Ferroptosis inducer based on fungal mannan and application thereof

Abstract

The invention discloses a ferroptosis inducer based on fungal mannan and application of the ferroptosis inducer. Specifically, the invention provides an application of an active component, the active component is selected from Flo8 gene deletion type candida albicans sourced mannan (FM), or pharmaceutically acceptable salts thereof, or active derivatives thereof, the active component is used for preparing a preparation, a medicine or a composition, and the preparation, the medicine or the composition is used for inducing cell ferroptosis. Or the active component is used for preparing a ferroptosis inducer; or the active ingredients are used for preparing health-care products; or the active ingredient is used for preparing a medical device. The FM provided by the invention can activate SYK kinase to regulate and control TRIM25-mediated GPX4 ubiquitination degradation, and specifically induce tumor cell ferroptosis, thereby playing a role in inhibiting the occurrence and development of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly, to an iron death inducer based on fungal mannan and its applications. Background Art

[0002] Ferroptosis is a newly discovered type of programmed cell death in recent years, which is characterized by the accumulation of intracellular iron and lipid peroxidation during the cell death process. The morphological characteristics of ferroptotic cells are that mitochondria become smaller, the membrane ruptures, and the cristae disappear, which is significantly different from necrosis, apoptosis, and autophagy. More and more evidence shows that triggering ferroptosis has great potential as an effective anti-cancer therapy for eradicating malignant tumors. In recent years, ferroptosis has gradually been recognized as a potential anti-cancer treatment target and plays an important role in inhibiting the growth of tumor cells. A large number of studies have found that the occurrence of ferroptosis in various cancer types (such as lung cancer, liver cancer, breast cancer) is closely related to the drug resistance of tumor cells. Ferroptosis can be induced by regulating key molecules related to iron metabolism and lipid metabolism (such as GPX4, SLC7A11, 7-DHC), thereby enhancing the sensitivity of tumors to chemotherapy. Existing studies have demonstrated that the inhibition of GPX4 in regulatory T cells can enhance the anti-tumor immune response; CDK1 inhibitors improve oxaliplatin resistance in colon cancer through a ferroptosis-dependent mechanism. However, currently, there are problems with various existing ferroptosis inducers (such as RSL3, Fin56, and ML210) non-specifically triggering cellular ferroptosis, which not only induces ferroptosis in tumor cells but also causes damage to normal cells, leading to a series of side effects.

[0003] Therefore, screening and developing anti-cancer strategies and preparations that specifically induce ferroptosis in tumor cells are urgently needed to be solved. Summary of the Invention

[0004] In view of the above problems, the object of the present invention is to provide the use of a gene mutant fungal mannan in the preparation of an iron death inducer and an anti-tumor drug.

[0005] In the first aspect of the present invention, there is provided the use of an active ingredient selected from mannan derived from Candida albicans with Flo8 gene deletion, or a pharmaceutically acceptable salt thereof, or an active derivative thereof, and the active ingredient is used to prepare a preparation, a drug, or a composition for inducing cellular ferroptosis; or the active ingredient is used to prepare a ferroptosis inducer; or the active ingredient is used to prepare a health product; or the active ingredient is used to prepare a medical device.

[0006] In another preferred example, the cell is a tumor cell.

[0007] In another preferred example, the active ingredient is used to prepare a drug for preventing and / or treating neurodegenerative diseases, tumors, strokes, intracerebral hemorrhages, traumatic brain injuries, ischemia-reperfusion injuries, and diseases related to renal failure.

[0008] In another preferred example, the neurodegenerative diseases include Alzheimer's disease, Huntington's disease, and Parkinson's syndrome.

[0009] In another preferred example, the active ingredient is used to prepare a drug or kit for preventing and / or treating tumors.

[0010] In another preferred example, the tumors include (but are not limited to): colon cancer, gastric cancer, lung cancer, and bladder cancer.

[0011] In another preferred example, the tumors include: gastric cancer cells, lung cancer cells, bladder cancer cells, excluding colorectal cancer cells; The medical devices include but are not limited to: medical dressings; The health products include but are not limited to: oral health products, function-oriented health products, and health products with special forms; The medical dressings include but are not limited to: band-aids, chronic wound dressings; The dosage forms of the oral health products include but are not limited to: tablets, capsules, oral liquids, powders, or granules; The function-oriented health products include but are not limited to: health products for enhancing immunity; The health products with special forms include but are not limited to: health drinks and health candies.

[0012] In another preferred embodiment, the preparation or composition is also used for the following purposes: (y1) Inhibiting the activity of tumor cells; (y2) Inducing ferroptosis in tumor cells; (y3) Activating SYK kinase to regulate the E3 ubiquitin ligase TRIM25; (y4) Promoting the ubiquitination and degradation of GPX4 protein and reducing the expression level of GPX4 protein; (y5) Promoting the upregulation of Fe 2+ levels and MDA levels in tumor cells; (y6) Reducing the expression level of SLC7A11 protein in tumor cells; (y7) Reducing the GSH level in tumor cells; and / or (y8) Promoting mitochondrial membrane shrinkage, reduction or disappearance of mitochondrial cristae, and fragmentation of the outer membrane in tumor cells.

