CN118576617A - Application of heparin and its derivatives in the prevention and treatment of liver failure - Google Patents

Abstract

The present invention discloses the application of heparin and its derivatives in the prevention and treatment of liver failure. Heparin is degraded by a variety of methods to obtain a series of low molecular weight heparins in different molecular weight ranges; in addition, a series of heparin derivatives are obtained by selectively desulfating the surface of heparin and derivatizing the side chains of heparin. By screening the above-mentioned small compound library, a product with the effect of preventing and treating acute liver failure is obtained. After screening, Na-1, En-1, 6-DeH, N-Acetyl and H-PEG have the most significant effects, which can promote the transformation of pro-inflammatory macrophages to normal phenotypes of macrophages, reduce the inflammation of acute liver damage, and then prevent the occurrence of liver failure, and alleviate the inflammatory storm during the occurrence of liver failure. In addition, the anticoagulant activity of these compounds is greatly reduced, reducing the hemorrhagic side effects of heparin.

Description

Application of heparin and its derivatives in the prevention and treatment of liver failure

Technical Field

The present invention belongs to the field of biomedicine technology, and in particular relates to the application of heparin and its derivatives in preventing and treating liver failure.

Background Art

Acute liver failure is a sudden, severe liver injury caused by a variety of causes. It is a rare but highly fatal disease, with clinical features including overactivation of intrahepatic immune cells, extensive liver necrosis, coagulation disorders, and hepatic encephalopathy. An inflammatory storm forms and rapidly progresses to systemic inflammation, causing the patient's condition to deteriorate dramatically. If not controlled, it will lead to a high mortality rate. Currently, the only clinical treatment for acute liver failure is liver transplantation. How to prevent the progression of acute liver injury in the early stages plays an important role in reducing the occurrence of acute liver failure and preventing the development of systemic inflammation.

Macrophages in the liver play an important role in the transition from acute liver injury to liver failure. Macrophages are a type of inflammatory cell with strong plasticity, continuous functional states and rich phenotypes. During the occurrence of liver injury, liver macrophages are rapidly activated, the phenotype turns to an irregular state, and a large number of inflammatory factors are released, leading to the occurrence of inflammation. When liver damage expands, macrophages continue to be activated, amplifying the inflammation and leading to the occurrence of liver failure; when drugs are used to inhibit the secretion of macrophage inflammatory factors, the occurrence of liver failure can be avoided. Therefore, it is possible to obtain effective drugs for the prevention or treatment of liver failure by using the phenotypic conversion of macrophages and the secretion level of inflammatory factors as screening objects.

Heparin is the most important anticoagulant drug, but it has a wide range of biological activities.

Directly or indirectly, it changes the secretion of chemokines, cytokines and complement activity by monocytes, T cells and neutrophils, controls platelet activation and aggregation, and plays an anti-inflammatory role in the treatment of diseases such as pancreatitis, asthma and pneumonia. Previous studies have shown that heparin can regulate the production of macrophage inflammatory factors and prevent or treat a variety of acute inflammations.

In clinical practice, heparin is often used to intervene in the treatment of coagulation in liver failure. However, the common heparin molecule itself is not the best structure for the treatment of liver failure. This is because the strong anticoagulant effect of heparin often leads to the occurrence of hemorrhagic risks, while the anti-liver failure activity of heparin exists independently of the anticoagulant activity. Second, heparin is mainly composed of a long chain of disaccharide repeating units consisting of 2S-L-iduronic acid and NS-6S-acetylglucosamine connected by 1→4 glycosidic bonds. Due to the diverse number and position of the sulfonic acid and acetyl groups in the parent structure during biosynthesis, the conformation of uronic acid is different, and the possible constituent units of heparin are as high as dozens, so it has a highly micro-heterogeneous structural system. The molecular weight of heparin, the position and number of sulfonated groups on the surface determine the affinity of heparin to the ligand. Therefore, there is a corresponding relationship between the biological activity of heparin and the specific sequence structure.

