Application of monkshood polysaccharide in preparation of product for treating intestinal cancer

Abstract

The invention provides an application of monkshood polysaccharide in preparation of a product for treating intestinal cancer. The monkshood polysaccharide is applied to the research of intestinal cancer treatment for the first time. A mouse experiment proves that the monkshood polysaccharide has no obvious toxicity, and then the treatment effect of the monkshood polysaccharide on intestinal cancer is researched. Research shows that the monkshood polysaccharide can play a role in treating intestinal cancer, and a new idea and an effective means are provided for treating intestinal cancer with traditional Chinese medicines.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to an application of aconite polysaccharide in preparing a product for treating intestinal cancer. Background Art

[0002] Aconite root, first mentioned in the Shennong Bencao Jing (Shen Nong's Classic of Materia Medica), is a processed product of the lateral roots of the perennial herb Aconitum carmichaeli Debx (Ranunculaceae). It is now widely cultivated in Sichuan, Shaanxi, Guizhou, and other regions of my country. It is a traditional Chinese medicine with a long history of use. It has the effects of restoring yang, tonifying fire and yang, dispelling cold and relieving pain. It is used for symptoms of yang deficiency, collapse, weak pulse, and cold limbs. Its main chemical components include alkaloids, flavonoids, polysaccharides, saponins, sterols, fatty acids, and inorganic salts. Before the mid-20th century, research on the material basis of aconite root focused primarily on alkaloids. Only in recent years has research on aconite polysaccharides gradually attracted attention.

[0003] Recent studies have found that aconite polysaccharides, the active ingredient in aconite root, possess a wide range of pharmacological activities, including immunomodulatory, anti-tumor, antidepressant, organ protection, blood sugar regulation, anti-inflammatory, and antibacterial effects, with minimal adverse reactions and a high safety profile. As the primary active ingredient in aconite root, aconite polysaccharides have shown promising applications in the anti-tumor field. Significant progress has been made in recent years in research on their effects on liver and gastric cancers, but their application in other cancers remains under further exploration. Summary of the Invention

[0004] In view of the limited application scope of the existing technology, the present invention provides an application of aconite polysaccharide in the preparation of a product for treating intestinal cancer.

[0005] The technical solutions of the present invention are as follows: The present invention provides the use of aconite polysaccharide (also known as Fuzi Polysaccharide, FPS) in preparing a product for treating colorectal cancer.

[0006] In the present invention, the product has the function of treating at least one of colon cancer and rectal cancer. Here, colon cancer and rectal cancer may be referred to as colorectal cancer. Colon cancer is cancer that occurs in the colon, and rectal cancer is cancer that occurs in the rectum.

[0007] In the present invention, the product has the effect of protecting the liver in the treatment of colon cancer and rectal cancer.

[0008] In the present invention, the product has the effect of protecting the spleen in the treatment of colon cancer and rectal cancer.

[0009] In the present invention, the product has the effect of controlling blood in stool in the treatment of colon cancer and rectal cancer.

[0010] In the present invention, the product is a product that regulates the TGF-beta signaling pathway.

[0011] The TGF-beta signaling pathway is a large family of multifunctional cytokines encompassing numerous members, exerting its effects by regulating processes such as cell growth, proliferation, differentiation, migration, and apoptosis. Smads are important intracellular molecules that transmit and regulate TGF-beta signaling. Abnormal Smad function can affect TGF-beta signaling, leading to tumorigenesis. Studies have shown that Smad gene mutations have been found in tumors such as colon cancer and head and neck cancer, with Smad2 and Smad4 mutations being particularly common.

[0012] In the present invention, the product is a product that regulates extracellular matrix-receptor interaction.

[0013] Extracellular matrix-receptor interaction (ECM-receptor interaction) refers to the specific binding between the extracellular matrix (ECM) and cell surface receptors (such as integrins, CD44, etc.), which regulates biological processes such as cell adhesion, migration, differentiation and signal transduction, and plays a key role in tissue development, tumor metastasis and immune regulation. In the present invention, the preparation of the aconite polysaccharide comprises one of enzyme-assisted extraction, water extraction and alcohol precipitation, ultrasonic extraction, water leaching and microwave extraction.

[0014] Here, aconite polysaccharide may also be a commercially available product.