[0013] In another preferred embodiment, the dosage of the drug is 0.1-100 μg / mL, preferably 1-50 μg / mL, more preferably 1-10 μg / mL, based on the concentration of mannan derived from Flo8 gene-deleted Candida albicans.

[0014] In another preferred embodiment, the dosage of the drug is 1-1000 mg / kg / day, preferably 20-500 mg / kg / day, more preferably 25-200 mg / kg / day, based on the dosage of mannan derived from Flo8 gene-deleted Candida albicans.

[0015] In another preferred embodiment, the tumors include: colon cancer, gastric cancer, lung cancer, bladder cancer.

[0016] In another preferred embodiment, the tumors include: gastric cancer cells, lung cancer cells, bladder cancer cells.

[0017] In another preferred embodiment, the derivatives include prodrugs.

[0018] In another preferred embodiment, the drug is administered to a subject selected from the group consisting of rodents or primates (humans or non-human primates).

[0019] In another preferred embodiment, the subject has one or more characteristics selected from the group consisting of: (y1) The subject has a relatively high expression level of TRIM25; (y2) The subject has a relatively low phosphorylation level of SYK kinase; (y3) The subject has a relatively high expression level of GPX4 or SLC7A11; (y4) The subject has a relatively low level of Fe 2+ or MDA; (y5) The subject has a relatively high level of GSH.

[0020] In another preferred embodiment, the relatively low refers to that the phosphorylation or Fe 2+ or MDA level is significantly lower than the reference value; the relatively high refers to that the expression level or GSH level is significantly higher than the reference value.

[0021] In another preferred embodiment, the reference value is the corresponding protein expression level or phosphorylation or Fe 2+ or MDA or GSH level in normal subjects (subjects without tumors).

[0022] In another preferred embodiment, the significantly lower means that the expression level ≤ 2 / 3 of the reference value, preferably ≤ 1 / 2 of the reference value, more preferably ≤ 1 / 3 of the reference value.

[0023] In another preferred embodiment, the significantly higher expression level refers to an expression level ≥ 4 / 3 of the reference value, preferably ≥ 3 / 2 of the reference value, more preferably ≥ 2 times the reference value.

[0024] In another preferred example, the drug comprises: (z1) A first active ingredient, mannan derived from Flo8 gene-deleted Candida albicans, or a pharmaceutically acceptable salt or derivative thereof; (z2) Optionally, a second active ingredient, other drugs for preventing and / or treating tumors or other ferroptosis inducers; and (z3) A pharmaceutically acceptable carrier.

[0025] In another preferred example, in the drug, the component (z1) accounts for 1-99 wt% of the total weight of the drug, preferably 10-90 wt%, more preferably 30-70 wt%.

[0026] In another preferred example, the other drugs for preventing and / or treating tumors include (but are not limited to): fluorouracil, cisplatin, oxaliplatin, irinotecan, capecitabine, trastuzumab, ramucirumab, pembrolizumab.

[0027] In another preferred example, the other ferroptosis inducers include (but are not limited to): RSL3, Erastin, IKE, FIN56, ML210, and Sorafenib.

[0028] In another preferred example, the drug can be a single compound or a pharmaceutical composition composed of a mixture of multiple compounds.

[0029] In another preferred example, the drug is used for preparing a drug or preparation for preventing and / or treating tumors.

[0030] In another preferred example, the dosage form of the drug is an oral dosage form or a non-oral dosage form.

[0031] In another preferred example, the oral dosage form is tablets, powders, granules, capsules, emulsions, or syrups.

[0032] In another preferred example, the non-oral dosage form is an injection or a syringe.

[0033] In another preferred example, the drug is selected from the group consisting of: injections, inhalants, tinctures, powders, granules, capsules, oral liquids, tablets, pills, suspensions, emulsions, lozenges, or dripping pills.

[0034] In another preferred example, the administration method of the drug is intravenous injection or intraperitoneal injection.

[0035] In another preferred embodiment, the subject to which the drug is administered is a human or non-human mammal.

[0036] In another preferred embodiment, the kit comprises: (f1) A first pharmaceutical composition, the pharmaceutical composition containing (i) mannan derived from Candida albicans lacking the Flo8 gene, or a pharmaceutically acceptable salt thereof, or a derivative thereof, as a first active ingredient; and (ii) a pharmaceutically acceptable carrier; and (f2) A reagent for detecting cell viability or Fe 2+ level or MDA level or GSH level.

[0037] In a second aspect of the present invention, there is provided an ferroptosis inducer, the ferroptosis inducer comprising mannan derived from Candida albicans lacking the Flo8 gene, or a pharmaceutically acceptable salt thereof, or a derivative thereof.

[0038] In a third aspect of the present invention, there is provided a method for inducing ferroptosis of cells for non-diagnostic or therapeutic purposes in vitro, comprising the steps of: (a) Culturing cells in the presence of mannan derived from Candida albicans lacking the Flo8 gene, thereby inducing ferroptosis of the cells.

[0039] In another preferred embodiment, the concentration of the mannan derived from Candida albicans lacking the Flo8 gene is 0.1 - 100 μg / mL, preferably 1 - 50 μg / mL, more preferably 1 - 10 μg / mL.

