CN118557561A - 9-Hexadecenoic acid in preparation of product for preventing and treating colorectal tumor and application of product as synergist of chemotherapeutic medicine - Google Patents

Abstract

The invention discloses a 9-hexadecenoic acid in the preparation of a product for preventing and treating colorectal tumor and the application of the 9-hexadecenoic acid as a synergist of a chemotherapeutic drug. The invention provides a novel application of 9-hexadecenoic acid in controlling colorectal cancer precancerous lesions, reducing intestinal inflammation, and inhibiting intestinal adenoma and the progression and deterioration of intestinal cancer. The 9-hexadecenoic acid not only has the effect of preventing and treating colorectal tumor, but also has obvious positive regulation effect on intestinal microecological disorder caused by colorectal tumor. In addition, the invention also provides a combined scheme of 9-hexadecenoic acid and a chemotherapeutic drug, the 9-hexadecenoic acid obviously improves the chemotherapeutic effect of colorectal tumor, simultaneously reduces the toxic and side effects of the chemotherapeutic drug, and provides a new technical choice for solving the difficult problems of intolerance and impaired curative effect of clinical chemotherapy.

Description

Application of 9-hexadecenoic acid in the preparation of products for the prevention and treatment of colorectal tumors and as a synergist of chemotherapy drugs

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to the use of 9-hexadecenoic acid in the preparation of products for preventing and treating colorectal tumors and as a synergist of chemotherapy drugs.

Background Art

Colorectal cancer (CRC) is the third most common malignant tumor and the second most deadly in the world. The occurrence and development of colorectal cancer is considered to be a complex mucosal epithelial pathology and microbial-host interaction process involving multiple factors, multiple stages and multiple genes. The heritability of colorectal cancer is 12-35%. Environmental factors play a greater role in inducing sporadic colorectal cancer, such as long-term chronic intestinal inflammation, disturbed intestinal flora and its metabolism, and intestinal mucosal barrier damage. Among them, the damaged intestinal epithelial cells, disturbed intestinal flora and its metabolism, and uncontrollable chronic inflammation in the occurrence and development of colorectal cancer promote each other and form a complex pathological feedback system. They are the core pathological causes that determine the dysfunction of the intestinal mucosal barrier and thus affect the development and deterioration of colorectal cancer and the impairment of chemotherapy efficacy.

Although adjuvant treatments such as targeted therapy and immunotherapy have made great progress in recent years, there are still the dilemmas of small audiences, low response rates, and limited improvement in survival. At present, the treatment model for colorectal cancer is still a comprehensive sequential treatment based on surgical resection and chemoradiotherapy. The tumor at the primary site is surgically removed, and adjuvant treatment is performed with chemotherapy, radiotherapy, immunotherapy, and other means after surgery. Effective chemotherapy can not only kill residual cancer cells, but also prevent further spread and metastasis of cancer cells. Chemotherapeutic drugs (mainly inhibiting the replication and proliferation of tumor cells), such as capecitabine and oxaliplatin, which are commonly used in clinical practice, play a key role in tumor treatment. However, treatment methods such as chemoradiotherapy are not targeted, and while killing tumor cells, they also damage normal cells. In addition, frequent drug resistance and drug toxicity and side effects, such as gastrointestinal reactions, seriously affect the quality of life of patients. Therefore, it is urgent to find new anti-colorectal cancer methods such as targeted therapy and improve the current clinical treatment effect, and to develop safer, more effective, economical, less toxic and side effects, and more convenient drug administration methods.

Summary of the invention

In order to solve the problems existing in the prior art of severe intestinal mucosal barrier damage, intestinal microecological disorder, uncontrollable chronic inflammation, poor clinical efficacy and frequent side effects in colorectal tumors, the present invention provides the use of 9-hexadecenoic acid in the preparation of products for the prevention and treatment of colorectal tumors and as a synergist of chemotherapeutic drugs.

The first object of the present invention is to provide the use of 9-hexadecenoic acid in preparing products for preventing and treating colorectal tumors.

The second object of the present invention is to provide the use of 9-hexadecenoic acid in the preparation of a product for repairing the mucosal barrier of colorectal tumor patients and/or alleviating intestinal mucosal damage in colorectal tumor patients.

The third object of the present invention is to provide the use of 9-hexadecenoic acid in the preparation of a product for preventing and/or treating intestinal flora imbalance in patients with colorectal tumors.

The fourth object of the present invention is to provide the use of 9-hexadecenoic acid in the preparation of a product for improving the chemotherapy effect of colorectal tumors.

The fifth object of the present invention is to provide the use of 9-hexadecenoic acid in the preparation of a product for alleviating the side effects of chemotherapy for colorectal tumors.

The sixth object of the present invention is to provide the use of 9-hexadecenoic acid as a synergist of chemotherapeutic drugs in the preparation of products for preventing and treating colorectal tumors.

The seventh object of the present invention is to provide the use of 9-hexadecenoic acid in combination with chemotherapeutic drugs in the preparation of products for preventing and treating colorectal tumors.

The eighth object of the present invention is to provide a pharmaceutical composition.

In order to achieve the above object, the present invention is implemented by the following scheme:

The present invention combines metabolomics, transcriptomics, animal models, molecular detection and other technical means to clarify that 9-hexadecenoic acid effectively controls colorectal precancerous lesions, reduces intestinal inflammation, inhibits the progression and deterioration of intestinal adenomas and intestinal cancer in colorectal precancerous lesions and in situ colorectal cancer models, and has a therapeutic and preventive effect on colorectal tumors. It also reveals that 9-hexadecenoic acid can significantly reduce the secretion of inflammatory factors IL-6 and TNF-α and thus inhibit the proliferation of colorectal cancer cells, and can also inhibit the phosphorylation level of inflammatory pathways, control the progression and deterioration of colorectal tumors, and the occurrence and development of colorectal cancer-related diseases.

The present invention requests protection for the following contents:

Application of 9-hexadecenoic acid in the preparation of products for preventing and treating colorectal tumors.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the prevention and treatment of colorectal tumors includes inhibiting the growth of colorectal tumors, prolonging the overall survival of colorectal tumor patients, prolonging the progression-free survival, improving the quality of life, and reducing any one or more of weight loss, organomegaly, diarrhea and perianal bleeding associated with colorectal tumors.

More preferably, the inhibition of colorectal tumor growth includes one or more of inhibiting the volume growth of colorectal tumors, reducing the number of colorectal tumors, and inhibiting the expression of inflammatory factors closely related to colorectal tumors or the activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

Application of 9-hexadecenoic acid in the preparation of products for repairing the mucosal barrier of patients with colorectal tumors and/or alleviating intestinal mucosal damage in patients with colorectal tumors.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the reduction of intestinal mucosal damage includes inhibiting one or more of the expression of inflammatory factors or activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

More preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

In the host, the colorectum is a place where intestinal bacteria live and move in large numbers. The complex molecular and chemical regulatory network between the intestinal trophic group and the flora plays a key role in regulating the homeostasis of the intestinal mucosal barrier and tumors. As an important driving force for nourishing the mucosal epithelium and reshaping the microbiome and its metabolic function, the intestinal trophic group interacts with the microbiome to change the adaptability of specific bacterial genera, thereby affecting the host mucosal barrier function, mucosal immune response and intestinal health. Domestic and foreign studies have shown that in the process of normal colon epithelium developing into cancerous tissue, the intestinal mucosal barrier is damaged and the intestinal permeability increases, resulting in a large number of pathogenic bacteria and harmful substances such as LPS influx into the mucosal layer to induce abnormal activation of the adaptive immune system, causing the physical barrier of the colon epithelium to collapse; the flora characteristics are mainly manifested in the reduction of beneficial bacteria with anti-inflammatory properties, especially Akkermansia, and the increase in the abundance of pro-inflammatory harmful bacteria. The present invention combines 16S sequencing technology to clarify the positive regulatory effect of 9-hexaenoic acid microorganisms on intestinal microecology.

