Application of trigonelline in preparation of medicine for treating myocardial hypertrophy

Abstract

The invention discloses an application of trigonelline in preparation of a medicine for treating myocardial hypertrophy. Angiotensin II is used for inducing an in-vitro H9c2 cell hypertrophy model, and research results show that the trigonelline can remarkably reduce the H9c2 cell hypertrophy area, reduce the total protein content of H9c2 cells and lower the expression level of H9c2 cell hypertrophy gene BNP mRNA. It is found for the first time that trigonelline can effectively relieve myocardial hypertrophy.

Description

Technical Field

[0001] The invention relates to application of trigonelline in preparing a medicine for treating myocardial hypertrophy. Background Art

[0002] Trigonelline (Trig) is a water-soluble pyridine alkaloid that can be metabolized from nicotine in plant cells and undergo N-demethylation in rats to produce nicotinic acid and its parent epoxide. [1] , is the main active ingredient in many traditional Chinese medicines and is also one of the alkaloids contained in Trichosanthes peel injection [2] Pharmacological studies have confirmed that during myocardial cell hypoxia / reoxygenation injury, trigonelline can reduce lipid peroxidation damage, stabilize mitochondrial membrane potential, and inhibit myocardial cell apoptosis. [3] ; In a hyperlipidemia rat model, trigonelline combined with aerobic exercise can improve lipid metabolism by inhibiting PCSK9 expression and upregulating LDLR (low-density lipoprotein receptor-related protein) expression, thereby exerting a lipid-lowering effect [4] In a heart failure mouse model constructed by aortic arch constriction, trigonelline can activate the PI3K / Akt pathway to reduce the accumulation of collagen fibers in the myocardial interstitium and perivascular space, inhibit myocardial cell apoptosis, and thus improve cardiac function [5] .

[0003] In the development of cardiovascular disease, a compensatory change often occurs, namely pathological myocardial hypertrophy, which is an independent risk factor for the occurrence and development of cardiovascular disease. [6,7] Pathological myocardial hypertrophy refers to the adaptive changes of myocardial cells to maintain normal ejection function under the pressure of cardiac preload and postload. It is characterized by an increase in myocardial cell surface area without an increase in cell number, an increase in myocardial cell protein synthesis rate and total protein content, and an upregulation of the expression of B-type (or brain-type) natriuretic peptide associated with hypertrophy. [8,9] Therefore, improving and reversing myocardial hypertrophy is an important part of the treatment and prevention of cardiovascular diseases. However, the use of trigonelline in anti-myocardial hypertrophy has not been reported in the literature.

[0004] References

[0005] [1]PERCHAT N,SAAIDIP L,DARII E,et al.Elucidation of the trigonellinedegradation pathway reveals previously undescribed enzymes and metabolites[J].PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OFAMERICA,2018,115(19):E4358-E67.

[0006] [2]TAO

[0007] [3] Han Kun, Wang Danping, Yuan Lei. Protective effect of trigonelline on hypoxia / reoxygenation neonatal rat cardiomyocytes[J]. Chongqing Medicine, 2015, 44(31): 4333-5.

[0008] [4] Jiang Haibin, Jiang Rui, Yang Lijie. Effect of trigonelline combined with aerobic exercise on lipid-lowering effect and PCSK9 / LDLR pathway in hyperlipidemia rats[J]. Chinese Journal of Clinical Pharmacology, 2024, 40(12): 1794-8.

[0009] [5] Xin Tingting, Li Hongchao, Zang Fucheng, et al. Trigonelline improves myocardial fibrosis and cardiac function in mice with decompensated heart failure by activating the phosphatidylinositol 3-kinase / protein kinase B signaling pathway [J]. Chinese Journal of Hypertension, 2024, 32(05): 470-82.

[0010] [6]MICHEAL AM,DANIEL AS,MALATHI SID,et al.High-ResolutionTranscriptomic Profiling of the Heart During Chronic Stress Reveals CellularDrivers of Cardiac Fibrosis and Hypertrophy[J].Circulation,2020:1448-63.

