Traditional Chinese medicine compound for preventing and treating colorectal cancer precancerous lesions and animal model verification method

Abstract

The invention relates to a traditional Chinese medicine compound for preventing and treating colorectal cancer precancerous lesions and an animal model verification method. The traditional Chinese medicine compound comprises the following components in parts by weight: 25-35 parts of semen coicis, 8-10 parts of radix aconiti carmichaeli, 12-18 parts of herba patriniae, 10-15 parts of radix et rhizoma rhei, 2-4 parts of cortex moutan, 8-10 parts of semen persicae, 25-35 parts of wax gourd kernels and 8-10 parts of mirabilite. The traditional Chinese medicine compound is based on the classical prescription of the coix lacryma-jobi radix aconiti carmichaeli dahurian patrinia powder and is combined with the prescription of the radix et rhizoma rhei and peony soup, so that the defect that the coix lacryma-jobi radix aconiti carmichaeli dahurian patrinia powder is insufficient in blood stasis removing and stagnation eliminating effects is overcome; finally, a spontaneous colorectal cancer mouse model shows a good inhibition effect on colorectal cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and in particular relates to a traditional Chinese medicine compound for preventing and treating colorectal precancerous lesions and an animal model verification method. Background Art

[0002] Colorectal cancer (CRC) is one of the common malignant tumors of the digestive system. Its incidence rate ranks second in my country and its mortality rate ranks fourth, showing an increasing trend year by year. About 85% to 90% of colorectal cancers are the result of the evolution of normal glandular cells-proliferative adenoma-tubular adenoma-villous adenoma-early cancer pattern. Colorectal adenomas (CRA), as the most important precancerous lesions of colorectal cancer, are prone to malignant transformation and recurrence, and are considered to be a key stage in the primary prevention of colorectal cancer.

[0003] Traditional Chinese medicine classifies colorectal adenoma as "intestinal tumor", "intestinal cyst", "polyp hemorrhoids" and other categories. The disease is located in the large intestine and is related to the liver and stomach. The key to the pathogenesis is the deficiency of qi in the middle, qi stagnation, phlegm and blood stasis in the large intestine, forming adenoma, which becomes turbid and toxic over time and turns into cancer. Traditional Chinese medicine often uses Chinese medicines that replenish qi and strengthen the spleen, promote blood circulation and remove blood stasis, and remove dampness and turbidity to treat colorectal adenoma.

[0004] Yiyifuzibaijiangsan is a classic anti-tumor Chinese medicine compound. It comes from the Synopsis of the Golden Chamber and is a classic prescription for treating intestinal carbuncle by Zhang Zhongjing. It is used to discharge pus, reduce swelling, and invigorate Yang Qi. In addition, studies have shown that Yiyifuzibaijiangsan can inhibit the occurrence and development of colorectal cancer by improving the diversity of intestinal flora, reducing NF-κB pathway activation, and regulating cytokine release. However, in actual use, although Yiyifuzibaijiangsan has the effect of tonifying deficiency and dispersing nodules, its ability to remove blood stasis and relieve stagnation is insufficient, and the complex pathogenic factors make Yiyifuzibaijiangsan alone insufficient to meet the needs of disease prevention and treatment. Moreover, there is a lack of direct and effective verification of the inhibitory and preventive effects of related Chinese medicine prescriptions on colorectal cancer.

[0005] Therefore, a new type of Chinese herbal compound that can prevent and treat colorectal precancerous lesions remains to be developed, and its efficacy must be specifically verified. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a Chinese medicine compound for preventing and treating colorectal precancerous lesions and an animal model verification method.

[0007] The purpose of the present invention can be achieved by the following technical solutions:

[0008] The present invention provides a traditional Chinese medicine compound for preventing and treating precancerous lesions of colorectal cancer. The traditional Chinese medicine compound comprises the following components in parts by weight: 25-35 parts of coix seed, 8-10 parts of aconite, 12-18 parts of patrinia scabiosaefolia, 10-15 parts of rhubarb, 2-4 parts of moutan bark, 8-10 parts of peach kernel, 25-35 parts of semen benincasae, and 8-10 parts of mirabilite.

[0009] Further, the traditional Chinese medicine compound comprises the following components in parts by weight: 28-32 parts of coix seed, 8-10 parts of aconite, 14-16 parts of patrinia scabiosaefolia, 11-13 parts of rhubarb, 2-4 parts of moutan bark, 8-10 parts of peach kernel, 28-32 parts of semen benincasae, and 8-10 parts of mirabilite.

[0010] Further, the traditional Chinese medicine compound comprises the following components in parts by weight: 30 parts of coix seed, 9 parts of aconite, 15 parts of patrinia scabiosaefolia, 12 parts of rhubarb, 3 parts of moutan bark, 9 parts of peach kernel, 30 parts of semen benincasae, and 9 parts of mirabilite.

[0011] The traditional Chinese medicine compound of the present invention is based on the classical prescription of Yiyiren Fuzibaijiang Powder and combines with the prescription of Dahuang Mudan Decoction, making up for the deficiency of Yiyiren Fuzibaijiang Powder in promoting blood circulation and dredging stasis. The two prescriptions are combined to form the traditional Chinese medicine prescription of the present invention, which can dredge stasis and replenish deficiency, treating both excess and deficiency simultaneously, and finally showing a good inhibitory effect on colorectal cancer.