Therefore, the present invention also requests protection for the following contents:

Application of 9-hexadecenoic acid in the preparation of products for preventing and/or treating intestinal flora imbalance in patients with colorectal tumors.

Preferably, the prevention and/or treatment of intestinal flora imbalance includes any one or more of improving the composition structure of intestinal flora, promoting the growth of beneficial bacteria, alleviating flora disorder and restoring flora homeostasis.

More preferably, the beneficial bacteria include Akkermansia muciniphila.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the prevention and treatment of colorectal tumors includes inhibiting the growth of colorectal tumors, prolonging the overall survival of colorectal tumor patients, prolonging the progression-free survival, improving the quality of life, and reducing any one or more of weight loss, organomegaly, diarrhea and perianal bleeding associated with colorectal tumors.

More preferably, the inhibition of colorectal tumor growth includes one or more of inhibiting the volume growth of colorectal tumors, reducing the number of colorectal tumors, and inhibiting the expression of inflammatory factors closely related to colorectal tumors or the activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

The present invention also clarifies in an in situ colorectal cancer model that 9-hexadecenoic acid improves the efficacy of colorectal cancer chemotherapy drugs and reduces the gastrointestinal toxic side effects of chemotherapy drugs.

Therefore, the present invention also requests protection for the following contents:

Application of 9-hexadecenoic acid in the preparation of products for improving the chemotherapy effect of colorectal tumors.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, improving the chemotherapy effect of colorectal tumors includes enhancing the inhibitory effect of chemotherapy drugs on the growth of colorectal tumors.

More preferably, the inhibitory effect on colorectal tumor growth includes one or more of inhibiting the volume growth of colorectal tumors, reducing the number of colorectal tumors, and inhibiting the expression of inflammatory factors closely related to colorectal tumors or the activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

More preferably, the chemotherapy drug is a 5-fluorouracil chemotherapy drug or a cisplatin chemotherapy drug.

More preferably, the 5-fluorouracil chemotherapy drug is capecitabine.

More preferably, the cisplatin-based chemotherapy drug is oxaliplatin.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

Application of 9-hexadecenoic acid in the preparation of products for alleviating the side effects of chemotherapy for colorectal tumors.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the relief of the side effects of colorectal tumor chemotherapy includes any one or more of repairing the mucosal barrier, reducing intestinal mucosal damage, and preventing and/or treating intestinal flora imbalance.

More preferably, the reducing of intestinal mucosal damage comprises inhibiting one or more of the expression of inflammatory factors or activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

More preferably, the prevention and/or treatment of intestinal flora imbalance includes any one or more of improving the composition structure of intestinal flora, promoting the growth of beneficial bacteria, alleviating flora disorder and restoring flora homeostasis.

Further preferably, the beneficial bacteria include Akkermansia muciniphila.

Preferably, the chemotherapy drug used for colorectal tumor chemotherapy is a 5-fluorouracil chemotherapy drug or a cisplatin chemotherapy drug.

More preferably, the 5-fluorouracil chemotherapy drug is capecitabine.

More preferably, the cisplatin-based chemotherapy drug is oxaliplatin.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

Application of 9-hexadecenoic acid as a synergist of chemotherapy drugs in the preparation of products for preventing and treating colorectal tumors.

Preferably, the chemotherapy drug is a 5-fluorouracil chemotherapy drug or a cisplatin chemotherapy drug.

More preferably, the 5-fluorouracil chemotherapy drug is capecitabine.

More preferably, the cisplatin-based chemotherapy drug is oxaliplatin.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the prevention and treatment of colorectal tumors includes inhibiting the growth of colorectal tumors, prolonging the overall survival of colorectal tumor patients, prolonging the progression-free survival, improving the quality of life, and reducing any one or more of weight loss, organomegaly, diarrhea and perianal bleeding associated with colorectal tumors.

More preferably, the inhibition of colorectal tumor growth includes one or more of inhibiting the volume growth of colorectal tumors, reducing the number of colorectal tumors, and inhibiting the expression of inflammatory factors closely related to colorectal tumors or the activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

Application of 9-hexadecenoic acid in combination with chemotherapy drugs in the preparation of products for preventing and treating colorectal tumors.

Preferably, the chemotherapy drug is a 5-fluorouracil chemotherapy drug or a cisplatin chemotherapy drug.

More preferably, the 5-fluorouracil chemotherapy drug is capecitabine.

More preferably, the cisplatin-based chemotherapy drug is oxaliplatin.

The colorectal tumor includes but is not limited to one or more of familial multiple intestinal polyposis, colorectal adenoma and colorectal cancer; the colorectal adenoma is one or more of tubular adenoma, villous adenoma and tubulovillous adenoma; the colorectal cancer is one or more of colorectal adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma and undifferentiated carcinoma.

Preferably, the colorectal tumor includes benign colorectal tumor and/or malignant colorectal tumor.

More preferably, the benign colorectal tumor includes one or more of colon adenoma, rectal adenoma and colorectal adenoma.

More preferably, the malignant colorectal tumors include but are not limited to colon cancer, rectal cancer, colorectal cancer, inflammation-related colorectal cancer and colorectal cancer-related precancerous lesions.

Preferably, the prevention and treatment of colorectal tumors includes inhibiting the growth of colorectal tumors, prolonging the overall survival of colorectal tumor patients, prolonging the progression-free survival, improving the quality of life, and reducing any one or more of weight loss, organomegaly, diarrhea and perianal bleeding associated with colorectal tumors.

More preferably, the inhibition of colorectal tumor growth includes one or more of inhibiting the volume growth of colorectal tumors, reducing the number of colorectal tumors, and inhibiting the expression of inflammatory factors closely related to colorectal tumors or the activation of inflammatory pathways.

More preferably, the inflammatory factors include TNF-α and IL-6.

Further preferably, the inflammatory pathway includes NF-κB pathway, IL-17 pathway and TNF-α pathway.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

A pharmaceutical composition comprises an active ingredient 1 and an active ingredient 2; the active ingredient 1 is 9-hexadecenoic acid; and the active ingredient 2 is a chemotherapy drug for treating colorectal tumors.

Preferably, the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolite forms thereof.

Preferably, the pharmaceutical composition is in the form of an injection, tablet, lyophilized powder injection, capsule or patch.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a new use of 9-hexadecenoic acid in controlling colorectal precancerous lesions, reducing intestinal inflammation, inhibiting intestinal adenomas and the progression and deterioration of intestinal cancer. 9-hexadecenoic acid not only has the effect of treating and preventing colorectal tumors, but also has a significant positive regulatory effect on the intestinal microecological imbalance caused by colorectal tumors. In addition, the present invention also provides a combination scheme of 9-hexadecenoic acid and chemotherapeutic drugs. 9-hexadecenoic acid significantly improves the chemotherapeutic efficacy of colorectal tumors, while reducing the toxic and side effects of chemotherapeutic drugs, providing a new technical option for solving the problem of clinical chemotherapy intolerance and impaired efficacy.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of metabolomics identification of abnormal lipid metabolism in colorectal tumor tissues; A is a volcano plot of metabolites detected in cancer tissues (T) and normal tissues far from cancer (N), up in tumor indicates metabolites significantly increased in cancer tissues, down in tumor indicates metabolites significantly decreased in cancer tissues, and no sig indicates metabolites with no differential expression; B is KEGG pathway analysis showing that differential metabolites are mainly enriched in lipid metabolism pathways; C is the relative abundance of 9-hexadecenoic acid in colorectal cancer patients with low TNM stage (Low stage) and high TNM stage (High stage); *p<0.05.

