Application of isolithocholic acid in preparation of medicine for preventing or treating colorectal cancer induced by high fat diet

Abstract

The invention discloses an application of isolithocholic acid in preparation of a medicine for preventing or treating colorectal cancer induced by high fat diet. The invention proves that the isolithocholic acid can be accurately replenished by intragastric administration of 100g / kg of isoLCA at one time, and meanwhile, the isolithocholic acid can obviously inhibit the occurrence of obesity-related colon tumors, reduce the proliferation index of tumor cells and repair the intestinal barrier structure. Isolithocholic acid is endogenous secondary bile acid, can be chemically synthesized or semi-synthesized, is high in safety and good in patient compliance through oral administration, is expected to reduce the occurrence rate of colorectal cancer of obese people, and has important significance in treatment of colorectal cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to the application of isolithocholic acid in the preparation of a drug for preventing or treating high-fat diet-induced colorectal cancer. BACKGROUND

[0002] Colorectal cancer (CRC) is one of the malignant tumors with high morbidity and mortality worldwide. In recent years, with the increase of high-fat diet (HFD) intake and the rise of the proportion of obese population, the incidence of high-fat diet-related colorectal cancer has increased significantly, becoming an important public health problem.

[0003] Studies have shown that high-fat diet can increase the risk of colorectal cancer by changing the structure of intestinal flora, inducing chronic inflammation, and promoting bile acid metabolism disorder. Among them, bile acid metabolism disorder is considered to be one of the key links of high-fat diet-induced colorectal cancer. Under normal circumstances, bile acids are synthesized in the liver and maintained in dynamic balance after being metabolized by intestinal flora. However, high-fat diet can significantly change the composition and concentration of bile acids, disrupt the balance of intestinal microecology, and thus promote tumor occurrence.

[0004] Isolithocholic acid (isoLCA) is a secondary bile acid, and studies have reported that it has certain antibacterial effect. However, there is no research report on the use of isolithocholic acid in preventing or relieving high-fat diet-induced colorectal cancer.

[0005] Therefore, there is a lack of a safe, effective, and orally available intervention method in the prior art, which can prevent or relieve high-fat diet-induced colorectal cancer by regulating bile acid metabolism and restoring intestinal microecological balance. SUMMARY

[0006] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide the application of isolithocholic acid in the preparation of a drug for preventing or treating high-fat diet-induced colorectal cancer.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: In a first aspect of the present application, the application of isolithocholic acid or its derivative in the preparation of a drug for preventing or treating cancer is provided.

[0008] In some embodiments of the present application, the dosage form of the drug is a capsule, a tablet, an oral preparation, a microcapsule preparation, an injection, a suppository, a spray, or an ointment.

[0009] In some embodiments of the present application, the cancer includes a cancer of the digestive system caused by high-fat diet.

[0010] In some embodiments of the application, the cancer of the digestive system comprises at least one of colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, and gallbladder cancer.

[0011] In some embodiments of the application, the colorectal cancer refers to a collective term of tumors occurring in the colon or rectum.

[0012] In some embodiments of the application, the colorectal cancer comprises colon adenocarcinoma and mucous adenocarcinoma.

[0013] In some embodiments of the application, the prevention or treatment of colorectal cancer comprises at least one of reducing tumor number, reducing lesion ratio, and inhibiting tumor cell proliferation.

[0014] In some embodiments of the application, the cancer-preventing or -treatment drug comprises at least one of a drug for reducing colorectal tumor number, reducing lesion ratio, and inhibiting tumor cell proliferation.

[0015] In some embodiments of the application, the prevention or treatment of colorectal cancer comprises improving colorectal cancer.

[0016] In some embodiments of the application, the prevention or treatment of colorectal cancer comprises modulating the immune microenvironment of cancer.

[0017] In some embodiments of the application, the prevention or treatment of colorectal cancer comprises improving intestinal barrier function.

