inflammatory response. Figure 2 As shown in C, immunofluorescence results confirmed that a large amount of IBA1 was present in the liver of ALI model mice. + CLEC4F - Monocyte-derived macrophages. Transwell chamber experiments found that ACM stimulation can upregulate the migration and chemotactic ability of RAW264.7 macrophages ( Figure 2 D). Silencing FASN expression using siRNA significantly inhibited the migration and chemotaxis of RAW264.7 macrophages ( Figure 2 E). Consistent with this, we found that Fasn △Mye The chemotactic ability of BMDM was also significantly lower ( Figure 2 F) These results suggest that increased fatty acid synthesis upregulates the inflammatory response of hepatic macrophages and the infiltration of peripheral monocytes during ALI.

[0055] 3. Confirm that conditional myeloid knockout of FASN can alleviate liver damage and inflammation in ALI model mice

[0056] It has been previously demonstrated that FASN-mediated fatty acid synthesis can upregulate the expression of inflammatory factors in liver macrophages and promote the infiltration of peripheral mononuclear macrophages. △Mye ) Further analyze the effect of FASN expression in monocytes and macrophages on liver damage and inflammation in ALI model mice.

[0057] in, Figure 3 The effect of myeloid conditional knockout of Fasn on liver injury and inflammation in ALI model mice; 8-10 weeks old myeloid conditional knockout of Fasn (Fasn ΔMye ) C57 / B6J male mice were intraperitoneally injected with APAP (300 mg / kg) to establish an ALI model. The mice were killed 24 hours later, and serum and liver tissues were collected.

[0058] Figure 3 A is mouse serum ALT / AST; Figure 3 B is H&E staining of mouse liver tissue; Figure 3 C is TUNEL staining of frozen sections of liver tissue and immunofluorescence analysis of mouse liver cell apoptosis; Figure 3D is qPCR analysis of the mRNA expression levels of inflammatory factors IL-1β and TNF-α in liver tissue; Figure 3 E is immunofluorescence analysis of liver macrophage subsets, IBA1 + CLEC4F + Liver Kupffer cells, IBA1 + CLEC4F - monocyte-derived macrophages.

[0059] Compared with the control group, Fasn △Mye The serum ALT / AST levels of mice were significantly reduced, and liver tissue necrosis was significantly improved ( Figure 3 A, B) TUNEL staining of liver cell apoptosis revealed Fasn △Mye Apoptosis of mouse liver cells was significantly reduced ( Figure 3 C) qPCR results showed that Fasn △Mye The mRNA levels of inflammatory factors TNFα and IL-1β in the liver tissue of mice decreased significantly ( Figure 3 D) In ​​vitro results suggest that FASN expression regulates the migration and chemotaxis of mononuclear macrophages. Immunofluorescence analysis of liver macrophage subsets revealed that FASN expression was higher in the liver than in the control group. △Mye IBA1 infiltrates the mouse liver + The number of CLEC4F-monocyte-derived macrophages was significantly reduced. These results suggest that increased FASN expression upregulates the inflammatory response and migration and chemotactic ability of macrophages, promoting the occurrence of ALI.

[0060] 4. Orlistat intervention can alleviate liver damage and inflammation in ALI model mice

[0061] Orlistat is an approved clinical weight loss drug that acts on FASN to inhibit its activity and fatty acid synthesis. To further clarify the role of fatty acid synthesis mediated by macrophage FASN expression in ALI, we administered orlistat at a dose of 10 mg / kg to mice before model establishment.

[0062] in, Figure 4 Orlistat intervention can alleviate liver damage and inflammation in ALI model mice; C57 / B6J male mice were given 10 mg / kg orlistat by gavage, fasted for 12 hours, and injected intraperitoneally with APAP (300 mg / kg). They were killed 24 hours later, and serum and liver tissue were collected.

