Application of atractylodes lactone I in the preparation of drugs for inhibiting lung metastasis of breast cancer

Abstract

This invention discloses the application of atractylodes lactone I in the preparation of drugs for inhibiting lung metastasis of breast cancer. This invention provides a novel use for atractylodes lactone I, which can reduce the lung metastasis ability of breast cancer cells. When atractylodes lactone I is used as a drug, experiments in breast cancer-bearing mice show that atractylodes lactone I can significantly inhibit the occurrence and development of lung metastasis of breast cancer, and has no significant effect on the body weight of mice, exhibiting high safety. Therefore, atractylodes lactone I shows promise for development into a new drug for inhibiting lung metastasis of breast cancer.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to application of atractylodes lactone I in preparing a medicine for inhibiting lung metastasis of breast cancer. Background Art

[0002] The process of tumor metastasis is highly selective and contains a series of orderly and interrelated steps. However, changes in tumor cells themselves are not enough to cause the occurrence, development and metastasis of tumors. Changes in the organ microenvironment where the tumor is located play an important role in tumor metastasis. There is a close relationship between organ-specific metastasis and organ tissue microenvironment. A suitable organ microenvironment is a good foundation for the proliferation and growth of metastatic tumors.

[0003] At present, most of the research on drugs that inhibit cancer cell migration is focused on in vitro experiments at the cellular level. In vitro experiments have a single influencing factor and only examine the direct effect of drugs on cells. In vivo, due to differences in administration routes and organ microenvironment, the effects of drugs can vary greatly.

[0004] Although chemotherapy, hormone therapy and targeted therapy have significantly improved the efficacy of breast cancer patients, the current clinical treatment of breast cancer is prone to toxic side effects and drug resistance, and also faces problems such as increased mortality caused by tumor metastasis, which brings great trouble to the treatment of breast cancer. Therefore, a low-side effect drug that can inhibit breast cancer metastasis is imminent.

[0005] Atractylenolide is mainly produced in Zhejiang and Anhui. The best one is produced in Yuqian, Zhejiang, which is called "Yushu". It is one of the "Eight Flavors of Zhejiang" authentic medicinal materials. The medicinal part of Atractylenolide is the dried rhizome, which is warm in nature, sweet and bitter in taste, and belongs to the spleen and stomach meridians. It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. Atractylenolide compounds are the main active ingredients of Atractylenolide. Atractylenolide mainly includes Atractylenolide I, Atractylenolide II and Atractylenolide III. Among them, Atractylenolide I has anti-tumor effects on many cancers and can inhibit the development of tumors by inhibiting tumor cell proliferation and inducing tumor cell apoptosis. However, there are no reports on the effect of Atractylenolide I on lung metastasis of breast cancer. Summary of the invention

[0006] The purpose of the present invention is to provide a new use of Atractylodes lactone I, namely, to prepare a drug for inhibiting lung metastasis of breast cancer. The present invention provides the following technical solutions:

[0007] Atractylodes lactone I structural formula:

[0008]

[0009] The present invention provides application of atractylodes lactone I in preparing a medicine for inhibiting lung metastasis of breast cancer.

[0010] The present invention also provides the use of atractylodes lactone I in the preparation of a drug for inhibiting the expression of fibronectin 1 (FN1) and osteopontin (SPP1) in patients with breast cancer lung metastasis.

[0011] Furthermore, in the above application, the drug is administered via the gastrointestinal tract.

[0012] Furthermore, in the above application, the drug consists of Atractylodes lactone I and a pharmaceutically acceptable excipient.

[0013] Furthermore, the drug is in the form of tablets, oral liquid, granules, traditional Chinese medicine, and capsules.

[0014] Furthermore, the tablet is one of ordinary compressed tablets, sugar-coated tablets, effervescent tablets, chewable tablets, multi-layer tablets, sustained-release tablets, and controlled-release tablets.

[0015] Furthermore, the oral liquid preparation is one of a solution, a syrup, an emulsion, and a suspension.

[0016] Furthermore, the granules are one of soluble granules, suspension granules and effervescent granules.

[0017] Furthermore, the Chinese medicine is one of decoction, powder, pill and pill.

