阅读说明：本技术 索拉胶在制备治疗急性药物性肝损伤药物中的应用 (Application of sorafenib gum in preparation of medicine for treating acute drug-induced liver injury ) 是由 张建法 顾旻昱 高岩 于 2019-11-06 设计创作，主要内容包括：本发明公开了索拉胶在制备治疗急性药物性肝损伤药物中的应用。索拉胶作为急性药物性肝损伤治疗药物使用时,降低对伤肝药物诱导的急性肝损伤中谷丙转氨酶和谷草转氨酶的水平,减少肝组织坏死面积,提高GSH水平,从而保护由伤肝药物服用过量所致的急性肝损伤。本发明首次发现索拉胶作为急性药物性肝损伤治疗药物使用时,能够显著提升急性药物性肝损伤后肝脏GSH的含量,降低炎症因子水平,从而达到抗氧化与调节免疫的作用,有效减轻药物引起的急性肝损伤,减少肝脏负荷,改善肝功能,降低肝移植率,降低个体死亡率。(The invention discloses application of sorafen gum in preparing a medicine for treating acute drug-induced liver injury. When the Sorana gum is used as a medicine for treating acute drug-induced liver injury, the levels of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in the acute liver injury induced by the liver injury medicine are reduced, the area of necrosis of liver tissues is reduced, and the GSH level is improved, so that the acute liver injury caused by excessive administration of the liver injury medicine is protected. The invention discovers for the first time that when the Soragel is used as the medicine for treating the acute drug-induced liver injury, the content of the GSH of the liver after the acute drug-induced liver injury can be obviously improved, and the level of inflammatory factors can be reduced, so that the effects of resisting oxidation and regulating immunity can be achieved, the acute liver injury caused by the medicine can be effectively reduced, the liver load can be reduced, the liver function can be improved, the liver transplantation rate can be reduced, and the individual mortality can be reduced.)

1. Application of Soraean gum in preparing medicine for treating acute drug-induced liver injury.

2. The use of claim 1, wherein the acute drug-induced liver injury is acute liver injury induced by a liver injury drug.

3. The use of claim 1, wherein the acute drug-induced liver injury is acetaminophen-induced acute liver injury.

4. The use of claim 1, wherein the sol is a sol gel having a preservation number of CCTCC NO: extracellular β -glucan produced by secretion from agrobacterium ZX09 of M2010020.

5. The use according to claim 1, wherein the dosage of the said medicine for treating acute drug-induced liver injury is 20mg/kg.b.w or more.

6. The use according to claim 1, wherein the amount of the used solarization accelerator gum in the medicament for the treatment of acute drug-induced liver injury is 40 mg/kg.b.w.

7. The use according to claim 1, wherein the subject is a mammal including a human.

Technical Field

The invention belongs to the technical field of medicines, and relates to application of Soraean gum in preparation of a medicine for treating acute drug-induced liver injury.

Background

The liver is an important organ mainly with metabolic function in human body, and has the functions of resisting oxidation, storing glycogen, synthesizing secretory protein and the like. Liver function is abnormal for a variety of reasons, and severe or persistent liver damage ultimately leads to liver failure. Paracetamol (APAP), also known as acetaminophen or N-acetyl p-aminophenol, is a drug used to treat fever, pain and inflammation, and APAP is normally safe and effective when used at normal therapeutic doses. The APAP is very widely applied and has huge consumption, and daily medicines such as Bai Ping, Bai Jia Black, Tai Nuo, 999 Ganmaoling, vitamin C Yinqiao tablets and the like all contain a large amount of APAP in China. Excessive APAP is the main reason of acute hepatic failure (ALF) in most industrialized countries, and due to the universality and universality of APAP medication, the hepatotoxicity caused by APAP is always widely concerned and regarded.

