CN111920953A

CN111920953A - Application of SAA inhibitor in preparing medicine for treating acute liver injury, acute liver failure or chronic acute liver failure

Info

Publication number: CN111920953A
Application number: CN202010772541.1A
Authority: CN
Inventors: 李尹雄; 游凯
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2020-08-04
Filing date: 2020-08-04
Publication date: 2020-11-13
Also published as: WO2022027793A1

Abstract

The invention relates to an application of a serum amyloid A inhibitor in preparing a medicament for treating acute liver injury, acute liver failure or chronic acute liver failure. Blocking SAA1/2 with serum amyloid A inhibitor can significantly reduce APAP induced liver injury degree, enhance repair process after injury, and reduce level of alanine Aminotransferase (ALT) and/or aspartate Aminotransferase (AST) in blood; inhibition of the effects of SAA1/2 reduces APAP-induced intrahepatic bleeding and platelet aggregation in the liver. Therefore, the serum amyloid A inhibitor can be applied to the preparation of medicines for treating acute liver injury, acute liver failure or chronic acute liver failure, a new strategy is provided for the treatment of acute liver injury, acute liver failure or chronic acute liver failure, the strategy can make up for the defects of short window period, large side effect, donor shortage and immune rejection in a liver transplantation method and the like of an NAC treatment method in the prior art to a certain extent, and the serum amyloid A inhibitor has the characteristics of low immunogenicity, low cost and the like.

Description

Application of SAA inhibitor in preparing medicine for treating acute liver injury, acute liver failure or chronic acute liver failure

Technical Field

The invention belongs to the technical field of biological medicines, relates to a new strategy for treating acute liver injury, acute liver failure or chronic acute liver failure, and particularly relates to application of a serum amyloid A inhibitor in preparation of a medicine for treating acute liver injury, acute liver failure or chronic acute liver failure.

Background

Acute liver failure is the terminal stage of serious liver damage caused by various factors, liver cell death causes dysfunction such as liver synthesis, detoxification, excretion and biotransformation to be irrepensible, and serious liver disease syndromes such as blood coagulation dysfunction, jaundice, hepatic encephalopathy and ascites occur. Acute liver failure is mainly caused by factors such as hepatitis virus (mainly hepatitis B virus HBV), drugs (mainly acetaminophen) and liver toxic substances (such as alcohol, chemical preparations and food poisoning). Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, and is safe and effective when APAP is used within the dosage range specified in the drug. However, excessive use of APAP can lead to severe liver damage and is likely to rapidly progress to acute liver failure, threatening life. Currently, there are very limited treatment means and drugs for APAP-induced liver injury, and N-acetylcysteine (NAC) is approved by FDA for treating acute liver injury caused by APAP, but its use is severely limited due to its disadvantages of large side effects, short use window, and the like. In addition, the process of acute liver injury caused by APAP and the mechanism of its occurrence remain to be further elucidated.

Serum Amyloid protein a (SAA) an acute phase response protein including injury-inducing genes SAA1 and SAA2 (hereinafter collectively referred to as SAA1/2), and a homeostatic expression gene SAA 4. In the case of pathogen infection or trauma to the body, the serum levels of the SAA1/2 protein are greatly increased, and most of the SAA1/2 secreted into the blood binds to HDL and replaces apoA-I therein, with a small proportion being present in free form. According to current studies, SAA1/2 has functions of participating in cholesterol transport and metabolism, regulating inflammatory responses, and playing a role in recruiting immune cells. However, no report has been found on the role and molecular mechanism studies of SAA1/2 in APAP-induced liver injury.

Aiming at the condition that the existing medicine for effectively treating acute liver injury or liver failure generally lacks clinically, the method is very significant if a new method or treatment means can be provided for treating acute liver injury or liver failure.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a new strategy for treating acute liver injury, acute liver failure or chronic acute liver failure, and particularly provides application of a serum amyloid A inhibitor in preparation of a medicine for treating acute liver injury, acute liver failure or chronic acute liver failure.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides the use of a serum amyloid a inhibitor for the manufacture of a medicament for the treatment of acute liver injury, acute liver failure or chronic plus acute liver failure.

The serum amyloid a (SAA1/2) inhibitor according to the present invention refers to a substance that can lower the level of serum amyloid a in blood or inhibit the function of serum amyloid a.

