Rhizobium exopolysaccharide and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to rhizobium exopolysaccharide and application thereof. By extracting and purifying the rhizobium extracellular polysaccharide and analyzing the yield and monosaccharide composition of the extracellular polysaccharide, the apoptosis action mechanism of the extracellular polysaccharide on the in-vitro liver cancer cell Hep-G2 is systematically researched. A mouse liver cancer model is constructed, a model group is used as a negative control, a specific drug group is used as a positive control, the liver cancer mouse model adopts concentration gradient exopolysaccharide intragastric administration as an experimental group, and the transcriptomics influence of the exopolysaccharide on liver cancer nude mouse blood and tumor samples is analyzed. The action effect of the exopolysaccharide in a Hep-G2 subcutaneous model is comprehensively characterized by various means such as liver and kidney toxicity test, Elisa experiment, HE staining and immunohistochemical experiment, and the regulation mechanism of the exopolysaccharide on nude mouse liver cancer tumor key protein is detected by adopting Western blot experiment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a rhizobium extracellular polysaccharide and its application in inhibiting liver cancer cells. Background Art

[0002] Exopolysaccharides (EPS) are a class of macromolecular carbohydrates composed of ten or more monosaccharides connected by glycosidic bonds. They are widely found in nature and can be either straight chains or branched chains. EPS can be classified into two major categories according to their chemical structure: homopolysaccharides and heteropolysaccharides. Homopolysaccharides: For example, dextran, which is composed of a single type of monosaccharide units, mainly undergoes synthesis outside the cell and relies on specific substrates such as sucrose. The sugar chains act as glycosyl donors and glycosyl acceptors, and polysaccharide sucrose transferase is also required for synthesis. Heteropolysaccharides: Regular repeating units are formed by the polymerization of two or more different monosaccharides, such as xanthan gum. The biosynthesis of heteropolysaccharides mainly includes the production of precursors in the cytoplasm, the synthesis of repeating units, and the polymerization of repeating units outside the cell. EPS plays a variety of important roles in organisms, including energy storage, cell recognition, cell signaling, and as part of the cell structure. In the fields of medicine and biology, polysaccharides have received extensive attention due to their unique biological functions and potential medical value. EPS also participates in various physiological activities in humans as antitumor, antiviral and anti-inflammatory agents, and can act as an inducer of interferon, platelet aggregation inhibitor and colony stimulating factor synthesis.

[0003] Extracellular polysaccharides are widely distributed in nature and play a key role in bacterial survival, adaptation to the environment, and interaction with the host. The composition, structure, and functional diversity of EPS reflect the adaptability and biodiversity of bacteria in different environments. The chemical composition and structure of EPS are very diverse, mainly including monosaccharides such as glucose, galactose, mannose, glucuronic acid, galacturonic acid, and their derivatives. This diversity gives EPS a wide range of physical and chemical properties, allowing it to function under different environmental conditions. Some bacteria can synthesize extracellular polysaccharides in vivo and secrete them outside the cell, providing a suitable microenvironment for bacterial growth and reproduction. Extracellular polysaccharides can also protect and promote the exchange of genes and metabolites within bacterial communities under extreme conditions. Studies have shown that the composition, conformation, side chain groups and other structural characteristics of extracellular polysaccharides vary, and researchers often find that they have physiological activities such as immune regulation and anti-tumor. Summary of the invention

[0004] The purpose of the present invention is mainly to solve the above-mentioned existing problems and provide a kind of Rhizobium exopolysaccharide and its use. By extracting and purifying Rhizobium exopolysaccharide, analyzing the yield and monosaccharide composition of exopolysaccharide, and then systematically studying the apoptosis mechanism of exopolysaccharide on in vitro hepatoma cell Hep-G2. A mouse liver cancer model was constructed, with the model group as the negative control and the special drug group as the positive control. The liver cancer mouse model used concentration gradient exopolysaccharide gavage as the experimental group to analyze the transcriptomic effects of exopolysaccharide on blood and tumor samples of nude mice with liver cancer. Through various means such as hepatotoxicity test, Elisa experiment, HE staining and immunohistochemistry experiment, the effect of exopolysaccharide in Hep-G2 subcutaneous model was fully characterized, and the regulatory mechanism of exopolysaccharide on key proteins of nude mouse liver cancer tumor was explored by Western blot experiment.

[0005] The above purpose is achieved by the following technical solutions:

[0006] A rhizobium exopolysaccharide, characterized in that: The molecular weight of the rhizobium extracellular polysaccharide is 691.01 kDa, and the polysaccharide is mainly composed of galactose, glucose, mannose and glucuronic acid. The molar ratio of galactose, glucose, mannose and glucuronic acid is 17.45:67.06:14.50:0.99.

[0007] Furthermore, the exopolysaccharide is prepared from Rhizobium yanglingensis ( Rhizobium yanglingense ) production is obtained.

[0008] Furthermore, the total sugar purity of the extracellular polysaccharide after purification by macroporous resin in S2 of the preparation method is at least 57.2% (mass fraction), and the total sugar purity of the polysaccharide after purification by gel in S3 is at least 84.5% (mass fraction).

