Medical use of AMPKγ2 protein in preventing or treating heart failure after myocardial infarction

Abstract

This invention discloses the medical use of AMPKγ2 protein for preventing or treating heart failure (HF) after myocardial infarction (MI), belonging to the field of pharmaceutical biotechnology. The expression level of AMPKγ2 protein or its active fragment is correlated with HF caused by abnormal cardiac remodeling due to excessive inflammatory response after MI. MI mice with systemic AMPKγ2 knockout significantly decreased left ventricular systolic function and significantly increased macrophage infiltration; whereas MI mice with systemic AMPKγ2 overexpression showed reduced cardiac dysfunction due to MI and macrophage infiltration. Furthermore, macrophage-specific knockout of AMPKγ2 exacerbated cardiac dysfunction due to MI and macrophage infiltration; conversely, macrophage-specific overexpression of AMPKγ2 alleviated cardiac dysfunction due to MI and macrophage infiltration. This invention provides a new target for the diagnosis and treatment of HF after MI and has significant clinical significance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and specifically relates to the medical use of the regulatory subunit AMPKγ2 in AMP-activated kinase (AMPK), and more specifically to the medical use of AMPKγ2 protein for preventing or treating heart failure after myocardial infarction. Background Art

[0002] Cardiovascular disease is a major health burden and remains the most common cause of death worldwide. The Global Burden of Disease (GBD) study estimates that global cardiovascular disease deaths increased by 21.1% between 2007 and 2017, and cardiovascular disease causes over 3.8 million deaths annually in Europe. The "China Cardiovascular Health and Disease Research Report 2021" also indicates that the incidence and prevalence of cardiovascular disease in my country continue to rise, becoming the leading cause of death among residents, with coronary heart disease accounting for a significant proportion. Acute myocardial infarction (AMI) is the most severe form of coronary heart disease, and the mortality rate from AMI has generally increased from 2002 to 2018. Although percutaneous coronary intervention and coronary artery bypass grafting can benefit patients with MI, multiple clinical trials have shown that heart failure (HF) after myocardial infarction (MI) remains an inevitable and difficult problem for several years. Most deaths occurring within approximately six years after MI are related to severe HF. HF is a common condition after MI, and its incidence increases sharply with age. Therefore, effective protection of the myocardium after MI is a necessary condition for preventing the occurrence of HF after MI and improving the prognosis of MI patients. Studies have shown that energy metabolism disorders, activation of pro-inflammatory pathways and changes in the extracellular matrix can lead to myocardial fibrosis, which extend the left ventricular remodeling after MI. "Adverse" or "pathological" remodeling after MI increases the risk of HF and significantly reduces patient survival, but its key regulatory mechanism has not yet been elucidated, and effective targeted therapeutic drugs have not yet been discovered. Therefore, clarifying the key mechanisms of heart failure after MI and finding new preventive and therapeutic targets are of great clinical and social significance.

[0003] MI triggers a robust inflammatory response that is crucial for cardiac repair. The extent of post-infarction remodeling depends on infarct size and the quality of cardiac repair. Inflammatory pathways contribute to the expansion and fibrotic remodeling of the infarcted heart, driving key events in the pathogenesis of post-infarction heart failure. After acute myocardial infarction (AMI), monocytes produced in the bone marrow and spleen enter the bloodstream and are recruited to the damaged cardiac tissue. Infiltrating monocytes then differentiate into M1 macrophages, responsible for clearing cellular debris from damaged tissue. Secretion of cytokines, chemokines, and growth factors in turn regulates M2 macrophages for tissue repair. Monocytes and macrophages are pleiotropic cells of the innate immune system and are essential for both the initial inflammatory response to injury and subsequent wound healing in the heart. However, excessive inflammatory responses can lead to myocardial necrosis, and how to regulate and target macrophage migration to inhibit myocardial inflammation remains under investigation. Monocytes and macrophages in the heart after AMI exhibit significant heterogeneity and plasticity. Therefore, elucidating the molecular mechanisms of monocyte-macrophage phenotypic transformation in ventricular remodeling and conducting targeted therapeutic interventions may be an important research direction for improving post-MI outcomes.

