CN114832109B

CN114832109B - Application of specific excited Mas receptor in preventing and treating acetaminophen induced drug-induced liver injury

Info

Publication number: CN114832109B
Application number: CN202210558974.6A
Authority: CN
Inventors: 杨长青; 李婧; 陈帅; 贾昊宇; 杨博
Original assignee: Shanghai Tongji Hospital
Current assignee: Shanghai Tongji Hospital
Priority date: 2022-05-21
Filing date: 2022-05-21
Publication date: 2023-04-18
Anticipated expiration: 2042-05-21
Also published as: CN114832109A

Abstract

The invention relates to an application of a specific excited Mas receptor in preventing and treating acetaminophen-induced drug-induced liver injury, which is realized by carrying out systemic knockout on a Mas1 gene (Mas 1) ^‑/‑ ) And wild type (Wildtype, WT) mice are injected with APAP in the abdominal cavity to construct a DILI in vivo research model, and the results show that the same dosage of APAP and Mas1 ^‑/‑ More severe hepatocyte damage and intrahepatic inflammatory responses were observed compared to WT mice; and the specific activation of Mas receptor before the APAP intervention of WT mice can obviously reduce the liver damage caused by APAP and reduce the death rate of mice. It was demonstrated that Mas receptor activation can induce lipid autophagy and fatty acid oxidation to exert protective effects on APAP-induced DILI. Based on the currently mastered in vivo research data, the Mas receptor can be used as an intervention target with great potential for drug-induced liver injury.

Description

Application of specific excited Mas receptor in preventing and treating acetaminophen induced drug-induced liver injury

Technical Field

The invention relates to the technical field of biology, in particular to application of specific agonism Mas receptor in prevention and treatment of acetaminophen-induced drug-induced liver injury.

Background

Drug-induced liver injury (DILI) is the most common adverse drug reaction and also an important obstacle that seriously affects the development and marketing of new drugs. APAP is one of the most widely used analgesic drugs in clinic and is the first pathogenic drug causing acute liver failure. Due to the availability of mouse models that are highly similar to human pathophysiology, APAP is currently the most widely studied hepatotoxic drug. The production of harmful metabolites by liver metabolism of excess APAP is the initiating link for APAP-induced liver damage, which can further induce mitochondrial damage through oxidative stress and ultimately hepatocyte death. In recent years, the role of lipid metabolism in DILI pathogenesis has received widespread attention.

After being activated, the Mas receptor can play multiple beneficial effects of improving lipid metabolism disorder, resisting inflammation, resisting oxidative stress and the like in liver tissues, so that the specific activation of the Mas receptor can play a role in protecting liver injury induced by APAP. We therefore by experiment: general knockout of Mas1 Gene (Mas 1) ^-/- ) And wild type (Wildtype, WT) mice are injected with APAP in the abdominal cavity to construct a DILI in vivo research model, and the result shows that the more serious hepatocyte injury and intrahepatic inflammatory reaction can be observed in the same dosage of APAP and Mas 1-/-compared with WT mice; and specifically activating a Mas receptor before performing APAP intervention on a WT mouse, for example, the small-molecule Mas agonist AVE0991 can obviously reduce liver injury caused by APAP and reduce the death rate of the mouse. Meanwhile, in the aspect of molecular mechanism discussion, the fact that Mas receptor activation can induce lipid autophagy and fatty acid oxidation to play a protective role on APAP-induced DILI is proved. Based on the currently mastered in vivo research data, the Mas receptor can be used as an intervention target with great potential for drug-induced liver injury. The application of the specific excited Mas receptor in preventing and treating acetaminophen induced drug-induced liver injury is not reported at present.

Disclosure of Invention

The invention aims to provide an application of a specific excited Mas receptor in preventing and treating acetaminophen-induced drug-induced liver injury, aiming at the defects of the existing drug intervention means.

In order to realize the purpose, the invention adopts the technical scheme that:

the invention provides an application of a Mas receptor stimulant in preparation of a medicine for preventing and/or treating liver injury.

Further, the invention provides an application of the Mas receptor agonist in preparing a medicament for preventing and/or treating drug-induced liver injury.

Further, the invention provides an application of the Mas receptor stimulant in preparing a medicine for preventing and/or treating acetaminophen-induced drug-induced liver injury.

Further, the invention provides an application of Mas receptor agonist AVE0991 in preparation of a medicine for preventing and/or treating acetaminophen-induced drug-induced liver injury.

Further, the invention provides an application of the Mas receptor agonist in preparing a medicament for enhancing lipid phagocytosis.

