Application of AMPK / mTOR signal channel activator in preparation of medicine for preventing and / or treating heart failure

Abstract

The invention discloses application of an AMPK / mTOR signal channel activator in preparation of a medicine for preventing and / or treating heart failure, and belongs to the technical field of biological medicine. In order to solve the problem that whether the heart function improvement of the body resistance strengthening and heart nourishing formula is related to the influence of AMPK / mTOR pathway regulation on autophagy related protein expression is not clear yet, an HFrEF rat model is constructed, and experiments verify that the body resistance strengthening and heart nourishing formula activates an AMPK / mTOR signaling pathway, down-regulates the p62 expression of autophagy protein, up-regulates the expressions of Becline1, LC3I and LC3II, relieves the pathological damage and fibrosis degree of myocardial tissue, and has the effect of strengthening the heart function. Therefore, the treatment effect of HFrEF is achieved. Experimental results show that the AMPK / mTOR signal channel can be used as a therapeutic target of the HFrEF, and the effect of treating the HFrEF is achieved by activating the AMPK / mTOR signal channel, so that a theoretical basis is provided for further clinical development of HFrEF therapeutic drugs and application of AMPK / mTOR signal channel activators.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of AMPK / mTOR signaling pathway activators in the preparation of drugs for the prevention and / or treatment of heart failure. Background Technology

[0002] Heart failure (HF) is the end-stage manifestation of various cardiovascular diseases. Its core characteristic is the heart's inability to effectively pump blood to meet the body's metabolic needs, leading to insufficient blood supply to all organs and tissues. HF is considered a cardiovascular disease affecting approximately 64 million people worldwide. Its prevalence is projected to increase due to population aging. Heart failure with reduced ejection fraction (HFrEF) is a more severe type. The one-year all-cause mortality rate for patients with heart failure with mid-range ejection fraction (HFmrEF) is 5.5%, while the mortality rate for patients with heart failure with preserved ejection fraction (HFpEF) is 4.7%.

[0003] Autophagy is a cellular degradation process that, through lysosome-mediated degradation of damaged proteins, organelles, and other macromolecules within the cell, maintains cellular homeostasis. Autophagy plays a central role in physiological processes by regulating metabolic homeostasis and maintaining energy balance; its dysfunction may be involved in pathological processes such as cardiovascular disease and is closely related to the pathogenesis of various diseases. Recent studies have revealed a dual role for autophagy in the pathophysiology of heart failure (HF). Under physiological conditions, autophagy maintains cardiomyocyte homeostasis by degrading damaged organelles; however, an imbalance in its activity (overactivation or inhibition) can induce programmed cell death in cardiomyocytes and accelerate the pathological process of cardiac decompensation. The AMPK / mTOR signaling pathway is one of the key molecular mechanisms regulating autophagy. AMPK (AMP-activated protein kinase), as an energy sensor, is activated by phosphorylation under energy stress, negatively regulating the cascade reactions mediated by the mTOR (Mechanistic Target of Rapamycin) signaling axis, ultimately initiating the autophagy program.

[0004] Traditional Chinese medicine (TCM) compound formulas can improve heart failure through multiple pathways, showing promising application prospects. The Fuzheng Yangxin formula can regulate the AMPK signaling pathway in HFrEF rats, reduce mitochondrial damage, and improve cardiac function. However, whether the improvement in cardiac function by the Fuzheng Yangxin formula is related to the regulation of the AMPK / mTOR pathway and its influence on the expression of autophagy-related proteins remains unclear. Summary of the Invention

[0005] To address the aforementioned problems, this invention aims to provide the application of AMPK / mTOR signaling pathway activators in the preparation of drugs for the prevention and / or treatment of heart failure. The AMPK / mTOR signaling pathway activators reduce myocardial tissue pathological damage and fibrosis by downregulating the expression of autophagy protein p62 and upregulating the expression of Becline1, LC3I, and LC3II.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides, in one aspect, the use of AMPK / mTOR signaling pathway activators in the preparation of drugs for the prevention and / or treatment of heart failure.

[0008] Optionally, the AMPK / mTOR signaling pathway activator includes a formula for strengthening the heart and regulating the body.

[0009] Optionally, AMPK / mTOR signaling pathway activators downregulate the expression of autophagy protein p62 while upregulating the expression of Becline1, LC3I, and LC3II, thereby achieving the effect of preventing and / or treating heart failure.

[0010] Optionally, the heart failure is heart failure with reduced ejection fraction.

