Application of methylhydroquinone homologues in improvement of cell ferroptosis

Abstract

The invention relates to the field of biological medicines, and particularly discloses application of methylhydroquinone homologues in anti-ferroptosis effect medicines. The methylhydroquinone homologues comprise hydroquinone, 2-methylhydroquinone, 2, 3-dimethyl hydroquinone and 2, 6-dimethyl hydroquinone. The methylhydroquinone homologues have the effect of improving cell ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of methyl hydroquinone homologues in anti-ferroptosis drugs. Background Art

[0002] Ferroptosis is a type of iron-dependent cell death pathway, which is different from other death pathways such as apoptosis, necrosis, and autophagy. Ferroptosis is manifested by comprehensive changes in multiple indicators such as cellular lipid peroxidation levels and increased expression of the marker gene Ptgs2, and can be specifically inhibited by iron ion chelators.

[0003] Currently available drugs for improving cell ferroptosis are mainly Ferrostatin-1, Liproxstatin-1, and Deferoxamine; Ferrostatin-1 is currently recognized as a ferroptosis inhibitor. [1] .

[0004] Methylhydroquinone homologues include hydroquinone, 2-methylhydroquinone, 2,3-dimethylhydroquinone, 2,6-dimethylhydroquinone, and the like.

[0005] Hydroquinone, also known as hydroquinone, is an organic compound formed by the replacement of two para-hydrogens of benzene by hydroxyl groups. Its chemical formula is C6H6O2. It is a white crystalline powder. It is currently reported to be mainly used in the preparation of black and white developers, anthraquinone dyes, azo dyes, rubber antioxidants, stabilizers and antioxidants. [2] .

[0006]

[0007] 2-Methylhydroquinone, methylhydroquinone, chemical formula C7H8O2, appears as white crystals or crystalline powder, soluble in water, alcohol and ether, with a strong aromatic smell. It is an important intermediate in the synthesis of different compounds, such as pharmaceuticals, dyes and polymers, and is currently reported to be used as an antioxidant and preservative. [3] .

[0008]

[0009] 2,3-Dimethylhydroquinone is widely used in organic synthesis. It is a colorless to pale yellow solid with a special aroma. It is soluble in some organic solvents such as alcohols, ethers and ketones at room temperature. The molecular formula is C8H 10 O2, it has strong oxidizing properties and can react with many substances. It is used as an oxidant and catalyst. It can also be used as a hydrogen transfer catalyst in organic synthesis reactions[4] .

[0010]

[0011] 2,6-Dimethylhydroquinone (2,6-DMHQ) is an organic compound with the molecular formula C8H 10 O2 is a colorless crystalline solid with a sweet taste and is easily soluble in water and ethanol. It can be used as an oxidant, reductant, acid-base indicator and bactericide. In organic synthesis, it can be used as an important catalyst. In addition, 2,6-DMHQ has been reported to reduce trivalent iron and mainly participate in the redox reaction of iron. [5] .

[0012]

[0013] However, no studies have reported the role of hydroquinone, 2-methylhydroquinone, 2,3-dimethylhydroquinone, and 2,6-dimethylhydroquinone in cellular ferroptosis.

[0014] The references mentioned above are as follows:

[0015] 1.Xuexian Fang et al.,The molecular and metabolic landscape of ironand ferroptosis in cardiovascular disease.Nat Rev Cardio.2023Jan;20(1):7-23;

[0016] 2. Mei-Chi Chang et al., Antiplatelet, antioxidative, and anti-inflammatory effects of hydroquinone. J Cell Physiol. 2019Aug; 234(10):18123-18130;

[0017] 3.Ezhaveni Sathiyamoorthi et al.,Hydroquinone derivatives attenuatebiofilm formation and virulence factor production in Vibrio spp.Int J FoodMicrobiol.2023Jan 2:384:109954;

[0018] 4.Lelde Krumina et al.,Environmental Science&Technology,2017Aug 15;51(16):9053-9061; 5. Gry Lyngsie et al., Scientific Reports, 2018Jul 17;8(1):10834. Summary of the invention

[0019] The technical problem to be solved by the present invention is to provide application of methyl hydroquinone homologues in improving cell ferroptosis.