[0011] [7] THOMAS GM, MIRANDA AJ, LESLIE A L. Regression of cardiachypertrophy in health and disease: mechanisms and therapeutic potential [J]. NATURE REVIEWS CARDIOLOGY, 2023: 347-63.

[0012] [8]NAKAMURA M,SADOSHIMA J.Mechanisms of physiological andpathological cardiac hypertrophy[J].Nature Reviews Cardiology,2018,15(7):387-407.

[0013] [9] Sun Na, Wang Hongxin, Lu Meili. Study on the inhibition of AngⅡ-induced cardiomyocyte hypertrophy in neonatal rats by ginsenoside Rg1 via SIRT1 / PGC-1α signaling pathway[J]. Pharmacology and Clinical Medicine of Traditional Chinese Medicine, 2019, 35(05): 69-73. Summary of the invention

[0014] The technical problem to be solved by the present invention is that the therapeutic effect of trigonelline on myocardial hypertrophy is unclear and the application of trigonelline is limited. A new use of trigonelline in the preparation of a drug for treating myocardial hypertrophy is provided, which not only expands the drug selection for treating myocardial hypertrophy, but also further expands the application scope of trigonelline. The present invention uses an in vitro H9c2 cell hypertrophy model to evaluate the anti-myocardial hypertrophy activity of trigonelline.

[0015] The invention provides an application of trigonelline in preparing a medicine for treating myocardial hypertrophy.

[0016] In the present invention, the CAS number of trigonelline is: 535-83-1; the molecular formula is C7H7NO2; the molecular weight is 137.14; and the chemical structural formula is shown in (1).

[0017]

[0018] In one embodiment of the present invention, the drug forms a pharmaceutical preparation for improving myocardial hypertrophy, and the pharmaceutical preparation comprises the trigonelline and a pharmaceutically acceptable excipient.

[0019] In one embodiment of the present invention, the application is the application of trigonelline as the sole active ingredient in the preparation of a drug for treating myocardial hypertrophy.

[0020] In the present invention, the myocardial hypertrophy may be concentric hypertrophy or eccentric hypertrophy.

[0021] In the present invention, AngⅡ refers to angiotensin Ⅱ, CAS No. 68521-88-0, molecular formula C 52 H 75 N 13 O 14 .

[0022] In the present invention, brain natriuretic peptide (BNP) is also called B-type natriuretic peptide (B-type Natriuretic Peptide).

[0023] In the present invention, the myocardial hypertrophy may be angiotensin II-induced myocardial hypertrophy.

[0024] In the present invention, the myocardial hypertrophy may be myocardial hypertrophy induced by hypertension, congenital heart disease, or aortic valve stenosis.

[0025] In the present invention, the cardiac hypertrophy may show a cardiac hypertrophy phenotype.

[0026] In the present invention, the myocardial hypertrophy may be myocardial hypertrophy caused by activation of atrial natriuretic peptide (ANF) and / or brain natriuretic peptide (BNP) genes.

[0027] In one embodiment of the present invention, the trigonelline is used to alleviate AngII-induced myocardial hypertrophy, including reducing the weight of the heart and / or reducing the size of myocardial cells.

[0028] In one embodiment of the present invention, the trigonelline is used to down-regulate the RNA expression of BNP, a marker of myocardial hypertrophy induced by AngII.

[0029] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0030] The reagents and raw materials used in the present invention are commercially available.

[0031] The positive and progressive effects of the present invention are:

[0032] The present invention uses AngⅡ to induce an in vitro H9c2 cell hypertrophy model. The research results show that trigonelline can significantly reduce the hypertrophy area of ​​H9c2 cells, reduce the total protein content of H9c2 cells, and downregulate the expression level of H9c2 cell hypertrophy gene BNP mRNA. The present invention first discovered that trigonelline can effectively alleviate myocardial hypertrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The effects of trigonelline and arginine on the hypertrophy of H9c2 cells; drug concentrations: AngⅡ, 1μM; Trig, 100μmol / L; Argi, 100μmol / L; XST, 10 -3 μM; F-actin and cell nucleus fluorescence staining.