The invention discovers that the expression level of SAA1/2 is obviously increased in the process of inducing liver injury of a mouse by APAP for the first time; in vitro experiments show that the SAA1/2 can enhance the damage effect of APAP on liver blood sinus endothelial cells and promote the adhesion capacity of platelets on the blood sinus endothelial cells. Since intrahepatic hemorrhage and enhanced coagulation response are one feature of APAP-induced liver injury, it is speculated that in the early stage of APAP-induced acute liver injury, SAA1/2 further promotes liver injury by destroying the integrity of hepatic sinus structure, leading to intrahepatic hemorrhage and platelet aggregation, suggesting that SAA1/2 can be a target for treating acute liver injury.

Through further experimental exploration, the following results are found: blocking SAA1/2 with serum amyloid A inhibitor can significantly reduce APAP induced liver injury degree, enhance repair process after injury, and reduce level of alanine Aminotransferase (ALT) and/or aspartate Aminotransferase (AST) in blood; inhibition of the effects of SAA1/2 reduces APAP-induced intrahepatic bleeding and platelet aggregation in the liver. Therefore, the serum amyloid A inhibitor can be applied to the preparation of medicines for treating acute liver injury, acute liver failure or chronic acute liver failure, a new strategy is provided for the treatment of acute liver injury, acute liver failure or chronic acute liver failure, the strategy can make up for the defects of short window period, large side effect, donor shortage and immune rejection in a liver transplantation method and the like of an NAC treatment method in the prior art to a certain extent, and the serum amyloid A inhibitor has the characteristics of low immunogenicity, low cost and the like.

Preferably, the serum amyloid a inhibitor comprises any one or a combination of at least two of an interfering RNA specifically targeting serum amyloid a mRNA sequence, a neutralizing antibody specifically targeting serum amyloid a, an antagonist peptide specifically targeting serum amyloid a, or a chemical molecule specifically targeting serum amyloid a.

The combination of at least two of the above-mentioned compounds, such as the combination of interfering RNA and neutralizing antibody, the combination of interfering RNA and antagonistic peptide, etc., can be selected in any combination manner, and thus, the detailed description thereof is omitted.

Preferably, the medicament reduces the level of glutamic-pyruvic transaminase and/or glutamic-oxalacetic transaminase in blood.

Preferably, the medicament reduces platelet aggregation at the site of liver injury.

In a second aspect, the present invention provides the use of a serum amyloid a inhibitor for the manufacture of a medicament for reducing the level of glutamic-pyruvic transaminase and/or glutamic-oxalacetic transaminase in blood.

In a third aspect, the present invention provides the use of a serum amyloid a inhibitor for the manufacture of a medicament for reducing platelet aggregation at a liver lesion.

In the application, the dosage form of the medicament comprises any one of suspension, granules, capsules, powder, tablets, emulsion, solutions, dripping pills, injection, suppositories, enemas, aerosols, patches or drops.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the auxiliary materials comprise any one or a combination of at least two of a carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrating agent, an emulsifier, a cosolvent, a solubilizer, an osmotic pressure regulator, a surfactant, a coating material, a coloring agent, a pH regulator, an antioxidant, a bacteriostatic agent or a buffering agent.

The combination of at least two of the above-mentioned components, such as the combination of diluent and excipient, the combination of binder and wetting agent, the combination of emulsifier and cosolvent, etc., can be selected in any combination manner, and will not be described in detail herein.

Compared with the prior art, the invention has the following beneficial effects:

Drawings

FIG. 1 is a graph showing the time-dependent change of the expression level of SAA1/2 detected by immunohistochemical staining of liver tissues after APAP-induced acute liver injury in mice;

FIG. 2 is a graph of SAA1/2mRNA levels in liver tissue over time following APAP-induced acute liver injury in mice;

FIG. 3 is a graph of SAA1/2 protein levels in liver tissue as a function of time following APAP-induced acute liver injury in mice;

FIG. 4 is a graph of the time course of the SAA1/2 protein level in serum after APAP-induced acute liver injury in mice;

FIG. 5 is a graph showing the time-dependent change of the expression level of SAA1/2 detected by immunohistochemical staining of liver tissues in a frozen-induced mouse acute liver injury model;

FIG. 6 is a graph of the time course of the SAA1/2 protein levels in liver tissue in a cryo-induced mouse model of acute liver injury;

FIG. 7 is a graph of the time course of the protein level of SAA1/2 in serum in a cryo-induced mouse model of acute liver injury;

