CN113069546A

CN113069546A - Macrophage-targeted RIPK1 and application of inhibitor thereof in screening and preparing liver injury diagnosis and treatment medicines

Info

Publication number: CN113069546A
Application number: CN202010009681.3A
Authority: CN
Inventors: 翁丹; 义玉国; 陶亮
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2020-01-06
Filing date: 2020-01-06
Publication date: 2021-07-06

Abstract

The invention discloses macrophage-targeting RIPK1 and application of an inhibitor thereof in screening and preparing a liver injury diagnosis and treatment medicine. According to the invention, the bone marrow of the mouse transplanted with RIPK1 kinase-inactivated bone marrow is transplanted into a normal C57 mouse, a non-alcoholic fatty liver, liver cirrhosis and liver fibrosis model is constructed, the result shows that the liver function of the mouse transplanted with RIPK1 kinase-inactivated bone marrow is obviously superior to that of the mouse transplanted with C57 mouse bone marrow, the contents of glutamic-pyruvic transaminase and triglyceride in serum are obviously reduced, and the pathological staining result of lipid components and the like show that the mouse transplanted with RIPK1 kinase-inactivated bone marrow can obviously relieve fatty liver pathological changes and obviously reduce lipid accumulation, and the inactivation of RIPK1 kinase can obviously reduce the inflammation and death of bone marrow macrophages induced by palmitic acid. The macrophage-targeted RIPK1 kinase active site can be used as a drug target for screening and treating liver injury diseases such as fatty liver, liver cirrhosis, hepatic fibrosis and the like, and the inhibitor can be used for preparing drugs for treating the liver injury diseases.

Description

Macrophage-targeted RIPK1 and application of inhibitor thereof in screening and preparing liver injury diagnosis and treatment medicines

Technical Field

The invention belongs to the technical field of gene function and application, and relates to application of a receptor-interacting serine/threonine kinase 1 (RIPK 1) and an inhibitor thereof in screening and preparing a liver injury diagnosis and treatment medicine.

Background

NAFLD (non alcoholic fatty liver disease) is a clinical pathological syndrome characterized by degeneration and accumulation of fat in parenchymal liver cells without excessive drinking history, and the disease spectrum shows non-uniform according to the progress of the disease process, and comprises simple fatty liver, non-alcoholic steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma. NAFLD is closely associated with obesity, metabolic syndrome and type 2 diabetes and is considered to be a major cause of end-stage liver failure.

Many patients with NAFLD develop a more inflammatory subtype-nonalcoholic steatohepatitis (NASH) and advanced liver fibrosis or cirrhosis. The pathophysiology of NASH is multifactorial and is not fully understood. However, liver-resident macrophages (kupffer cells) and the recruitment of macrophages play a major role in the progression of liver disease.

Resident Kupffer cells (Kupffer cells) and newly recruited monocyte-derived macrophages play a key role in the regulation of inflammation, fibrogenesis and fibrolysis at different stages of liver disease. After liver injury, kupffer cells recruit more immune cells, including neutrophils and inflammatory blood mononuclear cells, which differentiate into CD11b with phagocytic activity and secretion of pro-inflammatory cytokines and Reactive Oxygen Species (ROS)⁺F4/80⁺Classically activated macrophages (type M1) (Tacke and Zimmermann, 2014).

In disease models, hepatophagemid cell infiltration has been demonstrated in mouse diet models, such as High Fat Diet (HFD) and methionine-choline deficient diet (MCD), and is also associated with mouse age-growthAnd off. In the High Fat and High Cholesterol (HFHC) dietary NASH model, which replicates many of the pathophysiological characteristics of human NASH, F4/80 was observed⁺Infiltration of macrophages and activation of Kupffer cells and increased expression of proinflammatory cytokines (Tacke, 2017).

In addition to NAFLD and NASH, macrophages also play an important role in the development of hepatic immune homeostasis and other liver diseases, such as chronic viral hepatitis and alcoholic liver disease.

