Application of polysaccharide in aconite in preparation of products for treating intestinal cancer

Abstract

The application provides application of polysaccharide in aconite in preparation of a product for treating intestinal cancer. The application first applies the polysaccharide in aconite to the research on the treatment of intestinal cancer. Firstly, the polysaccharide in aconite is proved to have no obvious toxicity through a mouse experiment, and then the treatment effect of the polysaccharide in aconite on intestinal cancer is explored. Research shows that the polysaccharide in aconite can treat intestinal cancer, and provides a new thought and effective means for treating intestinal cancer by using traditional Chinese medicines.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of Aconitum carmichaelii polysaccharide in the preparation of products for treating colorectal cancer. Background Technology

[0002] Aconitum carmichaelii, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), is a processed product of the lateral roots of the perennial herbaceous plant *Aconitum carmichaeli* Debx, belonging to the Ranunculaceae family. It is currently widely cultivated in Sichuan, Shaanxi, Guizhou, and other regions of my country. It is a traditional Chinese medicine with a very long history of use, possessing the effects of restoring yang, tonifying fire and assisting yang, dispelling cold and relieving pain. It can be used for symptoms such as collapse due to yang deficiency, 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 Aconitum carmichaelii mainly focused on alkaloids; only in recent years has research on Aconitum carmichaelii polysaccharides gradually attracted attention.

[0003] Recent studies have found that Aconitum carmichaelii polysaccharides, as the active ingredient in Aconitum carmichaelii, possess a wide range of pharmacological activities, such as immunomodulation, antitumor, antidepressant, organ protection, glycemic regulation, anti-inflammatory, and antibacterial effects, with few adverse reactions and high safety. As the main active ingredient of Aconitum carmichaelii, Aconitum carmichaelii polysaccharides show broad application prospects in the field of antitumor therapy. In recent years, significant progress has been made in research on its application in liver cancer and gastric cancer, but its application in other cancers still needs further exploration. Summary of the Invention

[0004] This invention addresses the limitation of application scope in existing technologies by providing an application of Aconitum carmichaelii polysaccharide in the preparation of products for treating colorectal cancer.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides the application of Fuzi Polysaccharide (FPS) in the preparation of products for treating colorectal cancer.

[0007] In this invention, the product is a treatment for at least one of colon cancer and rectal cancer. Here, colon cancer and rectal cancer can be referred to as colorectal cancer. Colon cancer is cancer that occurs in the colon, and rectal cancer refers to cancer that occurs in the rectum.

[0008] In this invention, the product has a liver-protective effect in the treatment of colon and rectal cancer.

[0009] In this invention, the product has a protective effect on the spleen in the treatment of colon cancer and rectal cancer.

[0010] In this invention, the product has the effect of controlling rectal bleeding in the treatment of colon cancer and rectal cancer.

[0011] In this invention, the product is a product for regulating the TGF-beta signaling pathway.

[0012] The TGF-beta signaling pathway is a large family of multifunctional cytokines that plays a role in regulating cell growth, proliferation, differentiation, migration, and apoptosis. Smads are important intracellular TGF-beta signaling and regulatory molecules; abnormalities in their function can affect TGF-beta signaling, leading to tumorigenesis. Studies have shown mutations in Smad genes in tumors such as colon cancer and head and neck tumors, with mutations in Smad2 and Smad4 genes being particularly common.

[0013] In this invention, the product is a product that regulates extracellular matrix-receptor interactions.

[0014] Extracellular matrix-receptor interaction (ECM-receptor interaction) refers to the specific binding between the extracellular matrix (ECM) and cell surface receptors (such as integrins and CD44). It regulates biological processes such as cell adhesion, migration, differentiation, and signal transduction, playing a crucial role in tissue development, tumor metastasis, and immune regulation.

[0015] In this invention, the preparation of the Aconitum carmichaelii polysaccharide includes one of the following methods: enzyme-assisted extraction, water extraction and alcohol precipitation, ultrasonic extraction, water immersion extraction, and microwave extraction.

[0016] Here, Aconitum carmichaelii polysaccharide can also be a commercially available product.

[0017] The beneficial effects of this invention are:

[0018] This invention is the first to apply Aconitum carmichaelii polysaccharide to the treatment of colorectal cancer. Firstly, mouse experiments confirmed that Aconitum carmichaelii polysaccharide has no significant toxicity. Then, the therapeutic effect of Aconitum carmichaelii polysaccharide on colorectal cancer was investigated. The study shows that Aconitum carmichaelii polysaccharide can play a therapeutic role in colorectal cancer, providing a new approach and effective method for the treatment of colorectal cancer with traditional Chinese medicine. Attached Figure Description

[0019] Figure 1-1A This is a graph showing the daily average weight changes of female mice in each group during the observation period of Example 1.

[0020] Figure 1-1B This is a graph showing the daily average weight changes of male rats in each group during the observation period of Example 1;

[0021] Figure 1-1C This is a graph showing the daily average weight changes of female and male mice in each dosage group during the observation period of Example 1;

[0022] 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.

[0023] Figure 1-2B This is a graph showing the changes in the average daily food intake of male rats in each group during the observation period of Example 1.

