Application of atractylodes macrocephala polysaccharide in preparation of medicine for treating medicine-induced liver injury

Abstract

The invention provides application of atractylodes macrocephala polysaccharide in preparation of a medicine for treating drug-induced liver injury, and belongs to the field of traditional Chinese medicines. The rhizoma atractylodis macrocephalae polysaccharide can be used for treating acetaminophen-induced liver injury, reducing the number of apoptosis, improving inflammatory injury, improving the population diversity of intestinal flora, regulating key differential bacteria, up-regulating Roseburia of the family of hispidaceae, down-regulating Mogibacterium difficile and improving intestinal flora disorder caused by acetaminophen.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicines and relates to a kind of Atractylodes macrocephala polysaccharide, and specifically relates to the application of the Atractylodes macrocephala polysaccharide in preparing a medicine for treating drug-induced liver injury. Background Art

[0002] Drug-induced liver injury (DILI) is liver damage caused by the use of any prescription drugs, over-the-counter drugs, herbal medicines or dietary supplements. Its symptoms are elevated liver function indicators and even acute liver failure, leading to death.

[0003] Acetaminophen (APAP) is a common drug used to reduce fever and relieve pain. Overdose will cause it to accumulate and toxic to the liver in the body. During the pathogenesis of APAP-mediated drug-induced liver injury (AILI), the substance that first causes liver toxicity and direct damage to liver cells is mainly the drug metabolism byproduct NAPQI. After the GSH in the liver cells is completely consumed by it, it will form a large amount of covalent bonds on the mitochondrial membrane protein to form a complex. In this process, the osmotic pressure inside and outside the mitochondrial membrane is also disturbed, resulting in the opening of MPT, which is then broken and inactivated. Based on this, the ATP production drops sharply due to the mitochondrial strike, which also leads to damage to the energy production function. Finally, the liver cells are necrotic due to respiratory failure such as backstab. After cell necrosis, its various contents are released in large quantities, and the recognition receptors of damage-related molecular patterns are activated and play a role in making the above substances recognized by the corresponding receptors, thereby releasing cytokines that can activate pro-inflammatory factors (TNF-α, IL-1β, etc.) to induce inflammation; on the other hand, it will continue to cause apoptosis and necrosis of healthy liver cells. Apoptosis refers to programmed cell death regulated by genes and their transcription and translation products in the cell during the growth and development process or under other external influences. After cell apoptosis occurs, it has the following main characteristics: first, the overall size of the cell is significantly reduced, and it loses its connection with other cells and begins to detach. Then, various organelles including mitochondria continue to concentrate in the cytoplasm to increase the degree of accumulation of the entire cell. The nucleus is broken down into multiple membrane vesicles of uneven sizes accompanied by DNA fragmentation and fragmentation, and finally engulfed and degraded by phagocytes in the body.

[0004] Atractylodes macrocephala, a plant of the Asteraceae family, has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, calming the fetus and stopping sweating. It is often used in clinical Chinese medicine to treat spontaneous sweating due to qi deficiency, abdominal distension and diarrhea, poor appetite due to spleen deficiency, fetal movement disorder due to spleen deficiency, phlegm and fluid, dizziness and palpitations, edema, and leucorrhea.

[0005] Polysaccharide compounds are widely distributed, and many polysaccharides are derived from the roots, stems, leaves, flowers, fruits and seeds of various plants. Studies have shown that polysaccharides also have many biological effects in organisms, including: antioxidant, anti-tumor, immunomodulatory, anti-aging, anti-allergic, antiviral and other biological activities. At the same time, most polysaccharides have small toxic side effects and high safety. Many polysaccharides have been widely used in clinical practice.

[0006] The intestine is another important target organ for metabolic diseases and a container for the survival of intestinal flora. It involves various metabolic reactions of various hosts, including the metabolism of various substances such as sugar, amino acid, lipid metabolism, energy metabolism, and drug metabolism. It plays an important role in maintaining the body's digestive tract function, the perfect environmental balance, and the overall health of the human body. Enterohepatic circulation refers to the bidirectional material connection and exchange between the intestine and the liver through the bile duct and portal vein circulation. The prior art discloses the influence of intestinal flora on AILI. Intestinal microorganisms maintain the health and integrity of the microbial living environment in the intestine by promoting the digestion and metabolism of various nutrients, so that it tends to be steady. When the intestinal flora is disordered, the composition of the intestinal flora will change, causing the overgrowth of harmful bacteria, the reduction of the proportion of probiotics, and the decrease in the richness of the flora, resulting in damage to the intestinal mucosal barrier, resulting in bacterial translocation or harmful metabolites produced by harmful bacteria transferred to the liver through the "intestinal-liver axis", thereby inducing or deepening the liver's damage to APAP. Therefore, in recent years, the intestinal flora has gradually become an important "target" that can be regulated by AILI. The "gut-liver axis" is closely related to AILI, which is consistent with the fact that AILI in traditional Chinese medicine is located in the liver and has a close relationship with the gallbladder, spleen, and stomach. The manifestations of this deficiency are weak spleen qi and liver and kidney yin deficiency.

[0007] The invention patent with the publication number CN116726040A discloses the application of Atractylodes macrocephala polysaccharide in the preparation of drugs for preventing and treating psoriasis. Atractylodes macrocephala polysaccharide is obtained by water extraction and alcohol precipitation, which specifically includes the following steps: after drying and crushing Atractylodes macrocephala, adding water, extracting at 60-110°C for 0.5-8h, repeating the extraction several times, combining the extracts and filtering, concentrating, adding 70% or more ethanol solution for precipitation, standing at 4-30°C for 6-96h; filtering and collecting the precipitate to obtain a solution containing Atractylodes macrocephala polysaccharide, and freeze-drying to obtain Atractylodes macrocephala polysaccharide. Atractylodes macrocephala polysaccharide is composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose.

[0008] Since the extraction and purification methods have a significant impact on the composition and biological activity of polysaccharides, strategies need to be improved in order to seek effective, safe and low-toxic new therapeutic drugs for diabetic steatohepatitis. Summary of the invention

[0009] The present invention aims at the problems existing in the prior art and provides an application of Atractylodes macrocephala polysaccharide in the preparation of a drug for treating drug-induced liver injury. The present invention can improve acetaminophen-induced liver injury, reduce the number of apoptotic cells, improve inflammatory damage, increase the diversity of intestinal flora, and regulate the abundance of Roseburia and Mogibacteriaceae by using Atractylodes macrocephala polysaccharide.

[0010] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a use of Atractylodes macrocephala polysaccharide in the preparation of a medicament for treating drug-induced liver injury.

[0012] The drug-induced liver injury includes acetaminophen-induced liver injury, and the acetaminophen-induced liver injury includes one or more of liver injury, apoptosis, liver inflammation and intestinal flora disorder. In one embodiment, the acetaminophen-induced liver injury is intestinal flora disorder, and the intestinal flora includes one or two of Roseburia and Mogibateriaceae.

[0013] The Atractylodes macrocephala polysaccharide is prepared by a water extraction and alcohol precipitation method, which comprises extracting Atractylodes macrocephala and ultrapure water in a ratio of 1 g:15-25 mL at 85-95° C., extracting 1-3 times, each time for 1-3 hours, combining the extracts and concentrating to obtain a concentrated solution; adding 2-4 times the volume of a 90-100% ethanol solution to the concentrated solution, precipitating with alcohol to obtain a precipitate, and drying to obtain Atractylodes macrocephala polysaccharide.

[0014] In one embodiment, the water extraction and alcohol precipitation method is specifically:

[0015] Step 1): extract Atractylodes macrocephala and ultrapure water at a ratio of 1 g: 20 mL at 90°C for 2 h, centrifuge at 4000 rpm for 20 min, and collect the supernatant and residue;

[0016] Step 2): extract and centrifuge the residue for the second time according to the method of step 1), combine the supernatants of the two extractions and concentrate at 60° C. to obtain a concentrate;

[0017] Step 3): Add 3 times the volume of 95% ethanol solution to the concentrate, precipitate overnight at 4°C, collect the precipitate by centrifugation, wash the precipitate with ethanol and freeze-dry it to obtain Atractylodes macrocephala polysaccharide.

[0018] The atractylodes macrocephala polysaccharide is composed of glucose, arabinose, galactose, galacturonic acid and glucuronic acid.

[0019] Preferably, the Atractylodes macrocephala polysaccharide is composed of 82-85% glucose, 6-8% arabinose, 5-6% galactose, 2-4% galacturonic acid and 0.1-0.8% glucuronic acid.

[0020] In one embodiment, the Atractylodes macrocephala polysaccharide is composed of 83.92% glucose, 7.19% arabinose, 5.47% galactose, 3% galacturonic acid and 0.41% glucuronic acid.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the present invention can treat acetaminophen-induced liver injury, improve liver injury, cell apoptosis, liver inflammation and intestinal flora disorder by obtaining different Atractylodes macrocephala polysaccharides (BZ-W) with monosaccharide composition, regulate key differential bacteria, upregulate Roseburia of Lachnospiraceae, downregulate Mogibacteriaceae, and can be used to treat drug-induced liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the ion chromatogram of the monosaccharide composition of BZ-W;

[0023] Figure 2 is the Fourier-infrared spectrum of BZ-W;

[0024] Figure 3 The scanning electron microscope images of BZ-W at different magnifications; X5000, X10000, and X20000 are magnifications of 5k, 10k, and 20k times, respectively;

[0025] Figure 4 The diagrams are the detection of BZ-W's cytotoxicity to L02 cells and its effect on APAP-induced cell damage; A is the diagram of L02 cell activity detected by CCK8 at different concentrations after L02 cells were given BZ-W at different concentrations for 24 hours; B is the diagram of the detection of cell LDH release ratio under different concentrations of BZ-W treatment in the cell AILI (5mM APAP, 24h) model; C is the diagram of L02 cell morphology in each group in B (# indicates a significant difference between the APAP group and the CON group; #p<0.05, ##p<0.01, ###p<0.001; indicates a significant difference between the BZ-W (12.5μg / ml) / BZ-W (25μg / ml) / BZ-W (50μg / ml) group and the APAP group; p<0.05, p<0.01);

[0026] Figure 5The effect of BZ-W on APAP-induced inflammation and apoptosis of L02 cells; A is the result of fluorescence staining TUNEL for different doses of BZ-W groups after APAP (5mM) induced acute liver injury in L02 cells for 24 hours after cell crawling; B is the result of APAP (5mM) induced acute liver injury in L02 cells for 24 hours, and the apoptosis-related gene Bax induced by APAP in L02 cells under different concentrations of BZ-W treatment was detected mRNA level expression diagram; C is the expression of Bax and Bcl-2 (BCL2) protein levels in L02 cells induced by APAP after administration of BZ-W; D is the expression of inflammatory-related genes IL-1β and Tnf-α mRNA levels in L02 cells induced by APAP after administration of BZ-W (# indicates a significant difference between the APAP group and the CON group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; * indicates a significant difference between the BZ-W (6.25μg / ml) / BZ-W (12.5μg / ml) / BZ-W (25μg / ml) group and the APAP group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0027] Figure 6 Figure 1 is a diagram of the mouse modeling scheme;

[0028] Figure 7 Figure 1 is the effect of BZ-W on APAP-induced acute liver injury in mice; A is the H&E staining result of mice 12 hours after administration of APAP (10X); B is the statistical graph of the area of ​​liver pathological necrosis in the 12-hour model; C is the ALT and AST content in mouse serum detected by the kit; D is the H&E staining result of mice 24 hours after administration (10X); E is the statistical graph of the area of ​​liver pathological necrosis in the 24-hour model; F is the ALT content in mouse liver tissue detected by the kit (# indicates a significant difference between the APAP group and the CON group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; * indicates a significant difference between the BZ-WL / BZ-WH group and the APAP group, *p<0.05, **p<0.01);

