阅读说明：本技术 葫芦素b在制备铁死亡诱导剂及抗鼻咽癌药物中的应用 (Application of cucurbitacin B in preparation of iron death inducer and anti-nasopharyngeal carcinoma drug ) 是由 梁宝霞 严君 廖升荣 于 2020-06-05 设计创作，主要内容包括：本发明公开了葫芦素B在制备铁死亡诱导剂及抗鼻咽癌药物中的应用。本发明首次研究显示葫芦素B可诱导铁死亡的发生,以一种新型的促进细胞死亡的方式调控鼻咽癌细胞的死亡,进一步探究发现,葫芦素B在鼻咽癌细胞CNE1内能促使铁离子的累积,减少细胞内谷胱甘肽的含量,下调GPX4的表达,导致脂质过氧化物的增加,诱发细胞发生铁死亡。在体内外研究中,葫芦素B诱导CNE1细胞周期阻滞在G2/M期,抑制细胞的迁移和侵袭,此外,葫芦素B能显著抑制小鼠鼻咽癌肿瘤的生长,无明显毒副作用。本发明为鼻咽癌的治疗药物提供了一种新选择,即铁死亡诱导剂；同时也为铁死亡诱导剂及鼻咽癌的治疗提供了天然新型的药物来源即葫芦素B,高效、安全、无毒副作用。(The invention discloses an application of cucurbitacin B in preparation of an iron death inducer and a nasopharyngeal carcinoma resisting medicine. The invention shows that cucurbitacin B can induce iron death for the first time, and regulates the death of nasopharyngeal carcinoma cells in a novel cell death promoting mode, and further researches show that cucurbitacin B can promote the accumulation of iron ions in the nasopharyngeal carcinoma cells CNE1, reduce the content of glutathione in the cells, and regulate the expression of GPX4, so that the increase of lipid peroxide is caused, and the iron death of the cells is induced. In vitro and in vivo studies, cucurbitacin B induces CNE1 cell cycle to be blocked at G2/M stage, and inhibits migration and invasion of cells. The invention provides a new choice for the treatment medicine of nasopharyngeal carcinoma, namely an iron death inducer; meanwhile, a natural novel medicine source namely cucurbitacin B is provided for treating the iron death inducer and the nasopharyngeal carcinoma, and the cucurbitacin B is efficient, safe and free of toxic and side effects.)

1. Application of cucurbitacin B in preparing iron death inducer is provided.

2. Application of cucurbitacin B in preparing medicine for treating nasopharyngeal carcinoma is provided.

3. The use of claim 2, wherein said anti-nasopharyngeal carcinoma comprises increasing the intracellular iron concentration in the nasopharyngeal carcinoma.

4. The use of claim 2, wherein said anti-nasopharyngeal carcinoma comprises decreasing glutathione levels in cells of the nasopharyngeal carcinoma.

5. The use of claim 2, wherein said anti-nasopharyngeal carcinoma comprises inhibiting or down-regulating the expression of GPX4 in nasopharyngeal carcinoma cells.

6. The use of claim 2, wherein said anti-nasopharyngeal carcinoma comprises inducing cell cycle arrest of nasopharyngeal carcinoma at stage G2/M.

7. The use of claim 2, wherein said anti-nasopharyngeal cancer comprises inhibiting proliferation, migration and/or invasion of cells of nasopharyngeal cancer.

8. The use of claim 2, wherein said anti-nasopharyngeal carcinoma comprises increasing intracellular lipid reactive oxygen species in a nasopharyngeal carcinoma.

9. The use according to any one of claims 1 to 8, wherein said anti-nasopharyngeal cancer cell is a CNE1 nasopharyngeal cancer cell.

10. Application of iron death inducer in preparing medicine for treating nasopharyngeal carcinoma is provided.

Technical Field

The invention belongs to the technical field of medicines. More particularly, relates to the application of cucurbitacin B in preparing iron death inducers and anti-nasopharyngeal carcinoma medicines.

