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

Abstract

The invention discloses application of gastrodin in preparation of a medicine for inhibiting senescence of vascular endothelial cells, and belongs to the technical field of biological pharmacy. The invention finds that gastrodin has a specific effect on inhibition of doxorubicin-induced vascular endothelial cell senescence. By adjusting specific senescence markers (such as beta-gal staining, P16 and the like), activating an antioxidant pathway and other mechanisms, 50 [mu] mol / L gastrodin plays a role in a vascular endothelial cell senescence mechanism induced by 2 [mu] mol / L doxorubicin, generalized senescence is delayed, and drug-induced vascular endothelial cell senescence can be effectively improved. The application of gastrodin provided by the invention lays a foundation for research and development of related drugs for inhibiting senescence of vascular endothelial cells. The gastrodin disclosed by the invention can also be used as an effective chemical tool to provide a powerful means for further researching a mechanism of chemical small molecules for inhibiting senescence of vascular endothelial cells induced by doxorubicin.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to the application of gastrodin in the preparation of a medicine for inhibiting aging of vascular endothelial cells. Background Art

[0002] Vascular aging is a significant pathological basis for the degenerative changes in vascular structure and function with age. It is an independent risk factor for a variety of age-related vascular diseases (ARVDs) and an independent risk factor for increased mortality in the elderly. Vascular aging is manifested macroscopically as vascular lumen dilation, arterial wall thickening, and diffuse sclerosis. The senescence of vascular wall cells is the functional basis of vascular aging, which is mainly characterized by irreversible cell cycle arrest and the emergence of senescence-related secretory phenotypes, which has become an important factor in inducing aging, increasing mortality, and inducing a variety of chronic diseases. Endothelial cells, as an important component of vascular wall cells, play a key role in regulating nutrient intake, biomolecule delivery, maintaining tissue barrier function, and preventing immune cell adhesion and infiltration. Endothelial cell senescence is an important pathological feature of vascular aging. Therefore, exploring the mechanism of vascular endothelial cell senescence and drugs to delay its senescence are of great significance for preventing vascular aging.

[0003] Gastrodin is a natural organic compound extracted from the dried rhizome of Gastrodia elata, an orchid plant. In recent years, gastrodin has been shown to have good neuroprotective effects, and its mechanism involves inhibiting inflammation, oxidative stress, neuronal apoptosis and neuroautophagy. It is widely used in the clinic to treat neurological and vascular diseases such as epilepsy, convulsions, migraine, hypertension, etc. In terms of the anti-aging effect of gastrodin, although some studies have shown that it is widely used to slow down the aging process through antioxidant and anti-inflammatory mechanisms, and gastrodin has a wide range of neuroprotective effects, there are no public reports on the specific role of gastrodin in inhibiting doxorubicin-induced vascular endothelial cell senescence and the development of treatment methods and drugs targeting vascular aging. Summary of the invention

[0004] The purpose of the present invention is to provide the use of gastrodin in the preparation of drugs for inhibiting vascular endothelial cell senescence, so as to solve the problems existing in the above-mentioned prior art. The present invention particularly emphasizes its specific effect in inhibiting vascular endothelial cell senescence induced by doxorubicin. This effect is different from the broad anti-aging effect of gastrodin. Instead, it intervenes specifically on vascular endothelial cells through specific cellular mechanisms, such as inhibiting oxidative stress caused by doxorubicin, thereby improving vascular function and delaying the aging process. This specific effect is crucial for the development of treatments targeting vascular aging, which is different from its effects in other anti-aging fields.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of gastrodin (GAS for short) in preparing medicine for inhibiting senescence of vascular endothelial cells.

[0007] Preferably, the concentration of gastrodin is not less than 50 μmol / L.

[0008] Preferably, the vascular endothelial cell senescence is doxorubicin-induced vascular endothelial cell senescence.

[0009] Preferably, the concentration of doxorubicin is 2 μmol / L.

[0010] Preferably, the doxorubicin-induced vascular endothelial cell senescence includes an increase in intracellular β-gal.

[0011] Preferably, the vascular endothelial cell senescence induced by doxorubicin includes an increase in the expression levels of P16 and P53 proteins in the cells.

[0012] Preferably, the vascular endothelial cells are human umbilical vein endothelial cells.

[0013] The invention also provides a medicine for inhibiting vascular endothelial cell senescence, with gastrodin as the only effective ingredient.

[0014] Preferably, the vascular endothelial cell senescence is doxorubicin-induced vascular endothelial cell senescence.

[0015] Preferably, a pharmaceutically acceptable excipient is also included.