[0011] On the other hand, the present invention also provides a medicament for treating heart failure, wherein the active ingredient of the medicament includes an AMPK / mTOR signaling pathway activator.

[0012] Optionally, the drug may also include a pharmaceutically acceptable carrier or excipient.

[0013] The beneficial effects of this invention are:

[0014] This invention established a rat model of left anterior descending coronary artery ligation (HFrEF), randomly dividing rats into Sham, Model, FZYX, and ARNI groups, with continuous intervention for 28 days. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) were assessed by echocardiography. Serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) and inflammatory factors TNF-α, IL-1β, and IL-6 were detected by ELISA. Myocardial pathology and fibrosis were observed using HE and Masson staining. Western blot analysis was performed on AMPK / mTOR pathway proteins p-AMPK and p-mTOR, and autophagy markers Beclin1, LC3II, LC3I, and p62. Results showed that, compared with the Model group, FZYX significantly improved cardiac function, reduced left ventricular dilation and NT-proBNP levels, and inhibited the release of inflammatory factors. Pathological findings show that FZYX reduces cardiomyocyte disorder and fibrosis; at the molecular level, FZYX upregulates p-AMPK and inhibits p-mTOR, promoting autophagy activation.

[0015] The above experimental results indicate that Fuzheng Yangxin Formula improves cardiac function in HFrEF rats by regulating myocardial autophagy through activating the AMPK / mTOR signaling pathway. In other words, the AMPK / mTOR signaling pathway can serve as a therapeutic target for heart failure with reduced ejection fraction, and Fuzheng Yangxin Formula can act as an activator of the AMPK / mTOR pathway. By activating the AMPK / mTOR signaling pathway, it can alleviate myocardial cell disorder and fibrosis, and thus be applied to the treatment of heart failure with reduced ejection fraction. This provides a theoretical basis for further clinical development of drugs for the treatment of heart failure with reduced ejection fraction and the application of AMPK / mTOR signaling pathway activators. Attached Figure Description

[0016] Figure 1 HE staining results of the experimental rat heart in this invention;

[0017] Figure 2 The results of Masson staining of the heart of the experimental rats in this invention;

[0018] Figure 3 These are the AMPK / mTOR pathway expression bands in the heart tissues of rats in each group of this invention;

[0019] Figure 4 These are the expression bands of autophagy-related proteins in the heart tissues of rats in each group of this invention. Detailed Implementation

[0020] It should be noted that the following detailed description is exemplary and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention pertains.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0022] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0023] This research is derived from the general project of the National Natural Science Foundation of China (82374280). The specific research process and research results include the following contents.

[0024] 1 Materials and Methods

[0025] 1.1 Experimental Animals and Grouping

[0026] Thirty-six male Sprague-Dawley rats (SPF grade, body weight 180 - 200 g) were selected and purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Laboratory Animal Use License No. SCXK (Beijing) 2021-0006). The experimental animals were divided into four experimental groups by the random number table method: sham operation (Sham, n = 9), model construction (Model, n = 9), Fuzheng Yangxin Formula intervention (FZYX, n = 9), and sacubitril / valsartan treatment (ARNI, n = 9). The experimental animals were fed in the animal house of Xiyuan Hospital, China Academy of Chinese Medical Sciences. The room temperature was 20 - 26 °C, the humidity was 40% - 70%, and the light / dark cycle was 12 hours light / 12 hours dark. This experiment was approved by the Animal Ethics Committee (Ethical No.: 2024XLC011-1).

[0027] 1.2 Drugs, Reagents and Instruments

[0028] The formula for strengthening the body and nourishing the heart was prepared using the standardized preparation process of Xiyuan Hospital, China Academy of Chinese Medical Sciences (comprising eight Chinese herbs: raw ginseng, raw astragalus, angelica, raw rehmannia, lightly sliced ​​aconite, ophiopogon, rhodiola, and notoginseng powder). The median dose was determined based on pharmacodynamic pre-experiments and administered via gavage. The positive control group used ARNI (sacubitril / valsartan sodium, trade name: Noxinto, Novartis Pharmaceuticals, Beijing). The following enzyme-linked immunosorbent assay (ELISA) kits were used for rat N-terminal probrain natriuretic peptide (NT-proBNP), rat interleukin-1β (IL-1β), rat interleukin-6 (IL-6), and rat tumor necrosis factor-α (TNF-α) from Huangshi Ains Biotechnology Co., Ltd. (catalog numbers: INS-30112, INS-30206, INS-30219, and INS-31063, respectively). The p-mTOR, p-AMPK, LC3, P62, Beclin1, GAPDH antibodies, and HRP-goat anti-rabbit antibodies were all from Servicebio (catalog numbers: GB114686, GB114323, GB113801, GB11239-1, GB115741, GB15004, GB23303, respectively). The microplate reader was from BioTek (USA, model SYNERGY44), the electrophoresis apparatus was from Servicebio (model SVE-2), and the protein analysis and expression system was from Servicebio (model AIWBwell™).