[0040] In another preferred embodiment, the cells include cells related to neurodegenerative diseases, tumors, strokes, intracerebral hemorrhages, traumatic brain injuries, ischemia-reperfusion injuries, and renal failures.

[0041] In another preferred embodiment, the cells are tumor cells.

[0042] In another preferred embodiment, the tumors include: gastric cancer, lung cancer, bladder cancer.

[0043] In another preferred embodiment, the tumors do not include colorectal cancer.

[0044] In a fourth aspect of the present invention, there is provided the use of the ferroptosis inducer according to the second aspect of the present invention for preparing a drug for preventing and / or treating diseases related to neurodegenerative diseases, tumors, strokes, intracerebral hemorrhages, traumatic brain injuries, ischemia-reperfusion injuries, and renal failures, or for preparing a health product, or for preparing a medical device. In another preferred embodiment, the tumors include (but are not limited to): colon cancer, gastric cancer, lung cancer, bladder cancer.

[0045] In a fifth aspect of the present invention, there is provided a method for preventing and / or treating tumors, comprising the steps of: administering to a subject in need thereof a safe and effective amount of mannan derived from Flo8 gene-deleted Candida albicans, or a pharmaceutically acceptable salt thereof, or a derivative thereof.

[0046] In another preferred embodiment, the administration includes oral administration, intravenous injection or intraperitoneal injection.

[0047] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0048] In another preferred embodiment, the non-human mammal includes rodents and primates, preferably mice, rats, rabbits, monkeys.

[0049] Compared with the prior art, the present invention has the following technical effects: (1) The mannan derived from Flo8 gene-deleted Candida albicans (flo8 mutant-derived mannan, FM) provided by the present invention, as an ferroptosis inducer, can activate SYK kinase to regulate the ubiquitination and degradation of GPX4 mediated by TRIM25, specifically induce ferroptosis of tumor cells, and thus play a role in inhibiting the occurrence and development of tumors (especially colorectal tumors).

[0050] (2) The mannan derived from Flo8 gene-deleted Candida albicans (flo8 mutant-derived mannan, FM) provided by the present invention can significantly inhibit the growth of tumor cells.

[0051] (3) The mannan derived from Flo8 gene-deleted Candida albicans (flo8 mutant-derived mannan, FM) provided by the present invention can induce ferroptosis of various tumor cells.

[0052] (4) The mannan derived from Flo8 gene-deleted Candida albicans (flo8 mutant-derived mannan, FM) provided by the present invention can activate SYK kinase to regulate the E3 ubiquitin ligase TRIM25.

[0053] (5) It is known that TRIM25 is mainly expressed in tumor cells and not expressed or expressed at a very low level in normal cells. Therefore, the mannan (FM) derived from the Flo8 gene - deleted Candida albicans strain provided by the present invention activates the SYK / TRIM25 pathway, that is, it can selectively trigger the degradation of GPX4 protein mediated by TRIM25 through early ubiquitination in tumor cells, thereby inducing ferroptosis. At the same time, it will not cause ferroptosis in normal cells, including immune cells, and can achieve the ideal effect of inhibiting tumor cells without damaging anti - tumor immunity. Therefore, the FM provided by the present invention is undoubtedly a candidate anti - tumor preparation with high specificity. Brief Description of the Drawings

[0054] Figure 1 The figure shows the relevant results of FM (flo8 mutant derived mannan) in the examples of the present invention inducing ferroptosis in colon cancer cells. Among them, A: FM treatment significantly inhibits the viability of HT29 cells. B: Ferroptosis inhibitors DFO, Fer, and Lipro can block the inhibitory effect of FM on HT29 cells. Among them, DFO represents Deferoxamine, Fer - 1 represents Ferrostatin - 1, Lipro represents Liproxstatin - 1, CQ represents Chloroquine, ZAD represents Z - VAD - FMK, NSA represents Necrosulfonamide, and RSL3 is an agonist of ferroptosis. C: Electron microscopy observes the changes in cell mitochondria (red arrows) after FM treatment, where WM represents wild - type mannitol. D: After FM treatment, the levels of Fe 2+ and MDA in HT29 cells are up - regulated, and the level of GSH is down - regulated.

[0055] Figure 2 The figure shows the relevant results of FM (flo8 mutant derived mannan) in the examples of the present invention inducing ferroptosis in tumor cells. Among them: A: FM treatment significantly inhibits the viability of HGC27, H1299, and T24 cells. B: The level of Fe 2+ in HGC27, H1299, and T24 cells is up - regulated after FM treatment. C: FM treatment has no obvious inhibitory effect on HEK293T and THP1 cells. D: FM treatment has no effect on the level of Fe 2+ in HEK293T and THP1 cells.

[0056] Figure 3The figure shows the relevant results of the SYK kinase signaling pathway in the embodiments of the present invention participating in the specific induction of ferroptosis in tumor cells by FM. Among them, A: FM treatment time-dependently activates the SYK pathway. B-F: The SYK kinase inhibitor (Piceatannol, Pic-nol) can reverse the inhibitory effect of FM; among them, B: CCK8 is used to detect cell viability. C: Immunoblotting is used to detect the SYK kinase signaling pathway. D-F: The levels of Fe 2+ (D), MDA (E) and GSH (F).