Receptor-interacting protein 1 (RIP 1, also known as RIPK1) is a key regulatory molecule for cell death, plays an important role in regulating the processes of cell survival, cell death, inflammatory reaction and the like, and is an important sensor molecule for determining cell fate. In 1995, stanger et al discovered through yeast two-hybrid experiments that the first member of RIP family, RIPK1, whose C-terminus is a death domain, was able to interact with Fas, a member of the death receptor family, and was thus named receptor interacting protein. RIPK1 plays an essential role in the normal development of the body. The study finds that mice with the RIPK1 gene knocked out die 1-3 d after birth due to excessive apoptosis of lymphocytes and adipose tissues (Xu et al, 2018, Liu et al, 2017, Kaiser et al, 2014). In one aspect, RIPK1 is an important component of the TNF signaling pathway in which RIPK1 mediates NF- κ B activation and cell survival by ubiquitination, recruiting as a scaffold protein and binding to downstream signaling molecules (Kondylis et al, 2017, Dondelinger et al, 2015). On the other hand, when RIPK1 is de-ubiquinated, the cell apoptosis is mediated by the combination of caspase-8 and FADD protein; however, when caspase-8 is inhibited by chemical inhibitors or genetically deleted, RIPK1 promotes the cells to perform programmed necrosis (necrosis) by interacting with RIP3 (Newton et al, 2019, Dillon et al, 2014, Wang et al, 2008). Thus, RIPK1 is at the critical intersection for determining cell fate. Factors such as the level of RIPK1 expression, protein modification status, and interactions with other protein molecules determine the choice of cell fate, whether to continue survival or go to death (Newton,2015, christafferson et al, 2014, opengeim and Yuan, 2013).

The RIPK1 kinase, as a key molecule determining the trend of cell fate, is involved in regulating multiple important signal pathways and cellular effects including NF- κ B signaling pathway, apoptosis and necrosis, activation of inflammasome and apoptosis of cells, and thus is likely to be involved in the occurrence and development of various diseases, especially diseases closely related to cell death and inflammatory response. Some small molecule inhibitors have been developed against RIPK1 kinase, including Nec-1, Nec-1s, GSK547 and GSK963, among others. Studies have demonstrated that the inhibition of RIPK1 kinase activity by Nec-1 can reduce cell death and inflammation, and plays an important role in some inflammatory and necrotic diseases (Takahashi N et al 2012), but no relevant studies have been found in nonalcoholic fatty liver, liver fibrosis and liver cirrhosis diseases.

Disclosure of Invention

The invention aims to provide macrophage-targeted RIPK1 and application of an inhibitor thereof in screening and preparing a liver injury diagnosis and treatment medicine.

In one aspect, the invention provides the use of macrophage-targeting RIPK1 as a drug target in screening for a medicament for preventing, ameliorating and/or treating a liver injury disease.

In another aspect, the invention provides the use of a macrophage-targeting RIPK1 inhibitor in the manufacture of a medicament for the treatment of liver injury.

Specifically, in the present invention, the liver injury diseases are known to those skilled in the art, and include, but are not limited to, fatty liver, liver cirrhosis, liver fibrosis, and the like.

Specifically, in the invention, the macrophage-targeting RIPK1 inhibitor comprises but is not limited to a small molecule compound inhibitor capable of inactivating RIPK1 kinase, or siRNA capable of inhibiting RIPK1 gene expression and/or RIPK1 kinase inactivation.

More specifically, the siRNA is double-chain siRNA which takes RIPK1 as a target gene and interferes RIPK1 expression and kinase inactivation, and the siRNA is injected into a human body to inactivate RIPK1 kinase by an RNA interference method to treat liver injury diseases such as fatty liver, liver cirrhosis, hepatic fibrosis and the like.

More specifically, the small molecule compound inhibitor takes RIPK1 as a target point, can specifically inhibit the activation of RIPK1 kinase, and realizes the treatment of liver injury diseases such as fatty liver, liver cirrhosis and hepatic fibrosis. Including but not limited to, Nec-1s, GSK547, and GSK963, etc.

The inventor finds that the liver function of the mouse transplanted with RIPK1 kinase-inactivated bone marrow is obviously superior to that of the mouse transplanted with C57 mouse bone marrow, the content of glutamic-pyruvic transaminase (ALT) in serum is obviously reduced, and the pathological staining result of lipid components and the like all indicate that the mouse transplanted with RIPK1 kinase-inactivated bone marrow can obviously relieve fatty liver pathological changes and obviously reduce lipid accumulation. In vitro experiments primary bone marrow macrophages from RIPK1 kinase inactivated mice and C57 mice were isolated and cultured, and it was found that RIPK1 kinase inactivation significantly reduced palmitic acid-induced inflammation and death of bone marrow macrophages.

Drawings

FIG. 1 is a graph showing the levels of alanine Aminotransferase (ALT) and Triglyceride (TG) in the serum of mice, wherein A is the level of alanine Aminotransferase (ALT) in the serum; b is the content of Triglyceride (TG) in serum.

FIG. 2 is a graph of liver tissue Hematoxylin and Eosin (HE) staining of mice, A is liver tissue staining of transplanted C57 mice and RIPK1 kinase knock-out mice in MCD diet-induced model; b is liver tissue staining of bone marrow transplanted mice in MCD diet induced model.

FIG. 3 is a sirius staining pattern of liver tissues of mice, A is the liver tissue staining of transplanted C57 mice and RIPK1 kinase knockout mice in MCD diet-induced model; b is liver tissue staining of bone marrow transplanted mice in MCD diet induced model.

FIG. 4 is a graph of the results of inflammation and death of primary bone marrow macrophages induced by Palmitic Acid (PA) in mice, A is the expression of palmitic acid-induced bone marrow macrophage inflammatory factor IL-1 β; b is palmitic acid induced bone marrow macrophage mortality assay.

Detailed Description

The invention will be further described in detail below by way of examples and figures.

Example 1

The invention takes a C57 wild-type mouse and an RIPK1 gene knockout (kinase inactivation) mouse as experimental objects, constructs fatty liver, fatty hepatitis, hepatic fibrosis and liver cirrhosis models by feeding High Fat Diet (HFD) or methionine-choline deficiency feed (MCD), and researches the functions of macrophage and RIPK1 gene in relevant inflammatory diseases such as fatty hepatitis and the like by bone marrow transplantation.

The phosphorylation sites of RIPK1 genes are knocked out by using CRISPR Cas9 technology, and RIPK1 kinase inactivated mice are constructed. Normal C57 mice were transplanted with RIPK1 kinase inactivated mouse bone marrow and fed High Fat Diet (HFD) or methionine choline deficient diet (MCD) to construct non-alcoholic fatty liver, cirrhosis and liver fiber models.

1. Construction of bone marrow transplantation mice

8-week old wild-type C57BL/6 mice were lethally irradiated (9Gy), bone marrow from C57 and RIPK1 kinase-inactivated mice, respectively, was taken, and bone marrow cells harvested from wild-type or RIPK1 kinase-inactivated donor mice were injected via tail vein injection into irradiated C57 recipient mice (5X 10 mice)⁶Individual cells). Blood from chimeric mice was obtained 6 weeks after irradiation and Bone Marrow Transplantation (BMT) and analyzed by DNA sequencing to verify the efficiency of bone marrow reconstitution.

2. Construction of non-alcoholic fatty liver model

Non-alcoholic fatty liver models were constructed by feeding 8-week-old mice with a high-fat diet for 16 weeks or a methionine-choline deficient diet for 4 weeks.

3. Detection method

(3.1) hematoxylin and eosin staining

(1) Liver tissue section

After the mouse is killed, a small liver tissue is taken and put into 4% paraformaldehyde solution for fixation for 24 hours, the liver tissue is taken out and embedded by paraffin, a single slice is cut out by using a full-automatic slicer and is placed on a glass slide, an alcohol lamp is used for slightly baking to flatten the slice, the pasted slice is placed in a 60-degree oven for drying for 2 hours, and the slice is stored.

(2) Paraffin section dewaxing to water

Placing the slices in xylene I10 min-xylene II 10 min-absolute ethanol I5 min-absolute ethanol II 5 min-95% ethanol 5 min-90% ethanol 5 min-80% ethanol 5 min-70% ethanol 5 min-distilled water washing.

(3) Hematoxylin staining of cell nucleus

Slicing into Harris hematoxylin, staining for 3-8min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water.

(4) Eosin staining of cytoplasm

The sections were stained in eosin stain for 1-3 min.

(5) Dehydration seal

Placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum. Microscopic examination and image acquisition and analysis.

(3.2) enzyme-linked immunosorbent assay (ELISA)

(1) Taking out a new ELISA96 pore plate, adding IL-1 beta primary antibody into buffer solution according to a certain proportion, mixing uniformly, adding 50 mu L of each pore, sticking a film, shaking the plate to make the liquid spread over the pores, and putting the plate into a refrigerator at 4 ℃ for overnight incubation.

(2) The primary antibody was removed, decanted, and washed 3 times with PBST (250. mu.L). ELISA dilutions (20mL water +5mL dilutions (5XELISA/ELISPT DILUERT)) were prepared at 100. mu.L per well and the patch was left to stand for 1 h.

(3) The ELISA dilution was decanted, PBST washed 1 time, and the cell suspension added to a fresh plate (50. mu.L per well)

(4) Preparing a standard sample: add 140. mu.L of LELISA dilution and 10. mu.L of IL-1. beta. (protein) to a 1.5mL centrifuge tube and mix well. Adding 50 mu L of ELISA diluent into the 2 nd to 8 th holes, adding 50 mu L of protein diluent into the first hole and the second hole, uniformly stirring the second hole for 10 times, sucking 50 mu L of protein diluent into the third hole, repeating the steps, and finally sucking 50 mu L of protein diluent out of the eighth hole and discarding. Pasting the film and the like for 2 h.

(5) Poured off and washed 3 times with PBST.

(6) The secondary antibody was diluted, added to the ELISA diluent and diluted in proportion, 50 μ L per well, filmed, etc. for 1 h.

(7) Poured off and washed 3 times with PBST.

(8) Adding horseradish peroxidase (50 μ L per well), sticking membrane, and standing at room temperature for 30 min.