[0015] The beneficial effects of the present invention are: This study is the first to apply aconite polysaccharide to the treatment of colorectal cancer. First, experiments on mice confirmed the lack of significant toxicity of aconite polysaccharide, and then explored its therapeutic effects on colorectal cancer. The study showed that aconite polysaccharide can play a therapeutic role in colorectal cancer, providing new insights and effective approaches for the treatment of colorectal cancer with traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1-1A This is a graph showing the daily average body weight changes of female mice in each group during the observation period of Example 1; Figure 1-1B This is a graph showing the average daily body weight changes of male mice in each group during the observation period of Example 1; Figure 1-1C This is a graph showing the daily average body weight changes of female and male mice in each dosage group during the observation period of Example 1; Figure 1-2A This is a graph showing the changes in the average daily food intake of female mice in each group during the observation period of Example 1; Figure 1-2BThis is a graph showing the changes in the average daily food intake of male mice in each group during the observation period of Example 1; Figure 1-2C This is a graph showing the changes in average daily food intake of female and male mice in each dosage group during the observation period of Example 1; Figure 1-3A This is a graph showing the changes in the average daily water intake of female mice in each group during the observation period of Example 1; Figure 1-3B This is a graph showing the changes in the average daily water intake of male mice in each group during the observation period of Example 1; Figure 1-3C This is a graph showing the changes in average daily water intake of female and male mice in each dosage group during the observation period of Example 1; Figure 2-1 This is a diagram of the modeling cycle of the colorectal cancer mouse model in Example 2; Figure 2-2 This is a survival curve diagram of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-3 This is a graph showing the body weight coefficient of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-4 This is the stool index graph of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-5 This is a graph of hematochezia index of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-6 The diagram shows the length of the colorectum of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-7Figure 2 shows the statistical results of colorectal length of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2, where (* represents Model vs. Control); Figure 2-8 The figures are the colorectal HE staining results of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2; Figure 2-9 The spleen index graphs of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2, where * represents Model vs. Control; Figure 2-10 This is a graph of the thymus index of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2, where * represents Model vs. Control; Figure 2-11 The liver index graphs of the blank group (control), model group (model), FPS low-dose group (25 mg / kg), FPS medium-dose group (50 mg / kg), FPS high-dose group (100 mg / kg), and 5-Fu positive group in Example 2, where * represents Model vs. Control; # represents FPS vs. Model; Figure 2-12 This is the heat map of the correlation between samples in Example 2; Figure 2-13 The principal component analysis results of the blank group (control), model group (model), and FPS group in Example 2 are shown; Figure 2-14A This is the Model vs Control differential gene volcano plot of Example 2; Figure 2-14B This is the FPSvsModel differential gene volcano plot of Example 2; Figure 2-15This is a Venn diagram of differentially expressed genes in Example 2, wherein A represents UP.Model vs. Control; B represents DOWN.Model vs. Control; C represents UP.THC vs. Model; and D represents DOWN.THC vs. Model. Figure 2-16 This is the cluster heat map of differentially expressed genes in Example 2; Figure 2-17 GO enrichment analysis scatter plot; Figure 2-18 KEGG enrichment scatter plot. DETAILED DESCRIPTION

[0017] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0018] In the present invention, unless otherwise specified, all preparation raw materials are commercially available products well known to those skilled in the art.

[0019] Example 1 Toxicity test of aconite polysaccharide injection According to the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs," since the acute toxicity of most traditional Chinese medicines and natural medicinal products is likely relatively low, the maximum dose method can be used for acute toxicity studies of these medicines. Considering that aconite polysaccharides, as a traditional Chinese medicine, are likely to have low toxicity, mice were selected for acute toxicity studies after converting the maximum dose for clinical adults using a human-mouse conversion.

[0020] 1.1 Main instruments and reagents Intravenous visualization mouse tail injection holder; electronic balance; surgical instrument kit; ultrapure water; normal saline; aconite polysaccharide injection 1.2 Experimental animals and environment Fifty SPF Kunming (KM) mice, 8 weeks old, half male and half female, were housed separately and exposed to light for 12 hours per day.

[0021] 1.3 Administration Acute toxicity experiments were conducted by single-dose injection into the tail vein.

[0022] 1.4 Maximum dosage experiment Kunming mice were randomly divided into five groups according to body weight, with 10 mice in each group (half male and half female). The maximum dosage was calculated as the maximum dosage concentration (maximum solubility of Aconite polysaccharide) * the maximum dosage volume (0.5 mL for mice). The maximum dosage was set to 400 mg / kg. The dosing groups were set according to the Koch method, namely: (1) control group; (2) 400 mg / kg group; (3) 200 mg / kg group; (4) 100 mg / kg group; and (5) 50 mg / kg group.

[0023] The lowest dose in this trial was significantly higher than the clinical equivalent dose of 6 mg / kg (according to relevant experts, the recommended adult (60 kg) dose of Aconite Polysaccharide Injection is 40 mg. Based on the dose conversion coefficient, the equivalent dose for a single dose in mice is approximately 6 mg / kg). Therefore, no clinical equivalent dose group was established. Mice were fasted for 12 hours prior to intravenous injection. Each group of mice was injected with Aconite Polysaccharide Injection at the same dose volume of 0.5 mL and at different concentrations according to the above doses. The blank control group was given the same volume of solvent. After administration, the mice were observed continuously for approximately 4 hours, once every hour. They were then housed as usual, and the clinical symptoms and deaths of the mice were observed and recorded within 14 days.

[0024] 1.5 Observation of clinical manifestations During the experiment, all mice were housed in groups and fed once daily. The amount of feed given and remaining was recorded. Body weight was measured and food intake was calculated. ① Observe and record the mice's daily activities, fur color, secretions, feces, respiratory symptoms, and other clinical signs. ② Detailed records were made of the mice's body weight, food intake, poisoning reactions, and deaths. The cornea, iris, lens, and conjunctiva of the mice were observed for congestion or edema. (Any abnormalities were photographed and recorded.) 1.6 Organization Inspection On the 14th day after a single dose, the mice were euthanized and autopsied to observe pathological changes in the heart, liver, lungs, kidneys, spleen, gastrointestinal tract, testicles, ovaries and uterus of the mice, and to observe whether there was pleural or peritoneal effusion.