[0063] Figure 4 A is mouse serum ALT / AST; Figure 4 B is H&E staining of mouse liver tissue; Figure 4 C is TUNEL staining of frozen sections of liver tissue and immunofluorescence analysis of mouse liver cell apoptosis; Figure 4D is immunofluorescence analysis of liver macrophage subsets, IBA1 + CLEC4F + Liver Kupffer cells, IBA1 + CLEC4F - Monocyte-derived macrophages; Figure 4 E is qPCR analysis of the mRNA expression levels of inflammatory factors IL-1β and TNF-α in liver tissue; Figure 4 F was 600 mg / k and a survival curve was drawn to observe the survival status of mice. Figure 4 G: C57 / B6J male mice were given 10 mg / kg orlistat by gavage, fasted for 12 hours, and then injected intraperitoneally with APAP (300 mg / kg). The same dose of orlistat was given every 24 hours, and serum was collected 72 hours later for ALT / AST measurement.

[0064] Consistent with the results of FASN knockout, orlistat intervention significantly alleviated liver injury and necrosis in ALI model mice ( Figure 4 A, B). Immunofluorescence results showed that liver cell apoptosis in mice in the orlistat intervention group was significantly improved ( Figure 4 C), IBA1 + CLEC4F-monocyte-derived macrophages were significantly reduced ( Figure 4 D). qPCR results also showed that orlistat intervention could downregulate the mRNA levels of inflammatory factors TNFα and IL-1β in liver tissue ( Figure 4 E). A 600 mg / kg dose was administered to establish an acute liver failure model in mice. Survival curves were drawn and it was found that orlistat administration intervention significantly improved the survival of mice in the model group ( Figure 4 F) To analyze the potential effects of orlistat on liver function in mice, we administered orlistat continuously after model establishment. Analysis 72 hours later revealed no significant differences in serum ALT and AST levels compared to the control group.

[0065] These results suggest that orlistat, as a weight-loss drug, has new targets and efficacy, and can alleviate liver damage and inflammation in DILI mice.

[0066] In summary, our study revealed changes in fatty acid metabolism in liver macrophages during ALI, confirmed that increased FASN-mediated fatty acid synthesis regulates macrophage inflammatory responses and migratory chemotactic behavior, and demonstrated that targeting macrophage fatty acid synthesis can alleviate liver damage and inflammatory responses in ALI. This is the first discovery of a new target and new efficacy for the weight loss drug orlistat, which is expected to address the current treatment dilemma of DILI, which lacks specific drugs. This study has important scientific value and clinical significance.

[0067] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Use of a fatty acid synthase inhibitor in the preparation of a medicament for preventing and / or treating drug-induced liver injury, characterized in that: The fatty acid synthase inhibitor inhibits fatty acid synthesis by inhibiting fatty acid synthase activity, thereby preventing and / or treating drug-induced liver injury; the drug-induced liver injury is acetaminophen-induced drug-induced liver injury; The fatty acid synthase inhibitor includes orlistat or a pharmaceutically acceptable salt thereof.

2. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating drug-induced liver injury, characterized in that: The pharmaceutical composition comprises a fatty acid synthase inhibitor; the drug-induced liver injury is acetaminophen-induced drug-induced liver injury; The fatty acid synthase inhibitor includes orlistat or a pharmaceutically acceptable salt thereof.

3. The use according to claim 2, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. Use of a pharmaceutical preparation in the preparation of a drug for preventing and / or treating drug-induced liver injury, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 2 to 3; and the drug-induced liver injury is acetaminophen-induced drug-induced liver injury.

5. The use according to claim 4, characterized in that The pharmaceutical preparation includes a solid dosage form, a semisolid dosage form, a liquid dosage form or a gaseous dosage form.

6. The use according to claim 5, characterized in that The solid dosage forms include powders, pills, tablets, and capsules; the semisolid dosage forms include ointments, suppositories, and gels; the liquid dosage forms include lotions, mixtures, solutions, and injections; and the gaseous dosage forms include aerosols and sprays.

Images