[0057] Figure 4 The figure shows the relevant results of FM activating SYK kinase to regulate the degradation of GPX4 mediated by (E3 ubiquitin ligase) TRIM25 in the embodiments of the present invention. Among them, A: Mass spectrometry analysis of candidate proteins interacting with SYK. B: Co-immunoprecipitation verifies the interaction between SYK phosphorylation and TRIM25. C: FM treatment promotes the ubiquitination and degradation of GPX4. D: FM-induced ubiquitination and degradation of GPX4 are not affected by MG132.

[0058] Figure 5 The figure shows the relevant results of FM activating SYK kinase to regulate the degradation of GPX4 mediated by TRIM25 in the embodiments of the present invention. Among them, A: TRIM25 is highly expressed in colon cancer cells. B: FM treatment cannot induce ferroptosis in HEK293T cells. C: After overexpressing TRIM25, FM treatment can significantly inhibit the viability of HEK293T cells. D: Overexpression of TRIM25 and FM treatment significantly reduce the expression of GPX4.

[0059] Figure 6 The figure shows the relevant results of the physicochemical properties and structural analysis of FM in the embodiments of the present invention. Among them, A: Gas chromatography analysis shows the monosaccharide composition of FM. B: Methylation experiments suggest the glycosidic bond linkage mode. C: High-performance liquid chromatography analysis reveals the molecular weight distribution characteristics of FIM sugar. D: Nuclear magnetic resonance and structural analysis are used to explore the sugar structure and its linkage mode. Detailed implementation manners

[0060] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. All kinds of commonly used reagents used in the embodiments are commercially available products.

[0061] Terms Cell ferroptosis Ferroptosis is an iron-dependent form of cell death that is closely related to the accumulation of lipid peroxides. The essence of ferroptosis is the depletion of glutathione, the decline in the activity of glutathione peroxidase (GPX4), and the inability of lipid oxides to be metabolized through the glutathione reductase reaction catalyzed by GPX4. Subsequently, divalent iron ions oxidize lipids to produce reactive oxygen species, thus promoting the occurrence of ferroptosis. Different from apoptosis, necrosis, and autophagy, ferroptosis has unique biochemical and morphological characteristics.

[0062] Ferroptosis mainly has the following characteristics: ① During the process of cell death, there is a large accumulation of iron ions, accompanied by lipid peroxidation, an increase in ROS, and changes in some genes regulating iron homeostasis and lipid peroxidation metabolism.

[0063] ② In the fine structure of cells, there are mitochondria smaller than normal cells, with mitochondrial membrane shrinkage, a reduction or disappearance of mitochondrial cristae, and fragmentation of the outer membrane, but there are no obvious morphological changes in the nucleus.

[0064] Ferroptosis is a regulated form of cell death characterized by the iron-dependent accumulation of lipid peroxidation reaching a lethal level. The sensitivity of ferroptosis is closely related to many biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, as well as the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. It is also associated with pathological cell death related to mammalian degenerative diseases (such as Alzheimer's disease, Huntington's disease, and Parkinson's syndrome), tumors, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and renal failure.

[0065] Ferroptosis inducers Ferroptosis inducers are a class of compounds that can trigger ferroptosis through specific mechanisms and are widely used in fields such as cancer research and neurodegenerative diseases. The following are common ferroptosis inducers and their mechanisms of action: Erastin inhibits system Xc⁻ (cystine / glutamate antiporter), resulting in reduced cystine uptake and impaired glutathione (GSH) synthesis, ultimately leading to lipid peroxidation and ferroptosis.

[0066] IKE (Imidazole Ketone Erastin) is a novel ferroptosis inducer, a derivative of Erastin, with higher selectivity and potency. Its mechanism of action mainly revolves around inhibiting system Xc⁻ (cystine / glutamate antiporter), thereby triggering ferroptosis.

[0067] RSL3 directly inhibits glutathione peroxidase 4 (GPX4), leading to the accumulation of lipid peroxides and triggering ferroptosis.

[0068] As a multi-kinase inhibitor, sorafenib can also inhibit system Xc⁻ and induce ferroptosis.

[0069] FIN56 induces ferroptosis by depleting GPX4 and activating squalene synthase.

[0070] ML210 indirectly inhibits GPX4 and induces ferroptosis.

[0071] Ferroptosis inhibitors Mechanism of action of ferroptosis inhibitors: By inhibiting lipid peroxidation or regulating iron metabolism to prevent ferroptosis. Commonly used ferroptosis inhibitors include Deferoxamine (DFO), Ferrostatin-1 (Fer-1) and Liproxstatin-1 (Lipro).

[0072] Chloroquine may promote ferroptosis by inhibiting autophagy and increasing intracellular iron ion levels. At high concentrations, chloroquine may induce ferroptosis, but in some cases, it may also reduce ferroptosis by inhibiting autophagy.

[0073] Z-VAD-FMK widely inhibits caspase activity and prevents apoptosis. Z-VAD-FMK does not directly inhibit ferroptosis, but can make cells more susceptible to ferroptosis by blocking apoptosis.