Background

Nasopharyngeal carcinoma is a malignant tumor occurring at the top and side walls of the nasopharyngeal cavity, one of high-incidence malignant tumors in China, and the incidence of the malignant tumor is the first of otorhinolaryngological malignant tumors. Nasopharyngeal carcinoma mainly occurs in five provinces in south China, namely Guangdong, Guangxi, Hunan, Fujian and Jiangxi, and accounts for the first part of local head and neck malignant tumors, and southeast Asia countries are high incidence areas. At present, no particularly effective medicine is available for treating the nasopharyngeal carcinoma, and radiotherapy is the first choice treatment method for the nasopharyngeal carcinoma, but for patients with high differentiated cancer, late course and relapse after radiotherapy, surgical excision and chemotherapy are needed.

Dixon in 2012 reported that iron death (ferroptosis) was an iron ion-dependent non-apoptotic cell necrosis. The iron death is the regulated cell necrosis caused by lipid peroxidation induced by iron ions and active oxygen, and is obviously different from other forms of regulated cell necrosis such as apoptosis, necrosis, autophagy and the like on the morphological, biological and gene levels, and has become a hot point of research in recent years. The essence of iron death is metabolic disorder of lipid oxide in cells, and then abnormal metabolism is performed under the catalysis of iron ions, so that a large amount of lipid is produced, oxidation-reduction balance in the cells is destroyed, biomacromolecules are attacked, and cell death is triggered. Iron death is closely related to diseases such as nervous system diseases, tumors, ischemia-reperfusion injury, kidney injury, atherosclerosis, diabetes, heart disease and the like. A large number of research reports indicate that iron death plays a key negative regulatory role in the development of tumors. There is increasing evidence that induction of iron death can serve as an important mechanism for anti-tumor therapy, and therefore, induction of iron death in tumor cells is a novel anti-tumor therapeutic strategy. Iron death can be induced by two classes of small molecule substances: one is system Xc^-Inhibitors, such as Erastin; another class is GPx4 inhibitors, such as RSL 3. Patent CN110279697A provides application of iron death inducers Erastin and RSL3 in medicines for treating or relieving allergic airway inflammation, but the medicines often have obvious side effects, and artemisinin is the main discovered natural product capable of inducing iron death(Yi ren Xin, Wang and admire, Wang Handong, artemisinin and its derivatives through iron death pathway play the role of antitumor research progress [ J]Scientific and technological innovation, 2020(08):31-32.) and dangshen ketone derivatives, and natural products have more practical value due to the advantages of rich medicine sources, small side effect, multi-link integral treatment and the like.

Cucurbitacin compounds are tetracyclic triterpenoids, are commonly present in high-grade plants and large fungi of Scrophulariaceae, Cruciferae, Elaeagnaceae, Tetradaceae, Begoniaceae and the like, and have many pharmacological activities proved by experiments, such as anti-tumor, anti-inflammatory and the like. Cucurbitacin B (Cucurbitacin B, CuB) is one of the most abundant Cucurbitacin derivatives, and research reports that Cucurbitacin B can be used for treating various tumors, and compared with conventional chemotherapeutic drugs, Cucurbitacin B has the advantages of small bone marrow inhibition effect, low liver damage and small destructive power of an immune system, has important potential as an antitumor drug, but has no related research on treatment of nasopharyngeal carcinoma, and the main action mechanism of Cucurbitacin B is unclear. Therefore, the research and development of novel medicaments for treating nasopharyngeal carcinoma are urgently needed by taking the traditional Chinese medicine theory and the modern pharmacology and pharmacodynamics as guidance.

Disclosure of Invention

The invention aims to provide application of a natural product cucurbitacin B in preparation of an iron death inducer and a medicine for resisting nasopharyngeal carcinoma. Provides a natural novel medicine source with high efficiency, safety, no toxic or side effect for treating nasopharyngeal carcinoma.

The invention aims to provide the application of cucurbitacin B in preparing a ferrodeath inducer.

The invention also aims to provide application of cucurbitacin B in preparation of anti-nasopharyngeal carcinoma medicaments.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the invention relates to application of cucurbitacin B in preparation of an iron death inducer.