[0016] The present invention discloses the following technical effects:

[0017] In addition to the general anti-aging effect of gastrodin, the present invention particularly highlights its specific effect in inhibiting the aging of vascular endothelial cells induced by doxorubicin. By regulating specific aging markers (such as β-gal staining, P16, etc.), gastrodin plays a role in the specific aging mechanism of vascular endothelial cells, thereby not only delaying generalized aging, but also effectively improving the aging of vascular endothelial cells induced by drugs, and the specific mechanism includes activating antioxidant pathways, etc. The application of gastrodin provided by the present invention lays a foundation for the research and development of drugs for inhibiting the aging of vascular endothelial cells. The GAS involved in the present invention can also be used as an effective chemical tool to provide a powerful means for further studying the mechanism of chemical small molecules inhibiting the aging of vascular endothelial cells induced by doxorubicin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] Figure 1 The graph shows the observation results of GAS inhibiting doxorubicin-induced vascular endothelial cell senescence; A is the result of microscopic observation; B is the statistical result of the positive staining rate; Dox is doxorubicin; GAS is gastrodin; a means P<0.05 compared with the control group, b means P<0.05 compared with the Dox group; the scale length is 100 μm;

[0020] Figure 2 The figure shows the results of GAS reducing the level of ROS in doxorubicin-induced vascular endothelial cells and quantitative analysis; A is the result of fluorescence microscopy observation; B is the statistical result of the relative DCFH-DA fluorescence intensity of the cells; C is the statistical result of the relative DHE fluorescence intensity of the cells; DAPI is 4',6-diamidino-2-phenylindole; DCFH-DA is 2,7-dichlorofluorescein diacetate; DHE is dihydroethidine; Dox is doxorubicin; GAS is gastrodin; a means P<0.05 compared with the control group, b means P<0.05 compared with the Dox group; the scale length is 100μm;

[0021] Figure 3 The results of the experiment in which GAS inhibits doxorubicin-induced senescence of vascular endothelial cells by inhibiting p16 and p53; A is the result of Western blot experiment; B is the statistical result of SIRT1 expression level; C is the statistical result of Nrf2 expression level; D is the statistical result of P53 expression level; E is the statistical result of P16 expression level; Dox is doxorubicin; GAS is gastrodin; a means P<0.05 compared with the control group, b means P<0.05 compared with the Dox group;

[0022] Figure 4 The experimental results of GAS increasing the fluorescence intensity of SIRT1 in Dox-induced HUVECs; A is the result of confocal fluorescence microscopy observation; B is the result of relative fluorescence intensity observation; Dox is doxorubicin; GAS is gastrodin; a means P<0.05 compared with the control group, b means P<0.05 compared with the Dox group;

[0023] Figure 5The figure shows the experimental results of GAS improving cell senescence by activating the antioxidant pathway SIRT1 / Nrf2; A is the Western blot experimental result; B is the statistical result of SIRT1 expression level; C is the statistical result of Nrf2 expression level; D is the statistical result of P53 expression level; E is the statistical result of P16 expression level; Dox is doxorubicin; GAS is gastrodin; a means P<0.05 compared with the Dox+GAS group. DETAILED DESCRIPTION

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The structural formula of Gastrodin (GAS) described in the present invention is:

[0030]

[0031] Molecular formula: C13 H 18 O 7 ; Molecular weight: 286.28; Properties: white crystalline powder.

[0032] The DHE used in the present invention is dihydroethidium, which is a superoxide anion fluorescent probe and emits red fluorescence.

[0033] The DCFH-DA used in the present invention is 2,7-dichlorofluorescein diacetate, which is a cell-permeable active oxygen detection fluorescent probe and emits green fluorescence.

[0034] The composition of the endothelial cell-specific basal culture medium used in the present invention is as follows: 433.4 mL of M199 basal culture medium, 56 mL of 5% fetal bovine serum, 5 mL of endothelial cell growth supplement (ECGS) and 5.6 mL of penicillin-streptomycin solution (100×) are added to every 500 mL of endothelial cell-specific basal culture medium.

[0035] The conditions for culturing cells in the present invention are: 37°C, 5% CO 2 Incubator.

[0036] Example 1 GAS inhibits doxorubicin-induced vascular endothelial cell senescence

[0037] Human umbilical vein endothelial cells (HUVEC) were used as model cells and cultured in vitro.

[0038] Human umbilical vein endothelial cells were seeded into six-well plates, and a control group, a Dox group, and a Dox+GAS treatment group were set up for the following experiment.

[0039] Control group: cultured in basal medium dedicated to endothelial cells;

[0040] Dox group: containing doxorubicin (2 μM), cultured in basal medium dedicated to endothelial cells;

[0041] Dox+GAS group: containing doxorubicin (2 μM), endothelial cell-specific basal culture medium was pretreated with 50 μM GAS.