[0074] Necrosulfonamide inhibits MLKL and prevents necroptosis. Necrosulfonamide does not directly act on ferroptosis, but may indirectly affect the occurrence of ferroptosis by inhibiting necroptosis.

[0075] Tripartite motif 25 (TRIM25) TRIM25 is an E3 ubiquitin ligase, which consists of a RING domain, two B-box domains, a coiled-coil domain (CCD), and a C-terminal SPRY domain. As a key regulator of innate immunity, TRIM25 is involved in the regulation of natural immune signals such as NF-κB and IFN, and is closely related to stress responses such as tumor cell differentiation, proliferation, apoptosis, and immune response. In recent years, TRIM25 has been found to play an important role in malignant tumors such as lung cancer, prostate cancer, and liver cancer. TRIM25 is highly expressed in human lung cancer tissues. Knockdown of TRIM25 expression can inhibit the proliferation and in vitro migration of lung cancer cells. In addition, the expression of TRIM25 is positively correlated with the TNM stage and lymph node metastasis of non-small cell lung cancer (NSCLC), and may affect lung cancer cell apoptosis by regulating the expression of p53. High expression of TRIM25 is significantly associated with poor prognosis in prostate cancer patients. Moreover, TRIM25 can interact with GTPase-activating protein-binding protein 2 to form a complex, regulate p53 nuclear export, and promote the proliferation and migration of prostate cancer cells, indicating that TRIM25 is a potential therapeutic target for prostate cancer. TRIM25 is highly expressed in breast cancer. It promotes the growth of breast cancer cells by targeting the cell cycle regulator 14-3-3σ for proteasomal degradation. TRIM25 is highly expressed in both gastric cancer and colorectal cancer. And TRIM25 affects the progression of gastric cancer and colorectal cancer by regulating TGF-β. TRIM25 can affect the migration and invasion of gastric cancer cells and promote the proliferation and metastasis of colorectal cancer cells. Therefore, TRIM25 may be a potential target for the treatment of gastric cancer and colorectal cancer. It is worth noting that TRIM25 is mainly highly expressed in tumor cells, but not expressed or lowly expressed in normal cells (including immune cells), suggesting that TRIM25 can be used as a target for specific anti-tumor therapy.

[0076] Example 1: Preparation of mannan from Flo8 gene-deleted Candida albicans (flo8 mutant mannan, FM) The Candida albicans standard strain SC5314 (ATCC MYA-2876) was provided by the laboratory of Department of Microbiology and Immunology, Georgetown University, Washington, U.S.A. The Candida albicans with Flo8 gene deletion was provided by the research group of Jiangye Chen from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The mannan derived from Flo8 gene-deleted Candida albicans (flo8 mutant derived mannan, FM) used in the examples can be prepared by the traditional alkaline lysis method.

[0077] The extraction method of mannan derived from Flo8 gene deletion Candida albicans (flo8 mutant derived mannan, FM) is as follows: Use a pipette tip to pick up the Flo8 gene deletion Candida albicans colony on the 4°C plate, add it to 2 mL of YPD (yeast extract pentose dextrose) liquid medium, and culture it overnight at 30°C with shaking at 200 rpm. Dilute the Flo8 gene deletion Candida albicans 1:50 into YPD + 10% FBS medium, culture it in a 37°C + 5% CO2 incubator for 3 h, centrifuge to collect the cells, and wash them twice with PBS.

[0078] Resuspend the cells according to the ratio of 1 g of wet cells to 4 mL of 2% NaOH aqueous solution, and place them in a 100°C water bath for 1 h. Centrifuge at 7000 rpm to collect the supernatant, and dropwise add Fehling's reagent (mix equal volumes of solution A and solution B and mix immediately before use) to precipitate the polysaccharide until no more precipitation occurs in the solution. Centrifuge at 8000 rpm, dissolve the lower precipitate in the minimum volume of 3M HCl, and dropwise add it to 45 mL of a mixed solution of methanol:acetic acid = 8:1. After washing out the precipitate and centrifuging again, dissolve it in 3M HCl and repeat the addition to the methanol-acetic acid mixed solution. Repeat the operation 3 times to completely remove protein contamination. Finally, dissolve the white precipitate at the bottom of the tube in 5 - 10 mL of double-distilled water, place it in a 7 kDa dialysis bag and dialyze for 24 h. After lyophilization, store it in a -20°C refrigerator. Dissolve the mannan in sterile water to a 5 mg / mL solution for use.

[0079] Example 2: Specific induction of ferroptosis in tumor cells by FM The specific experimental procedure is as follows: Cell culture: Cultivate the cells in MaCoA5 supplemented with 10% FBS and antibiotics, and maintain them at 37°C, appropriate humidity, and 5% CO2 conditions.

[0080] The cells used in this example include: HT29, HGC27, H1299, T24, HEK293T, and THP1 cells.

[0081] CCK8 cell viability assay: Culture the above cells in a 96-well plate, with 1×10 cells per well 4After treatment with FM (5 μg / mL) for 24 h, cells were lysed and cell viability was detected using a CCK8 assay kit (Proteintech, Wuhan, China). The doses of other reagents were as follows: liproxstatin-1 (Lipro-1, 1 μM), ferrostatin-1 (Fer-1, 10 μM), deferoxamine (DFO, 100 μM), Chloroquine (CQ, 50 μM), necrosulfonamide (NSA, 1 μM), Z-VAD-FMK (Z-VAD, 10 μM).

