Application of oxypeucedanin in preparation of medicine for preventing or treating intestinal inflammation caused in anti-tumor process

Abstract

The invention provides an application of oxypeucedanin (OPD) in preparation of a medicine for preventing or treating intestinal inflammation caused in an anti-tumor process. According to the application, the drug combination of the oxypeucedanin and the chemotherapeutic drug is applied to preparation of the anti-tumor drug. In the combined medication process, the strong toxic effect of a single drug on the body is reduced through the synergistic effect of the drugs in the combined use of the oxypeucedanin and the chemotherapeutic drugs, and the oxypeucedanin reduces the activation of the chemotherapeutic drugs on the pyroptosis of normal tissues, so that the side effects are reduced. It is found for the first time that OPD plays a role in controlling inflammatory injury by relieving pyroptosis caused by chemotherapeutic drugs. When OPD and chemotherapeutic drugs are combined for use, pyroptosis induced by chemical drugs can be remarkably inhibited, and meanwhile damage to normal tissue of an organism is relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a pharmaceutical composition for treating cancer, in particular, to a synergistic anti-tumor pharmaceutical composition and its application. Background Art

[0002] Cisplatin is one of the most widely used and effective chemotherapy drugs. Although the anti-tumor mechanism of cisplatin has not been fully elucidated, it is generally believed that its mechanism of action mainly involves DNA cross-linking. This mechanism of action leads to the obstruction of DNA synthesis and replication and induces template defects. In rapidly dividing cells, especially tumor cells, DNA damage caused by DNA cross-linking is more serious, leading to irreversible cell death. At the same time, oxidative stress is also one of the important mechanisms of cisplatin toxicity. Cisplatin is metabolized in the body to produce toxic metabolites, which in turn promote the increase of reactive oxygen species (ROS) production. This process triggers mitochondrial dysfunction, thereby inducing a series of inflammatory responses. Considering that tumor cells have relatively high basal oxidation levels, they usually show stronger oxidative stress responses. Cisplatin tends to act on these tumor cells, inducing them to produce high levels of reactive oxygen species, which in turn leads to tumor cell death. For a long time, it has been generally believed that programmed cell death induced by chemotherapy drugs is mainly apoptosis. However, recent studies have found that several chemotherapy drugs achieve their anti-tumor effects by inducing cell pyroptosis. Cisplatin activates caspase-3 and specifically cleaves Gasdermin-E (GSDME) protein to produce perforated GSDME-N fragments, causing some dying cells to show cell pyroptosis characteristics such as cell swelling, rupture, and release of pro-inflammatory factors. During tumor treatment, repeated administration can cause cisplatin to accumulate in the body, causing serious damage to normal tissues such as the gastrointestinal tract, liver, and kidneys. Although cisplatin has always been a mainstream drug for cancer treatment, its severe toxicity limits its use and efficacy.

[0003] As a traditional Chinese edible medicinal material, Angelica sinensis has been playing an important role in protecting human health and curing diseases. So far, researchers have discovered more than 300 chemical components in Angelica sinensis. Among them, coumarin is one of the most important active compounds in Angelica sinensis, and it is also a class of natural compounds present in various plants. Oxypeucedanin (OPD) is a kind of furanocoumarin derivative, which has the characteristics of low molecular weight, simple structure, high bioavailability and low toxicity, which ensures that OPD and its semi-synthetic derivatives have the potential as new therapeutic agents. Evidence has shown that the combination of natural compounds and chemotherapeutic drugs can protect non-target organs and effectively reduce the toxicity of cisplatin. OPD has significant anti-inflammatory, antibacterial, antioxidant, anti-resistance, antidepressant and anti-tumor activities. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of oxidized desperadoes in the field of tumor treatment, thereby providing a pharmaceutical composition for treating tumors, and reducing the toxic side effects of chemotherapy drugs used alone on the body by combining OPD with chemotherapy drugs. Oxidized desperadoes and chemotherapy drugs reduce the strong toxic effects of chemotherapy drugs on the body through the synergistic effect between the drugs, and oxidized desperadoes reduces the activation of pyroptosis of normal tissues by chemotherapy drugs, thereby reducing the toxic side effects.

[0005] In order to achieve the above object, the present invention provides the use of oxidized desperadolin in the preparation of a drug for preventing or treating intestinal inflammation caused during the anti-tumor process.

[0006] Preferably, the combination of oxidized proserine and a chemotherapeutic drug is used in the preparation of a drug for preventing or treating intestinal inflammation caused during the anti-tumor process.

[0007] Any of the above items is preferably that the chemotherapy drug is a platinum-based chemical drug, and the oxidized procerasin reduces the level of cell pyroptosis caused by the chemotherapy drug. The oxidized procerasin reduces the killing of the chemotherapy drug to normal cells by reducing the level of cell pyroptosis, while the killing effect of the chemotherapy drug on tumor cells remains unchanged.

[0008] Preferably, any of the above items is that the chemotherapy drug is a platinum drug such as cisplatin.

[0009] Preferably, any of the above items is that the combination is a combination of two separate preparations of oxidized procerasin and a chemotherapeutic drug; the two separate preparations are administered simultaneously or the two separate preparations are administered sequentially.

[0010] Preferably, any of the above items is that the administration route includes intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection.

[0011] In any of the above, preferably, the concentration of oxidized desperadoxin is 6.25 to 100 μM, more preferably 6.25, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 μM and the range therebetween. Preferably, the concentration of oxidized desperadoxin is 12.5 to 100 μM. Preferably, the concentration of oxidized desperadoxin is 12.5 to 50 μM.

[0012] The present invention also provides the use of oxidized desperadoesin and chemotherapy drugs in the preparation of anti-tumor drugs. Oxidized desperadoesin and chemotherapy drugs reduce the strong toxicity of a single drug to the body through the synergistic effect between the drugs, and oxidized desperadoesin reduces the activation of pyroptosis of normal tissues by chemotherapy drugs, thereby reducing side effects.

[0013] The present invention is achieved on the basis of the following research findings:

[0014] 1. It was found that the addition of OPD pretreatment to a variety of cells, including normal human colon epithelium (NCM460) and melanoma (B16), can reduce the level of cell pyroptosis and reduce the killing of cells by the chemotherapy drug cisplatin.

[0015] 1) After OPD treatment in various cell lines, chemical drugs were used to treat them, and the killing of tumor cells was observed under a microscope and the release of LDH was detected. It was found that the proportion of pyroptotic cells was significantly reduced and the release of LDH was significantly reduced.

[0016] 2) Cells were pretreated with OPD and then treated with chemical drugs: Western blot detected that the GSDME cleavage activation level was significantly weakened, confirming that the pyroptosis level had decreased.

[0017] 2. It was found that in animal models, compared with the use of cisplatin alone, the combined use of OPD can significantly reduce chemical-induced intestinal damage in mice.

[0018] 1) After using OPD, the mice were treated with chemical drugs and the H&E stained sections of the intestinal tissue were observed under a microscope, which showed that the loss of the tips of the small intestinal villi and the damage to the small intestinal mucosa were significantly improved.

[0019] 2) After OPD, the mice were treated with chemical drugs and the cleavage level of GSDME in the intestinal tissue was detected by Western blot.

[0020] Based on observations and studies on molecular mechanisms, we speculate that the combined use of OPD and chemical treatment can negatively regulate the level of cell pyroptosis by inhibiting GSDME activation signals, which helps to reduce the side effects of chemical drugs.

[0021] Preclinical experiments on mouse tumor models further demonstrated the role of OPD in inhibiting cell pyroptosis and the relationship between it and the anti-tumor effects of chemotherapeutic drugs.