[0029] Figure 8Figure 2 is the effect of BZ-W on APAP-induced mouse hepatocyte apoptosis; A is the result of TUNEL fluorescent probe staining of mouse liver tissue paraffin section; B is the result of qPCR detection of mouse apoptosis-related gene transcription level expression; C is the expression level of mouse apoptosis-related proteins BCL-XL, BAX, and BCL-2; D is the expression level of mouse apoptosis-related protein CYTO C detected by WB experiment (# indicates significant difference between APAP group and CON group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; * indicates significant difference between BZ-W group and APAP group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0030] Fig. 9 Figure 2 is the effect of BZ-W on APAP-induced liver inflammation in mice; A is the result of CD68 immunohistochemical staining 12 hours after APAP administration in mice; B is the statistical result of CD68-positive area (CD68-positive area, %) in Figure A; C is the result of CD68 immunohistochemical staining 24 hours after APAP administration in mice; D is the statistical result of CD68-positive area (CD68-positive area, %) in Figure C; E is the result of WB experiment on the expression of inflammatory factor IL-1β protein (# indicates significant difference between APAP group and CON group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001; * indicates significant difference between NAC / BZ-WL / BZ-WH group and APAP group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0031] Fig.10 Figure 2 is the effect of BZ-W on the drug metabolism of APAP and the oxidative stress process induced by it; A is the result of ROS fluorescent probe staining of paraffin sections of small liver tissue; B is the result of qPCR detection of the mRNA expression level of genes related to phase II drug metabolism enzymes in mice; C is the result of Western blot detection of the protein expression level of drug metabolism enzyme CYP2E1 in mice (* indicates significant differences among the three groups, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0032] Fig.11 This is a schematic diagram of the fecal microbiota transplantation experiment plan;

[0033] Fig.12 The effect of BZ-W on AILI intestinal flora; A is WT FMT(WT)Group and WT FMT(BZ-W) Figure B is the H&E staining result of liver tissue paraffin sections of mice in group A 24 hours after APAP administration (10X), B is the statistical result of liver pathological necrotic area (NECROTIC AREA, %), C is the result of ALT content in mouse liver detected by kit, where FMT(WT) means WT FMT(WT) Group mice, FMT(BZ-W) means WT FMT(BZ-W) group mice (* indicates significant difference between FMT(BZ-W) and FMT(WT), *p<0.05);

[0034] Fig.13 The effect of BZ-W on intestinal flora in improving hepatocyte apoptosis in the process of AILI; A is WT FMT(WT) Group and WT FMT(BZ-W) A is the result of TUNEL immunohistochemistry staining of paraffin sections of liver tissues of mice in group A after administration of APAP, B is the statistical result of TUNEL positive area, and C is the result of qPCR detection of mRNA expression level of apoptosis-related genes in mouse liver (* indicates significant difference between FMT (BZ-W) group and FMT (WT) group, **p<0.01);

[0035] Fig.14 The figure shows the effect of BZ-W on the Chao1 index and Shannon index of α diversity among bacterial groups in intestinal flora disorder caused by AILI; A is the result diagram of the Chao1 index of α diversity among bacterial groups, B is the result diagram of the Shannon index of α diversity among bacterial groups, C is the result diagram of the Chao1 index dilution curve among groups, and D is the result diagram of the Shannon index dilution curve among groups;

[0036] Fig.15 Figure 2 is the effect of BZ-W on intestinal flora disorder caused by AILI, where A is the PCoA analysis result of β diversity between groups, B is the NMDS analysis result of β diversity between groups, and C is the accuracy analysis result of model prediction results;

[0037] Fig.16 This is the effect of BZ-W on the composition of the intestinal flora of mice (species composition stacked bar chart, with class as the horizontal classification);

[0038] Fig.17 This is the effect of BZ-W on the composition of the intestinal flora of mice (species composition stacked bar chart, classified at the family level);

[0039] Fig.18 This is the effect of BZ-W on the composition of the intestinal flora of mice (heat map of species composition of intestinal flora, classified at the class level);

[0040] Fig.19 The figure shows the screening of significant bacteria in the intestinal flora of AILI mice by BZ-W; A is the Lefse analysis result figure, the color represents the grouping, and the length represents the LDA value; B is the comparison of the relative abundance of species with significant differences between groups (n=5) (* indicates a significant difference between the BZ-W group and the APAP group, *p<0.05, **p<0.01);

[0041] Fig. 20 The figure shows the effect of BZ-W on APAP-induced mouse liver inflammation-related genes, where graphs AD are the changes in the mRNA expression levels of IL-1β, IL-6, TNF-α, and Nlrp3;

[0042] Fig.21 The results of the GSH content test in mouse liver tissue

[0043] Fig. 22 The results of the mRNA expression levels of liver apoptosis-related genes in mice after administration of APAP, where Figures AC are the changes in the mRNA expression levels of Bax, Bcl-xl and Bcl2, respectively. DETAILED DESCRIPTION

[0044] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0045] The following sources of raw materials are provided for illustrative purposes:

[0046] L02 cells: The trade name is Human Liver Cell, a human liver-derived cell line (L02), and the cell source is the Animal Center of Sun Yat-sen University (number: Clone 2.5B1 (CRL-12461)).

[0047] The main reagents are shown in Table 1:

[0048] Table 1 Main reagents

[0049]

[0050]

[0051] The main instruments and equipment are shown in Table 2.

[0052] Table 2 Main instruments and equipment

[0053]

[0054]

[0055] Data statistical analysis: All data are expressed as mean ± standard deviation (Mean ± SD). All data were calculated in Excel and then entered into Graphpad Prism 9.0 software for statistical analysis. The method used for comparing the differences between the two groups in this chapter was t-test, and the method used for comparing the differences between multiple groups was one-way ANOVA. In the results, p < 0.05 indicated that the difference was statistically significant.

[0056] Example 1

[0057] Preparation and analysis of Atractylodes macrocephala polysaccharide

[0058] 1. Preparation of Atractylodes macrocephala polysaccharide:

[0059] Atractylodes macrocephala was purchased from Kangmei Pharmaceutical. The main reagents included ethanol, methanol, potassium bromide, trifluoroacetic acid (TFA), and 13 monosaccharide standards: fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), and guluronic acid (Gul-UA). All reagents used were of analytical grade.

[0060] After the Atractylodes macrocephala was powdered, 10.0 g of Atractylodes macrocephala powder was accurately weighed, and 200 mL of ultrapure water was added and stirred and extracted at 90 ° C for 2 hours to obtain an extract. The extract was centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge at 0-5 ° C, and the supernatant and residue of the first extraction were collected. The residue was added to ultrapure water and mixed, and the above steps were repeated for the second extraction and centrifugation. The supernatants of the two extractions were combined, vacuum rotary evaporated at 60 ° C to concentrate to 1 / 3 of the original volume, and tested with iodine-potassium iodide reagent, indicating that there was no starch. The obtained concentrated solution was dissolved in 95% ethanol solution for dilution 4 times, stirred and mixed, and precipitated overnight at 4 ° C. After that, the collected mixed solution was centrifuged at 4000 rpm for 10 minutes and the precipitate was collected. The collected precipitate was then washed twice with ethanol of equal volume fraction to make the polysaccharide component more uniform. Finally, the precipitate was evenly dissolved in ultrapure water and vacuum freeze-dried for 48 hours to obtain a Atractylodes macrocephala polysaccharide (BZ-W) sample.

[0061] 2. Analysis of total sugar content of Atractylodes macrocephala polysaccharide

[0062] The total polysaccharide content of BZ-W was determined by the phenol-sulfuric acid method. Accurately weigh 2-5 mg of sample, add 1 mL of sterile water, ultrasonically extract for 10 min, and centrifuge at 12000 rpm for 10 min. Pipette 0.4 mL of supernatant into a new test tube and dilute to a certain volume with dd water (double distilled water). Then, add the diluted sample solution to the test tube and mix with phenol-concentrated sulfuric acid to react, and measure the absorbance at 490 nm. The total polysaccharide content is calculated by the absorbance obtained through the glucose standard curve. The extraction rate of Atractylodes macrocephala polysaccharide and its total sugar content are shown in Table 3.

[0063] Table 3 Extraction rate and total polysaccharide content of Atractylodes macrocephala polysaccharide

[0064]

[0065] The results in Table 3 showed that the extraction rate of Atractylodes macrocephala polysaccharide was 20.07% and the total sugar content was 87.06%.

[0066] 3. Monosaccharide composition analysis

[0067] The Thermo ICS5000 ion chromatography system was used to determine the monosaccharide composition of BZ-W using an electrochemical detector. 6.0 mg of BZ-W was hydrolyzed with trifluoroacetic acid (2.0 mol / L) at 121°C for 2 h, connected to nitrogen, and blown dry. Then 99.99% methanol was added for washing, blown dry, and the washing was repeated 3 times. Sterile water was added to dissolve and transferred to a chromatographic bottle for testing. The sample was set up for 3 replicates (PBZ-1, PBZ-2, PBZ-3). The mixed standard was prepared into a series of concentration gradients in proportion by fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA) and guluronic acid (Gul-UA). Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm) liquid chromatography column. The monosaccharide composition of BZ-W is as follows Figure 1 As shown in Table 4, Figure 1 This is the ion chromatogram of the monosaccharide composition of BZ-W.

[0068] Table 4 Monosaccharide composition percentage of BZ-W

[0069]

[0070] Note: Ara, arabinose; Fuc, fucose; Gal-UA, galacturonic acid; Gal, galactose; Glc-UA, glucuronic acid; Glc, glucose; Man, mannose; Xyl, xylose.

[0071] Figure 1 The results in Table 4 show that BZ-W is mainly composed of glucose (83.92%). In addition, it also contains a small amount of arabinose (7.19%), galactose (5.47%), galacturonic acid (3.00%) and glucuronic acid (0.41%). The structure of BZ-W is relatively pure, and the main type of sugar in BZ-W is dextran.

[0072] 4. Infrared spectroscopy analysis

[0073] The infrared spectrum of BZ-W was analyzed using a Fourier transform infrared spectrometer to identify the functional group characteristics. First, a small amount of BZ-W was weighed and mixed with 200 mg of potassium bromide, then pressed into a sheet with a thickness of 1 mm and then tested on the machine. A Fourier transform infrared spectrometer (Nicolet iZ-10) was used in the frequency range (4000-400 cm -1 ) to perform scanning analysis and record the infrared spectrum. Figure 2 As shown, Figure 2 This is the Fourier infrared spectrum of BZ-W.

[0074] Depend on Figure 2 As shown, at 3200cm -1 To 3600cm -1 The vibration region shown between 3000-2800cm -1 Within the range (2930cm -1 ) is the CH absorption peak, which includes CH, CH 2 and CH 3 Stretching vibration. At 1000-1200cm -1 The range of vibrations that appeared was caused by the vibration of the ester sugar group (CO-C) and the pyranose ring (COH), which proved that BZ-W contained a six-membered ring pyranose structure. Specifically, 905-876 cm -1 The vibration range that appears in the range is caused by the β-configuration of glucan. 855-833cm -1 The vibration range that appears in the range is caused by the α-configuration of glucan.

[0075] 5. Scanning electron microscope (SEM) analysis

[0076] A high-resolution field emission scanning electron microscope (Zeiss Merlin Compact) was used to perform electron microscopic scanning analysis of Atractylodes macrocephala polysaccharides. The polysaccharide sample was passed through a 100-mesh sieve, and a small amount was taken on a conductive carbon tape. After gold spraying, it was scanned and photographed using an electron microscope with a magnification of 100 to 4000 times. Representative polysaccharide structures were observed at different magnifications (5k, 10k, and 20k). The results are shown in Figure 2. Figure 3 As shown, Figure 3 These are scanning electron microscope images of BZ-W at different magnifications, where X5000, X10000, and X20000 represent magnifications of 5k, 10k, and 20k times, respectively.

[0077] Figure 3 The results show that the surface of BZ-W is loose and rough, with irregular multi-layer flakes or multi-layer rose petal-like distribution.

[0078] Example 2

[0079] Study on the effect of BZ-W on APAP-induced hepatocyte injury

[0080] 1. Experimental methods

[0081] Human hepatocytes L02 were used to establish a cell injury model induced by APAP, and then treated with different mass concentrations of BZ-W. Then, the cell morphology was observed and the cell viability and apoptosis-related evaluation indicators were detected to explore the effect of BZ-W in inhibiting the toxicity of APAP on L02 hepatocytes in vitro.

[0082] 1.1 Cryopreservation of L02 cells

[0083] The steps are as follows:

[0084] A. Change the cell culture medium once a day (24 hours) before freezing the cells.