By degrading the heparin sugar chain to obtain low molecular weight heparin, or by performing various chemical modifications on the heparin molecule, it is possible to remove structures irrelevant to the anti-liver failure activity and remove the strong anticoagulant activity, thereby obtaining the minimum unit structure effective for the treatment of acute liver failure, or a more effective structure. However, due to different degradation methods and modification routes, the intervention effects of the obtained products on acute liver failure are also very different. It is possible to obtain the best product by conducting activity screening and structure-activity relationship studies on these compounds.

Summary of the invention

To solve the above problems, the present invention degrades heparin by various methods to obtain a series of low molecular weight heparins with different molecular weight ranges; in addition, a series of heparin derivatives are obtained by selectively desulfating the surface of heparin and derivatizing heparin. By screening the above small compound library, products with the effect of preventing and treating acute liver failure are obtained.

The first object of the present invention is to provide a heparin and its derivatives for preventing and treating liver failure, comprising at least one of the following:

(1) low molecular weight heparin, wherein the weight average molecular weight of the low molecular weight heparin is 1000-4000;

(2) desulfated heparin, wherein the desulfated heparin is 6-desulfated heparin or N-reacetylated heparin having a weight average molecular weight of 12000-20000;

(3) Polyethylene glycol-modified heparin, wherein the weight average molecular weight of the heparin is 12,000-20,000.

Furthermore, the low molecular weight heparin is obtained by degradation by heparinase or hydrogen peroxide.

Furthermore, the structure of low molecular weight heparin obtained by heparinase degradation is shown in the following formula:

Wherein, R ₁ , R ₂ and R ₄ are independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^- , H or COCH ₃ ^- , and n is an integer of 3-8.

Furthermore, the structure of the low molecular weight heparin obtained by hydrogen peroxide degradation is one of the following formulae:

Wherein, R ₁ and R ₂ are independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^- , H or COCH ₃ ^- , and n is an integer of 3-8.

Furthermore, the molecular weight of the polyethylene glycol is 200-1,000,000.

Furthermore, the structure of the polyethylene glycol-modified heparin is shown in the following formula:

Wherein, R ₁ , R ₂ and R ₄ are independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^- , H or COCH ₃ ^- , and m is an integer of 20-30.

The second object of the present invention is to provide an anti-liver failure drug, which comprises the above-mentioned heparin and its derivatives.

Furthermore, the liver failure is acute liver failure.

The third object of the present invention is to provide the use of the above heparin and its derivatives in the preparation of anti-liver failure drugs.

Furthermore, it has been verified that the anticoagulant activity of the low molecular weight heparin, desulfated heparin or heparin derivatives provided by the present invention is greatly reduced, indicating that they are not used to prevent liver failure through anticoagulant activity, which is completely different from existing reports. It is further verified that these compounds are used to promote the transformation of pro-inflammatory macrophages to normal macrophage phenotypes, reducing the hemorrhagic side effects of current traditional heparin drugs.

Beneficial effects of the present invention:

The present invention degrades heparin by various methods to obtain a series of low molecular weight heparins in different molecular weight ranges; in addition, a series of heparin derivatives are obtained by selectively desulfating the surface of heparin and derivatizing the side chains of heparin. By screening the above-mentioned small compound library, products with the effects of preventing and treating acute liver failure are obtained. After screening, Na-1, En-1, 6-DeH, N-Acetyl and H-PEG have the most significant effects, which can promote the transformation of M1 macrophages to M0 type, reduce the inflammation of acute liver injury, and thus prevent the occurrence of liver failure and alleviate the inflammatory storm during the occurrence of liver failure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the animal experiment method;

Figure 2 shows that the drug group regulates the transformation of macrophages stimulated by liver supernatant to normal morphology;

FIG3 shows the effect of heparin and its derivatives on the aspect ratio of activated macrophages;

FIG4 shows the effect of heparin and its derivatives on the roundness of activated macrophages;

FIG5 shows the effect of heparin and its derivatives on the area of activated macrophages;

FIG6 shows that heparin, low molecular weight heparin and heparin derivatives inhibited the transcription of IL-1β to varying degrees;

FIG7 shows that heparin, low molecular weight heparin and heparin derivatives inhibited the transcription of IL-6 to varying degrees;

FIG8 shows that heparin, low molecular weight heparin and heparin derivatives inhibit the transcription of TNF-α to varying degrees;

FIG9 shows the changes in liver appearance caused by acute liver failure induced by ConA protein and the comparison of various drug groups;

FIG10 shows the changes in liver index caused by acute liver failure induced by ConA protein and the comparison of various drug groups;

FIG11 is H&E staining of liver tissues of mice with acute liver failure induced by ConA protein and each treatment group;

FIG. 12 is an analysis of the number of neutrophils in plasma.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

A small library of heparin derivatives was constructed by the following reaction:

1. Construction of a low molecular weight heparin library by combining heparinase degradation with alkaline degradation

2. Prepare various desulfated heparins by selectively desulfating heparin;

3. Obtain heparin derivatives by modifying the carboxyl group of heparin

Heparin and its derivatives were labeled by the following method:

Table 1 Nomenclature of heparin and its derivative samples

The above-mentioned small compound library was subjected to in vivo and in vitro biological activity screening against acute liver failure and anticoagulant activity determination.

Example 1 Construction of low molecular weight heparin library

Low molecular weight heparin prepared by heparinase I/III: 1 mL of heparinase I and heparinase III with an enzyme activity of 15 IU/mL was added to 100 mL of substrate solution (50 g/L heparin, 20 mM Tris, 200 mM NaCl, 5 mM CaCl ₂ , pH=7.4) for reaction, and the light absorption value of A232 nm was used as the main basis for controlling the end point of the reaction. The reaction was stopped when the value of A232 nm reached 10, 40 and 100. The weight average molecular weight (Mw) of the low molecular weight heparin was determined by gel exclusion high performance liquid semi-preparative chromatography (High Performance Liquid Chromatography, HPLC), and the detailed method can be found in EP 7.0. The chromatographic column was TSK-GEL G2000SWXL (TOSOH, Japan), the flow rate was 0.5 mL/min, and from the final product with an A232 absorption value of 100, products with a weight average molecular weight distribution of 1000-4000 were obtained by separation and purification; from the final product with an A232 absorption value of 40, products with a weight average molecular weight of 4001-8000 were obtained by separation and purification; from the final degradation product with an A232 value of 10, products with a weight average molecular weight of 8001 to 12000 and products with a weight average molecular weight of 12000 to 20000 were obtained by separation and purification.

Low molecular weight heparin prepared by heparinase II/III: 1 mL of heparinase II and heparinase III with an enzyme activity of 15 IU/mL was added to 100 mL of substrate solution (50 g/L heparin, 20 mM Tris, 200 mM NaCl, 5 mM CaCl ₂ , pH=7.4) for reaction, and the light absorption value of A232 nm was used as the main basis for controlling the end point of the reaction. When the value of A232 nm reached 35 and 66, the degradation was stopped, and the obtained product was ethanol precipitated and vacuum dried to obtain the final product.

Low molecular weight heparin obtained by NaOH/H ₂ O ₂ degradation method: Weigh 1g of fine heparin, make a 2% solution, add 30% H ₂ O ₂ , and adjust the pH value to pH=7 with NaOH, and degrade the heparin by ultrasonic treatment (450W) and controlling the temperature (60°C). The degradation time is regulated to be 10min, 30min, and 75min respectively. After the product is freeze-dried, it is washed with anhydrous ethanol. A small amount of water is added to redissolve to obtain the degradation product. The weight average molecular weight (Mw) of the low molecular weight heparin is determined by HPLC. The chromatographic column was TSK-GEL G2000SWXL (TOSOH, Japan), the flow rate was 0.5 mL/min, and from the final product with a degradation time of 75 min, a product with a weight average molecular weight distribution of 1000-4000 was obtained by separation and purification; from the final product with a degradation time of 30 min, a product with a weight average molecular weight of 4001-8000 was obtained by separation and purification; from the final degradation product with a degradation time of 10 min, a product with a weight average molecular weight of 8001 to 12000 was separated and obtained, and a product with a weight average molecular weight distribution range of 12001-20000.

Example 2 Preparation of Selectively Desulfated Heparin

The specific steps are as follows: First, heparin sodium is dissolved in water (10 g, 50 mL), and the solution is desalted by Amberlite IR-120-H+ ion exchange resin. The resin is washed with 2 column volumes of water, and the obtained product is neutralized with excess pyridine. The reaction product is dialyzed to remove excess pyridine molecules, and then freeze-dried to prepare heparin pyridinium salt.

Heparin pyridinium salt (2 g) was dissolved in a mixed solution of dimethyl sulfoxide and water (25 mL, 95:5), incubated at 50°C for 3 h, and then diluted with water (25 mL). The pH of the reaction solution was adjusted to about 9.0 with 0.1 M NaOH solution, and the solution was dialyzed with ddH ₂ O. The dialyzed product was freeze-dried to obtain N-desulfated heparin (N-DeH).

Dissolve N-desulfated heparin (1 g) in a saturated NaHCO ₃ solution (10 mL) at 4°C, then add acetic anhydride (625 μL) dropwise and stir for 2 h. Add saturated NaHCO ₃ solution to the reaction solution and adjust the pH value of the solution to between 8 and 9. Then, add excess hydrochloric acid to terminate the reaction. The product is dialyzed with water and freeze-dried to obtain N-acetylated heparin (N-AceH).

Heparin pyridinium salt (1 g) and BTSA (5 mL) were reacted in anhydrous pyridine (5 mL) at 80°C for 18 h, and then 2 mL of water was added to terminate the reaction. The solvent after the reaction was removed by vacuum distillation. The product was dialyzed and freeze-dried to obtain 6-desulfated heparin (6-O-Desulfated heparin, 6-DeH).

Heparin (1 g) and NaBH ₄ (200 mg) were dissolved in NaOH (1 M, 2 mL) solution at the same time. The mixture was neutralized to pH 7 with hydrochloric acid solution in an ice-water bath. The reaction product was dialyzed with water and freeze-dried to obtain 2-desulfated heparin (2-O-Desulfated heparin, 2-DeH).

Example 3 Preparation of heparin derivatives

Preparation of heparin-PEI/PEG. Weigh 200 mg PEI/PEG and dissolve it in 2 mL PBS; dilute the solution to 1 mg/mL with PBS; weigh 40 mg heparin and fully dissolve it in 20 mL PBS; weigh 25 mg (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimidehydrochloride, EDC), 15 mg N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS), quickly add them to the heparin solution, place it in an ice bath, and insert a cell disruptor probe into the solution for ultrasound. During the ultrasound process, slowly add 5 mL of 1 mg/mL PEI to the heparin solution, the ultrasound power is 200 W, the ultrasound is turned on for 5 seconds, the interval is 3 seconds, and the total time is 15 minutes. The obtained sample was dialyzed and freeze-dried for subsequent experiments.

Example 4 Regulatory Effects of Heparin/Low Molecular Weight Heparin and Heparin Derivatives on Macrophage Phenotype

Cell culture: RAW264.7 cells were placed in DMEM high-glucose medium containing 10% FBS and 1% penicillin/streptomycin (penicillin and streptomycin concentrations were 100 U/mL and 100 μg/mL, respectively) (hereinafter referred to as "culture medium") and cultured in a 37°C, 5% CO ₂ incubator (the culture conditions were the same). Observation under an inverted microscope showed that the cells were round and had good refractive index, and some cells had protrusions. Cells in the logarithmic growth phase (fusion degree reached 70% to 80%) were taken for subsequent experiments.

Phenotypic changes of macrophage RAW264.7: Cells in logarithmic growth phase were inoculated in 12-well plates at 4×10 ⁶ /mL. After culturing for 24 hours, the cells were randomly divided into groups. 50uL of liver supernatant was added to the model group, and the corresponding drugs were first added to each drug group to make the final concentration of the drug 500ug/mL, and cultured for 30 minutes. Then, an equal amount of liver supernatant solution from liver failure mice was added for stimulation, and microscopic observation was performed after culturing for 24 hours.

Compared with the blank group, the model group changed from highly regular round cells to many irregular antenna-type cells, indicating that there was a large number of polarization phenomena in macrophages, indicating that our modeling was successful. Compared with the model group, each drug-treated group inhibited cell polarization to varying degrees. In order to more intuitively compare the phenotypic changes of each group, we processed the data from three dimensions and selected 50 cells in each field of view for comparison, as shown in Figures 3 to 5. The degree of macrophage polarization was evaluated from the aspect ratio, roundness and area of the cells. After induction, the cells in the model group showed an increase in aspect ratio, a decrease in roundness and an increase in cell area, while all drug-treated groups were able to reduce the aspect ratio, increase cell roundness and reduce cell area, indicating that each group of drugs can regulate the transformation of activated macrophages to the normal group. Among them, in the low molecular weight heparin group, Na-1 and En-1 were significantly active and may be the smallest structural unit that inhibits macrophage activation; in addition, in the selective desulfation group, the 6-DeH group and the N-Acetyl group had significant therapeutic effects; and among the side chain modifications of heparin, the H-PEG group was closest to the normal form.

Statistical processing methods

The measurement data were expressed as mean ± standard deviation. The data were analyzed using GraphPad Prism version 8.0 software. One-way ANOVA was used for statistical analysis of variance for each measurement item. Bonferroni correction was used, and P < 0.05 was considered statistically significant.

In the text, ^* P means P＜0.05 compared with the Control group, ^** P means P＜0.01 compared with the Control group, ^*** P means P＜0.001 compared with the Control group, ^**** P means P＜0.0001 compared with the Control group; in the text, ^# P means P＜0.05 compared with the Model group, ^## P means P＜0.01 compared with the Model group, ^### P means P＜0.001 compared with the Model group.

Example 5 Effects of heparin/low molecular weight heparin and heparin derivatives on the transcriptional levels of inflammatory factors in activated RAW264.7 cells

(1) Sample preparation

Cells in logarithmic growth phase were inoculated in 6-well plates at 8×10 ⁶ /mL cells. After culturing for 24 hours, the cells were randomly divided into groups. 50uL liver tissue supernatant solution was added to the model group, and the corresponding drugs were first added to each drug group to make the final drug concentration of 500ug/mL, and cultured for 30min. Then an equal amount of liver supernatant was added, and samples were collected after culturing for 24 hours. 1mL Trizol reagent was added to each well of the cells in the 6-well plate to extract total cell RNA. According to the RNA isolation steps, RNA was extracted from the sample grinding solution, and the concentration was measured to dilute the concentration to 200-400ng/μL.

(2) Reverse transcription into cDNA

According to the RNA concentration in each sample, take the corresponding volume of RNA, add enzyme-free _ddH2O to make the total RNA to 1000ng, add 4μL 4×g DNA wipermix, mix well, and place in a 42℃ metal bath for 2min, then add 4μL 5×HiScriptⅡqRT SuperMixⅡ, mix well, and place in a 37℃ metal bath for 15min, and then place in a 85℃ metal bath for 5sec.

Table 2 RNA reverse transcription system

(3) Sample addition

First, add 180 μL ddH ₂ O to 20 μL cDNA, mix well, and centrifuge. Then, according to the system in each q-PCR tube, 5 μL diluted cDNA, 10 μL 2×ChamQ Universal SYBR qPCR Master Mix, 0.4 μL Primer 1 (10 μM), 0.4 μL Primer 2 (10 μM), and 4.2 μL ddH ₂ O were prepared according to the required amount of each solution, added to the q-PCR tube, covered the tube, flicked off the bubbles, centrifuged to make the liquid accumulate at the bottom, and sent to the fluorescent quantitative PCR instrument (CFX Controlnect) in the dark for q-PCR reaction.

The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

Table 3 Upstream and downstream primer sequences

Table 4 q-PCR system

Table 5 q-PCR reaction conditions and time

Statistical processing methods

The measurement data were expressed as mean ± standard deviation. The data were analyzed using GraphPad Prism version 8.0 software. One-way ANOVA was used for statistical analysis of variance for each measurement item. Bonferroni correction was used, and P < 0.05 was considered statistically significant.

In the text, ^* P means P＜0.05 compared with the Control group, ^** P means P＜0.01 compared with the Control group, ^*** P means P＜0.001 compared with the Control group, ^**** P means P＜0.0001 compared with the Control group; in the text, ^# P means P＜0.05 compared with the Model group, ^## P means P＜0.01 compared with the Model group, ^### P means P＜0.001 compared with the Model group.

Next, the real-time fluorescence quantitative polymerase chain reaction (q-PCR) method was used to detect the transcription levels of IL-6, IL-1β, and TNF-α mRNA, key inflammatory factors in activated macrophages. As shown in Figures 6 to 8, compared with the blank group, the expression levels of IL-6, IL-1β, and TNF-α mRNA in the model tissue increased significantly, and the expression levels of IL-6, IL-1β, and TNF-α mRNA in each medication group decreased significantly. The above experimental results show that heparin/low molecular weight heparin and heparin derivatives inhibit the occurrence of inflammation. The Na-1, En-1, 6-DeH, N-Acetyl and H-PEG groups obtained by cell morphology screening have more significant therapeutic effects.

Example 6 Determination of the Inhibitory Activity of Heparin/Low Molecular Weight Heparin and Heparin Derivatives in Liver Failure in Vivo

1. Experimental Animals

SPF BALB/c male mice (weight 20 ± 2 g, age 6-8 weeks) were purchased from Suzhou Sibeifu Experimental Animal Co., Ltd., license number SCXK (Su) 2022-0006. The animal experimental procedures in this experiment were approved by the ethics review of Jiangnan University, with the ethics number JN.No20210915i0401030[279].

2. Formula of main experimental reagents

Table 6 Main experimental reagent formula

3. Animal experiments

As shown in Figure 1, blank group: sterile saline was injected into the tail vein twice in a row, with a dose of 10 mg/kg each time, with an interval of 72 hours. Model group: Con A was injected into the tail vein 10 mg/kg each time, twice in a row, with an interval of 72 hours, to induce the ALF model of Con A secondary attack; heparin group: the modeling was the same as the model group, and the corresponding heparin was injected intraperitoneally 1 hour after each Con A injection, with a dose of 10 mg/kg, for a total of two injections, and the mice were killed 96 hours after the first injection of ConA protein.

4. Serum and tissue collection

(1) Serum collection: Blood was collected from the eyeballs and placed in anticoagulant tubes. The inflammatory cells in each group of mice were counted using a plasma biochemical analyzer.

(2) Tissue sampling: All mice were killed, dissected, spleen and liver were taken, wet weight of fresh liver was weighed, and liver index was calculated. Liver index = liver weight/body weight.

The liver tissues at the same site and all spleen tissues were cut and fixed in 4% paraformaldehyde for pathological sections. The remaining liver tissues were wrapped in tin foil and stored in a -80°C refrigerator.

5. Preparation of pathological sections

(1) Paraffin embedding; (2) HE staining; (3) Photographing and scoring of acute liver failure. Observe the mouse liver tissue pathological sections using CaseViewer and score according to the scoring criteria in Table 2-4.

Next, we used two injections of ConA protein to induce acute liver injury in mice, and induced acute liver failure after the second injection. After modeling, all groups of mice showed curling, lack of energy, drowsiness and other conditions during the modeling process. After the mice were killed, the mouse liver tissue was separated and the changes in the appearance of the mouse liver were observed. As shown in Figure 9, the liver of the blank group was bright red, uniform in color, smooth and flat on the surface, and no abnormality; the liver of the model group was reddish brown, uneven in color, no longer smooth and flat on the surface and granular, and the liver had obvious large-area necrosis. The liver of the drug-treated group was bright red, smooth on the surface, and no large-area necrosis occurred, which was close to the normal state, and was better than the model group. In addition, as shown in Figure 10, compared with the blank group, the liver index of the model group was significantly increased, proving that the mouse liver tissue had significant edema and inflammation, while each treatment group could significantly reduce the liver index, proving that each group of drugs could alleviate the swelling and congestion during liver necrosis.