Establishing a suitable experimental liver injury animal model has very important significance for preventing and treating liver diseases, researching pathogenesis and screening liver-protecting medicines. The mechanism of various liver injury models is different, and the specific introduction is as follows:

drug induced liver injury: the major metabolites of APAP in the liver are glucuronic acid and sulfate conjugates, which are converted in small amounts by CYP2E1 to a highly active toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Normally, NAPQI is rapidly activated due to the decrease of Glutathione (GSH) in the liver and then excreted as cysteine and mercaptoacid in bile and urine. When the APAP is excessively ingested, the glucuronolactone and sulfate pathways are saturated, the excessive APAP is metabolized through a CYP system to generate a large amount of NAPHI, GSH in the liver is consumed, the NAPHI which is not covalently bonded with the GSH can also be covalently bonded with cell protein thiol, the oxidation of liver cells is promoted, the anti-oxidation capability of the liver is reduced, and acute liver necrosis is caused. The tissue morphology of the model of liver injury induced by APAP in mice is characterized by necrosis of the central region of the lobule, which is a marker of APAP toxicity. The model is convenient and cheap, and the metabolic mechanism approaches the clinic, so the model gradually becomes an animal model for researching the screening of the treatment drug for acute liver injury caused by excessive taking of antipyretic analgesics in recent years.

Chemical liver injury: CCl₄Is a common chemical inducer and is widely used for establishing an animal model with liver injury. CCl₄The cytochrome P4502E1(CYP2E1) is metabolized in the liver, producing highly reactive trichloromethyl radicals that interfere with the redox homeostasis in the liver, causing oxidative stress. Free radicals can also react with unsaturated fatty acids in cellsThe oxidation reaction promotes lipid peroxidation, induces DNA damage in hepatocytes, causes apoptosis and necrosis of hepatocytes, and finally causes damage to hepatocytes. CCl₄The induced mouse liver damage model is similar to human liver diseases in the aspects of pathophysiology and the like, is easy to replicate, and is an ideal mouse liver damage model. However CCl₄Strong toxicity, damage to liver and other organs, and CCl₄The liver injury belongs to toxic liver injury, and has weak relevance with the immune regulation reaction of an organism, so the model is not suitable for screening immune regulation type liver protection medicines and researching on the immune mechanism.

Alcoholic liver injury: this is the result of a cascade of events that clinically leads first to alcoholic fatty liver and then primarily through alcoholic steatohepatitis or alcoholic hepatitis and hepatocellular carcinoma that may lead to cirrhosis. 80-90% of alcohol is metabolized in liver. When alcohol is oxidized, a large amount of reduced coenzyme I is produced and becomes a raw material for synthesizing fatty acid, thereby promoting the synthesis of fat. Acetaldehyde and a large amount of reduced coenzyme I can inhibit the function of mitochondria to cause the fatty acid oxidation to be obstructed, and the formation of fatty liver is caused. The ethanol dehydrogenase in the liver cytoplasm is catalyzed to be oxidized into acetaldehyde, and the acetaldehyde is catalyzed to be converted into acetic acid by the acetaldehyde dehydrogenase to finally form carbon dioxide. In the ethanol oxidation process, a large amount of hydrogen ions are removed and combined with coenzyme I. The coenzyme I is reduced into reduced coenzyme I, so that the ratio of the reduced coenzyme I to the coenzyme I is increased, the oxidation and reduction reactions of cells are changed, the substance metabolism depending on the reduced coenzyme I/the coenzyme I is changed, and the reduced coenzyme I/the coenzyme I is used as the basis of metabolic disorder and disease. Meanwhile, acetaldehyde has direct toxic effect on liver cells. Alcohol causes hyperlactacidosis, and proline is increased by stimulating the activity of proline hydroxylase and inhibiting the oxidation of proline, so that the formation of collagen in liver is increased, and the liver cirrhosis process is accelerated. And it is considered that the blood hypervalactia and the hyperprolinemia can be used as the markers of the hepatic fibrosis of the alcoholic liver disease.

At present, various liver-protecting and enzyme-reducing medicines are various, such as antiviral treatment medicines and immunoregulation medicines, and a plurality of medicines need to be taken for a long time and have higher dosage, and no medicine capable of completely blocking liver injury is available, so that the death rate of acute liver failure caused by acute liver failure due to large-area hepatocyte necrosis cannot be effectively prevented from being reduced. The pharmaceutical industry needs to find a drug for alleviating acute liver injury caused by liver injury drugs urgently.