[0025] 2 Experimental results One mouse in the 50 mg / kg-♀ group died of cervical fracture due to excessive activity in the rat cage during dosing. During the 14-day observation period following dosing, no deaths or toxic reactions occurred in any of the groups, and the mice showed no abnormalities in activity. The fur of the mice remained smooth and their eyes were normal. Dissections of the mice at the end of the observation period revealed no abnormal pathological changes in any tissue, and no pleural or peritoneal effusions. During the experiment, daily changes in weight, food intake, and water intake were observed and recorded for each group. Figure 1-1A 、 Figure 1-1B 、 Figure 1-1C 、 Figure 1-2A 、 Figure 1-2B 、 Figure 1-2C 、 Figure 1-3A 、 Figure 1-3B 、 Figure 1-3C The t-test showed that the water intake of the treatment group (subdivided by gender) was significantly different from that of the control group (P value < 0.05).

[0026] like Figure 1-2A As shown in Figure 2, the average daily food intake of female mice in each group during the observation period changed; Figure 1-2B As shown, the average daily food intake of male mice in each group during the observation period; Figure 1-2C The following table shows the average daily food intake of male and female mice in each dose group during the observation period. Values ​​are expressed as mean ± standard deviation. Note: Except for the 50 mg / kg female group (n = 4), all other groups (n = 5) were included.

[0027] like Figure 1-3A As shown in the figure, the average daily water intake of female mice in each group during the observation period; Figure 1-3B As shown in the figure, the average daily water intake of male mice in each group during the observation period; Figure 1-3C The following table shows the average daily water intake of male and female mice in each dose group during the observation period. Values ​​are expressed as mean ± standard deviation. Note: Except for the 50 mg / kg female group (n = 4), all other groups (n = 5) were included.

[0028] 3 Experimental Conclusions During the observation period, no mice died or suffered from poisoning. Their daily diet, with only occasional fluctuations, remained generally stable, with no changes in body weight or activity. Subdividing the treated group by sex revealed differences in water intake compared with the control group, suggesting that the aconite polysaccharide injection may have some effect on the normal physiological activities of mice. However, further animal studies are needed to confirm this. In summary, the aconite polysaccharide injection showed no significant toxicity in mice.

[0029] Example 2 Experiment on Colorectal Cancer of Aconite Polysaccharide 1 Experimental Materials 1.1 Experimental Animals SPF-grade C57BL / 6 male mice, 74 in total, were purchased from Beijing Huafukang Experimental Animal Co., Ltd., with 12 in the blank group, 20 in the model group, 12 in the positive group, and 10 in each of the three drug-treated groups, for a total of 6 groups.

[0030] 1.2 Experimental drugs and instruments 1mL syringe; mouse gavage needle; electronic balance; ultrapure water; aconite polysaccharide powder; azoxymethane / dextran sulfate sodium (AOM / DSS); 5-fluorouracil (5-Fu).

[0031] 2 Experimental methods 2.1 Preparation of modeling reagents After removing the AOM reagent, centrifuge at 1000×g for 5 minutes to allow the AOM powder to settle at the bottom. Then, place the ampoule in a fume hood, break open the cap, and add 0.9% saline to a 2 mg / mL AOM stock solution. Aliquot and store in a -80°C freezer. DSS solution should be prepared immediately before use. To prepare a 2% DSS solution, weigh a certain amount of DSS reagent into a beaker and add 100 times the amount of sterile aqueous solution. Stir thoroughly with a glass rod, then sonicate in an ultrasound machine until the DSS solids disappear. The solution is then evenly distributed into the water bottles of the five groups of mice, excluding the blank group.

[0032] 2.2 Establishment of colorectal cancer model mice and grouping and drug administration The standard model of colorectal cancer in mice was established based on the AOM / DSS animal model. Six-week-old SPF-grade male C57BL / 6 mice were purchased from Beijing Huafukang Co., Ltd. Figure 2-1 After one week of adaptation and feeding, the mice were randomly divided into six groups: blank control group, AOM / DSS model group, 25 mg / kg aconite polysaccharide group, 50 mg / kg aconite polysaccharide group, 100 mg / kg aconite polysaccharide group, and 20 mg / kg 5-Fu group. Mice in the blank control group were intraperitoneally injected with 100 g / mL of 0.9% saline according to body weight, and mice in the other five groups were intraperitoneally injected with 12 mg / kg of AOM. After one week of feeding, mice in the five groups except the blank group were given 2% DSS sterile aqueous solution for one week and DSS-free sterile water for two weeks. This cycle was repeated three times. Mice in the blank group were given DSS-free sterile water ad libitum throughout the entire process. During three cycles, mice in the blank control and model groups were gavaged daily with 100 g / mL of 0.9% saline. The three groups receiving Aconite Polysaccharide received corresponding doses of the drug. Starting with the second DSS cycle, the 5-Fu group received intraperitoneal injections of 20 mg / kg 5-Fu twice weekly. After the completion of three cycles, mice were eye-enucleated and blood was collected, and then sacrificed by cervical dislocation. During the dosing period, mouse body weights were recorded daily, and hematochezia and fecal scores were recorded weekly. After colorectal sampling, the spleen, liver, and thymus were individually weighed and analyzed for organ index.