[0022] 3. It was found that in the animal model of transplanted tumors, compared with cisplatin alone, the combination of OPD can significantly reduce cisplatin-induced tumor and normal tissue pyroptosis, and has no effect on the anti-tumor ability of cisplatin.

[0023] The present invention has beneficial technical effects:

[0024] 1) The present invention is the first to discover that OPD plays a role in controlling inflammatory damage by alleviating cell pyroptosis induced by chemotherapy drugs.

[0025] 2) The present invention discovered for the first time that when OPD is used in combination with chemotherapeutic drugs, it can significantly inhibit drug-induced cell pyroptosis and reduce damage to normal tissues of the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result that OPD in the preferred embodiment 1 of the present invention inhibits the activity of tumor cells in a dose-dependent manner.

[0027] Figure 2 These are the results of pre-treatment with OPD followed by treatment with chemotherapeutic drugs in NCM460 cells and B16 cell lines in preferred embodiment 1 of the present invention.

[0028] Figure 3 In the preferred embodiment 2 of the present invention, in the mouse intestinal injury model, when OPD and cisplatin were used in combination, the cisplatin-induced small intestinal inflammatory response was significantly inhibited.

[0029] Figure 4 In the preferred embodiment 2 of the present invention, in the small intestinal tissue of mice, the activation level of GSDME was significantly reduced after treatment with chemical drugs after using OPD.

[0030] Figure 5 In the preferred embodiment 3 of the present invention, in the mouse transplant tumor model, there was no significant difference in the tumor inhibition effect between the combination of OPD and cisplatin and the use of cisplatin alone.

[0031] Figure 6 In the preferred embodiment 2 of the present invention, in a mouse transplant tumor model, when OPD and cisplatin were used in combination, frozen sections observed that (A) the proportion of cells undergoing pyroptosis in the tumor was significantly reduced; and (B) the PI positive signal in the small intestine was weakened. DETAILED DESCRIPTION

[0032] The present invention is described in detail by specific examples below. The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The test methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used are all commercially available products.

[0033] Test Materials:

[0034] Melanoma cell line B16 and human normal colon epithelial cell line NCM460 are conventional cell lines in the prior art and can be purchased through commercial channels.

[0035] 6-week-old male BALB / c mice and 6-week-old male C57 mice were purchased from Liaoning Changsheng Experimental Animal Co., Ltd.;

[0036] Oxypeucedanin, cisplatin, and propidium iodide (PI) were purchased from Meilun Company.

[0037] Example 1

[0038] By pre-treating tumor cells with OPD and then treating them with chemical drugs, it was found that the proportion of cells undergoing pyroptosis decreased and the activation level of GSDME was significantly reduced.

[0039] The specific steps are:

[0040] NCM460 cells, B16 cells and CT26 cells were seeded in 12-well plates. When the cell density was about 70-80%, different concentrations of OPD (0, 6.25, 25, 100 μM) were added to the culture medium for 24 h. EdU staining was performed to test the effect of OPD on cell proliferation activity, indicating that OPD inhibited the activity of tumor cells in a dose-dependent manner, as shown in Figure 2. Figure 1 shown.

[0041] NCM460 cells and B16 cells were inoculated in 6-well plates. When the cell density was about 70-80%, OPD (12.5 μM) was added to the culture medium for pretreatment for 6 hours, and then the cells were treated with cisplatin (40 μM) for 20 hours. After the drug treatment, propidium iodide dye was added and observed under a microscope. Compared with the treatment with chemical drugs alone, the proportion of cells undergoing pyroptosis decreased after OPD combined with OPD; the supernatant was collected, and LDH release was also reduced. The results are as follows Figure 2 As shown, A shows that the proportion of cells undergoing pyroptosis is significantly reduced; B shows that the release of LDH is significantly reduced.