Disclosure of Invention

Aiming at the high morbidity of the existing drug-induced liver injury, the invention provides the application of the Soraean gum in preparing the drug for treating the acute drug-induced liver injury.

The acute drug-induced liver injury refers to acute liver injury induced by various liver injury drugs, for example, acute liver injury induced by acetaminophen.

The Sorana gum is prepared from the following components in percentage by weight: extracellular β -glucan produced by M2010020 secretion from Agrobacterium (Agrobacterium sp.) ZX09, see chinese patent 201010146371.2.

In the drug for treating acute drug-induced liver injury, the dosage of the solarium album is more than 20mg/kg.b.w, preferably 40 mg/kg.b.w.

The application object of the invention, namely the population suffering from acute drug-induced liver injury, is a mammal including a human.

The invention discovers for the first time that when the water-soluble beta-glucan and the Soraean gum are used as the medicine for treating the acute drug-induced liver injury, the content of the GSH of the liver after the acute drug-induced liver injury can be obviously improved, and the level of inflammatory factors can be reduced, so that the effects of resisting oxidation and regulating immunity can be achieved, the acute liver injury caused by the medicine can be effectively reduced, the liver load can be reduced, the liver function can be improved, the liver transplantation rate can be reduced, and the individual mortality can be reduced.

Drawings

FIG. 1 is a graph showing the results of reducing acetaminophen (APAP, 300mg/kg) -induced liver injury by low-dose and high-dose sola, wherein A is a graph showing the results of reducing ALT levels in mice in low-dose and high-dose sola groups 24 hours after liver injury is induced by APAP, and B is a graph showing the results of reducing AST levels in mice in low-dose and high-dose sola groups 24 hours after liver injury is induced by APAP.

FIG. 2 is a graph showing the effect of solasonine on hepatocyte necrosis.

FIG. 3 is a graph showing the effect of low-dose and high-dose Soragel on liver-related gene expression levels of APAP (300mg/kg) induced acute liver failure models in mice, wherein A is a graph showing the mouse liver Tnf-alpha mRNA expression level 24 hours after APAP-induced liver injury, and B is a graph showing the mouse liver IL-6mRNA expression level 24 hours after APAP-induced liver injury.

FIG. 4 is a graph of changes in GSH and MDA content in the liver of a mouse model of acute liver failure induced by APAP (300mg/kg) with low and high doses of Soragel.

Detailed Description

The present invention will be described in more detail with reference to the following examples and the accompanying drawings. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The sorafen gum is used as a medicine for treating acute drug-induced liver injury, is suitable for mammals including human beings, and is suitable for the condition of acute drug-induced liver injury caused by taking a large amount of hepatotoxic medicines such as acetaminophen. The liver injury is characterized in that the necrosis or the apoptosis of liver parenchymal cells can be distinguished pathologically, and complications caused by the liver injury comprise infectious complications, liver wound surface biliary leakage, secondary hemorrhage and acute liver and lung dysfunction; "liver failure" refers to severe liver damage caused by various factors, resulting in severe disorders or decompensations of its functions such as synthesis, detoxification, excretion and biotransformation, and a group of clinical syndromes mainly manifested by blood coagulation disorder, jaundice, hepatic encephalopathy, ascites, etc.

The Sorax gum (Salecan) is prepared from the following components in percentage by weight, wherein the preservation number is CCTCC NO: extracellular β -glucan produced by M2010020 secreted by Agrobacterium (Agrobacterium sp.) ZX09, see chinese patent 201010146371.2, whose molecular structure consists of the following repeating units:

→3)-β-D-Glcp-(1→3)-[β-D-Glcp-(1→3)-β-D-Glcp-(1→3)]₃-α-D-Glcp-(1→3)-α-D-Glcp-(1→。

when the preparation is used as a medicine for treating acute drug-induced liver injury, the low dose of the Soragel is 20mg/kg.b.w, and the high dose is 40 mg/kg.b.w.