[0012] Among them, the disease treated by Yiyiren Fuzibaijiang Powder belongs to the syndrome caused by deficiency of yang qi and internal binding of cold dampness and blood stasis. In the prescription, coix seed is used in large doses to strengthen the spleen, promote diuresis, and eliminate swelling and discharge pus; combined with patrinia scabiosaefolia to clear heat, activate blood circulation, and remove stasis and discharge pus, and aconite is used in small doses to warm and tonify the spleen and kidney, assist yang to promote qi stagnation. The whole prescription plays the role of warming yang and dissipating stasis, removing stasis and reducing swelling. In Dahuang Mudan Decoction, rhubarb and moutan bark are used as the main drugs to purge heat, remove stasis, cool blood and disperse blood stasis. Then, mirabilite is used to soften hardness and disperse stasis, break retained blood and disperse evil blood, peach kernel is used to break blood and promote blood circulation, and semen benincasae is used to clear heat and promote pus discharge. The whole prescription plays the role of purging heat, removing stasis, and dissipating swelling and mass.

[0013] Further, the dosage form of the traditional Chinese medicine compound is one of decoction, tablet, granule, capsule, pill or paste.

[0014] Further, the preparation method of the decoction comprises the following steps: weighing each traditional Chinese medicine component according to the parts by weight, soaking in clear water for later use except for mirabilite; boiling with strong fire and then continuing to decoct with slow fire, pouring out the decoction for standby; adding mirabilite to the medicinal residues, adding water again for decoction, and pouring out the decoction for standby; mixing the two decoctions evenly to obtain the traditional Chinese medicine compound.

[0015] Furthermore, the soaking time in clear water is 25-35 minutes, preferably 30 minutes.

[0016] Furthermore, the decocting time with slow fire is 30-50 minutes, preferably 40 minutes.

[0017] Furthermore, the medicinal liquids obtained from the two decoctions are mixed evenly at a ratio of 1:1.

[0018] The present invention also provides a method for verifying an animal model of a traditional Chinese medicine compound. The construction of the animal model includes the following steps:

[0019] S1: Select genetically engineered mice Villin-CreERT2 Kras G12D / + Apc flox / + as experimental subjects, and use a combination of antibiotics to eliminate the intestinal flora to construct pseudo-axenic mice;

[0020] S2: Establish a total of four experimental groups, namely, a group of genetically engineered mice, a group of genetically engineered mice administered with drugs, a group of pseudo-axenic mice, and a group of pseudo-axenic mice administered with drugs;

[0021] S3: Inject tamoxifen into the tail veins of the mice in the above experimental groups to induce the spontaneous appearance of tumors in the colorectal region;

[0022] S4: Administer drugs in groups. Among them, the group of genetically engineered mice and the group of pseudo-axenic mice are administered with normal saline by gavage, and the group of genetically engineered mice administered with drugs and the group of pseudo-axenic mice administered with drugs are administered with the traditional Chinese medicine compound by gavage;

[0023] S5: Administer drugs for 8 weeks, observe the conditions of the mice, dissect the intestinal tissues of the mice, and verify the therapeutic effect.

[0024] Furthermore, in step S1, the combination of antibiotics is composed of 1 mg / mL ampicillin, 1 mg / mL neomycin sulfate, 1 mg / mL metronidazole, and 0.5 mg / mL vancomycin.

[0025] Furthermore, in step S3, the injection concentration of tamoxifen is 1 mg / mL.

[0026] Furthermore, in step S4, the traditional Chinese medicine compound is a concentrated decoction of water decoction with a concentration of 0.35 - 0.4 g / ml.

[0027] Furthermore, the period of gavage is 8 weeks, 0.4 ml each time, once a day.

[0028] Furthermore, the traditional Chinese medicine compound inhibits the occurrence of colorectal cancer by reducing the infiltration of M2 macrophages and histone deacetylase mediated by the intestinal flora metabolite butyric acid.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The traditional Chinese medicine compound of the present invention is based on the classic prescription of Yiyifu Baojiang Powder and combines with the prescription of Dahuang Mudan Decoction, making up for the deficiency of Yiyifu Baojiang Powder in promoting blood circulation and dredging stasis. The combination of the two forms the traditional Chinese medicine prescription of the present invention, which can dredge stasis and replenish deficiency, combining attacking and tonifying, and finally shows a good inhibitory effect on colorectal cancer.

[0031] (2) The present invention constructs pseudo-sterile mice and a spontaneous colorectal cancer model by using genetically engineered mice to verify the therapeutic effect of the traditional Chinese medicine compound of the present invention, further confirming the therapeutic potential of the traditional Chinese medicine compound of the present invention.