[0033] 2.3 Colorectal length measurement and sampling The abdominal cavity of the mouse was opened after being killed by dislocation of the neck. The anus and the lower end of the cecum were found and cut open. Scissors were used to cut the intestine longitudinally along the anus. The intestinal tube was picked up with forceps and placed in a pre-chilled PBS solution to clean the feces. Take out the prepared blue photography background plate, lay the cleaned colorectum with the inside facing up on the background plate, with the zero scale of the ruler facing the colon, measure the length of the colorectum, and take photos to record. After taking photos and recording, take about 1 cm of the colorectum for subsequent qRT-PCR measurement, about 1 cm of the colorectum for subsequent Western Blot experimental measurement, about 2 cm of the intestinal section near the rectal end for transcriptomic measurement and analysis, and about 2 cm of the colorectum for pathological section observation.

[0034] 2.4 Scoring criteria for blood stool index and stool index While recording the weight of the mice, each mouse was examined for defecation and blood in the stool. Stool that was complete and granular and not sticking to the anus was scored as 0, semi-formed stool that was not sticking to the anus was scored as 1, mushy stool that stuck to the anus was scored as 2, and liquid stool that flowed out of the anus was scored as 3. At the same time, the presence of blood in the feces of each group of mice was observed. Stool without blood was scored as 0, stool with slight blood streaks or spots was scored as 1, stool with severe bleeding was scored as 2, and severe bleeding and extensive blood stains around the anus was scored as 3.

[0035] 2.5 HE staining Visually inspect the selected tissue. Remove the fixed tissue from the fixative and, in a fume hood, trim the desired section of tissue using a scalpel according to the desired section plan (note the largest section, transverse section, or longitudinal section). Place the trimmed tissue into a numbered embedding frame. After washing, place the embedding frame containing the tissue into the basket of a dehydrator. Dehydrate the tissue using a gradient of alcohol in the dehydrator and then wax-impregnate it. Embed the wax-impregnated tissue in an embedding machine. First, place the melted wax into the embedding mold. Once the wax solidifies slightly, carefully remove the tissue from the embedding frame with forceps and place it into the embedding mold according to the desired embedding surface. Gently press the tissue with the bottom of the forceps to ensure it is completely flat within the mold. Place the embedding frame over the mold containing the tissue and gently transfer it to a -20°C freezer to cool. Once the wax solidifies, remove the wax block from the mold and trim it. Place the trimmed paraffin block on a paraffin pathology microtome and slice to a thickness of 4 μm. Float the slices on a 42°C warm water slide to flatten the tissue. Lift the tissue vertically with a glass slide, drain slightly, and bake the slides in a 60°C slide oven for 30 min-1 h. Remove and store at room temperature. Perform HE staining according to the following steps. The procedure is shown in Table 1-1.

[0036] Table 1-1 HE staining process 2.5 Transcriptome Experiment Process 2.5.1 Sample testing Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA): Accurately detect RNA integrity and total amount.

[0037] 2.5.2 Library construction and quality control NEB general database construction mRNA is enriched from total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA is synthesized using random hexamer primers, followed by second-strand cDNA synthesis. After end repair, A-tailing, adapter ligation, fragment selection, amplification, and purification, the library is ready. The library is quantified using a Qubit instrument and real-time fluorescence quantitative PCR, and fragment size distribution is determined using a bioanalyzer.

[0038] Chain-specific library construction mRNA is enriched from total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA is synthesized using random hexamer primers. Second-strand cDNA is then synthesized using dUTP instead of dTTP. Directed libraries are prepared after end repair, A-tailing, adapter ligation, fragment selection, USER enzyme digestion, amplification, and purification. The libraries are quantified using a Qubit instrument and real-time fluorescence quantitative PCR, and fragment size distribution is determined using a bioanalyzer.

[0039] 2.5.3 Sequencing After passing the library inspection, different libraries are pooled according to effective concentration and target data volume requirements before Illumina sequencing, generating 150 bp paired-end reads. The basic principle of sequencing is sequencing by synthesis (SBS). Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. As each sequencing cluster extends the complementary chain, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescent signal and converts it into sequencing peaks through computer software, thereby obtaining the sequence information of the fragment to be tested.

[0040] 2.6 Transcriptome analysis 2.6.1 Data Quality Control Raw reads were first processed using fastp software. This step removed reads containing adapters, reads containing ploy-N, and low-quality reads to generate clean reads. Q20, Q30, and GC content were calculated for the clean data. All downstream analyses were performed on this high-quality clean data.

[0041] 2.6.2 Sequence alignment to the reference genome The reference genome and gene model annotation files were downloaded directly from the genome website. HISAT2 v2.0.5 was used to index the reference genome, and paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5. We chose HISAT2 as the alignment tool because it can generate a spliced ​​database based on the gene model annotation files, thus providing better alignment results than other non-spliced ​​alignment tools.