[0024] Figure 1-2C This is a graph showing the changes in the average daily food intake of female and male mice in each dosage group during the observation period of Example 1;

[0025] Figure 1-3A This is a graph showing the changes in the average daily water consumption of female mice in each group during the observation period of Example 1.

[0026] Figure 1-3B This is a graph showing the changes in the average daily water consumption of male rats in each group during the observation period of Example 1.

[0027] Figure 1-3C This is a graph showing the changes in the average daily water intake of female and male mice in each dosage group during the observation period of Example 1;

[0028] Figure 2-1 This is a diagram illustrating the modeling process of the colorectal cancer mouse model in Example 2.

[0029] Figure 2-2 Survival curves for the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2;

[0030] Figure 2-3 The figure shows the body weight coefficients of the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2.

[0031] Figure 2-4 The following is a fecal index graph for the blank group (control), model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2.

[0032] Figure 2-5 The graph shows the hematochezia index of the blank group (control), model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2.

[0033] Figure 2-6 The diagram shows the length of the colon and rectum in the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group of Example 2.

[0034] Figure 2-7 The graph shows the statistical results of colorectal length in the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2. (* indicates Model vs Control).

[0035] Figure 2-8 The images show the colorectal HE staining results of the blank group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2.

[0036] Figure 2-9 The spleen index graphs for the blank group (control), model group (model), low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2 are shown. * indicates Model vs Control.

[0037] Figure 2-10 The image shows the thymus index of the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2. * indicates Model vs Control.

[0038] Figure 2-11 The liver index graphs for the control group, model group, low-dose FPS group (25 mg / kg), medium-dose FPS group (50 mg / kg), high-dose FPS group (100 mg / kg), and 5-Fu positive group in Example 2 are shown. * indicates Model vs. Control; # indicates FPS vs. Model.

[0039] Figure 2-12 This is a heatmap of inter-sample correlations from Example 2;

[0040] Figure 2-13The graph shows the principal component analysis results for the control group, model group, and FPS group in Example 2.

[0041] Figure 2-14A This is a Model vs. Control differential gene volcano plot from Example 2;

[0042] Figure 2-14B This is a volcano plot of differentially expressed genes from FPSvsModel in Example 2;

[0043] Figure 2-15 This is a Venn diagram of differentially expressed genes in Example 2, where A represents UP.ModelvsControl; B represents DOWN.ModelvsControl; C represents UP.THCvsModel; and D represents DOWN.THCvsModel.

[0044] Figure 2-16 This is a heatmap of differentially expressed genes clustering from Example 2.

[0045] Figure 2-17 GO enrichment analysis scatter plot;

[0046] Figure 2-18 KEGG enrichment scatter plot. Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0049] Example 1 Toxicity test of Aconitum carmichaelii polysaccharide injection

[0050] According to the "Technical Guidelines for Single-Dose Toxicity Studies of Drugs," since most traditional Chinese medicines and natural drugs may have relatively low acute toxicity, the maximum dose method can be used for acute toxicity studies. Considering that Aconitum carmichaelii polysaccharide is a type of traditional Chinese medicine and its toxicity may be low, the maximum clinical adult dose was converted using human-mouse conversion, and mice were selected for acute toxicity studies.

[0051] 1.1 Main Instruments and Reagents

[0052] Intravenous visual mouse tail injection fixation device; electronic balance; surgical instrument kit; ultrapure water; physiological saline; Aconitum carmichaelii polysaccharide injection

[0053] 1.2 Laboratory Animals and Environment

[0054] Fifty SPF-grade Kunming (KM) mice, 8 weeks old, half male and half female, were housed separately and provided with 12 hours of light per day.

[0055] 1.3 Administration method

[0056] Acute toxicity tests were conducted using a single-dose tail vein injection.

[0057] 1.4 Maximum Dosage Experiment

[0058] Kunming mice were randomly divided into 5 groups of 10 mice each (half male and half female) according to their body weight. The maximum dosage was calculated as: maximum dosage concentration (maximum solubility of Aconitum carmichaelii polysaccharide) * maximum dosage volume (0.5 mL for mice). The maximum dosage was set at 400 mg / kg. The dosage groups were set according to the Kohl method as follows: (1) control group; (2) 400 mg / kg group; (3) 200 mg / kg group; (4) 100 mg / kg group; (5) 50 mg / kg group.

[0059] The lowest dose group in this experiment was already far greater than the clinically equivalent dose of 6 mg / kg (based on expert estimates, the recommended adult dose of Aconitum carmichaelii polysaccharide injection (60 kg) is 40 mg, and the equivalent dose for mice calculated using the dosage conversion factor is approximately 6 mg / kg), therefore no clinically equivalent dose group was set up. Mice were fasted for 12 hours prior to intravenous administration but allowed free access to water. Each group of mice was injected with Aconitum carmichaelii polysaccharide injection at the same volume (0.5 mL) at different concentrations, according to the above dosage. The blank control group received the same volume of solvent. After administration, mice were observed continuously for approximately 4 hours, once per hour. They were then fed normally, and clinical symptoms and mortality were observed and recorded within 14 days.

[0060] 1.5 Clinical Manifestations Observation

[0061] During the experiment, all mice were housed in groups, fed once daily, and the amount of feed given and any uneaten feed was recorded. Mice were weighed once daily to calculate their food intake. ① Observe and record the mice's daily activities, fur color, secretions, feces, and respiratory symptoms. ② Record in detail the mice's body weight, food and water intake, poisoning reactions, and mortality. Observe the mice's cornea, iris, lens, and bulbar conjunctiva for congestion, edema, etc. (If any abnormalities are found, photograph and record them).

[0062] 1.6 Organizational Inspection

[0063] Mice were euthanized on day 14 after a single dose and subjected to necropsy to observe pathological changes in the heart, liver, lungs, kidneys, spleen, gastrointestinal tract, testes, ovaries, and uterus, as well as the presence of pleural or peritoneal effusion.

[0064] 2 Experimental Results

[0065] One mouse in the 50 mg / kg female group died from cervical dislocation due to excessive activity in the mouse container during administration. No deaths or toxic reactions occurred in any group during the 14-day observation period after administration, and the mice exhibited normal activity levels. During the observation period, the mice's fur was smooth, and their eyes showed no abnormalities. Dissection of the mice after the observation period revealed no abnormal pathological changes in any tissues, and no pleural or peritoneal effusion. Daily changes in body weight, food intake, and water consumption were observed and recorded for each group during the experiment. The results are as follows: 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 As shown in the figure. The t-test revealed a significant difference in water intake between the treatment group (after subgrouping by gender) and the control group (P < 0.05).

[0066] like Figure 1-2A The figure shows the changes in the average daily food intake of female mice in each group during the observation period; as shown... Figure 1-2B The figure shows the changes in the average daily food intake of male rats in each group during the observation period; as shown... Figure 1-2C The figure shows the changes in the average daily food intake of female and male mice in each dosage 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.

[0067] like Figure 1-3A The figure shows the changes in the average daily water intake of female mice in each group during the observation period; Figure 1-3B The figure shows the changes in the average daily water intake of male rats in each group during the observation period; as shown... Figure 1-3C The figure shows the changes in average daily water intake of female and male mice in each dosage 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 excluded.

[0068] 3. Experimental Conclusions

[0069] During the observation period, no mice died or showed signs of poisoning. Their daily diet fluctuated only occasionally but remained relatively stable, their weight was unaffected, and their activity was normal. However, subgrouping the treated group by sex revealed differences in water intake compared to the control group, suggesting that Aconitum carmichaelii polysaccharide injection may have some impact on the normal physiological functions of mice. Further confirmation with a larger number of animal experiments is needed. In conclusion, Aconitum carmichaelii polysaccharide injection showed no significant toxicity to mice.

[0070] Example 2: Experiment with Aconitum carmichaelii polysaccharide in colorectal cancer

[0071] 1. Experimental Materials

[0072] 1.1 Laboratory Animals

[0073] SPF-grade C57BL / 6 mice, male, 74 in total: 12 in the control group, 20 in the model group, 12 in the positive control group, and 10 in each of the three drug-treated groups, for a total of 6 groups. Purchased from Beijing Huafukang Experimental Animal Co., Ltd.

[0074] 1.2 Experimental drugs and instruments

[0075] 1mL syringe; mouse gavage needle; electronic balance; ultrapure water; Aconitum carmichaelii polysaccharide powder; azomethane / dextran sulfate sodium (AOM / DSS); 5-fluorouracil (5-Fu).

[0076] 2 Experimental Methods

[0077] 2.1 Preparation of modeling reagents

[0078] After removing the AOM reagent, spin 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 mouth of the AOM reagent ampoule, and add 0.9% physiological saline to prepare a 2 mg / mL AOM stock solution. Aliquot and store at -80℃. DSS solution should be prepared fresh for use. The preparation method for 2% DSS solution is as follows: Weigh a certain amount of DSS reagent and add it to a beaker, then add 100 times the amount of sterile aqueous solution of DSS. Stir well with a glass rod, and sonicate in an ultrasonic machine until the DSS solid disappears. Then evenly distribute the solution to the water collection bottles of the other 5 groups of mice, excluding the blank group.

[0079] 2.2 Establishment and grouping of colorectal cancer mouse models for drug administration

[0080] The standard mouse model for colorectal cancer was established according to the AOM / DSS animal model. Six-week-old SPF-grade male C57BL / 6 mice purchased from Beijing Huafukang Co., Ltd. were used. Figure 2-1After one week of acclimatization, mice were randomly divided into six groups: a blank control group, an AOM / DSS model group, a 25 mg / kg Aconitum carmichaelii polysaccharide group, a 50 mg / kg Aconitum carmichaelii polysaccharide group, a 100 mg / kg Aconitum carmichaelii polysaccharide group, and a 20 mg / kg 5-Fu group. Mice in the blank control group were intraperitoneally injected with 100 g / mL of 0.9% saline solution, while the other five groups were intraperitoneally injected with 12 mg / kg AOM. After one week of feeding, the other five groups (excluding the blank control group) were given 2% DSS sterile water for one week, followed by sterile water without DSS for two weeks, repeating this cycle three times. Mice in the blank control group were given free access to sterile water without DSS throughout the entire cycle. During the three cycles, mice in the blank control group and model group were administered 100 g / mL of 0.9% saline by gavage daily. The three groups receiving Aconitum carmichaelii polysaccharide were injected with the corresponding doses of the drug. Starting from the second DSS cycle, the 5-Fu group received intraperitoneal injections of 20 mg / kg 5-Fu twice weekly. After the three cycles, mice were euthanized by enucleation, blood collection, and cervical dislocation. Throughout the administration period, mouse weight was recorded daily, and fecal blood score and stool score were recorded weekly. After colonic and rectal tissue collection, the spleen, liver, and thymus were harvested and weighed separately for organ index analysis.

[0081] 2.3 Measurement and sampling of colorectal length

[0082] The abdominal cavity of mice euthanized by cervical dislocation was opened, and the anus and lower end of the cecum were located and cut off. The intestine was longitudinally cut along the anus, and the segment was held in pre-cooled PBS solution to clean the feces. A prepared blue photographic background was used, and the cleaned colon and rectum were laid flat on the background with the inside facing upwards. The zero mark of a ruler was aligned with the colon area, and the length of the colon and rectum was measured and photographed. After photographing, approximately 1 cm of colon and rectum was used for subsequent qRT-PCR assays, approximately 1 cm of colon and rectum was used for subsequent Western blotting experiments, approximately 2 cm of the intestine from the rectum was used for transcriptomics analysis, and approximately 2 cm of colon and rectum was used for pathological section observation.

[0083] 2.4 Scoring criteria for rectal bleeding index and fecal index

[0084] While recording mouse weight, the defecation and blood in the stool of each mouse were examined. A score of 0 was given for stool that was intact and not attached to the anus; a score of 1 was given for partially formed stool that was not attached to the anus; a score of 2 was given for stool that was pasty and attached to the anus; and a score of 3 was given for stool that flowed down the anus and was liquid. Simultaneously, the presence of blood in the feces of each group of mice was observed. A score of 0 was given for stool without blood; a score of 1 was given for stool with slight streaks or spots of blood; a score of 2 was given for stool with severe bleeding; and a score of 3 was given for severe bleeding and extensive bloodstains around the anus.

[0085] 2.5 HE staining

[0086] Visually inspect the selected tissue. Remove the fixed tissue from the fixative and, in a fume hood, trim the tissue at the target site with a scalpel according to the tissue sectioning requirements (note the maximum surface, cross section, and longitudinal section). Place the trimmed tissue into a pre-labeled embedding frame. After washing, place the embedding frame containing the tissue into the basket of a dehydrator and dehydrate and impregnate it with paraffin using a gradient of alcohol. Embed the paraffin-impregnated tissue in the embedding machine. First, pour the melted paraffin into the embedding mold. Once the paraffin at the bottom of the mold has slightly solidified, carefully remove the tissue from the embedding frame with forceps and place it into the embedding mold according to the embedding surface requirements. Gently press the tissue with the bottom of the forceps to make it completely flat within the mold. Cover the embedding frame with the tissue and gently move it to a -20°C freezing stage to cool. After the paraffin has solidified, remove the paraffin block from the mold and trim it. The prepared paraffin blocks were placed in a paraffin microtome for sectioning, with a section thickness of 4 μm. The sections were then floated on a 42°C warm water spreader to flatten the tissue. The tissue was then vertically lifted using a glass slide, slightly drained, and baked in a 60°C slide oven for 30 min–1 h. After baking, the slides were stored at room temperature. HE staining was then performed according to the following steps. The procedure is shown in Table 1-1 below.

[0087] Table 1-1 HE staining procedure

[0088]

[0089] 2.5 Transcriptome Experimental Procedure

[0090] 2.5.1 Sample Testing

[0091] Agilent 2100 bioanalyzer (Agilent Technologies, CA, USA): Precisely detects RNA integrity and total amount.

[0092] 2.5.2 Library Construction and Quality Inspection

[0093] NEB Standard Database Creation

[0094] mRNA was enriched from total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA was 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 was ready. The library was quantified using a Qubit analyzer and real-time quantitative PCR, and fragment size distribution was detected using a bioanalyzer.

[0095] Chain-specific library construction

[0096] mRNA was enriched from total RNA using Oligo dT magnetic beads. After fragmentation, first-strand cDNA was synthesized using random hexamer primers. Second-strand cDNA was then synthesized using dUTP instead of dTTP. Following end repair, A-tailing, adapter ligation, fragment selection, USER digestion, amplification, and purification, the oriented library was ready. The library was quantified using a Qubit analyzer and real-time quantitative PCR, and fragment size distribution was detected using a bioanalyzer.

[0097] 2.5.3 Sequencing

[0098] After the libraries pass the library inspection, different libraries are pooled according to the 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. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers are added to the sequencing flow cell for amplification. When each sequencing cluster extends its complementary strand, each added fluorescently labeled dNTP releases corresponding fluorescence. The sequencer captures the fluorescence signal and converts it into sequencing peaks using computer software, thereby obtaining the sequence information of the fragment to be sequenced.

[0099] 2.6 Transcriptome Analysis

[0100] 2.6.1 Data Quality Control

[0101] The raw data (raw reads) are first processed using the FASTP software. In this step, clean reads are obtained by removing reads containing headers, reads containing poly-N pairs, and low-quality reads. Q20, Q30, and GC content are then calculated on the clean data. All downstream analyses are based on this high-quality clean data.

[0102] 2.6.2 Sequence alignment to the reference genome

[0103] The reference genome and gene model annotation files were downloaded directly from the genome website. An index of the reference genome was built using HISAT2 v2.0.5, and paired-end cleanreads were aligned with 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.

[0104] 2.6.3 Quantitative analysis of gene expression levels

[0105] featureCounts (1.5.0-p3) is used to calculate the reads mapped to each gene. Then, the FPKM for each gene is calculated based on its length, and the reads mapped to that gene are also calculated. FPKM refers to the expected number of fragments per thousand base pairs of sequenced transcript sequences per million base pairs. It considers both sequencing depth and gene length on the read count and is currently the most commonly used method for estimating gene expression levels.

[0106] 2.6.4 Differential Expression Analysis

[0107] Differential expression analysis between the two comparison combinations was performed using DESeq2 software (1.20.0). DESeq2 provides statistical procedures for identifying differential expression in digital gene expression data using a model based on a negative binomial distribution. The Benjamini and Hochberg method was used to adjust the resulting p-values ​​to control for false discovery rates. Genes with adjusted p-values ​​<= 0.05 were identified by DESeq2 as differentially expressed.

[0108] 2.6.5 Differential gene enrichment analysis

[0109] GO enrichment analysis of differentially expressed genes was performed using clusterProfiler (3.8.1) software, with gene length bias corrected. GO terms with corrected p-values ​​less than 0.05 were considered significantly enriched by differentially expressed genes. KEGG is a database resource used to understand the higher functions and utilities 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 in KEGG pathways.

[0110] 3 Experimental Results

[0111] 3.1 Number of mice that survived and died

[0112] The number of mice that survived and died after ten weeks of modeling and drug administration is shown in Table 1-2:

[0113] Table 1-2 Mouse survival status

[0114]

[0115] 3.2 Mouse survival curve

[0116] The survival status of mice was recorded starting with intraperitoneal injection of AOM solution. No deaths occurred in the control group; in the model group, 1 animal died on May 17, 1 on May 20, 1 on June 3, 1 on June 6, 1 on June 10, and 1 on July 1, for a total of 6 deaths. All deaths were accompanied by diarrhea, with the animal that died on June 10 also exhibiting hair loss and signs of being eaten; in the 5-Fu positive group, 2 animals died on May 20, both with diarrhea; in the low-dose FPS group, 1 animal died on May 20, 1 on May 23, and 1 on May 27, for a total of 3 deaths, all with diarrhea; in the medium-dose FPS group, 1 animal died on May 16, 1 on May 20, and 1 on June 24, for a total of 3 deaths, with symptoms of diarrhea, hair loss, and signs of being eaten; in the high-dose FPS group, 1 animal died on May 20 and 1 on May 27, for a total of 2 deaths, with symptoms of diarrhea, hair loss, and signs of being eaten. Survival curves were plotted using 7-day (W) intervals, as shown below. Figure 2-2 As shown: Comparing the survival curves and the survival rates in Table 1-2, it can be seen that the FPS-treated group increases the survival rate of colorectal cancer mice with increasing dosage.

[0117] 3.3 Status of surviving mice

[0118] (1) The mice in the blank group showed no abnormal activity during the modeling and drug administration period. After the modeling and drug administration, anatomical observation revealed no abnormal pathological changes in any tissues, and no pleural or peritoneal effusion;

[0119] (2) During the modeling and drug administration period, the mice in the model group gained weight slowly, and their weight decreased in the three cycles. The activity level of the mice also decreased. Starting from the second cycle, the mice began to have loose stools and bloody stools. The loose stools and bloody stools worsened in the third cycle. After the modeling was completed, the dissection revealed that the length of the colon and rectum of the mice in the model group was reduced, a large amount of tumor tissue was present, the spleen was enlarged, and there were no obvious changes in other tissues and organs. There was no pleural or peritoneal effusion.

[0120] (3) The weight gain of mice in the 5-Fu positive group was slow before drug administration during the modeling period. After drug administration at the beginning of the second cycle, the weight gain increased and the mice were not abnormal in their activity. From the beginning of the second cycle, the mice began to show mild loose stools and bloody stools. After the modeling was completed, the dissection revealed that the colorectal length of the mice in the 5-Fu positive group was reduced, there was a small amount of tumor tissue, the spleen was enlarged, and there were no obvious changes in other tissues and organs. There was no pleural or peritoneal effusion.

[0121] (4) During the modeling period, FPS mice were induced by 2% DSS solution to decrease their body weight and activity level. After the induction was stopped, their body weight slowly increased within two weeks, and their activity level returned to normal. From the second cycle onwards, the mice began to show symptoms such as loose stools and bloody stools, which worsened in the third cycle. After the modeling was completed, the autopsy revealed that the colorectal length of the FPS mice was shortened, there was a small amount of tumor tissue, the spleen was enlarged, and there were no obvious changes in other tissues and organs. There was no pleural or peritoneal effusion.

[0122] 3.4 Effect of FPS on body weight in mice with colorectal cancer

[0123] A cycle lasting three weeks was conducted, with a total of three cycles. During the animal experiment, the mice's weight was measured weekly, and the weight data over the 10 weeks was statistically analyzed using GraphPad software to obtain the average weight change data, which was then plotted as a weight change graph. Figure 2-3 As shown: In the first week, there was no significant difference in weight among the groups; in the first cycle (weeks 2-4), in the second week, there was no significant difference in weight among the groups; in the third week, compared with the model group, the weight changes in the blank group and the 5-Fu positive group were significantly different, while the model group was not significantly different from the FPS-treated group; in the fourth week, compared with the model group, the weight changes in the blank group, the FPS-treated group, and the three groups were significantly different, while the model group was not significantly different from the 5-Fu positive group; in the second cycle (weeks 5-7), in the fifth week, the weight changes in the blank group were significantly different from the model group, while the model group was not significantly different from the three FPS-treated groups and the positive group; in the sixth week, the weight changes in the blank group were significantly different from the model group, the model group was significantly different from the high-dose FPS group, and not significantly different from the low- and medium-dose FPS groups; the model group was significantly different from the 5-Fu positive group; in the seventh week, the weight changes in the model group were significantly different from all other groups, and the weight changes in the FPS-treated group increased with increasing dose. Mice in the treatment groups gained weight. In the third cycle (weeks 8-10), at week 8, the weight changes in the control group and the model group were significantly different. The weight changes in the model group and the low-dose FPS group were significantly different, but there was no significant difference compared with the medium- and high-dose FPS groups. The weight changes in the model group and the 5-Fu positive group were significantly different. At week 9, the weight changes in the control group and the model group were significantly different. The weight changes in the model group and the medium- and high-dose FPS groups were significantly different, but there was no significant difference compared with the low-dose FPS group. The weight changes in the model group and the 5-Fu positive group were significantly different. At week 10, the weight changes in the control group and the model group were significantly different. The weight changes in the model group and the medium- and high-dose FPS groups were significantly different, but there was no significant difference compared with the low-dose FPS group. The weight changes in the model group and the 5-Fu positive group were significantly different. Overall, as the FPS concentration increased, the weight changes between the FPS-treated mice and the model group gradually increased, showing a significant difference.

[0124] 3.5 Fecal index and bloody stool index

[0125] During the animal experiments, fecal index and fecal blood index were measured once a week. The average data of the changes in fecal index and fecal blood index were obtained by statistical analysis using Graphpad software and plotted as a graph of the changes in fecal index and fecal blood index.

[0126] like Figure 2-4 The fecal indices shown indicate the following: In the first week, all groups had normal fecal conditions. In the first cycle (2-4 weeks), in the second week, there was no significant difference in fecal indices between the groups and the model group. In the third week, the control group had normal fecal conditions but showed a significant difference from the model group. The low and medium dose FPS groups showed significant differences from the model group, while the high dose group showed no significant difference, and the fecal indices in the high dose group were more severely unformed than in the model group. The 5-Fu positive group showed no significant difference compared to the model group. In the second cycle (5-7 weeks), in the fifth week, there were no significant differences between the groups. In the sixth week, the control group had normal fecal conditions but showed a significant difference from the model group. The medium dose FPS group showed a significant difference from the model group, while the low and high dose groups showed no significant difference, and the low dose group had more severely unformed fecal conditions than in the model group. In the seventh week, except for the low dose FPS group which showed no significant difference compared to the model group, the other groups... Compared with the model group, all groups showed significant differences, with the medium-dose FPS group showing the best results and stool condition close to that of the control group. In the third cycle (8-10 weeks), in week eight, all groups showed significant differences compared with the model group, with the low- and medium-dose FPS groups showing the best stool condition. In week nine, the control group had normal stool condition, showing a significant difference from the model group. The medium- and high-dose FPS groups showed significant differences from the model group, while the low-dose group showed no significant difference, and the stool condition was more unformed than in the model group. The medium-dose group had slightly better stool condition, approaching that of the positive group. The 5-FU positive group showed a significant difference compared with the model group. In week ten, the control group and the 5-FU positive group had normal stool condition, showing a significant difference from the model group. The three FPS groups showed no significant differences from the model group, and the medium- and high-dose groups had more unformed stool condition than the model group, while the low-dose group was slightly better. Figure 2-5The bloody stool index showed that: in the first week, all groups had normal bloody stool; in the first cycle (2-4 weeks), the model group had severe bloody stool in the third week, showing significant differences compared to other groups, with the medium-dose FPS group showing the least bloody stool; in the second and fourth weeks, there were no significant differences between the model group and other groups, and no bloody stool; in the second cycle (5-7 weeks), the model group had no significant differences compared to other groups in the fifth week, and no bloody stool; in the sixth week, the blank group had no bloody stool and showed significant differences compared to the model group; the other four groups had no significant differences compared to the model group, with some mice in the low-dose FPS group experiencing rectal prolapse; in the seventh week, the blank group had normal fecal condition, showing significant differences compared to the model group; the model group had severe bloody stool; the low- and medium-dose FPS groups showed significant differences compared to the model group, with the low-dose FPS group showing improved bloody stool and no bloody stool; the model group had no significant differences compared to the 5-Fu positive group and the high-dose FPS group; in the third cycle ( (8-10 weeks) In week 8, except for the high-dose FPS group, all groups showed significant differences compared to the model group. The rectal bleeding status in the low- and medium-dose FPS groups was consistent with that in the 5-FU positive group, with no rectal bleeding. In week 9, there were significant differences between the blank group and the model group, significant differences between the low-dose FPS group and the model group, no significant differences between the medium- and high-dose FPS groups, and significant differences between the 5-FU positive group and the model group. Among them, the low-dose group had the mildest rectal bleeding after FPS administration. In week 10, there were significant differences between the blank group and the model group; there were significant differences between the 5-FU positive group and the model group. Compared with the model group, the low-dose FPS group showed a significant difference with the mildest rectal bleeding. The medium- and high-dose FPS groups showed some improvement in rectal bleeding compared to the model group, but there was no significant difference. Overall, FPS administration exacerbated loose stools caused by intestinal stimulation in mice with increasing dosage; however, it gradually improved rectal bleeding caused by colorectal cancer with increasing dosage.

[0127] 3.6 Effect of FPS on colorectal tissue length

[0128] After the C57 mouse model was established and drug administration was completed, the mice were dissected, and samples were taken from each group to measure and photograph the rectal condition for comparison. Figure 2-6 As shown: In the control group, the colorectal length was normal, and the intestinal wall was transparent and gelatinous; in the model group, the tumor volume was large and the number of tumors was high, and the intestinal wall transparency was decreased; in the 5-Fu positive group, the tumor volume was small and the number was low, and the intestinal wall transparency was close to that of the control group; in the FPS-treated group, the tumor volume was small and the number was low, and the intestinal wall transparency was higher than that of the model group; the medium-dose group had the lowest tumor volume and number among the treated groups, and the intestinal wall transparency was close to that of the positive group. The statistical results of the colorectal length of C57 mice are as follows: Figure 2-7 As shown: the blank group had the longest colorectal length, while the model group had a significantly shorter colorectal length compared to the blank group; the 5-Fu positive group had an increased colorectal length compared to the model group; and FPS had little effect on the colorectal length of mice with colorectal cancer compared to the model group.

[0129] 3.7 Effects of FPS on colorectal histopathology in mice with colorectal cancer

[0130] HE staining of mouse colorectal tissue yielded the following results: Figure 2-8 As shown, compared with the control group, the model group showed a large number of tumor cells under the microscope, with high basophilicity, high nucleus-to-cytoplasm ratio, and varied morphology. Many tumor cells formed glandular sieve-like structures, and a few necrotic exudates were visible within the glandular lumens. Numerous goblet cell proliferations were observed, with scattered lymphocyte infiltration around the cells. After FPS administration, these phenomena improved in mice with increasing concentration; inflammatory infiltration decreased, and the number of tumor cells decreased, with the lowest number of tumors observed in the colorectal region at the medium dose.

[0131] 3.8 Effects of FPS on organ indices in mice with colorectal cancer

[0132] Spleen index of C57 mice as follows Figure 2-9 As shown: Compared with the control group, the spleen index of colorectal cancer mice was significantly increased in the model group; after administration, the spleen index of the 5-Fu positive group was reduced compared with the model group; the spleen index of all three groups of FPS-treated mice 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. The thymus index of C57 mice is shown below. Figure 2-10 As shown: Compared with the control group, the model group significantly increased the thymus index in colorectal cancer mice; after drug administration, the 5-Fu positive group decreased the thymus index compared with the model group; FPS had no effect on the thymus index. Liver indices of C57 mice are shown below. 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 liver index of the 5-Fu positive group was reduced; the liver index of mice in the medium-dose and high-dose FPS groups was significantly reduced, and the liver index of the medium-dose group was close to that of the blank group.

[0133] 3.9 Transcriptome Analysis Results

[0134] 3.9.1 Sample Correlation Analysis

[0135] Biological replication is generally essential for any biological experiment, and most mainstream journals now require it. Biological replication serves two main purposes: first, to demonstrate that the experimental procedures involved are not accidental but reproducible; and second, to ensure more reliable results in subsequent differential gene analysis. The correlation of gene expression levels between samples is an important indicator of experimental reliability and the appropriateness of sample selection. The closer the correlation coefficient is to 1, the higher the similarity of expression patterns between samples; generally, an R² value greater than 0.8 is required for biological replication. Our results show that... Figure 2-12 As shown in the figure, the horizontal and vertical axes represent the squares of the correlation coefficients of each sample. The correlation coefficients of each group are all greater than 0.8, indicating good biological repeatability.

[0136] 3.9.2 Principal Component Analysis

[0137] Principal component analysis (PCA) is also commonly used to assess between-group differences and within-group sample replication. PCA employs linear algebraic calculations to reduce the dimensionality and extract principal components from 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 a PCA plot, between-group samples should be dispersed, and within-group samples should cluster together. For example... Figure 2-13 As shown, the horizontal axis represents the first principal component and the vertical axis represents the second principal component. The PCA analysis results show that the model group and the blank group are clearly clustered, and after FPS administration, the model group is closer to the blank group.

[0138] 3.9.3 Differential Gene Screening

[0139] After quantifying gene expression, statistical analysis of the expression data is required to screen for genes with significantly different expression levels under different states. DESeq was used to standardize the original read count, and differentially expressed genes were screened using a threshold of |log2(FoldChange)|>=0 and pvalue<=0.05. The results are as follows: Figure 2-14A and Figure 2-14B As shown, compared with the control group, 2849 genes were upregulated and 2222 genes were downregulated in the model group; compared with the model group, 795 genes were upregulated and 746 genes were downregulated in the FPS group. The intersection of the genes upregulated in the model group and those downregulated after drug administration with the genes upregulated after drug administration in the model group was used to obtain the key differentially expressed genes for drug treatment. The results are as follows: Figure 2-15 As shown in the Venn diagram, there is an intersection of 361 genes upregulated by ModelvsControl and genes downregulated by FPSvsModel, and an intersection of 493 genes downregulated by ModelvsControl and genes upregulated by FPSvsModel. Therefore, after taking the union, there are a total of 854 differentially expressed genes for FPS treatment of colorectal cancer.

[0140] Figure 2-15 The Venn of differentially expressed genes (A represents UP.ModelvsControl; B represents DOWN.ModelvsControl; C represents UP.THCvsModel; D represents DOWN.THCvsModel).

[0141] 3.9.4 Differential Gene Clustering

[0142] Cluster analysis was performed on the differentially expressed gene set to group genes with similar expression patterns together. We used mainstream hierarchical clustering to perform cluster analysis on the FPKM values ​​of the genes, and normalized the rows (Z-score). The results of the differentially expressed gene heatmap are shown below. Figure 2-16 As shown, the gene expression patterns of the Model group are significantly different from those of the Control group. After FPS administration, the expression patterns are closer to those of the Control group.

[0143] 3.9.5 GO Functional Enrichment Analysis

[0144] Gene Ontology (GO) is a comprehensive database describing gene function, 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 are selected and a scatter plot is generated for display, as shown below. Figure 2-17 As shown in the figure, the horizontal axis represents the ratio of the number of differentially expressed genes annotated on GOTerm to the total number of differentially expressed genes, and the vertical axis represents GOTerm. The size of the point represents the number of genes annotated on GOTerm, and the color from red to blue represents the significance of enrichment.

[0145] 3.9.6 KEGG Functional Enrichment Analysis

[0146] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a comprehensive database integrating genomic, chemical, and systemic functional information. KEGG pathway enrichment uses a padj value less than 0.05 as the threshold for significant enrichment. The enrichment results are shown below. Figure 2-18As shown, the PI3K-Akt signaling pathway, ECM-receptor interaction, Cytokine-cytokine receptor interaction, and TGF-beta signaling pathway 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 signaling pathways in the immune system. Cytokines are small protein molecules 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 not only participate in the initiation and regulation of immune responses but also involve inflammatory responses, self-tolerance, and the formation of immune memory. Therefore, the therapeutic effect of FPS on AOM / DSS-induced colorectal cancer mice and its role in immune regulation are relevant.

[0147] 4. Conclusion

[0148] During the experiment, some mice died. The control group remained normal. In the model group, mice showed reduced water and food intake, weight loss, and decreased activity levels during each round of 2% DSS solution induction, gradually recovering after induction was stopped. Except for fecal indices, all indicators in the medium-dose administration group showed significant trends compared to the model group, with a significant difference in liver indices; indicating that FPS gavage administration may have a protective effect on the liver and spleen of mice with colorectal cancer; it may have a certain controlling and slowing effect on the development of colorectal cancer, but its protective effect on the length of colorectal cancer may be insignificant; simultaneously, the FPS administration group showed good control of fecal hemorrhage in mice with colorectal cancer, but the loose stool condition gradually worsened with increasing dosage. Transcriptome results showed 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 FPS on colorectal cancer mice were as follows: medium dose > high dose > low dose; FPS had a good inhibitory effect on the development of colorectal cancer in mice; at the same time, FPS had an immunomodulatory effect on colorectal cancer mice.

Claims

1. The application of Aconitum carmichaelii polysaccharide in the preparation of drugs for treating colorectal cancer, characterized in that, The drug has the effect of treating at least one of colon cancer and rectal cancer; The drug has a liver-protective effect in the treatment of colon and rectal cancer; The drug has a protective effect on the spleen in the treatment of colon and rectal cancer; The drug has the effect of controlling rectal bleeding in the treatment of colon and rectal cancer.

2. The application according to claim 1, characterized in that, The preparation of the aconite polysaccharide includes one of the following methods: enzyme-assisted extraction, water extraction and alcohol precipitation, ultrasonic extraction, water immersion extraction, and microwave extraction.

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