Poplar phellinus polysaccharide SVP-1, and preparation method and application thereof

Abstract

The application discloses poplar phellinus polysaccharide SVP-1, a preparation method and application thereof, and belongs to the field of biological medicines. The poplar phellinus polysaccharide SVP-1 provided by the application has significant antitumor activity, can effectively inhibit the proliferation, invasion and migration of tumor cells, and thus blocks the malignant progression of tumors. The structure is clear, the preparation method is controllable, the stability and biological activity of the polysaccharide component are ensured, and a reliable foundation is provided for the development of antitumor drugs. The combination with radiotherapy and chemotherapy drugs can play a synergistic effect, can not only improve the sensitivity of tumors to treatment, but also reduce the toxic and side effects of radiotherapy and chemotherapy, and realizes the effect of 'attenuation and synergism'. The combined treatment strategy can significantly improve the curative effect of the existing tumor therapy, while reducing the adverse reactions, and provides a new optimization scheme for clinical tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a poplar phellinus polysaccharide SVP-1 and a preparation method and application thereof. BACKGROUND

[0002] At present, cancer has become a major disease threatening human health, although the development of medical technology has improved the early detection rate of cancer, but there are still a large number of patients who are diagnosed as being in the middle and late stages, resulting in poor treatment effect. The current mainstream cancer treatment methods all have obvious technical defects: surgery and radiotherapy have certain effect on early solid tumors, but the effect on patients with metastatic advanced cancer is limited; the immune checkpoint therapy represented by PD-1 / PD-L1 inhibitor has low objective remission rate, and there are problems such as super progression and immune-related adverse reactions, and some patients are difficult to benefit from it; although the targeted drugs can accurately kill tumor cells with specific gene mutations, drug resistance occurs universally due to tumor heterogeneity; as a common treatment for terminal cancer, chemotherapy has low overall efficiency, although it can improve the palliative symptoms and prolong the survival period to a certain extent, it cannot achieve radical cure. Even though CAR-T cell therapy has achieved good results in the treatment of hematological tumors, it still faces the technical bottleneck of low penetration efficiency in the treatment of solid tumors.

[0003] Although the combination of chemotherapy and immunomodulatory drugs provides a new idea for cancer treatment and can improve the remission rate of some cancer patients to a certain extent, there are still problems such as immune exhaustion rebound. Therefore, it is urgent to find new treatment methods and drugs. As a traditional medicinal fungus, the polysaccharide component of phellinus has many potential biological activities, and the research on phellinus polysaccharide and its preparation method and the exploration of its application in cancer treatment are expected to provide a new way for cancer treatment and solve the problems existing in the current treatment technology. SUMMARY

[0004] The purpose of the present application is to provide a poplar phellinus polysaccharide SVP-1 and a preparation method and application thereof, so as to solve the problems existing in the prior art. The poplar phellinus polysaccharide SVP-1 provided by the present application has significant antitumor activity, can effectively inhibit the proliferation, invasion and migration of tumor cells, thereby blocking the malignant progression of tumor, and can play a synergistic effect when combined with radiotherapy and chemotherapy drugs.

[0005] To achieve the above purpose, the present application provides the following scheme:

[0006] The present application provides a poplar phellinus polysaccharide SVP-1, the structure of the poplar phellinus polysaccharide SVP-1 is composed of 192 repeating units;

[0007] The main chain repeating unit is composed of four sugar residues of →6)-beta-D-Glcp-(1→, →3,2)-alpha-L-Manp-(1→, →6)-beta-D-Glcp-(1→ and →6,3)-beta-D-Glcp-(1→, which are sequentially connected by (1→3), (1→6), (1→3) glycosidic bonds, respectively.

[0008] The branched chain alpha-L-Manp-(1→ and beta-D-Galp-(1→ are sequentially connected to the →3,2)-alpha-L-Manp-(1→ and →6,3)-beta-D-Glcp-(1→ residues of the main chain by (1→2), (1→6) glycosidic bonds, respectively, and the structure of the sugar chain is as follows:

[0009]

[0010] The application further provides a preparation method of the Populus Populus polysaccharide SVP-1, comprising the following steps:

[0011] After the fruiting body of Populus Populus is crushed, distilled water is added for decoction and extraction, and the extraction filtrate is concentrated, precipitated by an ethanol solution, and then the precipitate is decolorized, impurity-removed and separated by a DEAE cellulose column to obtain the Populus Populus polysaccharide SVP-1.

[0012] Optionally, the mass-volume ratio of the powder of the fruiting body to the distilled water is 1g:10mL.

[0013] Optionally, the volume concentration of the ethanol solution in the precipitation system is 70%.

[0014] The application further provides application of the Populus Populus polysaccharide SVP-1 in preparation of an immunomodulatory drug for resisting tumors, and the immunomodulatory drug is used in combination with an anti-tumor chemotherapy and / or radiotherapy drug.

[0015] Optionally, the tumor includes liver cancer.

[0016] Optionally, the chemotherapy drug includes sorafenib.

[0017] The application further provides an immunomodulatory drug for resisting tumors, and the effective component includes the Populus Populus polysaccharide SVP-1.

[0018] The application discloses the following technical effects:

[0019] The Populus Populus polysaccharide SVP-1 provided by the application has significant anti-tumor activity, can effectively inhibit the proliferation, invasion and migration of tumor cells, and thus blocks the malignant progression of tumors. The structure is clear, the preparation method is controllable, the stability and biological activity of the polysaccharide component are ensured, and a reliable basis is provided for development of an anti-tumor drug.

[0020] The poplar phellinus polysaccharide SVP-1 provided by the application can regulate tumor microenvironment, reverse the immunosuppressive state, and enhance the anti-tumor immune response of the body. By remodeling the immune microenvironment, the immune microenvironment is changed from an immunosuppressive state to an immune activation state, thereby synergistically enhancing the killing effect of immune cells on tumors. This characteristic has important application potential in the field of tumor immunotherapy.

[0021] The poplar phellinus polysaccharide SVP-1 provided by the application can play a synergistic effect when combined with radiotherapy and chemotherapy drugs, not only can improve the sensitivity of tumors to treatment, but also can reduce the toxic and side effects of radiotherapy and chemotherapy, and achieve the effect of 'attenuation and synergism'. This combined treatment strategy can significantly improve the efficacy of existing tumor therapy, while reducing adverse reactions, and provides a new optimization scheme for clinical tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0023] Figure 1 For the purification, molecular weight and monosaccharide composition determination of SVP-1, (A) DEAE cellulose-52 elution curve; (B) HPGPC chromatogram (ELSD detector) of SVP-1; (C) High performance liquid chromatography (HPLC) analysis of monosaccharide standard after PMP derivatization; (D) High performance liquid chromatography (HPLC) analysis of SVP-1 hydrolysate after PMP derivatization;

[0024] Figure 2 For the characteristic analysis of SVP-1, (A) UV spectrum of SVP-1; (B) Fourier transform infrared spectrum of SVP-1; (C) Thermogravimetric analysis of SVP-1; (D) Scanning electron microscope images of SVP-1 at 1000 times, 2500 times, 5000 times and 10000 times magnification;

[0025] Figure 3 For the methylation analysis and one-dimensional nuclear magnetic resonance spectrum analysis of SVP-1, (A) Total ion gas chromatogram of SVP-1 in gas chromatography-mass spectrometry analysis; (B) 1 H NMR nuclear magnetic resonance spectrum; (C) 13 C NMR nuclear magnetic resonance spectrum;

[0026] Figure 4 For the hydrogen-hydrogen correlation spectrum of SVP-1 (H- 1 H- 1 H COSY spectrum);