[0034] Figure 2 The effects of trigonelline and arginine on the hypertrophy of H9c2 cells; drug concentrations: AngⅡ, 1μM; Trig, 10μmol / L; Argi, 10μmol / L; XST, 10 -3 μM; F-actin and cell nucleus fluorescence staining.

[0035] Figure 3 The effects of trigonelline and arginine on the hypertrophic area of ​​H9c2 cells (n=3); drug concentrations: AngⅡ, 1μM; Trig, 100μmol / L, 10μmol / L; Argi, 100μmol / L, 10μmol / L; XST, 10 -3 μM; ###P<0.001vsControl, ***P<0.001vs AngⅡ.

[0036] Figure 4 Effects of trigonelline and arginine on total protein content in H9c2 cells (n=6); drug concentrations: AngⅡ, 1μM; Trig, 100μmol / L, 10μmol / L; Argi, 100μmol / L, 10μmol / L; XST, 10 -3 μM; ##P<0.01vsControl, **P<0.01vs AngⅡ.

[0037] Figure 5The expression of BNP mRNA in each group during H9c2 cell hypertrophy (n=3); drug concentrations: AngⅡ, 1μM; Trig, 100μmol / L, 10μmol / L; Argi, 100μmol / L, 10μmol / L; XST, 10 -3 μM; ##P<0.01vs Control, ***P<0.001vs AngⅡ, **P<0.01vs AngⅡ. DETAILED DESCRIPTION

[0038] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0039] 1. Experimental Materials

[0040] Table 1

[0041]

[0042]

[0043] Trigonelline: CAS number is 535-83-1; molecular formula is C7H7NO2; molecular weight is 137.14; chemical structure is:

[0044] Arginine: 2-amino-5-guanidine valeric acid, molecular formula is C6H 14 N4O2, molecular weight 174.20, chemical structure formula:

[0045] (II) Experimental methods

[0046] 1. Cell Culture

[0047] The frozen rat cardiomyocyte cell line H9c2 was revived and cultured in DMEM containing 10% fetal bovine serum and 1% double antibody in a 37°C, 5% CO2 saturated humidity incubator, and the cells in the logarithmic growth phase were used for the experiment.

[0048] 2. Cell Grouping and Drug Administration

[0049] H9c2 cells in the logarithmic growth phase were taken and prepared into a cell suspension with complete culture medium. 5×10 4Cells were inoculated in 96-well plates or 6-well plates and cultured in a 5% CO2, 37°C incubator for 24 hours. Then, they were randomly divided into a control group (Control), a model group (AngⅡ), a trigonelline group (Trig), an arginine group (Argi), and a positive control group (valsartan, XST). The culture medium was changed according to the grouping and the cells were cultured for 24 hours. The control group was cultured in complete culture medium; the model group was cultured in complete culture medium containing 1μmol / L AngⅡ; the drug group was cultured in complete culture medium containing 1μmol / L AngⅡ+10μmol / L trigonelline, 1μmol / L AngⅡ+10μmol / L arginine, 1μmol / L AngⅡ+100μmol / L trigonelline, or 1μmol / L AngⅡ+100μmol / L arginine; the positive control group was cultured in complete culture medium containing 1μmol / L AngⅡ and 10 -3 The cells were cultured in complete culture medium containing μmol / L valsartan.

[0050] 3. Detection of H9c2 cell hypertrophy index

[0051] 3.1 Determination of H9c2 cell surface area

[0052] H9c2 cardiomyocytes were stained with rhodamine-labeled phalloidin. H9c2 cells were rinsed three times with PBS and then fixed with 4% paraformaldehyde for 15 minutes. The cells were permeabilized with 0.5% Triton X-100 for 15 minutes. After washing three times with PBS, diluted phalloidin solution was added and the cells were incubated at room temperature for 30 minutes. The cells were then washed three times with PBS again and incubated with DAPI for 5 minutes in the dark. Finally, the cells were washed three times with PBS and images were collected using a high-content cell analyzer under a 20x objective. The cell surface area was calculated according to the method in the reference (CHU CH, TZANG BS, CHEN LM, et al. IGF-II / mannose-6-phosphate receptor signaling induced cell hypertrophy and atrial natriuretic peptide / BNP expression via Galphaq interaction and protein kinase C-alpha / CaMKIIactivation in H9c2 cardiomyoblast cells[J]. J Endocrinol, 2008, 197(2): 381-90.). Four observation fields were selected for each well, and 60 cardiomyocytes were randomly selected for quantitative analysis using Image J software.