[0009] Furthermore, the method for preparing the extracellular polysaccharide comprises the following steps: S1: Extraction method of crude exopolysaccharide: (1) The screened strains were quantitatively inoculated into YMA liquid fermentation medium and cultured in a sterile shaking incubator at 28°C overnight until the stable phase; (2) The cultured rhizobium fermentation liquid was centrifuged to remove the bacteria, concentrated to a certain volume by rotary evaporation, and then precipitated overnight with 4 times anhydrous ethanol. The precipitate was collected by centrifugation, dialyzed with deionized water for 48 h, and vacuum freeze-dried to obtain crude polysaccharides; S2: Macroporous resin purification of extracellular polysaccharides: (1) Grind the freeze-dried sample and pass it through a 100-mesh sieve; (2) Add an appropriate amount of anhydrous ethanol, stir to extract fat-soluble pigments and some impurities, centrifuge at 6000 × g for 10 min, and collect the precipitate; (3) Add an appropriate amount of pure water to the precipitate, extract in a constant temperature water bath at 55°C for 3 h, then remove and centrifuge at 6000×g for 10 min. Collect the supernatant extract and extract the precipitate residue in the centrifuge tube again according to the above steps; (4) Combine the extracts collected twice, concentrate by vacuum evaporation to 1 / 10 of the original volume, and then add four times the amount of anhydrous ethanol to precipitate at 4°C overnight; (5) Centrifugation at 8000 × g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract; (6) Add an appropriate amount of pure water and a small amount of papain to the solid and ferment overnight; (7) Add Sevage reagent to the aqueous phase, mix thoroughly, and collect the upper aqueous phase; (8) Add an appropriate amount of petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase; (9) Add an appropriate amount of macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight; (10) Collect the liquid, dialyze it in a 3000 Da dialysis bag for 48 h to remove small molecular components, and freeze-dry the polysaccharide solution; (11) The phenol-sulfuric acid method was used to determine the soluble total sugar content and purity of the crude polysaccharide. The specific operation was as follows: accurately weigh a small amount of polysaccharide freeze-dried powder, add water to dissolve it, add sulfuric acid-phenol reagent, react in the dark for 10 min, and then 490nm Measure the absorbance at S3: Gel purification of exopolysaccharides: (1) Take the polysaccharide sample after ion purification and add pure water to make a polysaccharide mother solution with a concentration of 15 mg / mL. Centrifuge at 10,000 × g for 10 min, take the supernatant, and separate and purify it using a gel chromatography column at a flow rate of 1 mL / min: elute with pure water for 1.5 times the column volume, collect one tube for every 12 mL, and collect all the eluate; (2) Determine the total sugar content of the eluate in each collection tube using the sulfuric acid-phenol method and draw a gel purification elution curve; (3) Select and confirm the elution peak (the main peak is selected by default); (4) Combine the collected eluates corresponding to the same elution peak and concentrate by rotary evaporation to 1 / 5 of the original volume; (5) Freeze-drying and using the sulfuric acid-phenol method to identify the content and purity of the polysaccharide after gel purification.

[0010] A use of rhizobium exopolysaccharide, characterized in that: The extracellular polysaccharide is used in preparing drugs related to liver cancer cell inhibition.

[0011] Oral administration of extracellular polysaccharides did not cause fluctuations in ALT (alanine transaminase), AST (aspartate transaminase), ALP (alkaline phosphatase) and bilirubin levels and maintained them at normal levels, proving that extracellular polysaccharides would not damage liver and kidney tissues and were non-toxic to mice.

[0012] Extracellular polysaccharides can significantly reduce the levels of cancer markers such as AFP (alpha-fetoprotein), CEA (carcinoembryonic antigen) and CA199, and significantly upregulate the expression of MYD88, CD14, TNF-ɑ, JUN, TGF-β1, TLR4, MCSF, P53, and BCL-2 genes and proteins in the MAPK signaling pathway. Tumor cells show phenomena such as lysis, inflammatory cell infiltration, small cell size, nuclear condensation, increased tissue necrosis area, vacuolar degeneration, and cell death, indicating that extracellular polysaccharides promote the apoptosis rate of tumor cells and have a strong inhibitory effect on mouse liver cancer.

[0013] This application explores the inhibitory effect and biosafety of rhizobium-derived bacterial exopolysaccharides (EPS) on Hep-G2 hepatocellular carcinoma lines and nude mouse liver cancer models, with special attention paid to its ability to induce cell apoptosis and related mechanisms. Through the comprehensive application of multiple biotechniques such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and Western blot, the biological basis and mechanism of EPS's inhibitory effect on the growth of liver cancer cells were deeply explored. Considering the significant public health challenges posed by the high incidence and high mortality of liver cancer worldwide, and the limitations of existing treatment methods, especially in the field of drug therapy, it has become an urgent need to develop new and effective anti-liver cancer treatment strategies. Bacterial exopolysaccharides have shown their potential as a new anti-liver cancer treatment strategy due to their significant biological activities in anti-inflammatory, antibacterial, and immunomodulatory aspects. This application aims to provide new scientific basis and experimental support for liver cancer treatment by extracting and studying the effects of rhizobium EPS on Hep-G2 liver cancer cell lines, promote the application and development of EPS in the field of anti-liver cancer, and promote a deeper understanding of the mechanism of liver cancer occurrence and development and provide materials for future precision treatment.

[0014] The beneficial effects of the present invention are: 1) The purity of the purified extracellular polysaccharide was 84.5%, mainly composed of four monosaccharide residues, namely glucose, galactose, mannose and glucuronic acid. The CCK-8 cytotoxicity test showed that when the polysaccharide concentration was 150 μg / μL, the cell activity reached the lowest level, indicating that the extracellular polysaccharide had a certain inhibitory effect on liver cancer cells. Flow cytometry detection showed that the extracellular polysaccharide could significantly promote the apoptosis rate of Hep-G2 cells; 2) Transcriptomic analysis results showed that the MAPK signaling pathway plays a key regulatory role in the process of exopolysaccharide-induced apoptosis of Hep-G2 cells, and confirmed CD14, TLR4, MYD88, JUN, TGF-β1 Isogenes are key regulatory genes; 3) The results of the liver and kidney toxicity test showed that after the administration of extracellular polysaccharides, indicators such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood uric acid (UA) and urea (UREA) returned to near normal levels, indicating that the extracellular polysaccharides had less liver and kidney toxicity to mice. Enzyme-linked immunosorbent assay showed that there was no significant difference in indicators such as glutamyl transpeptidase (GGT), creatinine, and liver regeneration enhancer (ALR) between the normal group and the administration group, further confirming the low toxicity of extracellular polysaccharides to mouse tissues and organs; 4) The results of gene expression analysis showed that the cancer markers CA199, CEA, and AFB in the model mice in the exopolysaccharide administration group were significantly reduced, indicating that exopolysaccharides have an inhibitory effect on cancer. HE staining results showed that the administration group showed partial lysis of tumor cells, infiltration of inflammatory cells, small cell size, nuclear condensation, increased tissue necrosis area, vacuolar degeneration, cell death and other phenomena, indicating that exopolysaccharides have a significant anti-cancer effect. Immunohistochemical detection of apoptosis proteins in tumor tissue cells found that the number of positive cells in the model mice administered with exopolysaccharides increased significantly, and the expression of proteins MYD88, CD14, TNF-ɑ, JUN, TGF-β1, TLR4, MCSF, P53, and BCL-2 was upregulated, indicating that exopolysaccharides can effectively inhibit the growth of subcutaneous transplanted tumors in the mouse liver cancer Hep-G2 model. Western blot and qRT-PCR results showed that the expression of CD14, TLR4, MYD88, JUN, TGF-β1, TNF-ɑ and other proteins were upregulated, and P53 and BCL-2 cancer-related proteins were upregulated, promoting tumor cell apoptosis; 5) The above in vitro and in vivo experiments showed that Rhizobium exopolysaccharides can effectively promote the apoptosis rate of Hep-G2 cells, regulate the expression of genes and proteins related to cancer cell apoptosis in liver cancer model mice, and have the physiological function of curing liver cancer in mice.

