Use of vanillin in the preparation of a drug for preventing or / and treating myocardial hypertrophy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of vanillin in preparation of a drug for preventing or / and treating myocardial hypertrophy. The application discloses a new use of vanillin, and can be used for preparing a drug for preventing or / and treating myocardial hypertrophy. In the application, H9c2 myocardial cells are used as an in-vitro model, angiotensin II is used to induce myocardial cell hypertrophy, the influence of vanillin on cell activity is detected by a CCK-8 method, the protein expression level of a myocardial hypertrophy marker BNP is detected by Western blot, and the changes of myocardial cell morphology and cell area are observed by a phalloidin staining, so that it is confirmed from cell toxicity, hypertrophy marker expression and cell morphological changes that vanillin has an improvement effect on myocardial hypertrophy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of vanillin in the preparation of drugs for the prevention and / or treatment of myocardial hypertrophy. Background Technology

[0002] Myocardial hypertrophy is an adaptive change in the heart in response to long-term pressure load, volume load, or neurohumoral stimulation. In the short term, myocardial hypertrophy can maintain the heart's pumping function; however, under long-term pathological stimulation, myocardial hypertrophy can gradually develop into myocardial remodeling, myocardial fibrosis, heart failure, and even heart failure. Myocardial hypertrophy is one of the important pathological changes in the progression of various cardiovascular diseases such as hypertension, coronary heart disease, and cardiomyopathy. Persistent pathological myocardial hypertrophy can further lead to myocardial remodeling, decreased cardiac function, and even heart failure. Currently, interventions for myocardial hypertrophy mainly include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and beta-blockers, but existing interventions still have problems such as limited applicable populations, insufficient long-term medication adherence, and unsatisfactory efficacy in some patients. Angiotensin II (AngII) is one of the important stimulating factors that induce pathological myocardial hypertrophy. AngII can promote the increase of cardiomyocyte volume through multiple intracellular signaling pathways and induce increased expression of hypertrophy-related markers such as ANP and BNP. Therefore, using AngII to stimulate H9c2 cardiomyocytes to establish an in vitro cardiomyocyte hypertrophy model is a common technique for evaluating the anti-cardiomyocyte hypertrophy effects of candidate active substances.

[0003] Vanillin, chemically known as 4-hydroxy-3-methoxybenzaldehyde, is a common aromatic aldehyde compound with certain antioxidant, anti-inflammatory, and cell-protective effects. Current research mainly focuses on the following effects of vanillin: (1) antioxidant activity; (2) anti-inflammatory activity; (3) anti-apoptotic activity; (4) metabolic regulation; and (5) cardiomyocyte protection. In cardiac-related studies, vanillin has been found to alleviate doxorubicin-induced H9c2 cardiomyocyte damage, reduce oxidative stress and apoptosis levels, suggesting that vanillin has a certain protective effect on cardiomyocytes. Other studies have shown that in an isoproterenol-induced rat model of myocardial injury, vanillin can improve myocardial injury-related indicators and may exert its effects by regulating oxidative stress, inflammation, apoptosis, and Akt / HIF-1α / VEGF-related signaling. The aforementioned studies indicate that vanillin has a certain research foundation in cardioprotection. However, current technologies mainly focus on doxorubicin-induced cardiomyocyte toxicity models, isoproterenol-induced myocardial injury models, or myocardial infarction-like injury models. Research emphasis is primarily on cardiomyocyte injury, oxidative stress, inflammatory responses, apoptosis, and angiogenesis. However, there are no studies on the use of vanillin in myocardial hypertrophy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of vanillin in the preparation of drugs for the prevention and / or treatment of myocardial hypertrophy.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides, in one aspect, the use of vanillin in the preparation of drugs for the prevention and / or treatment of myocardial hypertrophy.

[0007] As a preferred technical solution, the myocardial hypertrophy is induced by Ang II.

[0008] As a preferred technical solution, vanillin inhibits the expression of BNP protein, a marker of myocardial hypertrophy induced by Ang II.

[0009] As a preferred technical solution, the effective mass percentage concentration of vanillin is 10-50%.

[0010] As a more preferred technical solution, the effective mass percentage concentration of vanillin is 50%.

[0011] Another aspect of the present invention provides a pharmaceutical composition for preventing and treating myocardial hypertrophy, comprising at least vanillin.

[0012] As a preferred technical solution, pharmaceutically acceptable excipients are also included.

[0013] As a more preferred technical solution, the excipient is one or more of the following: diluent, filler, surfactant, absorption promoter, disintegrant, wetting agent, or dispersant.