[0053] 3.2 Determination of total protein content in H9c2 cells

[0054] (1) Preparation of working fluid

[0055] According to the number of standards and samples, add BCA reagent and Cu reagent (50:1) to prepare BCA working solution and mix thoroughly.

[0056] (2) Dilution of standard

[0057] Take 10 μL of BSA standard and dilute it to 100 μL with PBS to make the final concentration 0.5 mg / mL. Take out 0, 2, 4, 6, 8, 12, 16, and 20 μL respectively and add them to the protein standard wells of the 96-well plate, and then add PBS to make up to 20 μL.

[0058] (3) Extraction and content determination of total protein from H9c2 cells

[0059] The cells cultured for 24 hours were taken out of the incubator, and 70 μL of 0.1% Triton X-100 was added to each well for lysis for 1 hour to obtain lysis solution, and 20 μL of lysis solution was added to the sample wells of the 96-well plate. 200 μL of pre-mixed BCA working solution was added to the sample wells and protein standard wells, and placed at 37°C for 20 minutes. The OD value of each group was measured at 562nm using an ELISA reader, and the protein concentration of each group was calculated based on the standard curve.

[0060] 3.3 Determination of the expression level of mast gene BNP in H9c2 cells

[0061] After the group culture was completed, the cells of each group were collected, washed, and the total RNA of each group of cells was extracted using a cell / tissue total RNA extraction kit, the RNA concentration and purity were determined, and the cDNA was synthesized by a reverse transcription kit, PCR amplification was performed, and GAPDH was used as an internal reference. The relative expression level of BNP mRNA in each group was calculated according to the formula BNP mRNA / internal reference according to the kit operation method, and the relative expression level of BNP mRNA in the control group was calculated as 1.00. The primers are shown in Table 2 below.

[0062] Table 2

[0063]

[0064] 4. Statistical analysis

[0065] SPSS25.0 statistical analysis software was used for data processing. The results were expressed as mean ± standard deviation (x ± SD). One-way analysis of variance was used among the groups. The differences were statistically significant when P < 0.05, ###P < 0.001 vs Control, ##P < 0.01 vs Control, #P < 0.05 vs Control, ***P < 0.001 vs AngⅡ, **P < 0.01 vs AngⅡ, *P < 0.05 vs AngⅡ.

[0066] (III) Experimental results

[0067] 1. Results of H9c2 cell surface area determination

[0068] After 24 h of treatment with AngⅡ, trigonelline and arginine, the area of ​​H9c2 cells in the AngⅡ group was significantly increased compared with that in the blank control group, and the difference was statistically significant, indicating that the AngⅡ-induced H9c2 cell hypertrophy model was successfully established; administration of trigonelline (Trig, 100 μmol / L, 10 μmol / L) and valsartan (XST, 10 -3 After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the area of ​​H9c2 cells was significantly reduced compared with that of AngⅡ group. After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the area of ​​H9c2 cells was not significantly changed compared with that of AngⅡ group (see Figure 1 , Figure 2 , Figure 3 and Table 3), indicating that trigonelline had a significant inhibitory effect on AngⅡ-induced H9c2 cell hypertrophy, while arginine had no significant inhibitory effect on H9c2 cell hypertrophy.

