Application of edaravone in preparation of medicine for inhibiting senescence of vascular endothelial cells

Abstract

The invention discloses an application of edaravone in preparation of a medicine for inhibiting vascular endothelial cell senescence, and belongs to the field of biological medicine, the concentration of edaravone is 30-80 [mu] M, and vascular endothelial cell senescence is doxorubicin induced vascular endothelial cell senescence. Experiments prove that the positive rate of beta-galactosidase staining of an edaravone treatment group is remarkably reduced compared with that of an aging group, and edaravone can inhibit doxorubicin-induced vascular endothelial cell aging. Specifically, edaravone can activate an Nrf2 / HO-1 / NQO1 pathway to inhibit doxorubicin-induced vascular endothelial cell senescence by reducing expression of senescence markers P53 and P21, and a foundation is laid for research and development of chemical small molecule drugs for inhibiting vascular endothelial cell senescence.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of edaravone in the preparation of a drug for inhibiting aging of vascular endothelial cells. Background Art

[0002] The progressive aggravation of the aging process has led to an increase in age-related diseases, bringing new challenges to health care. Blood vessels are the first organs to develop in the embryo, providing nutrients and oxygen to all cells. As blood vessels gradually age, their structure and function usually become abnormal, leading to many age-related diseases, including ischemic heart and brain diseases, neurodegenerative diseases or cancer. Among them, cardiovascular disease (CVD) has the highest prevalence in the elderly population, and vascular aging has been shown to be an important independent predictor of CVD events. At the same time, the aging of vascular endothelial cells can lead to a variety of structural and functional changes such as vascular tone disorders, increased endothelial permeability, arterial stiffness, angiogenesis and repair disorders, which are directly related to vascular aging.

[0003] Endothelial cell senescence is an important biological process closely related to a variety of age-related diseases. Endothelial cell senescence refers to the phenomenon that endothelial cells gradually lose their ability to proliferate and become functionally impaired due to long-term oxidative stress, metabolic disorders, inflammatory responses, DNA damage and other factors. Endothelial cell senescence can lead to endothelial dysfunction, which is specifically manifested as decreased vasoconstriction, increased endothelial permeability, impaired angiogenesis and repair capacity, arterial stiffness, hemodynamic changes, etc. These changes are closely related to the occurrence of cardiovascular diseases (such as hypertension, arteriosclerosis, myocardial infarction, etc.), diabetes-related complications, neurodegenerative diseases, and aging-related cancers. The molecular mechanisms of endothelial cell senescence include oxidative stress, telomere shortening, cell cycle regulation disorders, and abnormal DNA damage and repair mechanisms. Further studies have shown that the occurrence of endothelial cell senescence is closely related to changes in the extracellular matrix, activation of inflammatory responses, and decreased microangiogenesis.

[0004] In recent years, research on anti-aging drugs and strategies has gradually made progress, and drug development targeting vascular endothelial cell aging has also become an important research direction in the biomedical field. Therefore, finding drugs that can alleviate vascular endothelial cell aging and analyzing their mechanisms of action are very important for preventing and alleviating vascular aging.

[0005] Doxorubicin (DOX) is a drug commonly used in chemotherapy. Its clinical use is often accompanied by serious side effects, especially cardiotoxicity and vascular damage. Doxorubicin can induce oxidative stress, leading to endothelial cell damage and ultimately endothelial cell senescence. Endothelial cell senescence caused by doxorubicin is closely related to vascular dysfunction, angiogenesis disorders and the occurrence of cardiovascular diseases.