[0014] As a preferred technical solution, the dosage form of the drug is any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule or oral liquid.

[0015] This invention has the following advantages: It discloses a novel use for vanillin, which can be used to prepare drugs for the prevention and / or treatment of myocardial hypertrophy. Using H9c2 cardiomyocytes as an in vitro model, this invention induces cardiomyocyte hypertrophy with angiotensin II (Ang II), and detects the effect of vanillin on cell viability using the CCK-8 assay. The protein expression level of the myocardial hypertrophy marker BNP is detected by Western blot, and changes in cardiomyocyte morphology and cell area are observed by phalloidin staining. From the aspects of cytotoxicity, hypertrophy marker expression, and cell morphological changes, this invention confirms that vanillin has an ameliorative effect on myocardial hypertrophy. Attached Figure Description

[0016] Figure 1Flowchart for experimental verification of vanillin's ability to improve angiotensin II-induced H9c2 cardiomyocyte hypertrophy.

[0017] Figure 2 The effect of different concentrations of vanillin on the viability of H9c2 cells is shown in the figure.

[0018] Figure 3 The figures show the Western blot results of the effect of vanillin on the expression of markers of AngII-induced myocardial hypertrophy. Figure a shows the representative bands of BNP protein and internal reference protein GADPH in H9c2 cells of each group. Figure b shows the gray-scale quantitative analysis of the relative expression level of BNP protein (BNP / GAPDH). In the figures, BNP represents B-type natriuretic peptide, a classic marker of myocardial hypertrophy; GADPH represents glyceraldehyde-3-phosphate dehydrogenase, an internal reference protein; C represents Control, i.e., the control group; M represents Model, i.e., the Ang II-induced myocardial hypertrophy model group; V represents Vanillin, i.e., the vanillin intervention group; * indicates P<0.01; ns indicates no statistically significant difference.

[0019] Figure 4 The images show the phalloidin staining results of vanillin on the morphological changes of Ang II-induced H9c2 cardiomyocyte hypertrophy. Figure a shows a laser confocal microscope image of H9c2 cells observed by phalloidin staining, and Figure b shows the quantitative statistical analysis of H9c2 cell surface area in each group. In the figures, red fluorescence represents phalloidin-labeled F-actin, and blue fluorescence represents DAPI-labeled cell nuclei; Cell Area represents cell area; Control represents the control group; Model represents the Ang II-induced cardiomyocyte hypertrophy model group; and Vanillin represents the vanillin intervention group. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments. The scope of protection of the present invention is not limited to the following description:

[0021] Example 1: Experimental verification of vanillin's effect on angiotensin II-induced H9c2 cardiomyocyte hypertrophy.

[0022] I. Experimental Methods

[0023] The experimental verification process is as follows: Figure 1 As shown, the specific method is as follows:

[0024] 1. H9c2 cell culture, resuscitation and passage

[0025] Remove the frozen H9c2 cells from the liquid nitrogen tank and quickly place them in a 37°C water bath, gently shaking to rapidly thaw the cell suspension. Once the cell suspension has mostly thawed, transfer them to sterile centrifuge tubes, add high-glucose DMEM complete medium containing 10% fetal bovine serum (FBS), and gently pipette to mix. Centrifuge at 1000 r / min for 5 min, discarding the supernatant to remove dimethyl sulfoxide (DMSO) from the cryopreservation solution. Resuspend the cells in fresh complete medium and seed them in 25 cm² cell culture flasks, incubating at 37°C, 5% CO2, and saturated humidity. Observe cell growth daily during culture. When cell confluence reaches 80%–90%, discard the old medium, gently wash the cells 1–2 times with sterile PBS buffer, add an appropriate amount of 0.25% trypsin digestion solution, and digest at 37°C. Once the cells become rounded and begin to detach under a microscope, immediately add complete medium containing 10% FBS to stop digestion. The cell suspension was collected into centrifuge tubes, centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in fresh complete culture medium. The cells were then passaged at a ratio of 1:3. H9c2 cells in good growth condition and in the logarithmic growth phase were used for subsequent experiments.

[0026] 2. Preparation of vanillin stock solution

[0027] Vanillin was prepared as a high-concentration stock solution using DMSO, and thoroughly vortexed before use. Before experimental use, the vanillin stock solution was diluted to the required working concentration with serum-free high-glucose DMEM medium. The final concentration of DMSO in all drug-treated groups and the solvent control group was controlled below 0.1% to eliminate the influence of DMSO on cell viability and experimental results.