[0085] B. Wash off the culture medium, add 0.25% trypsin digestion solution into the dish and add it along the wall of the dish to avoid directly washing the cells. After adding the liquid, shake it to spread it evenly on the bottom of the dish, and then quickly put the dish in 37℃, CO 2 Digestion was performed in an incubator. After incubation for 40 seconds to 1 minute, the culture dish was quickly removed and 1640 medium was added to terminate the digestion. The digested cells were collected by centrifugation for counting and the cell density was adjusted to 5×10 6 / m L.

[0086] C. Wash twice with PBS and remove the supernatant after centrifugation.

[0087] D. Add commercial cell freezing solution to make 1 mL of resuspended cells and transfer the cell suspension into cryopreservation tubes, with at least 1 mL per tube.

[0088] E. Place the cryovials in a programmed cooling box, transfer the sealed cooling box with a modified lid to a -80°C refrigerator for programmed step-by-step cooling. Take them out the next day after 24 hours, store them on ice, and quickly transfer the cryovials to a liquid nitrogen tank for long-term freezing.

[0089] 1.2L02 cell culture method

[0090] Fetal bovine serum, double antibody, and sodium pyruvate were added to 1640 complete medium at a volume ratio of 10%:1%:1% and mixed well to prepare cell growth medium. The frozen L02 cells were revived and inoculated into the above medium and incubated at 37°C, 5%, and CO 2 , culture in a cell culture incubator with saturated humidity, and record cell information (cell type, generation, etc.). Observe whether the cells are completely attached to the wall within 12-24 hours after cell inoculation, and continue to observe the density and cell state of cell proliferation to prevent cell contamination. When the density is above 90%, digest and centrifuge with 0.25% trypsin, collect and add growth medium to mix to make a cell suspension for passaging, and then transfer to other culture dishes at a ratio of 1 / 3 to continue culture.

[0091] 1.3CCK8 cytotoxicity assay

[0092] Effect of BZ-W on the activity of L02 cells detected by CCK-8

[0093] Prepare a clean 96-well plate and add 1640 medium for cell growth to culture L02 cells at 10 6 PBS was added to the outermost wells of the 96-well plate to prevent evaporation, and 100 μL of L02 cells were added to each well. The cells were cultured for 24 h in a normal cell culture environment. After observing that the cell density and morphology were normal, the culture medium was removed, and the cells were washed once with an appropriate amount of PBS, and the residual liquid was removed. Fresh 1640 complete culture medium without serum or phenol red was added, and then the cells were cultured under CO. 2 Normalize serum-free starved cells in the incubator for 12 hours. After that, remove the culture medium and rinse once with pre-cooled sterilized PBS. After absorbing the residual liquid, add the same volume of the seed plate, and prepare a serum-free and phenol red-free culture medium mixture containing different mass concentrations of BZ-W solution (0, 3.125, 6.25, 12.5, 25, 50, 100 μg / mL) in advance. After incubation in the incubator for 24 hours, remove the culture medium, and add the same volume of freshly prepared 1640 complete culture medium containing 10% CCK-8 reagent detection solution to each well. React in the dark for 3 hours, then wrap the 96-well plate with tin foil to avoid light and transfer it to the microplate reader for colorimetric detection. Incubate and detect absorbance at 37°C and 450nm. The formula for CCK8 detection of cell viability is as follows:

[0094]

[0095] OD (drug addition): absorbance value of the detection plate containing BZ-W solution (3.125, 6.25, 12.5, 25, 50, 100 μg / mL);

[0096] OD(blank): absorbance value of blank detection well plate;

[0097] OD (drug added): absorbance value of the detection plate containing BZ-W solution (0 μg / mL).

[0098] 1.4 LDH detection in APAP-induced L02 cell injury model

[0099] Detection of the effect of BZ-W on APAP-induced LDH release rate in L02 cells

[0100] Prepare a clean 96-well plate and add 1640 medium for cell growth to make 10 6 PBS was added to the outermost wells of the 96-well plate to prevent evaporation, and 100 μL of L02 cells were added to each well to be measured. The cells were cultured for 24 h in a normal cell culture environment. After observing that the cell density and morphology were normal, the culture medium was removed, and the cells were washed once with an appropriate amount of PBS and the residual liquid was removed. Fresh 1640 complete culture medium without serum or phenol red was added, and then the cells were cultured under CO. 2 The cells were cultured in a serum-free incubator for 12 h. During the normalization period of cell starvation, 1640 complete medium without serum and phenol red was used to prepare a 5 mM APAP mixed solution and the same culture medium mixed solution containing different mass concentrations of BZ-W (0, 6.25, 12.5 and 25 μg / ml) were used to treat cells (APAP, APAP+BZ-W-6.25 μg / ml, APAP+BZ-W-12.5 μg / ml and APAP+BZ-W-25 μg / ml groups, respectively). A blank control group (CON group, cells treated with 1640 complete culture medium) was set up. After 24 hours of modeling, the supernatant of each group was aspirated and collected in the corresponding sterilized EP tubes and inserted on ice for later use. After rinsing with PBS and removing the residual liquid, a cell lysis solution with an equal volume of the supernatant was added to lyse the cells, and the cells were placed on ice for 10 minutes to fully lyse the cells. After lysis, the lysate was collected into the corresponding clean sterilized EP tubes and inserted on ice for later use. Subsequently, the experiment was carried out according to the method in the instruction manual of the detection kit, and the LDH values ​​in the supernatant and lysate were detected respectively. The lactate dehydrogenase release ratio was calculated according to the standard curve and formula provided in the instruction manual:

[0101]

[0102] 1.5 TUNEL fluorescence staining detection of APAP-induced L02 cell damage

[0103] The steps are as follows:

[0104] A. Prepare six-well plates and cell climbing plates in advance. The subsequent cell modeling process is the same as "1.3 CCK8 cytotoxicity experiment". The experimental group is set up with 6.25, 12.5, and 25 μg / mL BZ-W solution (named APAP+BZ-W-6.25μg / ml, APAP+BZ-W-12.5μg / ml, and APAP+BZ-W-25μg / ml groups, respectively), and a blank control group (PBS) and a model group (APAP) are set up.

[0105] B. Wash twice with PBS (5 min each time).

[0106] C. After the residual liquid is sucked out, add 50 μl TUNEL detection solution (red fluorescence result) to each well of the six-well plate.

[0107] When the plate is fully covered, PBS should be added to the gaps between the six-well plate to prevent evaporation.

[0108] Transfer the six-well plate to a light-proof environment or wrap it with tin foil and react at 37°C for 60 minutes.

[0109] D. Add 50 μL of 1X DAPI reagent to the sample and incubate at room temperature in the dark for 10 minutes.

[0110] E. Wash with PBS for 10 min.

[0111] F. After removing the cell slide plate, seal the slide, store it in a humidified box away from light, and transfer it to a fluorescence microscope for observation. Observe the results within the wavelength range of 550nm to 570nm.

[0112] 1.6L02 cell total protein extraction steps:

[0113] A. Transfer the treated cells from the incubator to the clean bench. Aspirate all the cell culture medium, add an appropriate amount of pre-cooled PBS to rinse the culture dish once or twice, and tilt the culture dish to aspirate the remaining liquid.

[0114] B. Add 80 μL of lysis mixture to the dish, cover it with a lid to allow the bottom to react evenly, and place it on ice for 10 minutes for lysis.

[0115] C. (The whole process is performed on ice) Use a cell scraper that has been cleaned with pure water to scrape the adherent cells along the bottom of the dish in a clockwise direction. The whole process should be controlled in about one minute and liquid splashing should be avoided. Collect the liquid and transfer it to a sterilized EP tube and lyse it on ice for 30 minutes.

[0116] D. Set the parameters in advance (4°C, 12000 rpm), place the scraped cells in a centrifuge for centrifugation, and take out the sample after centrifugation for 30 minutes.

[0117] E. Place the EP tube on ice after centrifugation, and transfer 60 μL of supernatant into a new sterilized EP tube as the protein supernatant stock solution for subsequent operations.

[0118] 1.7 Steps for detecting cell protein concentration using BCA kit:

[0119] A. Prepare the detection solution according to the ratio in the instructions of the protein quantification kit. For each sample, add 200 μL of detection solution to each replicate well.

[0120] B. Dilute the protein supernatant stock solution obtained previously with PBS ten times for concentration detection, and keep the rest on ice for later use. Prepare a clean 96-well plate, add the diluted test sample and BCA quantitative detection solution in sequence, and shake gently to mix after adding. Wait for it to fully react for 30 minutes (add an equal volume of PBS to the sample to the control well).

[0121] C. Use an ELISA reader to detect protein absorbance A at 37°C and 570 nm.

[0122] D. Calculate the protein content c of each sample based on the standard curve in the kit, and then calculate the required protein loading volume based on the WB experiment loading amount. The calculation results will unify the protein loading amount for each channel to be 30μg.

[0123] E. Calculate the amount of RIPA that needs to be added based on the calculation results, and prepare the protein mixed working solution for electrophoresis loading.

[0124] F. According to the calculation results, add the above sample at a ratio of bromophenol blue volume to one-fourth of the total sample volume. Vortex thoroughly and place in a metal bath at 98°C for 10 minutes before use.

[0125] 1.8 Western blotting

[0126] A. Preparation of stacking gel and separation gel

[0127] Clean the glass plate with distilled water and a test tube brush, then dry it until it is completely dry. After the glass plate cools down, clamp it on the glue rack to prevent glue leakage. Prepare a small beaker, a gun tip, and filter paper to prepare the separation glue. The preparation method of 15% separation glue is shown in Table 5.

[0128] Table 5 Preparation method of 15% separation gel

[0129] Material (15ml) 2 pieces of 1.5cm separation gel (30ml) 4 pieces of 1.5cm separation gel Ultrapure water 3.45mL 6.9mL 30%ACR / Bic 7.5mL 15mL 1.5 mol / L Tris·HCl 3.75mL 7.5mL 10% SDS 150μL 300μL 10% AP 300μL 300μL TEMED 6μL 12μL

[0130] Note: After preparing the glue, you can use a rotor to mix the solution at a uniform speed, then quickly use a 5mL pipette tip to uniformly add separation glue along one side until it reaches two-thirds of the height, and then quickly check whether each piece of glue is leaking. Then use anhydrous ethanol to add multiple times at a uniform speed from left to right to eliminate bubbles and seal the glue. The amount of glue added to each piece should not exceed 200μL. After 10 minutes, check if there is no abnormality (leakage, bubbles in the glue), and then let it stand at room temperature for 30 minutes. The time can be extended if the room temperature is low.

[0131] After the separation gel is completely solidified, pour out the sealing gel with anhydrous ethanol and wash it with filter paper to ensure that there is no other liquid on the gel for later use.

[0132] The preparation method of concentrated gel is shown in Table 6 below:

[0133] Table 6 Preparation method of 10% concentrated gel

[0134]

[0135] Wash and dry the comb in advance. The preparation steps of concentrated gel are similar to those of separation gel. After preparation, use a rotor to stir at low speed to mix evenly to avoid bubbles and place on ice for later use. After the comb cools down, use a 1mL pipette to add liquid in the same way as when adding separation gel and let it overflow to the highest point of the glass plate. Then insert the comb vertically and let it stand at room temperature for 30 minutes, and check the overall abnormality of the gel. After it is completely solidified, take out the glass plate and use it directly for subsequent experiments or soak it in the recovered electrophoresis solution and store it at 4°C.

[0136] B. Sample pretreatment:

[0137] Use the "1.6L02 total protein extraction procedure" to take the extracted protein sample and remove it from the metal bath for 3 minutes. After the sample cools to room temperature, vortex the sample to mix it; use a 20μL pipette to absorb the electrophoresis solution and gently rinse each sample channel to ensure that there is no floating gel in the channel.

[0138] C. Sample loading: Pipette 5 μL of Marker and add it to the first and last channels respectively.

[0139] μL volume was added into the channel at a constant speed to prevent it from rushing out of the channel.

[0140] D. Electrophoresis: During the concentrated gel electrophoresis stage, set the voltage condition to 75-80V for 15-20 minutes. The specific time depends on whether the marker has been completely separated and run through the concentrated gel. Then switch the voltage condition to 120V for electrophoresis of the separation gel until the bottom marker approaches the bottom of the separation gel.