[0020] In order to solve the above technical problems, the present invention provides the use of methyl hydroquinone homologues in the preparation of drugs for improving cell ferroptosis (anti-ferroptosis effect).

[0021] As an improvement of the application of the present invention: methyl hydroquinone homologues include hydroquinone, 2-methyl hydroquinone, 2,3-dimethylhydroquinone, and 2,6-dimethyl hydroquinone.

[0022] As a further improvement of the application of the present invention: improving the decrease of cell viability induced by RSL3.

[0023] During the invention process, the inventors introduced hydroquinone (HQ), 2-methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (2,3-DMHQ) and 2,6-dimethylhydroquinone (2,6-DMHQ) into the classical in vitro cell ferroptosis model, and found that the above-mentioned methylhydroquinone homologues all have the effect of improving cell ferroptosis.

[0024] When hydroquinone, 2-methylhydroquinone, 2,3-dimethylhydroquinone, and 2,6-dimethylhydroquinone are used as drugs, the in vivo administration method of Fer-1 can be referred to: intraperitoneal injection, once a day, a dose of 1 mg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0026] Figure 1 These are the results of cell viability tests on four compounds induced by ferroptosis.

[0027] A blank control group (Control group), RSL3 treatment group, RSL3+Fer-1 intervention group, and RSL3+compound group were set up.

[0028] Using hydroquinone (HQ), 2-methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (2,3-DMHQ) and 2,6-dimethylhydroquinone, it was found that the above-mentioned methylhydroquinone homologues all have the effect of improving cell ferroptosis. ** represents p < 0.01, the difference is statistically significant; ns represents not significant, the difference is not statistically significant.

[0029] Figure 2 The cell viability test results are shown when the four compounds were used alone. DETAILED DESCRIPTION

[0030] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0031] 1. Materials and Methods

[0032] 1.1 Cell experiment design

[0033] The inventors used the human cervical cancer Hela cell line, which is frequently used in the field of ferroptosis, to conduct experiments in a 96-well cell culture dish, with 5,000 adherent cells in each well. The cells were given the classic ferroptosis drug RSL3 (which mainly promotes ferroptosis by inhibiting the antioxidant protein GPX4), and the anti-ferroptosis drug Ferrostatin-1 (Fer-1) as the positive control group. Hydroquinone (HQ), 2-methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (2,3-DMHQ) and 2,6-dimethylhydroquinone (2,6-DMHQ) compounds were used as the experimental group. The drug treatment time was 24 hours.

[0034] 1.2 Drugs and treatment

[0035] Compound monomers RSL3, Ferrostatin-1, hydroquinone (HQ), 2-methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (2,3-DMHQ) and 2,6-dimethylhydroquinone (2,6-DMHQ) were purchased from MCE Company and prepared into stock solutions using dimethyl sulfoxide as solvent.

[0036] The preparation methods of various drug stock solutions are as follows:

[0037] RSL3 was dissolved in DMSO to prepare a 1 mM RSL3 stock solution;

[0038] Fer-1 was dissolved in DMSO to prepare a Fer-1 stock solution with a concentration of 2 mM;

[0039] Hydroquinone (HQ) was dissolved in DMSO to prepare a 50 mM HQ stock solution;

[0040] 2-Methylhydroquinone (MHQ) was dissolved in DMSO to prepare a 5 mM MHQ stock solution;

[0041] 2,3-Dimethylhydroquinone (2,3-DMHQ) was dissolved in DMSO to prepare a 5 mM 2,3-DMHQ stock solution;

[0042] 2,6-Dimethylhydroquinone (2,6-DMHQ) was dissolved in DMSO to prepare a 2,6-DMHQ stock solution with a concentration of 5 mM.

