Application of caulerpin and composition thereof in preparation of medicine with ovarian injury resisting effect and ovarian fibrosis resisting effect

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of caulerpin and a composition thereof in preparation of a medicine with an ovarian injury resisting effect and an ovarian fibrosis resisting effect. The ovarian injury is chemotherapy ovarian injury caused in the breast cancer chemotherapy process. The ovarian fibrosis is chemotherapy ovarian fibrosis caused in the breast cancer chemotherapy process. The invention also provides a composition which comprises cyclophosphamide and caulerpin. The composition is prepared from cyclophosphamide and caulerpin. Results show that the combination of caulerpin and cyclophosphamide has a remarkable treatment effect on tumor growth of tumor-bearing nude mice, and can be used for treating breast cancer. In addition, the caulerpin and the combination of the caulerpin and the cyclophosphamide can significantly improve ovarian toxicity effects induced by cyclophosphamide, such as the estrus cycle, the follicle reduction number, ovarian injury, ovarian fibrosis and the like of tumor-bearing nude mice.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of phycoerythrin and a composition thereof in the preparation of a drug having an anti-ovarian damage effect and an anti-ovarian fibrosis effect. Background Art

[0002] With the continuous advancement of tumor chemotherapy technology, the survival rate of breast cancer patients has been significantly improved. However, damage to the ovaries during breast cancer chemotherapy is still a major problem. Cyclophosphamide is a widely used chemotherapy drug that can effectively improve the survival rate of breast cancer patients, but its potential toxicity can lead to ovarian fibrosis, which in turn affects the patient's ovarian reserve function, reducing fertility and quality of life. Especially in breast cancer patients, the protection of reproductive function has become a focus of widespread attention during treatment. However, there is currently no standardized method in clinical practice to prevent or treat chemotherapy-induced ovarian fibrosis. Therefore, during cyclophosphamide treatment, repairing ovarian fibrosis is of great significance for protecting the reproductive function of breast cancer patients. This not only helps maintain the patient's fertility, but also improves their overall quality of life, which is one of the challenges that need to be urgently addressed in the current reproductive field.

[0003] Caulerpin is composed of five leucine residues and two n-methyl groups. It is a bisindole alkaloid with multiple biological activities such as anti-infection, anti-tumor and anti-inflammatory. Its structural formula is C24H18N2O4, and its molecular weight is 398.411. Its structural formula is as follows:

[0004]

[0005] However, the prior art does not report that Caulerpin has anti-ovarian damage and anti-ovarian fibrosis effects. Summary of the invention

[0006] In order to overcome at least one technical problem existing in the prior art, the present invention provides the use of phycoerythrin and a composition thereof in the preparation of a drug having an anti-ovarian damage effect and an anti-ovarian fibrosis effect.

[0007] The technical solution of the present invention is as follows:

[0008] The present invention firstly provides an application of phycoerythrin in preparing a medicine with an anti-ovarian damage effect.

[0009] Preferably, the ovarian damage is chemotherapy-induced ovarian damage.

[0010] Further preferably, the ovarian damage is chemotherapy-induced ovarian damage caused during breast cancer chemotherapy.

[0011] More preferably, the chemotherapy is chemotherapy using cyclophosphamide.

[0012] The present invention also provides an application of phycoerythrin in preparing a medicine having an anti-ovarian fibrosis effect.

[0013] Preferably, the ovarian fibrosis is chemotherapy-induced ovarian fibrosis.

[0014] Further preferably, the ovarian fibrosis is chemotherapy-induced ovarian fibrosis caused during breast cancer chemotherapy.

[0015] More preferably, the chemotherapy is chemotherapy using cyclophosphamide.

[0016] The present invention also provides a composition comprising cyclophosphamide and phycoerythrin.

[0017] Preferably, the mass ratio of cyclophosphamide to phycoerythrin in the composition is 10-15:1.

[0018] Most preferably, the mass ratio of cyclophosphamide to phycoerythrin in the composition is 12.5:1.

[0019] The present invention also provides an application of the above composition in preparing a medicine having an anti-breast cancer effect.

[0020] Further preferably, the composition is used in the preparation of a drug having both an effect against breast cancer and an effect against chemotherapy-induced ovarian damage caused during breast cancer chemotherapy.

[0021] Further preferably, the composition is used in the preparation of a drug having both anti-breast cancer and anti-chemotherapy ovarian fibrosis caused by breast cancer chemotherapy.

[0022] Beneficial effects: The present invention establishes a breast cancer cell (MDA-MB-231) tumor-bearing mouse model, adopts three experimental schemes of using cyclophosphamide and caulerpin alone, and using cyclophosphamide and caulerpin simultaneously, and uses H&E staining, Sirius red staining, Masson staining, Western blotting and other methods to observe the tumor growth of tumor-bearing nude mice, body weight, ovarian coefficient, evaluate the estrous cycle of mice, ovarian tissue and follicle morphology, and changes in the expression of fibrosis-related markers. The results show that caulerpin combined with cyclophosphamide has a significant therapeutic effect on the tumor growth of tumor-bearing nude mice, and can be used to treat breast cancer. In addition, caulerpin and caulerpin combined with cyclophosphamide can significantly improve the estrous cycle of tumor-bearing nude mice, the number of follicles, ovarian damage, ovarian fibrosis and other cyclophosphamide-induced ovarian toxic effects.