[0141] E. Transfer: Prepare the transfer solution in advance (prepared in advance and stored at 4°C), calculate the size of the membrane according to the position of the target protein, cut the PVDF membrane into the corresponding size, and mark it. Before transfer, pinch a corner of the prepared membrane with clean tweezers to peel off the outer dustproof paper, soak it in methanol to activate the PVDF membrane, soak it for 5 minutes, and then put it in the electrotransfer solution to wash until there are no oil drops; use new clean gloves to take out the glass plate, cut off the separation gel and excess concentrated gel, and transfer the gel to the electrotransfer sandwich, and place the sponge, four newly cut filter papers, and gel symmetrically in order, and repeat the other half symmetrically. Take the PVDF membrane, gently attach the membrane to the cut gel and require no bubbles between the veneers, then soak the remaining part of the sandwich in the electrotransfer solution and cover it in order, clamp it, and put the electrotransfer sandwich in the correct direction into the electrotransfer tank, put an ice bag in the gap in the tank, set the program to 250mA, and the time is 90min. Put the entire electrotransfer tank in ice for electrotransfer.

[0142] F. Blocking incubation: Take out the transferred PVDF membrane from the electrotransfer tank and place it in a membrane washing box to avoid placing the membranes face to face. Slowly add TBST buffer solution along the membrane washing box to slightly cover the membrane. Shake and soak on a shaker for 3 times, each time for 10 minutes. During this period, prepare non-specific antigen blocking solution with skim milk powder and TBST buffer solution (2.5g milk powder + 25ml TBST solution → two membranes). Put the membrane face up into the membrane washing box, add blocking solution along the wall of the box and block at room temperature for 1h (shake at the slowest speed on the shaker). After incubation, wash three times in the same way to remove the milk powder.

[0143] G. Primary antibody binding reaction: Take a 50mL centrifuge tube, add 10mL of primary antibody diluent, select the dilution ratio according to different antibodies (generally 1:1000) and add the corresponding primary antibody; stick the back of the PVDF membrane to the wall, put the side with the marker into the centrifuge tube, avoid cross-covering when putting multiple membranes in, put the centrifuge tube in a rotating shaker and incubate overnight at 4℃. After 1 to 2 days, take it out and put it in the membrane washing box, add TBST buffer and rinse three times on a shaker at room temperature, 10 minutes each time.

[0144] H. Secondary antibody binding reaction: Take the washed membrane and dilute it in a new 50ml centrifuge tube with TBST as the solvent to prepare the secondary antibody working solution (generally at a ratio of 1:10000), and then operate the same antibody binding reaction. The secondary antibody reaction condition is to react at room temperature for 1 hour.

[0145] I. Development: Take out the membrane bound to the secondary antibody and rinse it in the same way for later use. Take out the membrane and dry it with filter paper. Place the detection surface upside down in the prepared developer and keep it away from light. After the reaction time varies from 30s to 2min, stop the color development. Take out the membrane and dry it with filter paper. Place it in the developing equipment for development and photography.

[0146] J. The data were then saved and analyzed.

[0147] 1.9 Quantitative PCR (qPCR)

[0148] (1) Cell RNA extraction

[0149] A: After the cell modeling treatment, the culture medium was discarded with a sterilized rubber-tipped pipette, and the cells were washed three times with pre-cooled PBS.

[0150] B: After absorbing the residual liquid by tilting the dish, add an appropriate amount of Trizol lysis buffer and lyse at room temperature for 15 minutes. Then use a cell scraper washed with pure water to mix the liquid in the dish and scrape it off to avoid splashing. Then use a pipette to transfer the cell lysate to a sterilized EP tube.

[0151] C: 15 minutes after completing the above operation, add 200 μL of chloroform solution to each tube, and all samples are repeatedly inverted for 30 seconds at the same time and then allowed to stand at room temperature for 5 minutes.

[0152] D: First place the EP tube into a refrigerated centrifuge, centrifuge for 15 minutes at 0-5°C and 12,000 rpm (precooling required), then take out the original EP tube and transfer the upper liquid phase to a new sterilized EP tube.

[0153] E: Add isopropanol solution to the newly collected colorless liquid phase to precipitate RNA, shake it slowly to mix, and then let the EP tube stand at room temperature for 15 minutes.

[0154] F: Take out after centrifugation under the same conditions, and a precipitate should appear at the bottom of the tube.

[0155] G: After aspirating the residual liquid as much as possible and collecting the precipitate at the bottom, add 75% ethanol (diluted with DEPC water), repeatedly tilt and wash the precipitate and place it in a bowl for 5 minutes. Then centrifuge again under the same conditions for 5-10 minutes and aspirate the supernatant.

[0156] I: Place the above EP tube in a clean area at room temperature and open it to air dry naturally until it is translucent (no more than 15 minutes). Then, depending on the amount of precipitation, add DEPC water (usually 20 μL; tissue RNA is generally 30-40 μL) to dissolve the RNA at 55-60°C. The sample can be stored for later use or transferred to an ultra-low temperature refrigerator at -80°C.

[0157] (2) Reverse transcription reaction

[0158] A: To remove DNA:

[0159] Add various DNA removal reagents to the high temperature and high pressure sterilized EP tube for denuclease and gently shake to mix the DNA removal working solution. Prepare 10 μL of reaction solution, the RNA content of 10 μL of reaction solution is 1 μg. Specifically, prepare the reaction solution according to the sequence in Table 7.

[0160] Table 7 Preparation of 10 μL reaction solution

[0161]

[0162] For the SYBR Green method, 1 μg RNA was added to 10 μL of the system and then reacted at 42°C for 5 minutes.

[0163] B: Reverse transcription reaction

[0164] According to the instructions of the reverse transcription kit, as shown in Table 8.

[0165] Table 8 Reverse transcription reaction system

[0166]

[0167] Reaction conditions: 37°C, 15 min; 85°C, 5 s; 4°C, ∞.

[0168] C. Fluorescence quantitative PCR system:

[0169] SYBR Green Master MIX, 5 μL; Forward Primer, 0.5 μL; Reverse Primer, 0.5 μL; cDNA, 0.5 μL; DEPC water, 3.5 μL; Total, 10 μL.

[0170] Reaction procedure:

[0171] 1. Pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 30 s;

[0172] 5. React for 40 cycles and analyze the melting curve. D. Export the data and calculate the relative expression of each target gene compared with the internal reference gene.

[0173] 2. Experimental results

[0174] 2.1 Cytotoxicity of BZ-W on L02 cells and its effect on APAP-induced cell damage

[0175] The experimental results are as follows Figure 4 As shown, Figure 4 The figures are the cytotoxicity test of BZ-W on L02 cells and the effect on APAP-induced cell damage, wherein A is the activity of L02 cells detected by CCK8 at different concentrations after administration of BZ-W to L02 cells at different concentrations for 24 hours; B is the LDH release ratio of cells treated with different concentrations of BZ-W in the AILI (5mM APAP, 24h) model; C is the morphology of L02 cells in each group in B.

[0176] In order to determine whether BZ-W itself has a certain cytotoxic effect on L02 cells, CCK-8 reagent was used to detect whether BZ-W itself has a drug toxic effect on L02 cells by detecting cell activity after simple administration of different mass concentrations of BZ-W (0, 3.125, 6.25, 12.5, 25, 50, 100 μg / ml). According to the observation results, it showed that there was little effect on cell activity in the range of 3.125-50 μg / ml ( Figure 4 Therefore, we chose the optimal concentration of 12.5 μg / ml due to its relatively high cell activity, and set three safe doses (6.25, 12.5, and 25 μg / ml) around it for subsequent experiments.

[0177] After it was confirmed that BZ-W itself had no significant effect on the activity of L02 cells, in order to continue to test whether the effect of BZ-W against APAP was clear, the lactate dehydrogenase (LDH) release rate of L02 cells was tested. The results showed that the LDH release rate of the model group and the APAP group was significantly increased (p < 0.001); in contrast, the LDH release rate of the medium and high dose BZ-W treatment groups decreased significantly to varying degrees (p < 0.01, p < 0.05), among which the medium dose decreased most significantly ( Figure 4 (B) The results showed that after administration of BZ-W, the release of LDH into the extracellular space due to APAP-induced damage of L02 hepatocytes was significantly alleviated, indicating the protective effect of BZ-W against AILI at the cellular level.

[0178] At the same time, through the observation of cell morphology, it was found that the APAP group had more serious apoptosis, the number of floating apoptotic cells increased significantly, and the volume of most surviving cells also shrank. Compared with the APAP group, the cell state of the medium-dose group of BZ-W was significantly better, and the number of apoptotic cells was significantly reduced, while the number of apoptotic cells in the high-dose group did not change significantly ( Figure 4 Middle C).

[0179] In summary, BZ-W can improve APAP-induced L02 cell injury and has a significant anti-AILI protective effect at the cellular level.

[0180] 2.2 Effects of BZ-W on APAP-induced L02 hepatocyte injury and anti-apoptosis

[0181] The results of the anti-inflammatory and anti-apoptotic effects of low, medium and high doses of BZ-W (BZ-W(L) group, 6.25μg / ml; BZ-W(M) group, 12.5μg / ml, BZ-W(H) group, 25μg / ml) on APAP-induced L02 hepatocyte injury are shown in Figure 5 As shown, Figure 5Figure 3 is the effect of BZ-W on APAP-induced inflammation and apoptosis of L02 cells, where A is the results of fluorescence staining TUNEL for different doses of BZ-W groups after cell crawling, APAP (5mM) induced acute liver injury in L02 cells for 24 hours; B is the expression of Bax mRNA level of apoptosis-related gene induced by APAP in L02 cells under different concentrations of BZ-W treatment after APAP (5mM) induced acute liver injury in L02 cells for 24 hours; C is the expression of Bax and Bcl-2 protein levels in APAP-induced L02 cells after BZ-W administration; D is the expression of IL-1β and Tnf-α mRNA levels of inflammation-related genes in APAP-induced L02 cells after BZ-W administration.

[0182] Inflammation and apoptosis are the key pathogenesis of AILI and are also important factors that aggravate AILI liver injury. In order to determine the effect of BZ-W on inflammation and apoptosis in APAP-induced L02 cell injury, we used TUNEL fluorescent probe detection kit to perform fluorescent staining on cells, such as Figure 5 As shown in A, the red fluorescence apoptosis ratio increased significantly after APAP administration, while the purple fluorescence positive ratio after BZ-W administration was significantly reduced after merging with the nuclear dye DAPI in the low and medium dose groups, indicating that cell apoptosis was significantly improved. We also detected the mRNA transcription level of the pro-apoptotic gene BAX ( Figure 5 B) and protein expression levels as well as the protein expression level of the anti-apoptotic gene BCL2 ( Figure 5 C), and the mRNA transcription levels of IL-1β and TNF-α, genes related to pro-inflammatory factors in inflammation, were also detected ( Figure 5 (middle D).

[0183] The results showed that at the mRNA level, compared with the CON group, the mRNA levels of BAX, IL-1β, and TNF-α in the model group were significantly increased (p<0.0001); while in BZ-W, the high-dose group significantly reduced the trend of BAX (p<0.01, p<0.001), and also significantly reversed the trend of IL-1β and TNF-α (p<0.05, p<0.01, p<0.001). At the protein level, BAX slightly increased after APAP administration, and the three dose groups of BZ-W significantly reduced its protein expression level, while BCL2 significantly decreased after APAP administration. After medium and high doses of BZ-W, the protein expression of BCL2 was significantly upregulated. The results showed that BZ-W can significantly reduce the inflammation and apoptosis of L02 cells induced by APAP; it is speculated that BZ-W can improve AILI by improving cell inflammation and apoptosis.

[0184] In summary, after clarifying that BZ-W has no significant effect on the cell viability of L02, cells were cultured in a medium containing 5 mM APAP for 24 h to establish a model, and cells were administered with three screening doses of BZ-W. The results showed that through cell morphology observation and detection of the LDH release ratio of cells, it was found that compared with the APAP group, BZ-W administration significantly alleviated the damage of L02 cells. On the other hand, through the observation of the results of cell TUNEL fluorescence staining, it was observed that the degree of cell apoptosis was significantly alleviated, and qPCR and WB experiments detected that BZ-W administration group could significantly reduce inflammation and apoptosis at the transcriptional and translational levels. Therefore, at the cellular level, BZ-W significantly alleviated cell AILI injury by reducing cell inflammation and improving cell apoptosis, clarifying the direct protective effect of BZ-W on cell AILI.