[0042] After culturing for 24 hours, each group discarded the old culture medium, washed twice with PBS, fixed with freshly prepared 1×Fixative Solution at room temperature for 15 minutes, discarded the fixative, washed twice with PBS, 5 minutes each time, added 1 mL of β-gal staining working solution to each well, sealed with paraffin film and protected from light, incubated in a 37°C constant temperature incubator for 24 hours, discarded the staining solution, washed twice with PBS, observed under a microscope, and counted the number of positive cells.

[0043] The results showed that doxorubicin can induce senescence of endothelial cells, while GAS treatment can alleviate doxorubicin-induced senescence of endothelial cells. Cell senescence was determined by counting the proportion of β-gal positive cells (senescent cells) (see Figure 1 A and B).

[0044] Example 2 GAS reduces ROS levels in doxorubicin-induced senescent endothelial cells

[0045] Human umbilical vein endothelial cells were seeded into 6 cm dishes, and a control group, a Dox group, and a Dox+GAS treatment group were set up for the following experiment.

[0046] Control group: cultured in basal medium dedicated to endothelial cells;

[0047] Dox group: containing doxorubicin (2 μM), cultured in basal medium dedicated to endothelial cells;

[0048] Dox+GAS group: containing doxorubicin (2 μM), endothelial cell-specific basal culture medium was pretreated with 50 μM GAS.

[0049] After 24 hours of culture, each group discarded the old culture medium, added 1 mL of DHE (5 mmol / L) or DCFH-DA (10 μmol / L) to each 6 cm dish, and then placed the cells in a 37 ° C cell culture incubator for 20 minutes. Then the cells were incubated with 4', 6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI) at room temperature for 10 minutes, and photographed using a fluorescence microscope (OLYMPUS, Japan), and the relative fluorescence intensity was quantitatively analyzed using IPWIN60 software (Media Cybernetics, Inc.).

[0050] The results showed that in the Dox group, DHE emitted strong red fluorescence, DCFH-DA emitted green fluorescence, and DAPI emitted blue fluorescence. Figure 2 As shown in A. However, the fluorescence intensity of DHE and DCFH-DA in the Dox+GAS group was lower. Figure 2 Quantitative analysis of fluorescence density in Figures B and C showed that the ROS content was higher in the Dox group (P<0.01) and lower in the Dox+GAS group (P<0.01).

[0051] Example 3 GAS inhibits doxorubicin-induced senescence of vascular endothelial cells by inhibiting p16 and p53

[0052] Control group: cultured in basal medium dedicated to endothelial cells;

[0053] Dox group: containing doxorubicin (2 μM), cultured in basal medium dedicated to endothelial cells;

[0054] Dox+GAS: Containing doxorubicin (2 μM), endothelial cell-specific basal culture medium was pretreated with 50 μM GAS.

[0055] After 24 hours of culture, the cells of each group were collected after the group treatment, and the cells were lysed with RIPA buffer containing protease and phosphatase inhibitors, and the protein content was determined using a BCA kit. After the protein was boiled and denatured, 20 μL was loaded into each well and the protein was separated by SDS-PAGE electrophoresis, and the embedded protein was transferred to a PVDF membrane. The membrane was then blocked with 5% skim milk powder for 2 hours at room temperature, and blocked overnight with primary antibodies β-actin, Nrf2, SIRT1, P53 and P16 (all diluted 1:1000). After washing 3 times with TBST, P53 was incubated with goat anti-mouse IgG, and the rest were incubated with goat anti-rabbit IgG (1:1000) at room temperature for 1 hour. Chemiluminescent reagent was added to detect the bands. The grayscale value of the relevant bands was read, and the results were analyzed using ImageJ software. The β-actin expression level was used as the internal reference, and the grayscale value ratio of each protein / internal reference was used to calculate the relative expression of each protein. The experiment was repeated 3 times. Western blot results are shown in Figure 3 As shown in A.

[0056] The results showed that compared with the control group, the expression levels of aging-related proteins P53 and P16 in the Dox group were significantly increased (P<0.05), while the expression levels of P53 and P16 in the Dox+GAS group were significantly decreased compared with the Dox group (P<0.05). Figure 3 D and E. This shows that Dox can induce HUVEC senescence and the aging model was successfully established. The addition of GAS can improve HUVEC senescence. In addition, compared with the Dox group, the expression levels of Nrf2 and SIRT1 proteins in the Dox+GAS group were significantly increased (P<0.05). Figure 3 Figures B and C suggest that GAS may alleviate aging by upregulating the SIRT1 / Nrf2 pathway.

[0057] Example 4 GAS increases SIRT1 fluorescence intensity in DOX-induced HUVEC

[0058] HUVECs were seeded in confocal culture dishes, and each group of cells was treated after the cells adhered and proliferated.

[0059] Control group: cultured in basal medium dedicated to endothelial cells;

[0060] Dox group: containing doxorubicin (2 μM), cultured in basal medium dedicated to endothelial cells;

[0061] Dox+GAS group: containing doxorubicin (2 μM), endothelial cell-specific basal culture medium was pre-treated with 50 μM GAS.