[0042] 2.6.3 Quantification of gene expression levels featureCounts (1.5.0-p3) is used to count the number of reads mapped to each gene. The FPKM is then calculated for each gene based on its length and the number of reads mapped to that gene. FPKM refers to the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced. It accounts for the effects of sequencing depth and gene length on read counts and is currently the most commonly used method for estimating gene expression levels.

[0043] 2.6.4 Differential Expression Analysis Differential expression analysis between two comparison combinations was performed using DESeq2 software (1.20.0). DESeq2 provides statistical procedures for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values ​​were adjusted using the method of Benjamini and Hochberg to control the false discovery rate. Genes with adjusted P values ​​<= 0.05 found by DESeq2 were assigned as differentially expressed.

[0044] 2.6.5 Differential gene enrichment analysis GO enrichment analysis of differentially expressed genes was performed using clusterProfiler (3.8.1) software, which corrected for gene length bias. GO terms with a corrected P-value of less than 0.05 were considered significantly enriched by differentially expressed genes. KEGG is a database resource used to understand the high-level functions and utility of biological systems, such as cells, organisms, and ecosystems, from molecular-level information, particularly large-scale molecular datasets generated by genome sequencing and other high-throughput databases. We used clusterProfiler (3.8.1) software to analyze the statistical enrichment of differentially expressed genes within KEGG pathways.

[0045] 3 Experimental results 3.1 Number of mice surviving and dying The number of mice surviving and dying after ten weeks of modeling and drug administration is shown in Table 1-2: Table 1-2 Survival of mice 3.2 Mouse survival curve The survival status of mice was recorded starting from the intraperitoneal injection of AOM solution. There was no death in the blank group; in the model group, 1 died on 5.17, 5.20, 6.3, 6.6, 6.10, and 7.1, with a total of 6 deaths. All deaths were accompanied by diarrhea, and the one that died on 6.10 was also accompanied by hair loss and being eaten; in the 5-Fu positive group, 2 died on 5.20, with both deaths accompanied by diarrhea; in the low-dose FPS group, 1 died on 5.20, 5.23, and 5.27, with a total of 3 deaths. All deaths were accompanied by diarrhea; in the medium-dose FPS group, 1 died on 5.16, 5.20, and 6.24, with a total of 3 deaths. All deaths were accompanied by diarrhea, hair loss, and being eaten; in the high-dose FPS group, 1 died on 5.20 and 5.27, with a total of 2 deaths. All deaths were accompanied by diarrhea, hair loss, and being eaten. The survival curve is drawn in units of 7 days (W). Figure 2-2 As shown: By comparing the survival curve and the survival rate in Table 1-2, it can be seen that the survival rate of colorectal cancer mice in the FPS administration group increases with increasing dose.

[0046] 3.3 Condition of surviving mice (1) The blank group mice had no abnormal activity during the modeling and drug administration period. After the modeling and drug administration, the anatomical observations showed that there were no abnormal pathological changes in the tissues, and no pleural or peritoneal effusions. (2) The model group mice gained weight slowly during the modeling and drug administration period, and lost weight after three cycles, and their activity level decreased. Starting from the second cycle, the mice began to have loose stools and bloody stools, and the loose stools and bloody stools became more severe after three cycles. After the modeling was completed, the autopsy found that the colorectal length of the model group mice was shortened, a large amount of tumor tissue was present, and the spleen was enlarged. There were no significant changes in other tissues and organs, and there was no pleural or peritoneal effusion.

[0047] (3) The weight of mice in the 5-Fu positive group increased slowly before drug administration during the modeling period. After the second cycle of drug administration, the weight increased and the mice were active and normal. From the second cycle onwards, the mice began to have mild loose stools and blood in the stool. After the modeling was completed, the autopsy revealed that the length of the colorectum of the mice in the model group was reduced, the tumor tissue was small, the spleen was enlarged, and there were no significant changes in other tissues and organs. There was no pleural or peritoneal effusion.

[0048] (4) During the modeling period, the FPS mice were induced to lose weight and their activity level decreased. After the induction was stopped, their weight slowly increased within two weeks, and their activity level returned to normal. Starting from the second cycle, the mice began to have loose stools and blood in their stools, and the symptoms worsened after the third cycle. After the modeling was completed, the autopsy revealed that the colorectal length of the mice in the model group was shortened, the tumor tissue was small, and the spleen was enlarged. There were no significant changes in other tissues and organs, and there was no pleural or peritoneal effusion.