FIG. 8 is a graph showing the results of flow cytometry to detect APAP-induced primary liver sinusoid endothelial cell death at different concentrations;

FIG. 9 is a graph showing the results of flow cytometry to detect death of primary liver sinus endothelial cells induced by co-stimulation with APAP and SAA 1/2;

FIG. 10 is a statistical plot of cell counts of dead cells induced in primary liver sinus endothelial cells by co-stimulation with APAP and SAA1/2 detected by flow cytometry;

FIG. 11 is a microscopic image of platelets after co-culturing and washing with sinus endothelial cells;

FIG. 12 is a graph showing the fluorescence intensity statistics of adherent platelets;

FIG. 13 is a schematic flow chart of the reduction of APAP-induced liver damage in example 4 using neutralizing antibodies specifically targeting serum amyloid A to block the function of SAA 1/2;

FIG. 14 is a graph of the change in concentration of SAA1/2 protein in liver tissue after different times following APAP-induced liver injury, injected with anti-SAA neutralizing antibody;

FIG. 15 is a graph of the change in the levels of the liver injury markers ALT and AST in serum after different times following APAP-induced liver injury, injected with anti-SAA neutralizing antibody;

FIG. 16 is a graph of statistical results of liver lesion area after different times following APAP-induced liver injury with anti-SAA neutralizing antibody injection;

FIG. 17 is a representative graph of HE staining of liver tissue after various times following APAP-induced liver injury by injection of anti-SAA neutralizing antibody;

FIG. 18 is a graph of mouse survival;

FIG. 19 is a graph of blood platelets in liver tissue at various times after damage was detected by immunohistochemical staining in example 4;

FIG. 20 is a statistical graph of the number of platelets in liver tissue at different times after detection of lesions by immunohistochemical staining in example 4;

FIG. 21 is a schematic flow chart of the reduction of APAP-induced liver damage in example 5 using antagonistic peptides specifically targeting serum amyloid A to block the function of SAA 1/2;

FIG. 22 is a graph of the change in the level of the liver injury marker ALT in serum after various times following APAP-induced liver injury, injected with antagonistic peptide;

FIG. 23 is a representative graph of HE staining of liver tissue at various times post-APAP-induced liver injury with injected antagonist peptide;

FIG. 24 is a graph of statistics of liver injury lesion area at various times after APAP-induced liver injury with injection of antagonistic peptides;

FIG. 25 is a graph of blood platelets in liver tissue at various times after the detection of injury by the immunohistochemical staining method of example 5;

FIG. 26 is a statistical chart of the number of platelets in liver tissue at different times after detection of lesions by the immunohistochemical staining method in example 5;

FIG. 27 is a schematic flow chart of the reduction of freeze-induced liver injury in example 6 using neutralizing antibodies specifically targeting serum amyloid A to block the function of SAA 1/2;

FIG. 28 is a graph of the change in the level of the liver injury marker ALT in serum after different times following freezing induced liver injury with injection of anti-SAA neutralizing antibody;

FIG. 29 is a graph of the appearance of liver after various times following freezing induced liver injury with anti-SAA neutralizing antibody injected;

FIG. 30 is a graph of the thickness change at the site of liver tissue damage after various times following freezing induced liver damage by injection of anti-SAA neutralizing antibody;

FIG. 31 is a graph of blood platelets in liver tissue at various times after the detection of injury by the immunohistochemical staining method of example 6;

FIG. 32 is a graph showing statistics of platelet counts in liver tissues at different times after damage was detected by the immunohistochemical staining method in example 6.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

The procedures, conditions, reagents, test methods and the like for carrying out the present invention are those generally known in the art and are not specifically limited except for the contents specifically mentioned below. The test methods in each example, in which the specific conditions are not specified, are generally carried out under the conventional conditions or under the conditions recommended by the manufacturer.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, but in the event of conflict, the present specification, including definitions, will control.

The test animals used in the examples described below were BALB/c mice (WM: 23-27 g) purchased from Experimental animals technology, Inc., Viton, Beijing.

Paracetamol (APAP) used in the following examples was purchased from sigma (Cat. A5000), manufactured in Batch No. 113K011081, and specified at 100 mg.