[0032] (3) The traditional Chinese medicine compound of the present invention has a significant inhibitory effect on intestinal tumors in mice with a spontaneous colorectal cancer model, plays a certain protective role in intestinal tissues, and experimental verification shows that the inhibitory effect of the traditional Chinese medicine compound of the present invention on colorectal cancer may be achieved by down-regulating the expression of HDAC1, inhibiting the activation of the TLR4 / MyD88 / NF-κB signaling pathway, and preventing the polarization of M2-type TAMs, which also provides a new idea for further studying the therapeutic targets of colorectal cancer. Brief Description of the Drawings

[0033] Figure 1 It is the test result of the in vitro cell experiment of the traditional Chinese medicine compound of the present invention.

[0034] Figure 2 It is the schematic diagram of the establishment of the animal model and the grouping of mice of the present invention.

[0035] Figure 3 It is the colorectal state diagram of each group of mice in the experimental verification of the present invention.

[0036] Figure 4 It is the number of colorectal tumors of each group of mice in the experimental verification of the present invention.

[0037] Figure 5 It is the number of tumors in the distal colon of each group of mice in the experimental verification of the present invention.

[0038] Figure 6 It is the number of tumors in the proximal colon of each group of mice in the experimental verification of the present invention.

[0039] Figure 7 It is the HE staining diagram of intestinal tumors of each group of mice in the experimental verification of the present invention.

[0040] Figure 8 It is the pathological score of intestinal tumor tissues of each group of mice in the experimental verification of the present invention.

[0041] Figure 9 It is the flow cytometry analysis diagram of intestinal tumors of each group of mice in the experimental verification of the present invention.

[0042] Figure 10F4 / 80 of intestinal tumor tissues of mice in each group during the experimental verification of the present invention + CD206 + Expression levels.

[0043] Figure 11 Protein expression graphs of HDAC1, TLR4, MyD88, and NF-κB in mice in each group during the experimental verification of the present invention.

[0044] Figure 12 Analysis graphs of protein expression values of HDAC1, TLR4, MyD88, and NF-κB in mice in each group during the experimental verification of the present invention.

[0045] Figure 13 Schematic diagrams of mRNA levels of HDAC1, TLR4, MyD88, and NF-κB genes in the intestines of mice in each group during the experimental verification of the present invention.

[0046] Figure 14 PLS-DA score graph in the experimental verification of metabolites of the present invention. In the figure, Comp1 represents the interpretation degree of the first principal component, and Comp2 represents the interpretation degree of the second principal component.

[0047] Figure 15 Heat map of cluster analysis of differential metabolites in the experimental verification of metabolites of the present invention. Each column in the figure represents a sample, each row represents a metabolite, and the colors in the figure represent the relative expression levels of metabolites in this group of samples.

[0048] Figure 16 Comparison of VIP value differences in the heat map of cluster analysis of differential metabolites. Each row represents a metabolite, the color represents the relative expression level of the metabolite in this group of samples, and the corresponding relationship between the color gradient and the numerical size can be seen in the gradient color block.

[0049] Figure 17 KEGG compound cluster analysis graph in the experimental verification of metabolites of the present invention. The vertical coordinate is the KEGG compound classification, the horizontal coordinate is the number of compounds annotated to this type; the color of the bar represents the first-level classification category of the compound. Detailed implementation manners

[0050] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation manners and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0051] Unless otherwise specified, the reagents, methods, instruments, and equipment used in the present invention are conventional reagents, methods, instruments, and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Among them, the Chinese herbal pieces in the following examples are all sourced from Anhui Puren Chinese Herbal Pieces Co., Ltd. and have passed the inspection of crude drugs.

[0052] Example 1:

[0053] This example provides a traditional Chinese medicine compound for preventing and treating precancerous lesions of colorectal cancer, which includes the following components in parts by weight: 30 parts of coix seed, 9 parts of aconite, 15 parts of patrinia scabiosaefolia, 12 parts of rhubarb, 3 parts of moutan bark, 9 parts of peach kernel, 30 parts of semen benincasae, and 9 parts of mirabilite.

[0054] The dosage form of the traditional Chinese medicine compound in this example is a decoction, and the preparation method of the decoction includes the following steps: Weigh each Chinese medicine component according to the parts by weight. Except for mirabilite, soak it in clear water for 30 minutes and set aside; bring to a boil over high heat and then continue to simmer for 40 minutes, pour out the soup and set aside. Subsequently, add mirabilite to the medicinal residues, add water again, bring to a boil over high heat and simmer for 40 minutes, pour out the soup. Finally, mix the two decocted medicinal liquids evenly in equal proportions to obtain the decoction product of the traditional Chinese medicine compound.

[0055] The traditional Chinese medicine compound in this example can also be made into a freeze-dried powder. The specific preparation method is as follows: Mix, wash, dry, and crush the Chinese medicinal materials into coarse powder according to the above ratio, add 8 - 10 times the volume of ethanol, reflux and extract 2 - 3 times at 60 - 70 °C, filter and combine the extraction liquid, concentrate under reduced pressure through a rotary evaporator to remove most of the ethanol to obtain a thick paste-like extract. Freeze the thick paste-like extract to below -40 °C, place it in a freeze dryer for freeze-drying, crush it into fine powder after drying, and store it sealed in a frozen storage device. Dissolve the traditional Chinese medicine freeze-dried powder in PBS to make a 10 mg / mL mother liquor for subsequent cell and animal experiments.