[0049] Effect of FPS on the body weight of mice bearing colorectal cancer Three weeks constituted one cycle, with a total of three cycles. During the animal experiment, the weight of the mice was measured once a week, and the weight data for 10 weeks was statistically analyzed using Graph Pad software. The average weight change data was obtained and plotted into a weight change graph. Figure 2-3As shown: there was no significant difference in body weight between the groups in the first week; in the first cycle (2-4 weeks), there was no significant difference in body weight between the groups in the second week; in the third week, there were significant differences in body weight changes between the blank group and the 5-Fu positive group compared with the model group, and there was no significant difference between the model group and the FPS administration group; in the fourth week, there were significant differences in body weight changes between the blank group and the FPS administration group compared with the model group, and there was no significant difference between the model group and the 5-Fu positive group; in the second cycle (5-7 weeks), there was a significant difference in body weight changes between the blank group and the model group in the fifth week, and there was no significant difference in body weight changes between the model group and the three FPS administration groups and the positive group; in the sixth week, there was a significant difference in body weight changes between the blank group and the model group, there was a significant difference in body weight changes between the model group and the FPS high-dose group, and there was no significant difference between the FPS low- and medium-dose groups, and there was a significant difference in body weight changes between the model group and the 5-Fu positive group; in the seventh week, there were significant differences in body weight changes between the model group and each group, and with the increase of the FPS administration dose, the FPS The weight of mice in the drug-treated groups increased; in the third cycle (8-10 weeks), in the eighth week, there was a significant difference in weight change between the blank group and the model group, there was a significant difference in weight change between the model group and the low-dose FPS group, and there was no significant difference between the drug-treated groups with medium and high doses of FPS, and there was a significant difference in weight change between the model group and the 5-Fu positive group; in the ninth week, there was a significant difference in weight change between the blank group and the model group, there was a significant difference in weight change between the model group and the medium and high-dose FPS groups, and there was no significant difference between the drug-treated groups with low doses of FPS, and there was a significant difference between the model group and the 5-Fu positive group; in the tenth week, there was a significant difference in weight change between the blank group and the model group, there was a significant difference in weight change between the model group and the medium and high-dose FPS groups, and there was no significant difference between the drug-treated groups with low doses of FPS, and there was a significant difference between the model group and the 5-Fu positive group; generally speaking, with the increase of FPS drug concentration, the weight change between the FPS drug-treated mice and the model group gradually increased, and there was a significant difference.

[0050] 3.5 Stool Index and Blood in Stool Index During the animal experiment, the fecal index and blood-feces index were measured once a week. The average values ​​of the fecal index and blood-feces index changes were obtained by statistical analysis using Graph pad software and plotted into fecal index and blood-feces index change graphs.

[0051] like Figure 2-4The stool index shown can be seen as follows: the stool status of each group was normal in the first week; in the first cycle (2-4W), there was no significant difference in stool index between each group and the model group in the second week, the stool status of the blank group was normal in the third week, and there was a significant difference between the model group and the low- and medium-dose FPS groups, but no significant difference in the high-dose group, and the stool status was more severe than the model group, and there was no significant difference between the 5-Fu positive group and the model group; in the second cycle (5-7W), there was no significant difference between each group in the fifth week, the stool status of the blank group was normal in the sixth week, and there was a significant difference between the model group and the medium-dose FPS group, but no significant difference in the low- and high-dose groups, and the stool status of the low-dose group was more severe than the model group, and there was no significant difference between the low-dose FPS group and the model group in the seventh week. There were significant differences compared with the model group, among which the FPS medium-dose group was the best, and the stool status was close to the blank group; in the third cycle (8-10W), there were significant differences between the groups and the model group in the eighth week, among which the FPS low- and medium-dose groups had the best stool status. In the ninth week, the stool status of the blank group was normal, which was significantly different from the model group. There were significant differences between the FPS medium- and high-dose groups and the model group. There was no significant difference in the low-dose group, and the stool status was more severe than that of the model group in terms of shapelessness. The stool status of the medium-dose group was slightly better and close to that of the positive group. There was a significant difference between the 5-Fu positive group and the model group. In the tenth week, the stool status of the blank group and the 5-Fu positive group was normal, which was significantly different from the model group. There was no significant difference between the three FPS-administered groups and the model group, and the stool status of the medium- and high-dose groups was more severe than that of the model group in terms of shapelessness, and the low-dose group was slightly better. From Figure 2-5From the hematochezia index, we can see that: the hematochezia of each group was normal in the first week; in the first cycle (2-4W), the hematochezia of the model group was severe in the third week, which was significantly different from that of the other groups, among which the FPS medium-dose group had the mildest hematochezia, and there was no significant difference between the model group and the other groups in the second and fourth weeks, and there was no hematochezia; in the second cycle (5-7W), there was no significant difference between the model group and the other groups in the fifth week, and there was no hematochezia; in the sixth week, there was no hematochezia in the blank group, which was significantly different from the model group, and there was no significant difference between the other four groups and the model group, among which some mice in the FPS low-dose group had prolapse of the anus; in the seventh week, the feces of the blank group was normal, which was significantly different from that of the model group, and the hematochezia of the model group was severe. The FPS low- and medium-dose groups were significantly different from the model group, among which the hematochezia of the FPS low-dose group was improved, and there was no hematochezia, and there was no significant difference between the model group and the 5-Fu positive group and the FPS high-dose group; in the third cycle ( 8-10W), in the eighth week, except for the high-dose FPS group, all groups showed significant differences compared with the model group. The bloody stool status of the low- and medium-dose FPS groups was consistent with the 5-Fu positive group, with no bloody stool phenomenon. In the ninth week, there was a significant difference between the blank group and the model group, a significant difference between the low-dose FPS group and the model group, no significant difference between the medium- and high-dose FPS groups, and a significant difference between the 5-Fu positive group and the model group. Among them, the low-dose group had the mildest bloody stool after FPS administration. In the tenth week, there was a significant difference between the blank group and the model group; there was a significant difference between the 5-Fu positive group and the model group. Compared with the model group, the low-dose FPS group had a significant difference and the mildest bloody stool. Compared with the model group, the medium- and high-dose FPS groups had improved bloody stools, but there was no significant difference. Overall, FPS administration aggravated the loose stools caused by intestinal irritation in mice with increasing doses; but the bloody stool condition caused by colorectal cancer gradually improved with increasing doses.

[0052] Effect of 3.6 FPS on colorectal tissue length After the C57 mice were modeled and administered, the rectal status of each group of mice was measured and photographed. Figure 2-6 As shown: the blank group had normal colorectal length and a transparent, gelatinous intestinal wall; the model group had large tumors with large numbers and decreased intestinal wall transparency; the 5-Fu positive group had small tumors and small numbers, and the intestinal wall transparency was close to that of the blank group; the FPS-treated group had small tumors and small numbers, and the intestinal wall transparency was higher than that of the model group; the medium-dose group had the smallest tumors and the smallest number, and the intestinal wall transparency was close to that of the positive group. Figure 2-7 As shown: the blank group had the longest colorectal length, and the colorectal length was significantly shortened compared with the model group and the blank group; the colorectal length of the 5-Fu positive group was increased compared with the model group; compared with the model group, FPS had little effect on the colorectal length of colorectal cancer mice.

[0053] Effects of FPS on colorectal histopathology in colorectal cancer-bearing mice HE staining was performed on the colorectal tissue of mice. Figure 2-8 As shown, compared with the blank group, the model group showed a large number of tumor cells under the microscope, with high nuclear basophilia, a high nuclear-to-cytoplasmic ratio, and a variety of morphologies. Tumor cells often formed glandular sieve-like structures, with a small amount of necrotic exudate visible in the glandular cavity, and a large number of goblet cell proliferations, surrounded by scattered lymphocyte infiltration. After FPS administration, these phenomena improved in mice with increasing concentrations, with a decrease in inflammatory infiltration and tumor cells. The number of colorectal tumors was the lowest in the medium-dose group.

[0054] 3.8 Effects of FPS on Organ Indexes in Colorectal Cancer Mice C57 mouse spleen index Figure 2-9 As shown in the figure: the model group significantly increased the spleen index of colorectal cancer mice compared with the blank group; after administration, the 5-Fu positive group had a lower spleen index compared with the model group; the spleen index of the three groups of mice in the FPS administration group was lower than that of the model group, especially the spleen index of the medium dose group showed a trend compared with the model group. Figure 2-10 As shown in the figure: the model group significantly increased the thymus index of colorectal cancer mice compared with the blank group; after administration, the 5-Fu positive group could reduce the thymus index compared with the model group; FPS had no effect on the thymus index. Figure 2-11 As shown: compared with the blank group, the liver index of the model group was significantly increased; compared with the model group, the 5-Fu positive group was able to reduce the liver index; the liver index of mice with medium and high doses of FPS was significantly reduced, and the liver index of the medium dose group was close to that of the blank group.

[0055] 3.9 Transcriptome Analysis Results 3.9.1 Sample Correlation Analysis Biological replication is usually necessary for any biological experiment, and mainstream journals currently also basically require biological replication. Biological replication has two main uses: one is to prove that the biological experimental operations involved are not accidental, but repeatable. The other is to ensure that subsequent differential gene analysis obtains more reliable results. The correlation of gene expression levels between samples is an important indicator for testing the reliability of the experiment and whether the sample selection is reasonable. The closer the correlation coefficient is to 1, the higher the similarity of the expression patterns between samples. It is generally required that the R2 between biological replicate samples be at least greater than 0.8. Our results show that Figure 2-12 As shown in the figure, the horizontal and vertical axes are the squares of the correlation coefficients of each sample. The correlation coefficients of each group are all greater than 0.8, and the biological repetition is good.

[0056] 3.9.2 Principal Component Analysis Principal component analysis (PCA) is also commonly used to evaluate inter-group differences and intra-group sample duplication. PCA uses linear algebraic calculation methods to reduce the dimension and extract principal components of tens of thousands of gene variables. We performed PCA analysis on the gene expression values ​​(FPKM) of all samples, such as Figure 2-13 As shown. Under ideal conditions, in the PCA graph, samples between groups should be dispersed, and samples within groups should be clustered together. Figure 2-13 As shown, the horizontal axis is the first principal component, the vertical axis is the second principal component, and the PCA analysis results show that the model group and the blank group are clearly clustered, and after FPS administration, they are closer to the blank group.

[0057] 3.9.3 Differential gene screening After the gene expression quantification is completed, the expression data needs to be statistically analyzed to screen genes with significant expression differences in different samples. DESeq is used to normalize the original read count and use |log2(FoldChange)|>=0 & pvalue<=0.05 as the threshold to screen differentially expressed genes. The results are as follows Figure 2-14A and Figure 2-14B As shown in Figure 2, 2849 genes were upregulated and 2222 genes were downregulated in the model group compared with the blank group; 795 genes were upregulated and 746 genes were downregulated in the FPS group compared with the model group. The intersection of genes upregulated in the model group and downregulated after drug administration and genes downregulated in the model group and upregulated after drug administration was taken to obtain the key differentially expressed genes in drug treatment. The results are shown in Figure 2. Figure 2-15 As shown, the Venn diagram results show that there are 361 intersections of genes upregulated by Model vs Control and genes downregulated by FPS vs Model, and there are 493 intersections of genes downregulated by Model vs Control and genes upregulated by FPS vs Model. Therefore, after taking the union, there are a total of 854 differentially expressed genes in FPS treatment of colorectal cancer.