In the invention, the reaction mechanism of the sorafen for protecting acute drug-induced liver injury is as follows: when the APAP is excessively taken, a large amount of APAP is oxidized by CYP450 enzyme to form N-acetyl-p-benzoquinone imine (NAPHI), the NAPHI causes Glutathione (GSH) to be exhausted, and animal experiments prove that the Soraoke can obviously improve the GSH content of the liver of an animal after acute liver injury and reduce the level of inflammatory factors, thereby achieving the effects of resisting oxidation and regulating immunity.

Example 1

1. Animal model

Experimental animals: eight weeks old male C57BL/6(WT) mice. Feeding under standard experimental conditions: 12 hours light-12 hours dark cycle, free intake of water and food.

APAP is used for establishing a mouse acute liver failure model, and APAP (300 mg/kg.b.w.) is administrated through gastric lavage. WT mice were randomly divided into four groups, the first group was a control group (Saline) and treated with corresponding drug media (physiological Saline) during the experiment; the second group is APAP group (APAP), and APAP is administered by intragastric administration; the third group was a low dose group (APAP + LS) and the mixed solution of sora gum (20mg/kg.b.w) and the corresponding dose of APAP was administered by gavage. The fourth group was a high dose group (APAP + LS) and the mixed solution of sora gum (40mg/kg.b.w) and APAP at the corresponding dose was administered by intragastric gavage. After 24 hours of APAP intragastric administration, blood and liver were collected for evaluation of liver injury.

2. Measurement of serum enzyme Activity

Collecting whole blood, naturally coagulating at room temperature, centrifuging at 3000r/min for 15min, and measuring activity of glutamic-oxaloacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) with serum. The enzymatic activities of AST and ALT were determined by enzymatic chromogenic quantitative assay, following the protocol in the kit instructions, and finally the concentration was determined colorimetrically at a wavelength of 340 nm.

FIG. 1 is a graph showing the results of low and high dose sorafen in reducing acetaminophen (APAP, 300mg/kg) induced liver injury. Data in graph A show that the serum ALT level of the mice is obviously increased after APAP induces liver injury for 24 hours: in contrast, the serum ALT level of the low-dose Soragel group (APAP + LS) mice is reduced compared with that of the APAP model group, but no significant difference exists. The serum ALT level of the high-dose Soragel group (APAP + HS) mice is remarkably reduced compared with that of the APAP model group. The data in the B picture show that the serum AST level of the mice is obviously increased after the liver is injured by APAP for 24 hours: the serum AST level of the mice in the low-dose Soragel group (APAP + LS) is reduced compared with that in the APAP model group, but no significant difference exists. Serum AST levels of mice in the high dose sola group (APAP + HS) were significantly reduced compared to the APAP model group. In conclusion, C57BL/6WT mice were modeled for acute liver failure by gavage acetaminophen (APAP, 300 mg/kg). After the APAP is modeled for 24 hours, compared with a control group, the serum AST and ALT enzyme activity of the APAP group mouse is obviously improved; compared with the APAP group, the serum AST and ALT enzyme activity of the mice in the low-dose Soragel group is reduced by about one third, and the activity of the mice in the high-dose Soragel group is slightly reduced by the lower-dose Soragel group.

3. Histological analysis of liver

Liver tissues were fixed with 10% formalin, then paraffin-embedded sections, and stained with hematoxylin-eosin (HE). The necrosis of the hepatocytes was observed by a microscope.

FIG. 2 is a graph showing the effect of soracan on hepatocyte necrosis. C57BL/6WT mice were modeled for acute liver failure by gavage acetaminophen (APAP, 300 mg/kg). After 24 hours of APAP modeling, liver tissue lesions were detected. H & E staining (100 ×) of liver histopathological sections showed that hepatocytes of mice in the APAP group exhibited severe necrosis compared to the control group 24 hours after APAP modeling; compared with the APAP group, the hepatocyte necrosis degree of mice in the low-dose Sorana gum group (APAP + LS) is obviously reduced, and the hepatocyte necrosis degree of mice in the high-dose Sorana gum group (APAP + HS) is slightly reduced in the low-dose group.