[0056] Example 2:

[0057] This example provides a traditional Chinese medicine compound for preventing and treating precancerous lesions of colorectal cancer, which includes the following components in parts by weight: 25 parts of coix seed, 10 parts of aconite, 12 parts of patrinia scabiosaefolia, 15 parts of rhubarb, 4 parts of moutan bark, 10 parts of peach kernel, 25 parts of semen benincasae, and 10 parts of mirabilite.

[0058] Example 3:

[0059] This example provides a traditional Chinese medicine compound for preventing and treating precancerous lesions of colorectal cancer, which includes the following components in parts by weight: 35 parts of coix seed, 10 parts of aconite, 18 parts of patrinia scabiosaefolia, 10 parts of rhubarb, 2 parts of moutan bark, 8 parts of peach kernel, 35 parts of semen benincasae, and 8 parts of mirabilite.

[0060] The present invention first conducted in vitro cell experiments on the traditional Chinese medicine compound of Example 1 (hereinafter abbreviated as DFYYBJS). The CCK-8 method was used in the in vitro cell experiments to detect the effects of DFYYBJS on the proliferation ability of tumor cells and macrophages. The specific test method is as follows:

[0061] The test cells selected were mouse colon cancer cells MC-38 and mouse macrophages RAW264.7. MC-38 cells were placed in McCoy's 5A + 10% FBS medium, and RAW264.7 cells were placed in DMEM + 10% FBS medium, and cultured at 37 °C and 5% CO2. When the cells grew to 90%, they were digested with 1 ml of 2.5 g / L Tryspin for 1 minute, and the cell suspension was collected by pipetting and centrifuged (at room temperature, 1000 r / min, 5 minutes). After centrifugation, the cell suspension was taken for cell counting under a microscope; 10 4 cells / 100 μl density was inoculated into a 96-well plate and cultured for 24 h. Starved for 2 h using a basal medium without FBS (fetal bovine serum); treated with different concentrations of DFYYBJS (62.5, 125, 250, 500, 1000 μg / mL) for 24 h, 48 h, and 72 h; at different time points, 10 ml / L of CCK-8 solution was diluted into the cell culture medium and added to the 96-well plate in turn, and the absorbance value was measured at 450 nm with an enzyme-labeled instrument after incubation for 1.5 h.

[0062] In order to deeply study the effects of DFYYBJS on different cells in the intestinal environment, mouse colon cancer cells MC-38 and mouse macrophages RAW264.7 were selected in this study, and the effects of DFYYBJS on the proliferation of the two types of cells were analyzed by CCK-8 technology. The results showed that with the increase of the DFYYBJS dose, the proliferation rates of MC-38 cells and RAW264.7 cells were inhibited. However, when the concentration of DFYYBJS was lower than 250 μg / ml, neither MC-38 cells nor RAW264.7 cells reached IC20, indicating that DFYYBJS had no toxic effect on the two types of cells at low doses. In addition, with the increase of time, the inhibitory effect of DFYYBJS on the proliferation of the two types of cells was also significantly enhanced, suggesting that DFYYBJS could inhibit the proliferation of colon cancer cells and macrophages, and showed time- and dose-dependence, with statistical significance (P < 0.01).

[0063] The present invention conducted the following animal experiments on the traditional Chinese medicine compound of Example 1 to verify its effect in treating colorectal cancer. The specific experimental procedure is as follows:

[0064] (1) Selection of experimental animals:

[0065] B6.Cg-Kras<tm4Tyj>Apctm1TnoTg(CDX2-cre / ERT2)<752Erf> / MaraJ, abbreviated as Villin-Cre ERT2 Kras G12D / + Apc flox / + , is a mouse with a hybrid background generated by the University of Michigan laboratory. They express Cre-ERT2 downstream of the promoter / enhancer region of the human caudal type homeobox 2 (CDX2) gene. Currently donated to the Jackson Laboratory, the strain number is #035169.

[0066] Mouse co-housing method: Mate male mice with tm1Tno Apc tm4Tyj female mice with ERT2 Kras G12D / + and identify the genotypes of the offspring mice. Mice with the genotype of Villin-Cre flox / + Kras

[0067] (2) Grouping and drug administration:

[0068] Divide the mice into a genetically engineered mouse group (subsequently denoted as the Control group), a genetically engineered mouse drug administration group (subsequently denoted as the DFYYBJS group), a pseudo-sterile mouse group (subsequently denoted as the ABX group), and a pseudo-sterile mouse drug administration group (subsequently denoted as the ABX+DFYYBJS group), with 8 mice in each group. At the same time, concentrate and intragastrically administer the mixed traditional Chinese medicine soup (DFYYBJS), with a drug concentration of 0.38 g / ml, intragastrically for 8 weeks, 0.4 ml each time, once a day; the Control group and the ABX group are given normal saline by gavage, 0.4 ml each time, once a day. Observe the general conditions of the mice such as body weight, diarrhea, bloody stools, and rectal prolapse every day, and record the Disease Activity Index (DAI) score of the mice. After 8 weeks, decapitate and sacrifice the mice, dissect the intestinal tissues of the mice, count the number of intestinal adenomas, collect the intestinal tumors of the mice, and freeze them at -80 °C for standby.