Further, the invention provides an application of the Mas receptor agonist in preparing a medicament for enhancing fatty acid oxidation.

Further, the invention provides an application of the Mas receptor agonist AVE0991 in preparation of a medicament for enhancing lipid phagocytosis.

Further, the invention provides an application of the Mas receptor agonist AVE0991 in preparation of a medicament for enhancing fatty acid oxidation.

The invention has the advantages that:

the medicine in China is various, chinese herbal medicines and health care products are commonly abused, and medical staff and the public are still insufficient in understanding of the safety problem of the medicine, the incidence rate of DILI in China is on the trend of increasing year by year in recent years, and at present, although a few medicines can be applied to DILI treatment, the curative effect is limited, the basic mechanism research and medicine innovation are urgently needed to be strengthened, and the prevention and treatment of DILI in China are further promoted. The invention focuses on the G protein-coupled receptor Mas coded by proto-oncogene Mas1, fully proves that activating the Mas receptor can obviously relieve liver injury in an APAP-induced mouse acute liver injury model in vivo, and antagonizing the Mas receptor can aggravate liver injury (firstly, on an APAP-induced mouse DILI model, the protective effect of Mas receptor activation is proved, namely the inflammatory reaction in liver cell injury and liver tissues is obviously relieved, and the fact that the protective effect is related to lipid autophagy activation and fatty acid beta oxidation enhancement is technically clarified, so that an important early data basis is provided for the research and development of new drugs based on specific activation of the Mas receptor), and is expected to provide an important animal experimental basis for the later development of new drugs aiming at the Mas receptor.

Drawings

FIGS. 1A-F show that the systemic knockout of Mas1 gene can significantly aggravate the acute liver injury induced by APAP.

FIGS. 2A-L show that specific activation of the Mas receptor significantly reduced APAP-induced acute liver injury by enhancing lipophagocytosis and fatty acid oxidation.

Detailed Description

The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the appended claims.

Example 1

1 laboratory animals and methods

1.1a. Transgenic mice, mouse disease model construction and experimental grouping: (1) adopted transgenic mouse Mas1 ^-/- The gene background of both (systemic Mas1 gene knockout) and WT mice was C57BL/6. (2) Disease models and experimental groups: APAP (300 mg/kg) is injected into the abdominal cavity to construct an acute drug-induced liver injury mouse model.

b. Experiment design: (1) collecting and separating serum samples, detecting ALT by ELISA, and evaluating the degree of liver injury; (2) preparing a paraffin section of the liver tissue, and carrying out HE staining and TUNEL staining to evaluate the damage degree of parenchymal cells in the liver tissue; MPO and F4/80 staining to assess the degree of intrahepatic inflammatory (neutrophil and macrophage) infiltration; (3) preparing a liver tissue frozen section, and respectively marking lipid droplets and lysosomes by BODIPY and LAMP1 staining to evaluate the autophagy degree of the lipids; (4) collecting a fresh liver tissue specimen, observing by a transmission electron microscope, calculating the number of autophagy vesicles, and evaluating autophagy activity; western blot detection of expression of autophagy-related protein (LC 3B, P), apoptosis (Bax, bcl2, cleared-caspase 3) and inflammation-related protein (IL-1 beta); sequencing detection of transcriptome, proteome and metabolome Mas1 ^-/- And differences in expression of mRNA, protein and metabolites in WT mouse disease models; (5) mas1 ^-/- And WT mice were given a lethal dose (650 mg/kg) of APAP, respectively, and survival curves were observed and recorded;

1.2a. Experimental grouping: (1) WT mice in the background of the C57BL/6 gene; (2) before an APAP is injected into an abdominal cavity to induce a mouse acute liver injury model, a small molecule Mas receptor specific agonist (AVE 0991) and a solvent control are respectively given.

b. Experiment design: (1) collecting and separating serum samples, detecting ALT by ELISA, and evaluating the degree of liver injury; (2) respectively preparing paraffin sections of liver tissues of mice of an intervention group and a control group, and performing HE staining and TUNEL staining to evaluate the degree of liver injury; MPO and F4/80 staining to evaluate the inflammatory reaction in the liver; (3) respectively preparing liver tissue frozen sections of mice of an intervention group and a control group, and staining by BODIPY and LAMP1 to evaluate lipid autophagy; (4) respectively collecting fresh liver tissues of mice in an intervention group and a control group, observing autophagy conditions by using a transmission electron microscope, and calculating the number of autophagy vesicles; western blot detection of expression of autophagy-related protein (LC 3B, P), apoptosis (Bax, bcl2, cleared-caspase 3) and inflammation-related protein (IL-1 beta); transcriptome sequencing, proteome sequencing and metabolome sequencing are used for detecting the expression difference of genes, proteins and metabolites before and after intervention of the Mas receptor specific agonist AVE 0991; (5) the intervention group and the control group are respectively given APAP with lethal dose (650 mg/kg) of intraperitoneal injection, and the survival curve is observed and recorded; (6) the liver inflammatory cell infiltration of the intervention group and the control group is observed by using DAOSLIMIT, and the degree of inflammation in the liver is evaluated.