[0028] 3. Effect of vanillin on H9c2 cell viability as determined by the CCK-8 assay.

[0029] Logarithmically growing H9c2 cells were digested and a single-cell suspension was prepared. After adjusting the cell concentration, the cells were seeded into 96-well plates at a density of 5 × 10³ cells / well, with an appropriate amount of complete culture medium added to each well. The cells were incubated at 37°C in a 5% CO2 incubator for 24 h to allow for full cell adhesion. After cell adhesion, the original culture medium was discarded, and the cells were gently washed with PBS. Serum-free, high-glucose DMEM medium containing different concentrations of vanillin was then added. Experimental groups included: control group, DMSO solvent control group, 10 μmol / L vanillin group, 25 μmol / L vanillin group, 50 μmol / L vanillin group, 100 μmol / L vanillin group, and 200 μmol / L vanillin group. Each group was configured with 3 replicates, and the experiment was independently repeated 3 times. After culturing for another 24 hours, 10 μL of CCK-8 solution was added to each well, gently mixed, and incubated in a 37°C incubator in the dark for 2 hours. After incubation, the absorbance value (OD450) of each well was measured using a microplate reader at a wavelength of 450 nm. The control group consisted of wells containing only culture medium and CCK-8 solution and no cells.

[0030] Cell survival rate was calculated using the following formula: Cell survival rate (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group − OD value of blank group) × 100%. By comparing the survival rates of H9c2 cells treated with different concentrations of vanillin, a safe dosing concentration with no obvious toxicity to cells was screened for subsequent anti-cardiomyopathy experiments.

[0031] 4. Establishment of an Ang II-induced H9c2 cell mast model and vanillin intervention

[0032] Logarithmic growth phase H9c2 cells were seeded into 25 cm² culture flasks or 24-well plates according to different detection purposes. Cells used for protein extraction were seeded at a ratio of 2.5 × 10⁻⁶ cells / well. 6 Cells were seeded per T25 culture flask; cells used for cell morphology staining were seeded at a ratio of 2 × 10⁶ cells / flask. 4 Cells were seeded per well in 24-well plates pre-loaded with poly-L-lysine-coated climbing sheets. After seeding, cells were incubated at 37°C, 5% CO2 for 24 h to allow for cell adhesion and recovery to a healthy growth state. After cell adhesion, the complete culture medium was discarded, and the cells were gently washed with PBS, followed by synchronization treatment with serum-free, high-glucose DMEM medium. Cells were divided into three groups according to the experimental objectives:

[0033] (1) Control group: Add serum-free high-glucose DMEM medium and continue culturing for 24 h;

[0034] (2) Ang II model group: H9c2 cells were cultured in serum-free high-glucose DMEM medium containing 1 μmol / L Ang II for 24 h to induce hypertrophy;

[0035] (3) Vanillin treatment group: First, add serum-free high-glucose DMEM medium containing 50 μmol / L vanillin for 2 h pretreatment, then add Ang II to make the final concentration of Ang II 1 μmol / L, and continue to culture for 24 h.

[0036] All experiments were independently repeated three times. The effect of vanillin on Ang II-induced cardiomyocyte hypertrophy was evaluated by comparing the expression of cell hypertrophy markers and changes in cell morphology among the groups.

[0037] 5. Western blot detection of BNP protein expression

[0038] After treatment, the culture medium was discarded, and the cells were washed twice with pre-cooled PBS. Then, RIPA lysis buffer containing PMSF and a phosphatase inhibitor was added to fully cover the cell surface. Cells were scraped off with a cell scraper and collected in centrifuge tubes, placed on ice for lysis for 40 min, with intermittent vortexing to promote complete cell lysis. After lysis, the sample was centrifuged at 12000 r / min for 15 min at 4℃, and the supernatant was collected as the total protein extract. The protein concentration of each group was determined using the BCA protein quantification method. Based on the quantification results, the protein concentrations of each group were adjusted to be consistent, and 5× protein loading buffer was added to mix the protein sample with the loading buffer at a 4:1 ratio. The sample was then boiled in a 95℃ metal bath or water bath for 5 min to ensure complete protein denaturation. An equal amount of protein sample was added to each well; in this experiment, the protein amount loaded per well was 55 μg. Electrophoresis was performed using a 10% SDS-PAGE gel. After electrophoresis, the proteins in the gel were transferred to a PVDF membrane using a wet transfer method at 200 mA for 120 min. After transfer, the PVDF membrane was placed in 5% skim milk blocking buffer and blocked at room temperature for 1 h to reduce non-specific binding. After blocking, the PVDF membrane and primary antibody incubation solution were incubated overnight on a shaker at 4°C. The primary antibodies included BNP antibody (B-type natriuretic peptide, also known as brain natriuretic peptide, a classic marker protein for myocardial hypertrophy) and internal control antibody GAPDH. The BNP antibody was diluted 1:1000, and the internal control antibody was diluted according to the manufacturer's instructions. The next day, the PVDF membrane was removed and washed three times with TBST for 10 min each time. Then, HRP-labeled secondary antibody was added and incubated at room temperature for 1 h. The secondary antibody was diluted 1:5000. After incubation, the membrane was washed three more times with TBST for 10 min each time. After washing, ECL chemiluminescence reagent was added for development and exposure. Protein band images were acquired using an imaging system, and the grayscale values ​​of the protein bands in each group were analyzed using ImageJ software. BNP protein expression levels were corrected using the internal reference protein GAPDH, and relative protein expression levels were calculated. The effect of vanillin on the expression of myocardial hypertrophy markers was evaluated by comparing the differences in BNP protein expression among the control group, the Ang II model group, and the vanillin treatment group.

[0039] 6. Observation of H9c2 cell morphology and cell area by phalloidin staining.

[0040] H9c2 cells were seeded in 24-well plates pre-filled with poly-L-lysine-coated climbing sheets and subjected to Ang II modeling and vanillin intervention according to the groupings described above. After treatment, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS for 10 min each time. Then, 4% paraformaldehyde fixative was added, and the cells were fixed at room temperature. After fixation, the fixative was discarded, and the cells were washed twice with pre-chilled PBS for 10 min each time. After washing, 0.1% Triton X-100 solution was added, and the cells were permeabilized at room temperature for 5 min to allow sufficient entry of the staining reagent. After permeabilization, the cells were washed twice with pre-chilled PBS for 10 min each time. Then, 5 μg / mL phalloidin-TRITC staining solution was added, and the cells were incubated at room temperature in the dark for 30 min to label F-actin. After staining, the cells were washed with PBS to remove unbound dye. The slides were then removed from the 24-well plates, placed cell-side down on glass slides, and stained with DAPI staining solution (working concentration: 1 μg / mL). After staining, the slides were mounted with anti-fluorescence quenching mounting medium and the edges were sealed with nail polish. After mounting, the slides were observed and photographed under a laser confocal microscope. The TRITC channel was used to observe F-actin at an excitation wavelength of 543 nm; the DAPI channel was used to observe the cell nucleus at an excitation wavelength of 405 nm. Five fields of view were randomly selected from each group for photographing, maintaining consistent microscope parameters across groups, including laser intensity, exposure time, and magnification. Cell surface area was measured using ImageJ software, and the long axis / short axis ratio was calculated. By comparing changes in cell area and morphology among the groups, the effect of vanillin on improving the morphological changes of Ang II-induced H9c2 cell hypertrophy was evaluated.

[0041] 7. Data Statistical Analysis

[0042] All experiments were replicated independently at least three times. Experimental data are expressed as mean ± standard deviation. A t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. If the ANOVA results showed statistical significance, further multiple comparisons between groups were performed. P < 0.05 indicated a statistically significant difference, and P < 0.01 indicated a highly statistically significant difference.

[0043] II. Results and Analysis

[0044] 1. Effects of vanillin on H9c2 cell viability

[0045] To determine the safe dosing concentration of vanillin, the results are as follows: Figure 2As shown, the cell viability rates of the 10, 25, 50, 100, and 200 μmol / L vanillin treatment groups were 94.5%, 92.7%, 91.9%, 65.8%, and 59.7%, respectively, which were not significantly different from the control group (100%) (P>0.05). The cell viability rate of the 100 μmol / L group decreased to 59.7%, significantly lower than that of the control group (P<0.01). Therefore, in subsequent experiments, 50 μmol / L was selected as the safe concentration for vanillin administration, and the final concentration of DMSO was controlled within 0.1%. There was no significant difference between the solvent control group and the control group (P>0.05), indicating that DMSO had no effect on cell viability.

