A polysaccharide complex of Dictyophora shortskirt and Phellinus linteus with efficacy of alleviating colorectal cancer and its application

Abstract

The present invention discloses a polysaccharide complex of short-skirted bamboo fungus and poplar mulberry lindera with the efficacy of alleviating colorectal cancer and its application, belonging to the field of biomedical technology. The preparation method of the polysaccharide complex comprises the following steps: mixing an aqueous extract of short-skirted bamboo fungus and an aqueous extract of poplar mulberry lindera in equal volumes, concentrating under reduced pressure, removing the protein and organic layers to obtain a purified concentrate, precipitating the purified concentrate with an ethanol aqueous solution, collecting the precipitate and drying to obtain the polysaccharide complex. The present invention confirms through cell experiments and animal experiments that the polysaccharide complex of short-skirted bamboo fungus and poplar mulberry lindera has a significantly better inhibitory effect on colorectal cancer than single short-skirted bamboo fungus polysaccharide and poplar mulberry lindera polysaccharide, confirming that the combination of short-skirted bamboo fungus polysaccharide and poplar mulberry lindera polysaccharide has a synergistic effect in alleviating colorectal cancer, providing theoretical support for further developing colitis-related colorectal cancer drugs with effectiveness and biosafety.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a polysaccharide complex of Dictyophora serrata and Phellinus igniarius with the efficacy of alleviating colorectal cancer and its application. Background Art

[0002] Colorectal cancer (CRC), as a common malignant disease in human diseases, is listed as the third most common cancer in the world and the second leading cause of cancer death in humans. In recent years, with economic development, population aging and changes in lifestyle, its incidence and mortality have been on the rise. CRC may be caused by multifactorial perturbations, such as diet, obesity, heavy drinking, genetic and epigenetic factors, nutrient depletion and cellular metabolic disorders, diabetes, etc. Inflammation caused by intestinal damage, oxidative stress and inflammatory bowel disease is a major risk factor for cancer evolution. Colitis-associated colorectal cancer (CAC) is a type of CRC with clinically detectable inflammatory bowel diseases (IBDs) as the first symptom. Chronic inflammation plays an active role in the occurrence and development of CAC. Patients with IBDs are considered to be at risk of colorectal cancer and have a poor prognosis. The treatment of colorectal cancer includes endoscopic and surgical local resection, preoperative radiotherapy and systemic therapy, extensive surgery for local and metastatic disease, local ablative therapy for metastasis, as well as palliative chemotherapy, targeted therapy and immunotherapy. The vast majority of CAC patients are not diagnosed until the late stage of the disease, and chemotherapy is the main treatment for CAC at this stage. Traditional therapies are not only expensive, but patients often develop chemotherapy resistance and other serious side effects, resulting in poor prognosis and increased mortality. Therefore, there is an increasing need to find effective and biosafe alternative strategies to clinically reduce and control colorectal cancer.

[0003] Most carbohydrates in nature exist in the form of polysaccharides, which are considered to be the first biopolymers formed on Earth. Polysaccharides from different natural sources (such as large fungi, plants and bacterial extracellular polymers) are increasingly recognized as supplements to regulate the health of the body and have great potential. Dictyophora short-skirted is a edible and medicinal fungus with high medicinal value. Studies have found that Dictyophora short-skirted polysaccharides have antioxidant activity, anti-tumor and anti-cancer effects, anti-inflammatory and immunomodulatory effects, antibacterial effects, improvement of intestinal inflammation, and anti-aging effects. Phellinus igniarius is a general term for a class of precious perennial large traditional medicinal wood-rotting fungi. The polysaccharides, flavonoids, triterpenes, phenols, etc. in them have been reported to have multiple biological activities, including anti-cancer, antioxidant, anti-angiogenic, and antiviral activities. It has been reported that poplar mulberry igniarius polysaccharides have the activity of regulating the occurrence and development of colorectal cancer.