[0027] Figure 5 Carbon-hydrogen heteronuclear single quantum correlation spectrum of SVP-1 13 C- 1 H HSQC spectrum

[0028] Figure 6 Carbon-hydrogen heteronuclear multiple bond correlation spectrum of SVP-1 13 C- 1 H HMBC spectrum

[0029] Figure 7 Predicted chemical structure of SVP-1

[0030] Figure 8 Effect of SVP-1 combined with sorafenib on the proliferation ability of HepG-2 cells, (A, C) Effect of SVP-1 combined with sorafenib on the proliferation of HepG-2 cells was detected by EdU detection method; (B) Effect of SVP-1 combined with sorafenib on the viability of HepG-2 cells was detected by CCK-8 detection method; (D, E) Effect of SVP-1 combined with sorafenib on the colony formation of HepG-2 cells

[0031] Figure 9 Effect of SVP-1 combined with sorafenib on the invasion and migration ability of HepG-2 cells, (A, B) Effect of SVP-1 combined with sorafenib on the migration of HepG-2 cells; (A, C) Effect of SVP-1 combined with sorafenib on the invasion of HepG-2 cells

[0032] Figure 10 Effect of SVP-1 combined with sorafenib on the tumor, body weight, immune organ index, inflammatory factor, total bilirubin (TBIL) and aspartate aminotransferase (AST) levels of H22 tumor-bearing mice, (A) Tumor image; (B) Tumor volume change histogram; (C) Tumor weight histogram; (D) Tumor inhibition rate histogram; (E) Body weight increase before and after tumor-bearing

[0033] Figure 11 Hematoxylin-eosin (H&E) staining of tumor tissue and heart, liver, spleen, lung, kidney.

[0034] Figure 12 SVP-1 transcriptome data analysis chart, (A) Volcano plot of differentially expressed genes (DEGs) between SVP-1 group and model group; (B) Heat map of significantly differentially expressed genes between SVP-1 high dose group and model group

[0035] Figure 13Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the significantly differentially expressed genes between the SVP-1 high-dose group and the model group.

[0036] Figure 14 For SVP-1 to affect the key protein level change analysis of tumor-bearing mice, (A) Western blotting of CD48, CD244, LAT, PLCG1, PPP3CA and NFATC2 in mouse tumor tissues; (B-G) statistical results of CD48, CD244, LAT, PLCG1, PPP3CA and NFATC2 protein expression;

[0037] Figure 15 For the effect of SVP-1 on the expression of TNF-α, GM-CSF and IFN-γ in mouse tumor tissues, (A) immunohistochemical results of TNF-α, GM-CSF and IFN-γ in mouse tumor tissues, (B) statistical results of TNF-α, GM-CSF and IFN-γ expression. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present application will now be described in detail, which should be considered in a descriptive sense only and not for purposes of limitation to the application described. Rather, it is understood that certain aspects, particular features and embodiments of the application can be directed to one or more of the technical solutions described herein.

[0039] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for any numerical ranges that follow, it is intended that every number within the range and each end point is specifically recited. It is also understood that the end points of the ranges can be included or excluded. Any open range concepts within the described ranges are also intended to be specifically recited herein. The use of any open-ended number range concepts such as "between" are intended to cover any and all elastic ranges subsumed within the open-ended range.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application.

[0041] Many modifications and variations of this application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only.

[0042] As used herein, "comprise", "comprising", "having", "including", "contain", "containing", "include" and the like are open-ended terms that are intended to mean including but not limited to.

[0043] The Sanghuangporus vaninii used in the embodiments of the present application is purchased from Yucheng Changbai Mountain Wild Ganoderma Technology Co., Ltd. (Baishan, China). The H22 mouse hepatoma cell line is purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The human hepatoma cell line (HepG-2) is purchased from Beijing Beina Link Biological Technology Institute (Beijing, China). SD rats (body weight 180-220 g) and Kunming (KM) mice (body weight 18-22 g) are purchased from Changchun Yis Experimental Animal Technology Co., Ltd., license number SYXK(Ji)2018-0023.

[0044] Other reagents and biological materials are routinely purchased unless otherwise specified.

[0045] Example 1 Screening of Anti-cancer Active Fraction of Sanghuangporus vaninii

[0046] 1. Preparation of each extraction and separation fraction of Sanghuangporus vaninii

[0047] After crushing 5 kg of Sanghuangporus vaninii fruiting bodies, 10 times the amount of 70% methanol solution (50 L) was used for ultrasonic extraction for 3 times, 1 hour each time. The extraction solutions were combined and concentrated to dryness to obtain the methanol extract (denoted as SV-Me). Another 5 kg of Sanghuangporus vaninii fruiting bodies were crushed and 10 times the amount of hot water (50 L) was used for decoction for 3 times, 2 hours each time. The decoction solutions were combined and concentrated to 1 / 100 of the original volume (part of the concentrated solution was dried to obtain crude polysaccharides from Sanghuangporus vaninii, denoted as SVP).

[0048] Alcohol precipitation: ethanol was added stepwise to the concentrated decoction solution to achieve ethanol concentrations of 30%, 50%, 70% and 90%, respectively, to precipitate polysaccharides at different levels.

[0049] Decolorization: the polysaccharides precipitated at different levels were completely dissolved in water, respectively, and loaded onto a D101 macroporous resin column. Distilled water was used to elute the column for 8 times the column volume to remove pigments (pigments were adsorbed and removed by the resin). The eluate was collected and concentrated under reduced pressure, respectively.

[0050] Impurity removal: after removing proteins by the Savag method, the protein-free supernatant was concentrated and dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da to remove small molecular impurities. After lyophilization, Sanghuangporus vaninii crude polysaccharides precipitated at 30%, 50%, 70% and 90% ethanol (denoted as SVP-30%, SVP-50%, SVP-70% and SVP-90%, respectively) were obtained.

[0051] 2. Anti-hepatoma activity of each fraction of Sanghuangporus vaninii

[0052] The clean level SD rats were randomly divided into 7 groups (6 rats in each group), namely normal control group (Con), SV-Me group, SVP group, SVP-30% group, SVP-50% group, SVP-70% group, SVP-90% group, and were raised in SPF level environment (temperature 24±2℃, relative humidity 55±5%, 12 hours light / dark cycle, free feeding and drinking water), and the experimental scheme was approved by the experimental animal ethics committee of Jilin Agricultural University. After adaptive feeding for 1 week, each group was given intragastrically with equal volume of normal saline (Con) and 200 mg / kg of SV-Me (SV-Me group), SVP (SVP group), SVP-30% (SVP-30% group), SVP-50% (SVP-50% group), SVP-70% (SVP-70% group), SVP-90% (SVP-90% group) for 7 days, 2 mL per day. After the last administration, the rats were fasted for 12 hours, anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta, centrifuged at 4000 r / min for 15 minutes to separate serum, and stored in aliquots.

[0053] HepG-2 cells were subcultured at a density of 5×10 4 / mL in 96-well plates, incubated for 24 hours, and then divided into groups: control group; sorafenib single-drug group (Sor, 5 μM); SV groups (containing 10% rat serum containing SV-Me / SVP / SVP-30% / SVP-50% / SVP-70% / SVP-90%); each combination group (each 5% containing drug serum + 2.5 μM Sor). The drug concentration was determined by pre-experiment, and after the treatment of each group, 10 μL of CCK-8 reagent was added to each well, incubated at 37℃, 5% CO2 for 2 hours, and the absorbance (OD value) at 450 nm was determined by enzyme-labeled instrument.