(9) Poured off, washed 5 times with PBST, and 50. mu.L of color reagent (1X TMB ELISA Substrate solution) was added to each well and allowed to stand at room temperature for 15 min.

(10) Add 1M phosphoric acid (stop solution) 25. mu.L per well, break the bubble with a needle, read 450nm with microplate reader.

(3.3) cell death assay

According to the size and growth speed of the cells, a proper amount of cells are inoculated into a 96-well cell culture pore plate, so that the cell density to be detected is not more than 80-90% full. Different drugs were added for treatment and appropriate controls were set. After drug stimulation was complete, the cell culture plates were centrifuged for 5min at 400g in a multi-well plate centrifuge. The supernatant was aspirated as much as possible, 150. mu.l of LDH release reagent provided in a kit diluted 10-fold with PBS (1 volume of LDH release reagent was added to 10 volumes of PBS and mixed well), mixed well by shaking the plates appropriately, and then incubated for 1 hour in a cell incubator. The cell culture plates were then centrifuged for 5min at 400g in a multi-well plate centrifuge. 120. mu.l of the supernatant from each well was added to the corresponding well of a new 96-well plate, and then the sample assay was performed.

a. 60 μ l of LDH detection working solution was added to each well.

b. Mixing, and incubating at room temperature (about 25 deg.C) in dark for 30min (wrapping with aluminum foil, and slowly shaking on horizontal shaking table or side shaking table). The absorbance was then measured at 490 nm. The two-wavelength measurement is performed using either 600nm or a wavelength greater than 600nm as a reference wavelength.

c. Calculation (absorbance measured for each group should be subtracted from background blank wells).

d. Cytotoxicity or mortality (%) × (treated sample absorbance-sample control well absorbance)/(absorbance for maximum enzyme activity of cells-sample control well absorbance) × 100.

e. Cytotoxicity curves can be plotted: the ordinate is actual absorbance, and the abscissa is drug concentration; from this, the semi-lethal dose LD50 of the drug for a specific time can be calculated.

4. The results show that

The invention uses RIPK1^K45AGene knockout miceAnd wild type C57 mouse transplantation RIPK1^K45ABone marrow of the mouse is knocked out to construct a non-alcoholic fatty liver and liver fibrosis model. The results show that the inactivation of RIPK1 kinase can obviously improve the liver function of mice and reduce the fatty liver level, and the levels of glutamic-pyruvic transaminase and triglyceride in serum are obviously reduced (as shown in figure 1).

A fatty liver and hepatic fibrosis model is successfully constructed by feeding a mouse with MCD feed, and the liver tissue of the mouse is taken to carry out HE staining and sirius staining to observe the fatty liver and fibrosis degree of the liver of the mouse. The results show that RIPK1^K45AKnockout mice and transplantation of RIPK1^K45AThe wild type C57 mouse with gene knockout mouse bone marrow has obviously reduced degree of fatty liver and liver fibrosis compared with normal mouse, wherein the improvement effect of bone marrow transplantation mouse on fatty liver and fibrosis is better than that of the general RIPK1 knockout mouse (as shown in figure 2 and figure 3), therefore, the inhibitor can be developed aiming at the RIPK1 kinase activity in monocyte/macrophage to treat liver injury diseases such as fatty liver, liver cirrhosis, liver fibrosis and the like.

Respectively extracting C57 mouse and RIPK1^K45ABone marrow of knockout mice was induced to differentiate into bone marrow macrophages in vitro, treated with palmitic acid (PA300, PA400) for 24 hours, and examined for cell death and inflammation. RIPK1 was found^K45AThe knockout bone marrow macrophages were able to significantly reduce palmitic acid-induced death and inflammation (as shown in figure 4).

Claims

1. The application of macrophage-targeted RIPK1 as a drug target in screening drugs for preventing, relieving and/or treating liver injury diseases.

2. Application of macrophage-targeted RIPK1 inhibitor in preparation of liver injury treatment medicines.

3. The use according to claim 1 or 2, wherein the liver injury disease is fatty liver, liver cirrhosis or liver fibrosis.

4. The use of claim 2, wherein the macrophage-targeting RIPK1 inhibitor is a small molecule compound inhibitor that inactivates RIPK1 kinase, or an siRNA that inhibits RIPK1 gene expression and/or RIPK1 kinase inactivation.

5. The use of claim 4, wherein the siRNA is a double-stranded siRNA targeting RIPK1 and interfering with RIPK1 expression and kinase inactivation.

6. The use of claim 4, wherein the small molecule compound inhibitor is a small molecule compound that targets RIPK1 and specifically inhibits the activation of RIPK1 kinase.

7. The use of claim 6, wherein the small molecule compound inhibitor is selected from the group consisting of Nec-1, Nec-1s, GSK547 and GSK 963.