[0004] Although both Dictyophora short-skirted polysaccharide and Poplar mulberry ignia polysaccharide have been reported to play a role in improving intestinal inflammation and related anti-tumor effects, due to factors such as the uncertain risks of combined drug use and drug interactions, no research has been found on the combined use of Dictyophora short-skirted polysaccharide and Poplar mulberry ignia polysaccharide in the treatment of colorectal cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a polysaccharide complex of short-skirted bamboo fungus and poplar mulberry linterus with the efficacy of alleviating colorectal cancer and its application, so as to solve the problems existing in the above-mentioned prior art. The polysaccharide complex of short-skirted bamboo fungus and poplar mulberry linterus provided by the present invention has an outstanding effect in alleviating colitis-related colorectal cancer, and provides theoretical support for further developing colitis-related colorectal cancer drugs with effectiveness and biosafety.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polysaccharide complex of Dictyophora serrata and Phellinus linteus with the efficacy of alleviating colorectal cancer. The preparation method of the polysaccharide complex comprises the following steps:

[0008] The water extract of Dictyophora shortskirt and the water extract of Phellinus igniarius were mixed in equal volumes, concentrated under reduced pressure, and the protein and organic layers were removed to obtain a purified concentrate, which was precipitated with ethanol aqueous solution, and the precipitate was collected and dried to obtain the polysaccharide complex.

[0009] Furthermore, the short-skirted bamboo fungus water extract and the poplar mulberry lindera water extract are both prepared by the following method: the short-skirted bamboo fungus or the poplar mulberry lindera is mixed with water, extracted in a water bath for a total of 2 extractions, and the extracts are combined to obtain the short-skirted bamboo fungus water extract or the poplar mulberry lindera water extract.

[0010] Furthermore, the water bath extraction temperature is 80° C., and each extraction time is 2 hours.

[0011] Furthermore, the solid-liquid ratio of the short-skirted bamboo fungus or poplar mulberry linterus mixed with water is 1g:30mL.

[0012] Furthermore, the colorectal cancer is colitis-associated colorectal cancer.

[0013] The present invention also provides application of the polysaccharide complex in preparing medicine for treating colorectal cancer.

[0014] Furthermore, the colorectal cancer is colitis-associated colorectal cancer.

[0015] The present invention also provides a medicine for treating colorectal cancer, wherein the effective ingredient of the medicine comprises the polysaccharide complex.

[0016] Furthermore, the medicine also includes a pharmaceutically acceptable carrier substance and / or adjuvant.

[0017] The present invention discloses the following technical effects:

[0018] The polysaccharide complex of Dictyophora shortskirt and Poplar mulberry lindera provided by the present invention can specifically alleviate colitis-related colorectal cancer. It is further confirmed through cell experiments and animal experiments that the polysaccharide complex of Dictyophora shortskirt and Poplar mulberry lindera has a significantly better inhibitory effect on colorectal cancer than single Dictyophora shortskirt polysaccharide and Poplar mulberry lindera polysaccharide, confirming that the combination of Dictyophora shortskirt polysaccharide and Poplar mulberry lindera polysaccharide has a synergistic effect in alleviating colorectal cancer.

[0019] The polysaccharide complex of Dictyophora serrata and Phellinus igniarius provided by the present invention has an outstanding effect in alleviating colitis-related colorectal cancer, and provides a theoretical support for further developing colitis-related colorectal cancer drugs with effectiveness and biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The effects of three polysaccharides on the viability of SW480 cells, among which A is the effect of different doses of short-skirted bamboo fungus polysaccharide (DD) freeze-dried powder on the viability of SW480 cells; B is the effect of different doses of poplar mulberry linterus polysaccharide (SV) freeze-dried powder on the viability of SW480 cells; C is the effect of different doses of short-skirted bamboo fungus poplar mulberry linterus composite polysaccharide (DD+SV) freeze-dried powder on the viability of SW480 cells; D is a comparative analysis of the effects of the three polysaccharides on the viability of SW480 cells, among which # p<0.05, ## p<0.05, ### p<0.001 compared with Ctrl group, ** p<0.01, *** p<0.001 compared with DD+SV group;