[0054] The results of HepG2 cell viability detection are shown in Table 1.

[0055] Table 1 Effect of each SV-containing drug serum sample alone and in combination with Sor on HepG-2 cell viability

[0056]

[0057]

[0058] Note: The data are expressed as mean ± standard deviation (n=5 in each group). Compared with the model group, #P<0.05, ##P<0.01; compared with the Sor single-drug group, $P<0.05, $$P<0.01.

[0059] From the data of Table 1, compared with the model control group (Con), the positive drug sorafenib (Sor) group significantly inhibited the cell viability of cancer cells, and in the Phellinus baumii sample, only the 70% alcohol precipitated part of the crude polysaccharide of Phellinus baumii (SVP-70%) significantly inhibited the cell viability of cancer cells, and part of the crude polysaccharide samples such as SVP-30% and SVP-50% drug-containing serum even enhanced the cell viability of cancer cells. And SVP-70% showed obvious synergistic effect with Sor, which was significantly better than Sor alone. Therefore, the 70% alcohol precipitated part of the crude polysaccharide of Phellinus baumii was selected for further study.

[0060] Example 2 Preparation and structure characterization of Phellinus baumii polysaccharide SVP-1

[0061] 1. Preparation of Phellinus baumii polysaccharide SVP-1

[0062] The SVP-70% prepared in Example 1 was further separated, and the steps were as follows:

[0063] SVP-70% was dissolved in water at a concentration of 25 mg / mL, and was separated by DEAE cellulose-52 column (30 cm x 2.5 cm), and was eluted with water and different concentrations of NaCl solution (0.1 M, 0.3 M, 0.5 M, 0.7 M, 1 M) as mobile phase in turn, at a flow rate of 1 mL / min, and each tube was collected for 10 minutes. The polysaccharide content of each fraction (tube) was determined by phenol-sulfuric acid method, and the polysaccharide concentration of each tube was determined by detecting the absorbance at 490 nm. The fraction with the highest polysaccharide content was collected, dialyzed (molecular weight cut-off 3500) and lyophilized, and HPGPC (high performance gel chromatography) was used to analyze the purity using Tsk Gel 4000 PWXL chromatographic column (7.8 mm x 300 mm) and Waters 2424 evaporative light scattering detector (ELSD). The chromatographic conditions were: detector gain 100, pressure 30 MPa, drift tube temperature 90°C, carrier gas compressed air (pressure 5 bar), column temperature 40°C, mobile phase H2O, flow rate 1 mL / min. The results showed a single retention time chromatographic peak, indicating a homogeneous polysaccharide, which was named SVP-1.

[0064] 2. Structure characterization of Phellinus baumii polysaccharide SVP-1

[0065] (1) Polysaccharide purification and chemical composition

[0066] SVP-70% was separated by DEAE cellulose-52 ion exchange chromatography column to obtain three components Figure 1The polysaccharide content (A) was 73.36%, SVP-2 (15.26%), and SVP-3 (11.38%). Among these, SVP-1, obtained by water elution, exhibited a symmetrical peak and high purity, indicating the highest polysaccharide content. HPGPC evaporative light scattering (ELSD) analysis showed that SVP-1 chromatogram contained only a single symmetrical peak (…). Figure 1 (B) proves that it is a homogeneous polysaccharide. The linear equation fitted based on the dextran standard is (log(Mw)=-0.6122X+7.59, R0). 2 =0.9982) The average molecular weight of SVP-1 was calculated to be 186.6 kDa. Based on the retention time and concentration curves of monosaccharide standards ( Figure 1 The monosaccharide composition and molar ratio of SVP-1 (C, D) are: glucose (49.96%), mannose (33.39%), and galactose (16.65%).

[0067] (2) Ultraviolet and Fourier transform infrared (FT-IR) spectroscopy analysis

[0068] The ultraviolet spectroscopy showed that SVP-1 did not exhibit absorption peaks at either 260 nm or 280 nm. Figure 2 (A). 280nm is typically used to detect the absorbance of proteins, while 260nm is used for nucleic acid detection, indicating that purified SVP-1 does not contain nucleic acids or proteins. The infrared spectrum of SVP-1 shows typical characteristics of polysaccharides (A). Figure 2 B): 3250cm -1 The broad peak at 2932 cm⁻¹ is the stretching vibration peak of OH in polysaccharides. -1 The absorption band is attributed to the CH stretching vibrations of methyl and methylene groups in carbohydrates, confirming that SVP-1 has a characteristic peak of polysaccharides; 1643 cm⁻¹ -1 The absorption band corresponds to the stretching vibration of the hemiacetal group; 1411 cm⁻¹ -1 and 1326cm -1 The peaks are the bending vibration absorption peaks of methyl and methylene, respectively; 1146 cm⁻¹ -1 The absorption peak in this region is due to the bending vibration of CO on the pyran ring; 1073 cm⁻¹ -1 1025cm -1 and 985cm -1 The absorption peak originates from the bending vibration of the alcohol hydroxyl group, suggesting the presence of a pyranose structure; 956 cm⁻¹ -1 The peak represents the side-chain vibration of the terminal methylene group of the pyran ring; 824 cm⁻¹ -1 and 801cm -1 The nearby weak absorption bands indicate the presence of both α- and β-glycosidic bonds. In summary, SVP-1 contains both α- and β-glycosidic bonds.

[0069] (3) Thermogravimetric (TG) and scanning electron microscopy (SEM) analysis

[0070] Thermogravimetric analysis showed that the thermogravimetric process of SVP-1 could be divided into four stages ( Figure 2 (C) Stage 1 (35.28–175.33℃): Mass loss of 11.06%, possibly caused by SVP-1 dehydration; Stage 2 (175.30–438.61℃): Mass loss of 44.41%, the main stage of polysaccharide chemical structure degradation; Stage 3 (438.55–618.69℃): Mass loss of 22.71%, indicating further molecular chain breakage; Stage 4 (618.70–790.04℃): Mass loss of 4.88%, the residue is a carbonized product with high thermal stability. Stages 2 and 3 are the main stages of thermal weight loss, reflecting the influence of polysaccharide molecular weight, monosaccharide composition, glycosidic bond type, and branching degree on its thermal stability. The results show that SVP-1 has good thermal stability.

[0071] The microstructure of SVP-1 was observed using scanning electron microscopy. Figure 2 At magnifications of ×1000, ×2500, ×5000, and ×10000, its surface is rough and wrinkled, with irregular layered and flocculent structures. The flocculent structures are interspersed among the layered structures, and the folds are tightly arranged but without a fixed direction, resulting in a rather chaotic overall morphology.

[0072] (4) SVP-1 methylation analysis

[0073] Methylation analysis of SVP-1 was performed to obtain the total ion chromatogram of the sample. Figure 3 The secondary mass spectrometry fragment peaks of A and each sugar residue were analyzed. Based on literature and ion fragment information from the University of Georgia's Complex Carbohydrate Research Center (CCRC) database, the glycosidic bond types were inferred. Five glycosidic bond fragments were ultimately identified in SVP-1 (Table 2): T-Manp (terminal mannopyranosyl, 15.563%); 2,3-Manp (2,3-linked mannopyranosyl, 15.704%); T-Galp (terminal galactopyranosyl, 18.148%); 6-Glcp (6-linked glucopyranosyl, 33.257%); and 3,6-Glcp (3,6-linked glucopyranosyl, 17.328%).

[0074] Table 2. Methylation analysis data of SVP-1 and corresponding glycosidic bond linkage modes

[0075]

[0076]

[0077] (5) NMR analysis of SVP-1

[0078] Based on the results of methylation analysis of the sugar residue fragments, combined with 1 H NMR, 13 C NMR and 1 H- 1 H COSY spectrum Figure 3 B, C, Figure 4 ), five sugar residue fragments (A-E) were identified.

[0079] Anomeric proton region analysis: 1 The anomeric proton region (δ 5.0-5.2 ppm) of the H NMR spectrum detected five characteristic peaks with chemical shifts of δ 5.13, 4.57, 5.13, 4.55, 5.16 ppm, respectively.

[0080] 1 H- 1 H COSY spectrum further resolved the H signal chain of each residue:

[0081] Residue A: δ 5.13, 3.57, 3.72, 3.50, 3.99, 3.78 / 4.02 ppm;

[0082] Residue B: δ 4.57, 3.97, 4.02, 3.87, 3.80, 3.80 / 3.87 ppm;

[0083] Residue C: δ 5.13, 3.68, 3.87, 3.60, 3.85, 3.77 / 3.78 ppm;

[0084] Residue D: δ 4.55, 3.95, 3.81, 3.69, 3.76, 3.76 / 3.87 ppm;

[0085] Residue E: δ 5.16, 3.74, 3.61, 3.71, 3.70, 3.59 / 3.58 ppm.

[0086] 1 H- 13 C HSQC spectrum analysis Figure 5 , Table 3):

[0087] Residue A (δ 5.13 / 93.28, 3.57 / 72.58, 3.72 / 73.63, 3.50 / 70.35, 3.99 / 71.09, 3.78 / 4.02 / 63.26 ppm) was deduced as →6)-β-D-Glcp-(1→;

[0088] Residue B (δ 4.57 / 95.78, 3.97 / 77.31, 4.02 / 71.05, 3.87 / 75.80, 3.80 / 72.20, 3.80 / 3.87 / 60.60 ppm) was deduced to be →3,2)-α-L-Manp-(1→;

[0089] Residue C (δ 5.13 / 93.28, 3.68 / 70.92, 3.87 / 76.84, 3.60 / 70.92, 3.85 / 72.96, 3.77 / 3.78 / 63.12 ppm) was deduced to be →6,3)-β-D-Glcp-(1→;

[0090] Residue D (δ 4.55 / 95.93, 3.95 / 70.57, 3.81 / 72.46, 3.69 / 68.57, 3.76 / 74.06, 3.76 / 3.87 / 60.60 ppm) was α-L-Manp-(1→ (terminal);

[0091] Residue E (δ 5.16 / 92.13, 3.74 / 69.34, 3.61 / 69.75, 3.71 / 69.75, 3.70 / 71.09, 3.59 / 3.58 / 62.99 ppm) was β-D-Galp-(1→ (terminal).

[0092] 1 H- 13 C HMBC spectrum verified the connection order ( Figure 6 ) :

[0093] The signals at δ 5.13 / 71.05 and 4.57 / 63.26 ppm indicated that H1 of residue A was related to C3 of residue B, and H1 of residue B was related to C6 of residue A, i.e. A→B→A sequential connection.

[0094] The signals at δ 5.13 / 76.84 and 5.13 / 63.26 ppm indicated that H1 of residue A was related to C3 of residue C, and H1 of residue C was related to C6 of residue A, i.e. A→C→A sequential connection.

[0095] The signals at δ 4.55 / 77.31 and 5.16 / 63.12 ppm indicated that H1 of residue D (terminal α-L-Manp) was related to C2 of residue B, and H1 of residue E (terminal β-Galp) was related to C6 of residue C, i.e. D→B and E→C formed branches.

[0096] Conclusion: The main chain structure of SVP-1 is →A-B-A-C→, in which:

[0097] Residue D (α-L-Manp) is connected to C2 of residue B as a branch;

[0098] Residue E (β-Galp) is linked to residue C at C6 as a branch.

[0099] Based on the methylation and NMR data, the structure of SVP-1 is shown as Figure 7 , i.e. →A-B(D)-A-C(E)→.

[0100] Table 3. Main sugar residue of SVP-1 1 H and 13 C NMR chemical shift data

[0101]

[0102] Example 3 Therapeutic effect of Populus polysaccharide SVP-1 combined with chemotherapeutic drugs on cancer cell experiment

[0103] I. Methods

[0104] 1. Preparation of rat drug-containing serum

[0105] Five clean grade SD rats were selected and raised in SPF grade environment (temperature 24±2℃, relative humidity 55±5%, 12 hours light / dark cycle, free feeding and drinking water), and the experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin Agricultural University. After 1 week of adaptive feeding, SVP-1 (200mg / kg, 2mL per day) was administered by gavage for 7 consecutive days. After the last administration, the rats were fasted for 12 hours, anesthetized with sodium pentobarbital, and blood was collected from the abdominal aorta. The serum was separated by centrifugation at 4000r / min for 15 minutes and stored in aliquots.

[0106] 2. Cell viability detection (CCK-8 method)

[0107] HepG-2 cells were seeded in a 96-well plate at a density of 5×10 4 / mL, incubated for 24 hours to adhere, and then divided into 6 groups: (1) control group; (2) sorafenib monotherapy group (5μM); (3) SVP-1 low-dose group (containing 5% rat drug-containing serum); (4) SVP-1 high-dose group (containing 10% rat drug-containing serum); (5) SVP-1 low-dose combination group (2.5% drug-containing serum + 2.5μM sorafenib); (6) SVP-1 high-dose combination group (5% drug-containing serum + 2.5μM sorafenib). The drug concentration was determined by pre-experiment, and after the treatment of each group, 10μL CCK-8 reagent was added to each well, incubated at 37℃, 5% CO2 for 2 hours, and the absorbance (OD value) at 450nm was measured by a microplate reader. All cell experiments were set according to this 6-group concentration.

[0108] 3. 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment

[0109] HepG-2 cells (1×105 HepG-2 cells were seeded in 6-well plates at a density of 1 x 10 3 cells / well and incubated for 24 h. Different concentrations of drugs were added and incubated for 24 h. An equal volume of pre-prepared EdU working solution (20 mM) was added and incubated for 2 h. The medium was discarded, and 1 mL of 4% paraformaldehyde was added to fix the cells at room temperature for 20 min. After washing with PBS containing 3% BSA, 0.3% Triton X-100 was added and incubated for 20 min. After washing, 0.5 mL of Click reaction buffer (containing CuSO4, Azide 488 and Click additives) was added and incubated at room temperature for 30 min in the dark. The reaction solution was discarded, and Hoechst 33342 staining solution was added and incubated for 10 min in the dark. After washing, the proliferation of HepG-2 cells was observed under a fluorescence microscope (EdU-positive green fluorescent labeling of proliferating cells, blue fluorescent labeling of cell nuclei).

[0110] 4. Cell colony formation experiment

[0111] HepG-2 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 1 x 10 3 cells / well and incubated for 24 h. Different concentrations of drugs were added and incubated for 48 h. The medium was replaced with drug-free medium, and the medium was replaced every 3 days. The cells were cultured for 10-15 days until colonies were formed. The medium was discarded, and the cells were washed twice with PBS and fixed with methanol for 20 min. 0.1% crystal violet staining solution was added and incubated at room temperature for 20 min (1 mL per well). After washing with PBS until no residual staining solution was present, the number of colonies (cell clusters with a diameter of ≥ 50 pm) was counted using ImageJ software.