[0043] Preparation of drug working solution:

[0044] RSL3-containing medium: 1 mM RSL3 stock solution was diluted to 1 μM with culture medium (complete DMEM medium), which was named RSL3-containing medium;

[0045] RSL3+Fer-1 treatment solution: dilute the 2 mM Fer-1 stock solution with the above RSL3-containing medium to a Fer-1 concentration of 2 μM;

[0046] RSL3+HQ treatment solution: 50 mM HQ stock solution was diluted with the above RSL3-containing medium to an HQ concentration of 50 μM;

[0047] RSL3+MHQ treatment solution: dilute the 5 mM MHQ stock solution with the above RSL3-containing medium to a MHQ concentration of 5 μM;

[0048] RSL3+2,3-DMHQ treatment solution: 5 mM 2,3-MHQ stock solution was diluted with the above RSL3-containing medium to a 2,3-MHQ concentration of 5 μM;

[0049] RSL3+2,6-DMHQ treatment solution: 5 mM 2,6-DMHQ stock solution was diluted with the above RSL3-containing culture medium to a 2,6-DMHQ concentration of 5 μM.

[0050] The experiment was carried out in a 96-well cell culture dish, with 5,000 human cervical cancer Hela cells placed in each well (after the cells adhered overnight, the culture medium was discarded); then 100 μl of the above-mentioned treatment solution / culture medium containing RSL3 was added to obtain the corresponding experimental groups. The details are as follows:

[0051] Blank control group (Control group): add 100 μl of culture medium (complete DMEM culture medium) containing 0.1% DMSO;

[0052] RSL3-treated group: 100 μl of medium containing RSL3 was added;

[0053] RSL3+Fer-1 treatment group: add 100 μl of RSL3+Fer-1 treatment solution;

[0054] RSL3+HQ treatment group: add 100 μl of RSL3+HQ treatment solution;

[0055] RSL3+MHQ treatment group: add 100 μl of RSL3+MHQ treatment solution;

[0056] RSL3+2,3-DMHQ treatment group: 100 μl of RSL3+2,3-DMHQ treatment solution was added;

[0057] RSL3+2,6-DMHQ treatment group: add 100 μl of RSL3+2,6-DMHQ treatment solution.

[0058] The cells were cultured in an incubator at 37°C and 5% carbon dioxide for 24 h, and the cell viability was determined by CCK8 method, and the absorbance value of each well was determined by OD450.

[0059] 1.3 Statistical methods

[0060] All data were expressed as mean ± standard error (mean ± SEM), and statistical analysis was performed using GraphPad Prism (version 8.0, GraphPad software; San Diego, CA, USA) statistical software. ** represents p < 0.01, the difference is statistically significant; ns represents not significant, the difference is not statistically significant.

[0061] 2. Results

[0062] Figure 1 The results showed that HQ, MHQ, 2,3-DMHQ, and 2,6-DMHQ could improve the ferroptosis of Hela cells induced by RSL3. The specific manifestations are:

[0063] HQ, MHQ, and 2,6-DMHQ all significantly improved the decline in cell viability induced by RSL3; the improvement effects of HQ, MHQ, and 2,6-DMHQ on cell ferroptosis were no different from those of the positive control group Fer-1 treatment group.

[0064] 2,3-DMHQ significantly improved the decline in cell viability induced by RSL3; compared with the positive control group Fer-1 treated group, 2,3-DMHQ cells had a certain improvement effect on ferroptosis.

[0065] Another: Figure 2As described above, the present invention also proves that HQ (50 mM), MHQ (5 mM), 2,3-DMHQ (5 mM), and 2,6-DMHQ (5 mM) are non-toxic to Hela cells when used alone.

[0066] It should be noted that: as a common knowledge, Fer-1 has the effect of improving ferroptosis of cells in vivo, so it can be deduced that the methyl hydroquinone homologues of the present invention also have the effect of improving ferroptosis of cells in vivo.

[0067] Based on the above experiments, methyl hydroquinone homologues can be used as a new type of drug to prepare anti-cell ferroptosis effects.

[0068] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of methyl hydroquinone homologues in the preparation of drugs to improve cell ferroptosis.

2. The use according to claim 1, characterized in that: Methylhydroquinone homologues include hydroquinone, 2-methylhydroquinone, 2,3-dimethylhydroquinone, and 2,6-dimethylhydroquinone.

3. The use according to claim 1 or 2, characterized in that: Improved RSL3-induced decrease in cell viability.

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