[0023] It can be seen that the use of caulerpin in the preparation of drugs for preventing ovarian damage and ovarian fibrosis has important application value; in particular, the use of caulerpin in the preparation of drugs for preventing chemotherapy-induced ovarian damage and ovarian fibrosis caused by breast cancer chemotherapy has more important application value. Furthermore, the composition obtained by combining cyclophosphamide and caulerpin can significantly reduce chemotherapy-induced ovarian damage and ovarian fibrosis when used for treating breast cancer; and significantly reduce the ovarian toxicity induced by cyclophosphamide chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Caulerpin inhibits breast cancer tumor growth in nude mice and reduces chemotherapy-induced ovarian damage.

[0025] Figure 2 Effects of Caulerpin on ovarian fibrosis and ECM protein expression in mice with chemotherapy-induced breast cancer

[0026] Figure 3 Effect of Caulerpin on ROS levels in ovarian tissue of mice with chemotherapy-induced breast cancer

[0027] Figure 4 Effects of Caulerpin on CTX-induced ROS accumulation and ECM-related protein activation in ovarian granulosa cells in vitro. DETAILED DESCRIPTION

[0028] The present invention is further explained below in conjunction with specific examples, but the examples do not limit the present invention in any form.

[0029] Example 1 Effect of Caulerpin on Inhibiting Tumor Growth and Alleviating Chemotherapy-Induced Ovarian Damage

[0030] Cyclophosphamide combined with caulerpin to treat breast cancer cell (MDA-MB-231) tumor-bearing mouse model Experimental animals were specific pathogen-free (SPF) grade female BALB / C-Nude nude mice (n=32, 4-6 weeks old, 18.1±0.1g), purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd., and raised in the Animal Experiment Center of Jinan University, under conditions of 22±2°C, relative humidity of 50±10%, 12-hour light / dark cycle, and free access to food and water. MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% glutamine-penicillin-streptomycin in a 5% CO2 incubator at 37°C. The passage time was confirmed according to the cell state and growth rate, and cells with good growth status and in the logarithmic growth phase were used for the experiment. MDA-MB-231 cells (5×107cells / mL) were collected aseptically and transplanted orthotopically into the mammary fat pad of each mouse. After successful modeling, the tumor-bearing mice were randomly divided into 4 groups (n=8): saline group, cyclophosphamide group (50 mg / kg), caulerpin treatment group (4 mg / kg) and cyclophosphamide combined with caulerpin treatment group (caulerpin 4 mg / kg + cyclophosphamide 50 mg / kg).

[0031] The mice were weighed before each administration, the development of primary xenograft tumors in the transplanted mice was monitored, and the mice were sacrificed when the tumors reached 10% of their body weight. The ovaries were weighed after carefully removing adipose tissue and other tissues, and the ovarian index was calculated by dividing the wet weight of both ovaries by the body weight.

[0032] The results showed that caulerpin could significantly inhibit tumor growth in vivo ( Figure 1 AB). Compared with the control group, the cyclophosphamide combined with caulerpin group had a significant inhibitory effect on tumor growth. After 20 days of administration, the tumor volumes of each group were 605.39±17.49mm 3 (control group), 372.76±18.53mm 3 (caulerpin), 245.95±16.32mm 3 (cyclophosphamide + caulerpin) and 112.76 ± 12.57 mm 3 (Cyclophosphamide) (Table 1). The body weight of mice in the cyclophosphamide group was significantly reduced compared with the other groups, indicating that cyclophosphamide was toxic to mice. However, there was no significant difference in the body weight of mice in the cyclophosphamide combined with caulerpin group, the caulerpin group, and the control group ( Figure 1C). Compared with control mice, mice bearing cyclophosphamide-induced tumors had smaller ovarian size and lower ovarian index (ovary / body weight). Compared with the cyclophosphamide group, mice treated with caulerpin had larger ovarian size and weight ( Figure 1 DE). This indicates that caulerpin synergistically enhances the anti-tumor effect of cyclophosphamide, while reducing the toxic side effects of cyclophosphamide and protecting ovarian tissue.