[0029] 1.3 Model Establishment

[0030] Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), cannulated, and the ribs were dissected to expose the heart. A myocardial infarction model was induced by ligation of the left anterior descending (LAD) coronary artery; in a sham surgery, the thoracic cavity was opened without arterial ligation. One week after ligation, cardiac function was assessed using echocardiography, and a left ventricular ejection fraction (LVEF) <40% was considered a successful model. All functional measurements were performed and analyzed in unaware researchers in the treatment group. After successful model establishment, the rats were continuously administered the drug for 28 days. Following functional assessment, the rat hearts were dissected for histological analysis.

[0031] 1.4 Observation Indicators and Detection Methods

[0032] Staining: Heart sections were fixed with 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, and then cut into 6 μm thick sections. HE staining: After routine dewaxing and hydration, sections were stained with hematoxylin and eosin (HE), differentiated, dehydrated, cleared with xylene, air-dried, and mounted with neutral resin. Masson staining: Paraffin sections were dewaxed, stained with reagent AF according to the instructions, differentiated, and then mounted with neutral resin.

[0033] ELISA: Serum and tissue samples from each group were used as the research subjects. The NT-proBNP, TNF-α, IL-1β and IL-6 ELISA kits were operated according to the instructions. The optical density (OD) of each well was measured at the specified wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

[0034] Western blot (WB): SDS-PAGE is used to separate proteins into equal amounts. Proteins are transferred to a PVDF membrane using a semi-dry method. After constant current transfer, the membrane is blocked with skim milk powder at room temperature for 1-2 hours, followed by the addition of diluted primary antibodies (p-AMPK, p-mTOR, P62, LC3I, LC3II, Beclin1) and incubation overnight at 4°C. After blocking, diluted secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) is added, and the membrane is incubated on a shaker at room temperature for 1 hour. After rinsing, enhanced chemiluminescence autoradiography is used. The absorbance values ​​of the bands are measured using a densitometer, representing the relative expression level of the target protein.

[0035] 1.5 Statistical Analysis

[0036] Experimental data were processed using SPSS 25.0 statistical software. Quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0037] 2 Experimental Results

[0038] 2.1 Effects of FZYX on Cardiac Pathological Morphology

[0039] The results of HE staining in experimental rats are attached. Figure 1 As shown, attached Figure 1 The results showed that cardiomyocytes in the Sham group were intact and neatly arranged, while those in the Model group were structurally disordered, loosely arranged, and showed compensatory hypertrophy. Furthermore, the Model group exhibited nucleus pyknosis or fragmentation. After FZYX treatment, cell damage was reduced, and the integrity of cell structure and arrangement was preserved. Compared to the ARNI group, while ARNI treatment improved the disordered arrangement of cardiomyocytes, mild nuclear pyknosis remained in some areas; whereas the FZYX group showed cardiomyocyte morphology similar to the Sham group, with regular nuclear morphology and no obvious pyknosis or fragmentation, and a more significant restoration of intercellular uniformity.

[0040] The results of Masson staining in experimental rats are attached. Figure 2 As shown, attached Figure 2The results showed that the Sham group had almost no collagen deposition and the lowest degree of fibrosis, while the Model group had significant collagen fiber deposition in the myocardial infarction area, more severe fibrosis, and cardiomyocyte hypertrophy. FZYX treatment alleviated these myocardial damage and fibrosis. Compared to the ARNI group, ARNI treatment reduced collagen fiber deposition, but focal fibrosis still existed in the periphery of the myocardial infarction; while the FZYX group showed a collagen fiber distribution more similar to the Sham group, a significantly smaller fibrotic area, and a more significant improvement in cardiomyocyte hypertrophy.