[0022] Figure 2 The experimental process (A) of DD+SV treatment of CAC mice, as well as the changes in mouse body weight (B), colon morphology (C), tumor number (D) and colon index (E), among which, ## p<0.01, ### p<0.001 compared with Ctrl group, ** p<0.01, ***p<0.001 compared with the Model group;

[0023] Figure 3 The results of histopathological staining of colon tissues of mice in different groups are shown, magnification 400×, scale bar: 20 μm;

[0024] Figure 4 The results of histopathological staining of organ tissues of mice in different groups are shown, magnification 200×, scale bar: 100 μm;

[0025] Figure 5 are the organ indexes of different organs of mice, where A is the organ index of the heart, B is the organ index of the liver, C is the organ index of the spleen, D is the organ index of the kidney, and E is the organ index of the thymus;

[0026] Figure 6 It is a diversity index diagram, where A is Chao1 index, B is Observed species index, C is Shannon index, D is Simpson index, E is Faith's PD index, F is Pielou_e index, and G is Good's coverage index;

[0027] Figure 7 Venn diagram (A), NMDS analysis (B), genus-level heat map (C), and metabolic pathway statistics (D) of ASV / OTUs in the gut microbiota of AOM / DSS mice regulated by DD+SV;

[0028] Figure 8 Venn diagram (A), differential metabolite heat map (B), metabolic pathway statistics (C), and combined analysis of intestinal flora metabolites (D) of the metabolites of AOM / DSS mice regulated by DD+SV;

[0029] Fig. 9 Venn diagram (A) and heat map of significantly differentially expressed proteins (B) showing the regulation of protein levels in intestinal tissues of AOM / DSS mice by DD+SV;

[0030] Fig.10 GO enrichment analysis of differentially expressed proteins in the intestinal tissues of AOM / DSS mice regulated by DD+SV;

[0031] Fig.11 KEGG pathway analysis of differentially expressed proteins in the intestinal tissues of AOM / DSS mice regulated by DD+SV;

[0032] Fig.12 Immunofluorescence images of HIF-1α and VEGF in mouse intestinal tissue (A) and immunoblotting results of HPSE, P-ERK1 / 2, and SDHB in mouse intestinal tissue (B). DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Example

[0039] 1. Experimental Methods

[0040] 1. Preparation of composite polysaccharides from Dictyophora shortskirt and Phellinus linteus

[0041] The fruiting bodies of Dictyophora short-skirt were dried and crushed at 40°C. The hot water extraction method was used with a solid-liquid ratio of 1g:30mL, and the supernatant was filtered out. The extraction was performed twice, each time for 2 hours, and the extracts were combined to obtain the Dictyophora short-skirt water extract. The poplar mulberry lindera water extract was prepared by the same method.

[0042] The water extract of the short-skirted bamboo fungus and the water extract of poplar mulberry lindera were concentrated to 1 / 10 of the original volume, mixed in a volume ratio of 1:1, mixed at 4°C and 60r / min for 24-48h, allowed to fully interact, and concentrated under reduced pressure to obtain a complex of the water extract of the short-skirted bamboo fungus and poplar mulberry lindera. 1 / 4 volume of sevage reagent (chloroform: n-butanol = 4:1) was added to the water extract of short-skirted bamboo shoot polysaccharide, the water extract of poplar mulberry lindera, and the complex of the short-skirted bamboo fungus and poplar mulberry lindera water extract to remove protein, remove the organic layer after sufficient reaction, repeat 3 times, dialyze at 4°C for 72h, add anhydrous ethanol to 80% in the polysaccharide solution, stand for 24h, centrifuge to obtain the precipitate and freeze-dry, and obtain freeze-dried powder of short-skirted bamboo fungus polysaccharide (DD), freeze-dried powder of poplar mulberry lindera polysaccharide (SV), and freeze-dried powder of short-skirted bamboo fungus and poplar mulberry lindera composite polysaccharide (DD+SV), respectively.