The growth inhibition effect of cucurbitacin B on nasopharyngeal carcinoma cells CNE1 is detected by an MTT method, the fact that cucurbitacin B can obviously inhibit the proliferation of nasopharyngeal carcinoma cell strains in vitro is found, the change of the morphology of nasopharyngeal carcinoma cells CNE1 induced by cucurbitacin B is observed by an electron microscope, and the feature that the nasopharyngeal carcinoma cell morphology shows iron death under the action of cucurbitacin B is found.

In order to further verify that cucurbitacin B has the effect of inducing iron death, the cucurbitacin B is subjected to intracellular iron ion content detection after being subjected to dosing treatment, and the result shows that the cucurbitacin B can promote the accumulation of iron ions in nasopharyngeal carcinoma cells CNE 1; because glutathione in cells mainly plays an anti-oxidation role and is an important molecule for resisting peroxidation pressure in the cells, the exhaustion of the glutathione can cause lipid peroxidation accumulation, and iron death can be induced to a certain degree, the invention further researches show that cucurbitacin B can reduce the content of glutathione in the cells and promote the accumulation of lipid active oxygen in nasopharyngeal carcinoma cells CNE 1; the expression of GPX4 has an important regulation effect on iron death, GPX4 can degrade small molecular peroxides and certain lipid peroxides under normal physiological conditions and inhibit lipid peroxidation, so that the inhibition of the activity of GPX4 or the reduction of the expression of GPX4 is beneficial to promoting the occurrence of iron death. In order to further evaluate the in vivo anti-tumor effect of cucurbitacin B, the influence of cucurbitacin B on the cell cycle is detected in cells, the cell cycle is detected by adopting a flow method, and the result shows that the cucurbitacin B can induce the cell cycle to block in the G2/M phase in CNE1 cells; in the research of further evaluating the in vivo anti-tumor effect of cucurbitacin B, the influence of cucurbitacin B on cell migration and invasion is detected, and the result shows that cucurbitacin B can effectively inhibit the cell migration and invasion; in order to evaluate the in vivo anti-tumor effect of cucurbitacin B, a mouse transplantation tumor model is constructed, and the result shows that cucurbitacin B can obviously inhibit the growth of nasopharyngeal carcinoma tumors and has no obvious toxic or side effect.

The invention is creatively researched, and the result shows that cucurbitacin B can induce the occurrence of iron death, regulate and control the death of nasopharyngeal carcinoma cells in a novel cell death promoting mode, is an effective iron death inducer, and can be used for preparing and developing anti-nasopharyngeal carcinoma medicaments.

Therefore, the invention also claims the application of cucurbitacin B in preparing the anti-nasopharyngeal carcinoma medicine.

Preferably, the anti-nasopharyngeal cancer comprises inhibiting proliferation of cells of the nasopharyngeal cancer, inhibiting migration and/or invasion of cells of the nasopharyngeal cancer.

Preferably, the anti-nasopharyngeal cancer comprises inducing cell cycle arrest of the nasopharyngeal cancer at stage G2/M.

Preferably, the anti-nasopharyngeal cancer comprises inhibiting or down-regulating expression of a nasopharyngeal cancer cell GPX 4.

Preferably, the anti-nasopharyngeal cancer comprises increasing the intracellular concentration of iron ions in the nasopharyngeal cancer.

Preferably, the anti-nasopharyngeal cancer comprises decreasing glutathione levels in cells of the nasopharyngeal cancer.

Preferably, the anti-nasopharyngeal cancer comprises increasing intracellular lipid reactive oxygen species in the nasopharyngeal cancer.

As a preferred example, the nasopharyngeal cancer cell is a CNE1 nasopharyngeal cancer cell.

In addition, the inventive research shows that the iron death inducer has better nasopharyngeal carcinoma tumor resisting effect, so the invention also requests to protect the application of the iron death inducer in preparing the nasopharyngeal carcinoma resisting medicine.