[0069] Table 3 Surface area of ​​H9c2 cells in each group

[0070]

[0071] 2. Results of total protein content determination of H9c2 cells

[0072] After 24 h of treatment with AngⅡ, trigonelline and arginine, the total protein content of H9c2 cells in the AngⅡ group was significantly increased compared with that in the blank control group, and the difference was statistically significant, indicating that the AngⅡ-induced H9c2 cell hypertrophy model was successfully established; administration of trigonelline (Trig, 100 μmol / L, 10 μmol / L) and valsartan (XST, 10 -3 After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the total protein content of H9c2 cells was significantly reduced compared with the AngⅡ group. After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the total protein content of H9c2 cells did not change significantly compared with the AngⅡ group (see Table 4 and Figure 4), indicating that trigonelline had a significant inhibitory effect on AngⅡ-induced H9c2 cell hypertrophy, while arginine had no significant inhibitory effect on the total protein content of H9c2 cells.

[0073] Table 4 Total protein content of H9c2 cells in each group

[0074]

[0075]

[0076] 3. Results of the determination of the expression level of mast gene BNP in H9c2 cells

[0077] After 24 h of treatment with AngⅡ, trigonelline and arginine, the expression level of BNP in H9c2 cells in the AngⅡ group was significantly upregulated compared with that in the blank control group, and the difference was statistically significant, indicating that the AngⅡ-induced H9c2 cell hypertrophy model was successfully established; administration of trigonelline (Trig, 100 μmol / L, 10 μmol / L) and valsartan (XST, 10 -3 After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the expression level of BNP in H9c2 cells was significantly downregulated compared with that in the AngⅡ group. After administration of arginine (Argi, 100 μmol / L, 10 μmol / L), the expression level of BNP in H9c2 cells did not change significantly compared with that in the AngⅡ group (see Table 5 and Figure 5 ), indicating that trigonelline had a significant inhibitory effect on AngⅡ-induced H9c2 cell hypertrophy, while arginine had no significant inhibitory effect on the expression level of BNP in H9c2 cells.

[0078] Table 5 BNP mRNA expression levels of H9c2 cells in each group

[0079]

[0080] 4. Conclusion

[0081] The present invention proves for the first time that trigonelline has a significant inhibitory effect on myocardial cell hypertrophy. Although literature reports that trigonelline can reduce lipid peroxidation damage by inhibiting reactive oxygen species (ROS), inhibiting the production of ROS does not mean that myocardial hypertrophy can be inhibited. For example, literature studies have shown (WANG Z, YANG N, HOU Y, et al. L-Arginine-Loaded Gold Nanocages Ameliorate Myocardial Ischemia / Reperfusion Injury by Promoting Nitric Oxide Production and Maintaining Mitochondrial Function [J]. Advanced Science, 2023, 10 (26): 2302123.) that arginine can significantly protect H9c2 cell toxicity and apoptosis induced by oxygen-glucose deprivation / reoxidation (OGD / R) by blocking the generation and release of ROS, improving mitochondrial membrane potential, and restoring ATP synthase activity. However, the present invention proves that arginine has a significant protective effect on AngⅡ-induced H9c2 cell hypertrophy area, total protein content and hypertrophy gene BNP. There was no significant difference in the expression of mRNA (P>0.05).

Claims

1. A use of trigonelline in the preparation of a drug for treating myocardial hypertrophy.

2. The use according to claim 1, characterized in that The myocardial hypertrophy is angiotensin II-induced myocardial hypertrophy.

3. The use according to claim 1, characterized in that The myocardial hypertrophy is myocardial hypertrophy induced by hypertension, congenital heart disease, and aortic valve stenosis.

4. The use according to claim 1, characterized in that The myocardial hypertrophy is caused by activation of atrial natriuretic peptide and / or brain natriuretic peptide genes.

5. The use according to claim 1, characterized in that The trigonelline is used for alleviating the cardiac hypertrophy induced by angiotensin II, including reducing the weight of the heart and / or the size of the cardiac muscle cells.

6. The use according to claim 1, characterized in that The trigonelline is used for down-regulating the RNA expression of BNP, a marker of myocardial hypertrophy induced by angiotensin II.

7. The use according to claim 1, characterized in that The drug forms a drug preparation for improving cardiac hypertrophy, and the drug preparation comprises the trigonelline and a pharmaceutically acceptable auxiliary material.

8. The use according to claim 1, characterized in that The application is the application of the trigonelline as the sole active ingredient in the preparation of a drug for treating myocardial hypertrophy.

Images