[0043] 2. DD+SV alleviates colorectal cancer in vitro cell experiment

[0044] (1) Cell culture

[0045] SW480 cells were cultured in complete medium in an incubator set at 37°C and 5% CO. 2 After the cells have grown to about 80% of the bottom area of ​​the culture flask, they can be passaged. Add 1 mL of trypsin to the culture flask and digest for 3 minutes at 37°C. After terminating the cell digestion, pipette the cells to make them single cells. The collected cells are passaged at a ratio of 1:4 or 1:5.

[0046] (2) Cell viability assay

[0047] The effects of DD, SV, and DD+SV groups on the viability of CRC tumor cells were detected by MTT method. The sw480 cells in good condition were digested to obtain a cell suspension, which was inoculated in a 96-well plate at a density of 1000 cells / well. PBS was added to the outermost circle of wells of the 96-well plate to prevent the evaporation of the liquid solution, and 100 μL of cell suspension was added to each well in the middle, and the plate was placed in an incubator for overnight culture. After the cells adhered to the wall, sw480 cells were treated with different concentrations of DD (0-8 mg / mL), SV (0-8 mg / mL), and DD+SV (0-8 mg / mL) and incubated together. After 24 hours, 100 μL of MTT solution was added to each well and incubated for another 4 hours. The supernatant solution in the well plate was discarded, and 100 μL of DMSO was added to each well to dissolve the formazan precipitate. After the well plate was shaken to fully dissolve the precipitate, the absorbance value was measured at a wavelength of 490 nm using an ELISA reader. Then, 2 mg / mL of DD and SV and 1.5 mg / mL of DD+SV were used to treat sw480 cells and incubated together. The steps were the same as above.

[0048]

[0049] 3. Study on the activity of DD+SV in alleviating colitis-related colorectal cancer

[0050] (1) Mouse grouping and drug administration

[0051] 45 SPF-grade 6-week-old healthy male C57BL / 6 mice were prepared, kept at a temperature of 23±1°C and a relative humidity of 55±5%, and fed with free food and water for 7 days. They were then randomly divided into three groups, with 15 mice in each group. The food intake and body weight of the mice were recorded during the experiment.

[0052] The first group was the normal control group, referred to as the Ctrl group. On the first day of modeling, a single intraperitoneal injection of 10 mg / kg sterile saline was given, fresh drinking water was provided during the modeling period, and 100 mg / kg saline was gavaged at a fixed time every day starting from the 28th day.

[0053] The second group was the AOM / DSS+normal saline model group, referred to as the AOM / DSS group. On the first day of modeling, a single intraperitoneal injection of 10 mg / kg AOM was given; 2% DSS solution was given on days 7-14, 28-35, and 49-58, and normal saline was used for the rest of the time; starting from the 28th day, 100 mg / kg normal saline was gavaged at a fixed time every day.

[0054] The third group was the AOM / DSS+DD experimental group, referred to as the DD group. The AOM / DSS modeling scheme was the same. From the 28th day, 100 mg / kg of DD was intraperitoneally administered at a fixed time every day.

[0055] The fourth group was the AOM / DSS+SV experimental group, referred to as the SV group. The AOM / DSS modeling scheme was the same. From the 28th day, 100 mg / kg of SV was intraperitoneally administered at a fixed time every day.

[0056] The fifth group was the AOM / DSS+DD+SV experimental group, referred to as the DD+SV group. The AOM / DSS modeling scheme was the same. From the 28th day, 100 mg / kg of DD+SV was intraperitoneally administered at a fixed time every day.

[0057] (2) Sample preparation method

[0058] After the last administration, mice in each group were fasted but not watered for 12 hours, and the fasting weight of the mice was recorded. Blood was collected from the tail vein, left to stand at room temperature for 30 minutes, centrifuged at 3000r / min for 10 minutes, and the supernatant was taken and stored in a -80℃ refrigerator for later use. The experimental animals were then killed, and the contents of the cecum were first taken, and the heart, liver, spleen, kidney, thymus, and colon tissues were collected. The organs were weighed, and the colon weight and colon length were recorded, and the supernatant was stored in a -80℃ refrigerator for later use.