2 results of the experiment

2.1 the results of the experiment can be seen in FIGS. 1A-F.

FIGS. 1A-F: systemic Mas1 knockdown exacerbates APAP-induced liver injury in mice.

(A, B, D-F) WT and Mas1 ^-/- Mice were given intraperitoneal APAP and biological samples were collected for testing 24 hours later (n =6 per group). Representative pictures of immunohistochemical staining of liver tissues: HE (necrotic area of hepatocytes is circled with white dotted line), TUNEL (apoptosis), F4/80 (macrophage), MPO (neutrophil). Arrows point to positive staining. Scale bar 100 μm. (B) serum ALT levels. (C) For WT and Mas1 ^-/- Mice were injected with a lethal dose of APAP (650 mg/kg, n =12 per group, log-rank test) and survival curves were plotted. (D) Representative liver tissue Western blot detection bands. (E) mouse liver tissue mRNA level: thermographic format showing expression of genes associated with apoptosis, necrosis, iron death and inflammatory pathways =(n =3 per group). (F) DAOSLIMIT measures the dynamic changes of neutrophils (Ly 6G, red) and macrophages (F4/80, green) inside and outside the blood vessels (WGA, blue) in the liver of live mice. Representative in vivo subcellular imaging images were taken at 3 and 22 hours post-APAP dosing and the results showed mean migration rates of neutrophils, as well as a trace of neutrophil numbers over time within 3-4 hours post-dose.

2.2 the results of the experiment can be seen in FIGS. 2A-L.

FIGS. 2A-L: in vivo activation of the mouse Mas receptor induces lipophagocytosis and fatty acid oxidation, thereby reducing the extent of APAP-induced liver damage.

(a-L) an APAP-induced acute liver injury model was constructed based on WT mice, and Mas receptor-specific agonist (AVE 0991) and solvent control were pre-administered in vivo before modeling, and biological samples were collected 24 hours later for testing (n =6 per group). (A) representative picture of immunohistochemical staining of liver tissue: HE (necrotic area of hepatocytes is encircled with white dotted line), TUNEL (apoptosis), F4/80 (macrophage), MPO (neutrophil). Arrows indicate positive staining. Scale bar 100 μm. (B) serum ALT levels. (C) WT mice were previously given AVE0991 and solvent control in vivo, followed by intraperitoneal injection of a lethal dose of APAP (650 mg/kg) and a survival curve was plotted (n =12 per group). (D) Representative liver tissue Western blot detection bands. (E) DAOSLIMIT observed representative images of the number of neutrophils and the average migration velocity in the liver of mice. (F) level of autophagy in mouse liver tissue: representative electron micrographs (left) and statistical analysis (right) (black arrows point to autophagosomes). Scale bar 2 μm. (G) level of lipophagy in liver tissue of mice: representative confocal photographs (top panel) of BODIPY (red, labeled lipid droplets)/LAMP 1 (green, labeled lysosomes) and quantification of co-localization (bottom panel). (H) Representative liver tissue Western blot detection bands. (I) The heat map shows mRNA levels of genes associated with pathways such as fatty acid degradation, autophagy, and lipid droplets in liver tissue of mice. (J) Spearman correlation analysis was used to determine correlations between metabolomics (fatty acids) and transcriptomics (fatty acid degradation and autophagy pathway related genes) data and to perform clustering analysis. The heatmap shows the expression levels of the target type of fatty acid. (K) hepatic tissue TG concentration. (L) mRNA level of a specific gene in liver tissue.

3 conclusion of the experiment

3.1 knockout of Mas receptor significantly aggravates the severity of APAP-induced acute liver injury, and significant inhibition of lipophagocytosis and fatty acid beta-oxidation can be observed in vivo.

3.2 activation of Mas receptor significantly reduced the severity of APAP-induced acute liver injury, with a significant upregulation of both lipophagocytosis and fatty acid beta-oxidation observed in vivo.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and additions can be made without departing from the principle of the present invention, and these should also be considered as the protection scope of the present invention.

Claims

Application of Mas receptor agonist AVE0991 in preparation of drugs for preventing and/or treating acetaminophen-induced drug-induced liver injury.