Application of farrerol in preparation of intestinal micro-ecological regulator and medicine for treating colorectal cancer

Abstract

The invention belongs to the technical field of biology, and discloses application of farrerol in preparation of an intestinal micro-ecological regulator and a medicine for treating colorectal cancer. The farrerol provided by the invention can effectively enhance the tumor immune killing effect of a body by changing the intestinal microecology, inhibiting the growth of tumor cells, regulating and controlling the expression of cell factors, regulating the inflammation level of a tumor tissue microenvironment and other biological activities; the compound has good potential of being used as a medicine for treating the colorectal cancer and / or an enhancer of ICIs for treating the colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of farrerol in the preparation of intestinal microecological regulators and drugs for the treatment of colorectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, and its global incidence has shown an upward trend in recent years. The latest statistical data shows that CRC ranks third in the global incidence of tumors in both men and women. Among newly diagnosed CRC patients, approximately 25% of patients have metastatic lesions, and even after radical surgery, approximately 30% of patients will develop metastasis or recurrence within 5 years after surgery.

[0003] In recent years, immune checkpoint inhibitors (ICIs) have attracted much attention in the field of tumor treatment as a potential systemic treatment method. Immunotherapy drugs such as pembrolizumab and nivolumab have been used in the treatment of CRC and have achieved good therapeutic effects. However, the therapeutic effect of ICIs is limited by the immunosuppression of the tumor microenvironment (TME). A variety of immunosuppressive cells and immunosuppressive cytokines such as regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages present in the TME lead to the exhaustion of tumor-infiltrating lymphocytes (TILs), thereby affecting the immunotherapy effect.

[0004] Therefore, there is an urgent need to explore a drug that can reduce the immune therapy resistance of ICIs. Summary of the Invention

[0005] Based on the problem of poor immunotherapy effect caused by the immunosuppression of the tumor microenvironment in the existing ICIs immunotherapy for colon cancer, the inventors of the present invention, through extensive and in-depth research and a large number of experiments, found that: Farrerol, a typical flavanone isolated from the Chinese medicinal plant Rhododendron dauricum L., can not only directly act on tumor cells to promote apoptosis and inhibit death, directly achieving the anti-tumor effect; but also has the biological activity of regulating the intestinal microecology, by changing the intestinal flora structure and metabolic conditions, to change the colorectal tumor microenvironment, improve the inflammation level of the tumor tissue, thereby enhancing the body's tumor immune killing effect. It is a highly potential enhancer for ICIs in the treatment of colorectal cancer, and can effectively solve the problem of ICIs immunotherapy drug resistance; moreover, Farrerol can also weaken the dominance of the imbalanced intestinal flora in the tumor microenvironment. When used in combination with fecal microbiota transplantation (FMT), it can effectively improve the colonization success rate of healthy donor intestinal flora, and through the synergistic cooperation of the two, correct the flora imbalance and repair the intestinal barrier, improve the intestinal flora metabolic state, regulate the expression of inflammatory factors and cell cycle factors in the tumor tissue, improve the inflammation level of the tumor tissue and inhibit the proliferation of tumor cells, while increasing the expression level of intestinal indole-3-propionic acid (IPA), which acts on the process of increasing the inflammation level of the tumor tissue in reverse, to optimize the inflammation level of the colorectal tumor microenvironment, avoid the occurrence of tumor immune escape while achieving effective tumor killing, and has the potential to be directly applied as a drug for the treatment of colorectal cancer or an ICIs enhancer, which is of great significance for achieving targeted therapy of colorectal cancer. Based on this, the technical solution of the present invention is obtained.

[0006] The first object of the present invention is to provide the use of Farrerol or a pharmaceutically acceptable salt thereof in the preparation of an intestinal microecology regulator for the treatment of colorectal cancer.

[0007] The second object of the present invention is to provide the use of Farrerol or a pharmaceutically acceptable salt thereof in the preparation of an enhancer for ICIs in the treatment of colorectal cancer.

[0008] The third object of the present invention is to provide a probiotic preparation. Specifically, the probiotic preparation includes healthy donor intestinal flora and Farrerol or a pharmaceutically acceptable salt thereof.

[0009] The fourth object of the present invention is to provide the use of the above probiotic preparation in the preparation of an indole-3-propionic acid expression promoter.

[0010] The fifth object of the present invention is to provide the use of the above probiotic preparation in the preparation of a regulator for the inflammation level of the tumor microenvironment

[0011] The sixth object of the present invention is to provide the use of the above probiotic preparation in the preparation of a tumor cell apoptosis promoter.

[0012] The seventh object of the present invention is to provide the application of the above-mentioned microecological preparation in the preparation of a tumor cell proliferation inhibitor.

[0013] The eighth object of the present invention is to provide the application of the above-mentioned microecological preparation in the preparation of an agent for improving the mucosal protection effect of colorectal tissue.

[0014] The ninth object of the present invention is to provide the application of the above-mentioned microecological preparation in the preparation of a drug for the treatment of colorectal cancer.

[0015] The tenth object of the present invention is to provide the application of the above-mentioned microecological preparation in the preparation of an enhancer for ICIs for the treatment of colorectal cancer. Description of the Drawings

[0016] Figure 1 It is a graph showing the experimental results of the change in body weight of mice provided in Example 1 of the present invention;

[0017] Figure 2 It is a graph showing the experimental results of the change in tumor volume of mice provided in Example 1 of the present invention;

[0018] Figure 3 It is a photo of the tumor of mice after the administration ended in Example 1 of the present invention;

[0019] Figure 4 It is a graph showing the experimental results of the tumor mass of mice after the administration ended in Example 1 of the present invention;

[0020] Figure 5 It is a graph showing the experimental results of IHC detection of tumor tissue sections of mice after the administration ended in Example 1 of the present invention (scale bar is 100 μm);

[0021] Figure 6 It is a graph showing the experimental results of IHC scoring of tumor tissue sections of mice after the administration ended in Example 1 of the present invention;

[0022] Figure 7 It is a graph showing the experimental results of HE staining of liver and kidney tissue sections of mice after the administration ended in Example 1 of the present invention (scale bar is 200 μm);

[0023] Figure 8 It is a graph showing the experimental results of species annotation analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0024] Figure 9 It is a graph showing the experimental results of Alpha diversity analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0025] Figure 10Experimental result graph of ANOSIM index comparison analysis for the between-group differences in Beta diversity indices of the intestinal flora of mice provided in Example 1 of the present invention;

[0026] Figure 11 Experimental result graph of PCA similarity analysis for the between-group differences in Beta diversity indices of the intestinal flora of mice provided in Example 1 of the present invention;

[0027] Figure 12 Experimental result graph of PCoA similarity analysis for the between-group differences in Beta diversity indices of the intestinal flora of mice provided in Example 1 of the present invention;

[0028] Figure 13 Experimental result graph of the phylum-level flora species composition of the differential species analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0029] Figure 14 For Figure 13 Experimental result graph of the comparison of the characteristic differential bacterial phylum levels in the intestinal flora of mice in the control group and experimental group 2 provided in

[0030] Figure 15 Experimental result graph of the class-level flora species composition of the differential species analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0031] Figure 16 For Figure 15 Experimental result graph of the comparison of the characteristic differential bacterial class levels in the intestinal flora of mice in the control group and experimental group 2 provided in

[0032] Figure 17 Experimental result graph of the order-level flora species composition of the differential species analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0033] Figure 18 For Figure 17 Experimental result graph of the comparison of the characteristic differential bacterial order levels in the intestinal flora of mice in the control group and experimental group 2 provided in

[0034] Figure 19 Experimental result graph of the family-level flora species composition of the differential species analysis of the intestinal flora of mice provided in Example 1 of the present invention;

[0035] Figure 20 For Figure 19 Experimental result graph of the comparison of the characteristic differential bacterial family levels in the intestinal flora of mice in the control group and experimental group 2 provided in

[0036] Figure 21 For Figure 19Relative abundance map of the Muribaculaceae family of bacteria in the intestinal flora of the control group and experimental group 2 mice provided;

[0037] Figure 22 Experimental result graph of the genus-level flora species composition of the differential species analysis of the intestinal flora of the mice provided in Example 1 of the present invention;

[0038] Figure 23 For Figure 22 Experimental result graph of the comparison of the characteristic differential bacterial genera in the intestinal flora of the control group and experimental group 2 mice provided;

[0039] Figure 24 For Figure 22 Relative abundance map of the Muribaculum genus of bacteria in the intestinal flora of the control group and experimental group 2 mice provided;

[0040] Figure 25 For Figure 22 Relative abundance map of the Escherichia_Shigella genus of bacteria in the intestinal flora of the control group and experimental group 2 mice provided;

[0041] Figure 26 Relative abundance map of the Enterococcus_faecium bacterial species in the intestinal flora of the mice provided in Example 1 of the present invention;

[0042] Figure 27 Histogram of LDA values of the differential species analysis of the intestinal flora of the mice provided in Example 1 of the present invention;

[0043] Figure 28 Phylogenetic tree diagram of the differential species analysis of the intestinal flora of the mice provided in Example 1 of the present invention;

[0044] Figure 29 Schematic diagram of the evolutionary relationship between the intestinal flora species of the mice provided in Example 1 of the present invention;

[0045] Figure 30 Experimental result graph of the difference in the metabolic pathways of the intestinal flora of the mice provided in Example 1 of the present invention based on KEGG analysis;

[0046] Figure 31 Experimental result graph of the difference in the metabolic pathways of the intestinal flora of the mice provided in Example 1 of the present invention based on COG analysis;

[0047] Figure 32 Experimental result graph of the change in the tumor diameter of the mice provided in Example 2 of the present invention;

[0048] Figure 33 Tumor photos of the mice after the administration in Example 2 of the present invention;

[0049] Figure 34 It is the experimental result graph of the tumor weight of the mice provided in Example 2 of the present invention after the administration ends;

[0050] Figure 35 It is the experimental result graph of the IHC detection of the tumor tissue section provided in Example 2 of the present invention (the scale bar is 50μm);

[0051] Figure 36 It is the experimental result graph of the IHC score of CD3 in the tumor tissue section provided in Example 2 of the present invention;

[0052] Figure 37 It is the experimental result graph of the IHC score of CD4 in the tumor tissue section provided in Example 2 of the present invention;

[0053] Figure 38 It is the experimental result graph of the IHC score of CD8 in the tumor tissue section provided in Example 2 of the present invention;

[0054] Figure 39 It is the experimental result graph of the IHC score of Cleaved Caspase-3 in the tumor tissue section provided in Example 2 of the present invention;

[0055] Figure 40 It is the experimental result graph of the IHC score of Cleaved Caspase-9 in the tumor tissue section provided in Example 2 of the present invention;

[0056] Figure 41 It is the experimental result graph of the relative transcription levels of genes related to the intestinal barrier function of the colorectal tissue provided in Example 2 of the present invention;

[0057] Figure 42 It is the experimental result graph of the relative transcription levels of inflammatory cytokines in the tumor tissue provided in Example 2 of the present invention;

[0058] Figure 43 It is the volcano graph of the differential expression of mouse gut microbiota metabolites provided in Example 2 of the present invention;

[0059] Figure 44 It is the clustering heat map of differential metabolites of mouse gut microbiota provided in Example 2 of the present invention;

[0060] Figure 45 It is the experimental result graph of the PCA analysis of differential metabolites of mouse gut microbiota provided in Example 2 of the present invention;

[0061] Figure 46 It is one of the OPLS-DA validation graphs of differential metabolites of mouse gut microbiota provided in Example 2 of the present invention;

[0062] Figure 47 The second OPLS-DA validation graph of the differential metabolites of the mouse intestinal flora provided in Example 2 of the present invention;

[0063] Figure 48 The bar graph of the differential multiples of the differential metabolites of the mouse intestinal flora provided in Example 2 of the present invention;

[0064] Figure 49 The differential abundance score graph of the differential metabolites of the mouse intestinal flora provided in Example 2 of the present invention;

[0065] Figure 50 The bubble graph of the enrichment degree of KEGG analysis of the differential metabolites of the mouse intestinal flora provided in Example 2 of the present invention;

[0066] Figure 51 The bubble graph of the enrichment degree of GO analysis of the differential metabolites of the mouse intestinal flora provided in Example 2 of the present invention;

[0067] Figure 52 The bar graph of the differential metabolic pathways of the mouse intestinal flora provided in Example 2 of the present invention;

[0068] Figure 53 The clustering heat map of the differential metabolic pathways of the mouse intestinal flora provided in Example 2 of the present invention;

[0069] Figure 54 The relative concentration of IPA in the fecal samples of the control group mice provided in Example 2 of the present invention;

[0070] Figure 55 The relative concentration of IPA in the serum samples of the control group mice provided in Example 2 of the present invention;

[0071] Figure 56 The relative concentration of IPA in the fecal samples of the experimental group mice provided in Example 2 of the present invention;

[0072] Figure 57 The relative concentration of IPA in the serum samples of the experimental group mice provided in Example 2 of the present invention;

[0073] Figure 58 The experimental result graph of the correlation analysis of the IPA expression level and the IHC score of CD8 provided in Example 2 of the present invention;

[0074] Figure 59 The experimental result graph of the correlation analysis of the IPA expression level and the relative transcription level of IL-1B provided in Example 2 of the present invention;

[0075] Figure 60It is the experimental result diagram for the correlation analysis of the IPA expression level and the relative transcription level of THF-α provided in Example 2 of the present invention;

[0076] Figure 61 It is the experimental result diagram for the correlation analysis of the IPA expression level and the relative transcription level of IL-6 provided in Example 2 of the present invention;

[0077] Figure 62 It is the experimental result diagram for the correlation analysis of the IPA expression level and the relative transcription level of IL-10 provided in Example 2 of the present invention;

[0078] Figure 63 It is the experimental result diagram for the correlation analysis of the IPA expression level and the relative transcription level of IL-18 provided in Example 2 of the present invention. Detailed implementation mode

[0079] In the present invention, the farrerol refers to a typical flavanone compound isolated from the traditional Chinese medicine plant Rhododendron dauricum L., and its structure is specifically shown in formula (1).

[0080]

[0081] In the present invention, specific examples of the pharmaceutically acceptable salts include but are not limited to: one or more of hydrochloride, sulfate, hydrobromide, hydroiodide, formate, acetate, and oxalate.

[0082] In the first aspect, based on the biological activity of farrerol in regulating the intestinal microecology, the present invention provides the use of farrerol or its pharmaceutically acceptable salt in the preparation of an intestinal microecology regulator for the treatment of colorectal cancer.

[0083] In the present invention, the farrerol can significantly change the intestinal flora morphology, increase the abundances of bacteria such as Escherichia-Shigella, Lachnospiraceae_NK4A136_group, and Bacteroides, and decrease the abundances of bacteria such as Muribaculaceae, Ruminococcaceae_UCG-014, and Akkermansia, so as to correct the intestinal flora imbalance, and can enhance the immune killing effect on tumors, thereby achieving a good therapeutic effect on colorectal cancer.

[0084] In the present invention, the farrerol can significantly change the intestinal flora metabolism, enhance metabolic pathways such as Biosynthesis of vancomycin group antibiotics, Polyketide sugar unit biosynthesis, and Flp pilus assembly protein TadD, contains TPR repeats, etc., so as to increase the inflammatory level of the tumor microenvironment and enhance the immune killing effect on tumors, thereby achieving good therapeutic effects for colorectal cancer.

[0085] In a second aspect, based on the biological activity of farrerol in regulating the expression of inflammatory cytokines and increasing the inflammatory level of tumor tissues, the present invention provides the use of farrerol or a pharmaceutically acceptable salt thereof in the preparation of an enhancer for ICIs for the treatment of colorectal cancer.