Figure 2 shows the results of 9-hexadecenoic acid controlling the occurrence and development of colorectal adenomas in Apc ^{min/+ mice, which are spontaneous colorectal precancerous lesions; A is a schematic diagram of the construction of the spontaneous colorectal precancerous lesion model of Apc min/+} mice and the process of grouping treatment; B is a colon endoscopy image of mice in the model group (Apc) and the 9-hexadecenoic acid treatment group (Apc+9-HA); C is a colon anatomical diagram of the two groups of Apc ^min/+ mice, with arrows indicating colon adenomas; D is a statistical diagram of the number of tumors in the two groups of Apc ^min/+ mice; E is a statistical diagram of the tumor burden scores in the two groups of Apc ^min/+ mice; *p<0.05, **p<0.01.

Figure 3 shows that 9-hexadecenoic acid inhibits the proliferation of colorectal adenomas and repairs mucosal damage in Apc ^min/+ mice. A is hematoxylin & eosin staining (H&E) showing adenoma pathology and Alcian blue-periodic acid-Schiff staining (AB-PAS) showing colonic goblet cells; B is the statistical results of pathological analysis, Normal represents normal intestinal tissue, LGD represents low-grade atypical hyperplasia, HGD represents high-grade atypical hyperplasia, and Carcinoma represents lesion cancer tissue; C is Ki-67 immunohistochemical staining to show tumor proliferation and malignancy, and E-cadherin immunohistochemical staining to show colon mucosal barrier integrity; D is the quantitative analysis result of Ki-67 index; E is the quantitative analysis result of E-cadherin index; F is transmission electron microscopy showing intestinal epithelial cell heterogeneity, intercellular tight junction structure and intercellular gap, arrows represent cell tight junction structure, and circles represent tight junction structure destruction; **p<0.01.

Figure 4 shows that 9-hexadecenoic acid significantly inhibits the number and development of tumors in colorectal cancer model mice; A is a schematic diagram of the process of constructing a colorectal cancer model using azomethane (AOM)/dextran sulfate sodium (DSS) and the grouping treatment process; B is the perianal bleeding of mice in the colorectal cancer group (AOM) and the 9-hexadecenoic acid treatment group (AOM+9-HA); C is a colon endoscopy image of the two groups of colorectal cancer model mice; D is a colon anatomical diagram of the two groups of colorectal cancer model mice, with arrows indicating colon tumors; E is a statistical graph of the number of tumors in the two groups of colorectal cancer model mice; F is a statistical graph of the tumor burden scores in the two groups of colorectal cancer model mice; **p<0.01; ***p<0.001.

Figure 5 shows that 9-hexadecenoic acid can significantly reduce the secretion of pro-inflammatory factors, alleviate intestinal barrier damage, and inhibit the occurrence and development of colorectal cancer. A is hematoxylin & eosin staining (H&E) showing adenoma pathology and Alcian blue-periodic acid-Schiff staining (AB-PAS) showing colonic goblet cells; B is Ki-67 immunohistochemical staining showing tumor proliferation and malignancy, and E-cadherin immunohistochemical staining showing colon mucosal barrier integrity; C is the quantitative analysis result of Ki-67 index; D is the quantitative analysis result of E-cadherin index; E is transmission electron microscopy showing intestinal epithelial cell heterogeneity, intercellular tight junction structure and intercellular gap, arrows indicate cell tight junction structure, and circles indicate tight junction structure destruction; F is serum LPS concentration reflecting intestinal mucosal barrier permeability; G is the concentration of IL-6, an inflammatory factor closely related to colorectal cancer; H is the concentration of TNF-α, an inflammatory factor closely related to colorectal cancer; *p<0.05, **p<0.01.

Figure 6 shows that 9-hexadecenoic acid inhibits the proliferation of colorectal cancer cells; A to C are the CCK8 detection results of DLD1, HCT116 and NCM 460 cells under 9-hexadecenoic acid treatment, respectively; D is the plate clone formation result of DLD1, HCT116 and NCM 460 cells under 9-hexadecenoic acid treatment; E is the statistical histogram of the cell clone number (%) in D; *p<0.05, **p<0.01; ***p<0.001.

Figure 7 is a heat map and functional analysis diagram of differentially expressed genes in the human colorectal cancer cell line DLD1 after treatment with 9-hexadecenoic acid; A is a volcano plot of differentially expressed genes, up indicates differentially expressed genes that are significantly upregulated after treatment with 9-hexadecenoic acid, down indicates differentially expressed genes that are significantly downregulated, and no sig indicates genes that are not differentially expressed; B is the KEGG enrichment pathway of significantly differentially expressed genes, where the ordinate is the KEGG signaling pathway and the abscissa is the enrichment rate (Rich Factor), representing the ratio of differentially expressed genes enriched in a certain KEGG signaling pathway to the proportion of all genes enriched in functional and signaling pathways.

Figure 8 shows the results of 9-hexadecenoic acid inhibiting NF-κB phosphorylation and destroying the mucosal barrier cascade; A is the effect of 9-hexadecenoic acid on the phosphorylation of NF-κB P65, a key transcription factor in the NF-κB signaling pathway, detected by Western blot; B is the effect of 9-hexadecenoic acid on the MLCK/MLC pathway of the mucosal barrier mediated by NF-κB signaling in the TNF-α-induced cell barrier damage model detected by Western blot; C is the immunofluorescence detection results of the NF-κB P65 phosphorylation level in the colon tissues of the model group (Apc) and the 9-hexadecenoic acid treatment group (Apc+9-HA) of the spontaneous colorectal cancer precancerous lesion model of Apc ^min/ + mice in Example 2, and the intestinal cancer group (AOM) and the 9-hexadecenoic acid treatment group (AOM+9-HA) of the in situ colorectal cancer model in Example 3; D is the NF-κB of AOM and AOM+9-HA in C Analysis results of P65 phosphorylation level; E is the analysis results of NF-κB P65 phosphorylation level of Apc and Apc+9-HA in C; *p<0.05.

Figure 9 shows the results of 9-hexadecenoic acid regulating the intestinal microecology of precancerous lesion model Apc ^min/+ mice; A is a bar graph of the relative abundance of species at the phylum level of the spontaneous colorectal cancer precancerous lesion mouse model group (Apc) and the 9-hexadecenoic acid treatment group (Apc+9-HA) in Example 2 of the present invention; B is a bar graph of the relative abundance of species at the genus level of the two groups; C is a result graph of LEfSe analysis of differentially enriched species in the two groups; D is a statistical graph of the intestinal flora imbalance index (MDI) of the two groups; wherein MDI is an index to determine the degree of microbial ecological imbalance, and the larger the value, the greater the degree of flora disorder.

Figure 10 shows the results of 9-hexadecenoic acid regulating the intestinal microecology of colorectal cancer model mice. A is a bar graph of the relative abundance of species at the phylum level in the intestinal cancer group (AOM) and the 9-hexadecenoic acid treatment group (AOM+9-HA) in Example 3 of the present invention; B is a bar graph of the relative abundance of species at the genus level in the two groups; C is a result graph of LEfSe analysis of differentially enriched species in the two groups; D is a statistical graph of the intestinal flora dysbiosis index (MDI) of the two groups.

Figure 11 shows that 9-hexadecenoic acid improves the efficacy of chemotherapy drugs (capecitabine or oxaliplatin) in the treatment of colorectal cancer; A is a schematic diagram of the process of colorectal cancer model construction and chemotherapy drug (capecitabine or oxaliplatin) treatment, 9-hexadecenoic acid (9-HA), capecitabine (Cap), oxaliplatin (Oxa); B to E are colorectal cancer model group (AOM), 9-hexadecenoic acid treatment group (AOM+9-HA), capecitabine treatment group (AOM+Cap), capecitabine and 9-hexadecenoic acid combination treatment group (AOM+ Figure 2: Colon endoscopy images, colon anatomy, tumor quantity statistics and tumor burden score statistics of mice in each group (AOM, 9-hexaenoic acid treatment group (AOM+9-HA), oxaliplatin treatment group (AOM+Oxa), and oxaliplatin and 9-hexaenoic acid combination treatment group (AOM+Oxa+9-HA), respectively. Arrows indicate colon tumors, and circles indicate a large number of tumors. *p<0.05, **p<0.01; ***p<0.001.