The molecular formula of cucurbitacin B is C₃₂H₄₆O₈Molecular weight 558.71, can be isolated from plants by various methods known in the art, or can be obtained directly from commercial sources.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers that the natural product cucurbitacin B has the function of inducing iron death in nasopharyngeal carcinoma cells for the first time, can be used as a high-efficiency iron death inducer, and shows better nasopharyngeal carcinoma tumor resisting effect in vivo and in vitro, and provides a new choice for a nasopharyngeal carcinoma treatment medicament, namely the iron death inducer; meanwhile, a natural novel medicine source namely cucurbitacin B is provided for treating the iron death inducer and the nasopharyngeal carcinoma, and the cucurbitacin B is efficient, safe and free of toxic and side effects.

Drawings

FIG. 1 shows the result of MTT method for detecting the growth effect of cucurbitacin B on nasopharyngeal carcinoma cell CNE 1.

FIG. 2 shows the results of experiments on the morphological effect of cucurbitacin B on nasopharyngeal carcinoma cells CNE 1.

FIG. 3 shows the effect of cucurbitacin B on the level of iron ion in nasopharyngeal carcinoma cells CNE 1.

FIG. 4 shows the effect of cucurbitacin B on the content of total glutathione in nasopharyngeal carcinoma cells CNE 1.

FIG. 5 shows the effect of cucurbitacin B on the content of lipid reactive oxygen species in nasopharyngeal carcinoma cells CNE 1.

FIG. 6 shows the effect of cucurbitacin B on the expression of GPX4 in nasopharyngeal carcinoma cells CNE 1.

FIG. 7 shows the results of experiments on the blockade of CNE1 cycle by cucurbitacin B in nasopharyngeal carcinoma cells.

FIG. 8 shows the results of experiments on the inhibition of cucurbitacin B migration to CNE 1.

FIG. 9 shows the inhibition experiment results of cucurbitacin B on the invasion of nasopharyngeal carcinoma cell CNE 1.

FIG. 10 shows the results of experiments on the inhibition of cucurbitacin B growth in mice on nasopharyngeal carcinoma transplantable tumors.

FIG. 11 shows the toxicity test results of cucurbitacin B to mouse organs.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Natural products have been an important source of pharmaceutical research due to the diversity and biocompatibility of natural chemical molecular structures. Since the eighties of the last century, research and development of natural product-based drugs have accounted for over 50% of all new drug sources, and natural product-based antitumor drugs account for 65% of antitumor drug sources. The terpenoid is one of three major secondary metabolites in the biological world, and has the structural characteristics that the structural unit of isoprene is formed, and the number of carbon atoms is multiple of 5. Triterpenes are distributed widely in nature, and are distributed in fungi, ferns, monocotyledons, dicotyledons, animals and marine organisms, particularly in the dicotyledons. Is mainly distributed in Caryophyllaceae, Araliaceae, Leguminosae, Hippocastanaceae, Polygalaceae, Campanulaceae and Scrophulariaceae. A few triterpene components are also present in animals, such as lanolin alcohol and squalene. Triterpenoids are of many structural types, most triterpenes being tetracyclic triterpenes and pentacyclic triterpenes, and few being chain, monocyclic, bicyclic and tricyclic triterpenes. Many highly oxidized new framework-type triterpenoids with complex structures, which are generated by oxidation, ring cleavage, methyl translocation, rearrangement and degradation, have also been discovered in recent decades. They exist in a free state or are combined with aglycone to form glucoside, have remarkable biological activity and play an important role in the life activities of animals and plants. The natural product cucurbitacin B is a tetracyclic triterpenoid and has multiple pharmacological activities.

The statistical treatment used was: statistical analysis was performed using SPSS 24.0 statistical software. Independent sample t test is adopted for the comparison of the metering data between two groups, and a one-factor ANOVA analysis of variance method is adopted for the comparison of three groups (or more than three groups). The results are expressed as

When P is present<At 0.05, the difference is significant, P<0.01 indicates that the difference is very significant, and both have certain statistical significance.

The structural formula of cucurbitacin B is shown as follows: