Application of atractylodes macrocephala koidz polysaccharide in preparation of medicine for preventing / treating melanoma

Abstract

The invention relates to the technical field of medicines, in particular to application of atractylodes macrocephala polysaccharide in preparation of a medicine for preventing / treating melanoma. The invention provides a new application of atractylodes macrocephala polysaccharide as an anti-melanoma drug, and experiments show that the atractylodes macrocephala polysaccharide has a good inhibition effect on melanoma and can obviously delay the growth speed of tumors, reduce the content of Treg cells in vivo and increase the content of CD8 + T cells and NK cells. Therefore, the atractylodes macrocephala polysaccharide plays an anti-tumor role by enhancing in-vivo immune response. According to the invention, a new application of the atractylodes macrocephala polysaccharide is developed, and a new candidate medicine is provided for clinically treating melanoma diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a novel use of Atractylodes macrocephala polysaccharide in the preparation of drugs for the prevention / treatment of melanoma. Background Technology

[0002] Malignant melanoma is an aggressive skin cancer characterized by rapid progression, high mortality, and extremely poor prognosis. Approximately 48,000 melanoma patients die globally each year, posing a serious threat to human health. Clinically, treatment strategies such as radiotherapy, chemotherapy, targeted therapy, and immunotherapy have been developed. However, these treatments have problems such as side effects and drug resistance, therefore, there is an urgent need to find new, highly effective, and low-toxicity anti-melanoma drugs.

[0003] Currently, the core bottleneck in melanoma treatment lies in immune escape mediated by the immunosuppressive tumor microenvironment. Regulatory T cells (Tregs) are considered important cells in maintaining immune tolerance homeostasis in the tumor microenvironment and weakening the efficacy of immunotherapy. They suppress effector T cells (CD8+) by highly expressing immune checkpoint molecules such as CTLA-4 and PD-1, and secreting anti-inflammatory cytokines such as IL-10 and TGF-β. + The anti-tumor functions of T cells and natural killer cells (NK cells) mediate tumor immune escape and promote tumor progression.

[0004] Traditional Chinese medicine has advantages such as abundant sources, low toxicity and side effects, and low price. Due to its strong immunomodulatory activity and low toxicity and side effects, polysaccharides of traditional Chinese medicine have shown broad prospects in the field of adjuvant cancer treatment and have attracted increasing attention from researchers.

[0005] Atractylodes macrocephala Koidz. Polysaccharide (AMP) is the main active ingredient in Atractylodes macrocephala, possessing pharmacological effects such as antioxidant, anticancer, hepatoprotective, immunomodulatory, and digestive system regulatory properties. However, research on Atractylodes macrocephala polysaccharide as a drug for the prevention / treatment of melanoma has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to study novel anti-melanoma drugs, using Atractylodes macrocephala polysaccharide as the research object, and to provide new uses for Atractylodes macrocephala polysaccharide in the preparation of anti-melanoma drugs.

[0007] In a first aspect, the invention provides the use of Atractylodes macrocephala polysaccharide in the preparation of a medicament for the prevention / treatment of melanoma.

[0008] Furthermore, the aforementioned drug for preventing / treating melanoma inhibits the growth of melanoma tumors, reduces the content of immunosuppressive Treg cells in tumor tissue, and increases the content of immune killer cells CD8 in tumor tissue. + Drugs containing high levels of T cells and NK cells.

[0009] Furthermore, the aforementioned drug for the prevention / treatment of melanoma uses Atractylodes macrocephala polysaccharide as the sole active monomeric component, or Atractylodes macrocephala polysaccharide is used in combination with other drugs to form a pharmaceutical composition.

[0010] Furthermore, in the aforementioned drug for the prevention / treatment of melanoma, the dosage of Atractylodes macrocephala polysaccharide is 250 mg / kg.

[0011] The advantages of this invention are:

[0012] This invention provides a novel use of Atractylodes macrocephala polysaccharide as an anti-melanoma drug, specifically, the use of Atractylodes macrocephala polysaccharide in the preparation of drugs for the prevention / treatment of melanoma, in vivo inhibition of tumor growth in melanoma-bearing mice, reduction of the content of immunosuppressive Treg cells in tumor-bearing mice, and increase of CD8+ cytotoxic cells. + Application of T cell and NK cell content.