Figure 12 shows the combined use of 9-hexadecenoic acid and chemotherapy drugs (capecitabine), which can better maintain mucosal damage, inhibit the proliferation and lesions of colorectal cancer, and improve intestinal microecology; A is the Ki-67 immunohistochemical staining image showing the tumor proliferation and malignancy of each group of mice; B is the quantitative analysis result of the Ki-67 index; C is the E-cadherin immunohistochemical staining image showing the integrity of the colon mucosal barrier; D is the quantitative analysis result of the E-cadherin index; E is the result of LEfSe analysis of differentially enriched species in the capecitabine group (AOM+Cap) and the capecitabine and 9-hexadecenoic acid combined treatment group (AOM+Cap+9-HA); *p<0.05, **p<0..01; ***p<0.001.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

The 9-hexadecenoic acid referred to in the present invention (molecular formula: C ₁₆ H ₃₀ O ₂ , CAS No.: 373-49-9, structural formula: ), including but not limited to any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable carriers, esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystal forms and metabolites thereof. The pharmaceutically acceptable carriers include but are not limited to one or more of glucose, sucrose, sorbitol, mannose, trehalose, starch, skim milk, lactose, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.

It should be noted that the 9-hexadecenoic acid and 9-hexadecenoic acid derivatives or the pharmaceutical composition containing the 9-hexadecenoic acid of the present invention can be applied to the above-mentioned indications and exhibit the functions described in the present invention after being administered to a subject. The formulation form of the pharmaceutical composition includes but is not limited to injection, tablet, freeze-dried powder injection, capsule and patch. All dosage forms within the scope of the present invention have been tested. Hereinafter, only a small part of them is described in the examples for illustration only, but it should not be understood as a limitation of the present invention.

Example 1 Metabolomics identified abnormal lipid metabolism in colorectal tumor tissue and severe deficiency of 9-hexadecenoic acid in cancer tissue of critically ill patients

1. Experimental Methods

1. Preparation of sequencing samples

Tumor tissues (all medical waste) from 20 patients with a clear pathological diagnosis of colorectal cancer were collected from the clinic, including 20 cancer tissues (T) and 20 paired normal tissues (N) far from the tumor 5 cm away from the tumor collected during colorectal cancer resection. All subjects were informed of the nature of the sampling and experiment and their consent was obtained. The above samples have excluded samples from patients with a history of other cancers, other major systemic or digestive system diseases, chronic viral infections, preoperative bacterial infections, and patients receiving immunosuppressive treatment (such as chemotherapy, oral steroids, etc.) or patients who have received other cancer-related treatments before surgery.

2. Non-targeted metabolomics sequencing and analysis

According to conventional methods, non-targeted metabolomics analysis was performed on the above tissue samples. The main steps included: metabolite extraction → de-noising and smoothing, baseline correction, overlapping peak identification → normalization, data conversion, standardization → multivariate statistical analysis, multiple algorithm VIP value analysis, correlation analysis, and KEGG pathway analysis.

2. Experimental Results

As shown in Figure 1A, a total of 306 differential metabolites were screened to be enriched in tumor tissues, and 117 metabolites were reduced in tumor tissues. The metabolic pathway annotation was performed through the KEGG database (https://www.kegg.jp/kegg/pathway.html) to obtain the pathways involved in the differential metabolites. As shown in Figure 1B, the KEGG metabolic pathway found that the differential metabolites were mainly enriched in lipid metabolism, indicating abnormal lipid metabolism in tumor tissues. As shown in Figure 1C, the 9-hexadecenoic acid in the tumor tissues of patients with severe colorectal cancer (High stage) was lower than that of patients with mild disease (Low stage), indicating that the reduction of 9-hexadecenoic acid is closely related to the occurrence and development of colorectal cancer.

Example 2 9-hexadecenoic acid controls the development of spontaneous colorectal adenomas in Apc ^min/+ mice with colorectal cancer precancerous lesions

1. Experimental Methods

(1) Modeling and drug administration

The development and progression of colorectal cancer are affected by both intrinsic genetic factors and extrinsic environmental factors. The tumor suppressor gene Apc is mutated in most colorectal cancer patients, resulting in partial or complete loss of its function to inhibit abnormal proliferation of tumor cells. Therefore, Apc ^min/+ mice with Apc gene mutations that can spontaneously develop adenomatous polyps are excellent animal models for studying the mechanism of colorectal cancer.

In order to further analyze the effect of 9-hexadecenoic acid on colorectal precancerous lesions, this example first refers to the prior art (Moser AR, Luongo C, Gould KA, McNeley MK, Shoemaker AR, Dove WF. ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer. 1995 Jul-Aug; 31A (7-8): 1061-4.) to construct an Apc ^min/+ mouse spontaneous colorectal cancer precancerous lesion model, and drug treatment was performed at the same time. The operation process is shown in A of Figure 2, and the specific steps are as follows: 21 7-8 week old Apc ^min/+ female mice were randomly divided into two groups, one of which had 10 Apc ^min/+ mice, recorded as the model group (i.e., Apc); the other group had 11 Apc ^min/+ mice, recorded as the 9-hexadecenoic acid treatment group (i.e., Apc+9-HA). The two groups of mice were allowed to drink water containing 2.5W/V% dextran sulfate sodium (DSS) for five days, and then given normal drinking water until day 30 to induce a spontaneous colorectal cancer precancerous lesion model. From the first 5 days of DSS induction, the Apc group was gavaged with sterile water, and the Apc+9-HA group was gavaged with 9-hexadecenoic acid (0.5g/kg) every 2 days until day 30, during which the anal blood and anal tumor conditions of the mice were continuously recorded.

(2) Detection

At the end of the experiment, all mice were dissected, and the entire colon of the mice was longitudinally cut open and photographed, and the number of adenomas and tumor burden scores (burden score, tumor diameter ≤ 1 mm, score = 1; 1 mm ≤ tumor diameter ≤ 2 mm, score = 2; 2 mm ≤ tumor diameter, score = 3) were counted.

Colorectal tissues were fixed with 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and analyzed by transmission electron microscopy. Hematoxylin-eosin (H&E), Alcian blue-periodic acid-Schiff (AB-PAS) and immunohistochemical staining were performed to analyze the pathological conditions, cancerous infiltration, tumor proliferation, and mucosal damage of colorectal tissues.

2. Experimental Results

Colorectal cancer is a heterogeneous disease of intestinal epithelial carcinogenesis, a digestive tract tumor with chronic inflammation and intestinal mucosal barrier damage as typical pathological features. Intestinal mucosal barrier dysfunction is a key factor in the occurrence and development of colorectal cancer, and the increased permeability of the intestinal mucosal epithelium caused by internal and external factors inducing intestinal mucosal inflammatory response, down-regulation of tight junctions between cells such as ZO-1 and Occludin, and destruction of intercellular adhesive junctions such as E-cadherin are the key links leading to intestinal mucosal dysfunction.

As shown in Figure 2B, the mice in the model group (Apc) had obvious perianal adenomas, while the mice in the 9-hexadecenoic acid group (Apc+9-HA) had no obvious perianal adenomas or bleeding, indicating that 9-hexadecenoic acid treatment can significantly delay adenoma growth and inhibit the occurrence and development of adenoma.

As shown in Figure 2 C to E, the mice in the model group developed large-segment spontaneous adenomas in the colorectum and rectal redness and swelling, while the size, number and tumor burden scores of the adenomas in the intestines of the mice in the 9-hexadecenoic acid group were significantly less than those in the model group.