[0059] The calculation formula of mouse organ index is as follows:

[0060]

[0061] The calculation formula of mouse intestinal index is as follows:

[0062]

[0063] (3) Histopathological observation

[0064] Organs and colon tissues were fixed in 4% paraformaldehyde, routinely paraffin-embedded, sectioned, stained with hematoxylin and eosin (H&E), and observed under an optical microscope for pathological analysis.

[0065] (4) Analysis of intestinal microbial communities

[0066] The collected intestinal contents were subjected to DNA extraction according to the instructions, and the DNA purity was analyzed using a NanoDrop spectrophotometer (ThermoFisher Scientific), and the quality of the extracted DNA was analyzed using 1.2% agarose gel electrophoresis. The V3-V4 region of the bacterial 16SrRNA gene was amplified by polymerase chain reaction. The amplified products were quantitatively analyzed and mixed according to sample requirements. The cecal content DNA was sequenced using the Illumina platform. Sequence denoising and clustering were performed based on the results of the original sequencing data. The two main methods used were DADA2 and Vsearch. High-quality sequence clustering was based on 97% similarity to obtain an OTU abundance table for subsequent analysis. Based on the OTU sequences, alpha diversity, and beta diversity indices of different samples, the diversity of species within and between habitats was analyzed respectively.

[0067] (5) Serum non-targeted metabolomics

[0068] The collected serum samples were placed in 5 mL vacuum tubes containing ethylenediaminetetraacetic acid and centrifuged for 15 min (1500 g, 4 °C). 150 μL of plasma samples were stored at -80 °C until LC-MS analysis. The samples were thawed at 4 °C, and 100 μL of the samples were centrifuged with 400 μL of pre-cooled methanol / acetonitrile (1:1, v / v) for 20 min (14000 g, 4 °C). The supernatant was dried in a vacuum centrifuge. For LC-MS analysis, the samples were redissolved in 100 μL of acetonitrile / water (1:1, v / v) solvent, centrifuged at 14000 g, 4 °C for 15 min, and the supernatant was analyzed. Ultra-high performance liquid chromatography system (Agilent 1290 Infinity LC) and HILIC columns were used for separation, and the samples were placed in an autosampler at 4 °C throughout the process. After separation, serum samples were analyzed by mass spectrometry (TripleTOF 6600 mass spectrometer), and electrospray ionization was used for detection in positive and negative ion modes, respectively. The obtained raw data were subjected to data preprocessing, and after the structural identification of the metabolites, the experimental data were subjected to quality control analysis and processing.

[0069] (6) Proteomics

[0070] Colon tissue samples were lysed and protein extracted using SDT (4% SDS, 100 mM Tris-HCl, pH 7.6) buffer. Protein digestion with trypsin was performed in the filter-assisted sample preparation (FASP) procedure. The enzymatic peptides of each sample were loaded onto a C18 column (Empore TM The samples were desalted on SPE cartridges C18 (standard density, 7 mm id, 3 mL volume, Sigma), concentrated by vacuum centrifugation, and reconstituted in 40 μL 0.1% (v / v) formic acid.

[0071] LC-MS / MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific) with peptides loaded onto a C18 reverse phase analytical column (Thermo Scientific Easy Column, 25 cm length, 75 μm inner diameter, 1.9 μm resin) in 95% buffer A (0.1% formic acid in water) and separated with a linear gradient of buffer B (99.9% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl / min. The mass spectrometer was operated in positive ion mode. The applied electrospray voltage was 1.5 kV. Parent ions and fragments were analyzed on a TOF detector with a mass range of m / z 100–1700. The TimsTOF Pro was operated in parallel accumulation sequential fragmentation (PASEF) mode with PASEF mode data acquisition based on the following parameters: Ion mobility coefficient (1 / K0) values ​​were set to 0.6 to 1.6 Vs cm 2; 1MS and 10MS / MS PASEF scans. Active exclusion was enabled and the release time was 24s. The MS raw data of each sample were merged and retrieved for identification and quantitative analysis using MaxQuant 1.6.14 software.