[0112] 5. Cell migration experiment (Transwell method)

[0113] HepG-2 cells were seeded in 6-well plates and cultured to an appropriate density. After digestion, the cells were resuspended in serum-free medium (containing the corresponding drugs, and an equal volume of blank rat serum was added to the sorafenib group and the model group to balance the effects of the drug-containing serum group) and adjusted to a uniform concentration (1 x 10 5 cells / well). 100 pL of cell suspension was added to the upper chamber of the Transwell, and 600 pL of medium containing 30% fetal bovine serum (FBS) was added to the lower chamber. The cells were incubated at 37°C for 24 h. After incubation, the chamber was removed, and the medium in the well was discarded. The cells were gently washed twice with PBS, fixed with methanol for 30 min, and naturally air-dried. The cells were stained with 0.1% crystal violet for 20 min, and the non-migrated cells in the upper chamber were carefully wiped off with a cotton swab. The cells were washed three times with PBS. The number of migrated cells was counted under a microscope in five randomly selected fields.

[0114] 6. Cell invasion experiment (Matrigel method)

[0115] Transwell upper chamber was pre-coated with Matrigel matrix glue (50 μL / chamber), and the glue was polymerized at room temperature for 3 hours. After pretreatment according to the migration experiment method, 1×10 5 The cell suspension was added to the upper chamber pre-coated with the matrix glue, 600 μL of the culture medium containing 30% FBS was added to the lower chamber, and it was incubated at 37°C for 12 hours. The culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 minutes, and then stained with 0.1% crystal violet for 20 minutes. The number of cells that had invaded the lower chamber was counted under a microscope.

[0116] Data analysis was performed on all results, and the results were expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparison between groups, and Student's t-test was used for pairwise comparison. P<0.05 was considered to be statistically significant.

[0117] II. Results

[0118] 1. Effect of SVP-1 combined with sorafenib on the proliferation of HepG2 cells

[0119] The CCK-8 method was used to evaluate the activity of HepG-2 cells in vitro. The results showed that, compared with the model group, all the drug groups (containing different concentrations of SVP-1 rat drug-containing serum combined with sorafenib) could significantly inhibit the proliferation of HepG-2 cells. Compared with the sorafenib monotherapy group, the inhibition of HepG-2 cell activity in the SVP-1 combined with sorafenib group was significantly enhanced. Among them, the high-dose group of SVP-1 combined with sorafenib had the most significant effect, which increased the inhibition rate of sorafenib monotherapy (32%) to 78% (Fig. 1B). Further EdU experiment and colony formation experiment (Fig. 1A, C-E) were used to verify the effect of combined administration on cell proliferation. The results were consistent with the CCK-8 method: the proliferation ability of HepG-2 cells in the SVP-1 combined with sorafenib group was significantly lower than that in the sorafenib monotherapy group; in the colony formation experiment, the number of cell clusters formed in the combined administration group was significantly reduced, indicating a synergistic inhibitory effect. Conclusion: SVP-1 combined with sorafenib can significantly enhance the anti-tumor activity and may inhibit the proliferation of hepatocellular carcinoma cells through synergistic effect. Figure 8 Figure 8 2. Effect of SVP-1 combined with sorafenib on the migration and invasion of HepG-2 cells

[0120] Tumor cells will migrate and invade during the progression, therefore, the present application explores the effect of SVP-1 combined with sorafenib on the migration and invasion of HepG-2 cells by Transwell chamber experiment (Fig. 2A, B).

[0121] Tumor cells will migrate and invade during the progression, therefore, the present application explores the effect of SVP-1 combined with sorafenib on the migration and invasion of HepG-2 cells by Transwell chamber experiment (Fig. 2A, B). Figure 9 ​The results show that: compared with the model group, all the administration groups can significantly inhibit the migration and invasion of HepG-2 cells. Compared with the sorafenib monotherapy group, the inhibition of cell migration and invasion in the SVP-1 combined with sorafenib group is significantly enhanced, and the effect of the high-dose SVP-1 combined with sorafenib administration group is the most significant.

[0122] Example 4 Efficacy of Populus polysaccharide SVP-1 combined with chemotherapy drugs on cancer in animal experiments

[0123] I. Methods

[0124] Male Kunming (KM) mice were raised in a SPF level environment (temperature 24 ± 2℃, relative humidity 55 ± 5%, 12 hours light / dark cycle, free access to food and water). The experimental protocol was approved by the Experimental Animal Ethics Committee of Jilin Agricultural University.

[0125] 1. Establishment and treatment of H22 tumor-bearing mouse model

[0126] The mice were randomly divided into 7 groups (8 mice per group): (1) blank control group; (2) model group; (3) sorafenib monotherapy group (Sor: 30 mg / kg); (4) SVP-1 low-dose group (200 mg / kg); (5) SVP-1 high-dose group (400 mg / kg); (6) sorafenib combined with SVP-1 low-dose group (Sor: 15 mg / kg, SVP-1: 100 mg / kg); (7) sorafenib combined with SVP-1 high-dose group (Sor: 15 mg / kg, SVP-1: 200 mg / kg). Except for the blank control group, the remaining groups of mice were injected with H22 ascites tumor cells subcutaneously in the right axilla to establish a tumor-bearing model. The tumor volume was monitored daily, and when the tumor grew to about 100 mm 3 times a day: sorafenib and SVP-1 were administered once a day by gavage; the blank control group and the model group were given the same volume of normal saline. The drug doses were determined according to the literature and pre-experiments. During the treatment period, the long diameter (L) and short diameter (W) of the tumor were measured daily, and the tumor volume was calculated according to the formula V = 1 / 2 × L × W 2 The tumor volume was calculated, and the mice were sacrificed on day 20.

[0127] 2. Determination of tumor inhibition rate and immune organ index

[0128] After the last administration, the body weight of the mice was measured using an electronic balance. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and after eye blood collection, the serum was separated by centrifugation at 4℃ and 3500 r / min for 10 minutes. Then the mice were sacrificed by cervical dislocation, dissected and the thymus, spleen and tumor tissues (the remaining tissues were frozen for later use) were removed, rinsed with sterile normal saline and weighed after being absorbed with filter paper. The tumor inhibition rate and immune organ index were calculated according to the following formula:

[0129] Tumor inhibition rate (%) = (mean tumor weight in the model group - mean tumor weight in the treatment group) / mean tumor weight in the model group / (mean tumor weight in the model group - mean tumor weight in the treatment group) / mean tumor weight in the model group × 100%,

[0130] Immune organ index (mg / g) = thymus or spleen weight (mg) / mouse body weight (g).

[0131] 3. Hematoxylin-eosin (H&E) staining of tissues and organs

[0132] Tumor tissues, as well as tissues from the heart, liver, spleen, lungs, and kidneys, were collected and fixed in 4% paraformaldehyde for 24 hours. After dehydration with graded ethanol, clearing with xylene, and embedding in paraffin, the sections were prepared to a thickness of 4 μm. Sections were stained with hematoxylin for 5 minutes, differentiated with hydrochloric acid and ethanol, counterstained with eosin for 2 minutes, and mounted with neutral resin. Histopathological changes (such as tumor necrosis areas, inflammatory cell infiltration, and organ damage) were observed under a light microscope and photographed.

[0133] 4. Transcriptomics analysis

[0134] Total RNA was extracted from tumor tissue using the Trizol method, and RNA concentration and purity were detected using a Nanodrop 2000 (Thermo Scientific). Hieff NGS was used. TM MaxUp Dual-Mode mRNA Library Construction Kit RNA sequencing libraries were prepared (compatible with standard sequencing) and sequenced using an Illumina HiSeq2000 sequencer. Sequencing data were aligned to the mouse reference genome (GRCm38) using Hisat2 (v2.1.0), with splicing site information based on the Ensembl 92 database. Gene expression levels were expressed as FPKM (kilobase fragments per million aligned exons) and converted to log2 values ​​for subsequent analysis. Differentially expressed genes (DEGs) were screened using the edgeR package, and multiple tests were corrected for false discovery rate (FDR) (threshold FDR < 5%). Hierarchical clustering analysis was performed using R, and gene ontology (GO) functional enrichment analysis was conducted using topGO (v2.24.0), followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using clusterProfiler (v3.0.5). GO entries and KEGG pathways with P < 0.05 were considered significantly enriched.