[0082] Mitochondrial observation: Embedding, section preparation, and photographing of HT29 cell samples were all completed by Wuhan Sevier Biotechnology Co., Ltd. (Wuhan, China). The transmission electron microscope used was a HITACHI HT 7800 at 80 kv, resolution: 1.0 nm (acceleration voltage 120 kV, lattice image); magnification: 200 - 200,000 high-contrast mode; acceleration voltage: 40 - 120 KV.

[0083] ELISA detection of intracellular Fe 2+ 、MDA, and GSH levels: According to the manufacturer's instructions, an iron ion detection kit, a lipid peroxidation (MDA) detection kit, and a GSH / GSSG ratio detection kit (Elabscience, China) were used to measure the relative concentrations of intracellular Fe 2+ 、MDA, and GSH.

[0084] Immunoblotting: The total proteins of each group of cells were electrophoresed using a 10% SDS-PAGE denaturing gel and then transferred to a PVDF membrane (Millipore). After sealing and washing, the PVDF membrane was incubated with primary antibodies (SYK, p-SYK, GPX4, SLC7A11, etc.) overnight at 4 °C. After thorough washing, the PVDF membrane was incubated with a secondary antibody (horseradish peroxidase-labeled anti-rabbit antibody) for 45 minutes at room temperature. The PVDF membrane was washed 5 times with TBST (Tris-salt buffer + 1% Tween 20) for 5 minutes each time. Then, signals were collected after development using an enhanced chemiluminescence ECL kit (Pierce Biotechnology).

[0085] The results showed that FM could significantly inhibit the growth viability of colon cancer cell line HT29 ( Figure 1 A). The ferroptosis inhibitors DFO, Fer-1, and Lipro could block the inhibitory effect of FM on HT29 cells ( Figure 1 B). Electron microscopy showed that after treatment with FM, the mitochondria of HT29 cells shrank, and the cristae decreased or disappeared ( Figure 1(As indicated by the red arrow in C). Further studies have found that FM treatment can induce ferroptosis in tumor cells, as shown by the increase in lipid MDA and Fe 2+ The level of ferroptosis was upregulated, the level of GSH was downregulated, and the expression levels of GPX4 and SLC7A11, the key proteins of ferroptosis, were decreased ( Figure 1 D). In addition to colon cancer cells, FM can also significantly inhibit the cell viability of gastric cancer cell line (HGC27), lung cancer cell line (H1299), and bladder cancer cell line (T24) ( Figure 2 A) and increase intracellular Fe 2+ level( Figure 2 B), but had no inhibitory effect on normal cells HEK293T and THP1 ( Figure 2 C and 2D). The results indicate that FM is highly selective for tumor cells and exerts inhibitory effects.

[0086] Example 3: FM specifically activates the SKY / TRIM25 pathway in tumor cells to induce ferroptosis The specific experimental process is as follows: Immunoprecipitation and mass spectrometry analysis: HT29 cell samples before and after FM treatment were immunoprecipitated using activated spleen tyrosine kinase (SYK) antibody combined with ProteinA / G magnetic beads (MCE, China). The collected protein samples were subjected to 10% SDS-PAGE denaturing gel electrophoresis. After cutting the gel strip, Lianchuan Biotechnology Co., Ltd. (Hangzhou, China) was commissioned to perform full protein spectrum (LC-MS) identification and bioinformatics analysis. Other experimental procedures are as described in Example 2.

[0087] In the human colon cancer cell line HT29, FM promoted SYK phosphorylation and downregulated the expression of ferroptosis-related proteins SLC7A11 and GPX4 ( Figure 3 A), upregulating intracellular Fe 2+ and MDA levels, but down-regulated GSH levels. The SYK kinase inhibitor (Piceatannol, abbreviated as Pic-nol) can reverse these effects of FM ( Figure 3 C-3F), but had no significant effect on other ferroptosis inducers RSL3, erastin and IKE ( Figure 3 B). Therefore, it is suggested that the SYK kinase signaling pathway plays an important role in FM-induced ferroptosis.

[0088] Mass spectrometry analysis of candidate proteins interacting with SYK ( Figure 4 A). Immunoprecipitation confirmed the interaction between SYK phosphorylation and TRIM25 ( Figure 4B). Additionally, FM is a polysaccharide and has no structural similarity to the currently widely used ferroptosis inducers RSL3, Fin56, IKE, and Erastin (the existing RSL3, Fin56, and Erastin are all small molecule compounds, while FM is a fungal polysaccharide), but they can all cause the degradation of GPX4 protein ( Figure 4 C). The degradation of GPX4 protein induced by FM is not affected by the proteasome inhibitor (MG132), while the other ferroptosis inducers RSL3 and Fin56 are regulated by the proteasome inhibitor during this process. ( Figure 4 D).

[0089] Thus, it can be seen that the mechanism of FM specifically inducing ferroptosis in tumor cells is unique. Treatment of tumor cells with FM can activate the SYK signaling pathway. By using immunoprecipitation combined with mass spectrometry analysis, it was first discovered that the SYK kinase interacts with the E3 ubiquitin ligase TRIM25 ( Figure 4 A-4B), and treatment of colon cancer cell line HT29 with FM can promote the autophosphorylation of SYK kinase and induce the ubiquitination and degradation of GPX4. The above results suggest that FM may induce ferroptosis in colon cancer cells by activating the TRIM25 pathway and promoting the ubiquitination and degradation of GPX4.

[0090] TRIM25 is mainly expressed in tumor cells, and it is highly expressed in colon cancer cells and not expressed or expressed at a very low level in normal cells (such as HEK293T cells) ( Figure 5 A). Treatment with FM cannot induce ferroptosis in HEK293T cells ( Figure 5 B). After overexpressing TRIM25, treatment with FM can significantly inhibit the viability of HEK293T cells ( Figure 5 C). After overexpressing TRIM25, treatment with FM significantly reduces the expression of GPX4 ( Figure 5 D). Therefore, FM activates the SYK / TRIM25 pathway, which can selectively trigger the early ubiquitination and degradation of GPX4 protein mediated by TRIM25 in tumor cells to induce ferroptosis, while not causing ferroptosis in immune cells. Therefore, FM is undoubtedly a candidate anti-tumor preparation with high specificity.

[0091] Example 4: Physicochemical Properties and Structural Analysis of FM The specific experimental process is as follows: The physicochemical properties and structural analysis of FM were entrusted to Brilliant Glycobiotechnology Co., Ltd. (Yangzhou, China) to complete. The specific experimental steps are as follows: Experiment for determining monosaccharide composition: The monosaccharide composition was determined using an ion chromatograph (ICS5000, Thermo Fisher). 5 mg of the sample was precisely weighed and placed in an ampoule bottle. 2 mL of 3M TFA was added, and hydrolysis was carried out at 120 °C for 3 h. An accurate volume of the acid hydrolysis solution was transferred to a tube and dried by nitrogen blowing. 5 mL of water was added and vortexed thoroughly. 20 μL was taken and added to 980 μL of deionized water, and centrifuged at 12000 rpm for 5 min. The supernatant was taken for instrumental analysis. Chromatographic column: Dionex CarbopacTM PA20 (3*150 mm); Mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH & 100 mM NaAc; Flow rate: 0.3 mL / min; Injection volume: 25 μL; Column temperature: 30 °C; Gradient elution, detected by an electrochemical detector.

[0092] Experiment for determining the linkage mode of polysaccharides: The linkage mode of the polysaccharide sample after methylation and other derivatizations was determined by GC-MS. The polysaccharide sample (2 - 3 mg) was weighed and placed in a glass reaction flask. 1 mL of anhydrous DMSO was added, and then the methylation reagent solution A was quickly added and sealed. It was dissolved under ultrasonic action, and then the methylation reagent solution B was added. The reaction was carried out at 30 °C for 60 min under magnetic stirring in a water bath. Finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction. The methylated polysaccharide was taken, 1 mL of 2M trifluoroacetic acid (TFA) was added for hydrolysis for 90 min, and it was evaporated to dryness using a rotary evaporator. The residue was added with 2 mL of double-distilled water, reduced with 60 mg of sodium borohydride for 8 h, neutralized with glacial acetic acid, rotary evaporated, dried in an oven at 101 °C, and then acetylated with 1 mL of acetic anhydride at 100 °C for 1 h and cooled. Then 3 mL of toluene was added, and it was concentrated under reduced pressure and evaporated to dryness, repeating 4 - 5 times to remove the excess acetic anhydride. The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added and shaken well, and the upper aqueous layer was removed, repeating this 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid-phase vial. Analysis was carried out using an Agilent GCMS 6890 - 5973 gas chromatography - mass spectrometry instrument to determine the acetylated product sample; GC-MS conditions: RXI-5 SIL MS chromatographic column 30 m * 0.25 mm * 0.25 μm; The programmed temperature condition was: initial temperature 120 °C, heated to 250 °C at 3 °C / min; Held for 5 min; Injection port temperature was 250 °C, detector temperature was 250 °C, carrier gas was helium, and flow rate was 1 mL / min.

[0093] Molecular weight determination: The molecular weight and purity of the polysaccharide were determined by high performance liquid chromatography. The standard sample was placed on the injection tray, and the above-mentioned chromatographic method was selected for analysis to obtain the retention time. The standard curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), and lgMn-RT (Mn number average molecular weight) were plotted to obtain the molecular weight calculation formula. The sample was placed on the injection tray, and the above-mentioned chromatographic method was selected for analysis to obtain the chromatogram and retention time. Substituting the retention time into the formula, the molecular weight (Mp, Mw, Mw) could be obtained.

[0094] NMR spectrum analysis and interpretation: 50 mg of the polysaccharide sample was weighed and dissolved in 0.5 ml of heavy water and freeze-dried. Subsequently, the lyophilized powder was dissolved in 0.5 ml of heavy water again and freeze-dried. The above process was repeated to fully exchange the labile hydrogens. Then the sample was dissolved in 0.5 ml of heavy water, and 1H NMR spectrum, 13C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum were measured at room temperature of 25 °C using a nuclear magnetic resonance spectrometer (Bruker 600M, NMR).