[0018] In some embodiments of the application, the reduction of lesion ratio comprises reducing the ratio of colon adenocarcinoma and dysplasia.

[0019] In some embodiments of the application, the isofucoidan derivative comprises at least one of an isofucoidan pharmaceutically acceptable salt and ester.

[0020] In some embodiments of the application, the isofucoidan pharmaceutically acceptable salt comprises at least one of a sodium salt, a potassium salt, and a choline salt of isofucoidan.

[0021] In some embodiments of the application, the drug further comprises a pharmaceutically acceptable excipient.

[0022] In some embodiments of the application, the pharmaceutically acceptable excipient comprises at least one of an excipient, a diluent, a stabilizer, an osmotic pressure regulator, a pH regulator, a preservative, and an antioxidant.

[0023] In some embodiments of the application, the excipient comprises at least one of sucrose, trehalose, glycine, histidine, dextran, polyethylene glycol, and cyclodextrin.

[0024] In some embodiments of the present application, the suspending agent comprises at least one of sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, and microcrystalline cellulose.

[0025] In some embodiments of the present application, the iso-lithocholic acid is used in an amount of 50-100 µg per kg of body weight of the patient.

[0026] In some embodiments of the present application, the medicament further comprises a substance having an anti-inflammatory component for enhancing the barrier repair and anti-inflammatory effects.

[0027] In some embodiments of the present application, the medicament further comprises at least one of a prebiotic and a probiotic for enhancing the barrier repair and anti-inflammatory effects.

[0028] In a second aspect of the present application, there is provided use of a substance for promoting in vivo synthesis of iso-lithocholic acid in the preparation of a medicament for preventing or treating cancer.

[0029] In some embodiments of the present application, the substance for promoting in vivo synthesis of iso-lithocholic acid comprises a substance for promoting expression of an iso-lithocholic acid gene.

[0030] In some embodiments of the present application, the substance for promoting expression of an iso-lithocholic acid gene comprises: (1) an expression vector for promoting synthesis of iso-lithocholic acid; (2) a recombinant cell containing the expression vector of (1).

[0031] The present application has the following beneficial effects: The present application provides use of iso-lithocholic acid in the preparation of a medicament for preventing or treating high-fat-diet-induced colorectal cancer. The present application proves through experiments that oral administration of 50-100 µg / kg of iso-LCA once can achieve accurate replenishment of iso-lithocholic acid. Meanwhile, the present application also proves that iso-lithocholic acid can significantly inhibit the occurrence of obesity-related colon tumors, reduce the proliferation index of tumor cells, and repair the intestinal barrier structure. Iso-lithocholic acid is an endogenous secondary bile acid, which can be chemically or semi-synthetically synthesized and has high safety. As an oral administration, the patient has good compliance, and the present application provides a "nutrition-metabolism-immunity" integrated intervention strategy, which is expected to reduce the incidence of colorectal cancer in obese people and reduce the cost of late-stage surgery and chemotherapy, and has important significance for the treatment of colorectal cancer. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Figure is a result graph of the iso-lithocholic acid gavage dose screening experiment.

[0033] Figure 2 Figure is a flowchart of the experiment of AOM / DSS method for inducing colon tumor formation.

[0034] Figure 3 Figure for the number of colon tumor in mice after isoLCA treatment.

[0035] Figure 4 Figure for the histopathological analysis in mice after isoLCA treatment.

[0036] Figure 5 Figure for the cell proliferation detection in mice after isoLCA treatment.

[0037] Figure 6 Figure for the intestinal barrier function detection in mice after isoLCA treatment.

[0038] Figure 7 Figure for the immune microenvironment regulation detection in mice after isoLCA treatment. DETAILED DESCRIPTION

[0039] The content of the present application is further illustrated in detail by specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels, or can be obtained by prior art methods, unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0040] Example 1 A high-fat diet can cause bile acid metabolism disorder, resulting in lower concentration of isoLCA in the intestines of obese model mice than in normal mice. This example provides a dose screening of isoLCA for gavage in obese model mice. The specific experimental steps are as follows.

[0041] To determine the optimal intervention dose of isoLCA in obese mice, 7-8 week old C57BL / 6 male mice were selected and fed with a high-fat diet for 10 weeks to induce an obesity model.

[0042] HFD-isoLCA group: obese model mice were gavaged with isoLCA (Shanghai Haoyuan Biomedicine Technology Co., Ltd.) at the following doses according to the ratio of isoLCA usage and mouse body weight: 0, 5, 10, 25, 50 and 100 µg / kg.

[0043] ND group (normal diet group): 7-8 week old C57BL / 6 male mice were also selected for normal diet.

[0044] Fecal samples were collected from each group of mice at 18 hours after administration, and then homogenized with PBS buffer (pH 7.2-7.4, 0.01 mol / L) at a ratio of 1 g of feces to 9 mL of PBS buffer. The obtained liquid was centrifuged at 5000 rpm for 15 minutes, and the supernatant was taken for detection of isoLCA content in feces using an isoLCA ELISA detection kit (Shanghai Zymo Biosciences Co., Ltd., #MM-47307M1) according to the instructions.

[0045] The results, as shown in Figure 1 , showed that the fecal isoLCA concentration increased with increasing gavage dose, and reached the physiological level of normal diet mice (≥98% of the reference value) at 100 µg / kg. A single gavage at this dose can restore the fecal isoLCA concentration of obese mice to the physiological level of normal diet mice within 18 hours. High concentrations of isoLCA in the intestine can cause cytotoxicity, increase metabolic stress, and cause signal pathway disorders in the body. In order to avoid the physiological overload of isoLCA in the intestine, 100 µg / kg was used as the intervention dose in subsequent experiments.

[0046] Example 2 This example provides the effect of isoLCA on colon tumor occurrence in high-fat diet-induced obese mice.

[0047] Based on the dose screening results of Example 1, the intervention effect of isoLCA in an obesity-related colon tumor model was further evaluated. Seven to eight-week-old C57BL / 6 male mice were given a high-fat diet for 10 weeks to establish an obesity model, and then the AOM / DSS method (AOM was purchased from Sigma-Aldrich, and the dosage used was 10 mg / kg of body weight, which was dissolved in 0.9% sodium chloride solution (normal saline) when prepared; DSS was purchased from MP Biomedicals) was used to induce colon tumor formation. The mice in different treatment groups were randomly divided into two groups, and the specific treatments are shown below. Figure 2 HFD+isoLCA group: After 10 weeks of high-fat diet to establish an obesity model, 1 injection of AOM was given by intraperitoneal injection at a dosage of 10 mg / kg of body weight, and after the 12th week, 2% (w / v) DSS drinking water was freely drunk for 7 days, and then isoLCA was given by gavage at a dosage of 100 µg / kg every two days on the basis of the high-fat diet for two weeks, followed by the alternation of free drinking of 2% (w / v) DSS drinking water for 7 days and continuous gavage of isoLCA for two weeks, which lasted until the end of the experiment (21st week).

[0048] ​HFD+DMSO group: after the obesity model was established by high-fat diet for 10 weeks, 10 mg / kg AOM was injected intraperitoneally once, and after the 12th week, 2% (w / v) DSS drinking water was freely drunk for 7 days, and after the 13th week, DMSO was given by gavage on the basis of high-fat diet every two days (the amount of gavage was the same as the amount of cholic acid), which lasted for two weeks, and then the treatment of free drinking of 2% (w / v) DSS drinking water for 7 days and gavage of DMSO for two weeks was alternately carried out, which lasted until the end of the experiment (21st week).

[0049] After the end of the experiment, the colon tissue was taken for subsequent pathological and immunological analysis, and the specific experiments are shown as follows: 1. HE staining: (1) Sample preparation and fixation: after the intestinal tissue was rolled, it was placed in 4% (v / v) paraformaldehyde fixing solution, and fixed at room temperature for 24-48 hours. After fixation, the intestinal tissue was washed with running water to remove the surface fixing solution.

[0050] (2) Dehydration and transparency: the tissue block was dehydrated in gradient alcohol: 70% alcohol→80% alcohol→90% alcohol→95% alcohol→100% alcohol (1-2 hours for each concentration). After dehydration, the tissue block was transparent in xylene (2 times, 30 minutes each time) until the tissue block became transparent.

[0051] (3) Wax immersion and embedding: the transparent tissue block was placed in melted paraffin (60-65°C) for 2-3 times, 1-2 hours each time; the tissue block was embedded into a wax block with an embedding box, and cooled to solidify.

[0052] (4) Sectioning and mounting: the wax block obtained in (3) was trimmed, and a 4 μm thick slice was cut by a microtome. The slice was floated in 40°C warm water to flatten, and then was taken out with a glass slide and dried in a 65°C oven.

[0053] (5) De-waxing and hydration: the slice was placed in xylene I, II (10 minutes each)→100% alcohol→95% alcohol→90% alcohol→80% alcohol→70% alcohol (5 minutes for each concentration). Finally, it was washed with distilled water for 5 minutes.

[0054] (6) Hematoxylin staining: the slice was placed in hematoxylin staining solution for 5-10 minutes, washed with running water for 10 minutes, and returned to blue (or differentiated with 1% hydrochloric acid alcohol for a few seconds and then returned to blue again).

[0055] (7) Eosin staining: the slice was placed in eosin staining solution for 1-3 minutes (adjust according to the depth of staining), and then quickly washed with running water for a few minutes to stop the staining.

[0056] (8) Dehydration and transparency: sequentially and rapidly pass through 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, and then put into xylene for transparency twice, each for 5 minutes.

[0057] (9) Mounting and observation: add neutral gum, cover with cover glass, and avoid bubbles. After the mounting agent solidifies, observe under a microscope.

[0058] 2. Immunofluorescence, the specific steps are as follows: (1) Sample preparation and fixation: roll the intestinal tissue and put it into 4% (v / v) paraformaldehyde fixing solution, fix at room temperature for 24-48 hours. After fixation is completed, rinse the intestinal tissue with running water to remove the surface fixing solution.

[0059] (2) Dehydration and transparency: sequentially put the tissue block into gradient alcohol for dehydration: 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, and 100% alcohol (treat for 1-2 hours for each concentration). After dehydration, put into xylene for transparency treatment (2 times, each for 30 minutes) until the tissue block becomes transparent.

[0060] (3) Wax immersion and embedding: put the transparent tissue block into melted paraffin (60-65°C) for 2-3 times, each for 1-2 hours; embed the tissue block into a wax block with an embedding box, and cool to solidify after embedding.

[0061] (4) Sectioning and mounting: trim the wax block obtained in (3) and use a microtome to cut a 4 μm thick slice. Float the slice in 40°C warm water to flatten, then use a glass slide to pick it up and put it into a 65°C oven for drying.

[0062] (5) De-waxing and hydration: sequentially put the slice into xylene I and II (each for 10 minutes), 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol (treat for 5 minutes for each concentration). Finally, wash with distilled water for 5 minutes.

[0063] (6) Antigen repair: after the paraffin section is de-waxed with xylene and rehydrated with gradient alcohol in (5), place it in 0.01 M citric acid buffer (pH 6.0) in a 95°C water bath for 20 min of antigen repair; after cooling to room temperature, cover the tissue with 3% (v / v) H2O2 for 15 min to quench endogenous peroxidase, and rinse with PBS for 3 times, each for 5 min. Then, place the section again in 0.01 M citric acid buffer (pH 6.0) and perform 15 min of antigen repair at 95°C, and then take it out for natural cooling and PBS rinsing.