[0086] In the present invention, the farrerol can change the colorectal tumor microenvironment by altering the intestinal flora structure and metabolism, increase the inflammatory level of tumor tissues, and thus enhance the tumor immune killing effect of the body.

[0087] In a third aspect, based on the biological activity of farrerol in regulating the intestinal microecology, the present invention provides a microecological preparation.

[0088] In the present invention, the microecological preparation specifically includes the intestinal flora of a healthy donor and farrerol or a pharmaceutically acceptable salt thereof; the farrerol cooperates with the intestinal flora transplantation combined treatment and has the potential to achieve targeted treatment for colorectal cancer.

[0089] In the present invention, specific examples of the dosage form of the microecological preparation include, but are not limited to: one or more of tablets, capsules, oral liquids, granules, suspensions, injections, suppositories, and drops.

[0090] In the present invention, the intestinal flora of the healthy donor specifically refers to the intestinal flora isolated from the feces of a healthy donor screened based on strict criteria, and the specific screening and isolation and purification methods are a technical means commonly used in FMT, and the present invention does not particularly limit it.

[0091] In a fourth aspect, the present invention also provides the use of the above microecological preparation in the preparation of an indole-3-propionic acid expression promoter.

[0092] In the present invention, the simultaneous administration of farrerol and the intestinal flora of healthy donors, that is, the combined treatment of farrerol and FMT, can improve the intestinal flora structure and the metabolism of intestinal flora, strengthen metabolic pathways such as the tryptophan metabolism pathway and the short-chain fatty acid degradation pathway, so as to increase the abundance of indole-3-propionic acid (IPA)-producing bacteria or improve the IPA-producing ability of bacteria, thereby increasing the expression level of IPA, acting on the body's immune killing effect on tumors, and achieving excellent targeted treatment effects for colorectal cancer.

[0093] Fifthly, the present invention also provides the application of the above-mentioned microecological preparation in the preparation of a regulator for the inflammatory level of the tumor microenvironment.

[0094] In the present invention, the simultaneous administration of farrerol and the intestinal flora of healthy donors, that is, the combined treatment of farrerol and FMT, can, while increasing the inflammatory level of tumor tissues, avoid excessive inflammation of the tumor microenvironment through the increase in the IPA expression level, optimize the inflammatory state of the tumor microenvironment, and while achieving a good killing effect on tumor cells, reduce the occurrence of immune escape reactions, thereby achieving excellent targeted treatment effects for colorectal cancer.

[0095] Sixthly, the present invention also provides the application of the above-mentioned microecological preparation in the preparation of an agent for improving the mucosal protection effect of colorectal tissues.

[0096] In the present invention, the simultaneous administration of farrerol and the intestinal flora of healthy donors, that is, the combined treatment of farrerol and FMT, can significantly increase the expression levels of substances related to mucosal protection and barrier integrity such as MUC-2, Occludin, and ZO-1, improve the mucosal protection effect of colorectal tissues, and achieve excellent targeted treatment effects for colorectal cancer.

[0097] Seventhly, the present invention also provides the application of the above-mentioned microecological preparation in the preparation of a drug for the treatment of colorectal cancer.

[0098] Eighthly, the present invention also provides the application of the above-mentioned microecological preparation in the preparation of an enhancer for ICIs used in the treatment of colorectal cancer.

[0099] The following details the embodiments of the present invention. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those without specific technical or conditions noted in the examples, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments without the manufacturer noted, they are all conventional products that can be obtained through commercial purchase.

[0100] Example 1

[0101] This example is used to illustrate the application potential of farrerol in the preparation of drugs for the remission and / or treatment of colon cancer, specifically including:

[0102] 1. Preparation of mouse colon cancer cell suspension: Mouse colon cancer cells MC38 were inoculated into RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin, and cultured at 37 °C and 5% CO 2 until the cell density reached 80% - 90%. Then, trypsin digestion was carried out and a mouse colon cancer cell suspension with a density of 1×10 7 cells / mL was prepared.

[0103] 2. Construction of mouse model: (1) 6 - 8 - week - old male BALB / c mice (purchased from Shanghai Slac Laboratory Animal Center) were used as experimental subjects (N = 18), and were raised in a clean - grade animal breeding room. The breeding conditions included: average temperature of 22 ± 2 °C, relative humidity of 45 - 65%, 12h / 12h day - night alternation, and were adaptively raised for 7 days.

[0104] (2) After the adaptive breeding was completed (recorded as day 0), 100 μL of cell suspension per mouse was taken and inoculated subcutaneously into the right limb of the mouse, and the mice were raised for 14 days under the same breeding conditions as above (the tumor volume was about 100 mm 3 ). A CRC - bearing mouse model was constructed and randomly divided into three groups (6 mice in each group), and the following operations were carried out for treatment:

[0105] (i) Control group: 0.2 mL of 0.9% normal saline was used to gavage the mice once a day for 12 consecutive days;

[0106] (ii) Experimental group 1 - low - dose farrerol: 0.9% normal saline was mixed with farrerol to prepare a drug solution. 0.2 mL of the drug solution was taken according to the dosage of 10 mg / kg and used to gavage the mice once a day for 12 consecutive days;

[0107] (iii) Experimental group 2 - high - dose farrerol: 0.9% normal saline was mixed with farrerol to prepare a drug solution. 0.2 mL of the drug solution was taken according to the dosage of 30 mg / kg and used to gavage the mice once a day for 12 consecutive days.

[0108] 3. Mouse body weight and tumor growth: The mouse body weight was recorded before inoculating mouse colon cancer cells MC38. After administration, the body weight and tumor size of the nude mice were measured once every 3 days [tumor volume calculation formula: V=(long diameter × short diameter 2 ) / 2]. The nude mice were sacrificed on the 10th day after the gavage ended, and the tumor tissues were taken out and weighed. The results are as Figures 1 - 4 shown.

[0109] ByFigures 1 - 4 As can be seen from the results shown, compared with the control group, after treating mice with farrerol in experimental groups 1 and 2, the tumor growth of the mice was inhibited, and the tumor growth rate of the mice treated with high-dose farrerol was significantly slower than that of the low-dose group, that is, farrerol has significant anti-tumor activity, and its anti-tumor activity is dose-dependent.

[0110] 4. Immunohistochemical detection of mouse tumor tissues: After washing the tumor tissues with 0.9% normal saline and removing the residual moisture with filter paper, fix them with 4% paraformaldehyde, embed them in paraffin, and prepare tissue sections (4 μm) for immunohistochemical detection of cleaved caspase-3, CDK4, and CDK6. The results are as Figure 5 and 6 shown.

[0111] From Figure 5 and 6 As can be seen from the results shown, compared with the control group, after treating mice with farrerol in experimental groups 1 and 2, the expression level of Cleaved Caspase-3 was significantly increased, indicating that farrerol can promote the apoptosis of tumor cells by increasing the expression of Cleaved Caspase-3; while the expression levels of CDK4 and CDK6 were significantly decreased, indicating that farrerol can inhibit the progression of tumor cells from the G1 phase to the S phase by inhibiting the expression of CDK4 and CDK6, so as to inhibit the proliferation of tumor cells. That is, farrerol has excellent biological activities of promoting tumor cell apoptosis and inhibiting tumor cell cycle progression, and it is a very promising anti-colorectal cancer therapeutic drug.

[0112] Moreover, by comparing experimental groups 1 and 2, it can be seen that high-dose farrerol shows better effects in promoting tumor cell apoptosis and inhibiting tumor cell cycle. That is, the anti-tumor activity of farrerol is dose-dependent to a certain extent.

[0113] 5. HE staining of mouse kidney and liver tissues: Dissect the kidneys and livers from the abdominal cavity of the mice, wash the kidneys and livers with 0.9% normal saline, remove the residual moisture of the kidneys and livers with filter paper, fix them with 4% paraformaldehyde, embed them in paraffin, and prepare tissue sections (4 μm). Perform HE staining on the kidney tissue sections and liver tissue sections of the mice. The results are as Figure 7 shown.

[0114] From Figure 7As can be seen from the results shown, in the mice of experimental groups 1 and 2, no obvious pathological changes were observed in the liver and kidney tissues, the cell morphology was intact, the structure of the central vein and portal area was normal, and no obvious pathological changes such as hepatocyte swelling, vacuolar degeneration or inflammatory cell infiltration were seen, indicating that farrerol does not produce liver and kidney toxicity at therapeutic doses, has good safety, and can be well applied to the treatment of colorectal cancer mice.

[0115] 6. Bioinformatics analysis of the intestinal flora of mice: (1) After the administration was completed, the feces of the mice in the control group and experimental group 2 were collected with sterile forceps, and DNA was extracted from the mouse feces using a DNA extraction kit (QIAGEN, catalog number 51304) and referring to the instructions. After verifying the DNA purity, concentration and integrity by spectrophotometry and gel electrophoresis, PCR amplification was performed using specific 16S V3-V4 region primers with Barcode to obtain PCR products.

[0116] (2) After purifying the PCR products, Illumina MiSeq PE300 was used for sequencing. The obtained raw data was processed using QIIME software (v1.8.0), clustering analysis was performed on the representative sequences of operational taxonomic units OTUs at 97% similarity level, and species annotation analysis was performed in the Silva database (v123) according to each classification level. The results are as Figure 8 shown.

[0117] As Figure 8 can be seen from the results shown, there were 1371 identical OTUs in the feces of the mice in the control group and experimental group 2, and these OTUs represented the core bacterial community members that might exist in the intestines of all mice; while the intestinal flora of the control group mice contained 901 unique OTUs, and the intestinal flora of the mice treated with 30 mg / kg farrerol contained 522 unique OTUs, indicating that the administration of farrerol had a significant impact on the composition of the intestinal flora of mice.

[0118] (3) The Chao1, Shannon, Simpson, and Ace indices were calculated using Qiime 2 software, and Alpha diversity analysis was performed using R software. The results are as Figure 9 shown.

[0119] As Figure 9 can be seen from the results shown, compared with the control group, the α diversity of the intestinal flora in experimental group 2 was significantly reduced after the mice were treated with 30 mg / kg farrerol.

[0120] (4) Principal components analysis (PCA) and principle coordination analysis (PCoA) were performed using R software, and the between-group differences in beta diversity indices were analyzed. The results are as Figures 10 - 12 shown.

[0121] As can be seen from the Figures 10 - 12 results shown, there were significant differences in the beta diversity indices PCA and PCoA of the intestinal flora of mice in the control group and experimental group 2 (P < 0.05).

[0122] (5) Differential species analysis of the intestinal flora of mice in the control group and experimental group 2 was performed using R software. The results are as Figures 13 - 29 shown.

[0123] As can be seen from the Figure 13 and 14 results shown, at the phylum level, there were significant differences in the composition of the intestinal flora of mice in the control group and experimental group 2. After treatment with 30 mg / kg of farrerol, the abundances of bacteria in phyla such as Bacteroidetes and Verrucomicrobia in the intestinal flora of mice decreased, while the abundances of bacteria in phyla such as Firmicutes and Proteobacteria increased.

[0124] As can be seen from the Figure 15 and 16 results shown, at the class level, there were significant differences in the composition of the intestinal flora of mice in the control group and experimental group 2. After treatment with 30 mg / kg of farrerol, the abundances of bacteria in classes such as Bacteroidia and Verrucomicrobiae in the intestinal flora of mice decreased, while the abundances of bacteria in classes such as Clostridia and Gammaproteobacteria increased.

[0125] As can be seen from the Figure 17 and 18 results shown, at the order level, there were significant differences in the composition of the intestinal flora of mice in the control group and experimental group 2. After treatment with 30 mg / kg of farrerol, the abundances of bacteria in orders such as Bacteroidales and Verrucomicrobiales in the intestinal flora of mice decreased, while the abundances of bacteria in orders such as Clostridiales and Enterobacteriales increased.

[0126] As can be seen from the Figures 19 - 21As can be seen from the results shown, at the family level, there were significant differences in the intestinal flora composition of mice between the control group and experimental group 2. After treatment with 30 mg / kg of farrerol, the abundances of bacteria in families such as Muribaculaceae and Akkermansiaceae in the intestinal flora of mice decreased, while the abundances of bacteria in families such as Enterobacteriaceae and Lachnospiraceae increased.

[0127] From Figures 22 - 25 As can be seen from the results shown, at the genus level, there were significant differences in the intestinal flora composition of mice between the control group and experimental group 2. After treatment with 30 mg / kg of farrerol, the abundances of bacteria in genera such as Muribaculaceae, Ruminococcaceae_UCG-014, and Akkermansia in the intestinal flora of mice decreased, while the abundances of bacteria in genera such as Escherichia-Shigella, Lachnospiraceae_NK4A136_group, and Bacteroides increased.

[0128] From Figure 26 As can be seen from the results shown, at the species level, compared with the control group, the abundance of the bacterial species Enterococcus_faecium in the intestinal flora of mice treated with 30 mg / kg of farrerol was significantly increased.

[0129] From Figures 27 - 29 As can be seen from the results shown, there were 41 characteristic differences in the intestinal flora of mice in the control group, while there were 25 characteristic differences in the intestinal flora of mice in experimental group 2; that is, the administration of farrerol could significantly change the morphology of the intestinal flora of mice.

[0130] 7. Metabolomics analysis of the intestinal flora of mice: Take 0.3 g of mouse fecal samples, mix them thoroughly with 5 mL of 70% ethanol, centrifuge them at 4°C and 14,000 g, and collect the supernatant; then add an appropriate amount of 2-ethylbutyric acid solution, derivatize the samples, separate the components of the samples using high-performance liquid chromatography, and then each single component will enter the ion source of a high-vacuum mass spectrometer for ionization, separate them according to the mass-to-charge ratio (m / z) to obtain a mass spectrum, and perform microbial metabolic pathway analysis of the mass spectrometry data of the samples based on KEGG and COG. The conditions for processing, detecting, and analyzing the feces of the two groups of mice, the control group and experimental group 2, are kept consistent. The results are as Figure 30 and 31 shown in Table 1.

[0131] Table 1.

[0132]

[0133]

[0134] As can be seen from Figure 30 the results shown, metabolic pathways such as Biosynthesis of vancomycin group antibiotics and Polyketide sugar unit biosynthesis were significantly enriched in the intestinal flora of control group mice, while metabolic pathways such as Vibrio cholerae pathogenic cycle were significantly enriched in the intestinal flora of experimental group 2 mice.

[0135] As can be seen from Figure 31 the results shown in Table 1, metabolic pathways such as Flp pilus assembly protein TadD, contains TPR repeats (COG5010) were significantly enriched in the intestinal flora of control group mice, while metabolic pathways such as Proline dehydrogenase (COG0506) were significantly enriched in the intestinal flora of experimental group 2 mice.

[0136] Example 2

[0137] This example is used to illustrate the application potential of farrerol combined with fecal microbiota transplantation (FMT) in the treatment of colon cancer, specifically including:

[0138] 1. Preparation of fecal microbiota suspension of healthy mice: Collect fresh feces of healthy adult mice without any treatment and mix them into a sterile centrifuge tube; resuspend the collected fresh feces at a ratio of 120 mg: 1 mL in 0.9% sterile physiological saline; centrifuge at 4°C and 1200 rpm for 3 min, and take the supernatant to obtain the fecal microbiota suspension of healthy mice.

[0139] 2. Preparation of mouse colon cancer cell suspension: Inoculate mouse colon cancer cells MC38 in RPMI 1640 medium containing 10% (v / v) fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin, and culture at 37°C and 5% CO 2 under the condition until the cell density reaches 80% - 90%, and perform trypsin digestion and prepare a mouse colon cancer cell suspension with a density of 1×10 7 cells / mL.

[0140] 3. Construction of mouse model: (1) Use 6 - 8-week-old male BALB / c mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd.) as experimental subjects (N = 10), and raise them in a clean-grade animal breeding room. The breeding conditions include: average temperature of 22 ± 2°C, relative humidity of 45 - 65%, 12h / 12h day-night alternation, and perform adaptive breeding for 7 days.

[0141] (2) After the adaptive feeding ended, a broad-spectrum antibiotic mixture was administered by gavage for 14 consecutive days to eliminate endogenous flora, preparing for subsequent fecal microbiota transplantation (FMT). The cell suspension was taken at an inoculation volume of 100 μL per mouse and inoculated subcutaneously into the right hind limb of the mouse, and the mouse was raised for 7 days under the same feeding conditions as above to construct a CRC tumor-bearing mouse model. The mice were evenly divided into two groups (6 mice in each group) according to the average tumor volume of the mice, and the following operations were performed:

[0142] (i) Control group: 0.9% normal saline was mixed with farrerol to prepare a drug solution. 0.2 mL of the drug solution was taken at a dosage of 30 mg / kg and administered to the mice by gavage. At the same time, 0.2 mL of 0.9% normal saline was administered by gavage, once a day for 12 consecutive days;

[0143] (ii) Experimental group: 0.9% normal saline was mixed with farrerol to prepare a drug solution. 0.2 mL of the drug solution was taken at a dosage of 30 mg / kg and administered to the mice by gavage. At the same time, 0.2 mL of fecal bacteria solution from healthy mice was administered by gavage, once a day for 12 consecutive days.

[0144] 4. Mouse body weight and tumor growth: The body weight of the mice was recorded before inoculating mouse colon cancer cells MC38. After administration, the tumor size of the nude mice was measured every 3 days. On the 17th day after the gavage ended, the nude mice were sacrificed, and the tumor tissues were taken out and weighed. The results are as Figures 32 - 34 shown.

[0145] From Figures 32 - 34 the results shown, it can be seen that compared with the control group, in the experimental group, farrerol and FMT treatment were combined to treat the mice, and the tumor growth rate of the mice slowed down, and the diameter and weight of the finally formed tumors were significantly reduced, indicating that farrerol and FMT treatment can cooperate synergistically to achieve a better effect of alleviating or treating colorectal cancer.

[0146] 5. Immunohistochemical detection of mouse tumor tissues: After washing the tumor tissues with 0.9% normal saline and sucking off the residual moisture with filter paper, they were fixed with 4% paraformaldehyde and then embedded in paraffin to prepare tissue sections (4 μm), and immunohistochemical detection of CD3, CD4, CD8, cleaved caspase-3 and cleaved caspase-9 was performed. The results are as Figures 35 - 40 shown.

[0147] From Figures 35 - 40As can be seen from the results shown, compared with the control group, in the experimental group, treating mice with the combination of farrerol and FMT therapy, there were no significant changes in the expressions of CD3 and CD4 in the colon tissues of mice, while the expression levels of CD8, Cleaved Caspase-3, and Cleaved Caspase-9 were significantly increased, indicating that farrerol and FMT can cooperate synergistically to enhance CD8+ T cells and increase the expressions of Cleaved Caspase-3 and Cleaved Caspase-9 to achieve the effects of killing tumor cells and promoting tumor cell apoptosis, showing excellent anti-tumor treatment effects.

[0148] 6. Transcriptomic detection of mouse colorectal and tumor tissues: Dissect the colorectal tissues from the mouse abdominal cavity, wash them with 0.9% saline and blot the residual moisture with filter paper, then extract total RNA from the colorectal and tumor tissues using TRIzol reagent (Invitrogen, catalog number 15596018CN), perform specific qPCR reactions for genes related to intestinal barrier function and inflammatory cytokines using the Vazyme reverse transcription kit and referring to the instructions, and calculate the relative transcription levels according to the 2-ΔΔCt method. The results are as Figure 41 and 42 shown.

[0149] From Figure 41 the results shown, it can be seen that compared with the control group, after treating mice in the experimental group with the combination of farrerol and FMT therapy, the relative transcription levels of MUC-2, Occludin, and ZO-1 related to mucosal protection and barrier integrity in the colorectal tissues of mice were significantly increased, the relative transcription levels of anti-inflammatory factors such as COX2 and iNOS2 were significantly decreased, and the expression level of CALP related to the regulation of intracellular signal transduction pathways was also decreased.

[0150] From Figure 42 the results shown, it can be seen that compared with the control group, after treating mice in the experimental group with the combination of farrerol and FMT therapy, the relative transcription levels of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, IL-18, P21, and P27 in the tumor tissues of mice were significantly increased, the relative transcription levels of anti-inflammatory cytokines such as IL-10 were significantly decreased, the relative transcription levels of cell cycle factors such as P21 and P27 that inhibit tumor cell proliferation were significantly increased, and the relative transcription levels of cell cycle factors such as CDK4, CDK6, Cyclin D1, Cyclin B1, Ki67, and pHH3 that promote tumor cell proliferation were significantly decreased.

[0151] Comprehensively Figure 41 and 42As can be seen from the results shown, the combined treatment of farrerol and FMT can ultimately achieve excellent targeted therapeutic effects for colorectal cancer by improving the mucosal protection of colorectal tissues, increasing the inflammatory level of tumor tissues, and directly inhibiting the proliferation of tumor cells.

[0152] 7. Metabolomics analysis and metabolic pathway enrichment analysis of the intestinal flora of mice: (1) Take 0.3 g of mouse fecal samples and mix them thoroughly with 5 mL of 70% ethanol, centrifuge at 4°C and 14,000 g, and collect the supernatant; then add an appropriate amount of 2-ethylbutyric acid solution, derivatize the sample, separate the components of the sample using high-performance liquid chromatography, and then each single component will enter the ion source of a high-vacuum mass spectrometer for ionization, separate it according to the mass-to-charge ratio (m / z) to obtain a mass spectrum, and perform metabolomics analysis and metabolic pathway enrichment analysis on the mass spectrometry data of the sample. The conditions for processing, detecting, and analyzing the feces of mice in the control group and the experimental group are kept consistent. The results are as Figures 43 - 53 shown.

[0153] From Figures 43 - 48 the results shown, it can be seen that among the intestinal flora of mice in the control group and the experimental group, there are 638 significantly up-regulated metabolites, 705 significantly down-regulated metabolites, and 3,662 non-significantly different metabolites. And OPLA-DA is used to analyze and compare the metabolites between the control group and the experimental group. The results show that there is no overlapping phenomenon between the two groups of samples, and the separation effect of the two groups is good. The parameters of the permutation test for the OPLS-DA model are: R2Y = 1, Q2 = 0.938, and the model has very good reliability.

[0154] From Figures 49 - 53 the results shown, it can be seen that there are significant differences in metabolic pathways such as the tryptophan metabolism pathway and the short-chain fatty acid degradation pathway among the intestinal flora of mice in the control group and the experimental group.

[0155] 8. Detection of the concentration of indole-3-propionic acid (IPA) in the serum and feces of mice: (1) Take blood from the eyes of mice, immediately put the blood into a 1.5 mL centrifuge tube, let it stand at room temperature for 60 min, then centrifuge at 2,000 rpm for 15 min, and collect the supernatant to obtain serum samples; detect the IPA in the fecal samples and serum samples of mice. The results are as Figures 54 - 57 shown.

[0156] From Figures 54 - 57 the results shown, it can be seen that compared with the control group treated with 30 mg / kg farrerol alone, the concentration of IPA in the feces and serum of mice treated with the combined treatment of farrerol and FMT increased significantly.

[0157] (2) Further comparative analysis based on the IPA concentration, immunohistochemical detection, and transcriptomic detection results obtained the correlation between indole-3-propionic acid (IPA) and substances such as CD8 and inflammatory cytokines. The results are as Figures 58 - 63 shown.

[0158] As Figures 58 - 63 shown by the results, contrary to the current general view that IPA has an anti-inflammatory effect, in the mice treated with rhododendrol and FMT, IPA was significantly positively correlated with the expression of substances such as CD8, IL-1β, TNF-α, IL-6, and IL-18 in the tumor tissues of mice, and significantly negatively correlated with IL-10.

[0159] Based on Figures 43 - 63 the results shown, during the combined treatment of rhododendrol and FMT, through the synergistic cooperation of rhododendrol and FMT, it is possible to change the original intestinal flora structure of the body to correct the flora imbalance and repair the intestinal barrier, improve the metabolic state of the intestinal flora, and also regulate the expression of inflammatory factors and cell cycle factors in tumor tissues, increase the inflammatory level of tumor tissues and inhibit the proliferation of tumor cells. At the same time, the change in the metabolic state of the intestinal flora leads to an increase in the expression level of IPA, which acts on the process of increasing the inflammatory level of tumor tissues to optimize the inflammatory level of the tumor microenvironment, achieve effective killing of tumors while avoiding the occurrence of tumor immune escape, enhance the performance of ICIs in the body, and thus achieve a better therapeutic effect for colorectal cancer.

[0160] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. Use of rhododendron or a pharmaceutically acceptable salt thereof in the preparation of an intestinal microecological regulator for the treatment of colorectal cancer.

2. Use of rhododendrin or a pharmaceutically acceptable salt thereof in the preparation of an enhancer of ICIs for the treatment of colorectal cancer.

3. A microecological preparation, characterized in that: The microecological preparation comprises intestinal flora from healthy donors and rhododendron or a pharmaceutically acceptable salt thereof.

4. Use of the probiotic preparation according to claim 3 in the preparation of an indole-3-propionic acid expression promoter.

5. Use of the proecological preparation according to claim 3 in the preparation of a regulator of tumor microenvironment inflammation level.

6. Use of the probiotic preparation according to claim 3 in the preparation of a tumor cell apoptosis promoter.

7. Use of the probiotic preparation according to claim 3 in the preparation of a tumor cell proliferation inhibitor.

8. Use of the probiotic preparation according to claim 3 in the preparation of an agent for improving the protective effect of colorectal tissue mucosa.

9. Use of the probiotic preparation according to claim 3 in the preparation of drugs for treating colorectal cancer.

10. Use of the probiotic preparation according to claim 3 in the preparation of an enhancer of ICIs for the treatment of colorectal cancer.

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