[0062] After 24 hours of culture, wash with PBS three times, 5 minutes each time, fix the cells with 4% paraformaldehyde for 30 minutes, and wash with PBS three times. Treat the cells with 3mL / L Triton X-100 for 25 minutes and wash with PBS three times. Add 1.5mL of BSA blocking solution to each culture dish, block for 1 hour and then wash with PBS. Add 100μL of SIRT1 antibody diluted 1:200 and incubate overnight at 4℃. Wash with PBS three times, add 100μL of fluorescent secondary antibody diluted 1:200, and react at 37℃ in the dark for 1 hour. After washing with PBS, add DAPI staining solution for cell nucleus staining, treat at 37℃ in the dark for 30 minutes, and wash with PBS three times. Observe and take pictures using a confocal microscope. The experiments were repeated three times independently.

[0063] The results show that: Figure 4 As shown in A and B, compared with the control group, the fluorescence intensity of SIRT1 in the Dox group was decreased (P<0.05); compared with the DOX group, the fluorescence intensity of SIRT1 in the Dox+GAS group was increased (P<0.05).

[0064] Example 5 GAS improves cell senescence by activating the antioxidant pathway SIRT1 / Nrf2

[0065] The third generation HUVECs were cultured at 1×10 5The cells were inoculated in 6-well plates at a density of 100 μmol / well, and the cells were divided into the DOX+GAS group (50 μmol / L GAS pretreatment for 24 hours, followed by the addition of 2 μmol / L Dox and continued culture for 24 hours) and the Dox+GAS+ML385 group (50 μmol / L GAS+2.5 μmol / L Nrf2 specific inhibitor ML385 pretreatment for 24 hours, followed by the addition of 2 μmol / L Dox and continued culture for 24 hours). After the intervention, the cells in each group were collected, lysed with RIPA buffer containing protease inhibitors and phosphatase inhibitors, and the protein content was determined using a BCA kit. After the protein was boiled and denatured, 20 μL was loaded into each well and the protein was separated by SDS-PAGE electrophoresis, and the embedded protein was transferred to a PVDF membrane. The membrane was then blocked with 5% skim milk powder at room temperature for 2 hours, and incubated with primary antibodies β-actin, Nrf2, SIRT1, P53 and P16 (all diluted 1:1000) overnight. After washing 3 times with TBST, P53 was incubated with goat anti-mouse IgG, and the rest were incubated with goat anti-rabbit IgG (1:1000) at room temperature for 1 hour. Chemiluminescent reagent was added to detect and read the gray value of the relevant bands. The results were analyzed using ImageJ software. The β-actin expression level was used as the 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. Western blot results are shown in Figure 5 As shown in A.

[0066] The results showed that compared with the Dox group, the expression levels of SIRT1 and Nrf2 proteins in the Dox+GAS group were significantly increased (P<0.05). The Nrf2-specific inhibitor ML385 was introduced to reduce the expression of SIRT1 / Nrf2 pathway. After adding ML385, compared with the Dox+GAS group, the expression of SIRT and Nrf2 in the Dox+GAS+ML385 group was significantly reduced (P<0.05). Figure 5 As shown in B and C; the expression levels of aging-related proteins P53 and P16 in the inhibitor group were increased (P<0.05). Figure 5 As shown in D and E. These all indicate that GAS can alleviate Dox-induced senescence of human umbilical vein endothelial cells, and this protective effect may be achieved by activating the SIRT1 / Nrf2 pathway. And it reflects that by using the doxorubicin-induced vascular endothelial cell senescence model, it was observed that gastrodin has a significant effect in inhibiting oxidative stress and reducing the expression of aging markers. Different from general anti-aging compounds, gastrodin can specifically intervene in the aging process in vascular endothelial cells and show its unique anti-aging mechanism at the molecular level.

[0067] 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. Application of gastrodin in the preparation of drugs for inhibiting aging of vascular endothelial cells.

2. The use according to claim 1, characterized in that The concentration of the gastrodin is not less than 50 μmol / L.

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 3, characterized in that The concentration of doxorubicin is 2 μmol / L.

5. The use according to claim 3, characterized in that The doxorubicin-induced vascular endothelial cell senescence includes the increase of intracellular β-gal.

6. The use according to claim 3, characterized in that The vascular endothelial cell senescence induced by dobiprofen includes the increase of the expression levels of P16 and P35 proteins in the cells.

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

8. A drug for inhibiting vascular endothelial cell senescence, characterized in that: Gastrodin is the only active ingredient.

9. The drug according to claim 8, characterized in that The vascular endothelial cell senescence is vascular endothelial cell senescence induced by doxorubicin.

10. The drug according to claim 8, characterized in that Pharmaceutically acceptable excipients are also included.

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