As shown in A to B in Figure 3, pathological analysis showed that the colon tissue of the mice in the model group had multiple adenomas, infiltration of a large number of cancer cells and inflammatory cells, irregular arrangement of infiltrated cancer cells, and destruction and deformation of the basic structure of each layer of the colon; while the colon structure of the mice in the 9-hexaenoic acid group was relatively intact, with less inflammatory cell infiltration, and significantly less than that in the model group.

To analyze the proliferation of cancer cells and mucosal barrier function, immunohistochemical kits were used to detect the expression of nuclear proliferation antigen Ki-67 and intercellular adhesion junction E-cadherin. As shown in Figure 3C to E, the positive rate of Ki-67 in the colon tissue of mice in the 9-hexadecenoic acid group was significantly lower than that in the model group, and the expression of cell adhesion protein E-cadherin was significantly higher than that in the model group. The transmission electron microscopy results shown in Figure 3F also show that the microstructure of the intestinal mucosa of the precancerous lesion model mice in the 9-hexadecenoic acid treatment group was improved.

The above results indicate that 9-hexadecenoic acid treatment significantly inhibits the proliferation of colon tumors and repairs the intestinal mucosal barrier function.

Example 3 9-hexadecenoic acid can alleviate intestinal barrier damage, significantly reduce the secretion of pro-inflammatory factors, and inhibit the occurrence and development of colorectal cancer

1. Experimental Methods

(1) Modeling and drug administration

In this example, 21 5-6 week old SPF healthy female C57BL/6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd., with an average body weight of 18-22 g) were selected as experimental animals; after one week of adaptive feeding, the mice were randomly divided into two groups, which were named in sequence: a colon cancer group (AOM) of 10 mice and a 9-hexadecenoic acid treatment group (AOM+9-HA) of 11 mice.

Subsequently, referring to the prior art "DOI: 10.4103/1477-3163.78279", an in situ colorectal cancer model was constructed using azoxymethane (AOM) and dextran sulfate sodium (DSS), and drug treatment was performed at the same time. The operation process is shown in A in Figure 4, and the specific steps are: 10 mg/kg of azoxymethane was injected intraperitoneally on the first day, and 2.5W/V% dextran sulfate sodium (DSS) purified water was drunk on the fifth day. Freshly prepared DSS aqueous solution was replaced every day for five consecutive days, and then purified water was replaced for 14 consecutive days. This was one cycle. The above process was carried out for 3 consecutive cycles. On the 90th day, both groups of mice had anal bleeding and obvious tumors around the anus, and the colorectal cancer model was successfully constructed. Starting from the first day of AOM treatment, the AOM group was gavaged with sterile water, and the AOM+9-HA group was gavaged with 9-hexadecenoic acid (0.5 g/kg) once every 2 days until the 90th day. During this period, the anal blood and anal tumor conditions of the mice were continuously recorded.

(2) Detection

At the end of the experiment, all mice were dissected, and the entire colon of the mice was longitudinally cut open and photographed, and the number of adenomas and tumor burden scores (burden score, tumor diameter ≤ 1 mm, score = 1; 1 mm ≤ tumor diameter ≤ 2 mm, score = 2; 2 mm ≤ tumor diameter, score = 3) were counted.

The colorectal tissues were fixed with 4% paraformaldehyde, routinely embedded in paraffin, sliced, and analyzed by transmission electron microscopy. Hematoxylin-eosin (H&E), Alcian blue-periodic acid-Schiff staining (AB-PAS) and immunohistochemical staining were performed to analyze the pathological conditions, cancerous infiltration, tumor proliferation, and mucosal damage of the colorectal tissues. In addition, mouse serum was collected, and the concentrations of inflammatory factors TNF-α and IL-6, which are closely related to colorectal cancer, and the concentration of LPS, which is related to the permeability of the intestinal mucosal barrier, were detected by ELISA.

2. Experimental Results

Changes in mucosal barrier function and abnormal activation of inflammatory pathways are closely related to the occurrence and development of intestinal tumors. Inflammatory responses in colorectal tumors exacerbate the development, carcinogenesis, and metastasis of adenomas; excessive activation of inflammatory responses leads to the destruction of the intestinal mucosal barrier, causing the collapse of the colon epithelial physical barrier, leading to a chronic inflammatory environment and epithelial carcinogenesis.

As shown in Figure 4, B to F, the perianal tumors of mice in the intestinal cancer group (AOM) were more obvious, and multiple colorectal cancers were also observed under endoscopy. Compared with the intestinal cancer group, the perianal redness and swelling of mice in the 9-hexadecenoic acid treatment group (AOM+9-HA) were reduced, the number of colorectal tumors was reduced, and the tumor burden score was reduced. This shows that 9-hexadecenoic acid can significantly inhibit the occurrence and development of colorectal cancer.

The results of tumor pathological analysis are shown in Figure 5A. The colon tissues of mice in the intestinal cancer group had obvious infiltration of a large number of tumor cells, the basic structure of each layer of the colon was destroyed, the cells were irregularly arranged, the nuclear-cytoplasmic ratio was significantly increased, obvious inflammatory cell infiltration and ulceration were observed, and AB-PAS staining showed a decrease in goblet cells and destruction of epithelial cells. Compared with the intestinal cancer group, the colon tumors of mice in the 9-hexadecenoic acid treatment group (AOM+9-HA) were fewer and smaller, the colon mucosal cells tended to be normal, with only a few irregular arrangements, the nuclear-cytoplasmic ratio was significantly reduced, and the basic pathological morphology was significantly improved.

The results of immunohistochemical staining of cell proliferation antigen Ki-67 and cell adhesion protein E-cadherin are shown in Figure 5 B to D. The expression of Ki-67 in the colon tumor of mice in the 9-hexadecenoic acid treatment group was significantly reduced, and the expression of cell adhesion protein E-cadherin was significantly increased. The results of transmission electron microscopy shown in Figure 5 E also show that the microstructure of the intestinal mucosa of colorectal cancer mice in the 9-hexadecenoic acid treatment group was improved.

The above results indicate that 9-hexadecenoic acid maintains the intestinal mucosal barrier function and significantly inhibits the occurrence and development of colorectal cancer.

The results of the detection of inflammatory factors TNF-α and IL-6, which are closely related to the occurrence and development of colorectal cancer, and LPS levels reflecting the intestinal mucosal barrier function are shown in Figure 5F to H. Compared with the intestinal cancer group (AOM), the IL-6, TNF-α and LPS levels in the serum of mice in the 9-hexadecenoic acid treatment group were significantly reduced, indicating that 9-hexadecenoic acid can effectively reduce the secretion of pro-inflammatory factors closely related to colorectal cancer and protect the intestinal mucosal barrier.

The above results show that 9-hexaenoic acid significantly alleviates intestinal barrier damage, inhibits the occurrence and development of colorectal cancer, and inhibits the proliferation of colon tumors, restores and maintains the function of the intestinal mucosal barrier, reduces the carcinogenesis of colon epithelial cells, reduces the secretion of pro-inflammatory factors, and has stronger anti-colorectal tumor activity and function.

Example 4 9-hexadecenoic acid inhibits the proliferation of colorectal cancer cells and inhibits the cascade destruction of mucosal barriers by NF-κB phosphorylation

1. Experimental Methods

(1) Cell culture and CCK8 detection of the effect of 9-hexadecenoic acid on cell proliferation

In this experiment, human colorectal cancer cell lines (DLD-1 and HCT116) and human normal intestinal epithelial cells (NCM 460) were used as controls to detect the effect of 9-hexadecenoic acid on cell proliferation. The specific operation is as follows:

① DLD-1, HCT116 and NCM 460 cells were cultured separately using complete culture medium: DMEM high glucose medium (Gibco) containing 1% double antibody and 10% fetal bovine serum, cultured in a cell culture incubator at 37°C and 5% CO ₂ ; when the cell confluence reached 70-80%, trypsin was used to digest and prepare cell suspension. About 1000 cells/100 μL were added to each well of a 96-well plate;

② When cells were observed to be completely attached to the wall, the culture medium of each well was aspirated, and the complete culture medium containing 0.05 mM 9-hexadecenoic acid was added to the treatment group (i.e., 9-HA), and the same amount of complete culture medium was added to the control group (i.e., Ctrl), and then the 96-well plate was incubated in a cell culture incubator at 37°C and 5% _CO2 for 24, 48 or 72 hours;

③ At each time point, add 10 μl of CCK-8 solution directly to each well, and be careful to avoid bubbles during the addition process. Place the culture plate with CCK-8 in a 37°C, 5% CO2 cell culture incubator for 2 hours, take out the 96-well plate, and use an ELISA reader to detect the OD value of each well at a wavelength of 450 nm, and analyze and process the data.