[0004] AMP-activated protein kinase (AMPK) is a conserved serine / threonine kinase and a key cellular energy sensor regulating bioenergetic metabolism. AMPK is present in nearly all eukaryotic organisms and exerts anti-inflammatory and antioxidant activities. AMPK exists as a heterotrimeric complex consisting of a catalytic α subunit and regulatory β and γ subunits. Each subunit has multiple isoforms (two α, two β, and three γ), with the α2, β2, γ1, and γ2 subunits predominantly expressed in the heart. AMPK has been widely reported to possess cardioprotective effects in various cardiovascular diseases. For example, AMPK phosphorylates ACE2 at Ser680 in pulmonary endothelial cells, increasing ACE2 expression in the endothelium and protecting against pulmonary hypertension. AMPK activation improves insulin sensitivity by inhibiting lipogenesis (ACC1, SREBP1c), protein synthesis (mTORC1), and lipolysis (HSL), and activating FAO (ACC2), potentially benefiting the prevention and treatment of patients with type 2 diabetes. AMPK also protects against myocardial infarction and cardiac aging through both metabolic and non-metabolic pathways. The AMPKγ subunit plays a crucial role in AMPK activation. When AMP binds to the γ subunit, it can allosterically activate the complex, making it a more susceptible substrate for phosphorylation at threonine 172. This makes it more susceptible to phosphorylation by the primary upstream AMPK kinase in the activation loop of the α subunit. The γ subunit facilitates allosteric AMPK activation and the expression of downstream protective genes. Furthermore, studies have shown that AMPKγ2, through nuclear translocation, inhibits ribosome biogenesis, thereby protecting against myocardial ischemia / reperfusion (I / R) injury. Mutations in AMPKγ2 have been associated with the development of several diseases, including cardiomyopathy and Wolff-Parkinson-White syndrome. However, whether AMPKγ2 is involved in the development and progression of myocardial ischemia (MI) remains unclear. Summary of the Invention

[0005] To address the above issues, the present invention provides the medical use of AMPKγ2 protein for the prevention or treatment of post-MI heart failure (HF). Through extensive experiments, the inventors discovered that after MI in C57BL / 6 mice, expression of the inflammatory factor INF-γ increased, while both mRNA and protein expression of the PRKAG2 gene decreased significantly. In vitro cell experiments simulating inflammatory stimulation also revealed a significant decrease in both mRNA and protein expression of the PRKAG2 gene. Systemic knockout of AMPKγ2 in MI mice significantly decreased left ventricular systolic function and increased macrophage infiltration. However, systemic overexpression of AMPKγ2 in MI mice reduced cardiac dysfunction associated with MI and macrophage infiltration. Furthermore, macrophage-specific knockout of AMPKγ2 exacerbated cardiac dysfunction associated with MI and macrophage infiltration; conversely, macrophage-specific overexpression of AMPKγ2 alleviated cardiac dysfunction associated with MI and macrophage infiltration. Cellular studies revealed that si-AMPKγ2 exacerbated macrophage migration and increased expression of inflammatory-related proteins in response to INF-γ stimulation, while overexpression of AMPKγ2 reduced macrophage migration and expression of inflammatory-related proteins. These results suggest that AMPKγ2 protein can be used to prevent or treat HF caused by abnormal cardiac remodeling due to excessive inflammatory response after MI.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions.

[0007] The present invention provides use of a preparation specifically overexpressing AMPKγ2 protein or its active fragment in preparing a drug for preventing and / or treating heart failure caused by abnormal cardiac remodeling resulting from excessive inflammatory response after myocardial infarction.

[0008] Furthermore, the preparation specifically overexpressing AMPKγ2 protein or its active fragment includes:

[0009] (1) AMPKγ2 protein, or a modified AMPKγ2 protein derivative, or an AMPKγ2 protein analog;

[0010] (2) a polynucleotide encoding an AMPKγ2 protein;

[0011] (3) an expression construct comprising the AMPKγ2 protein described in (1) or the polynucleotide described in (2);

[0012] (4) An agonist of the AMPKγ2 protein described in (1);

[0013] (5) Nucleic acid molecules of AMPKγ2 protein or recombinant vectors or recombinant cells of its nucleic acid molecules;

[0014] (6) Reagents that can upregulate the expression of AMPKγ2 protein or its activity.