The neutralizing antibody specifically targeting serum amyloid A used in the following examples is specifically recombinant Anti-SAA1+ SAA2 antibody derived from rabbit monoclonal antibody (cat. ab199030) produced by Abcam corporation; the antagonistic peptide specifically targeting serum amyloid A is an artificially synthesized peptide segment, and the source of the antagonistic peptide is a customized peptide segment synthesized by Competition Biotechnology (Shanghai) corporation.

The primary sinusoidal liver endothelial cells used in the examples below were derived from primary cells isolated from BALB/c mice.

Ctrl in the figures of the specification and abstract of the present application represents the control group, h or hrs represents the unit hour, and d or days represents the unit day. i.v. for intravenous injection and i.p. for intraperitoneal injection. SAA, SAA1/2, Saa and Saa1/2 all represent serum amyloid A.

Example 1

This example explores the expression behavior of serum amyloid A (SAA1/2) in mice during acute liver injury, and operates as follows:

(1) an APAP-induced mouse acute liver injury model is constructed, and the specific method comprises the following steps: an APAP induced acute liver injury model can be established by adopting an 8-week-old BALB/c strain mouse, fasting for 6 hours before APAP medicine injection, and then injecting APAP into an abdominal cavity according to the dosage of 400 mg/kg. Subsequently, samples were harvested at 6 hours, day 1, day 3 and day 7 post-injury, including cardiac blood sampling, paraffin embedding and cryopreserving liver tissue for subsequent experiments.

(2) Detecting the expression level of SAA1/2 by a liver tissue immunohistochemical staining method, which comprises the following steps:

liver tissues were collected at various time points after APAP-induced liver injury in mice, fixed in paraformaldehyde solution for 24 hours, and subjected to dehydration, embedding and slicing experiments, with a slice thickness of 5 μm. Then, the tissue slices are baked in an oven at 55 ℃ for 30min, and then immediately placed in xylene for dewaxing twice for 10 min/time, and then sequentially placed in 100% ethanol-95% ethanol-80% ethanol-70% ethanol-ddH₂O, 3 min/time; then put in 0.3% H₂O₂Neutralizing for 10min, eliminating endogenous peroxidase activity, and adding ddH₂Washing in O for 3 times, 3 min/time. Then adopting citric acid buffer solution to carry out microwave heating method restoration on tissue antigen, naturally cooling to room temperature after restoration, then washing 3 times with 1 XPBS for 5 min/time, and dripping Anti-SAA1+ SAA on the tissue on the sliceAntibody 2 (1:1000 dilution, Cat. ab199030), placed in a wet box overnight at 4 ℃. The next day, the wet box was taken out, equilibrated at room temperature for 30min, primary antibody was aspirated, washed 3 times with 1 × PBS for 5 min/time, then secondary antibody with horseradish peroxidase (HRP) was added dropwise, and incubated at 37 ℃ for 30min in the dark. Then, the secondary antibody was aspirated, washed 3 times with 1 XPBS for 5 min/time, and subjected to color development by dropping DAB (DAKO), followed by color development in ddH₂Washing in O for 3 times, 3 min/time. Finally, cell nuclei are counterstained with hematoxylin for 2min, washed with 1 × PBS for 1 time and 3min, rinsed with tap water for 1 time and 3min, air-dried, sealed with neutral quick-drying glue, observed under a microscope and photographed. The results are shown in FIG. 1 (75 μm on the 20X scale and 25 μm on the 40X scale), and it can be seen from FIG. 1 that: during APAP-induced liver injury, the protein expression level of SAA in liver tissues is significantly up-regulated and reaches a peak 24 hours after injury;

detecting the change level of SAA1/2mRNA in liver tissue, which comprises the following steps: collecting liver tissue at different time points after APAP induced liver injury to extract RNA, performing qRT-PCR, and detecting the mRNA level of SAA1/2 by using primers: the forward primer is 5'-GCGAGCCTACACTGACATGA-3'; the reverse primer was 5'-TGCCGAAGAATTCCT GAAAG-3', and the results are shown in FIG. 2, which shows that: during APAP-induced liver injury, the mRNA level of SAA in liver tissue was significantly up-regulated and peaked 24 hours after injury and then gradually decreased;