[0069] (3) Hematoxylin-eosin (HE) staining

[0070] For pathological examination, take the diseased colon tissue, cut it along the longitudinal axis, fix it routinely, dehydrate it, embed it in paraffin, section it, make it transparent with xylene, dehydrate it with gradient ethanol, and then perform HE staining according to the HE kit instructions. After sealing the slices with neutral gum, observe the tissue morphology of the colon mucosa under an ordinary light microscope, and score it according to the severity of colon tissue inflammation, lesion depth, and crypt and epithelial damage, from mild to severe as normal (0 points), mild (1 point), moderate (2 points), and severe (3 points).

[0071] (4) Preparation of tumor single cell suspension

[0072] Place the mouse tumor tissue in a grinder, add grinding beads and 1ml PBS, grind thoroughly on the machine, 60HZ, 5min, stand on ice for 5min after grinding, filter with 70uM filter, centrifuge at 800g for 5min to remove the supernatant, add 4ml tissue diluent, prepare a single cell suspension, add 4mL Ficoll solution at 45° tilt, 800g, horizontal centrifuge for 20min (4min for speed increase, 4min for speed decrease). After centrifugation, obvious stratification can be seen. Use a pipette to insert into the cloud layer at the interface of the upper and middle layers to absorb mononuclear cells. Transfer to a 15mL centrifuge tube, add PBS to 10mL, invert to mix, 800g, centrifuge for 5min (9min for speed increase, 9min for speed decrease), discard the supernatant, add 1mL PBS and resuspend to obtain a single cell suspension.

[0073] (5) Flow cytometry to detect M2 macrophage infiltration in tumor tissue

[0074] Place the above extracted tumor single cell suspension on ice, count and dilute according to the required number of tubes, divide into EP tubes, wash twice with 1mL ice PBS, centrifuge at 4℃, 800g for 5min. Resuspend the cells with 200ul PBS, add 0.25ul cell death dye to each tube, incubate at 4℃ in the dark for 20min, add 1mL PBS to wash and centrifuge. Resuspend the cells with 200ul PBS, add 1ul FcR receptor blocker to each tube, incubate at 4℃ in the dark for 15min, add 1mL PBS to wash and centrifuge. Resuspend the cells with 200ul PBS, add 0.5ul fluorescent labeled antibody F4 / 80-APC to each tube, incubate at 4℃ in the dark for 20min, add 1mL PBS to wash and centrifuge. Cell fixation: add 100ul of fixative solution to the tube to resuspend the cells and vortex, incubate at 4℃ in dark for 30min, add 1mL of PBS to wash and centrifuge, add 100ul of permeabilization solution to resuspend the cells and add intracellular antibody of CD206-PerCP-Cyanine5.5, incubate at 4℃ in dark for 30min, add 1mL of PBS to wash and centrifuge twice, then add 200ul of PBS to resuspend the cells, and transfer the cell suspension to the flow cytometer for detection.

[0075] (6) Western blot analysis of HDAC1, TLR4, MyD88 and NF-KB protein expression

[0076] The obtained single-cell suspension was centrifuged at 1200 r / min at 4°C for 5 min. 100 μl of the prepared lysis buffer was added to each group. After pipetting evenly, it was left standing on ice for 30 min, then centrifuged at 12000 r / min at 4°C for 20 min. The supernatant was collected and left standing on ice for 10 min, then centrifuged again. The supernatant was taken for protein concentration determination. The protein samples were processed, polyacrylamide gels were prepared, and samples were loaded into groups. Electrophoresis was carried out at 100 V for 20 min and then transferred to 120 V for 60 min. After that, it was transferred to a PVDF membrane at 230 mA for 80 min. It was blocked with 5% skim milk powder for 2 h, and primary antibodies were added, including rabbit anti-HDAC1 antibody (1:1000), mouse anti-TLR4 antibody (1:1000), mouse anti-MyD88 antibody (1:1000), rabbit anti-NF-κB antibody (1:1000), and mouse anti-GAPDH antibody (1:2000), and incubated overnight on a shaker at 4°C. The membrane was washed with TBST for 5 min, three times, then incubated with HRP-labeled secondary antibody at room temperature for 2 h. The membrane was washed with TBST for 5 min, three times, and then developed on the machine. The results were expressed as the ratio of the corresponding protein level to GAPDH.