[0015] The present invention also provides the use of a reagent for detecting the expression level of AMPKγ2 protein or its active fragment in preparing a product, characterized in that the product is used for predicting excessive inflammatory response after MI and / or evaluating the therapeutic effect and prognosis.

[0016] Furthermore, AMPKγ2 protein or its active fragment is used as a marker for predicting excessive inflammatory response after MI and / or evaluating treatment effect and prognosis.

[0017] Furthermore, the product can predict myocardial injury and inflammatory response after MI, or assess its therapeutic efficacy or prognosis, by detecting that the expression level of AMPKγ2 protein or its active fragment in blood, tissue, or cells is lower than a reference value. Furthermore, the product is a chip, preparation, or kit.

[0018] The present invention also provides a pharmaceutical composition comprising a preparation for specifically overexpressing AMPKγ2 protein or an active fragment thereof and an optional pharmaceutically acceptable carrier or excipient.

[0019] Furthermore, the preparation specifically overexpressing AMPKγ2 protein or its active fragment includes:

[0020] (1) AMPKγ2 protein, or a modified AMPKγ2 protein derivative, or an AMPKγ2 protein analog;

[0021] (2) a polynucleotide encoding an AMPKγ2 protein;

[0022] (3) an expression construct comprising the AMPKγ2 protein described in (1) or the polynucleotide described in (2);

[0023] (4) An agonist of the AMPKγ2 protein described in (1);

[0024] (5) Nucleic acid molecules of AMPKγ2 protein or recombinant vectors or recombinant cells of its nucleic acid molecules;

[0025] (6) Reagents that can upregulate the expression of AMPKγ2 protein or its activity.

[0026] Furthermore, any of the above pharmaceutical compositions is used for preventing and / or treating HF caused by abnormal cardiac remodeling due to excessive inflammatory response after MI.

[0027] The present invention also provides the use of a preparation specifically over-expressing AMPKγ2 protein or an active fragment thereof in the preparation of an anti-inflammatory drug.

[0028] In the present invention, the active fragment of AMPKγ2 protein refers to a fragment having the function of AMPKγ2 protein, which can be a part of AMPKγ2 protein or a fragment obtained by deleting, adding or replacing the amino acid sequence of AMPKγ2 protein. Those skilled in the art can avoid sites that may affect the activity as needed and perform deletion, addition or replacement on other sites so that the modified AMPKγ2 protein still has the activity or function of AMPKγ2 protein.

[0029] In the present invention, the post-MI myocardial injury has a meaning well known in the art, and refers to left ventricular dysfunction occurring after MI.

[0030] In the present invention, the prevention and / or treatment of excessive inflammatory response after MI refers to inhibiting or slowing down the infiltration of macrophages and the increase of inflammatory factors.

[0031] In the present invention, the detection of the expression level of AMPKγ2 protein or its active fragment for prediction and / or evaluation means that when the expression level of AMPKγ2 protein or its active fragment in blood, tissue or cells is lower than the reference value, myocardial damage and inflammatory response after MI can be predicted, or its treatment effect or prognosis can be evaluated.

[0032] In the present invention, the expression level of AMPKγ2 protein or its active fragment can be detected by methods known in the art, such as amplifying PRKAG2 mRNA by polymerase chain reaction and performing a quantitative reaction, or detecting the expression level of AMPKγ2 protein by Western Blot.

[0033] In the present invention, the expression level of the protein refers to the level of mRNA or the level of protein.

[0034] In the present invention, upregulating / downregulating protein expression in tissues / cells refers to increasing or decreasing the protein level or mRNA level in tissues / cells by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by more than 100%, wherein the upregulation or downregulation is compared with untreated tissues or cells.

[0035] In the present invention, the reagent capable of inhibiting the down-regulation of the expression of AMPKγ2 protein or its active fragment or promoting the up-regulation of the expression of AMPKγ2 protein or its active fragment is well known in the art.