[0041] 2.2 Effects of FZYX on Cardiac Function

[0042] The cardiac function of experimental rats was assessed using echocardiography. The results are shown in Table 1 below. Table 1 shows that, compared with the Sham group, the Model group rats exhibited heart failure and altered cardiac structure, with decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and increased left ventricular internal dimension at end-systole (LVIDs) and left ventricular internal dimension at end-diastole (LVIDd), indicating severe damage to cardiac structure and function after myocardial infarction (MI) (p < 0.05). Treatment with medium-dose FZYX showed a significant increase in LVEF and LVFS (p < 0.05). Furthermore, the FZYX group showed a significant decrease in LVIDs and LVIDd (p < 0.05). Compared with ARNI treatment, FZYX, while maintaining the same level of LVEF improvement (48.07% vs 48.10%), not only significantly reduced the variability of LVFS index (standard deviation ±3.80 vs ±12.10), making the improvement of cardiac function more stable, but also reduced the individual differences in LVIDs (standard deviation 0.77 vs 1.65), achieving a more balanced ventricular reverse remodeling.

[0043] Table 1. Echocardiographic results of rats (mean ± standard deviation)

[0044] LVEF (%) LVFS (%) LVIDs(mm) LVIDd(mm) Sham 82.06±8.24# 53.32±10.06# 3.18±0.76# 6.81±0.45 Model 35.58±8.28* 15.65±2.73* 7.81±0.69* 9.42±0.79* FZYX 48.07±6.20#* 22.56±3.80#* 6.94±0.77#* 8.80±1.14*# ARNI 48.10±13.69#* 28.6±12.10 6.23±1.65* 8.62±1.18*

[0045] Note: *P < 0.05 compared to the Sham group; #P < 0.05 compared to the Model group;

[0046] 2.3 Effects of FZYX on NT-proBNP and inflammatory factors

[0047] Serum and tissue samples from each group were used as research subjects. The expression of rat cardiac function biomarkers was analyzed according to the instructions of the NT-proBNP, TNF-α, IL-1β and IL-6 ELISA kits.

[0048] The results of NT-proBNP in rats are shown in Table 2 below. Under physiological conditions, NT-proBNP is mainly located in the cytoplasm of cardiomyocytes, and its abnormally elevated serum levels can serve as a molecular diagnostic basis for HFrEF. Compared with the Sham group, the serum NT-proBNP level in the Model group rats was significantly increased (P < 0.05), and the NT-proBNP index was significantly reduced after treatment with FZYX and ARNI (P < 0.05).

[0049] Table 2. NT-proBNP results in rats (mean ± standard deviation)

[0050] Group NT-proBNP (pg / ml) Sham <![CDATA[226.18±16.83 # ]]> Model 316.27±25.65* FZYX <![CDATA[271.94±25.80 # *]]> ARNI <![CDATA[248.96±11.07 # *]]>

[0051] Note: Compared with the Sham group, *P < 0.05; compared with the Model group, # P < 0.05

[0052] The results of rat inflammatory factors are shown in Table 3 below. Compared with the Sham group, the release of inflammatory factors (TNF-α, IL-1β and IL-6) in the Model group was increased (P < 0.05), indicating the activation of the inflammatory cascade. After intervention with FZYX and ARNI, the levels of TNF-α, IL-1β and IL-6 were observed to be lower than those in the model group (P < 0.05).

[0053] Table 3. Results of rat confirmatory factors (mean ± standard deviation)

[0054] Group TNF-α (pg / ml) IL-1β (pg / ml) IL-6 (pg / ml) Sham 55.01±5.35# 6.88±0.75# 22.37±2.68# Model 89.21±7.29* 9.99±0.93* 33.52±2.51* FZYX 64.73±6.83#* 7.82±0.69*# 26.43±2.04*# ARNI 64.96±5.99#* 7.69±0.56# 22.79±2.26#

[0055] Note: Compared with the Sham group, *P < 0.05; compared with the Model group, #P < 0.05

[0056] 2.4 Effects of FZYX on the expression of AMPK / mTOR signaling pathway-related proteins in rat myocardial tissue

[0057] Western blot analysis was performed on the expression bands of the AMPK / mTOR signaling pathway and autophagy-related proteins in rat myocardial tissue. The results are shown in the attached figures. Figure 3 Table 4 and Appendix Figure 4 As shown in Table 5, among which, appendix Figure 3 The expression bands of the AMPK / mTOR pathway in the heart tissue of rats in each group are shown below. Figure 4 The bands representing the expression of autophagy-related proteins in the heart tissue of rats in each group.