[0018] In the drug, atractylodes lactone I is the only active ingredient, and its mass accounts for 1 to 99% of the total mass of the drug. The mouse dosage of atractylodes lactone I is at least 50 mg / kg / d (equivalent to a human dosage of at least 332.97 mg / kg / d). Its function of inhibiting breast cancer lung metastasis is achieved by inhibiting the expression of FN1 and SPP1.

[0019] The pharmacological experiments of the present invention show that Atractylodes lactone I has no obvious toxicity to the main organs (heart, liver, spleen, and kidney) of mice and has high safety; and can significantly inhibit the occurrence of lung metastasis of breast cancer.

[0020] The present invention verifies the inhibitory effect of atractylodes lactone I on lung metastasis of breast cancer. The purpose of the present invention is to provide a drug for inhibiting lung metastasis of breast cancer and to provide a new use of atractylodes lactone I. Beneficial Effects

[0021] (1) The present invention confirms that Atractylodes macrocephala lactone I can inhibit the number and area of ​​lung metastatic nodules in breast cancer-bearing mice, and inhibit the expression of metastasis-related genes FN1 and SPP1, providing a new idea for the treatment of breast cancer.

[0022] (2) The Atractylodes lactone I in the present invention has good safety and has no obvious toxicity to the main organs of mice (heart, liver, spleen, and kidney), and can reduce the toxic and side effects of drugs on the human body during cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the changes in body weight over time in mice in different treatment groups;

[0024] Figure 2 This is a graph showing the changes in tumor volume over time in mice in different treatment groups;

[0025] Figure 3 are the in vivo imaging images of mice in different treatment groups;

[0026] Figure 4 It is a statistical graph of the luminescence intensity of the lungs of mice in different treatment groups;

[0027] Figure 5 are photos of ex vivo tumors from mice in different treatment groups;

[0028] Figure 6 is a statistical graph of the in vitro tumor weights of mice in different treatment groups;

[0029] Figure 7 are the organ indexes of mice in different treatment groups;

[0030] Figure 8 are photos of lung tissues of mice in different treatment groups;

[0031] Fig. 9 is the statistical diagram of lung metastatic nodules in mice of different treatment groups;

[0032] Fig.10 are H&E sections of the lungs of mice in different treatment groups;

[0033] Fig.11 is a statistical diagram of lung metastasis area in mice in different treatment groups;

[0034] Fig.12 is the expression of SPP1 in lung tissues of mice in different treatment groups;

[0035] Fig.13 Figure 2 is the expression of FN1 in lung tissues of mice in different treatment groups. DETAILED DESCRIPTION

[0036] The following is a further detailed description of the inhibition of breast cancer lung metastasis by Atractylodes lactone I provided by the present invention in conjunction with the accompanying drawings and specific examples. The following examples do not limit the present invention in any form. The raw materials and equipment used in the specific implementation of the present invention are all known products. Unless otherwise specified, the reagents and materials used in the following examples are obtained by purchasing commercially available products.

[0037] The instruments and equipment in the present invention are specifically: ultrapure water machine (Millipore); cell counter (CouterStar); vortexer (Midwest); CO2 constant temperature cell culture box (Thermo Fisher Scientific); refrigerator (Haier); Zeiss stereo microscope (Semorr); low temperature centrifuge (Beckman); -80°C refrigerator (Thermo Fisher Scientific); fluorescence microscope (Olympus); clean bench (Shanghai Hujing Medical Instrument Co., Ltd.); multi-model animal living imaging system (Guangzhou Bolu Teng Biotechnology Co., Ltd.); fluorescence quantitative PCR instrument (Thermo Fisher, USA).

[0038] The reagent materials used in the present invention are specifically: RPMI-1640 culture medium, fetal bovine serum, penicillin-streptomycin double antibody, phosphate buffer (Gibco); dimethyl sulfoxide solution, formaldehyde (Sigma); D-luciferin potassium salt (Shanghai Yuanye Biotechnology Co., Ltd.); Atractylodes lactone I standard (Chengdu Munster Biotechnology Co., Ltd.).

[0039] All statistical analyses in the present invention were performed using SPSS, Graphpad Prism 5, and Adobe Illustrator software. Multiple group comparisons were performed using one-way analysis of variance (ANOVA). Data were expressed as the mean ± standard deviation of the number of independent experiments. P <0.05 was considered statistically significant. # represent P <0.05, ## represent P <0.01).