[0064] After blocking with 5% (w / v) BSA at room temperature for 1 h, rabbit anti-Ki67 antibody (Service, dilution ratio of 1:100), rabbit anti-ZO-1 antibody (Service, dilution ratio of 1:100), anti-IL-17A antibody (Abeam, dilution ratio of 1:100), and anti-CD4 antibody (Invitrogen, dilution ratio of 1:100) were incubated at 4°C overnight, respectively.

[0065] After overnight incubation, the slices were washed with PBS for 3 times, 5 min each time, and then sequentially subjected to signal amplification and labeling using a TSA (Tyramide Signal Amplification) multi-label fluorescent immunohistochemical staining kit (Abeam, #abs50014, HRP-secondary antibody + corresponding fluorescent tyramide) according to the instructions; microwave heat elution was performed after each TSA reaction (the slices were placed in citric acid buffer at 95°C for 10 min) to remove the previous antibody-enzyme complex. After the last round of TSA was completed, the slices were stained with DAPI (Thermo Fisher, #D3571) for 5 min, washed with PBS, and then mounted with an anti-fluorescence quenching mounting medium.

[0066] 3. Flow cytometry, the specific experimental steps are as follows: First, the obtained colon tissues were separated, and the specific experimental steps are as follows: (1) The mouse colon tissues after the above experiment were washed twice in 1x DPBS buffer until no obvious fecal residue was observed.

[0067] (2) The colon tissues were placed in a 1.5 mL EP tube, and the tissues were cut into a paste using sterile scissors and forceps.

[0068] (3) Digestion solution (2.5 mL RPMI (serum-free) medium containing 2 mg / mL collagenase II (Guangzhou Aiskin Biological Technology Co., Ltd.), 2 mg / mL dispase (neutral protease, Roche), and 0.05 mg / mL DNase I (Shanghai Macklin Biochemical Technology Co., Ltd.)) was added, and the tissues were digested for about 30 min.

[0069] (4) After digestion, the tissue suspension was sucked and hit using a Pasteur pipette until the tissues were obviously loose, and then passed through a 100 pm cell sieve, and the supernatant was collected into a 50 mL centrifuge tube.

[0070] (5) The supernatant collected in step (4) was centrifuged at 4°C and 500xg, and the supernatant was discarded.

[0071] (6) The precipitate in step (5) was resuspended with 1 mL of red blood cell lysis solution (Abeam (Shanghai) Biological Technology Co., Ltd.), and incubated on ice for 1 min.

[0072] (7) After the red blood cell lysis solution was cracked red, it was mixed and centrifuged at 4℃, 500xg for 3min, and the supernatant was discarded to obtain the colon tissue cells.

[0073] After obtaining the cells, subsequent flow cytometry was performed, and the specific experimental steps were as follows: (1) After the cells were harvested, they were first incubated with Zombie Violet TM fixable viability dye (Biolegend, 423113) at 4℃ for 15min in the dark to distinguish between dead and live cells; PBS was washed once.

[0074] (2) Surface markers: resuspend the cells in 100 µL staining buffer (PBS containing 2 % (v / v) FBS (fetal bovine serum)), add FITC anti-mouse CD3 antibody (Zhiyuan Biotechnology Co., Ltd., 100203, 1:100), APC anti-mouse CD4 antibody (Zhiyuan Biotechnology Co., Ltd., 100412, 1:100) in turn, incubate at 4℃ for 30min in the dark; wash twice.

[0075] (3) Fixation / membrane rupture: strictly follow the instructions of the Intracellular Fixation & Permeabilization Kit (Elabscience), first fix at room temperature for 40min with Fixation Buffer in the kit, then centrifuge at 600xg for 5min with Permeabilization in the kit.

[0076] (4) Intracellular staining: resuspend the cells after membrane rupture in 100 µL Perm Buffer in the kit, add PE / Cyanine7 anti-mouse IL-17A (Bailaiyuan (Tianjin) Biotechnology Co., Ltd., 506921, dilution ratio 1:50), incubate at room temperature for 30min; resuspend in 300 µL staining buffer after washing twice.

[0077] (5) Machine detection: samples were immediately collected on a Cytoflex S flow cytometer, with forward angle scatter (FSC) and side angle scatter (SSC) gate exclusion of debris, and CD3+ cell population was further selected in the live cell gate (Zombie Violet negative), and CD4+ IL-17A+ cell population was detected. At least 5x10 4 live cells were collected for each sample, and the data was analyzed using FlowJo v10.8 software.

[0078] The results are shown in Figures 3-7 , and the specific analysis is as follows: (1) Compared with the HFD group, the number of colon tumors in the isoLCA treatment group was significantly reduced (P < 0.05) Figure 3 ); (2) Histopathological analysis (HE staining) results showed that the proportion of adenocarcinoma and high-grade dysplasia (HGD) and low-grade dysplasia (LGD) in the colon tissue of the HFD group mice was higher, and the proportion of the above lesions was significantly reduced after isoLCA treatment ( Figure 4 ); (3) Cell proliferation detection (Ki-67 immunofluorescence) results showed that isoLCA gavage significantly reduced the number of Ki-67 positive cells in the colon tissue, indicating that it can inhibit tumor cell proliferation ( Figure 5 ); (4) Intestinal barrier function (ZO-1 immunofluorescence) results showed that isoLCA treatment can significantly improve the HFD-induced damage to the structure of the colon barrier and restore epithelial integrity ( Figure 5 ); (5) Immune microenvironment regulation (flow cytometry and CD4 and IL-17A immunofluorescence) results showed that the proportion of Th17 cells in the colon tumor tissue of the isoLCA treatment group was significantly reduced ( Figures 6-7 ), suggesting that it can regulate the local immune microenvironment and thereby inhibit the occurrence and development of obesity-related colon tumors.

[0079] In summary, isoLCA gavage treatment can effectively alleviate high-fat diet-induced colon tumor occurrence, and its mechanism of action may be related to restoring intestinal barrier function, inhibiting tumor cell proliferation, and regulating the immune microenvironment.

[0080] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of isolithocholic acid or a derivative thereof in the preparation of a medicament for preventing or treating cancer.

2. Use according to claim 1, characterized in that, The cancer includes a cancer of the digestive system caused by a high-fat diet.

3. Use according to claim 2, characterized in that, The cancer of the digestive system includes at least one of colorectal cancer, gastric cancer, esophageal cancer, pancreatic cancer, and gallbladder cancer.

4. Use according to claim 1, characterized in that, The isolithocholic acid derivative includes at least one of a pharmaceutically acceptable salt and an ester of isolithocholic acid.

5. The use according to claim 1, characterized in that, The pharmaceutically acceptable salt of isolithocholic acid includes at least one of a sodium salt, a potassium salt, and a choline salt of isolithocholic acid.

6. Use according to claim 1, characterized in that, The dosage form of the medicament includes a capsule, a tablet, an oral preparation, a microcapsule preparation, an injection, a suppository, a spray, or an ointment.

7. Use according to claim 1, characterized in that, The medicament further includes a pharmaceutically acceptable excipient.

8. Use according to claim 7, characterized in that, The pharmaceutically acceptable excipient includes at least one of an excipient, a diluent, a stabilizer, an osmotic pressure adjusting agent, a pH adjusting agent, a preservative, and an antioxidant.

9. Use of a substance for promoting in vivo synthesis of isolithocholic acid in the preparation of a medicament for preventing or treating cancer.

10. Use according to claim 9, characterized in that, The substance for promoting in vivo synthesis of isolithocholic acid includes a substance for promoting expression of an isolithocholic acid gene; preferably, the substance for promoting expression of the isolithocholic acid gene includes: (1) an expression vector for promoting synthesis of isolithocholic acid; (2) a recombinant cell containing the expression vector of (1).

Images