(2) Cell culture and detection of the effect of 9-hexadecenoic acid on cell clone formation ability

This experiment used human colorectal cancer cell lines (DLD-1 and HCT116) and human normal intestinal epithelial cells (NCM 460) to detect the effect of 9-hexaenoic acid on cell clone formation ability. The specific operation is as follows:

① DLD-1, HCT116 and NCM 460 cells were cultured separately in complete medium: DMEM high glucose medium (Gibco) containing 1% double antibody and 10% fetal bovine serum, and cultured in a cell culture incubator at 37°C and 5% _CO2 ; when the cell confluence reached 70-80%, the cells were digested with trypsin and made into cell suspension;

②After thorough mixing, take 2 μL of cell suspension to a cell counter to count the number of cells and obtain the cell density of the suspension;

③ Dilute the suspension to 300 cells/1000 μL with complete culture medium, mix thoroughly, and seed the cells in a 12-well plate at 1000 μL per well, and gently rotate to evenly disperse the cells;

④ After the cells are completely attached, discard the old culture medium, add complete culture medium containing 0.05 mM 9-hexadecenoic acid to the treatment group (i.e. 9-HA), and add an equal amount of complete culture medium to the control group (i.e. Ctrl); change the medium every three days, and terminate the culture after 7 to 10 days when visible clones appear in the culture plate;

⑤Discard the supernatant and carefully wash twice with PBS. Add 5mL of 4% paraformaldehyde to fix the cells for 15 minutes. Then remove the fixative, add an appropriate amount of crystal violet-methanol solution to stain, then slowly wash away the staining solution with running water and air dry. Use a plate scanner to scan and count the number of clones, and finally calculate the clone formation rate.

(3) Detection of the effects of 9-hexadecenoic acid on the transcriptome and inflammatory pathways of human colorectal cancer cells

Inflammatory factors in the tumor microenvironment, such as IL-6 and TNF-α, play a role in promoting the induction, promotion, invasion and metastasis of tumors. Long-term inflammatory signal stimulation leads to the activation of abnormal signal pathways such as NF-κB, which can lead to abnormal growth and invasion of colorectal tumors.

This experiment first used the human colorectal cancer cell line DLD-1 and used transcriptome sequencing technology to detect the effect of 9-hexadecenoic acid on the signaling pathways related to inflammatory factors in the cell transcriptome, and analyzed the mechanism of 9-hexadecenoic acid in inhibiting the proliferation of colorectal cancer cells. The specific operations are as follows:

① Cultivate DLD1 cells with complete medium: DMEM high-glucose medium (Gibco) containing 1% double antibody and 10% fetal bovine serum, and culture in a cell culture incubator at 37°C and 5% _CO2 ; when the confluence reaches 70-80%, digest with trypsin and make a cell suspension, mix thoroughly, take 2 μL of the cell suspension to a cell counter to count the number of cells and obtain the cell density of the suspension. Dilute the suspension with complete medium to 8×10 ⁵ cells/1000 μL, mix thoroughly, and seed the cells in a 12-well plate at 1000 μL per well;

② After the cells were completely attached, the old culture medium was discarded, and the treatment group (i.e., 9-HA) was added with complete culture medium containing 0.1 mM 9-hexadecenoic acid, and the control group (i.e., Ctrl) was added with an equal amount of complete culture medium, and then the cells were cultured in a 37°C, 5% _CO2 cell culture incubator; the cells were collected after 6, 12, and 24 hours respectively;

③A part of the cells were collected for RNA extraction and transcriptome sequencing analysis; the other part of the cells were lysed with RIPALysis Buffer and the cell protein was extracted. The expression of signal pathways and mucosal barrier proteins obtained by transcriptome sequencing analysis was identified by western blot. The antibodies used were: PPARγ (CST, 2435), E-cadherin (CST, 3195s), p-NF-κB p65 (CST, 3033s), NF-κB p65 (CST, 8242s) and GAPDH (proteintech, 10494-1-AP).

This experiment also used a cell barrier damage model constructed by TNF-α-induced human colorectal cancer cell line (DLD-1) to detect the effect of 9-hexaenoic acid on cell mucosal barrier damage in vitro. The specific operation is as follows:

① Cultivate DLD1 cells with complete medium: DMEM high-glucose medium (Gibco) containing 1% double antibody and 10% fetal bovine serum, and culture in a cell culture incubator at 37°C and 5% _CO2 ; when the confluence reaches 70-80%, digest with trypsin and make a cell suspension, mix thoroughly, take 2 μL of the cell suspension to a cell counter to count the number of cells and obtain the cell density of the suspension. Dilute the suspension with complete medium to 8×10 ⁵ cells/1000 μL, mix thoroughly, and seed the cells in a 12-well plate at 1000 μL per well;

② After the cells were completely attached, the old culture medium was discarded, and DLD-1 cells were treated with complete culture medium containing TNF-α (final concentration 50 ng/mL) for 24 hours to construct an intestinal epithelial cell barrier injury model. After that, the cells in the treatment group were added with complete culture medium containing 0.2 mM or 0.4 mM 9-hexaenoic acid, and the control group was added with an equal amount of complete culture medium. After incubation for 12 hours, the cells were collected and protein samples were extracted;

②To further explore the mechanism of action of 9-hexaenoic acid in improving TNF-α-induced intestinal epithelial tight junction damage, the expression of related signaling pathways (including NF-κB pathway), MLCK/MLC/p-MLC and mucosal barrier-related molecules (ZO-1, Occludin and E-cadherin, etc.) in protein samples was detected by Western Blot. The antibodies used were: p-NF-κB p65 (CST, 3033s), NF-κB p65 (CST, 8242s), E-cadherin (CST, 3195s), MLCK (Affinity, AF5314), p-MLC (Affinity, AF8618), ZO-1 (Affinity, AF5145), Occludin (Affinity, DF7504) and GAPDH (proteintech, 10494-1-AP).

This experiment also used the spontaneous colorectal cancer precancerous lesion model of Apc ^min/+ mice in Example 2 and the in situ colorectal cancer model in Example 3 to detect the effect of 9-hexadecenoic acid on the inflammatory pathway in the tumor microenvironment in vivo. The specific operation is as follows:

① Take colorectal tissue sections of the spontaneous colorectal cancer precancerous lesion model of Apc ^min/+ mice in Example 2 (Apc group, Apc+9-HA group) and the in situ colorectal cancer model in Example 3 (AOM group, AOM+9-HA group), and detect the phosphorylation of the NF-κB pathway in the paraffin tissue sections by immunofluorescence. The antibody used is p-NF-κB p65 (CST, 3033s).

2. Experimental Results

(1) Effect of 9-hexadecenoic acid on cell proliferation

As shown in Figure 6A to C, the proliferation of two human colorectal cancer cell lines (DLD-1 and HCT116) was significantly inhibited by 9-hexadecenoic acid treatment, while the proliferation of normal intestinal epithelial cells (NCM 460) was not affected. This indicates that 9-hexadecenoic acid can inhibit the proliferation of colorectal tumor cells and will not affect the growth and development of normal intestinal epithelial cells at the same time.