Pathomorphology

The changes in the pathological sections of mouse liver stained with HE are the most intuitive reflection of the disease progression of mice. As shown in Figure 11, the morphological structure of liver tissue in the blank group was normal, neatly arranged, clearly demarcated, the bile duct diameter was small, the liver cell structure was clear, there was no necrosis, and there were no inflammatory cells or inflammatory infiltration; the liver tissue in the model group was largely destroyed, the bile duct diameter was large, the liver cell structure was disordered, the cell nucleus disappeared, a large number of liver cells were necrotic, and a large number of inflammatory cells and inflammatory infiltration appeared. The pathological morphology of liver tissue in the drug-treated group changed to varying degrees compared with the model group, the degree of liver tissue damage was reduced, the bile duct diameter was reduced to a certain extent, the liver cell necrosis was less, and the inflammatory cells and inflammatory infiltration were alleviated to varying degrees, which inhibited the occurrence of liver necrosis.

6. Analysis of plasma inflammatory cells

Neutrophils are the most representative inflammatory cells of the inflammatory storm of liver failure. Figure 12 shows the changes in neutrophils in the peripheral blood of mice. As shown in Figure 12, compared with the blank group, the blood neutrophil (NEU) index of the model group mice was significantly increased, which means that the model mice transformed from acute liver injury to liver failure, and caused an inflammatory storm by recruiting a large number of neutrophils. The number of plasma neutrophils in the treatment group was improved to varying degrees compared with the model group. The efficacy of the Na-1, En-1, 6-DeH, N-Acetyl and H-PEG groups obtained in the first two rounds of screening remained stable, and could replace the heparin group, or more significantly inhibit the inflammatory storm caused by liver necrosis than the heparin group. In summary, the above compounds can prevent the occurrence of large-area liver necrosis and the excessive inflammation caused by it by inhibiting the activation of macrophages.

Example 7 Determination of anticoagulant activity of heparin/low molecular weight heparin and heparin derivatives

For details, please refer to the published patent: Low molecular weight heparin and use of heparin for preparing drugs for treating pulmonary fibrosis Application No. 201710374727X Application Date 2017-05-24 Publication/Announcement No. CN108117615B

Activity assays have shown that degradation or modification can remove most of the anticoagulant activity of heparin itself. Therefore, since it does not affect its activity in inhibiting liver failure, the products obtained through screening are safer than heparin and do not pose the risk of hemorrhagic diseases.

Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims (10)

1. Heparin and its derivatives for use in the prevention and treatment of liver failure, characterized in that they comprise at least one of the following:

(1) A low molecular weight heparin, the weight average molecular weight of the low molecular weight heparin being 1000-4000;

(2) Desulphated heparin, which desulphated heparin is 6-desulphated heparin or N-re-acetylated heparin with a weight average molecular weight of 12000-20000;

(3) Polyethylene glycol modified heparin, wherein the weight average molecular weight of heparin is 12000-20000.

2. Heparin and its derivatives according to claim 1, characterized in that: the low molecular weight heparin is obtained by degradation with heparinase or hydrogen peroxide.

3. Heparin and its derivatives according to claim 2, characterized in that the structure of the low molecular weight heparin obtained by heparinase degradation is shown in the following formula:

Wherein R ₁、R₂、R₄ is independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^-, H or COCH ₃ ^-, and n is an integer of 3-8.

4. Heparin and its derivatives according to claim 2, characterized in that the low molecular weight heparin obtained by hydrogen peroxide degradation has a structure represented by one of the following formulas:

Wherein R ₁、R₂ is independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^-, H or COCH ₃ ^-, and n is an integer of 3-8.

5. Heparin and its derivatives according to claim 1, characterized in that: the molecular weight of the polyethylene glycol is 200-1000000.

6. Heparin and its derivatives according to claim 1, characterized in that the structure of the polyethylene glycol modified heparin is shown as follows:

Wherein R ₁、R₂、R₄ is independently selected from SO ₃ ^- or H, R ₃ is selected from SO ₃ ^-, H or COCH ₃ ^-, and m is an integer of 20-30.

7. An anti-liver failure medicament, which is characterized in that: the anti-liver failure drug comprises heparin and derivatives thereof according to any one of claims 1-6.

8. The anti-liver failure drug according to claim 7, wherein: the liver failure is acute liver failure.

9. Use of heparin according to any one of claims 1-6 and derivatives thereof for the preparation of an anti-liver failure medicament.

10. The use according to claim 9, characterized in that: the anti-liver failure drug is used for promoting normal phenotype conversion of pro-inflammatory macrophages to macrophages.