detecting the change level of the SAA1/2 protein in the liver tissue, wherein the specific method comprises the following steps: liver tissues were collected at different time points after APAP-induced liver injury, and histones were extracted by a tissue homogenization method to perform Western blot experiments to detect the protein expression level of SAA1/2, wherein the primary antibody used was Anti-SAA1+ SAA2 antibody (1:1000 dilution, Cat. ab199030), and the results are shown in FIG. 3, and it can be seen from FIG. 3: during APAP-induced liver injury, the protein expression level of SAA in liver tissues is significantly up-regulated and reaches a peak 24 hours after injury;

detecting the change level of the SAA1/2 protein in serum, wherein the specific method comprises the following steps: mouse sera were collected at various time points after the APAP-induced liver injury, and then the serum concentration of SAA1/2 was measured using SAA ELISA kit (Cat. EK1190, Strobile Biotech, Inc., Wuhan doctor), the results of which are shown in FIG. 4 and are shown in FIG. 4: during APAP-induced liver injury, protein concentrations of SAA in serum increased significantly and peaked 24 hours after injury and then gradually decreased to normal levels.

The above tests all used normal mice as control group.

(3) A freezing induced mouse acute liver injury model is constructed, and the specific method comprises the following steps: adopting a BALB/c strain mouse of 8 weeks old, preparing anesthetics, surgical instruments, liquid nitrogen and a conical metal rod; firstly, injecting Tribromoethanol (TBE) into abdominal cavity according to the weight of the mouse (200 mg/kg), cutting a small opening with the length of about 1cm along the abdominal midline below the xiphoid process of the mouse after the mouse is completely anesthetized, pulling the left lobe of the liver out of the body as much as possible, precooling the metal bar with liquid nitrogen, frostbite the whole left lobe with the metal bar, putting the left lobe of the liver back into the body, and suturing the incision. Subsequently, samples were harvested on

days

1, 3 and 7 post-injury, including cardiac blood sampling, paraffin embedding and cryopreserving liver tissue for subsequent experiments.

(4) The expression level of SAA1/2 was detected by immunohistochemical staining of liver tissues, as described above, and the results are shown in FIG. 5 (scale: 50 μm), and it can be seen from FIG. 5 that: during freeze-induced liver injury, immunohistochemistry showed significant upregulation of SAA protein levels in liver tissue, and reached a peak at 12-24 hours, followed by a gradual decrease;

the level of change in SAA1/2 protein in liver tissue was determined by the same method as above, and the results are shown in fig. 6, and it can be seen from fig. 6 that: SAA protein levels in liver tissue were significantly upregulated during freeze-induced liver injury and peaked between 12-24 hours and then gradually decreased;

the change level of SAA1/2 protein in serum was detected by the same method, and the results are shown in FIG. 7, and it can be seen from FIG. 7 that: during freeze-induced liver injury, SAA protein concentration in serum increased significantly and peaked at 24 hours and then gradually decreased.

The above tests all used normal mice as control group.

Example 2

This example explores the effect of serum amyloid A (SAA1/2) in enhancing APAP damage to liver sinus endothelial cells by:

(1) the flow cytometry was used to detect the death of primary liver sinus endothelial cells induced by APAP at concentrations of 10, 20 and 30mM, respectively, according to the instructions of FITC Annexin V Apoptosis Detection Kit I (cat.556547) of BD, and the results are shown in fig. 8, which shows that: after the liver blood sinus endothelial cells are stimulated by APAP with different concentrations for 6 hours, the death of the liver blood sinus endothelial cells can be obviously induced, and the concentration dependence is realized, and DMSO is used as a control.

(2) The flow cytometry was used to detect the death of primary liver sinus endothelial cells induced by APAP at 10mM in combination with SAA1/2 (at 3. mu.g/mL), according to the FITC Annexin V Apoptosis Detection Kit I (Cat.556547) from BD company, as shown in FIG. 9, and as shown in FIG. 9: SAA can obviously promote APAP-induced liver blood sinus endothelial cell death, and DMSO, APAP alone and SAA1/2 alone are used as controls; the number of dead cells was counted and the proportion of dead cells was calculated, and the results are shown in FIG. 10.

Example 3

This example explores the ability of serum amyloid A (SAA1/2) to promote platelet adhesion to sinus endothelial cells, as follows:

separating primary blood sinus endothelial cells, culturing the primary blood sinus endothelial cells in vitro for 48 to 72 hours until the cell confluence reaches 100%, stimulating the blood sinus endothelial cells by using DMSO, APAP, SAA1/2 and APAP + SAA1/2 for 2 hours, co-culturing the separated blood platelets (marked by Di1 red dye) and the blood sinus endothelial cells for 2 hours, washing the non-adhered blood platelets by using PBS, and photographing the blood platelets as shown in figure 11 (the scale in the figure is 50 microns); as shown in fig. 12, the fluorescence intensity of the adhered platelets was counted, and as can be seen from fig. 12: SAA1/2 stimulation alone was able to promote platelet adhesion to sinus endothelial cells.

Example 4

This example reduces APAP-induced liver damage using neutralizing antibodies specifically targeting serum amyloid a to block SAA1/2 function as follows:

(1) in an APAP-induced mouse acute liver injury model (see example 1), 1 hour before APAP injection into abdominal cavity, neutralizing antibody anti-SAA (3 μ g/mouse) and control antibody IgG (3 μ g/mouse) of SAA1/2 are injected intravenously through a mouse tail, APAP (400mg/kg) is given 1 hour after the injection to induce acute liver injury, and then mouse blood and liver tissue samples are collected at different times after the injury to verify the influence of the function of the neutralizing SAA1/2 on liver injury and repair, and the flow chart is shown in FIG. 13;

(2) the changes in concentration of SAA1/2 protein in liver tissue after different times after injury with anti-SAA neutralizing antibody are shown in FIG. 14, and it can be seen from FIG. 14 that: neutralizing SAA function can reduce SAA protein expression levels in liver tissue; the changes in the levels of the liver injury markers ALT and AST in serum are shown in fig. 15, and it can be seen from fig. 15 that: neutralizing SAA function can reduce ALT and AST activity in serum; the statistical method of the area of the liver injury focus comprises the following steps: first, the liver tissue was subjected to HE staining and prepared, and then each section was randomly photographed under a 100-fold microscope for 8-10 visual fields, and the size of the necrotic foci was quantified and analyzed using image pro plus (version 6.0) software, and the result graph is shown in fig. 16, and it is known from fig. 16 that: neutralizing SAA function can reduce the area of necrotic foci in liver tissue; the HE staining pattern of the liver tissue is shown in fig. 17 (scale: 100 μm), and it can be seen from fig. 17 that: inhibition of SAA function can reduce the area of necrotic foci in liver tissue; the survival graph of the mouse is shown in fig. 18, and it can be seen from fig. 18 that: inhibition of SAA function can increase survival time in APAP-induced liver injury mice.

(3) The immunohistochemical staining method detects the labeled molecule Cd42b (anti-1: 1000 dilution, Cat. ab183345) of platelets, analyzes the number of platelets in liver tissue at different times after injury as shown in FIG. 19 (scale: 50 μm), and the statistical result chart is shown in FIG. 20, and it can be known from FIG. 20 that: blocking the function of SAA by adopting a neutralizing antibody mode can obviously inhibit the aggregation of platelets in APAP-induced acute injury liver tissues.

Example 5

This example uses antagonistic peptides specifically targeting serum amyloid a to block SAA1/2 function to reduce APAP-induced liver damage as follows:

(1) in an APAP-induced mouse acute liver injury model (see example 1), 1 hour before APAP is injected into an abdominal cavity, an antagonistic peptide (with a sequence of YIGSDKYFHARGNY and 50 mu g/mouse) of SAA1/2 and a control short peptide (with a sequence of NYAGRKFHYSGDYI and 50 mu g/mouse) are injected intravenously through a mouse tail, APAP (300mg/kg) is administered for inducing acute liver injury after 1 hour of injection, and then mouse blood and liver tissue samples are collected at different time after the injury to verify the influence of the SAA1/2 function on liver injury and repair, wherein the flow chart is shown in figure 21 (in the figure, SAA-pep refers to the antagonistic peptide of SAA 1/2; Scamble-pep refers to the control short peptide);

(2) the changes in serum levels of the liver injury marker ALT after various times post-injury with the injection of antagonist peptides are shown in fig. 22, from which fig. 22 it can be seen that: the antagonistic peptide of SAA1/2 can significantly reduce APAP-induced serum ALT level; the HE staining pattern of the liver tissue is shown in fig. 23 (scale: 100 μm), and it can be seen from fig. 23 that: the antagonistic peptide of SAA1/2 was able to significantly reduce the area of necrotic foci in APAP-induced liver tissue; fig. 24 shows a statistical result chart of the area of the liver lesion, and it can be seen from fig. 24 that: the antagonistic peptide of the SAA1/2 can obviously reduce the area of necrotic foci in APAP-induced liver tissues;

(3) the immunohistochemical staining method was used to detect the number of platelets in liver tissue at different times after the injury, as shown in FIG. 25 (scale: 50 μm), and the statistical result chart is shown in FIG. 26, from which it can be seen that: blocking the function of SAA by adopting an antagonistic peptide mode of SAA1/2 can obviously inhibit the aggregation of platelets in APAP-induced acute injured liver tissues.