(2) Effect of 9-hexadecenoic acid on cell clone formation ability

As shown in Figure 6D and E, 9-hexaenoic acid significantly inhibited the size and number of colony formation in human colorectal cancer cell lines (DLD-1 and HCT116) without affecting the colony formation of normal intestinal epithelial cells (NCM 460).

(3) Effects of 9-hexadecenoic acid on the transcriptome and inflammatory pathways of human colorectal cancer cells

The results of transcriptome sequencing analysis are shown in A and B in Figure 7. 9-hexadecenoic acid mainly inhibits the NF-κB signaling pathway and IL-17 signaling pathway of tumors. As shown in A in Figure 8, Western Blot results showed that 9-hexadecenoic acid inhibited the phosphorylation of NF-κB P65, a key transcription factor in the NF-κB signaling pathway. Further, in the TNF-α-induced cell barrier damage model, as shown in B in Figure 8, Western Blot results showed that 9-hexadecenoic acid can maintain the homeostasis of the intestinal mucosal barrier and thus inhibit the proliferation of colorectal cancer cells.

The detection results of the spontaneous colorectal cancer precancerous lesion model and the orthotopic colorectal cancer model of Apc ^min/+ mice are shown in Figure 8C to E. 9-Hexadecenoic acid inhibited the phosphorylation level of NF-κB P65 in the tumor tissues of mice.

Based on the above results, it can be shown that 9-hexaenoic acid exhibits excellent anti-tumor effects and anti-inflammatory properties both in vivo and in vitro, and does not affect the growth and development of normal intestinal epithelial cells.

Example 5 Effect of 9-hexadecenoic acid on intestinal microecology

1. Experimental Methods

(1) Sample collection

In this experiment, the colon contents of 4 groups of mice were collected, including the model group (Apc) and the 9-hexadecenoic acid treatment group (Apc+9-HA) of the spontaneous colorectal cancer precancerous lesion model of Apc ^min/+ mice in Example 2, and the intestinal cancer group (AOM) and the 9-hexadecenoic acid treatment group (AOM+9-HA) of the in situ colorectal cancer model in Example 3. DNA was extracted using SoilDNA Kit (Omega Bio-Tek) to obtain fecal DNA.

(2) 16S amplification

For the extracted fecal DNA, the upstream primer 27F (5'-AGRGTTYGATYMTGGCTCAG-3') and the downstream primer 1492R (5'-RGYTACCTTGTTACGACTT-3') were used to amplify the 16S V3-V4 region. The PCR product was then excised and recovered using the AxyPrep DNA gel recovery kit (AXYGEN), eluted with Tris-HCl buffer, and then detected by 2% agarose electrophoresis.

(3) PacBio library construction and sequencing

① End repair: first change the sticky end of the fragment into a flat end; connect the circular linker: connect the two ends of the circular single chain respectively, and connect the two ends of the single chain to the double-stranded positive and negative chains respectively, to obtain a dumbbell-like structure ("horse ring"), called SMRTBell;

②Remove the sequence that is not connected to the adapter;

③ Anneal the single-stranded loop of the library with the primer and bind it to the polymerase fixed at the bottom of the ZMW (zero-mode waveguides).

(4) Analysis of sequencing results

The PacBio offline data is obtained through basecall to obtain the original data file, and the HiFi sequence file is obtained through PacBio's SMRTLinkv11.0 analysis software, which is in fastq format. After distinguishing the samples by barcode, species taxonomy analysis is performed. Based on the taxonomic information, statistical analysis of community structure can be performed at various taxonomic levels. Based on the above analysis, a series of in-depth statistical and visual analyses such as community structure and phylogeny can be performed.

The analysis and statistical methods used in this experiment are as follows: count the species with the highest maximum abundance at the phylum level and genus level, and generate a cumulative bar graph of species relative abundance to evaluate the relative abundance of intestinal flora species; LEfSe (LDA Effect Size) performs difference tests at multiple levels, analyzes multi-level differential species, and uses LDA values to measure the size of the species' influence on the differential effect. The larger the LDA value, the greater the difference, the more stringent the conditions, and the more reliable the results; calculate the intestinal dysbiosis index (MDI) to determine the degree index of microbial ecological imbalance. The larger the MDI value, the greater the degree of flora disorder.

2. Experimental results

(1) Effects of 9-hexaenoic acid on the intestinal microecology of the spontaneous colorectal cancer precancerous lesion model of Apc ^min/+ mice

The relative abundance of intestinal flora species is shown in A and B in Figure 9. It can be seen that in the model group (Apc) and the 9-hexadecenoic acid treatment group (Apc+9-HA), the proportion of the top five species at the phylum level is ranked as follows: Firmicutes, Bacteroidota, Proteobacteria, Verrucomicrobia and Actinobacteria, accounting for more than 97%; among them, red and blue have the highest proportion, and the difference is the most intuitive. It can be clearly seen from the bar chart of group statistics that the red color is Firmicutes (Firmicutes) with a lower proportion in the model group, and the proportion in the precancerous lesion mice after 9-hexadecenoic acid treatment has increased. Blue is Bacteroidetes (Bacteroidetes), which has the highest proportion in the model group (Apc), while the proportion of Bacteroidetes in the 9-hexadecenoic acid treatment group (Apc+9-HA) gradually decreases; In addition, at the genus level, it can be seen that the 9-hexadecenoic acid treatment group (Apc+9-HA) significantly changed the composition structure of the intestinal flora of precancerous lesion mice.

The results of LEfSe analysis are shown in C of Figure 9. The enriched bacteria in the model group (Apc) include Muribaculum intestinale, Odoribacter laneus, Dubosiella newyorkensis, Prevotellaceae and other bacteria that promote the development of colorectal tumors. In the 9-hexadecenoic acid-treated group (Apc+9-HA), Akkermansia muciniphila, Escherichia fergusonii, Desulfovibrio desulfuricans and other bacteria increased significantly after treatment with 9-hexadecenoic acid. Among them, the beneficial bacteria Akkermansia muciniphila has been shown to significantly inhibit the occurrence and development of tumors in colorectal cancer and precancerous lesions.

The MDI statistical results are shown in D of FIG9 , which shows that the MDI value of the 9-hexadecenoic acid treatment group (Apc+9-HA) is significantly lower than that of the model group (Apc), indicating that 9-hexadecenoic acid can significantly alleviate the flora disorder of mice with precancerous lesions.

(2) Effects of 9-hexadecenoic acid on the intestinal microecology of an orthotopic colorectal cancer model

The relative abundance of intestinal flora species is shown in Figure 10A and B. It can be seen that in the intestinal cancer group (AOM) and the 9-hexadecenoic acid treatment group (AOM+9-HA), the proportion of the top four species at the phylum level is ranked as follows: Firmicutes, Verrucomicrobia, Bacteroidota, Actinobacteria, accounting for more than 99%. The comparison shows that the treatment with 9-hexadecenoic acid significantly changed the composition structure of the intestinal flora of colorectal cancer mice.

The results of LEfSe analysis are shown in Figure 10C. It can be seen that compared with the intestinal cancer group (AOM), the beneficial bacteria Akkermansia muciniphila in the 9-hexadecenoic acid treatment group (AOM+9-HA) increased significantly after 9-hexadecenoic acid treatment.

The MDI statistical results are shown in Figure 10D, which shows that the MDI value of the 9-hexadecenoic acid treatment group (AOM+9-HA) is significantly lower than that of the intestinal cancer group (AOM), indicating that 9-hexadecenoic acid can significantly alleviate the flora disorder of colorectal cancer mice.

Taken together, the above results indicate that 9-hexaenoic acid can regulate the intestinal microecology of colorectal cancer mice, improve the flora structure, increase the proportion of beneficial bacteria, alleviate flora disorder, restore flora homeostasis, and thereby improve and control the occurrence and development of colorectal cancer.