[0006] Edaravone is commonly used to treat acute ischemic stroke and acute cerebral infarction. Antioxidation mainly refers to the removal of reactive oxygen species (ROS) in the body, thereby reducing the damage to cells caused by oxidative stress. Anti-aging specifically refers to delaying or reversing the aging process in cells caused by genetic or external factors. The aging of vascular endothelial cells induced by doxorubicin is not a single oxidative stress process, but also involves the cell cycle, DNA damage repair and activation of aging genes. There are no reports on the use of edaravone as a drug to inhibit the aging of vascular endothelial cells. Summary of the invention

[0007] The purpose of the present invention is to provide an application of edaravone in the preparation of a drug for inhibiting vascular endothelial cell senescence, so as to solve the problems existing in the above-mentioned prior art. Edaravone can inhibit doxorubicin-induced vascular endothelial cell senescence by reducing the expression of senescence markers P53 and P21 and activating the Nrf2 / HO-1 / NQO1 pathway. The present invention lays a foundation for the research and development of chemical small molecule drugs for inhibiting vascular endothelial cell senescence.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides application of edaravone in preparing a medicine for inhibiting senescence of vascular endothelial cells.

[0010] Optionally, the concentration of edaravone is 30-80 μM.

[0011] Optionally, the vascular endothelial cell senescence is doxorubicin-induced vascular endothelial cell senescence.

[0012] Optionally, the vascular endothelial cells include human umbilical vein endothelial cells.

[0013] Optionally, the edaravone inhibits the senescence of vascular endothelial cells by reducing the expression of P53 and P21.

[0014] Optionally, the edaravone inhibits vascular endothelial cell senescence by activating the Nrf2 / HO-1 / NQO1 pathway.

[0015] The present invention also provides a drug for inhibiting vascular endothelial cell senescence, the drug comprising edaravone and a pharmaceutically acceptable excipient;

[0016] The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0017] Optionally, the concentration of edaravone is 30-80 μM.

[0018] Optionally, the vascular endothelial cell senescence is doxorubicin-induced vascular endothelial cell senescence.

[0019] Optionally, the dosage form of the drug includes a solid dosage form, a semisolid dosage form or a liquid dosage form.

[0020] The present invention discloses the following technical effects:

[0021] The present invention provides the use of edaravone in the preparation of a drug for inhibiting the senescence of vascular endothelial cells. Experimental verification shows that the positive rate of β-galactosidase staining in the edaravone-treated group is significantly lower than that in the aging group, and edaravone can inhibit the senescence of vascular endothelial cells induced by doxorubicin. Specifically, edaravone can inhibit the senescence of vascular endothelial cells induced by doxorubicin by reducing the expression of senescence markers P53 and P21 and activating the Nrf2 / HO-1 / NQO1 pathway. The present invention lays a foundation for the development of drugs for inhibiting the senescence of vascular endothelial cells by chemical small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Effects of edaravone on the level of reactive oxygen species in HUVECs; A: Fluorescence image, green fluorescence is the DCFH-DA staining site, red fluorescence is the DHE staining site, and blue fluorescence is the DAPI staining site; B: Comparison of relative fluorescence intensity of DCFH-DA in each group; C: Comparison of relative fluorescence intensity of DHE in each group; Compared with the control group, a means P < 0.05; Compared with the aging group, b means P < 0.05;

[0024] Figure 2β-galactosidase staining of HUVEC in each group; A: Fluorescence image, blue-stained cells are positively stained cells, scale bar = 100 μm; B: HUVEC β-galactosidase staining positive rate in each group; compared with the control group, a means P < 0.05; compared with the aging group, b means P < 0.05;

[0025] Figure 3 The expression levels of Nrf2, HO-1, NQO1, P21, and P53 proteins in HUVECs in each group; A: Western blot of Nrf2, HO-1, NQO1, P21, and P53; BF: Western blot quantitative comparison of Nrf2, HO-1, NQO1, P21, and P53, respectively; compared with the control group, a means P < 0.05; compared with the aging group, b means P < 0.05;

[0026] Figure 4 The graphs show the expression levels of Nrf2, HO-1, NQO1, P21, and P53 proteins in HUVECs of each group; A: Western blot of Nrf2, HO-1, NQO1, P21, and P53; BF: Quantitative comparison of Western blot of Nrf2, HO-1, NQO1, P21, and P53, respectively; compared with the aging + edaravone group, a indicates P < 0.05. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] The edaravone used in the present invention is a white crystalline powder, and its structural formula is shown below. The molecular formula is C 10 H 10 N2O, molecular weight 174.199, was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0033]