[0072] (7) Immunofluorescence analysis

[0073] The colon tissue sections were washed with xylene, anhydrous ethanol and distilled water respectively. After completion, antigen repair was performed. After cooling, the sections were washed three times with PBS, incubated in hydrogen peroxide in the dark, and washed three times with PBS. After BSA was added to the slide for blocking for half an hour, HIF-1α was added to the sections for incubation and placed flat at 4°C overnight. After incubation, it was washed three times with PBS and incubated with the corresponding fluorescent secondary antibody at room temperature for 50 minutes. After washing three times with PBS, TSA was added after the sections were slightly dry, incubated at room temperature in the dark for 10 minutes, and washed three times with PBS after completion. The tissue sections were then subjected to antigen repair and incubated with VEGF and fluorescent secondary antibodies in the same steps. After the sections were slightly dry, DAPI staining solution was added to counterstain the cell nuclei, incubated at room temperature in the dark for 10 minutes, and washed three times with PBS after completion. An autofluorescence quencher was added, and the sections were sealed after rinsing with running water. The sections were placed under a scanner to collect images and analyzed.

[0074] (8) Western Blot

[0075] Accurately weigh 20 mg of colon tissue into a 1.5 mL EP tube, add 200 μL of RIPA lysis buffer and fully homogenize, then place it on ice for 20 min. After it is fully lysed, centrifuge it twice at 12,000 rpm at 4°C for 8 min. Take the supernatant to determine the protein concentration, calculate it, add loading buffer and lysis buffer, place it in a metal bath at 95°C for 10 min to fully denature the protein, then divide it into portions and store it at -50°C for later use.

[0076] Add 40μg of sample to the 12% SDS-PAGE sample tank and start electrophoresis. Adjust the voltage to 90V until the protein separation gel is pressed into a line, then adjust the voltage to 120V. After the electrophoresis is completed, transfer the protein to the PVDF membrane. After the transfer is completed, block it in 5% BSA for 6h. Place the PVDF membrane in a centrifuge tube containing the diluted primary antibody solution, incubate the primary antibody for 15h at 4℃, and then wash the membrane with TBST 5 times, 8min each time. Then put it in the secondary antibody solution and incubate it at 4℃ for 4h. After washing 5 times with TBST buffer, detect the bands through the gel imaging system, and use Image J software to perform semi-quantitative analysis of the corresponding proteins.

[0077] 2. Experimental Results

[0078] 1. Inhibitory effect of DD+SV on sw480 cell viability

[0079] sw480 cells were treated with three groups of polysaccharides at concentrations of 0-8 mg / mL, and the cell viability was detected using the MTT method. Figure 1 AC shows that the three polysaccharides have a significant inhibitory effect on the viability of SW480 cells in a dose-dependent manner (p<0.001). In order to evaluate the synergistic effect of the drugs, whether the combination of short-skirted bamboo fungus polysaccharide and mulberry linterus polysaccharide has a better inhibitory effect on SW480 cells was investigated. 2 mg / mL of short-skirted bamboo fungus polysaccharide and poplar mulberry linterus polysaccharide, as well as 1.5 mg / mL of the composite polysaccharide were used to treat SW480 cells for 24 hours. Figure 1 D showed that DD+SV had the best inhibitory effect on sw480 cells (p<0.01).

[0080] 2. Study on the activity of DD+SV in alleviating colitis-related colorectal cancer

[0081] 2.1. Study on the protective activity of DD+SV in CAC mice

[0082] After preliminary cell screening, it has been verified that the composite polysaccharides of Dictyophora shortskirt and Phellinus igniarius can effectively inhibit the growth of colorectal cancer cells and have a synergistic effect. In order to further verify its mechanism, Figure 2 As shown in the figure, an AOM / DSS-induced mouse CAC model was adopted. After 42 days of oral administration, the CAC model showed that the weight loss of the CAC model could be significantly restored (p<0.01), among which the weight of the mice in the DD+SV group was closer to the Ctrl group. After euthanasia, the colorectum of each group of mice was dissected and removed. The length of the colorectum in the CAC model was significantly shortened, and there were a large number of large tumor tissues in the colorectum; this phenomenon was significantly inhibited after DD+SV administration (p<0.001), and the effect was better than that of the DD and SV groups. The data show that DD+SV has a protective effect on the colorectum and inhibits colorectal lesions.