Example 6

This example uses neutralizing antibodies specifically targeting serum amyloid a to block the function of SAA1/2 to reduce freeze-induced liver damage as follows:

(1) in a freezing-induced mouse acute liver injury model (see example 1), 1 hour before freezing treatment, neutralizing antibody anti-SAA (3 mug/mouse) and control antibody IgG (3 mug/mouse) of SAA1/2 are injected into the tail vein of the mouse, acute liver injury is induced after 1 hour of injection, then mouse blood and liver tissue samples are collected at different times after injury to verify the effect of neutralizing SAA1/2 function on liver injury and repair, and the flow chart is shown in FIG. 27;

(2) the level of the liver injury marker ALT in serum after different times post injury with anti-SAA neutralizing antibody injected is shown in fig. 28, from which fig. 28 it can be seen that: the neutralizing antibody is adopted to inhibit the SAA function, so that the activity of ALT in blood can be obviously inhibited; fig. 29 shows the appearance of the liver at different time points, and the white dotted line region is the lesion region, as can be seen from fig. 29: the neutralizing antibody is utilized to inhibit the SAA function, so that the repair process of the liver injury focus can be remarkably accelerated; the depth change of the damaged portion of the liver tissue is shown in fig. 30, and it can be seen from fig. 30 that: the thickness of the damaged part can be obviously reduced by utilizing the neutralizing antibody to inhibit the SAA function, and the damage repairing process is accelerated;

(3) the immunohistochemical staining method was used to detect the number of platelets in liver tissue at different times after the injury, as shown in FIG. 31 (scale: 50 μm), and the statistical result chart is shown in FIG. 32, from which it can be seen that: in freezing induced acute liver injury tissue, inhibition of SAA function by neutralizing antibodies can also reduce platelet accumulation in the liver.

The above results suggest that in the early stage of acute liver injury, SAA may further contribute to liver injury by disrupting the integrity of the hepatic sinus structure, leading to intrahepatic hemorrhage and platelet aggregation.

The applicant states that the application of the serum amyloid a inhibitor of the present invention in the preparation of a medicament for treating acute liver injury, acute liver failure or chronic plus acute liver failure is described by the above examples, but the present invention is not limited to the above examples, which does not mean that the present invention is necessarily implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. Application of serum amyloid A inhibitor in preparation of medicine for treating acute liver injury, acute liver failure or chronic acute liver failure.

2. The use of claim 1, wherein the serum amyloid A inhibitor comprises any one or a combination of at least two of an interfering RNA specifically targeting serum amyloid AmRNA sequence, a neutralizing antibody specifically targeting serum amyloid A, an antagonist peptide specifically targeting serum amyloid A, or a chemical molecule specifically targeting serum amyloid A.

3. The use of claim 1 or 2, wherein the medicament reduces the level of glutamic-pyruvic transaminase and/or glutamic-oxalacetic transaminase in the blood.

4. The use of any one of claims 1 to 3, wherein the medicament reduces platelet aggregation at a site of liver injury.

5. Use of a serum amyloid a inhibitor for the manufacture of a medicament for reducing the level of glutamic-pyruvic transaminase and/or glutamic-oxalacetic transaminase in blood.

6. Use of a serum amyloid a inhibitor for the manufacture of a medicament for reducing platelet aggregation in liver lesions.

7. The use of any one of claims 1 to 6, wherein the medicament is in a dosage form selected from the group consisting of a suspension, granules, a capsule, a powder, a tablet, an emulsion, a solution, a drop pill, an injection, a suppository, an enema, an aerosol, a patch or a drop.

8. The use of any one of claims 1 to 7, wherein the medicament further comprises a pharmaceutically acceptable excipient.

9. The use of claim 8, wherein the adjuvant comprises any one or a combination of at least two of a carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, tonicity modifier, surfactant, coating material, colorant, pH adjuster, antioxidant, bacteriostatic agent, or buffer.