Determination of GSH

(1) Pretreatment of liver samples

Preparation of liver 10% homogenate: weighing the tissue weight according to the weight (g): volume (ml) ═ 1: 9, adding 9 times of physiological saline, homogenizing by an electric homogenizer under the condition of ice-water bath, 2500r/min, centrifuging for 10min, taking 10 mu l to 1.5ml of Eppendorf tubes, storing at-80 ℃ for BCA protein detection, and carrying out subsequent GSH detection on the rest supernatant.

(2) GSH assay procedure in the liver

The protocol of the GSH kit of Nanjing institute of bioengineering is built by reference, and the specific steps are as follows:

(2.1) preparation of supernatant: taking 0.1ml of sample to be detected, adding 0.4ml of reagent application liquid, mixing uniformly (equivalent to 5 times of dilution), centrifuging for 10min at 3500-.

(2.2) color reaction:

mixing, standing for 5min, and measuring O.D. value at 450nm with enzyme labeling instrument.

(2.3) calculating:

calculating according to a standard substance:

(3) BCA kit for detecting protein content

Protocol for determining protein content by referring to a triumph BCA kit.

(3.1) taking out the protein sample from-80 ℃, melting at normal temperature, adding 90. mu.l ddwater, adding 4. mu.l 10% SDS, and boiling water bath for 30s-1 min.

(3.2) for the liver histones, 400. mu.l of ultrapure water was added to dilute 50-fold. 10 μ l was taken for subsequent measurement.

(3.3) protein standards were diluted in proportion, ref 2.1.2.7(4)

(3.4) according to the number of samples, preparing a proper amount of BCA working solution by adding 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and fully mixing.

(3.5) Add 100. mu.l BCA working solution in 37 ℃ water bath for 30 min.

(3.6) O.D. value was measured at 562nm using a microplate reader.

Determination of MDA

(1) Pretreatment of liver samples

Preparation of liver 10% homogenate: weighing the tissue weight according to the weight (g): volume (ml) ═ 1: 9, adding 9 volumes of physiological saline, homogenizing by an electric homogenizer under the condition of ice-water bath, 2500r/min, centrifuging for 10min, taking 10 mu l to 1.5ml of Eppendorf tubes, storing at-80 ℃ for BCA protein detection, and carrying out subsequent MDA determination on the rest supernatant.

(2) Liver MDA assay procedure

The protocol of the MDA kit of the Nanjing institute of bioengineering is built by reference, and the specific steps are as follows:

(2.1) preparation of working solution: a first reagent: and a second reagent: reagent three ═ 0.1 ml: 0.9 ml: 0.3ml, how much is used, and the measurement is carried out on the day after preparation.

(2.2) ratio of operation to reagent:

	blank tube	Standard tube	Measuring tube
				Absolute ethyl alcohol (ml)	0.1
10nmol/L MDA standard		0.1
				Test sample (ml)			0.1
Working solution (ml)	1.2	1.2	1.2

(2.3) uniformly mixing by using a vortex mixer, tightly covering a cover of an Eppendorf tube, burning the sharp long needle to be red by using an alcohol lamp, covering the tube with 2-3 small holes to prevent the tube from being burst when the tube is boiled, and boiling the tube for 40min by using a pot cover.

(2.4) taking out, cold cutting with running water, and centrifuging for 10min at 3500-4000 r/min.

(2.5) taking 100. mu.l of the supernatant, and measuring the OD value at 532nm by using a microplate reader.

(2.6) calculating:

calculating according to a standard substance:

6. determination of expression level of related Gene in liver

Mouse liver RNA extraction mouse liver tissue (about 50mg) was taken, 600. mu.L of TRIzol reagent was added thereto, and homogenized at high speed. To the homogenate was added 120. mu.L of chloroform, and shaken vigorously for 15 s. After standing for 10min, centrifuging 11000g for 15min at 4 ℃. Aspirate approximately 300. mu.L of supernatant into a sterile 1.5mL EP tube (take care not to aspirate the protein layer), add an equal volume of isopropanol, and mix by gentle inversion. After standing for 10min, centrifuging 11000g for 10min at 4 ℃.