[0083] The colorectal histopathological analysis of AOM / DSS model mice was performed by H&E staining. Figure 3As shown. From the cross-section of the colorectum of the Ctrl group mice, it can be seen that the structure of each layer of the tissue is relatively easy to distinguish and observe, the cells are arranged neatly and tightly, the tissue is relatively complete, the intestinal gland morphology is normal, and the intestinal mucosal epithelium has no lesions or inflammatory cell infiltration. Large areas of cancerous tissue can be observed in the colorectal tissue of the AOM / DSS group mice, accompanied by severe inflammatory cell infiltration, the crypt structure and goblet cells disappeared, and the cell structure was irregular. Compared with the AOM / DSS group, normal goblet cells and crypt structures can be observed in the intestinal structure of the DD group, SV group and DD+SV group. There was no polyp or cancer tissue formation in the DD+SV group. Compared with the DD and SV groups, the DD+SV group tissue was closer to the blank group, indicating that DD+SV can inhibit the occurrence of tumor tissue better and inhibit the development of CAC.

[0084] The pathological changes of organs (heart, liver, spleen, kidney and thymus tissue) of AOM / DSS mice were compared by H&E staining and organ index. Figure 4-Figure 5 ), DD+SV had little effect on the physiological structure of various organs, indicating that DD+SV had no pathological effect on organs and no toxic side effects.

[0085] 2.2 Effect of DD+SV on regulating the intestinal microbiota structure of CAC model mice

[0086] The role of the intestinal flora in the development of colorectal cancer is twofold. A balanced intestinal flora environment promotes immunity and inhibits cancer cells, while an unbalanced ratio and excess of malignant bacteria promote the development and progression of colorectal cancer. The present invention uses 16SrRNA sequencing to examine whether the improvement of DD+SV on CAC tumorigenesis in mice is related to the intestinal microbiota.

[0087] Alpha diversity mainly evaluates the diversity of intestinal microbiota in a local uniform habitat, including indicators such as diversity, richness and uniformity. Figure 6 It can be seen that the intestinal flora environment of the AOM / DSS group changed significantly, and the richness and diversity of microorganisms were improved after administration of DD+SV.

[0088] Venn diagrams were used to evaluate the number of ASVs at the fecal microbiome level in the three groups. Of the 5185 ASVs detected in all groups in this experiment, 364 were common in all groups. The number of specific ASVs in the Ctrl group was 1771, the Model group was 1399, and the DD+SV group was 1319, indicating that there were large differences in the composition of the microbial community between the groups ( Figure 7 A).

[0089] Beta diversity (inter-habitat diversity) analyzed the differences in microbial community structure between different samples, and NMDS showed ( Figure 7B), the confidence intervals of the Ctrl and Model groups were completely separated, and DD+SV treatment significantly changed the microbiota structure affected by AOM / DSS and partially tended towards the Ctrl group.

[0090] The top 15 microorganisms at the genus level were subjected to row standardization and then a heat map was drawn to show the differences in the intestinal microbiome structure at the genus level. The abundance of signal pathways involved in the differentially expressed microorganisms was also analyzed. Figure 7 The results showed that the abundance of various microorganisms such as Parabacteroides and Sutterella decreased after AOM / DSS treatment, and the abundance could be increased after DD+SV administration; the abundance of microorganisms such as Allobaculum and Desulfovibrio increased after AOM / DSS treatment, and DD+SV could inhibit their increase. Parabacteroides can increase the colonic expression of tight junction proteins and reduce the occurrence of colorectal tumors.