[0058] From the appendix Figure 3 As shown in Table 4 below, compared with the Sham group, the expression of p-AMPK / GAPDH in the myocardium of the Model group rats was decreased (P<0.05), and the expression of p-mTOR / GAPDH was increased (P<0.05). This indicates that the autophagy pathway AMPK / mTOR was inhibited after modeling. After FZYX intervention, compared with the Model group, the expression of p-AMPK / GAPDH in the FZYX group was increased (P<0.05), and the expression of p-mTOR / GAPDH was decreased (P<0.05). The above data indicate that FZYX can effectively promote the activation of the autophagy pathway AMPK / mTOR.

[0059] Table 4. Effects of FZYX on protein expression of p-AMPK / GAPDH and p-mTOR / GAPDH in rat myocardial tissue (mean ± standard deviation, n = 3)

[0060] Group p-AMPK / GAPDH p-mTOR / GAPDH Sham 0.62±0.02# 0.21±0.03# Model 0.24±0.03* 0.79±0.01* FZYX 0.37±0.02#* 0.52±0.02*# ARNI 0.49±0.04#* 0.35±0.02#

[0061] By detecting the expression levels of autophagy-related proteins, it was found (see appendix) Figure 4 As shown in Table 5 below, compared with the Sham group, Beclin1 expression in the myocardial tissue of the Model group was significantly downregulated, while LC3II and LC3I were significantly decreased, and the expression of the autophagy substrate p62 was significantly increased, suggesting that autophagy activity was inhibited in the HFrEF model. In the drug intervention groups, Beclin1 expression in the FZYX group and ARNI group was partially restored compared with the Model group (P<0.05), and LC3II and LC3I were significantly increased, while p62 expression decreased (P<0.05).

[0062] Table 5. Effects of FZYX on the relative expression of autophagy-related proteins Becline1, LC3I, LC3II, and p62 in rat myocardial tissue (mean ± standard deviation, n = 3)

[0063] Group Becline1 LC3I LC3II p62 Sham 0.70±0.08# 1.24±0.15# 1.14±0.15# 0.36±0.04# Model 0.27±0.03* 0.44±0.05* 0.03±0.01* 0.93±0.09* FZYX 0.52±0.04#* 0.96±0.11#* 0.76±0.09#* 0.77±0.03#* ARNI 0.43±0.06#* 0.76±0.10#* 0.47±0.06#* 0.73±0.08#*

[0064] Note: Compared with the Sham group, *P < 0.05; compared with the Model group, #P < 0.05

[0065] In summary, this study administered FZYX to HFrEF rats via gavage for 28 days. The results showed that FZYX could improve cardiac function in rats, mainly by increasing LVEF and LVFS, decreasing LVIDs and LVIDd, significantly reducing the serum cardiac failure marker NT-proBNP level, alleviating myocardial tissue pathological damage and fibrosis, and decreasing inflammatory markers. This was accompanied by a decrease in autophagy protein p62 and an upregulation of Becline1, LC3I, and LC3II.

[0066] Furthermore, this study also showed that, compared with the model group, the myocardial tissue of rats in the sham-operated group exhibited a significant downregulation of p-AMPK protein expression and a significant upward trend of p-mTOR expression, suggesting that myocardial ischemia-induced injury can significantly inhibit the activity of the AMPK / mTOR pathway. After FZYX intervention, compared with the model group, the treatment group showed increased p-AMPK expression and decreased p-mTOR expression, indicating that FZYX can regulate the autophagy level of cardiomyocytes to the physiological range by activating the AMPK / mTOR signaling pathway, thereby maintaining cellular energy metabolism homeostasis and alleviating pathological damage caused by excessive autophagy.

[0067] Therefore, this study shows that FZYX can improve cardiac function in HFrEF rats, and its mechanism may be related to the activation of the AMPK / mTOR signaling pathway-mediated cardiomyocyte autophagy.

[0068] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of AMPK / mTOR signaling pathway activators in the preparation of drugs for the prevention and / or treatment of heart failure.

2. The application according to claim 1, characterized in that: The AMPK / mTOR signaling pathway activator includes the Fuzheng Yangxin formula.

3. The application according to claim 1, characterized in that: AMPK / mTOR signaling pathway activators downregulate the expression of autophagy protein p62 while upregulating the expression of Becline1, LC3I, and LC3II, thereby preventing and / or treating heart failure.

4. The application according to claim 1, characterized in that: The heart failure described is heart failure with reduced ejection fraction.

5. A drug for treating heart failure, characterized in that: The active ingredient of the drug includes an AMPK / mTOR signaling pathway activator.

6. A medicament for treating heart failure according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable carriers or excipients.

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