[0046] 2. Effects of vanillin on the expression of AngII-induced markers of myocardial hypertrophy

[0047] BNP is a classic biomarker of myocardial hypertrophy, expressed at low levels in normal adult myocardium, and reactivated during hypertrophy. Western blot results, as shown in Figure 3, indicate that compared to the blank control group, the BNP protein expression level in the AngII model group was significantly increased, reaching 1.9 times that of the control group (P<0.01). Compared to the model group, the BNP expression in the 50 μmol / L vanillin treatment group was reduced by 24.3% (P<0.05), suggesting that vanillin can effectively inhibit the expression of AngII-induced myocardial hypertrophy markers and improve the myocardial hypertrophy phenotype.

[0048] 3. Effects of vanillin on AngII-induced morphology of H9c2 cells

[0049] Phalloidin can specifically bind to F-actin, label the cytoskeleton, and be used to observe changes in cell morphology. Figure 4 In the figure, 'a' represents a laser confocal microscopy image (red: F-actin, blue: cell nucleus, scale bar = 50 μm): Control group cells exhibit a typical spindle shape with neatly arranged myofibrils and small cell volume; AngII model group cells are significantly larger, irregular in shape, with disordered myofibrils and pseudopodia-like protrusions; 50 μmol / L vanillin treatment group cells have a morphology similar to the control group, with a clear spindle structure and relatively regular myofibrils. Quantitative analysis results are as follows... Figure 4 As shown in b, the cell surface area in the AngII model group was (1183.47±98.7) μm², which was 24.7% higher than that in the control group (949.16±65.2) μm² (P<0.01); the cell surface area in the 50 μmol / L treatment group was (942.81±53.4), which was 20.3% lower than that in the model group. These results further confirm that vanillin can improve AngII-induced cardiomyocyte hypertrophy.

[0050] III. Results Analysis

[0051] This embodiment evaluated the ameliorative effect of vanillin on Ang II-induced myocardial hypertrophy using an in vitro H9c2 cell experimental system. First, CCK-8 assay results showed that treatment with 10–50 μmol / L vanillin for 24 h had no significant effect on H9c2 cell viability, while high concentrations of vanillin (100 μmol / L and 200 μmol / L) reduced cell survival, suggesting that vanillin may exhibit cytotoxicity at higher concentrations. Therefore, this embodiment selected 50 μmol / L as the subsequent intervention concentration, which is both safe and experimentally feasible.

[0052] Secondly, Ang II treatment significantly increased BNP protein expression in H9c2 cells, indicating the successful establishment of the myocardial hypertrophy model. Vanillin intervention significantly reduced BNP protein expression compared to the model group, suggesting that vanillin can inhibit the expression of Ang II-induced myocardial hypertrophy markers. The decrease in BNP indicates that vanillin can alleviate the hypertrophic response of cardiomyocytes at the molecular level.

[0053] Furthermore, phalloidin staining results showed that Ang II-induced H9c2 cells exhibited typical hypertrophic changes such as increased cell area, disordered cytoskeleton, and irregular morphology. In contrast, vanillin intervention significantly reduced cell area, improved cytoskeleton arrangement, and resulted in cell morphology approaching normal. These results further demonstrate from a cellular morphology perspective that vanillin can improve Ang II-induced cardiomyocyte hypertrophy.

[0054] In summary, this embodiment demonstrates, through cell viability detection, myocardial hypertrophy marker expression detection, and cell morphology observation, that within a safe dosage range, vanillin can significantly reduce Ang II-induced BNP expression, alleviate H9c2 cell enlargement and cytoskeleton disorder, indicating that vanillin has a clear ameliorative effect on Ang II-induced cardiomyocyte hypertrophy. Therefore, vanillin can be used to prepare drugs for the prevention and / or treatment of myocardial hypertrophy, and has potential application value.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, are covered within the scope of protection of the present invention.

Claims

1. The use of vanillin in the preparation of drugs for the prevention and / or treatment of myocardial hypertrophy.

2. The application according to claim 1, characterized in that, The myocardial hypertrophy was induced by Ang II.

3. The application according to claim 1, characterized in that, Vanillin inhibits the expression of BNP protein, a marker of Ang II-induced myocardial hypertrophy.

4. The application according to claim 1, characterized in that, The effective mass percentage concentration of vanillin is 10-50%.

5. The application according to claim 4, characterized in that, The effective mass percentage concentration of vanillin is 50%.

6. A pharmaceutical composition for preventing and treating myocardial hypertrophy, characterized in that, It includes at least vanillin.

7. The pharmaceutical composition according to claim 6, characterized in that, It also includes pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The excipients are one or more of the following: diluent, filler, surfactant, absorption promoter, disintegrant, wetting agent, or dispersant.

9. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the drug is any one of injection, emulsion, tablet, powder, granule, gel, ointment, capsule or oral liquid.

Images