[0042] Example 2

[0043] The reversal of chemical-induced intestinal damage in mice by OPD was verified by intraperitoneal injection of OPD (15 mg / kg) 1 hour in advance and then oral administration of chemical drugs (20 mg / kg) for 7 consecutive days.

[0044] First, the intestinal tissues of mice were collected, and paraffin sectioning and H&E staining were performed. The results were observed under a microscope, confirming that OPD could effectively reverse the small intestinal mucosal damage and loss of villus tips caused by chemical treatment. Figure 3 shown.

[0045] The mouse intestinal tissue was collected and lysed, and Western blot experiments showed that the cleavage level of GSDME was also reduced after OPD pretreatment. Figure 4 shown.

[0046] Example 3

[0047] In mouse transplanted tumors, when OPD was used in combination with chemotherapy drugs, the combined therapy could significantly reduce cisplatin-induced pyroptosis of tumors and normal tissues, and had no effect on the anti-tumor ability of cisplatin.

[0048] 5 × 10 6 B16 or CT26 tumor cells. First, the cells were cultured in vitro. When the cell density reached 100%, the cells were digested with trypsin. After centrifugation to remove the trypsin, the cells were resuspended with PBS, and then the cell suspension was counted. The cell suspension was diluted with PBS according to the number of injected cells and divided into centrifuge tubes, and then the cell suspension was placed on ice for later use. The mice were randomly and evenly divided into 4 groups, with 8 mice in each group. The four groups were: 1) control treatment group (PBS); 2) OPD alone group; 3) cisplatin alone group; 4) OPD and cisplatin combination group (intraperitoneal injection of OPD, cisplatin gavage 1 hour later). On the 6th day after tumor implantation, OPD was pretreated by intraperitoneal injection for 1 hour, and then the drugs were injected according to the needs of each group and administered separately. The dosage of OPD was 15 mg / kg, and the dosage of cisplatin was 20 mg / kg, and they were administered every day. According to the requirements of animal experimental ethics and experimental animal welfare, the volume of mouse tumors was greater than 2000mm 3 Or when the diameter of the tumor ulcer approaches 1 cm, the observation is terminated, and the mice are killed and deemed dead. Among them, the average tumor size of the control group mice reaches 2000 mm 3 The date was the cut-off point. By this time, each group of mice had been given the drug at least 6 times. From the experimental results, it can be seen that there was no significant difference in the effect of inhibiting tumor growth between the OPD and cisplatin combination group and the cisplatin group alone. Figure 5 As shown in the figure, the observation of frozen sections revealed that the positive signal of PI in the small intestine of the combined drug group was weakened compared with the cisplatin group alone. Figure 6 shown.

[0049] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Application of oxidized proserpine in the preparation of drugs for preventing or treating intestinal inflammation caused by anti-tumor process.

2. The use according to claim 1, characterized in that Application of oxidized proserine combined with chemotherapy drugs in the preparation of drugs for preventing or treating intestinal inflammation caused during anti-tumor treatment.

3. The use according to claim 2, characterized in that The chemotherapy drugs include platinum-based drugs, and the oxidized procerasin reduces the level of cell pyroptosis caused by the chemotherapy drugs.

4. The use according to claim 3, characterized in that The chemotherapy drug includes cisplatin.

5. The use according to claim 2 or 3, characterized in that: The combined use of oxidized desperadoesin and chemotherapeutic drugs is a combination of two separate preparations of oxidized desperadoesin and chemotherapeutic drugs; the two separate preparations are administered simultaneously or the two separate preparations are administered sequentially.

6. The use according to claim 5, characterized in that The administration routes include intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration or transdermal administration, preferably intraperitoneal injection.

7. The use according to any one of claims 1 to 6, wherein the combination of oxidized procerasin and a chemotherapeutic drug is used in the preparation of an anti-tumor drug.

8. A pharmaceutical composition of oxidized desperadoline and a chemotherapeutic drug prepared according to the use of any one of claims 1 to 7.

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