[0091] 2.3 Effects of DD+SV on the regulation of metabolites and metabolic pathways in CAC model mice

[0092] The present invention uses non-targeted metabolomics technology to measure the metabolite profiles of mouse serum samples in the three groups to determine how metabolites are involved in the development of CAC and the therapeutic effects of DD+SV. Figure 8 It can be seen that by comparing the two groups to screen the differential metabolites, the Venn diagram shows that a total of 9 metabolites changed in level after AOM / DSS treatment, and DD+SV can restore the level of metabolites to the same level as the blank. We found that the level of succinate in the Model group mice decreased significantly. In previous studies, the accumulation of succinate led to high levels of HIF-1α expression, triggering the activation of the VEGF pathway related to angiogenesis and promoting the malignant progression of tumors.

[0093] 2.4 Effects of DD+SV on protein expression in colorectal and tumor tissues of CAC model mice

[0094] Metabolomics is a phenotype-based omics that describes what has happened in the body, while proteomics describes what is happening in the body. Figure 9-11 In the study, mouse colon tissue and tumor samples were tested. After AOM / DSS treatment, the levels of 6 proteins were upregulated and 3 proteins were downregulated. After DD+SV administration, the levels were changed and close to the blank histone level. Combined with GO analysis and KEGG analysis, we found that among the differentially expressed proteins, the upregulation of heparanase (HPSE) in the AOM / DSS group led to the activation of phosphorylated ERK. The activated phosphorylated ERK inhibited the activity of succinate dehydrogenase, leading to the accumulation of succinate. This phenomenon was reversed by DD+SV administration.

[0095] In order to verify the results of multi-omics combined analysis, we selected colon tissues from the Ctrl group, AOM / DSS group, and drug-treated group, and verified the expression of each protein level by immunofluorescence staining and immunoblotting analysis. Fig.12 The results showed that combined induction of AOM / DSS would cause an increase in the level of HPSE, activate the ERK pathway, inhibit the activity of succinate dehydrogenase, trigger the accumulation of succinate, and increase the expression levels of HIF-1α and VEGF, while DD+SV would reverse this phenomenon, indicating that DD+SV can play a protective role by inhibiting the ERK pathway, reducing angiogenesis in colon tissue, and slowing down the progression of cancer.

[0096] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A polysaccharide complex of Dictyophora shortskirt and Phellinus linteus with the efficacy of alleviating colorectal cancer, characterized in that: The preparation method of the polysaccharide complex comprises the following steps: The water extract of Dictyophora shortskirt and the water extract of Phellinus igniarius were mixed in equal volumes, concentrated under reduced pressure, and the protein and organic layers were removed to obtain a purified concentrate, which was precipitated with ethanol aqueous solution, and the precipitate was collected and dried to obtain the polysaccharide complex.

2. The polysaccharide complex according to claim 1, characterized in that The short-skirted bamboo fungus water extract and the poplar mulberry lindera water extract are both prepared by the following method: the short-skirted bamboo fungus or the poplar mulberry lindera is mixed with water, extracted in a water bath, extracted twice in total, and the extracts are combined to obtain the short-skirted bamboo fungus water extract or the poplar mulberry lindera water extract.

3. The polysaccharide complex according to claim 2, characterized in that The water bath extraction temperature is 80° C., and each extraction time is 2 h.

4. The polysaccharide complex according to claim 2, characterized in that The solid-liquid ratio of the short-skirted bamboo fungus or poplar mulberry linterus mixed with water is 1 g:30 mL.

5. The polysaccharide complex according to claim 1, characterized in that The colorectal cancer is colitis-associated colorectal cancer.

6. Use of the polysaccharide complex according to any one of claims 1 to 5 in the preparation of a drug for treating colorectal cancer.

7. The use according to claim 6, characterized in that: The colorectal cancer is colitis-associated colorectal cancer.

8. A drug for treating colorectal cancer, characterized in that: The active ingredient of the drug includes the polysaccharide complex according to any one of claims 1 to 4.

9. The drug according to claim 8, characterized in that The medicine further comprises a pharmaceutically acceptable carrier substance and / or adjuvant.

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