[0015] Deposit of biological material: The Yangling Rhizobium involved in the present invention was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Committee on July 8, 2013, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC NO.7895, and the name is Yangling Rhizobium ( Rhizobium yanglingense ). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the ion chromatogram of the standard sample in Example 1 of the present invention; Figure 2 is the ion chromatogram of the polysaccharide sample in Example 1 of the present invention; Figure 3 This is a morphological diagram of nude mouse tumor cells in Example 2 of the present invention; the diagram is marked as: liver cancer model group (Model), positive drug group (Positive), high-dose group (High), medium-dose group (Middle), and low-dose group (Low); Figure 4 This is a comparison chart of the enzyme-linked immunosorbent assay for evaluating ALB, AFB, CEA, Creatinine, CA199, GTT and other indicators according to Example 2 of the present invention; the following are marked in the figure: normal control group (Normal), liver cancer model group (Model), positive drug group (Positive), high-dose group (High), medium-dose group (Middle), and low-dose group (Low). DETAILED DESCRIPTION

[0017] The following are the abbreviations that may be used:

[0018] [Example 1] Extraction and purification of extracellular polysaccharides.

[0019] In this example, the purpose is to obtain high-quality EPS samples through the extraction and purification of extracellular polysaccharides (EPS) for in-depth monosaccharide component analysis. EPS is composed of a variety of monosaccharides connected by glycosidic bonds, and its specific monosaccharide composition and structural arrangement directly affect its biological activity and application potential. Therefore, the use of accurate and efficient extraction and purification technology is a prerequisite for analyzing the molecular structure of EPS, which is of great significance for the subsequent exploration of its biological functions and the development of related biomedicine and industrial applications. EPS extraction and purification techniques including centrifugation, dialysis, macroporous resin adsorption, protein removal, fat removal and gel chromatography, as well as high performance liquid chromatography (HPLC) and other methods were used to qualitatively and quantitatively analyze EPS monosaccharide components.

[0020] 1. Materials and methods.

[0021] 1.1 Experimental materials.

[0022] Rhizobium, Yangling Rhizobium with the accession number of CGMCCN0.7895 ( Rhizobium yanglingense ). The rhizobium is recorded in Chinese patent document CN104277992A.

[0023] 1.2 Information on standard products and preparation of standard products. Standard products mainly come from Sigma.

[0024] .

[0025] Preparation of standard products. Weigh the glucose standard accurately and dry it to constant weight, add distilled water to make a 10 mg / mL glucose mother solution single standard, then take an appropriate amount of glucose mother solution single standard and mix it to make a glucose mixed standard with a maximum index concentration of 40 μg / mL, 50μg / mL or 60 μg / mL, and prepare the standard products required for the test according to the concentration gradient in the table below (monosaccharide mixed standard gradient concentration).

[0026] .

[0027] 1.3 Sample preparation.

[0028] 1) Solid sample extraction: Weigh an appropriate amount of the extracted polysaccharide sample, add 1 mL of 2 M TFA acid solution, and heat at 121°C for 2 h. Pass nitrogen and blow dry. Add 99.99% methanol to wash, blow dry again, repeat the methanol washing 2-3 times, and finally add sterile water to dissolve, transfer to a chromatographic bottle and wait for testing.

[0029] 2) Liquid sample extraction: Take an appropriate amount of sample and process it in the same way as solid samples.

[0030] 1.4 Instrument parameters.

[0031] The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector.

[0032] A Dionex™ CarboPac™ PA20 (150×3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μL. Mobile phase A (H2O), mobile phase B (0.1 M NaOH), mobile phase C (0.1 M NaOH, 0.2 M NaAc), flow rate 0.5 mL / min; column temperature 30℃; elution gradient: 0 min phase A / phase B / phase C (95∶5∶0, V / V / V), 26 min phase A / phase B / phase C (85∶5∶10, V / V / V), 42 min phase A / phase B / phase C (85∶5∶10, V / V / V), 42.1 min phase A / phase B / phase C (60∶0∶40, V / V / V), 52 min phase A / phase B / phase C (60∶40∶0, V / V / V), 52.1 min phase A / phase B / phase C (95∶5∶0, V / V / V), 60 min phase A / phase B / phase C (95∶5∶0, V / V / V).

[0033] 1.5 Qualitative and quantitative analysis of metabolites.

[0034] The quantitative analysis of this test uses standards of different concentrations as controls, with different concentrations of the standards as the horizontal axis and the peak area of ​​the standards as the vertical axis to obtain the relationship between the measured monosaccharides and their peak areas, and then calculate the concentration of the extracellular polysaccharides based on their peak area.

[0035] 1.6 Extraction and content determination of extracellular polysaccharides.

[0036] 1) Extraction method of extracellular polysaccharides: The selected strains were quantitatively inoculated into YMA liquid fermentation medium and cultured in a sterile shaking incubator at 28°C overnight until the stable period. The cultured rhizobium fermentation broth was centrifuged to remove the bacteria, concentrated to a certain volume by rotary evaporation, and then precipitated with 4 times anhydrous ethanol overnight. The precipitate was collected by centrifugation, dialyzed with deionized water for 48 h, and vacuum freeze-dried to obtain crude polysaccharides;

[0037] 2) Draw a glucose standard curve: Accurately weigh 10 mg of anhydrous glucose standard dried to constant weight at 105°C, dissolve in deionized water and dilute to a 100 mL volumetric flask to obtain a glucose standard solution with a mass concentration of 0.1 g / L. Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of glucose standard solution, place in stoppered test tubes, add water to 1.0 mL, add 1.0 mL of 5% phenol solution to each, shake well, place in an ice water bath, add 5 mL of concentrated H2SO4, shake well, place in a 30°C water bath and keep warm for 30 min, take out and cool to room temperature, and precipitate at OD 490nm The absorbance was measured, and a standard curve was drawn with the standard glucose concentration as the horizontal axis and the absorbance value as the vertical axis. The extracellular polysaccharide production was calculated by establishing a linear regression equation.