[0058] Figure 2-15 Venn of differentially expressed genes (A represents UP.Model vs. Control; B represents DOWN.Model vs. Control; C represents UP.THC vs. Model; D represents DOWN.THC vs. Model).

[0059] 3.9.4 Differential gene clustering We clustered the differentially expressed genes and grouped the genes with similar expression patterns together. We used the mainstream hierarchical clustering to cluster the FPKM values ​​of the genes and normalized the rows (Z-score). The heat map of differentially expressed genes is shown below. Figure 2-16As shown, the gene expression pattern of the Model group was significantly different from that of the Control group. After FPS administration, its expression pattern was closer to that of the Control group.

[0060] 3.9.5 GO functional enrichment analysis GO (Gene Ontology) is a comprehensive database describing gene functions, which can be divided into three parts: biological process, cellular component, and molecular function. GO functional enrichment uses a padj value less than 0.05 as the threshold for significant enrichment. From the GO enrichment analysis results, the 30 most significant terms were selected to draw a scatter plot for display, as shown below: Figure 2-17 The horizontal axis is the ratio of the number of differentially expressed genes annotated to GOTerm to the total number of differentially expressed genes, and the vertical axis is GOTerm. The size of the dots represents the number of genes annotated to GOTerm, and the colors from red to blue represent the significance of enrichment.

[0061] 3.9.6 KEGG functional enrichment analysis KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database that integrates genomic, chemical, and system functional information. KEGG pathway enrichment uses padj less than 0.05 as the threshold for significant enrichment. The enrichment results are as follows: Figure 2-18 As shown. Among them, the PI3K-Akt signaling pathway, ECM-receptor interaction, Cytokine-cytokine receptor interaction, TGF-beta signaling pathway, etc. are all related to immunity. The PI3K / Akt signaling pathway responds to changes in the internal and external environment by activating downstream effector molecules, thereby affecting the survival, proliferation, differentiation and function of immune cells. The Cytokine-cytokine receptor interaction pathway is one of the core signal transduction pathways in the immune system. Cytokines are small molecule proteins secreted by immune cells and other cells. They regulate the growth, differentiation, activation, proliferation, migration and apoptosis of immune cells by binding to specific cell surface receptors. These interactions are crucial for the normal functioning of the immune system because they are not only involved in the initiation and regulation of immune responses, but also involve the formation of inflammatory responses, self-tolerance and immune memory. Therefore, the therapeutic effect of FPS on AOM / DSS-induced colorectal cancer mice is related to immune regulation.

[0062] 4 Conclusion During the experiment, some mice died. The blank group was normal. During each round of 2% DSS solution induction, the mice in the model group drank less water and ate less, lost weight, and became less active, but gradually recovered after the induction was stopped. Except for the fecal index, all indicators of the mice in the medium-dose administration group showed a significant trend compared with the model group, and the liver index showed a significant difference; this indicates that oral administration of FPS may have a protective effect on the liver and spleen of mice with colorectal cancer; it has a certain control and slowing effect on the development of colorectal cancer, but may not have a significant protective effect on the length of colorectal cancer; at the same time, the FPS administration group has a good control effect on the blood in the stool of mice with colorectal cancer, but the loose stool condition gradually worsens with increasing doses. Transcriptome results show that the therapeutic effect of FPS on AOM / DSS-induced colorectal cancer in mice is related to the regulation of signaling pathways such as Cytokine-cytokine receptor interaction. In summary, the effects of the FPS administration group on colorectal cancer mice are: medium dose > high dose > low dose; FPS has a good inhibitory effect on the development of colorectal cancer in mice; at the same time, FPS has an immune regulatory effect on colorectal cancer mice.

Claims

1. Application of aconite polysaccharide in the preparation of products for the treatment of colorectal cancer.

2. The use according to claim 1, characterized in that The product has a product for treating at least one of colon cancer and rectal cancer.

3. The use according to claim 2, characterized in that The product has a liver-protecting effect in the treatment of colon cancer and rectal cancer.

4. The use according to claim 2, characterized in that The product has the effect of protecting the spleen in the treatment of colon cancer and rectal cancer.

5. The use according to claim 2, characterized in that The product has the effect of controlling blood in stool in the treatment of colon cancer and rectal cancer.

6. The use according to claim 1, characterized in that The product is a product that regulates the TGF-beta signaling pathway.

7. The use according to claim 1, characterized in that The product is a product that regulates extracellular matrix-receptor interactions.

8. The use according to any one of claims 1 to 7, characterized in that The preparation of the aconite polysaccharide comprises one of an enzyme-assisted extraction method, a water extraction and alcohol precipitation method, an ultrasonic extraction method, a water leaching method, and a microwave extraction method.

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