[0033] HE staining showed that the number of follicles in the cyclophosphamide-induced group decreased, while the Caulerpin-treated group restored the cyclophosphamide-induced ovarian morphological changes and increased the number of follicles ( Figure 1 F)

[0034] Table 1: Mouse weight and tumor size in the experimental and control groups

[0035]

[0036] Example 2 Effects of Caulerpin on fibrosis and oxidative status of ovarian tissue in mice with chemotherapy-induced breast cancer

[0037] Figure 2 Masson and Sirius red staining showed that the ovarian fibrosis area and collagen deposition increased in the cyclophosphamide group compared with the control group ( Figure 2 A), and immunohistochemical staining results showed that in the ovarian tissue of the cyclophosphamide group, the expression of extracellular matrix markers (FN1 and COL1A1) and TGF-β1 protein was higher than that of the control group ( Figure 2 B), indicating that cyclophosphamide induced ovarian fibrosis. Compared with the cyclophosphamide group, the positive areas of Masson and Sirius red staining in the caulerpin treatment group were significantly reduced ( Figure 2 A), and decreased expression of COL1A1 and FN1 in ovarian stroma ( Figure 2 B), indicating that caulerpin alleviates cyclophosphamide-induced ovarian fibrosis.

[0038] Ovarian tissues were collected and the antioxidant system in the ovaries, such as the content of superoxide dismutase (SOD) and glutathione (GSH), and the content of the oxidation product malondialdehyde (MDA) were measured using a colorimetric assay kit. Figure 3The results showed that compared with the control group, the levels of GSH (31.92±4.01 vs 18.35±2.39, P<0.0001) and SOD (299.3±90.53 vs 177.5±33.28, P<0.05) in the cyclophosphamide group were decreased, and the level of MDA was increased (18.59±5.09 vs 30.23±7.00, P<0.05), indicating that cyclophosphamide caused an imbalance between oxidation and antioxidant effects in ovarian tissue, resulting in oxidative stress. Compared with the cyclophosphamide group, the GSH (18.35±2.39 vs 23.76±2.26, P<0.01) and SOD (177.5±33.28 vs 315.4±34.16, P<0.05) levels in the caulerpin group increased significantly, while the MDA (30.23±7.00 vs 23.26±6.06, P<0.05) level decreased, indicating that caulerpin has a protective effect on cyclophosphamide-induced oxidative stress by inhibiting the production of reactive oxygen species (Table 2).

[0039] Table 2: SOD, GSH and MDA levels in mouse ovaries of different experimental groups

[0040]

[0041] Example 3 Effect of Caulerpin on CTX-induced ROS accumulation and ECM-related protein activation in ovarian granulosa cells

[0042] In vitro cell experiments were conducted using ovarian granulosa cells (COV-434). Two experimental schemes were used: using the active metabolite of cyclophosphamide (4-hydroperoxide cyclophosphamide, 4HC) alone or simultaneously with caulerpin. Immunofluorescence staining, Western blotting and other methods were used to observe ROS activity and fibrosis in ovarian granulosa cells.

[0043] The expression of AMH and FSHR was detected by immunofluorescence, and the viability of COV434 cells treated with 4HC was evaluated. Compared with the control group, the levels of AMH and FSHR decreased after 4HC treatment. Compared with the 4HC group, the levels of AMH and FSHR increased after caulerpin treatment, indicating that caulerpin can improve the active damage of ovarian granulosa cells induced by cyclophosphamide ( Figure 4 A). Intracellular ROS activity and DNA damage were assessed by analyzing the fluorescence intensity of DCFH-DA and γ-H2AX (sensitive markers of DNA double-strand breaks). In COV434 cells, 4HC treatment significantly increased intracellular ROS levels and DNA damage. In contrast, after treatment with caulerpin, a decrease in ROS levels was observed ( Figure 4B) These results suggest that caulerpin can alleviate cyclophosphamide-induced elevated reactive oxygen species levels and DNA damage.

[0044] Western blot results showed that in COV434 cells treated with 4HC, the protein levels of ECM-related genes (TGF-β1, COLA1, FN1) were increased, while caulerpin reduced the up-regulated expression of these genes ( Figure 4 C) These data suggest that caulerpin can alleviate ECM deposition and attenuate ovarian fibrosis.

Claims

1. Application of fern phycoerythrin in the preparation of drugs with anti-ovarian damage effects.

2. The use according to claim 1, characterized in that: The ovarian damage is chemotherapy-induced ovarian damage.

3. The use according to claim 1, characterized in that: The ovarian damage is chemotherapy-induced ovarian damage caused during breast cancer chemotherapy.

4. Application of fern phycoerythrin in the preparation of drugs with anti-ovarian fibrosis effects.

5. The use according to claim 4, characterized in that: The ovarian fibrosis is chemotherapy-induced ovarian fibrosis.

6. The use according to claim 4, characterized in that: The ovarian fibrosis is chemotherapy-induced ovarian fibrosis caused during breast cancer chemotherapy.

7. A composition, characterized in that Contains cyclophosphamide and phycoerythrin.

8. The composition according to claim 7, characterized in that The mass ratio of cyclophosphamide to phycoerythrin in the composition is 10-15:

1.

9. The composition according to claim 8, characterized in that The mass ratio of cyclophosphamide to phycoerythrin in the composition is 12.5:

1.

10. Use of the composition according to claim 7 in the preparation of a drug having an anti-breast cancer effect.

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