[0038] 3) Extracellular polysaccharide macroporous resin purification: (1) Grind the freeze-dried sample and pass it through a 100-mesh sieve; (2) Add an appropriate amount of anhydrous ethanol, stir to extract fat-soluble pigments and some impurities, centrifuge at 6000 × g for 10 min, and collect the precipitate; (3) Add an appropriate amount of pure water to the precipitate, extract in a constant temperature water bath at 55°C for 3 h, then remove and centrifuge at 6000×g for 10 min. Collect the supernatant extract and extract the precipitate residue in the centrifuge tube again according to the above steps; (4) Combine the extracts collected twice, concentrate by vacuum evaporation to 1 / 10 of the original volume, and then add four times the amount of anhydrous ethanol to precipitate at 4°C overnight; (5) Centrifugation at 8000 × g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract; (6) Add an appropriate amount of pure water and a small amount of papain to the solid and ferment overnight; (7) Add Sevage reagent to the aqueous phase, mix thoroughly, and collect the upper aqueous phase; (8) Add an appropriate amount of petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase; (9) Add an appropriate amount of macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight; (10) Collect the liquid, dialyze it in a 3000 Da dialysis bag for 48 h to remove small molecular components, and freeze-dry the polysaccharide solution; (11) The phenol-sulfuric acid method was used to determine the soluble total sugar content and purity of the crude polysaccharide. The specific operation was as follows: accurately weigh a small amount of polysaccharide freeze-dried powder, add water to dissolve it, add sulfuric acid-phenol reagent, react in the dark for 10 min, and then 490nm Measure the absorbance.

[0039] 4) Gel purification: (1) Take the polysaccharide sample after ion purification and add pure water to make a polysaccharide mother solution with a concentration of about 15 mg / mL. Centrifuge at 10,000 × g for 10 min, take the supernatant, and separate and purify it using a gel chromatography column at a flow rate of 1 mL / min: elute with pure water for 1.5 times the column volume, collect one tube for every 12 mL, and collect all the eluate; (2) Determine the total sugar content of the eluate in each collection tube using the sulfuric acid-phenol method and draw a gel purification elution curve; (3) Select and confirm the elution peak (the main peak is selected by default); (4) Combine the collected eluates corresponding to the same elution peak and concentrate by rotary evaporation to 1 / 5 of the original volume; (5) Freeze-drying and using the sulfuric acid-phenol method to identify the content and purity of the polysaccharide after gel purification.

[0040] 2. Results and analysis.

[0041] 2.1 Qualitative results of crude polysaccharides.

[0042] The chromatographic data were processed using the Chromeleon software. Figure 1 , Figure 2 Shown are the standard sample ion chromatogram and the sample ion chromatogram, respectively. The horizontal axis is the retention time (Time, min) of the detection, and the vertical axis is the response value (Response, nC) of the ion detection.

[0043] Quantitative results of each component in crude polysaccharide .

[0044] The exopolysaccharides of rhizobia stored in the laboratory were extracted and purified, and the components of the obtained exopolysaccharides were analyzed: (1) The molecular weight (or average molecular weight) of this extracellular polysaccharide is 691.01 kDa. The total sugar purity after the first macroporous resin purification is 57.2%, and the total sugar purity after the second gel purification is 84.5%; (2) The main components of this extracellular polysaccharide are galactose, glucose, mannose and glucuronic acid, with a molar ratio of 17.45:67.06:14.50:0.99.

[0045] [Example 2] The mechanism of action of extracellular polysaccharides on Hep-G2 cell-induced liver cancer in nude mice.

[0046] In this example, the effect of extracellular polysaccharide (EPS) on the Hep-G2 liver cancer model in vivo was studied in depth, as well as the preliminary exploration of its anti-tumor mechanism. By establishing a nude mouse liver cancer model induced by Hep-G2 cells, its potential biological mechanism of action and its biosafety were explored. By evaluating the effect of EPS treatment on the body weight of nude mice, performing HE staining and immunohistochemical analysis of tumor tissues, and exploring the changes in signaling pathways and the effects on related differentially expressed proteins under EPS intervention at the molecular level, the therapeutic potential of EPS for liver cancer was revealed, providing theoretical and experimental support for the future development of EPS-based liver cancer treatment strategies.

[0047] 1. Materials and methods.

[0048] 1.1 Construction of mouse liver cancer model.

[0049] 1) Experimental animal grouping: 60 nude mice were randomly divided into normal control group (Normal), liver cancer model group (Model), positive drug group (Positive), high-dose group (High), medium-dose group (Middle), and low-dose group (Low), with 10 mice in each group and numbered; 2) Modeling: Hep-G2 cells in the logarithmic growth phase were harvested and resuspended in PBS buffer with a pH of 7.4 to a final concentration of 6×10 7 Pipette 100 μL (containing 6×10 6 The cell suspension was injected into the skin of the left axillary area of ​​nude mice that had been adaptively fed for one week and disinfected with 75% ethanol. 3) Administration: Starting from one week after modeling, the experimental group was gavaged with bacterial extracellular polysaccharide extract solution: 300 μg in the high-dose group, 150 μg in the medium-dose group, and 75 μg in the low-dose group. The positive group was given about 150 μg of sorafenib according to body weight. The normal group was given purified water once a day for 30 consecutive days; 4) Observation indicators: Starting from the first day, each mouse was weighed every 6 days; 5) Sampling: On the 30th day, blood samples, tumor samples, and liver samples were collected from nude mice.

[0050] 1 2 Indicator detection and analysis.