[0077] (7) RT-PCR was used to detect the mRNA expression of HDAC1, TLR-4, MyD88, and NF-κB in tumor tissues

[0078] Total RNA of tumor tissues was extracted with Trizol. Using the total mRNA of the sample as a template, reverse transcription cDNA was carried out. Using the cDNA as a template, upstream and downstream primers of the target gene were added for amplification. Using GAPDH as an internal reference, the Ct value was measured on the machine. The reaction program was: 95°C for 3 min, 95°C for 12 s, 62°C for 30 s, and 72°C for 30 s, for a total of 40 cycles. The primer sequences were as follows: TLR4, F: 5'-ATGGCATGGCTTACACCACC-3', R: 5'-GAGGCCAATT TTGTCTCCACA-3'. HDAC1, F: 5'-AGTCTGTTACTACTACGACGGG-3', R: 5'-TGAGCAGCAAATTGTGAGTCAT-3'. MyD88, F: 5'-TCATGTTCTCCATACCCTTG GT-3', R: 5'-AAACTGCGAGTGGGGTCAG-3'. NF-κB, F: 5'-AGGCTTCTGGGCC TTATGTG-3', R: 5'-TGCTTCTCTCGCCAGGAATAC-3'. GAPDH, F: 5'-AGGTCGGTGTGAACGGATTTG-3', R: 5'-TGTAGACCATGTAGTTGAGGTCA-3'. The experimental data were processed according to 2 -ΔΔCr methods.

[0079] (8) Non-target metabolomics analysis of the changes in metabolites in mouse blood

[0080] Absorb 100 μL of the liquid sample into a 1.5 mL centrifuge tube, add 400 μL of the extraction solution (acetonitrile:methanol = 1:1) containing 0.02 mg / mL of the internal standard (L-2-chlorophenylalanine), vortex for 30 s, then perform ultrasonic extraction at low temperature for 30 min (5 °C, 40 KHz). Let the sample stand at -20 °C for 30 min. Centrifuge at 4 °C and 13000 g for 15 min, transfer the supernatant, dry it under nitrogen, re-dissolve it with 100 μL of the reconstitution solution (acetonitrile:water = 1:1), perform ultrasonic extraction at low temperature for 5 min (5 °C, 40 KHz), centrifuge at 4 °C and 13000 g for 10 min, and transfer the supernatant to an injection vial with an inner cannula for on-machine analysis.

[0081] Perform LC-MS / MS analysis on the sample using the Thermo Fisher Scientific ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Q Exactive HF-X system. Specifically: The sample mass spectrometry signal acquisition uses positive and negative ion scanning modes, and the mass scanning range is 70 - 1050 m / z. The sheath gas flow rate is 50 psi, the auxiliary gas flow rate is 13 psi, the auxiliary gas heating temperature is 425 °C, the positive mode ion spray voltage is set to 3500 V, the negative mode ion spray voltage is set to -3500 V, the ion transfer tube temperature is 325 °C, and the normalized collision energy is 20 - 40 - 60 V cyclic collision energy. The resolution of the first-stage mass spectrometry is 60000, the resolution of the second-stage mass spectrometry is 7500, and data is collected using the DDA mode.

[0082] (9) Statistical analysis:

[0083] Use SPSS 21.0 statistical software for data analysis. Measurement data conforming to a normal distribution are expressed as . For data with a normal distribution, one-way ANOVA is used for comparison among multiple groups, and non-parametric rank sum test is used for data that do not satisfy a normal distribution. P < 0.05 is considered statistically significant.

[0084] The construction of the above animal model and the results of experimental verification are as follows:

[0085] The present invention uses genetically engineered mice Villin-Cre ERT2 Kras G12D / + Apc flox / + . Through the Cre-loxp system, the Apc gene in mouse colorectal epithelial cells was conditionally knocked out, and the Kras gene was activated to spontaneously generate a colorectal tumor model, and its advantage is higher clinical similarity. For 6-week-old Villin-Cre ERT2 Kras G12D / + Apc flox / +Mice were injected with tamoxifen (1 mg / mL) via the tail vein. Four weeks later, Villin-Cre ERT2 Kras G12D / + Apc flox / + Mice began to spontaneously develop tumors in the colorectal region, and then were grouped and treated according to the experimental method. After 8 weeks, they were sacrificed by cervical dislocation. The specific process is as Figure 2 shown.

[0086] At 1 week, 4 weeks, and 8 weeks respectively, the body weight changes, stool characteristics, and stool bleeding conditions of each group of mice were recorded, and DAI scores were performed. The results showed (Table 1) that at 1 week of intervention, there were no significant differences among groups, and there was no statistical significance (P > 0.05). At 4 weeks and 8 weeks of intervention, compared with the Control group, the DAI scores of the DFYYBJS group decreased significantly, with statistical significance (P < 0.01). Compared with the DFYYBJS group, the DAI scores of the DFYYBJS + ABX group increased significantly (P < 0.05), indicating that DFYYBJS could improve the intestinal symptoms of Villin-Cre ERT2 Kras G12D / + Apc flox / + mice, but the addition of ABX weakened the efficacy of DFYYBJS. At the same time, compared with the DFYYBJS + ABX group, the DAI scores of the ABX group increased, but there was no statistical significance (P > 0.05), indicating that in the absence of intestinal flora and their metabolites, the effect of DFYYBJS was inhibited. It is suggested that DFYYBJS may exert its anti-tumor effect through intestinal flora or their metabolites.

[0087] Table 1 DAI scores of each group of mice

[0088]

[0089]

[0090] Compared with the Control group, *P < 0.05, compared with the DFYYBJS group, #P < 0.05.