[0040] S1. Acquisition of experimental cell lines and recipient animals

[0041] Mouse breast cancer cell line 4T1 was purchased from Wuhan Punosai Biotechnology Co., Ltd. and frozen in a cell storage liquid nitrogen tank in this laboratory. The mice required for the experiment were purchased from Shanghai Slake Animal Experimental Center, SPF-grade healthy and mature 4-6 week-old Balb / c female nude mice.

[0042] S2. Screening of experimental cell lines and recipient animals

[0043] Balb / c female nude mice, 4-6 weeks old, with an initial body weight between 18 and 22 grams. Collect 4T1 cells in good growth state, count and adjust the cell concentration, and take 4T1 cells (1×10 7 cells / 100 μL).

[0044] S3. Model grouping and processing

[0045] The mice were randomly divided into 5 groups.

[0046] ①Control group (NC): healthy female nude mice;

[0047] ②Model group (MC): 4T1 cells were injected orthotopically into the mammary tissue of female nude mice;

[0048] ③ Atractylodes lactone I low-dose group (ATI-L): 4T1 cells were injected orthotopically into the mammary tissue of female nude mice, followed by oral administration of low-dose Atractylodes lactone I (25 mg / kg);

[0049] ④ Atractylodes lactone I high-dose group (ATI-H): 4T1 cells were injected orthotopically into the mammary tissue of female nude mice, followed by oral administration of high-dose Atractylodes lactone I (50 mg / kg);

[0050] ⑤ Positive drug capecitabine group (CAP): 4T1 cells were injected orally into the mammary tissue of female nude mice, followed by gavage with capecitabine (CAP 600 mg / kg).

[0051] 4T1 cells were inoculated into the mammary gland of nude mice using a 1 mL sterile syringe on a clean bench. Before inoculation of 4T1 cells, Atractylodes lactone I was gavaged 2 days in advance and continued for 22 days; Capecitabine was gavaged for 7 days and then stopped for 3 days. The above treatments were repeated 5 times in each group, and the experimental duration was 24 days.

[0052] Atractylodes lactone I was dissolved with 0.5% sodium carboxymethylcellulose (CMC-Na). Capecitabine was diluted with normal saline. Mice in the treatment group were intragastrically administered with Atractylodes lactone I, and mice in the control group and model group were intragastrically administered with 0.5% CMC-Na.

[0053] S4. Measurement of mouse body weight and tumor volume

[0054] During the experiment, the mice were weighed every other day using an electronic scale, and the tumor volume of the mice was measured using a vernier caliper (the longest diameter (a) and shortest diameter (b) of the tumor, according to V = 1 / 2ab 2 Calculate the tumor volume and record it). Figure 1 and Figure 2 .

[0055] The eating and activity status of mice in each group were normal. After inoculation of breast cancer 4T1 cells, there was a significant difference in body weight between the control group and the model group, and 2 mice died in the model group, 1 mouse died in the low-dose group of Atractylodes lactone I, and there was no death in the high-dose group of Atractylodes lactone I and the control group. Atractylodes lactone I had no significant effect on the body weight and tumor volume of breast cancer-bearing mice.

[0056] S5. Perform in vivo imaging and measure lung fluorescence intensity

[0057] Before killing, each mouse was injected with 0.15 mg / g D-luciferin potassium salt. Five minutes later, the exposure was set for three seconds, and photos were collected to measure the fluorescence intensity of the mouse lungs. The fluorescence intensity of each group was statistically analyzed, such as Figure 3 and Figure 4 .

[0058] The results showed that the lung fluorescence intensity of the Atractylodes lactone I-administered group was significantly lower than that of the model group, indicating that Atractylodes lactone I can significantly inhibit the lung metastasis of breast cancer.

[0059] S6. Observation of general condition of mice

[0060] The mice were killed by CO2 asphyxiation and then rapidly dissected. Photos of the ex vivo tumors were kept and the weight of the ex vivo tumors was measured. Figure 5 and Figure 6 Weigh the viscera and calculate the organ coefficient by mouse organ weight / body weight × 100%, such as Figure 7 .