[0051] 1) Detection of liver and kidney function indicators: After the last administration, all nude mice were fasted (but not water-deprived) for 24 h, blood was collected from the eye sockets, centrifuged at 12,000 × g, 4°C for 15 min, and the supernatant was collected. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), and uric acid (UA) of nude mice were detected by fully automatic biochemical instrument; 2) Pathological histological examination: Immunohistochemistry was used to detect the expression of CSF1R, CD14, AP-1, Myd88, and TLR-4 proteins in tumor tissues of nude mice; Tumor HE staining: (1) Ethanol dehydration: The tissue was dehydrated in different concentrations of ethanol solution, 75%, 85%, 95%, and 100%, and each concentration gradient ethanol treatment was 40 min (Note: Bony and calcified tissues need to be immersed in decalcification solution for 24 h, and then the ethanol dehydration step can be performed after the tissue softens); (2) Transparency: The tissue was immersed in three xylene tanks in sequence, each tank for 30 min; (3) Wax immersion: The tissue is immersed in three paraffin tanks in sequence, the first tank for 1 h, the second tank for 1.5 h, and the third tank for 2 h; (4) Embedding: Pour liquid paraffin into the mold box, then place the wax-soaked tissue block flat on the bottom, with the cut surface facing downward. After the paraffin solidifies, remove the embedding frame. After it has completely cooled and hardened, trim the wax block. Keep the paraffin around the tissue to a moderate level for easy slicing. (5) Slice making: Fix the pre-cooled wax block on the paraffin slicer so that the cut surface of the wax block is parallel to the blade. The inclination of the knife is usually 15 degrees. Turn the rotary propeller to adjust the slice thickness to 3 μm and cut into slices of uniform thickness. Hold the brush in your left hand and rotate the handle of the slicer with your right hand. After the slice is brought out, gently lift it up with the brush, and then use tweezers to gently tweeze the wax slice and put it into the slide box with the front side. The water temperature is about 40°C. After it is spread flat, pick up the slice. Attach the slice. Hold one end of the glass slide with your left hand and put it vertically into the water to attach the slice. Use tweezers in your right hand to help push it and attach it to two-thirds of the way on the slide. After the slice is attached, place it in the air to dry slightly, bake it on a 60°C slide oven for 1 hour, and then bake it in an oven for 2 hours; (6) Dewaxing paraffin sections to water: Dewax the paraffin sections in xylene I for 15 min, xylene II for 15 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 10 min, and 85% ethanol for 10 min in a gradient dewaxing process; (7) HE staining: paraffin sections were stained with hematoxylin for 5-10 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, then blued with saturated aqueous solution of lithium carbonate for 1 min, rinsed with running water for a few seconds, stained with eosin stain for a few seconds, and rinsed with running water; (8) Dehydration and sealing: Paraffin sections are placed in 75% ethanol for 2 min, 85% ethanol for 2 min, anhydrous ethanol for 2 min, anhydrous ethanol for 2 min, and xylene for 2 min to make them transparent. Take the sections out of xylene and seal them with neutral gum. 3) Enzyme-linked immunosorbent assay: Immunohistochemical staining: (1) Dewaxing of paraffin sections: sequentially place the sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water; (2) Antigen repair: See the table above for repair. During this process, prevent excessive evaporation of the buffer and do not allow the slide to dry out. After natural cooling, place the slide in PBS (pH 7.4) and wash on a decolorizing shaker 3 times, 5 minutes each time (the repair solution and repair conditions are determined according to the tissue); (3) Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide solution and incubate at room temperature in the dark for 25 min. Place the slides in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 min. (4) Serum blocking: Add 3% BSA in the tissue circle to evenly cover the tissue, and block at room temperature for 30 minutes. (If the primary antibody is from goat, use rabbit serum to block, and if it is from other sources, use BSA to block); (5) Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS on the slices, and place the slices flat in a humidified box and incubate at 4°C overnight; (6) Adding secondary antibody: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. After the slices are slightly dried, add a secondary antibody (HRP-labeled) of the same species as the primary antibody to cover the tissue and incubate at room temperature for 50 min. (7) DAB color development: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time. After the slices are slightly dried, add freshly prepared DAB color development solution in the circle. Control the color development time under a microscope. The positive color is brown-yellow. Rinse the slices with tap water to stop the color development. (8) Re-staining of cell nuclei: Re-stain with hematoxylin for about 3 minutes, wash with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, blue with hematoxylin bluing solution, and rinse with running water; (9) Dehydration and sealing: Place the slices in 75% ethanol for 5 min, 85% ethanol for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min to dehydrate and make them transparent. Take the slices out of xylene and let them dry slightly, then seal them with sealing glue. (10) Microscopic examination: Place the sample under a white light microscope to interpret the results; Enzyme-linked immunosorbent assay was performed on ALB, AFB, CEA, Creatinine, CA199, GTT and other indicators: (1) Dilution of standard; (2) Sample addition: Set up blank wells and add the sample to the bottom of the well of the ELISA plate without touching the well wall. Gently shake to mix. (3) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min. (4) Solution preparation: dilute the 30-fold concentrated washing solution with 30-fold distilled water for later use; (5) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this process 5 times and pat dry. (6) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank well; (7) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 min. (8) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it. Repeat this process 5 times and pat dry. (9) Color development: First add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 10 min. (10) Termination: Add 50 μL of stop solution to each well to terminate the reaction; (11) Measurement: Use blank hole to adjust to zero, 450 nm The absorbance of each well is measured in sequence at different wavelengths. The measurement should be performed within 15 minutes after adding the stop solution; 4) Transcriptome analysis of nude mouse blood and tumor samples: ——4.1 Sample collection method The blood collection technique of retro-orbital vein is used: the operator fixes the mouse with the left hand, and gently squeezes the sides of the neck with the index finger and thumb to promote the congestion of the retro-orbital vein. At a 45° angle, a fine needle or a thin glass tube is inserted from the inner corner of the eye, and the blood is rotated and aspirated, and then the blood is transferred to a pre-cooled cryotube; ——4.2 Sample processing flow The collected samples were added with Trizol reagent (for erythrocyte enucleation) at a ratio of 1:3, and the samples were fully mixed by vigorous pipetting and vortexing, and incubated at room temperature for 5 min. The samples were then divided into RNase-free cryotubes at a ratio of 1 mL / tube and used immediately or quickly frozen in liquid nitrogen and transferred to -80°C for storage; ——4.3 RNA extraction process (1) The treated samples were allowed to stand at room temperature for 5 min and then centrifuged at 4°C and 12,000 rpm for 5 min. (2) Add chloroform to the sample, vortex mix for 15 seconds, and then let stand at room temperature; (3) Centrifuge again at 4°C and 12,000 rpm for 15 min and transfer the supernatant to a new centrifuge tube. (4) Add 1.2 times the volume of anhydrous ethanol to the supernatant and extract RNA using the magnetic bead method or centrifugal column method; The detailed steps of the magnetic bead method are as follows: (5) Add magnetic beads to the solution, vortex mix for 15 seconds, and let stand at room temperature for 5 minutes; (6) After a brief centrifugation, collect the residual liquid at the bottom of the tube and place the centrifuge tube on a magnetic rack for 3 min. After the magnetic beads are completely adsorbed, remove the supernatant; (7) Add 500 μL of washing buffer to the centrifuge tube, vortex to mix, centrifuge briefly, place on a magnetic rack for 3 min, and discard the supernatant; (8) Remove the centrifuge tube from the magnetic rack, open the lid and let it stand at room temperature for 3 min; (9) Add RNase-free water to the tube, vortex to mix, and let stand at room temperature for 5 min. After centrifugation, collect the solution into a new RNase-free centrifuge tube to obtain 45-95 μL of RNA solution; ——4.4 RNA sequencing Total RNA was extracted from the samples, and the concentration, purity and integrity of RNA were tested by agarose gel electrophoresis, Agilent 2100 and other methods. The total amount of RNA required for each library construction was no less than 1 μg, the concentration should be ≥35 ng / μL, and the OD 260 / 280 The value should be between 1.8 and 2.2. Then, mRNA is enriched, fragmented, reverse transcribed into cDNA, adapters are connected, fragments are screened, and the library is enriched, ready for sequencing; The differentially expressed genes were functionally analyzed using GO, KEGG and other databases, and the KEGG pathway diagram of the differentially expressed genes was constructed. Based on the results of differential gene analysis, the key genes related to liver cancer treatment and immune response were screened through differential gene GO enrichment and KEGG pathway analysis, such as MYD88, CD14, MCSF, TGF-β1, P53, JUN, BCL-2, TNF-α and TLR4Etc. This step is to further verify the protein expression data obtained by Western blot experiments and ensure the consistency of transcriptomics data with protein levels, thereby providing more comprehensive and reliable biological evidence for the research.

[0052] 2. Results and analysis.

[0053] 2.1 Effects of extracellular polysaccharides on liver and kidney function indicators of nude mice with Hep-G2 cell-induced liver cancer.

[0054] After analysis, we found that the alanine aminotransferase (ALT) level in the model group was significantly increased when compared with the normal control group, which indicates impaired liver function. However, it is worth noting that the ALT level in the high-dose treatment group was slightly higher than that in the positive control group, but there was no significant difference compared with the normal control group. In addition, the ALT levels in the medium-dose and low-dose treatment groups were lower than those in the positive control group, indicating that at these doses, the hepatotoxicity of bacterial exopolysaccharides to nude mice was maintained within a safe range ( P =0.0024). In further analysis of liver function, we observed that the levels of aspartate aminotransferase (AST) decreased in the high-dose and medium-dose treatment groups, indicating that liver toxicity was alleviated ( P =0.0468). Meanwhile, the positive control group had an increased uric acid (UA) level after taking the drug ( P =0.5234), which suggests that the kidneys may be slightly damaged. Nevertheless, the urea (UREA) level in the positive control group remained within the normal range after taking the drug ( P =0.05). Based on the above results, we can conclude that the bacterial exopolysaccharide has no adverse effects on the kidney and liver at moderate doses, thus demonstrating its excellent biosafety.

[0055] 2.2 HE staining results of nude mouse tumor tissue.

[0056] Analysis of the effects of extracellular polysaccharides on nude mouse tumor morphology revealed that the tumor tissue in the model group showed a heterogeneous distribution, typically manifested as a honeycomb or fibrous cord-like structure with increased density. The nuclear heterogeneity in this tissue was obvious, the continuity between cells was enhanced, there was obvious nuclear atypia and cell volume inconsistency, and many nuclei showed asymmetric mitosis and other morphologies. In addition, the tumor tissue proliferated actively, the nuclei were deeply stained, and there was infiltration of inflammatory cells. The invasion and infiltration growth of tumor tissue on the surrounding muscle tissue were also confirmed.

[0057] In contrast, all groups in the drug-treated group showed partial lysis of tumor cells, with reduced cell volume, increased intercellular spaces, contracted nucleoli, and significantly expanded necrotic areas. The positive control group and the medium-dose group showed significant therapeutic effects, accompanied by a large number of inflammatory cell infiltrations, vacuolar degeneration, and cell death. The low-dose group had fewer necrotic areas. In summary, the pathological morphological changes in the drug-treated group significantly indicated the significant inhibitory effect of rhizobium exopolysaccharides on tumor growth at medium doses. This finding emphasizes the potential of rhizobium exopolysaccharides as an effective antitumor agent, which shows strong biological activity by affecting the proliferation and survival mechanisms of tumor cells, providing a new strategic direction for tumor treatment.

[0058] 2.3 Effects of bacterial extracellular polysaccharides on Creatinine, CA199, GGT, CEA, ALB, AFB and other indicators in nude mice with liver cancer.

[0059] Biochemical indicators such as creatinine, carcinoembryonic antigen (CEA), gamma-glutamyl transferase (GGT), cancer antigen 199 (CA199), albumin (ALB) and alpha-fetoprotein (AFB) were evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that creatinine, GGT and ALB, as biochemical indicators of renal function, showed lower renal damage in the low-dose and medium-dose treatment groups ( P <0.0001). At the same time, CA199, CEA and AFB, as cancer biomarkers, decreased significantly after administration ( P <0.0001), indicating that the application of exopolysaccharides and positive control drugs (sorafenib) can effectively improve the overall health of tumor-bearing nude mice, and show a clear therapeutic effect, reducing liver and kidney damage, indicating that it has a high safety. This result provides an important experimental basis for the application of exopolysaccharides in anti-cancer treatment, and emphasizes its potential value in reducing the burden of tumors on the kidneys and protecting kidney function.

[0060] In this example, the effects of bacterial exopolysaccharides (EPS) from rhizobia on a nude mouse model of liver cancer and its potential mechanism of action were explored in vivo. By constructing a nude mouse model of liver cancer induced by Hep-G2 cells and administering exopolysaccharides, the inhibitory effect of EPS on liver cancer growth in nude mice and its effects on liver and kidney function indicators, tumor tissue pathological changes, and tumor markers were deeply analyzed. At the same time, the molecular mechanism of EPS's anti-liver cancer effect was further explored through transcriptomic analysis and protein expression detection.

[0061] The study found that EPS administration can significantly inhibit the growth of Hep-G2 liver cancer cells in nude mice, as shown by the recovery of body weight and condition after administration. The test results of liver and kidney function indicators showed that EPS administration can not only effectively inhibit the growth of liver cancer, but also has no significant toxic side effects on the liver and kidney function of mice, showing good biosafety. Pathological examination of tumor tissues further confirmed the anti-tumor activity of EPS, and a significant increase in cell apoptosis, infiltration of inflammatory cells, and destruction of tumor cell structure appeared in tumor tissues.

[0062] Through transcriptomic analysis and immunohistochemical detection, the molecular mechanism of EPS's anti-liver cancer effect was deeply explored. The results showed that EPS treatment significantly affected multiple signaling pathways closely related to the occurrence and development of liver cancer, especially the MAPK signaling pathway. In this pathway, EPS regulated the expression of multiple key genes and proteins, such as MYD88, CD14, MCSF, TGF-β1, P53, JUN, BCL-2, TNF-α and TLR4, and the regulation of these molecules is closely related to biological processes such as cell apoptosis, immune response and cell proliferation.

[0063] Discussion of the results The goal of this application is to explore the effects of rhizobia extracts on a nude mouse model of liver cancer and its mechanism of action. First, crude polysaccharides were extracted from bacterial fermentation broth using rotary evaporation, alcohol precipitation, deionized water dialysis, and freeze-drying techniques. Subsequently, the crude polysaccharides were further purified through a series of purification processes, including protein removal, defatting, decolorization, macroporous resin adsorption, and gel filtration, and the yield of the purified polysaccharides and their monosaccharide composition were determined. This application deeply explores the anticancer activity of exopolysaccharides (EPS) produced by rhizobia on a hepatocellular carcinoma (HCC) model and its potential mechanism. Through detailed in vitro and in vivo experiments and biochemical analyses, the significant ability of this exopolysaccharide in immunomodulation and promoting cancer cell apoptosis was revealed, and its safety was evaluated.

[0064] In summary, extracellular polysaccharides rich in glucose, galactose, mannose and glucuronic acid may participate in immune regulation through various mechanisms, thereby playing a positive role in enhancing the host's immunity and resistance. For example, glucose and galactose, as key structures recognized by cell surface receptors, may activate immune cells and promote immune responses through specific receptor-mediated signaling pathways, such as the Toll-like receptor (TLRs) pathway.

[0065] In the in vitro experiment, the CCK-8 method was first used to evaluate the effect of the extracted extracellular polysaccharides on Hep-G2 liver cancer cells in vitro. The results showed that when the polysaccharide concentration reached 150 μg / μL, the activity of Hep-G2 cells was observed to be reduced to the lowest, indicating that the extracellular polysaccharides have a certain inhibitory effect on early liver cancer cells. This finding echoes previous studies that Ganoderma lucidum deep fermentation intracellular polysaccharides have an inhibitory effect on human liver cancer cells Hep-G2 at lower doses in the early stages, but the effect is weakened in the later stages of the treatment. However, high doses of intracellular polysaccharides can induce apoptosis of Hep-G2 cells in the later stages of culture, and additional doses of intracellular polysaccharides can further enhance this apoptotic effect.

[0066] To further explore the effect of exopolysaccharides on apoptosis of Hep-G2 cells, cells treated with different concentrations of exopolysaccharides were analyzed by flow cytometry. Compared with the blank control group and the normal group, the proportion of apoptotic cells in the exopolysaccharide-treated group was significantly increased.

[0067] In the in vivo experiment, 4-5 week old BALB / c-nu nude mice were selected as experimental subjects to construct a liver cancer model and treated with gradient concentrations of bacterial exopolysaccharides in order to explore the role of the polysaccharide in mammals and its potential liver and kidney toxicity. At the end of the experiment, biochemical and histological analyses were performed from the collected serum and organ samples. Key biochemical indices included indicators of liver function, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and indicators of kidney function, such as blood uric acid (UA) and urea (UREA). The results of these indicators showed that compared with the normal control group, the nude mice treated with exopolysaccharide did not show statistically significant differences in the above biochemical indicators, indicating that the polysaccharide did not have a negative impact on the liver and kidney function of the experimental animals. Other important biochemical markers, including glutamyl transpeptidase (GGT), creatinine (Creatinine) and enhancer of liver regeneration (ALR), were then quantitatively analyzed using enzyme-linked immunosorbent assay (ELISA) technology. These data also showed that there was no significant difference between the polysaccharide-treated group and the normal control group, further verifying the safety of the exopolysaccharide. It is worth noting that the levels of cancer-related biomarkers such as CA199, CEA, and AFB showed a significant decrease after treatment, suggesting that bacterial exopolysaccharides have a potential effect in inhibiting the development of liver cancer. Compared with other treatment groups and the positive control group (sorafenib), polysaccharide treatment can effectively reduce liver and kidney damage and help restore the function of these organs, providing further evidence for the anti-liver cancer effect of bacterial exopolysaccharides.

[0068] The pathological morphological analysis of tumor tissue revealed that in the model group, the tumor tissue showed uneven distribution and high density, with significant nuclear atypia, different cell sizes, and most nuclei showed asymmetric division morphology. In addition, the tumor cells showed active growth, dark nuclear staining, and infiltration of inflammatory cells, and the invasion and infiltration of tumor tissue into the surrounding muscle tissue was obvious. In contrast, the tumor cells in the groups treated with extracellular polysaccharides showed partial dissolution, vacuolation, and cell miniaturization, widened intercellular spaces, condensation of nuclei, and significantly increased necrotic areas. This effect was particularly evident in the positive control group and the medium-dose treatment group, accompanied by a large number of inflammatory cell infiltrations and cell death. In contrast, the necrotic area in the low-dose group was relatively small. These results indicate that the studied extracellular polysaccharides have a significant inhibitory effect on tumor growth.

[0069] Based on transcriptomics to explore the mechanism of action of extracellular polysaccharides on Hep-G2 cell-induced liver cancer in nude mice, transcriptomics analysis of liver cancer nude mice samples showed that 33,115 genes were expressed in tumor samples, 103,238 transcripts were expressed, and 21,755 genes were expressed in blood samples, 68,621 transcripts were expressed. After in-depth analysis of the two sets of data, the MAPK signaling pathway was selected as the differentially expressed pathway of this study, and the differentially expressed genes in this pathway were CD14, TLR4, MYD88, JUN, TGF-β1 are key regulatory genes. CD14 Located in cholesterol- and sphingolipid-rich plasma membrane nanodomains, it is considered TLR4 Activation site. TLR4Disorders of endocytosis and dysfunction of the endosomal mechanism can affect the pro-inflammatory response triggered by TLR4, and can also affect MyD88-dependent signal transduction, while slowing down TLR4 degradation will increase the pro-inflammatory response. In this experiment, the expression of apoptotic proteins in tumor tissue cells was detected by immunohistochemistry. Compared with the model group, the number of positive cells in the drug-treated group increased significantly. The results showed that this exopolysaccharide can significantly upregulate the expression of proteins MYD88, CD14, TNF-ɑ, JUN, TGF-β1, TLR4, MCSF, P53, and BCL-2. Therefore, the selected exopolysaccharide can effectively inhibit the growth of Hep-G2 subcutaneous transplanted tumors. The key proteins such as CD14, TLR4, MYD88, JUN, TGF-β1, and TNF-ɑ were also confirmed to be activated in liver cancer cells and liver cancer nude mouse tissues in the western blot and QPCR results of this study. Exopolysaccharides exert their anti-liver cancer effects through specific molecular mechanisms, especially by activating and regulating the MAPK signaling pathway and its downstream apoptosis-related factors. The formation process of liver cancer involves the transformation of normal liver cells into tumor cells, which is mainly caused by DNA damage and excessive cell proliferation caused by carcinogenic factors. Western blot experiments verified that P53 and BCL-2 proteins were upregulated after administration, and in vivo experiments in liver cancer nude mice verified that P53 and BCL-2 proteins were upregulated after administration, promoting tumor cell apoptosis.

[0070] In summary, this application reveals the molecular mechanism of the inhibitory effect of Rhizobium exopolysaccharides on Hep-G2 liver cancer cells, namely, promoting the apoptosis of liver cancer cells by activating the MAPK signaling pathway and its downstream apoptosis-related molecules. These findings not only provide a new mechanistic understanding of the anti-liver cancer activity of exopolysaccharides, but also provide a scientific basis for the future development of anti-cancer treatment strategies based on exopolysaccharides.

[0071] In addition, it is necessary to provide a scientific basis for the future development of anti-cancer therapeutic strategies based on extracellular polysaccharides. Several points worth emphasizing include: 1) In-depth analysis of the mechanism of action of polysaccharides: This application reveals that extracellular polysaccharides have significant inhibitory and apoptosis-inducing effects on Hep-G2 cells, which is closely related to the structural characteristics of extracellular polysaccharides. The specific monosaccharide composition and molecular weight in extracellular polysaccharides are key factors in their anti-liver cancer activity. Future studies can further explore the specific effects of these structural characteristics on their biological activity, and provide a theoretical basis for the synthesis of polysaccharides with specific anti-cancer activity; 2) Combined application of exopolysaccharides with other treatment methods: Considering the significant inhibitory effect of exopolysaccharides on Hep-G2 cells, combining them with existing treatment methods, such as chemotherapy and targeted therapy, may produce synergistic effects, enhance therapeutic effects, and reduce side effects. The potential for combined application of exopolysaccharides with other treatment strategies can be explored; 3) Production and optimization of exopolysaccharides: In order to realize the clinical application of exopolysaccharides, the development of large-scale production and purification methods is crucial. It is necessary to further improve the yield and purity of exopolysaccharides while reducing production costs, including optimizing fermentation conditions and exploring more efficient purification technologies; In summary, this application provides new insights into the application of exopolysaccharides in the field of liver cancer and demonstrates their great potential and biosafety as potential anticancer drugs. Through further mechanism research, safety assessment and production optimization, exopolysaccharides are expected to become an effective anti-liver cancer treatment strategy. Future work will be devoted to in-depth exploration of other potential anti-cancer mechanisms of exopolysaccharides and evaluation of their clinical application prospects, hoping to provide new solutions for the treatment of liver cancer.

[0072] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A rhizobium exopolysaccharide, characterized in that: The molecular weight of the rhizobium extracellular polysaccharide is 691.01 kDa, and the polysaccharide is mainly composed of galactose, glucose, mannose and glucuronic acid. The molar ratio of galactose, glucose, mannose and glucuronic acid is 17.45:67.06:14.50:0.

99.

2. The Rhizobium exopolysaccharide according to claim 1, characterized in that: The exopolysaccharide is prepared from Rhizobium yanglingensis ( Rhizobium yanglingense ) production is obtained.

3. The Rhizobium exopolysaccharide according to claim 1, characterized in that: The preparation method of the extracellular polysaccharide comprises the following steps: S1: Extraction of crude exopolysaccharide; S2: Macroporous resin purification of exopolysaccharides; S3: Gel purification of exopolysaccharides.

4. The Rhizobium exopolysaccharide according to claim 3, characterized in that: The preparation method of the extracellular polysaccharide comprises the following steps: S1: Extraction method of crude exopolysaccharide: (1) The screened strains were quantitatively inoculated into YMA liquid fermentation medium and cultured in a sterile shaking incubator at 28°C overnight until the stable phase; (2) The cultured rhizobium fermentation liquid was centrifuged to remove the bacteria, concentrated to a certain volume by rotary evaporation, and then precipitated overnight with 4 times anhydrous ethanol. The precipitate was collected by centrifugation, dialyzed with deionized water for 48 h, and vacuum freeze-dried to obtain crude polysaccharides; S2: Macroporous resin purification of extracellular polysaccharides: (1) Grind the freeze-dried sample and pass it through a 100-mesh sieve; (2) Add an appropriate amount of anhydrous ethanol, stir to extract fat-soluble pigments and some impurities, centrifuge at 6000 × g for 10 min, and collect the precipitate; (3) Add an appropriate amount of pure water to the precipitate, extract in a constant temperature water bath at 55°C for 3 h, then remove and centrifuge at 6000 × g for 10 min. Collect the supernatant extract and extract the precipitate residue in the centrifuge tube again according to the above steps; (4) Combine the extracts collected twice, concentrate by vacuum evaporation to 1 / 10 of the original volume, and then add four times the amount of anhydrous ethanol to precipitate at 4°C overnight; (5) Centrifugation at 8000 × g for 10 min to collect the precipitated solid, i.e., the crude polysaccharide extract; (6) Add an appropriate amount of pure water and a small amount of papain to the solid and ferment overnight; (7) Add Sevage reagent to the aqueous phase, mix thoroughly, and collect the upper aqueous phase; (8) Add an appropriate amount of petroleum ether to the liquid, mix thoroughly, and collect the lower aqueous phase; (9) Add an appropriate amount of macroporous resin AB-8 to the aqueous phase, mix thoroughly, and adsorb overnight; (10) Collect the liquid, dialyze it in a 3000 Da dialysis bag for 48 h to remove small molecular components, and freeze-dry the polysaccharide solution; (11) The phenol-sulfuric acid method was used to determine the soluble total sugar content and purity of the crude polysaccharide; S3: Gel purification of exopolysaccharides: (1) Take the polysaccharide sample after ion purification and add pure water to make a polysaccharide mother solution with a concentration of 15 mg / mL. Centrifuge at 10,000 × g for 10 min, take the supernatant, and separate and purify it using a gel chromatography column at a flow rate of 1 mL / min: elute with pure water for 1.5 times the column volume, collect one tube for every 12 mL, and collect all the eluate; (2) Determine the total sugar content of the eluate in each collection tube using the sulfuric acid-phenol method and draw a gel purification elution curve; (3) Select and confirm the elution peak; (4) Combine the collected eluates corresponding to the same elution peak and concentrate by rotary evaporation to 1 / 5 of the original volume; (5) Freeze-drying and using the sulfuric acid-phenol method to identify the content and purity of the polysaccharide after gel purification.

5. A rhizobium exopolysaccharide according to claim 3 or 4, characterized in that: The total sugar purity of the extracellular polysaccharide after purification by macroporous resin in S2 of the preparation method is at least 57.2%, and the total sugar purity of the polysaccharide after purification by gel in S3 is at least 84.5%.

6. The Rhizobium exopolysaccharide according to any one of claims 1 to 4, characterized in that: The extracellular polysaccharide is used in preparing drugs related to liver cancer cell inhibition.

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