[0013] Experiments have confirmed that Atractylodes macrocephala polysaccharide has a good inhibitory effect on melanoma. At 250 mg / kg, it can significantly delay the growth rate of tumors in B16-F10 tumor-bearing mice, reduce the content of Treg cells in vivo, and increase CD8. + The levels of T cells and NK cells indicate that Atractylodes macrocephala polysaccharides exert their anti-tumor effect by enhancing the immune response in melanoma-bearing mice.

[0014] This invention opens up new applications for Atractylodes macrocephala polysaccharide and provides new candidate drugs for the clinical treatment of melanoma. Attached Figure Description

[0015] Figure 1 Effects of AMP on tumor volume, tumor weight, and body weight changes in melanoma mice; (A) Visual representation of tumor tissue in each group; (B) Tumor mass changes; (C) Body weight changes in mice; (D) Tumor growth curve; Compared with the Model group, P < 0.05 P < 0.01.

[0016] Figure 2 The effect of AMP on the content of Treg cells in tumor tissue; compared with the Model group, P < 0.05 P < 0.01.

[0017] Figure 3Effects of AMP on the levels of CD8+ T cells and NK cells in tumor tissue; (A) CD8+ T cell content; (B) NK cell content; compared with the Model group, P < 0.05 P < 0.01. Detailed Implementation

[0018] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0019] Example 1: Effect of Atractylodes macrocephala polysaccharide on melanoma growth in tumor-bearing mice.

[0020] I. Materials and Methods

[0021] 1. Experimental reagents

[0022] Atractylodes macrocephala polysaccharide, anhydrous ethanol, physiological saline, DMEM high-glucose medium, fetal bovine serum, penicillin and antibiotics, trypsin, PBS solution, stain buffer, FVS, FC receptor antagonist, fixation and perforation solution, perforation buffer, flow cytometry antibodies: CD45-FITC, CD3-PER5.5, CD4-APC, CD25-PE, FOXP3-PE-CY7, CD8-PE-CY7, CD49b-APC.

[0023] 2. Cells

[0024] Melanoma B16-F10 was provided by the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in DMEM in an incubator at 37°C, 5% CO2, and saturated humidity.

[0025] 3. Laboratory animals

[0026] Ten healthy male C57BL / 6 mice, 5-6 weeks old and weighing 16-18g, were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and fed in an SPF-grade environment (constant temperature 25±2℃, constant humidity 40%~50%).

[0027] 4. Instruments

[0028]

[0029] 5. Preparation of B16-F10 cell suspension:

[0030] ① Perform the experiment in a clean bench according to aseptic operation rules. 4-5 days before the experiment, revive the cells, passage them in T25 cell culture flasks, and culture them 3 times.

[0031] ② When the cells have grown to 80%, discard the culture medium, add 4 mL of PBS to wash twice, discard the PBS, and perform routine digestion;

[0032] ③ Transfer to a 15 ml centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of pre-cooled PBS at 4℃, and prepare 1×10⁻⁶ ppm. 7 Prepare a cell suspension of cells per mL and store on ice for later use.

[0033] 6. Establish a C57BL / 6 subcutaneous tumor-bearing mouse model:

[0034] ① The right hind limb of the mouse was skinned the day before the experiment, and the day of the experiment was recorded as day0. The weight was recorded.

[0035] ②In a clean bench, mice were anesthetized by inhaling isoflurane through their mouth and nose;

[0036] ③ Use your left hand to grasp and lift the mouse by pinching the skin on the back of its neck, exposing the skin under its right armpit. Disinfect the skin at the transplant site with 75% alcohol. Use your right hand to hold the needle and insert it horizontally into the subcutaneous tissue of the mouse's right rib area. The needle tip should be able to swing horizontally after insertion to prevent it from entering the muscle layer.

[0037] ④ Subcutaneously inject 3 × 10⁶ B16F10 cells into each mouse. 5 Inject one cell slowly, then press the needle hole with an alcohol swab to prevent leakage;

[0038] ⑤ Continue feeding and observe the tumor formation.

[0039] 7. Grouping and administration method:

[0040] Two groups of mice were set up: a model group and an Atractylodes macrocephala polysaccharide (AMP) group, with 5 mice in each group. All mice were given AMP by gavage starting on day 1 after modeling, marked as day 1, once a day for 14 consecutive days. Model group: 0.2 mL of physiological saline per mouse by gavage; AMP group: 0.2 mL of physiological saline solution per mouse by gavage, at a dose of 250 mg / kg / day.

[0041] 8. Tumor growth detection and animal sampling

[0042] Tumor volume and weight were recorded, and tumor growth was compared among groups. From the date of tumor formation, the long axis (L) and transverse axis (W) of the subcutaneous tumors in mice were measured daily to calculate tumor volume and plot tumor growth curves. The calculation formula is: Tumor volume (cm²) = (L / W) * (W ... 3 = 1 / 2 × L × W 2 Mice were sacrificed 14 days after modeling, and tumor tissue was removed and weighed to compare the differences in tumor growth among the groups.

[0043] 9. Flow cytometry

[0044] Prepare tissue dissociation solution. Under aseptic conditions, collect mouse tumor tissue, thoroughly mince it with scissors, and place it in 5 mL of tissue dissociation solution. Incubate at 37°C on a shaker for 20 min. After dissociation, add 1640 units of complete culture medium to terminate the reaction. Filter through a 70 μm filter, centrifuge at 2000 rpm for 10 min at 4°C to obtain cell pellet, discard the supernatant, add erythrocyte lysis buffer, incubate at room temperature in the dark for 5 min, then add 3 times the volume of complete culture medium to terminate the reaction, centrifuge at 2000 rpm for 10 min at 4°C, discard the supernatant, resuspend the pellet in PBS, centrifuge at 2000 rpm for 10 min at 4°C, discard the supernatant, resuspend the pellet in 1 mL of PBS, and store on ice. Analyze the sample using a flow cytometer, sequentially detecting Treg and CD8 levels. + The proportion of T cells to total T cells and the proportion of NK cells to immune cells were analyzed using statistical data.

[0045] II. Experimental results are shown below Figures 1-3 .

[0046] 1. Atractylodes macrocephala polysaccharide (AMP) inhibits tumor growth in melanoma mice.

[0047] First, it was determined whether AMP had an anti-tumor effect on melanoma mice. A B16-F10 melanoma subcutaneous xenograft model was established using C57BL / 6 mice, divided into a Model group and an AMP group. Tumor volume, tumor weight, and mouse body weight changes were monitored in each group. The study found that AMP significantly inhibited tumor growth in melanoma mice. (See below) Figure 1 AD.

[0048] 2. AMP reduces the number of Treg cells in melanoma-bearing mouse tumor tissue.

[0049] Currently, the tumor microenvironment is one of the core factors leading to cancer treatment failure. Among these factors, Treg cells are considered important cells that maintain immune tolerance homeostasis in the tumor microenvironment and weaken the efficacy of immunotherapy. Multiple clinical studies have clearly demonstrated that high Treg cell infiltration in melanoma tissue is positively correlated with shortened patient survival. In this embodiment, flow cytometry was used to detect the Treg cell content in mouse tumor tissue. The results showed that the proportion of Treg cells in the AMP group was significantly lower than that in the Model group, indicating that AMP can enhance anti-tumor immunity by reducing the infiltration of immunosuppressive Treg cells. Figure 2 ).

[0050] 3. AMP increases NK cells and CD8+ in melanoma mouse tumor tissue. ＋ T cell count

[0051] Flow cytometry was used to detect NK cells and CD8+, immune killer cells that exert anti-tumor effects, in tumor tissue. ＋ T cell levels were found to be significantly increased by AMP in melanoma mouse tumor tissues, with NK cells and CD8+ being significantly increased. ＋ The presence of T cells indicates that AMP can increase NK cells and CD8+ in tumor tissue. ＋ T cell infiltration plays an anti-tumor role. Figure 3 ).

[0052] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. Application of Atractylodes macrocephala polysaccharide in the preparation of drugs for the prevention / treatment of melanoma.

2. The application of Atractylodes macrocephala polysaccharide according to claim 1 in the preparation of a drug for the prevention / treatment of melanoma, characterized in that, The aforementioned drugs for the prevention / treatment of melanoma inhibit the growth of melanoma tumors, reduce the content of immunosuppressive Treg cells in tumor tissue, and increase the content of CD8 immune killer cells in tumor tissue. + Drugs containing high levels of T cells and NK cells.

3. The application of Atractylodes macrocephala polysaccharide according to claim 1 in the preparation of a drug for the prevention / treatment of melanoma, characterized in that, The aforementioned drug for the prevention / treatment of melanoma uses Atractylodes macrocephala polysaccharide as the sole active monomer component, or Atractylodes macrocephala polysaccharide is used in combination with other drugs to form a pharmaceutical composition.

4. The application of Atractylodes macrocephala polysaccharide according to claim 1 in the preparation of a drug for the prevention / treatment of melanoma, characterized in that, The dosage of Atractylodes macrocephala polysaccharide in the drug for the prevention / treatment of melanoma is 250 mg / kg.

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