Example 6 9-hexadecenoic acid improves the efficacy of chemotherapy for colorectal cancer

1. Experimental Methods

(1) Modeling and drug administration

In the capecitabine and 9-hexadecenoic acid combined treatment experiment in this embodiment, 28 5-6 week old SPF healthy female C57BL/6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were selected, with an average body weight of 18-22 g; after 1 week of adaptive feeding, the mice were divided into four groups, named in sequence: 7 intestinal cancer model group (AOM), 7 9-hexadecenoic acid treatment group (AOM+9-HA), 7 capecitabine, a commonly used chemotherapy drug for intestinal cancer, was treated group (AOM+Cap), and capecitabine + 9-hexadecenoic acid treatment group (AOM+Cap+9-HA).

In the combined treatment experiment of oxaliplatin and 9-hexaenoic acid in this embodiment, 21 5-6 week old SPF healthy female C57BL/6 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were selected, with an average body weight of 18-22 g; after one week of adaptive feeding, the mice were divided into four groups, named in sequence: 5 mice in the intestinal cancer model group (AOM), 5 mice in the 9-hexaenoic acid treatment group (AOM+9-HA), 6 mice in the oxaliplatin treatment group (AOM+Oxa), and 5 mice in the oxaliplatin + 9-hexaenoic acid treatment group (AOM+Oxa+9-HA).

Subsequently, referring to the prior art "DOI: 10.4103/1477-3163.78279", an in situ colorectal cancer model was constructed with azoxymethane (AOM) and dextran sulfate sodium (DSS) and drug treatment was performed at the same time. The operation process is shown in A in Figure 11. On the first day, 10 mg/kg of azoxymethane was injected intraperitoneally, and on the fifth day, 2.5w/v% dextran sulfate sodium (DSS) purified water was drunk. Freshly prepared DSS aqueous solution was replaced every day for five consecutive days, and then purified water was replaced for 14 consecutive days. This was one cycle. The above process was carried out for 3 cycles. On the 90th day, both groups of mice had anal bleeding and obvious tumors around the anus. The colorectal cancer model was successfully constructed. Starting from the first day of AOM treatment, mice were gavaged with sterile water or 9-hexadecenoic acid (0.5 g/kg) once every 2 days, and chemotherapeutic drugs (capecitabine at a dose of 0.25 g/kg or oxaliplatin at 5 mg/kg by intraperitoneal injection) were administered twice a week; until the 90th day, the anal blood and anal tumor conditions of the mice were continuously recorded.

(2) Detection

At the end point, all mice were dissected, the entire colon of the mice was longitudinally cut open and photographed, and the number of adenomas and tumor burden scores (burden score, tumor diameter ≤ 1 mm, score = 1; 1 mm ≤ tumor diameter ≤ 2 mm, score = 2; 2 mm ≤ tumor diameter, score = 3) were counted.

The colorectal tissues of mice were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. Immunohistochemical staining was performed to analyze the pathological conditions, cancerous infiltration, tumor proliferation, and mucosal damage of the colorectal tissues.

The colonic fecal contents of the mice were collected, and fecal DNA was extracted according to the method in Example 5, and 16S amplification sequencing and LEfSe analysis were performed.

2. Experimental Results

As shown in B to E in Figure 11, the perianal tumors of mice in the intestinal cancer group (AOM) were obvious, and multiple colorectal cancers were also observed under endoscopy; compared with the intestinal cancer group, the perianal redness and swelling of mice in the 9-hexadecenoic acid treatment group (AOM+9-HA) and the capecitabine treatment group (AOM+Cap) were reduced, the number of colorectal tumors was reduced, and the tumor burden score was reduced; and compared with single drug treatment, the combined treatment of mice with capecitabine and 9-hexadecenoic acid (AOM+Cap+9-HA) further greatly reduced the number and burden of colorectal cancer in mice. The results of the combined treatment experiment of oxaliplatin and 9-hexadecenoic acid are shown in F to H in Figure 11, which shows that the combination of oxaliplatin and 9-hexadecenoic acid (AOM+Oxa+9-HA) greatly reduced the number and burden of colorectal cancer in mice compared with single drug treatment (AOM+9-HA or AOM+Oxa).

The results of immunohistochemical staining of cell nuclear proliferation antigen Ki-67 and cell adhesion protein E-cadherin are shown in A to D in Figure 12. Compared with the 9-hexadecenoic acid treatment group (AOM+9-HA) and the capecitabine treatment group (AOM+Cap), the expression of Ki-67 in the colon tumor of mice in the capecitabine and 9-hexadecenoic acid combination group (AOM+Cap+9-HA) was further reduced, and the expression of cell adhesion protein E-cadherin was significantly increased. This indicates that 9-hexadecenoic acid alleviates the damage of chemotherapy drugs to the intestinal mucosal barrier, and the combination of 9-hexadecenoic acid and chemotherapy drugs further promotes intestinal mucosal repair.

As shown in Figure 12E, linear discriminant analysis (LDA) combined with effect size measurement determined the differential species of AOM/DSS-induced intestinal cancer mice after treatment with capecitabine (AOM+Cap) or capecitabine and 9-hexadecenoic acid combination group (AOM+Cap+9-HA), and Akkermansia muciniphila in the box scored the highest among the significantly enriched species in the capecitabine and 9-hexadecenoic acid combination group. This indicates that the combination of 9-hexadecenoic acid and capecitabine can also alleviate the intestinal flora imbalance caused by chemotherapy drugs alone and promote the growth of probiotics.

The above results indicate that 9-hexaenoic acid not only inhibits the occurrence and development of colorectal cancer, but also improves the efficacy of colorectal cancer chemotherapy drugs, while reducing the gastrointestinal toxic side effects of chemotherapy drugs, such as chemotherapy-induced mucosal barrier damage and intestinal flora imbalance.

In summary, 9-hexadecenoic acid effectively controls colorectal precancerous lesions, reduces intestinal inflammation, inhibits the progression and deterioration of intestinal adenomas and colorectal cancer, has therapeutic and preventive effects on colorectal tumors, and can also improve the intestinal microecological imbalance caused by colorectal tumors, significantly increase the effect of chemotherapy drugs in treating tumors, and reduce the toxic side effects of chemotherapy drugs.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. Use of 1.9-hexadecenoic acid in the manufacture of a product for the prevention and treatment of colorectal neoplasms.
2. Use of 9-hexadecenoic acid in the manufacture of a product for repairing a mucosal barrier in a colorectal tumor patient and/or alleviating an intestinal mucosal lesion in a colorectal tumor patient.
3. Use of 9-hexadecenoic acid for the preparation of a product for the prevention and/or treatment of dysbacteriosis in the intestinal tract of a patient suffering from colorectal cancer.
4. Use of 4.9-hexadecenoic acid in the manufacture of a product for improving the chemotherapeutic effect of colorectal neoplasms.
5. Use of 9-hexadecenoic acid in the manufacture of a product for alleviating side effects of colorectal tumour chemotherapy.
6. 6.9-Hexadecenoic acid is used as a synergist of a chemotherapeutic drug in the preparation of a product for preventing and treating colorectal tumor.
7. Use of 7.9-hexadecenoic acid in combination with a chemotherapeutic agent for the manufacture of a product for the prevention and treatment of colorectal tumours.
8. 8. The use according to any one of claims 1 to 7, wherein the colorectal tumor comprises a benign colorectal tumor and/or a malignant colorectal tumor.
9. 9. The use according to any one of claims 1 to 7, wherein the 9-hexadecenoic acid is any one or more of the following: 9-hexadecenoic acid, pharmaceutically acceptable esters or salts, prodrugs, solvates, hydrates, stereoisomers, tautomers, geometric isomers, crystalline forms and metabolite forms thereof.
10. 10. A pharmaceutical composition comprising an active ingredient 1 and an active ingredient 2; the active ingredient 1 is 9-hexadecenoic acid; the active ingredient 2 is a chemotherapeutic drug for treating colorectal tumor.