The supernatant was discarded. 1mL of 75% ethanol in DEPC water was added to wash, bounce off the precipitate, centrifuge 7000g, 5min, 4 ℃. The supernatant was decanted, 100. mu.L of DEPC water was added to dissolve the precipitate thoroughly, 4. mu.L of 5M NaCl and 250. mu.L of absolute ethanol (added in a ratio of 100:4:250 depending on the amount of precipitate) were added, and the mixture was stored at-80 ℃.

Reverse transcription Using M-MLV reverse transcriptase with mRNA as template and random primer (Oligo dT)₁₈) Synthesizing a single-stranded DNA (cDNA) complementary to the template under the initiation of (1). The method comprises the following specific steps: the stored RNA was removed from-80 ℃ and mixed well, 10. mu.L of the mixture was added to a 1.5mL EP tube and centrifuged at 13000g for 10min at 4 ℃. The supernatant was completely aspirated. To the EP tube was added 12. mu.L of mix1 containing 7. mu.L of DEPC water, 1. mu.L of oligo dT₁₈And 4. mu.L of 2.5nM dNTPs, mixed well, metal bath 65 ℃, 5min, then placed on ice to cool. To the EP tube, 8. mu.L of mix2 containing 4. mu.L of 5 XFirst buffer, 1. mu.L of DEPC water, 2. mu.L of 0.1M DTT and 1. mu.L of reverse transcriptase M-MLV were added and mixed well. The EP tube was incubated at 37 ℃ for 50min, heated in a metal bath at 70 ℃ for 15min, and the remaining reverse transcriptase was inactivated and stored at-20 ℃.

PCR amplification and fluorescent quantitative PCR, and ordinary PCR amplification is carried out by taking the obtained cDNA as a template. The PCR reaction was performed using a primer beta-actin and was synthesized by Shanghai Sangni Biotech Co., Ltd. The 20. mu.L PCR reaction system is shown in the following table.

PCR reaction system

The PCR amplification reaction conditions are as follows: 95 ℃ for 5 min; at 95 ℃ for 30 s; 30s at 60 ℃; 72 ℃, 30s, 40 cycles; extension at 72 ℃ for 10 min. And detecting the PCR product through agarose gel electrophoresis to confirm whether the product meets the experimental result. The cDNA obtained by reverse transcription was diluted 30-fold and used for RT-PCR analysis. 20 μ L RT-PCR reaction system is shown in the table below.

RT-PCR reaction system

The RT-PCR reaction conditions are as follows: at 50 ℃ for 2 min; at 95 ℃ for 10 min; at 95 ℃ for 15s, at 60 ℃ for 1min, for 40 cycles; 95 ℃ for 15 s; 30s at 60 ℃; 95 ℃ for 15 s.

The relative expression quantity of the target gene is analyzed by a method, and beta-actin is selected as an internal reference. The gene primer sequences are shown in the following table.

Primer sequences

The statistical method comprises the following steps: all data are expressed as mean ± standard error means. Error between the two sets of data, using the Student's t test method of one-dimensional analysis of variance, was clearly different for the two sets of data when p < 0.05.

FIG. 3 is a graph showing the effect of low-dose and high-dose of Soragel on the expression level of liver-related genes in a mouse model of acute liver failure induced by APAP (300 mg/kg). The data in panel A shows that the expression of mouse liver-alpha mRNA is increased compared with the control group 24 hours after APAP induces liver injury: the liver TNf-TNf-alpha mRNA expression of the low-dose Sorana gum group (APAP + LS) mice is reduced compared with that of the APAP model group, but the two groups have no significant difference, and the TNf-alpha mRNA expression of the high-dose Sorana gum group (APAP + HS) mice is reduced compared with that of the APAP model group, but the two groups have no significant difference; the B picture data shows that after APAP induces liver injury for 24 hours, the IL-6mRNA expression of the mouse liver is increased compared with that of a control group: the liver IL-6mRNA expression of the low-dose Soragel group (APAP + LS) mice has a trend of decreasing compared with that of the APAP model group, but the two have no significant difference; the high-dose Soragel group (APAP + HS) mice have significantly reduced liver IL-6mRNA expression compared with the APAP model group.

FIG. 4 is a graph of changes in GSH and MDA content in the liver of a mouse model of acute liver failure induced by APAP (300mg/kg) with low and high doses of Soragel. The data of the A picture shows that the liver GSH content of the mouse with the APAP-induced acute liver injury is rapidly reduced for 0 to 4 hours, and the GSH content of the mouse with the APAP-induced acute liver injury is 1/2 which is less than the GSH value of the normal mouse liver at 4 hours; and the liver GSH of the mouse of the Sorax-Sorpa gum group (APAP + HS) basically recovers to the liver GSH value of the normal mouse at 4 h. The data of the B picture shows that the MDA content of the liver of the mouse is increased compared with that of the control group after the APAP induces the liver injury for 24 hours: the liver MDA content of the mice in the low-dose Soragel group (APAP + LS) is reduced compared with that in the APAP model group, but the liver MDA content of the mice in the low-dose Soragel group (APAP + LS) is not significantly different from that in the APAP model group, and the Tnf-alpha mRNA expression of the mice in the high-dose Soragel group (APAP + HS) is reduced compared with that in the APAP model group, but the liver MDA content of the mice in the high-.

7. Data analysis

Comparisons between groups were statistically analyzed using ANOVA statistics. P <0.05 was considered to be a significant difference.

8. Liver damage in acute liver failure

After 24 hours of stomach perfusion of APAP, acute liver failure symptoms can be generated, the activity of AST and ALT enzyme of serum is strongly increased, and the change of liver tissue structure is accompanied, obvious liver parenchymal cell necrosis is shown through HE staining microscopy, and APAP can generate obvious liver injury.

9. Effect of adenosine on ameliorating liver injury in acute liver failure

By treating WT mice with a mixture of adenosine and APAP, liver damage due to APAP can be alleviated. Significant reduction in serum AST, ALT enzyme activity, reduction in necrotic area of hepatocytes and restoration of GSH of hepatocytes was observed.

TABLE 1 comparison of indications of sorafen in protecting acetaminophen against acute liver injury

Group of	ALT reduction ratio (%)	AST reduction ratio (%)
			Low dose of carrageenan/model set	24.80	23.99
High dose of solarization rubber/model set	43.70	40.91
			Schisandra chinensis alcohol extract group/model group	7.93	4.19
Auricularia auricula polysaccharide group/model group	6.31	3.65
			Compatible high dose/model group	21.28	19.89

Table 1 shows the index comparison of sorafen in protecting acetaminophen against acute liver injury. In Xuxiu flowers, Yangpo et al, protection of Auricularia polysaccharide and Schizandrae fructus alcohol extract against acetaminophen induced mouse acute liver injury (hereinafter, denoted by group A, and group B as the patent), we performed the following comparisons: ALT reduction ratio: the high dose of Sorana gum group (APAP + LS) has the highest numerical value, the low dose of Sorana gum group (APAP + LS) follows, and the high dose group, the schizandrol extract group and the Auricularia auricular polysaccharide group are combined respectively. AST reduction ratio: and ALT. Specific values are shown in table 1.2. Dosage and mode of administration: the dose differences were as follows: the APAP concentration of the group A is 200mg/kg, the alcohol extract group concentration of the schisandra chinensis is 200mg/kg, the auricularia auricula polysaccharide group concentration is 250mg/kg, the compatible high-dose group concentration is 200mg/kg of the alcohol extract of the schisandra chinensis plus 250mg/kg of the auricularia auricula polysaccharide, the APAP concentration of the group B is 300mg/kg, the low-dose Soraya gum group concentration is only 20mg/kg, and the high-dose Soraa gum group concentration is 40 mg/kg; the administration mode comprises the following steps: the group A is administered by intragastric administration according to the measurement every day, continuously for 10 days, and after the last administration, 1h except the control group is subjected to abdominal cavity injection APAP molding; group B need not be advanced, and each group is administered by intragastric administration on the day. As can be seen from Table 1, compared with the three groups of the schizandrol extract group, the auricularia auricula polysaccharide group and the compatible high-dose group, the effect of the low-dose and high-dose solarium solaricoides gum on APAP-induced acute liver injury is better, the dose requirement is lower, and the administration in advance is not needed.