[0185] Example 3

[0186] Study on the Effect and Mechanism of BZ-W on APAP-Induced Liver Injury in Mice

[0187] 1. Experimental Methods

[0188] 1.1 Experimental Animals and Environment

[0189] The Ethics Committee of the Animal Experiment Center of Guangdong Pharmaceutical University reviewed all processes of the animal experiment and approved the conduct of the experiment. All animal experiment procedures were carried out in accordance with the regulations of the Chinese Animal Welfare Law. Male Balb / c mice (22 - 26 g, 6 - 8 weeks old, 34 mice) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Animal Production License No.: SCXK(Yue)2022 - 0063), and the second batch of mice of the same age and sex (14 mice) were purchased from the Guangdong Provincial Animal Experiment Center (Production License No.: SCXK(Yue)2022 - 0002).

[0190] The above mice were raised and experimented in the Guangyao Animal Experiment Center (SPF level) (SYXK(Yue)2022 - 0125). Mice had free access to drinking water and mouse food, the temperature was maintained at 25 - 26 °C, and the humidity was maintained at 55 - 65%.

[0191] 1.2 Establishment of a Mouse Acute Liver Injury Model Induced by Excessive APAP

[0192] The first batch of 34 mice was randomly divided into 5 groups, namely the normal control group for 12 h model CON group (n = 6), the 12 h model administration APAP group (n = 7), the 12 h model positive drug NAC group (n = 7), the 12 h model BZ-W low dose (50 mg / ml) group (named BZ-W(L), (n = 7)), and the 12 h model BZ-W high dose (100 mg / ml) group (named BZ-W(H), n = 7).

[0193] The second batch of 20 mice were randomly divided into 3 groups: normal control group 24h model CON group (n=6), 24h model APAP administration group (APAP-24h, n=7), 24h model BZ-W administration BZ-W group (BZ-W concentration was 100 mg / ml, BZ-W-24h, n=7).

[0194] The experimental steps are as follows Figure 6 As shown, Figure 6 This is a diagram of the mouse modeling scheme. Figure 6 The specific steps of the mouse modeling scheme are as follows: all Balb / c mice were adaptively fed for one week after quarantine, and mice aged 6-8 weeks (6-8W) were used. All mice in the BZ-W administration group were pre-administered by gavage (0.01ml / g) for 7 days (once every 24h) before APAP modeling. BZ-W was prepared with normal saline as the dissolution medium. At the same time, the mice in the other groups were gavaged with an equal volume of normal saline. On the last day of pre-administration, all mice were fasted but not watered for 22h; after 22h, the mice were restored to diet. The mice other than the CON group were injected with a volume of 50:1 (g / kg) according to the body weight ratio. / ml) was intraperitoneally injected with APAP (250mg / kg) solution, which was also prepared by dissolving in saline. The CON group was injected with an equal volume of saline. One hour after the intraperitoneal injection of APAP, the NAC group was intraperitoneally injected with 0.01ml / g of NAC solution (50mg / kg), which was prepared by dissolving in saline. The other groups were injected with the same volume of saline. After the 12h and 24h modeling time, the 7 groups of mice were intraperitoneally injected with 0.01ml / g of 5% chloral hydrate solution to anesthetize the mice for sampling. The steps are as follows:

[0195] (A) Remove the eyeball and collect blood, avoiding contact with the tube wall. Collect the blood in a sterilized EP tube and keep it at room temperature for use before continuing to collect other tissues. After half an hour, put the sample into a centrifuge and centrifuge it at 3500rpm for 15 minutes. Obvious plasma-serum stratification can be observed. Otherwise, repeat the operation. After centrifugation, carefully aspirate the upper serum layer and transfer it to a new sterilized EP tube. Dispense it for subsequent use or store it in a -80℃ refrigerator for later use.

[0196] (B) After blood was collected, the mice were killed by dislocation, fixed, and the abdomen was cut open to remove the liver, which was then cut into three parts. The larger part of the liver was used to make paraffin sections, which were then cut into 1X 1X 0.5 cm blocks and fixed in fixative. The second-to-last small part of the liver was added with RNA protective agent and quickly frozen with liquid nitrogen. The rest of the liver was quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for later use.

[0197] (C) The small intestine and colon of the mice were removed and the contents of the small intestine and colon were rinsed with PBS. The small intestine was cut into two parts. One part was used to make paraffin sections and fixed in neutral formalin at 4°C for 24 h, and the other part and the contents of the colon and cecum were quickly frozen with liquid nitrogen and stored in a -80°C refrigerator for use.

[0198] 1.3 H&E staining of mouse liver

[0199] (1) Tissue dehydration and embedding

[0200] (A) Mouse liver tissue fixed with neutral formalin at 4°C for 24 h was placed in a tissue fixation tank and rinsed with running water to remove the fixative and accumulated acetic acid.

[0201] (B) The embedding cassettes were filled into the mechanical basket of the automatic dehydrator and the mouse liver tissue was dehydrated according to Table 9.

[0202] Table 9 Mouse liver tissue dehydration steps

[0203]

[0204] (C): The dehydrated tissue was stored in a cool place and then embedded in a paraffin embedding machine. The largest section of the liver tissue was placed face down on the mold. After adding high melting point paraffin wax to the mold, the mold was transferred to a -20℃ freezer to solidify the wax block, which was then stored in a wax block cabinet in a cool place.

[0205] (2) Tissue sections

[0206] A: Place the paraffin block in a -20°C freezer overnight, take it out and fix it on the fixture in a semi-automatic paraffin sectioner, and trim it at a thickness of 10 μm until the largest section of the tissue is obtained.

[0207] B: Adjust the advance distance of the slicer to 4μm, refrozen the tissue and use a new blade or knife to slice, spread the slices on a 40℃ water surface, then scoop out the slices and air dry them at room temperature.

[0208] (3) Drying and dewaxing

[0209] A: Select white tissue slices with complete structure and good sectioning results and place them in a 65℃ oven for preliminary dewaxing and baking for 1 hour.

[0210] B: Place the baked tissue sections into the dewaxing station for dewaxing as soon as possible (complete the dewaxing and hydration process in the order of xylene, gradient diluted ethanol and distilled water).

[0211] (4) The H&E staining steps are shown in Table 10 below.

[0212] Table 10 H&E staining steps

[0213]

[0214] After sealing with neutral gum, the slides were observed under an Olympus microscope system, and the liver tissues were photographed under 10X and 20X lenses, while the intestinal tissues were photographed under 4X and 20X lenses.

[0215] 1.4 Determination of ALT and AST in mice

[0216] The serum collected after centrifugation or the serum taken out from -80°C was diluted 30-50 times with physiological saline for later use, and the remaining samples were stored in a -80°C refrigerator. Then, a clean 96-well plate was prepared, and the biochemical levels of the two types of enzymes in the mouse serum were determined according to the steps in the Nanjing Jiancheng ALT & AST test kit instructions, as shown in Table 11.

[0217] Table 11 ALT & AST test methods

[0218]

[0219] This kit can measure the ALT and AST activities in various animal serum (plasma), tissue and other samples. For specific experimental steps, reagents and operating procedures, please refer to the instructions above. Finally, the obtained data will be used to calculate the enzyme activity according to the standard curve in the instructions.

[0220] Note: When the enzyme activity of the sample exceeds 150 Karmen's units, the data calculated using the standard curve will be inaccurate and the sample must be further diluted with saline and re-measured.

[0221] 1.5 Liver tissue CD68 immunohistochemistry (IHC)

[0222] The steps of tissue dehydration, embedding and section dewaxing are the same as those in “H&E staining of mouse liver”. The subsequent operation procedures are shown in Table 12.

[0223] Table 12 Immunohistochemistry experimental steps

[0224]

[0225] After sealing with neutral gum, the slides were transferred for observation under a microscope and then photographed under a 10X or 20X lens of an Olympus microscope equipped with an imaging capture system.

[0226] 1.6 Reactive oxygen species detection ROS probe fluorescence staining

[0227] The steps of tissue dehydration, embedding and section dewaxing were the same as those in “H&E staining of mouse liver”. The subsequent operations were performed according to the instructions of the kit. The process is shown in Table 13.

[0228] Table 13 ROS fluorescent probe staining steps

[0229]

[0230] After sealing with anti-fading sealing agent, the slides were transferred to a light-proof humid box for storage, and then transferred to a fluorescence microscope for observation and filming. The optimal excitation wavelength of DCFH-DA is 550, 485 (500±15nm), and the emission wavelength is 525 (530±20nm) (green fluorescence).

[0231] 1.7 TUNEL fluorescence staining

[0232] The steps of tissue dehydration, embedding and section dewaxing were the same as those in “H&E staining of mouse liver”. The subsequent operation was carried out according to the instructions of the one-step TUNEL fluorescence kit. The process is shown in Table 14.

[0233] Table 14 One-step TUNEL fluorescence staining steps

[0234]

[0235] After sealing with anti-quenching agent, transfer to a light-proof humid box for storage, and then observe under a fluorescence microscope. Film the film at a wavelength of 550-570nm.

[0236] 1.8 Western Blot

[0237] (1) Tissue protein extraction

[0238] A. Take out the tissue from the -80℃ freezer and thaw it. Prepare sterilized scissors and other instruments. Wash with physiological saline to remove excess blood clots and then dry with filter paper. Cut the required sample of about 20-25mg tissue and put it into a sterilized EP tube. Add two steel beads to each tube and add 500μL tissue lysis solution.

[0239] B. Place the sample in a homogenizer and homogenize for 1 minute at 60 Hz. Homogenize twice. Remove the sample and place it on ice to continue lysis for 30 minutes.

[0240] C. Centrifuge at 4℃, 12000rpm for 30min, take out the sample and observe the obvious stratification. Transfer the upper liquid phase to a newly sterilized EP tube for protein quantification.

[0241] (2) BCA kit to detect protein concentration

[0242] The operation process is the same as "BCA kit for detecting cell protein concentration in Example 2"

[0243] (3) Western blotting

[0244] The subsequent operation process is the same as the "immunoprecipitation step of Example 2".

[0245] 1.9 Quantitative PCR (qPCR)

[0246] (1) Tissue RNA extraction

[0247] A. Take out the frozen tissue from the -80℃ freezer, take it out from the RNA protective solution after thawing, wash off the protective solution and residual blood in a large dish, dry it with filter paper, and use sterile tools to cut 20-25mg of tissue and put it into a new sterilized EP tube, put 2 sterilized steel beads in it and add 1mL Trizol (TAKARA), and then homogenize it with the homogenization steps in "3.3.9".

[0248] B. Add 100 μL of chloroform to the above EP tube, shake vigorously for 1 min, and leave at room temperature for 2-3 min.

[0249] C. Centrifuge at 4℃, 12000rpm for 15min, separate the layers, take the bottom layer of clear liquid and put it into a sterilized nuclease-free EP tube.

[0250] D. Add an equal volume of isopropanol to precipitate RNA, invert repeatedly for 30 seconds, and then place at room temperature for 10 minutes.

[0251] E. Centrifuge for 10 minutes under the same conditions; the bottom precipitate can be obtained after centrifugation.

[0252] F. Carefully discard the supernatant and add 1 mL of 75% ethanol diluted with DEPC water to wash the precipitate twice, then centrifuge under the same conditions for 5-10 minutes.

[0253] G. Use the nuclease-free pipette tip to discard the supernatant and air-dry at room temperature (no more than 15 minutes)

[0254] H. Add about 40-80 μL of DEPC water and heat at 55-60℃ to dissolve the RNA, then directly reverse transcribe or freeze at -80℃.

[0255] I. Determination of RNA content.

[0256] (2) Reverse transcription reaction

[0257] The subsequent operation process is the same as "2.3.8 Fluorescence quantitative PCR (qPCR)"

[0258] 2. Experimental results

[0259] Effect of 2.1BZ-W on APAP-induced acute liver injury in mice

[0260] The results are as follows Figure 7 As shown, Figure 7The figure shows the effect of BZ-W on APAP-induced acute liver injury in mice, where A is the H&E staining result of mice 12 hours after administration of APAP (10X); B is the statistical graph of the area of ​​liver pathological necrosis in the 12-hour model; C is the ALT and AST content in mouse serum detected by the kit; D is the H&E staining result of mice 24 hours after administration; E is the statistical graph of the area of ​​liver pathological necrosis in the 24-hour model; F is the ALT content in mouse liver tissue detected by the kit.