[0095] FM was analyzed by gas chromatography. The main components of WM and FM were both mannose. Compared with WM, the mannose component in FM decreased (from 100.0% to 98.5%), while a glucose component (1.5%) appeared ( Figure 6 A), indicating that the monosaccharide composition changed little. Methylation experiments found that the glycosidic bond linkage pattern in FM changed significantly ( Figure 6 B). High performance liquid chromatography analysis found that the molecular weight range of wild-type mannan WM was wide, concentrated at 270 KD and 43 KD, while the molecular weight distribution of FM was concentrated at 37 KD, indicating that the sugar structure of FM changed and the glycosidic bond linkage pattern tended to be unified ( Figure 6 C). Nuclear magnetic resonance and structural analysis indicated that the main chain unit of the sugar structure of FM was composed of mannose connected by 1,2-linkage, and the branches were composed of mannose connected by 1,4- and 1,6-linkage. The main chain unit structure was repeated 4 times, and the branches were connected to 1,6-glucose, which was different from the structure of WM. It was estimated that the molecular weight of the monosaccharide of FM was 7 kDa, while that of WM was 10.3 kDa. Therefore, it was speculated that the change in the monosaccharide linkage mode led to the change in the polymerization mode and degree of FM, thereby endowing it with a unique anti-tumor effect ( Figure 6 D).

[0096] The above-mentioned embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A use of an active ingredient, characterized in that: The active ingredient is selected from mannan derived from Flo8 gene-deficient Candida albicans, or a pharmaceutically acceptable salt thereof, or an active derivative thereof, and the active ingredient is used to prepare a preparation, a medicine or a composition, and the preparation, the medicine or the composition is used to induce cell ferroptosis; or the active ingredient is used to prepare a ferroptosis inducer; or the active ingredient is used to prepare a health product; or the active ingredient is used to prepare a medical device.

2. The use according to claim 1, characterized in that The active ingredients are used for preparing medicines for preventing and / or treating neurodegenerative diseases, tumors, stroke, cerebral hemorrhage, traumatic brain injury, local ischemia-reperfusion injury and diseases related to renal failure.

3. The use according to claim 2, characterized in that: The tumors include (but are not limited to): gastric cancer, lung cancer, bladder cancer, colon cancer; The medical devices include but are not limited to: medical dressings; The health products include, but are not limited to: oral health products, function-oriented health products and health products with special forms; The medical dressings include but are not limited to: Band-Aids, chronic wound dressings; The dosage forms of the oral health products include, but are not limited to: tablets, capsules, oral liquids, powders or granules; The function-oriented health products include but are not limited to: health products that enhance immunity; The health products with some special forms include but are not limited to: health drinks and health candies.

4. The use according to claim 1, characterized in that The preparation, medicine or composition is also used for the following purposes: (y1) inhibiting tumor cell activity; (y2) induce ferroptosis in tumor cells; (y3) Activation of SYK kinase regulates E3 ubiquitin ligase TRIM25; (y4) Promotes the ubiquitination and degradation of GPX4 protein and reduces the expression level of GPX4 protein; (y5) Promotes Fe 2+ levels and MDA levels were upregulated; (y6) reducing the expression level of SLC7A11 protein in tumor cells; (y7) reducing GSH levels in tumor cells; and / or (y8) Promotes mitochondrial membrane shrinkage, reduction or disappearance of mitochondrial cristae, and outer membrane fragmentation in tumor cells.

5. The use according to claim 1, characterized in that: The kit comprises: (f1) a first pharmaceutical composition comprising (i) as a first active ingredient, mannan derived from Flo8 gene-deficient Candida albicans, or a pharmaceutically acceptable salt thereof, or a derivative thereof; and (ii) a pharmaceutically acceptable carrier; and (f2) Detection of cell activity or Fe 2+ Level or MDA level or GSH level reagent.

6. A ferroptosis inducing agent, characterized in that The ferroptosis inducing agent includes mannan derived from Flo8 gene-deficient Candida albicans, or a pharmaceutically acceptable salt thereof, or a derivative thereof.

7. A method for inducing cell ferroptosis in vitro for non-diagnostic or non-therapeutic purposes, characterized in that: Includes steps: (a) Cells were cultured in the presence of mannan derived from Flo8 gene-deficient Candida albicans to induce ferroptosis of the cells.

8. The method according to claim 7, characterized in that The concentration of the mannan derived from the Flo8 gene-deficient Candida albicans is 0.1-100 μg / mL, preferably 1-50 μg / mL, and more preferably 1-10 μg / mL.

9. A use of the ferroptosis inducing agent according to claim 6, characterized in that: Used for preparing drugs for preventing and / or treating neurodegenerative diseases, tumors, stroke, cerebral hemorrhage, traumatic brain injury, local ischemia-reperfusion injury and renal failure-related diseases, or for preparing health products, or for preparing medical devices.

10. The use according to claim 9, characterized in that The tumors include gastric cancer, lung cancer, and bladder cancer.

Images