[0135] 5. Detection of soluble cytokines

[0136] Tumor tissues (muscle tissues of the same part instead of normal control group) were added with appropriate amount of physiological saline for grinding, centrifuged (4℃, 3000r / min, 15 minutes) to take the supernatant as the tissue homogenate. After detecting the total protein concentration of the spleen homogenate of each group of mice by BCA protein quantitative kit, the expression level of interleukin (IL-2, IL-4, IL-5, IL-10) in the spleen homogenate was detected according to the steps of mouse ELISA kit instructions, and the unit protein concentration (pg / mg) was converted according to the BCA detection results.

[0137] 6、CD8 + T, NK, Treg, Th2, M2 type macrophage proportion detection

[0138] Sample processing: collect tumor tissue single cell suspension (normal control group takes muscle tissue of the same part instead), adjust the cell density to 1×10 6 / mL.

[0139] Cell stimulation and blocking (for Th2 detection): add PMA (25 ng / mL) + ionomycin (1 μg / mL) for 4-6 hours, and use monensin (1.7 μg / mL) to block cytokine secretion.

[0140] Surface staining: add CD3, CD4, CD8, CD56, CD16, CD25, CD68 antibodies, incubate at 4℃ for 30 minutes, and wash with PBS.

[0141] Fixation and membrane rupture: use pre-cooled Fix / Perm reagent (such as BD Cytofix / Cytoperm TM ) to process the cells for FoxP3 and IL-4 / GATA3 intracellular staining.

[0142] Flow detection and analysis: set compensation control (single dye tube) and FMO (Fluorescence Minus One) control, and perform multi-parameter analysis by FlowJo or Diva software.

[0143] 7、Western blot analysis

[0144] The mouse tumor tissue was washed with pre-cooled PBS for 2-3 times to remove blood, homogenized under ice bath condition, and pre-cooled tissue protein lysis solution was added for lysis on ice for 30 minutes. The supernatant was obtained by centrifugation at 4°C and 12000 r / min for 5 minutes. The protein concentration was determined by BCA method, and 30 μg of protein sample was denatured at 95°C for 10 minutes, and then subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 1 hour, the primary antibodies CD48 (Proteintech, 83871-1-RR, 1:2000), CD244 (Invitrogen, PA5-106904, 1:2000), LAT (abcam, EPR26091-2, 1:1000), PLCG1 (Invitrogen, MA5-32276, 1:1000), PPP3CA (Thermo Fisher, A300-908A, 1:2000), NFATC2 (Proteintech, 22023-1-AP, 1:2000) and internal reference β-actin (Proteintech, 66009-1-Ig, 1:20000) were added, and incubated at 4°C overnight. After washing with TBST for 3 times, HRP-labeled goat anti-rabbit secondary antibody (abcam, ab205718, 1:10000) was added and incubated at room temperature for 2 hours. After ECL development, the image was obtained by a scanner, and the gray value of the protein band was analyzed by Image Pro Plus 6.0.

[0145] 8. Immunohistochemical (IHC) analysis

[0146] The tumor sample was fixed with formalin for more than 24 hours, transferred to 70% ethanol for storage, and sliced after paraffin embedding (thickness 4 μm), attached to a glass slide and baked. Immunohistochemical staining was performed using a Leica automatic staining platform, and the primary antibodies included TNF-α (Proteintech, 60291-1-Ig, 1:500), GM-CSF (Proteintech, 17762-1-AP, 1:250), and IFN-γ (Cell Signaling Technology, 8455T, 1:100). After DAB color development, hematoxylin was used for re-staining, and the distribution of positive signals (brown-yellow particles) was observed under a microscope and photographed.

[0147] Data analysis was performed on all results, and the results were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) was used for comparison between groups, and Student's t-test was used for pairwise comparison. P<0.05 was considered to have statistical significance.

[0148] II. Results

[0149] 1. Effect of SVP-1 combined with sorafenib on H22 tumor-bearing mice

[0150] In vitro experiments confirmed that SVP-1 can enhance the inhibitory effect of sorafenib on the proliferation of HepG-2 cells, so H22 tumor-bearing mice were further selected to study the in vivo anti-tumor activity of SVP-1. Model establishment and animal state: Before inoculation of H22 ascites passage cells, the body weights of mice in each group were similar and there was no difference between groups, all mice had bright fur, good mental state, and normal excretion. During the treatment period after modeling, mice in the sorafenib monotherapy group showed symptoms such as diarrhea, bloody stool, dark urine, disheveled hair, and listlessness, while mice in the other groups were in good condition. Tumor inhibition effect Figure 10 A-D): The tumor grew rapidly in the model group, and all drug-treated groups inhibited tumor growth. Tumor inhibition rate: the sorafenib monotherapy group was 46.16%, the SVP-1 low-dose group was 38.15%, and the SVP-1 high-dose group was 50.72%. Combination effect: the tumor inhibition rate of the SVP-1 combined with sorafenib high-dose group was 72.94%, which was significantly higher than that of the sorafenib monotherapy group (46.16%); the tumor inhibition rate of the SVP-1 combined with sorafenib low-dose group was 61.9%. Body weight change Figure 10 E): Compared with the model group, the body weight of mice in all treatment groups showed an upward trend. Compared with the sorafenib monotherapy group, the SVP-1 combined with sorafenib groups significantly improved the body weight of tumor-bearing mice, suggesting that combination therapy may reduce the toxic side effects of anticancer drugs. Conclusion: SVP-1 combined with sorafenib can significantly enhance anti-tumor activity through synergistic effect, while possibly reducing the toxicity of anticancer drugs, providing a potential strategy for liver cancer treatment.

[0151] 2. Immune organ index, serum TBIL and AST levels

[0152] The results of the effect of SVP-1 combined with sorafenib on immune organ indices and liver and gallbladder indices in H22 tumor-bearing mice are shown in Table 4.

[0153] Table 4 Effect of SVP-1 combined with sorafenib on immune organ indices and liver and gallbladder indices in H22 tumor-bearing mice

[0154] Note: Data are expressed as mean ± standard deviation (n = 5 per group). Compared with the control group, *P <0.05, **P <0.01; compared with the model group,

[0155] #P <0.05, ##P <0.01; compared with the sorafenib monotherapy group, $P <0.05, $$P <0.01.

[0156] From the data in Table 4, it can be seen that the immune organ index: model group: compared with the control group, the spleen index of the model group mice was significantly increased, and the thymus index was significantly decreased (tumor growth stimulates splenomegaly and thymus atrophy). Sorafenib monotherapy group: the spleen index and thymus index were significantly lower than those of the control group, suggesting that sorafenib may cause damage to the immune organs. SVP-1 treatment group: compared with the model group, the spleen index of the SVP-1 administration group was significantly reduced, and the thymus index was significantly increased. Combination administration group: compared with the sorafenib monotherapy group, the thymus and spleen indexes of the SVP-1 combined with sorafenib group were significantly up-regulated, indicating that SVP-1 can reduce the damage of sorafenib to the immune organs and play a protective role.

[0157] Serum TBIL (total bilirubin) and AST (aspartate aminotransferase) levels: model group: serum TBIL and AST levels were significantly higher than those of the control group (tumor caused abnormal liver function). Sorafenib monotherapy group: failed to significantly reduce the increase of TBIL and AST caused by tumors, which may be related to the hepatotoxic side effects of sorafenib. SVP-1 treatment group: significantly reduced TBIL and AST levels, indicating that SVP-1 can improve liver function. Combination administration group: compared with the sorafenib monotherapy group, the TBIL and AST levels of the SVP-1 combined with sorafenib group were further reduced, demonstrating that SVP-1 can reduce the hepatotoxicity of sorafenib.

[0158] Conclusion: SVP-1 combined with sorafenib not only enhances anti-tumor activity, but also achieves synergistic effect by protecting immune organ function and reducing liver damage, providing experimental evidence for improving the safety of liver cancer treatment.

[0159] 3. H&E staining of tumor tissue

[0160] The pathological changes of tumor tissue were observed by H&E staining. Figure 11 Hematoxylin stains the nucleus deep blue-purple, and eosin stains the cytoplasmic protein pink. Model group: tumor cells have complete morphology, dense nucleus and orderly arrangement, almost no necrotic cells, showing typical tumor cell growth characteristics. Sorafenib or SVP-1 monotherapy group: tumor cells show varying degrees of damage and apoptosis, with a decrease in the number of normal cells; nuclear fragmentation and lymphocyte infiltration can be seen (indicating activation of immune response); lipid droplets appear in the tissue (reflecting abnormal metabolism or damage of tumor cells). SVP-1 combined with sorafenib group: the number of blue-purple nuclei is significantly reduced, indicating that the nuclear components (such as DNA) are gradually degraded by proteases and nucleases; the areas of apoptosis and necrosis are enlarged, and the degree of tumor structure destruction is more significant than that of the monotherapy group. Conclusion: combined administration synergistically induces tumor cell nuclear lysis, exacerbates apoptosis and structural damage, significantly enhances anti-tumor effect, and has a better pathological improvement effect compared with monotherapy.

[0161] 4. H&E staining of heart, liver, spleen, lung, and kidney tissues

[0162] H&E staining of heart, liver, spleen, lung, and kidney tissues of H22 tumor-bearing mice was performed, and the results are shown in Figure 11 .

[0163] As can be seen from Figure 11 , SVP-1 monotherapy has no obvious toxicity to the heart, liver, spleen, lung, and kidney. In combination with sorafenib, it can reduce the cardiotoxicity of sorafenib (interstitial widening, capillary hyperemia); improve liver lobular structure, reduce lymphocyte infiltration; repair the white pulp-red pulp boundary of the spleen; relieve lung interstitial thickening and bleeding; protect glomerular morphology and reverse sorafenib-induced kidney damage.

[0164] It is suggested that SVP-1 enhances the safety of sorafenib treatment through multi-organ protection.

[0165] 5. Differential gene screening

[0166] DESeq (1.12.4) was used for differential gene (DEGs) analysis. The screening conditions were set as follows: significance threshold: corrected P value (Q value) ≤ 0.05; difference threshold: |log2FoldChange| ≥ 1 (i.e., gene expression up-regulation or down-regulation ≥ 2-fold).

[0167] Results: Compared with the model group, 3039 DEGs were screened in the high-dose SVP-1 group: 1383 genes were up-regulated (promoting anti-tumor effect); 1656 genes were down-regulated (inhibiting pro-tumor signals) Figure 12 .

[0168] 6. GO and KEGG enrichment analysis of DEGs

[0169] Functional enrichment analysis was performed using clusterProfiler (3.0.5), and topGO (2.24.0) was used for GO enrichment analysis. The significance threshold was corrected P value (Q value) < 0.05.

[0170] (1) GO enrichment analysis:

[0171] Gene Ontology (GO) is an international standardized classification system for gene function, covering the following three dimensions: biological process (BP): biological activities in which genes are involved; cellular component (CC): cell structures where gene products are located; molecular function (MF): molecular activities of gene products.

[0172] Enrichment results of DEGs of SVP-1 group vs. model group Figure 13 :

[0173] BP: CD8+ T cell positive regulation; TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) production positive regulation; natural killer (NK) cell tolerance induction; positive regulation of antibody-dependent cell-mediated cytotoxicity (ADCC); positive regulation of type II hypersensitivity; antigen processing and presentation of exogenous peptides by MHC class Ib molecules.

[0174] CC: MHC class I peptide loading complex; mitochondrial respiratory chain complex; early endosome membrane.

[0175] MF: TAP1 / TAP2 binding (antigen presentation associated); CD8 receptor binding; MHC protein binding; NK cell Clec1-like receptor binding.

[0176] (2) KEGG enrichment analysis:

[0177] KEGG database integrates genomic, chemical and system functional information for pathway analysis.

[0178] KEGG enrichment results of SVP-1 group vs. model group: Figure 13

[0179] Most significant pathway: Natural killer cell mediated cytotoxicity, significantly higher than other pathways.

[0180] Key differential proteins: 6 significantly differentially expressed proteins were screened in this pathway: CD48 (NK cell activation ligand); CD244 (NK cell receptor); LAT (signal transduction linker protein); PLCG1 (phospholipase C gamma 1, regulates calcium signaling); PPP3CA (calcineurin subunit, pro-apoptotic); NFATC2 (nuclear factor activated T cell cytoplasmic protein 2, regulates cytokine expression).

[0181] (3) Mechanism analysis:

[0182] Positive activation of polysaccharide SVP-1 on CD8 + T cells and TRAIL-mediated apoptosis: by up-regulating MHC-I expression, promoting tumor antigen presentation, enhancing CD8 + T cell recognition and activation, promoting IL-2 secretion; activated CD8 + T cells secrete IFN-γ and TNF-α, stimulate tumor cells to express TRAIL and its receptors DR4 / DR5, trigger mitochondrial apoptosis pathway.

[0183] ​Antitumor effect of polysaccharide SVP-1 on NK cells and enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC): activation of PLCG1-IP3-Ca by up-regulating NK cell surface receptor CD48 / CD244 2+ - PPP3CA-NFATC2 pathway, promoting IFN-γ and GM-CSF secretion, enhancing cytotoxicity; increasing tumor cell surface antigen MHC-I expression, promoting IgG antibody binding, and enhancing NK cell-mediated ADCC effect through FcyRIIIa (CD16).

[0184] Polysaccharide SVP-1 promotes the reversal of tumor immunosuppressive microenvironment: by reducing IL-10 (Treg secretion) and IL-4 / IL-5 (Th2 secretion) levels, reducing M2 macrophage polarization, and thus relieving the inhibition of CD8 + T cells and NK cells.

[0185] Conclusion: RNA sequencing shows that polysaccharide SVP-1 may promote the positive activation of CD8 + T cells, TRAIL-mediated apoptosis, enhancement of NK cell antitumor effect and antibody-dependent cell-mediated cytotoxicity (ADCC), and reversal of immunosuppressive microenvironment are the core molecular mechanisms of its anti-hepatoma effect.

[0186] 7. Soluble cytokine detection

[0187] The results of soluble cytokine detection in tumor tissue are shown in Table 5.