[0261] All mice were sampled 12 and 24 hours after intraperitoneal injection and the pathological morphology of liver tissue paraffin sections was observed by H&E staining to evaluate the damage of liver tissue. Figure 7 As shown. A shows that after 12 hours of APAP administration, compared with the CON group, the APAP group had extensive liver cell damage (p < 0.0001), with extensive liver necrosis, sudden shrinkage or loss of cell nuclei, and extensive bleeding and congestion in the central venous region. The positive drug NAC significantly reduced the necrosis area (p < 0.001), while low and high doses of BZ-W also reduced the size of congestion damage, especially high doses of BZ-W significantly reduced the damage area, and the degree of improvement was close to that of the positive drug NAC group. Figure 7 B is correct Figure 7 The necrotic area statistics in A showed that the low- and high-dose BZ-W (L) groups significantly reduced the overall necrotic area of ​​the liver lobules in the APAP group (p < 0.05, p < 0.01).

[0262] Figure 7 Middle C is the result of the detection of the content of alanine aminotransferase ALT and aspartate aminotransferase AST in the serum of mice in each group of the APAP administration model for 12 hours. The results show that after the administration of APAP, the content of ALT and AST increased significantly. The BZ-W administration group significantly reversed this trend, especially the high-dose group (BZ-W-(H)), which significantly reduced this value, and its reduction effect was almost the same as that of the positive drug group (p < 0.05, p < 0.01). After observing the obvious therapeutic effect of the BZ-W high-dose group (BZ-W-(H)), APAP was continued to be administered at a high dose concentration for 24 hours to observe whether BZ-W still had a therapeutic effect.

[0263] Figure 7 D in the middle is the H&E staining result of the liver tissue of the 24h model mice (10X), and the results show that BZ-W (100mg / ml) still significantly reduces the area of ​​APAP-induced liver damage. Figure 7 China E statistics Figure 7 The results of D showed that BZ-W (100 mg / ml) significantly reduced the area of ​​APAP-induced liver injury (p < 0.05). Figure 7Figure F is the detection of ALT content in liver tissue of 24h model mice. The detection data showed that BZ-W (100mg / ml) significantly reduced ALT content (p<0.05), and the result was consistent with the pathological results and the statistical results of the damaged area.

[0264] In summary, BZ-W significantly reduced APAP-induced liver injury in mice and still had a therapeutic effect at different time points (12h) and (24h) of AILI, which clearly showed that BZ-W had a sustained and potent therapeutic effect on APAP.

[0265] Effect of BZ-W on APAP-induced apoptosis of mouse hepatocytes

[0266] The experimental results are as follows Figure 8 As shown, Figure 8 Figure 2 is the effect of BZ-W on APAP-induced mouse hepatocyte apoptosis; A is the result of TUNEL fluorescent probe staining of mouse liver tissue paraffin section; B is the result of qPCR detection of mouse apoptosis-related gene transcription level expression; C is the expression level of mouse apoptosis-related proteins BCL-XL, BAX, and BCL-2; D is the expression level of mouse apoptosis-related protein CYTO C detected by WB experiment.

[0267] In order to detect cell apoptosis, we used a one-step TUNEL fluorescent staining detection kit to detect mouse liver cell apoptosis. Figure 8 As shown in A, red fluorescence is the positive expression of the broken nuclear DNA part of TUNEL staining, and purple fluorescence is the positive apoptotic nucleus after merging with the nuclear dye DAPI. From the results, it can be observed that after the administration of APAP, a large number of hepatocytes in the injured area of ​​the central vein of the mouse liver appeared apoptotic, and the NAC positive drug significantly improved the apoptosis, while the BZ-W administration group (100mg / ml) also significantly alleviated the apoptosis, and the degree was almost the same as NAC. Therefore, we detected the expression of apoptosis-related genes at the transcription and translation levels, such as Figure 8 Medium B and Figure 8 As shown in C, at the transcriptional level, BZ-W (100 mg / ml) significantly reduced the upregulation of the pro-apoptotic gene Bax caused by APAP administration, and at the same time significantly upregulated the anti-apoptotic genes Bcl-2 and Bcl-xl, which had low transcriptional levels after APAP administration; Figure 8 D in the middle shows that the BZ-W group (100 mg / ml) reduced the protein content of CYTO C (cytochrome C) released after cell apoptosis, indicating that the cell apoptosis process was inhibited. At the same time, the protein expression levels of Bax, Bcl-2 and Bcl-xl were consistent with the qPCR results.

[0268] In summary, BZ-W (100 mg / ml) can significantly reduce APAP-induced hepatocyte apoptosis and play a strong anti-apoptotic role in the process of AILI injury.

[0269] Effect of BZ-W on APAP-induced liver inflammation in mice

[0270] Aseptic inflammation is a core mechanism in the AILI process. The continued aggravation of this process is also a key factor in AILI-induced liver cell death and liver failure, and is the main cause of liver damage in the "second hit" of AILI. During the aseptic inflammation period of APAP-mediated liver damage, a large number of immune monocytes tend to the liver and are recruited in the necrotic area near the central vein of the liver. CD68 is a biomarker for macrophages. Through immunohistochemistry experiments, we detected the inflammatory infiltration of macrophages in the damaged area of ​​liver tissue for 12 hours. The results are as follows: Fig. 9 As shown, Fig. 9 Figure 3 is the effect of BZ-W (100 mg / ml) on APAP-induced liver inflammation in mice, wherein A is the result of CD68 immunohistochemical staining 12 hours after APAP administration in mice, B is the statistical result of the CD68 positive area in Figure A, C is the result of CD68 immunohistochemical staining 24 hours after APAP administration in mice, D is the statistical result of the CD68 positive area in Figure C, and E is the result of WB experiment detecting the change of inflammatory factor IL-1β protein expression.

[0271] The results showed that compared with the CON group, the brown CD68 positive area increased significantly after 12 hours of APAP administration ( Fig. 9 In the middle A), the positive drug NAC group significantly reduced this trend, and the BZ-W group also significantly reduced the positive expression of CD68. After the positive area was counted ( Fig. 9 B), which also reflects that BZ-W significantly reduced the total CD68-positive area. Since inflammation mainly occurs in the late stage of AILI, we further detected the results of macrophage inflammatory infiltration 24 hours after APAP administration and statistically analyzed the results ( Fig. 9 Middle (C and D) also reflected that BZ-W significantly reduced the degree of inflammation after administration of APAP.

[0272] Immune cells stimulated by external factors will secrete inflammatory factors to activate and promote inflammation. The translation level changes of the inflammatory factor IL-1β were detected by WB ( Fig. 9 Middle E). WB results showed that APAP upregulated the protein expression of IL-β, while the protein expression of IL-1β in the BZ-W treatment group was significantly reduced.

[0273] In summary, BZ-W can reduce the transcription of NLRP3 inflammasome, thereby reducing the mRNA transcription and protein translation expression levels of various inflammatory factors, thereby reducing APAP-induced liver inflammation in mice in the middle and late stages of AILI, thereby playing a role in treating AILI.

[0274] Effects of BZ-W on APAP metabolic pathway and hepatic oxidative stress induced by HQ

[0275] Excessive APAP can lead to the accumulation of its toxic byproducts in the body and cause a significant increase in the release of ROS from liver mitochondria, leading to subsequent damage to mitochondrial structure and function and cell necrosis. Therefore, we examined whether BZ-W plays a protective role by affecting the drug metabolism pathway of APAP production, NAPQI, and oxidative stress. Fig.10 As shown, Fig.10 Figure 3 shows the effects of BZ-W on the drug metabolism of APAP and the oxidative stress process induced by it. A is the result of ROS fluorescent probe staining of small liver tissue paraffin sections, B is the result of qPCR detection of the mRNA expression level of genes related to phase II drug metabolic enzymes in mice, and C is the result of Western blot detection of the protein expression level of mouse drug metabolic enzyme CYP2E1.

[0276] The results are as follows Fig.10 The results in middle A show that the green fluorescent part is the ROS positive point. Compared with the CON group, the content of reactive oxygen in the tissue increased sharply after the administration of APAP, but BZ-W had little effect on it. At the same time, we aimed at the generation process of AILI metabolites that cause ROS at the mRNA level ( Fig.10 B) and protein levels ( Fig.10 Middle C) The expression changes of phase II metabolic enzymes CYP2E1, Sult1a1, and Ugt1a1 in the process of AILI were detected, and the results showed that BZ-W did not affect the phase II metabolism of APAP. The results showed that BZ-W treatment of AILI did not directly affect the in vivo drug metabolism of APAP and did not affect its subsequent oxidative stress process. It is speculated that the effect of BZ-W treatment of AILI is mainly exerted on the anti-apoptosis and anti-inflammatory mechanism in AILI.

[0277] The above results indicate that BZ-W can treat AILI by improving hepatocyte apoptosis and resisting liver inflammation, but does not alleviate liver damage in mice by affecting the APAP metabolic pathway and improving liver oxidative stress.

[0278] Example 4

[0279] Effects of BZ-W on gut-liver interaction in AILI

[0280] 1. Experimental methods

[0281] 1.1 Experimental animals, environment, and reagents

[0282] This experiment was approved by the Ethics Committee of the Animal Experiment Center of Guangdong Pharmaceutical University, and all animal experimental procedures were carried out in accordance with the provisions of the Animal Welfare Law of China. Male Balb / c mice (20-26g, 6-8 weeks old, 14 mice) purchased from the Guangdong Provincial Animal Experiment Center were used for fecal microbiota transplantation experiments. (Animal Production License No.: SCXK (Guangdong) 2022-0002). The above mice were raised and experimented in the Animal Experiment Center (SPF level) (SYXK (Guangdong) 2022-0125). Mice had free access to drinking water and mouse food, the temperature was maintained at 25-26°C, and the humidity was maintained at 55-65%.

[0283] Methanol, ethanol, acetonitrile, formic acid, and isopropanol were all of mass spectrometry grade (manufacturer: Thermo Fisher Scientific).

[0284] The treatment concentration of BZ-W was 100 mg / ml.

[0285] 1.2 Mouse fecal microbiota transplantation experiment

[0286] Schematic diagram of fecal microbiota transplantation experiment Fig.11 As shown, specifically:

[0287] (1) Fourteen mice (named WT, 6-8 weeks old) underwent fecal microbiota transplantation (FMT) and were randomly divided into two groups: a group receiving normal mouse FMT (named WT) and a group receiving normal mouse FMT (named WT). FMT(WT) , n=7) and the group receiving BZ-W mice FMT (BZ-W) (named WT FMT(BZ-W) ), n=7).

[0288] Stool Sample Collection:

[0289] Collect all feces of the BZ-W administration group after 7 days of pre-administration of BZ-W (100 mg / ml) in Example 3 (mouse modeling scheme) and all feces of the CON group mice after 7 days of pre-administration of physiological saline: put clean paper in a clean cage, grab the mice in the same group and put them in the cage immediately, grab each mouse in the same group, gently pat the mouse abdomen to promote defecation, and immediately use sterile tweezers to collect the mice in a sterile centrifuge tube after defecation, place them on ice, and transport them to the laboratory clean bench within 2 hours, and package them according to the subsequent grouping dosage requirements. If the fecal bacteria liquid cannot be made as soon as possible, the fecal sample is quickly transferred to a -80°C refrigerator for storage after being poured with liquid nitrogen.

[0290] The required feces volume is calculated as follows: 1 feces pellet is about 30 mg, the fecal microbiota concentration is 0.125 g / mL, and the intragastric administration is 200 uL per mouse. For example, a fecal microbiota transplant of 40 mice requires 8 mL of fecal microbiota solution, so at least 1 g of feces (more than 35 pellets) must be collected.

[0291] Preparation of fecal bacteria liquid:

[0292] 1) Place freshly collected feces or feces that have been thawed at room temperature for 10 minutes on ice, transfer and divide into groups.