[0091] Colorectal cancer includes colon cancer and rectal cancer, and the incidence of rectal cancer accounts for about 70% of the total number of colorectal cancers. For mice with spontaneous colorectal cancer, the probability of carcinogenesis is higher in proximal colorectal tumors (right side) compared to distal colorectal tumors (left side). Figures 3-4The colorectal status and tumor numbers of each group of mice after 8 weeks were shown respectively. Compared with the Control group, the colon length of the drug-administered group was significantly shortened, and the tumor number was 2.5±1.6, with a significant difference (P<0.01), indicating that the traditional Chinese medicine compound of the present invention has a significant curative effect on the inhibition of colorectal cancer. Colorectal cancer includes colon cancer and rectal cancer, and the incidence of rectal cancer accounts for about 70% of the total number of colorectal cancers. For mice with spontaneous colorectal cancer, the probability of carcinogenesis of proximal colorectal tumors is higher than that of distal colorectal tumors. In this study, as Figures 5-6 shown, compared with the Control group, the number of distal colon in the drug-administered group was significantly reduced, with a significant difference (P<0.01). However, there was no significant difference between the DFYYBJS+ABX group and the DFYYBJS group.

[0092] Histologically, as Figures 7-8 shown, the pathological examination of the colon tissue of mice in the Control group showed that the glands were dense, arranged in a cribriform structure, the adenomatous epithelial cells showed obvious dysplasia, the cell nuclei were significantly enlarged and pathological karyokinesis was visible, and there was infiltration of chronic inflammatory cells in the stroma, diagnosed as high-grade intraepithelial neoplasia or adenocarcinoma. Compared with the Control group, the tumors formed in the DFYYBJS group were pathologically evaluated as adenomas (possibly tubular adenomas), confined to the submucosa, the number of adenocarcinomas decreased, and the number of adenomas increased.

[0093] Studies have shown that M2-like macrophages can enhance the invasion of tumor cells in the TME, inhibit the anti-tumor immune response of T cells, and disrupt the interaction of immune cells, mainly manifested as: the increased expression levels of its markers F4 / 80 + CD206 + suggest that macrophages polarize towards the M2 phenotype, thus having the effect of promoting tumor proliferation and metastasis. As Figures 9-10 shown, the results of flow cytometry analysis found that after the intervention of the traditional Chinese medicine compound DFYYBJS, in Villin-CreERT2Kras G12D / + Apc flox / + mice intestine, the percentages of F4 / 80 + CD206 + cells were significantly down-regulated, with a significant difference (P<0.01). Compared with the DFYYBJS group, the percentages of F4 / 80 + CD206 + cells in the DFYYBJS+ABX group were significantly increased, with a significant difference (P<0.01). It is suggested that DFYYBJS can down-regulate the proportion of M2 macrophages, inhibit macrophage polarization, and is related to the participation of ABX.

[0094] In addition, a large number of studies have confirmed that the activation of the TLR / MyD88 / NF-κB signaling pathway can promote the polarization of macrophages into the M2 type. Therefore, in this study, Western blot detection found that compared with the Control group, the expression levels of HDAC1, TLR4, and MyD88 proteins in the DFYYBJS group were significantly decreased ( Figures 11-12 ), and the difference was statistically significant (P < 0.01). In addition, there was no significant difference in the expression of NF-κB between the two groups. It is speculated that non-phosphorylated NF-κB may not represent the activation degree of this signaling pathway, and its phosphorylated expression needs to be further analyzed.

[0095] The results of qRT-PCR are as Figure 13 shown. Compared with the Control group, the mRNA expression levels of HDAC1, TLR4, MyD88, and NF-κB in the DFYYBJS group were significantly decreased, and the difference was statistically significant (P < 0.05, P < 0.01). Compared with the DFYYBJS group, the mRNA expression levels of HDAC1, TLR4, and MyD88 in the DFYYBJS+ABX group were significantly increased (P < 0.01), suggesting that the addition of ABX increased the transcriptional levels of key genes in the HDAC / TLR / MyD88 signaling pathway, indicating that the absence of gut microbiota or their metabolites is an important reason for the failure of the traditional Chinese medicine compound DFYYBJS.

[0096] To further clarify how DFYYBJS targets the HDAC / TLR / MyD88 signaling pathway, we used non-targeted metabolomics to analyze the changes in various metabolites in the peripheral blood of Villin-CreERT2 Kras G12D / + Apc flox / + mice. The ropls package (Version 1.6.2) in R language was used to perform principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) on the preprocessed data matrix, and it was found that the separation degree of the two groups of samples in the DFYYBJS group and the Control group was relatively large, indicating a significant classification effect ( Figure 14 ). The clustering results of differential metabolites showed ( Figures 15-16 ) that compared with the Control group, the contents of butyric acid, caproic acid, and delta-guanidinovaleric acid in short-chain fatty acids in the DFYYBJS group were significantly increased, and their VIP values indicated that the difference was statistically significant (P < 0.01). The results of KEGG compound clustering analysis showed ( Figure 17), significant changes were observed in fatty acids, phospholipids, bioactive peptides, and secondary metabolites. This suggests that after DFYYBJS intervention, the changes in short-chain fatty acids in mice were significant, and the significant decrease in the HDAC / TLR / MyD88 signaling pathway mediated by its target HDAC may be the result of the action of butyric acid in short-chain fatty acids.