[0188] Table 5 Effect of SVP-1 combined with sorafenib on part of soluble cytokines in H22 tumor-bearing mice

[0189] Group Control group Model group Sorafenib group SVP-1 high-dose group High-dose combination group IL-2 (pg / mL) 26.5±3.4 11.7 ± 2.6 ** ]] 15.2 ± 2.5 # ]] 18.4 ± 2.9 ## ]] 28.7 ± 3.3 ##$$ ]] IL-4 (pg / mL) 15.4±2.5 42.5 ± 7.2 ** ]] 31.2 ± 5.3 ## ]] 20.6 ± 4.7 ## ]] 17.4 ± 4.7 ##$$ ]] IL-5 (pg / mL) 11.3±2.1 34.8 ± 5.8 ** ]] 21.4 ± 3.3 ## ]] 15.6 ± 2.6 ## ]] 13.4 ± 4.5 ##$$ ]] IL-10 (pg / mL) 221±41 485 ± 62 ** ]] 361 ± 52 ## ]] 295 ± 49 ## ]] 207 ± 37 ##$$ ]]

[0190] Note: Data are expressed as mean ± standard deviation (n = 5 per group). Compared with the control group, *P <0.05, **P <0.01; compared with the model group, #P <0.05, ##P <0.01; compared with the sorafenib monotherapy group, $P <0.05, $$P <0.01.

[0191] From the overall analysis of the data in Table 5, SVP-1 combined with sorafenib can more effectively regulate the levels of soluble cytokines such as IL-2, IL-4, IL-5, and IL-10 in H22 tumor-bearing mice compared with sorafenib monotherapy, and has potential advantages in improving immune function and inhibiting tumor growth, providing a new combined drug treatment approach for tumor treatment.

[0192] 8. CD8 + T, NK, Treg, Th2, and M2 macrophage proportion detection

[0193] The results of detecting the changes of the core cell groups of tumor microenvironment are shown in Table 6.

[0194] Table 6 Effects of SVP-1 combined with sorafenib on the core cell groups of tumor microenvironment of H22 tumor-bearing mice

[0195] Group Control group Model group Sorafenib group SVP-1 high-dose group High-dose combination group CD8 + T cells / PBMC 23.5±2.1 11.5 ± 3.4 ** ]]> 14.7 ± 2.8 # ]] 22.6 ± 3.1 ## ]]> 25.8 ± 3.8 ##$$ ]]> NK cells / PBMC 11.3±1.9 3.2 ± 0.8 ** ]] 5.5 ± 1.7 ## ]] 10.3 ± 2.5 ## ]] 12.2 ± 2.3 ##$$ ]] Treg cells / CD4 + T]] 6.3±0.7 15.6 ± 1.7 ** ]] 13.2 ± 3.4 ## ]] 8.7 ± 1.8 ## ]] 6.2 ± 1.3 ##$$ ]] Th2 cells / CD4 + T]]> 8.1±1.6 18.4 ± 3.2 ** ]] 14.2 ± 2.8 ## ]] 9.7 ± 1.8 ## ]]> 8.4 ± 1.5 ##$$ ]] M2 macrophages / monocytes 12.2±2.4 38.1 ± 5.1 ** ]]> 30.3 ± 4.5 ## ]] 15.8 ± 2.1 ## ]] 13.1 ± 2.1 ##$$ ]]

[0196] Note: The data are expressed as mean ± standard deviation (n = 5 per group). Compared with the control group, *P <0.05, **P <0.01; compared with the model group, #P <0.05, ##P <0.01; compared with the sorafenib monotherapy group, $P <0.05, $$P <0.01.

[0197] From the data in Table 6, it can be seen that CD8 + T cells are important anti-tumor effector cells, and an increase in their proportion means that the body's anti-tumor immune ability is enhanced. Both sorafenib and SVP-1 can promote the recovery of CD8 + T cells in tumor-bearing mice, and the effect is more pronounced when used in combination. NK cells can directly kill tumor cells without prior sensitization, and an increase in their proportion helps to enhance the body's anti-tumor ability. Sorafenib and SVP-1 can increase the number of NK cells in tumor-bearing mice, and the effect is better when used in combination. Treg cells have immunosuppressive function, and a decrease in their proportion is beneficial to relieve the inhibition of the body's anti-tumor immune response. Both sorafenib and SVP-1 can inhibit the expansion of Treg cells in tumor-bearing mice, and the inhibitory effect is more pronounced when used in combination. Th2 cells are mainly involved in humoral immunity, and an excessively high proportion of Th2 cells can inhibit cellular immunity. A decrease in their proportion is beneficial to enhance the body's cellular immune function. Sorafenib and SVP-1 can reduce the proportion of Th2 cells in tumor-bearing mice, and the effect is better when used in combination. M2-type macrophages have the functions of promoting tumor growth, angiogenesis, and immunosuppression, and a decrease in their proportion is beneficial to inhibit tumor development. This indicates that sorafenib and SVP-1 can reduce the number of M2-type macrophages in tumor-bearing mice, and the effect is better when used in combination.

[0198] In summary, compared with sorafenib monotherapy, SVP-1 combined with sorafenib can more effectively regulate the proportion of the core cell groups of tumor microenvironment of H22 tumor-bearing mice, enhance the body's anti-tumor immune ability, and inhibit the growth and development of tumors, thereby providing a more potential combined drug regimen for the treatment of tumors.

[0199] 9. Western blotting and immunohistochemistry (IHC) verification

[0200] (1) Western blotting (WB) results

[0201] SVP-1 group and combination group: compared with the model group, the expression of CD48, CD244, LAT, PLCG1, PPP3CA and NFATC2 proteins in the tumor tissues of mice was significantly increased (consistent with the transcriptomic results, Figure 14 ).

[0202] Sorafenib group: the expression of CD48, LAT, PLCG1 and NFATC2 proteins was significantly increased compared with the model group; the expression of CD244 and PPP3CA proteins had no significant change.

[0203] (2 Immunohistochemistry (IHC) results

[0204] SVP-1 group and combination group: compared with the model group, the expression of TNF-α (tumor necrosis factor-α), GM-CSF (granulocyte-macrophage colony-stimulating factor) and IFN-γ (interferon-γ) in the tumor tissues was significantly increased Figure 15 ).

[0205] Sorafenib group: the expression of TNF-α and IFN-γ was significantly increased; although the expression of GM-CSF was increased to a certain extent, it had no statistical significance.

[0206] Conclusion: WB and IHC experiments confirmed that SVP-1 synergistically enhanced the anti-tumor immune response by up-regulating the expression of key proteins (CD48, CD244, etc.) in the NK cytotoxicity pathway and pro-inflammatory factors (TNF-α, GM-CSF and IFN-γ), which was one of the core mechanisms of its inhibition of liver cancer growth. Although sorafenib partially activated this pathway, its effect was weaker than that of SVP-1 and the combination group.

[0207] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. The application of a polysaccharide SVP-1 from poplar tree in the preparation of an antitumor immunomodulatory drug, characterized in that, The immunomodulatory drugs are used in combination with antitumor chemotherapy drugs; The structure of the poplar mulberry polysaccharide SVP-1 consists of 192 repeating units; The main chain repeating unit is composed of four sugar residues, →6)-β-D-Glcp-(1→, →3,2)-α-L-Manp-(1→, →6)-β-D-Glcp-(1→ and →6,3)-β-D-Glcp-(1→), which are sequentially linked by glycosidic bonds of (1→3), (1→6), and (1→3), respectively. The side chains α-L-Manp-(1→) and β-D-Galp-(1→) are sequentially linked to the →3,2)-α-L-Manp-(1→) and →6,3)-β-D-Glcp-(1→ residues of the main chain via (1→2) and (1→6) glycosidic bonds, respectively. The sugar chain structure is shown below: 。 2. The application according to claim 1, characterized in that, The tumors include liver cancer.

3. The application according to claim 1, characterized in that, The chemotherapy drugs include sorafenib.

Images