[0293] 2) After sterilization with UV light for 30 minutes, turn on the clean bench and turn on the fan for 15 minutes. 3) Prepare and sterilize the required instruments and materials (centrifuge tubes, mesh filters, etc.) and put them in the clean bench for use. 4) Transfer the processed fecal sample to the clean bench for operation, dissolve it with sterile saline at a ratio of 1:10 (g / ml) to prepare a fecal suspension, stir and refine the larger feces during the process, and then set it aside. 5) Use 200-1000 mesh sterile mesh filters to filter layer by layer. 6) Collect and transfer the filtrate into a new sterilized centrifuge tube. 7) Vortex for 5 minutes to obtain a heavy suspension. 8) Centrifuge at 3000rpm for 5-10 minutes. Note: The centrifugation speed should not be too high, 8000rpm will precipitate the bacteria. 9) The fecal suspension obtained after centrifugation is directly used for subsequent fecal microbiota transplantation experiments. Alternatively, the prepared fecal bacteria liquid can be added with 10% glycerol in a clean bench to preserve bacterial activity and then transferred to a -80°C refrigerator for storage (it should be aliquoted in advance to avoid freezing and thawing and taken out for use).

[0294] Preparation of pseudo-germ-free mouse animal model (recipient mice):

[0295] Vancomycin (100 mg / kg), thiomycin (200 mg / kg), metronidazole (200 mg / kg), and ampicillin (200 mg / kg) were dissolved in sterile water and administered intragastrically (10 μL / g) for 5 consecutive days to deplete intestinal microorganisms. Note: Observe the state of the mice during intragastrically administering antibiotics, and stop administering antibiotics in time when the mice are weak.

[0296] (2) Fecal microbiota transplantation:

[0297] All pseudo-germ-free mouse models were adaptively fed for one week after quarantine. Before APAP modeling, they were given a broad-spectrum antibiotic cocktail therapy (ABX) for 5 days by combined oral administration of antibiotics to deplete the existing microbiota, and then oral administration of the corresponding fecal microbiota solution for 3 days to complete the fecal microbiota transplantation. FMT(WT) The CON group of mice was transplanted with fecal bacteria, and the WT FMT(BZ-W)The first group consisted of mice transplanted with BZ-W-administered fecal bacteria. On the last day of oral gavage, the mice were fasted but not watered for 22 hours. After 22 hours, the mice were restored to diet. All mice were intraperitoneally injected with 20 μL of APAP (250 mg / kg) solution per gram of body weight, which was dissolved in physiological saline. After the 24-hour modeling period, the mice were anesthetized with 0.01 ml / g of 5% chloral hydrate solution by intraperitoneal injection to obtain samples.

[0298] 1.3 H&E staining of mouse liver

[0299] The steps are the same as those in Example 3 “H&E staining of mouse liver”.

[0300] 1.4 Determination of ALT and AST in mice

[0301] The steps are the same as those in Example 3 "Determination of mouse alanine aminotransferase and aspartate aminotransferase".

[0302] 1.5 TUNEL immunohistochemistry experiment in liver tissue

[0303] The steps of tissue dehydration, embedding and section dewaxing were the same as those in “Example 3 H&E staining of mouse liver”. Subsequent operations were performed according to the instructions in the kit. The process is shown in Table 15.

[0304] Table 15 TUNEL immunohistochemical staining steps

[0305]

[0306] 1.6 Quantitative PCR (qPCR)

[0307] The operation steps are the same as those in Example 3 “Fluorescence quantitative PCR (qPCR)”.

[0308] 1.7 16S amplicon sequencing

[0309] The 16S amplicon sequencing process is as follows:

[0310] (1) Collection of cecal contents samples

[0311] Use a sterilized scalpel and tweezers to remove the cecum and cut out 150-300 mg of the required contents on an ultra-clean workbench. Place the samples into cryopreservation tubes according to groups (50-100 mg per tube), and immediately freeze them in liquid nitrogen. Samples that need to be stored for a long time should be stored in a -80°C refrigerator.

[0312] (2) Microbiome total DNA extraction and target fragment amplification

[0313] By collecting the contents of mouse cecum and extracting the microbial DNA therein, the required primers are designed for target sequences such as microbial rRNA or specific gene fragments, and the selected gene fragments are amplified.

[0314] (3) Quantification of amplification products

[0315] The PCR amplification products were subjected to fluorescence quantitative analysis, and sample mixing was prepared in accordance with sequencing requirements.

[0316] (4) Sequencing library preparation

[0317] 1) Repair the ends of the desired amplified product samples; 2) Perform corresponding base modifications on the ends of the DNA fragments required for sequencing; 3) Add a sequencing adapter containing a library-specific tag (i.e., Index sequence) to the 5' end of the sequence so that the DNA molecule can be fixed on the Flow Cell; 4) Use BECKMAN BZ-Wure XP Beads to purify the library system after adding the adapter; 5) Amplify the DNA fragments after the desired tag is fixed; 6) Perform final fragment selection and purification of the library by agarose gel electrophoresis.

[0318] (5) Perform high-throughput sequencing on the machine

[0319] 1) Quality check before sequencing. 2) Quantify the library using a fluorescence quantitative system. 3) After the sample is diluted, denature the double-stranded structure under strong alkaline conditions for sequencing.

[0320] (6) Data screening and graphical analysis

[0321] 1) Export the data for quality inspection. 2) Divide the library and samples through the original sequence of the initial quality screening. 3) Perform sequence denoising or OTU clustering according to the QIIME2 dada2 analysis process or the Vsearch software analysis process. 4) Classify and group the bacterial communities of each sample from the kingdom to the species level and conduct systematic analysis and display (including multi-faceted and hierarchical analysis and calculation from species diversity, intra-group differences to differential species screening). 5) Use the QIIME2 dada2 visualization function to draw charts with paper publication standards, generate analysis reports and organize and package the original data.

[0322] 2. Experimental results

[0323] Effect of BZ-W on intestinal flora transmission of APAP-induced liver injury

[0324] The experimental results are as follows Fig.12 As shown, Fig.12 The effect of BZ-W on AILI intestinal flora, where A is WT FMT(WT) Group and WT FMT(BZ-W)Figure B is the H&E staining result of liver tissue paraffin sections of mice in group A 24 hours after APAP administration (10X), B is the statistical result of liver pathological necrotic area (NECROTIC AREA, %), C is the result of ALT content in mouse liver detected by kit, where FMT(WT) means WT FMT(WT) Group mice, FMT(BZ-W) represents WT FMT(BZ-W) Group of mice.

[0325] Hematoxylin-eosin (H&E) staining was used to observe liver morphology and assess liver tissue damage. Fig.12 As shown in Figure A, compared with mice receiving WT mouse fecal bacteria, mice receiving BZ-W group fecal bacteria fluid significantly reduced damage and congestion around the central vein and reduced large-area cell necrosis damage. After statistical analysis of the area of ​​​​the damaged area, the results ( Fig.12 Middle B) also showed that the lesion area of ​​mice receiving fecal microbiota transplantation from the BZ-W group was significantly reduced (p < 0.05). At the same time, we tested the ALT content in the liver of mice, and the results were as follows: Fig.12 As shown in C, compared with WT FMT(WT) Group mice, WT FMT(BZ-W) The ALT content of mice in the BZ-W group was significantly reduced, which was consistent with the pathological results. The results showed that the fecal microbiota of the donor mice in the BZ-W group had a certain significant therapeutic effect on APAP-induced liver injury, indicating that the protective effect of BZ-W on liver AILI can be transmitted through the intestinal flora, indicating that BZ-W can affect AILI by affecting the intestinal flora.

[0326] 2.2 Effect of BZ-W on intestinal flora to improve hepatocyte apoptosis in the process of AILI

[0327] The results are as follows Fig.13 As shown, Fig.13 The effect of BZ-W on intestinal flora in improving hepatocyte apoptosis in the process of AILI, where A is WT FMT(WT) Group and WT FMT(BZ-W) A is the result of TUNEL immunohistochemical staining of paraffin sections of liver tissues of mice in group A after administration of APAP, B is the statistical result of TUNEL positive area, and C is the result of qPCR detection of mRNA expression level of apoptosis-related genes in mouse liver.

[0328] TUNEL immunohistochemistry kit was used to detect apoptosis of hepatocytes in the liver of FMT recipient mice. Fig.13 As shown in A, compared with WT FMT(WT) Group mice, WT FMT(BZ-W) The number of apoptotic cells near the injured area of ​​the central vein of mice in the group was significantly reduced. The results of statistical analysis of the injured area showed that ( Fig.13 B) also shows that compared with WT FMT(WT) Group mice, WT FMT(BZ -W) The total area of ​​positive lesions in mice of the group was also significantly reduced (p < 0.01).

[0329] It can be concluded that BZ-W can resist APAP-induced cell apoptosis by regulating intestinal flora or producing protective metabolites through intestinal flora, interacting with the liver through the "gut-liver axis", thereby protecting APAP-induced liver damage.

[0330] Effect of BZ-W on intestinal flora disturbance caused by AILI

[0331] Since studies have shown that APAP can induce liver damage and excessive doses can also cause intestinal flora disorders, regulating intestinal flora may also be an important method for treating liver metabolic diseases such as AILI. In order to study the complex relationship and interaction between the effective effect of BZ-W in treating AILI and the intestinal microbiome, we used 16S rDNA intestinal bacteria sequencing technology to analyze the cecal contents of mice. Figure 14-15 As shown, Fig.14 Figure 3 is the effect of BZ-W on the Chao1 index and Shannon index of α diversity among bacterial groups in intestinal flora disorder caused by AILI, where A is the Chao1 index result diagram of α diversity among bacterial groups, B is the Shannon index result diagram of α diversity among bacterial groups, C is the Chao1 index dilution curve result diagram among groups, and D is the Shannon index dilution curve result diagram among groups. Fig.15 Figure 3 is the effect of BZ-W on intestinal flora disorder caused by AILI, where A is the PCoA analysis result of β diversity between groups, B is the NMDS analysis result of β diversity between groups, and C is the accuracy analysis result of model prediction results.

[0332] First, it was found that after administration of BZ-W, the diversity of the intestinal population of mice was significantly improved, and the Chao1 index (a numerical value used to reflect the richness of the biological community) was calculated. The box plot of α diversity between groups showed ( Fig.14 (A), compared with the APAP group, the richness of the BZ-W group was significantly increased, and the Shannon index (a value reflecting the richness and uniformity of the biological community) was calculated. The α diversity box plot between groups ( Fig.14 Middle B) shows that compared with the APAP group, the BZ-W group not only has a significantly higher abundance of bacteria, but also has a more uniform composition of bacteria with increased abundance. Fig.14 C) and ( Fig.14As shown in Figure D), the back end of the experimental data dilution curve is close to the parallel x-axis, indicating that the sample size is nearly saturated and sufficient to reflect the α diversity parameter. If the number of samples in each group is increased, the sequencing depth will not be able to detect a large number of new ASV / OTUs that have not yet been discovered. On the other hand, the principal coordinate component analysis results (PCoA) Fig.15 A) and NMDS non-metric multidimensional analysis results ( Fig.15 The β-diversity analysis in (B) found that the bacterial community distance between the BZ-W group and the APAP group was obvious, indicating that the bacterial community composition structure and abundance of the BZ-W and APAP groups were quite different. Fig.15 In the figure C, the ordinate is the true label of the sample, and the abscissa is the label predicted by the classifier; the depth of the color indicates the proportion of the group of samples predicted as the corresponding label (abscissa), and the depth of the color represents the proportion of the group of samples predicted as the corresponding label (abscissa) in this experimental model. If the arrangement order of the horizontal and vertical coordinates is consistent, the darker the color on the main diagonal, the lighter the color outside the main diagonal, the results show that the prediction accuracy of this experimental model is high and the model grouping is more accurate. The results show that BZ-W significantly improves the species uniformity and richness of the intestinal flora of mice, and largely alleviates the intestinal flora disorder of AILI mice.

[0333] Figure 16-18 The effect of BZ-W on the composition of mouse intestinal flora is shown in Figure 2. Fig.16 This is the effect of BZ-W on the composition of the intestinal flora of mice (species composition stacked bar chart, classified by class level). Fig.17 The effect of BZ-W on the composition of the intestinal flora of mice (stacked bar chart of species composition, classified at the family level). Fig.18 This is the effect of BZ-W on the composition of the intestinal flora of mice (heat map of intestinal flora species composition, classified at the class level). Figure 16-18 The results showed that due to the β diversity, the bacterial community composition between the two groups was quite different. Further observation of the class-level bacterial community abundance heat map ( Fig.18 ) and stacked column charts ( Fig.16 ) It can be found that BZ-W significantly changed the composition of the flora at the class level, among which the more obvious Bacteroidia was significantly downregulated, and Clostridia was also upregulated by BZ-W. At the family level, there are more obvious differences in the composition of the flora, such as ( Fig.17 ) decreased Bacteroidetes and significantly increased the abundance of beneficial Lachnospiraceae.

[0334] Fig.19The figure is a screening diagram of significant bacteria in the intestinal flora of AILI mice by BZ-W, wherein A is the Lefse analysis result diagram, the color represents the grouping, the length represents the LDA value, and B is a comparison diagram of the relative abundance of species with significant differences between groups (n=5).

[0335] To continue to compare and explore the differential bacterial flora that can be used as "target organs", we refer to the results of Lefse analysis. Fig.19 As shown in A, the ordinate is the taxonomic units with significant differences in the comparison between the two groups, and the values ​​of the abscissa and the bar graph show the logarithmic score of the LDA analysis of each taxonomic unit. The LDA value greater than 3 is used as the screening value to indicate that the abundance of each group is significantly higher than that of other groups of Biomarkers. It can be observed that compared with the APAP group, the abundance of CC_115 bacteria at the phylum level and Mogibateriaceae bacteria at the family level in the BZ-W group was significantly reduced, while the abundance of Firmicutes bacteria at the phylum level, Clostridia and Gammaproteobacteria at the class level, Clostridiales and Bacillales at the order level, and some bacterial species at the family and genus levels were significantly increased. After further comparison of the relative abundance of bacteria at the genus and family levels and detailed screening through literature research, it was found that BZ-W treatment of AILI may be related to a significant change in the abundance of some key bacterial genera. BZ-W significantly upregulated the abundance of Roseburia bacteria in the probiotic Lachnospiraceae family ( Fig.19 B), which is a butyrate-producing bacterium that can create an intestinal environment suitable for the survival of most probiotics and thereby improve intestinal biodiversity. It can also reduce obesity and improve related liver diseases by resisting inflammation. On the other hand, the abundance of harmful bacteria Mogibateriaceae was significantly reduced by BZ-W ( Fig.19 (B) According to the literature, harmful bacteria of the Mogibateriaceae family may be involved in the occurrence of inflammation and apoptosis, and their abundance is positively correlated with Bax.

[0336] The above results indicate that BZ-W can significantly improve the composition structure of the intestinal flora of AILI mice, increase the species richness of the flora, create a healthier flora ecological environment, and treat AILI by influencing and regulating key flora.

[0337] Comparative Example 1

[0338] Preparation of Atractylodes macrocephala polysaccharide (BZ-CM):

[0339] After the Atractylodes macrocephala was powdered, 10.0 g of Atractylodes macrocephala powder was accurately weighed and the first extraction was performed: at 80°C, the Atractylodes macrocephala powder and ultrapure water were stirred and extracted for 5 hours at a solid-liquid ratio of 1:15 (g / mL) to obtain an extract, and the extract was centrifuged at 4000 rpm for 20 minutes in a high-speed refrigerated centrifuge to collect the supernatant and residue of the first extraction. The residue was extracted for the second and third times according to the method of the first extraction, and the supernatants of the three extractions were combined and concentrated to 1 / 3 of the original volume by vacuum rotary evaporation at 60°C to obtain a concentrate. The iodine-potassium iodide reagent was used for detection, and the results showed that there was no starch in the concentrate. Three times the amount of 85% volume fraction ethanol aqueous solution was added to the concentrate, stirred and mixed, and placed at 4°C for alcohol precipitation overnight. The mixed solution after alcohol precipitation was centrifuged at 4000×r for 10 minutes, the supernatant was removed, and the precipitate was collected. The precipitate was then washed twice with an ethanol-water solution of equal volume fraction to make the polysaccharide component more uniform. Finally, the precipitate was evenly dissolved in ultrapure water and vacuum freeze-dried for 48 hours to obtain Atractylodes macrocephala polysaccharide (BZ-CM).

[0340] The monosaccharide composition of BZ-CM was analyzed according to the method of Example 1. The results are shown in Table 16.

[0341] Table 16 Monosaccharide composition percentage of BZ-CM

[0342]

[0343] Note: Ara, arabinose; Fuc, fucose; Gal-UA, galacturonic acid; Gal, galactose; Glc-UA, glucuronic acid; Glc, glucose; Man, mannose; Xyl, xylose.

[0344] The results showed that BZ-CM was composed of monosaccharides glucose, arabinose, galactose, galacturonic acid, and glucuronic acid.

[0345] Test Example 1

[0346] 1. Experimental methods

[0347] 1.1 Establish an acute liver injury model in mice induced by excessive APAP according to the method in 1.2 of Example 3

[0348] Among them, the mice were grouped as follows: normal control group 24h model CON group (n=6), 24h model APAP administration group (n=7), 24h model BZ-W administration BZ-W group (BZ-W concentration was 100 mg / ml, n=7), 24h model BZ-CM administration BZ-CM group (BZ-CM concentration was 100 mg / ml, n=7).

[0349] 1.2 Quantitative PCR (qPCR)

[0350] The operation process is the same as step 1.9 of fluorescent quantitative PCR (qPCR) in Example 3.

[0351] 1.3 Determination of GSH levels in mouse liver

[0352] A: Take out the frozen liver tissue in 1.2 and thaw it. Add physiological saline to a large dish to clean the blood clots. Use sterilized scissors to cut 50 mg of tissue and weigh it to record the weight. After blotting it dry on filter paper, transfer it to a sterilized EP tube and add two sterilized steel balls.

[0353] B: Add the corresponding volume of physiological saline according to the ratio of 1g tissue: 9mL physiological saline, place the sample in a fully automatic glass grinder, and grind for 1min x 2 times; centrifuge the tissue homogenate obtained after grinding at 2000-2500rpm in a centrifuge at 4℃ (precooling is required) for 15min, collect the supernatant of the tissue homogenate, discard the precipitate, transfer to a newly sterilized EP tube, and keep on ice for later use.

[0354] C: According to the steps in the kit instructions, add the precipitant in the kit to the above EP tube, centrifuge the precipitated sample at 2000rpm, 4℃, for 15min, collect the supernatant to be tested in a new EP tube and keep it on ice for later use

[0355] D: Follow the steps specified in the kit and measure the GSH content in the liver tissue using the ELISA colorimetric method at the corresponding wavelength.

[0356] 2. Experimental results

[0357] 2.1 qPCR detection of changes in the expression of inflammatory-related genes

[0358] The results are as follows Fig. 20 As shown, Fig. 20Figure 2 is the effect of BZ-W on APAP-induced mouse liver inflammation-related genes, among which AD is the expression change of mRNA level of IL-1β, IL-6, TNF-α and Nlrp3. The results showed that compared with the CON group, the mRNA expression of inflammatory factors IL-1β, IL-6, TNF-α and in the APAP group was significantly upregulated (p < 0.001, p < 0.0001), while the mRNA expression of IL-β, TNF-α and IL-6 in the BZ-W treatment group was significantly downregulated compared with the model group (p < 0.01, p < 0.001), but the mRNA expression of TNF-α and IL-6 in the BZ-CM treatment group was not significantly different from that in the model group, and the mRNA expression of IL-β in the BZ-CM treatment group was higher than that in the BZ-W treatment group. For the inflammasome Nlrp3, compared with the CON group, the mRNA of the APAP group was also significantly upregulated (p < 0.001), and the transcription level of NLRP3 was also significantly downregulated after administration of BZ-W (p < 0.001), and there was no significant difference between the BZ-CM treatment group and the APAP group. This shows that BZ-W can reduce the transcription of NLRP3 inflammasomes, thereby reducing the mRNA transcription and protein translation expression levels of various inflammatory factors, thereby reducing APAP-induced mouse liver inflammation in the middle and late stages of AILI, and its ability to reduce APAP-induced mouse liver inflammation is better than BZ-CM.

[0359] 2.2 Results of determination of GSH levels in mouse liver

[0360] The results are as follows Fig.21 As shown, Fig.21 This is the result of detecting GSH content in mouse liver tissue. Fig.21 The results showed that both BZ-W and BZ-CM had no significant effect on resisting ROS in the process of AILI.

[0361] Test Example 2

[0362] 1. Experimental methods

[0363] 1.1 The mouse fecal microbiota transplantation experiment was carried out according to the method in 1.2 of Example 4

[0364] Among them, the mice were grouped into: FMT (WT) group receiving normal mouse fecal microbiota transplantation (n=7), FMT (BZ-W) group receiving BZ-W mouse fecal microbiota transplantation (n=7), and FMT (BZ-CM) group receiving BZ-CM mouse fecal microbiota transplantation (n=7).

[0365] 1.2 Quantitative PCR (qPCR)

[0366] The operation process is the same as step 1.9 of fluorescent quantitative PCR (qPCR) in Example 3.

[0367] 2. Experimental results

[0368] Fig. 22 Figure 2 is the result of mRNA expression level of liver apoptosis-related genes after APAP administration in mice, among which AC is the expression change of mRNA level of Bax, Bcl-xl and Bcl2 respectively. The results showed that compared with the FMT (WT) group, the FMT (BZ-W) group significantly reduced the mRNA expression level of Bax with high expression; at the same time, the FMT (BZ-W) group also significantly upregulated the mRNA transcription level of Bcl-2 and Bcl-xl with low expression, which was consistent with the anti-apoptotic treatment results of BZ-W, while the mRNA expression level of Bcl-2, Bcl-xl and Bax in the FMT (BZ-CM) group was not significantly different from that in the FMT (WT) group. It can be concluded that BZ-W can resist APAP-induced apoptosis by regulating intestinal flora or producing protective metabolites through intestinal flora, interacting with the liver through the "intestinal-liver axis" to protect APAP-induced liver damage.

[0369] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An application of Atractylodes macrocephala polysaccharide in the preparation of a drug for treating drug-induced liver injury.

2. The use according to claim 1, characterized in that: The drug-induced liver injury includes acetaminophen-induced liver injury.

3. The application according to claim 2, characterized in that: The acetaminophen-induced liver damage includes one or more of liver damage, cell apoptosis, liver inflammation and intestinal flora disorder.

4. The application according to claim 3, characterized in that: The acetaminophen-induced liver injury is a disorder of intestinal flora, and the intestinal flora includes one or both of Roseburia and Mogibateriaceae.

5. The use according to claim 1, characterized in that: Atractylodes macrocephala polysaccharide was prepared by water extraction and alcohol precipitation.

6. The use according to claim 5, characterized in that: The water extraction and alcohol precipitation method comprises: extracting Atractylodes macrocephala and ultrapure water in a ratio of 1 g:15-25 mL at 85-95° C., extracting 1-3 times, each time for 1-3 hours, combining the extracts and concentrating to obtain a concentrated solution; adding 2-4 times the volume of 90-100% ethanol solution to the concentrated solution, precipitating with alcohol to obtain a precipitate, and drying to obtain Atractylodes macrocephala polysaccharide.

7. The use according to claim 6, characterized in that: The water extraction and alcohol precipitation method is specifically as follows: Step 1): extract Atractylodes macrocephala and ultrapure water at a ratio of 1 g: 20 mL at 90°C for 2 h, centrifuge at 4000 rpm for 20 min, and collect the supernatant and residue; Step 2): extract and centrifuge the residue for the second time according to the method of step 1), combine the supernatants of the two extractions and concentrate at 60° C. to obtain a concentrate; Step 3): Add 3 times the volume of 95% ethanol solution to the concentrate, precipitate overnight at 4°C, collect the precipitate by centrifugation, wash the precipitate with ethanol and freeze-dry it to obtain Atractylodes macrocephala polysaccharide.

8. The use according to claim 7, characterized in that: Atractylodes macrocephala polysaccharide is composed of glucose, arabinose, galactose, galacturonic acid and glucuronic acid.

9. The use according to claim 8, characterized in that: The Atractylodes macrocephala polysaccharide is composed of 82-85% glucose, 6-8% arabinose, 5-6% galactose, 2-4% galacturonic acid and 0.1-0.8% glucuronic acid.

10. The use according to claim 9, characterized in that: The Atractylodes macrocephala polysaccharide is composed of 83.92% glucose, 7.19% arabinose, 5.47% galactose, 3% galacturonic acid and 0.41% glucuronic acid.

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