[0097] From this, it can be seen that the results of metabolomics are consistent with the results of protein and gene detection, indicating that DFYYBJS can up-regulate the content of short-chain fatty acids (SCFA), especially the expression of butyric acid and its subtypes. Research has shown that SCFA is an HDAC inhibitor, and with the increase in the content of SCFA, the expression of HDAC is significantly inhibited. Therefore, DFYYBJS may inhibit the function of HDAC by up-regulating the content of SCFA.

[0098] In summary, the intestinal tumor suppression and intestinal tissue protection effects of the traditional Chinese medicine compound of the present invention on spontaneous colorectal cancer model mice may be achieved by down-regulating the expression of HDAC1, inhibiting the activation of the TLR4 / MyD88 / NF-κB signaling pathway, and preventing the polarization of M2-type TAMs. Further research found that compared with colorectal cancer mice with depleted intestinal flora, DFYYBJS has a more obvious tumor suppression effect on normal transgenic mice, suggesting that intestinal flora or its metabolites may be involved in the process of DFYYBJS inhibiting tumor growth, providing new ideas for further research on CRC treatment targets.

[0099] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A Chinese medicinal compound for preventing and treating colorectal precancerous lesions, characterized in that: The traditional Chinese medicine compound comprises the following components in parts by weight: 25-35 parts of coix seeds, 8-10 parts of aconite root, 12-18 parts of patrinia herb, 10-15 parts of rhubarb, 2-4 parts of peony bark, 8-10 parts of peach kernel, 25-35 parts of wax gourd kernel and 8-10 parts of mirabilite.

2. A Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 1, characterized in that: The traditional Chinese medicine compound comprises the following components in parts by weight: 28-32 parts of coix seeds, 8-10 parts of aconite root, 14-16 parts of patrinia herb, 11-13 parts of rhubarb, 2-4 parts of peony bark, 8-10 parts of peach kernel, 28-32 parts of wax gourd kernel and 8-10 parts of mirabilite.

3. A Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 1, characterized in that: The traditional Chinese medicine compound comprises the following components in parts by weight: 30 parts of coix seeds, 9 parts of aconite roots, 15 parts of patrinia herb, 12 parts of rhubarb, 3 parts of peony bark, 9 parts of peach kernel, 30 parts of wax gourd kernel and 9 parts of mirabilite.

4. A Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 1, characterized in that: The dosage form of the Chinese medicine compound is one of water decoction, tablet, granule, capsule, pill or paste.

5. A Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 4, characterized in that: The preparation method of the decoction comprises the following steps: Weigh each Chinese medicine component by weight, and soak them in clean water except for Glauber's salt for later use; boil over high heat, then continue to simmer over low heat, pour out the soup for later use; add Glauber's salt to the medicine residue, add water for decocting again, pour out the soup for later use; mix the medicinal liquids decocted twice evenly to obtain the Chinese medicine compound.

6. The Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 5, characterized in that: The soaking time in clear water is 25-35 minutes, and the decocting time over low heat is 30-50 minutes.

7. The Chinese medicinal compound for preventing and treating colorectal precancerous lesions according to claim 5, characterized in that: The medicinal liquids from the two decoctions were mixed evenly in a 1:1 ratio.

8. An animal model validation method for the Chinese medicinal compound according to any one of claims 1 to 7, characterized in that: The construction of the animal model includes the following steps: S1: Select genetically engineered mice Villin-CreERT2Kras G12D / + Apc flox / + As experimental subjects, the intestinal flora was eliminated by combined antibiotics to construct pseudo-germ-free mice; S2: Establish four experimental groups, namely, genetically engineered mice group, genetically engineered mice drug administration group, pseudo-germ-free mice group, and pseudo-germ-free mice drug administration group; S3: The mice in the above experimental groups were injected with tamoxifen via the tail vein to induce spontaneous tumors in the colorectal area; S4: Drug administration was carried out in groups, wherein the genetically engineered mice group and the pseudo-germ-free mice group were intragastrically administered with normal saline, and the genetically engineered mice group and the pseudo-germ-free mice group were intragastrically administered with a Chinese medicine compound; S5: After 8 weeks of drug administration, the mice were observed and their intestinal tissues were removed to verify the therapeutic effect.

9. The animal model validation method for the Chinese medicinal compound according to claim 8, characterized in that: In step S1, the combined antibiotic consists of 1 mg / mL ampicillin, 1 mg / mL neomycin sulfate, 1 mg / mL metronidazole and 0.5 mg / mL vancomycin; In step S3, the injection concentration of tamoxifen is 1 mg / mL.

10. The animal model validation method for a Chinese medicinal compound according to claim 8, characterized in that: In step S4, the Chinese medicine compound is a decoction concentrate with a concentration of 0.35-0.4 g / ml; The cycle of the intragastric administration is 8 weeks, 0.4 ml / time, once / day.

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