[0034] Many drugs have antioxidant effects, but not all antioxidant drugs have the effect of inhibiting aging. An ITP program led by the US NIA adds drugs to mouse feed for aging intervention to evaluate whether the drugs really have anti-aging effects. The program accepts up to six drugs for inclusion in the study each year. All tests are conducted simultaneously in multiple centers, following standard operating procedures, and the results are reproducible. Several drugs identified by ITP that have antioxidant effects but do not really have anti-aging effects include but are not limited to: curcumin, fish oil, inulin, oxaloacetic acid, resveratrol, and ursolic acid. The reason is that in the aging process, oxidative stress is one of the factors that cause cell damage. However, the mechanisms involved in aging are more complex than oxidative stress, involving gene expression, cell metabolism, inflammatory response, immune system, mitochondrial function and other aspects. Therefore, even if some drugs have antioxidant effects, they may not be enough to cover or affect other aging pathways, thereby inhibiting aging. The article "A Quantitative Tissue-Specific Landscape of Protein Redox Regulation during Aging, DOI: 10.1016 / j.cell.2020.02.012" states: "Antioxidation alone will reduce the ability of ROS to oxidatively modify macromolecules, which may disrupt physiological processes and cause adverse effects." The article measured the oxidation levels of cysteine ​​residues and ROS modification sites in mice. The results showed that compared with young mice (16 weeks), there was no significant difference in the global protein cysteine ​​oxidation level or the oxidation level of organelles in various tissues in old mice (80 weeks), indicating that there is no correlation between aging and the accumulation of ROS oxidative modification. It can also be seen from the content disclosed in the article that antioxidation may not alleviate aging. After a large number of experimental screenings, the present application finally found that edaravone can prevent and treat doxorubicin-induced aging of vascular endothelial cells, laying the foundation for the development of small chemical molecules to inhibit aging of vascular endothelial cells. The following is further explained in the form of examples.

[0035] Example 1 Edaravone can reduce the level of active oxygen

[0036] Human umbilical vein endothelial cells (HUVECs) were used as model cells and cultured in vitro.

[0037] The 4th generation HUVECs were seeded into six-well plates and divided into three groups: a control group (no special treatment, cultured in ECM medium for 24 h), an aging group (cultured in ECM medium containing 2 μmol / L doxorubicin for 24 h), and an aging + edaravone group (pretreated with ECM medium containing 80 μM edaravone for 24 h, followed by the addition of ECM medium containing 2 μM doxorubicin and 80 μM edaravone and continued culture for 24 h).

[0038] After the culture, the level of reactive oxygen species in each group was measured. The specific steps were as follows: dihydroethidium (DHE) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining was used to evaluate the intracellular ROS level, including superoxide anions and hydroxyl radicals. After adding appropriate volume of DHE (5mmol / L) or DCFH-DA (10μmol / L), the cells were placed in a 37℃ cell culture incubator for 20min. Then the cells were incubated with 4',6-diamidino-2-phenylindole (DAPI) at room temperature for 10min, and the photos were taken using a fluorescence microscope (OLYMPUS, Japan). The relative fluorescence intensity was quantitatively analyzed using IPWIN60 software (Media Cybernetics, Inc.).

[0039] The results are as follows Figure 1 As shown, the results showed that the reactive oxygen content in the aging group was significantly higher than that in the other two groups, indicating that doxorubicin can cause a significant increase in the reactive oxygen content in vascular endothelial cells, while edaravone treatment can significantly reduce the increase in the level of reactive oxygen caused by doxorubicin to a level similar to that of the control group.

[0040] Example 2 Edaravone can reduce the positive rate of β-galactosidase staining

[0041] The 4th generation HUVECs were seeded into six-well plates and divided into three groups: a control group (no special treatment, cultured in ECM medium for 24 h), an aging group (cultured in ECM medium containing 2 μmol / L doxorubicin for 24 h), and an aging + edaravone group (pretreated with ECM medium containing 80 μM edaravone for 24 h, followed by the addition of ECM medium containing 2 μM doxorubicin and 80 μM edaravone and continued culture for 24 h).

[0042] After the culture was completed, the senescent cells in each group were determined. The specific steps were as follows: when the cells covered 80% of the bottom of the well, the cell culture medium was removed, and 1 mL of SA-β-Gal staining fixative was added after washing with PBS. The fixative was discarded and washed with PBS. At the same time, 1 mL of SA-β-Gal staining working solution was added to each well. The 6-well plate was sealed with a sealing film, and the culture plate was placed in an incubator and incubated overnight. After the staining was completed, the blue-stained cells were counted under an ordinary optical microscope, and the cell senescence rate (blue-stained cells / total number of cells) was calculated.

[0043] The results are as follows Figure 2As shown, the results showed that a large number of cells in the aging group were senescent, indicating that doxorubicin can induce the aging of vascular endothelial cells; compared with the aging group, the staining positive rate of the aging + edaravone group was significantly reduced, indicating that edaravone treatment can alleviate the senescence of vascular endothelial cells induced by doxorubicin.

[0044] Example 3 Edaravone inhibits doxorubicin-induced senescence of vascular endothelial cells by reducing the expression of senescence markers P53 and P21 and activating the Nrf2 / HO-1 / NQO1 pathway

[0045] The 4th generation HUVECs were seeded into six-well plates and divided into three groups: a control group (no special treatment, cultured in ECM medium for 24 h), an aging group (cultured in ECM medium containing 2 μmol / L doxorubicin for 24 h), and an aging + edaravone group (pretreated with ECM medium containing 80 μM edaravone for 24 h, followed by the addition of ECM medium containing 2 μM doxorubicin and 80 μM edaravone and continued culture for 24 h).

[0046] After the culture, the expression levels of P21, P53, Nrf2, HO-1 and NQO1 in each group of cells were determined. The specific steps were as follows: the treated cells were added to RIPA lysis buffer containing PMSF for full lysis, centrifuged at 12,000 rpm for 10 min at 4°C (centrifugal radius 85 mm) to obtain total protein, and the protein concentration was quantified using a BCA protein assay kit. After electrophoresis, the membrane was transferred to a PVDF membrane and blocked with 5% skim milk for 1 h. After blocking, the membrane was placed in an antibody hybridization box containing specific primary antibodies β-actin (1:1000), P21 (1:1000), P53 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), and NQO1 (1:1000), and incubated overnight at 4°C. After washing the membrane with TBST, the secondary antibody was added and incubated at room temperature for 2 h. After washing the membrane with TBST, ECL reagent was added for color development and exposure, and quantification was performed using Image J software. The GAPDH expression level was used as an internal reference, and the gray value ratio of each protein / internal reference was used to calculate the relative expression of each protein. The experiment was repeated 3 times.

[0047] The results are as follows Figure 3 As shown in the figure, the results showed that edaravone treatment significantly changed the protein expression levels of Nrf2, HO-1 and NQO1; for the aging markers P53 and P21, doxorubicin significantly increased their expression levels. Compared with the aging group, the P53 and P21 protein expression levels in the aging + edaravone group were significantly decreased, indicating that edaravone treatment can alleviate doxorubicin-induced senescence of vascular endothelial cells, significantly reduce the expression of aging markers P53 and P21, and activate the Nrf2 / HO-1 / NQO1 pathway.

[0048] Example 4 Edaravone inhibits doxorubicin-induced senescence of vascular endothelial cells by activating the Nrf2 / HO-1 / NQO1 pathway

[0049] The 4th generation HUVECs were seeded into six-well plates and divided into four groups: a control group (no special treatment, cultured in ECM medium for 24 h), an aging group (cultured in ECM medium containing 2 μmol / L doxorubicin for 24 h), an aging + edaravone group (pretreated with ECM medium containing 80 μM edaravone for 24 h, and then added with ECM medium containing 2 μM doxorubicin and 80 μM edaravone and cultured for a further 24 h), and an aging + edaravone + ML385 group (pretreated with ECM medium containing 80 μM edaravone and 2.5 μM Nrf2-specific inhibitor ML385 for 24 h, and then added with ECM medium containing 2 μM doxorubicin, 2.5 μM Nrf2-specific inhibitor ML385 and 80 μM edaravone and cultured for a further 24 h).

[0050] After the culture, the expression levels of P21, P53, Nrf2, HO-1 and NQO1 in each group of cells were determined. The specific steps were as follows: the treated cells were added with RIPA lysis buffer containing PMSF to fully lyse, and the total protein was obtained by centrifugation at 12,000 rpm for 10 min (centrifugal radius 85 mm) at 4°C. The protein concentration was quantified using a BCA protein assay kit, and the membrane was transferred to a PVDF membrane after electrophoresis and blocked with 5% skim milk for 1 h. After blocking, the membrane was placed in an antibody hybridization box containing specific primary antibodies β-actin (1:1000), P21 (1:1000), P53 (1:1000), Nrf2 (1:1000), HO-1 (1:1000), and NQO1 (1:1000), and incubated overnight at 4°C. After washing the membrane with TBST, the secondary antibody was added and incubated at room temperature for 2 h. After washing the membrane with TBST, ECL reagent was added for color development and exposure, and quantification was performed using Image J software. The GAPDH expression level was used as an internal reference, and the gray value ratio of each protein / internal reference was used to calculate the relative expression of each protein. The experiment was repeated 3 times.

[0051] The results are as follows Figure 4 The results showed that after treatment with the Nrf2-specific inhibitor ML385, the expression of the Nrf2 / HO-1 / NQO1 pathway was reduced, while the expression of senescence markers P53 and P21 was increased, and the inhibitory effect of edaravone on senescence of vascular endothelial cells induced by doxorubicin was weakened, indicating that the inhibitory effect of edaravone on doxorubicin-induced senescence of vascular endothelial cells was achieved by activating the Nrf2 / HO-1 / NQO1 pathway.

[0052] Based on the above results, the positive rate of β-galactosidase staining in the edaravone-treated group was significantly lower than that in the aging group, and edaravone can inhibit the senescence of vascular endothelial cells induced by doxorubicin. Specifically, edaravone can inhibit the senescence of vascular endothelial cells induced by doxorubicin by reducing the expression of senescence markers P53 and P21 and activating the Nrf2 / HO-1 / NQO1 pathway. The present invention lays a foundation for the development of chemical small molecules to inhibit the senescence of vascular endothelial cells.

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of edaravone in the preparation of drugs for inhibiting vascular endothelial cell senescence.

2. The use according to claim 1, characterized in that The concentration of edaravone is 30-80 μM.

3. The use according to claim 1, characterized in that The vascular endothelial cell senescence is vascular endothelial cell senescence induced by doxorubicin.

4. The use according to claim 1, characterized in that The vascular endothelial cells include human umbilical vein endothelial cells.

5. The use according to claim 1, characterized in that The edaravone inhibits the senescence of vascular endothelial cells by reducing the expression of P53 and P21.

6. The use according to claim 1, characterized in that The edaravone inhibits the aging of vascular endothelial cells by activating the Nrf2 / HO-1 / NQO1 pathway.

7. A drug for inhibiting vascular endothelial cell senescence, characterized in that: The drug includes edaravone and a pharmaceutically acceptable excipient; The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

8. The drug according to claim 7, characterized in that The concentration of edaravone is 30-80 μM.

9. The drug according to claim 7, characterized in that The vascular endothelial cell senescence is vascular endothelial cell senescence induced by doxorubicin.

10. The drug according to claim 7, characterized in that The dosage form of the drug includes solid dosage form, semisolid dosage form or liquid dosage form.

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