[0061] Atractylodes lactone I had no significant effect on the in vitro tumor weight of breast cancer-bearing mice. The results of organ coefficient showed that compared with the model group, the high-dose group of Atractylodes lactone I could reduce the organ coefficient of lung tissue, and had no significant effect on the organ coefficient of other organs.

[0062] S7. Pathological observation of mouse lung tissue

[0063] S7.1. Keep photos of ex vivo lung tissue, such as Figure 8 As shown, the number of tumor nodules in the lung tissue after dissection was counted, as shown Fig. 9 As shown, it was found that the number of tumor nodules in the lungs of mice in the Atractylodes macrocephala lactone I group was significantly lower than that in the model group, indicating that Atractylodes macrocephala lactone I can significantly inhibit the lung metastasis of breast cancer.

[0064] S7.2. Mouse lung tissues were fixed with 4% paraformaldehyde, embedded in paraffin, routinely sectioned, and stained with hematoxylin and eosin (H&E) to observe histopathological changes under an optical microscope. Fig.10 and Fig.11 shown.

[0065] H&E staining of lung tissue showed that compared with the model group, the tumor metastasis area in the lungs of mice in the Atractylodes lactone I group was significantly smaller than that in the model group, indicating that Atractylodes lactone I can significantly inhibit the lung metastasis of breast cancer.

[0066] S8. Expression of FN1 and SPP1 in mouse lung tissue

[0067] FN1 is an important component in the ECM pathway and is closely related to the proliferation, invasion, drug resistance and metastasis of tumor cells; SPP1 is a secreted phosphorylated glycoprotein present in the extracellular matrix and plays an important role in the proliferation, invasion and progression of malignant tumors.

[0068] S8.1. Accurately weigh 20 mg of tissue sample into a 1.5 mL centrifuge tube containing 4 sterile steel beads, add 1 mL of RNAiso Plus reagent, and grind in a pre-cooled homogenizer at 70 Hz for 3 min to obtain tissue homogenate. Extract total RNA and perform reverse transcription. The obtained cDNA is used for subsequent experiments.

[0069] S8.2. Perform fluorescence quantitative PCR experiment. The primer sequences are shown in Table 1. GAPDH is used as internal standard. -△△CT Method to measure gene expression.

[0070] Table 1 Primer sequences

[0071]

[0072] like Fig.12 and Fig.13 As shown, the results showed that compared with the model group, atractylodes lactone I could significantly downregulate the expression of metastasis-related genes SPP1 and FN1 in lung tissue, indicating that the effect of atractylodes lactone I in inhibiting breast cancer lung metastasis is related to the inhibition of the expression of FN1 and SPP1.

[0073] It can be seen from the above examples that Atractylodes lactone I has a significant effect of inhibiting breast cancer lung metastasis. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. It is not necessary and impossible to list all the embodiments here. The obvious improvements and modifications derived from this should also be regarded as the protection scope of the present invention.

Claims

1. Application of atractylodes lactone I in the preparation of a drug for inhibiting lung metastasis of breast cancer, wherein atractylodes lactone I is the only active ingredient in the drug.

2. The use according to claim 1, characterized in that: Suppression of FN1 and SPP1 expression in patients with breast cancer lung metastasis.

3. The use according to claim 1, characterized in that: The administration route of the drug is through the gastrointestinal tract.

4. The use according to claim 1, characterized in that: The medicine consists of atractylodes lactone I and a pharmaceutically acceptable excipient.

5. The use according to claim 4, characterized in that: The drug is in the form of tablets, oral liquid, granules or capsules.

6. The use according to claim 5, characterized in that: The tablet is one of ordinary compressed tablets, sugar-coated tablets, effervescent tablets, chewable tablets, multi-layer tablets, sustained-release tablets and controlled-release tablets.

7. The use according to claim 6, characterized in that: The oral liquid preparation is one of a solution, a syrup, an emulsion and a suspension.

8. The use according to claim 7, characterized in that: In the medicine, atractylodes lactone I accounts for 1 to 99% of the total mass.

9. The use according to claim 1, characterized in that: The dosage of Atractylodes lactone I is at least 332.97 mg / kg / d.

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