[0036] Compared with the prior art, the present invention has the following beneficial effects.

[0037] The expression level of AMPKγ2 protein or its active fragment is associated with abnormal cardiac remodeling caused by excessive inflammatory response after MI, leading to heart failure (HF). Systemic AMPKγ2 knockout in MI mice significantly reduced left ventricular systolic function and increased macrophage infiltration. However, systemic overexpression of AMPKγ2 in MI mice reduced cardiac dysfunction associated with MI and macrophage infiltration. Furthermore, macrophage-specific knockout of AMPKγ2 exacerbated cardiac dysfunction associated with MI and macrophage infiltration; conversely, macrophage-specific overexpression of AMPKγ2 alleviated cardiac dysfunction associated with MI and macrophage infiltration. This study provides a new target for the diagnosis and treatment of HF after MI and has significant clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 After the C57BL / 6 mouse MI model was established, IFN-γ expression was upregulated and AMPKγ2 expression was downregulated during the early inflammatory activation stage. (A) ELISA was used to detect serum IFN-γ levels in MI mice (n=5, ** p<0.01, compared with the sham group); (B) ELISA method was used to detect the level of IFN-γ in the spleen of MI mice (n=5, * p<0.05, ** p<0.01, compared with the sham group); (CD) Western blot detection of AMPKγ2 protein expression in bone marrow-derived macrophages (BMDM) of MI mice at different time points (n=3, ** p<0.01, compared with the sham group); (E) Fluorescence quantitative PCR detection of PRKAG2 gene mRNA expression in BMDM of MI mice (n=3, * p < 0.05, compared with the control group).

[0039] Figure 2 In vitro simulated inflammatory stimulation decreased AMPKγ2 expression. (AB) are Western blots detecting the expression of representative inflammatory cytokines, including IL-6, MCP1, and AMPKγ2, in macrophages (RAW264.7) stimulated by IFN-γ (n=3, ** p<0.01, compared with the control group); (C) Fluorescence quantitative PCR detection of the mRNA expression of representative inflammatory factors proteins IL-6, MCP1 and PRKAG2 in macrophages (RAW264.7) from BMDM of MI mice under IFN-γ stimulation (n=3, ** p<0.01, *** P<0.001, compared with the control group); (DE) Western blot analysis of AMPKγ2 protein expression in BMDM of C57BL / 6 mice stimulated with different concentrations of IFN-γ (n=3,* p<0.05, compared with the control group); (FI) Western blot detection of AMPKγ2 protein expression in BMDM of C57BL / 6 mice stimulated by different concentrations of TNF-α and LPS and CoCl2 (n=3, * p<0.05, ** p < 0.01, compared with the control group).

[0040] Figure 3 AMPKγ2 knockout mice (AMPKγ2-KO) show aggravated myocardial insufficiency and macrophage infiltration. (A) is the left ventricular systolic function (EF%) assessed by small animal ultrasound (n=11, 10, 9, 9, * p<0.05, ** p < 0.01, compared with the control group; # p<0.05, compared with MI group); (B) FS% of left ventricular systolic function evaluated by ultrasound in small animals (n=11, 10, 8, 8, * p<0.05, ** p < 0.01, compared with sham; # p<0.05, compared with the MI group); (C) Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect myocardial cell hypertrophy (n=11, 10, 9, 9); (D) CD68 immunohistochemical staining was used to detect inflammatory cell infiltration (n=11, 10, 8, 8).

[0041] Figure 4 Systemic overexpression of AMPKγ2 in MI mice (AAV-AMPKγ2 MI) alleviates cardiac dysfunction caused by MI and macrophage infiltration. (A) is the left ventricular systolic function (EF%) assessed by small animal ultrasound (n=5, # p<0.05, compared with the MI control group); (B) FS% of left ventricular systolic function evaluated by ultrasound in small animals (n=5, # p<0.05, compared with the MI control group); (C) Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect myocardial cell hypertrophy (n=5); (D) CD68 immunohistochemical staining was used to detect inflammatory cell infiltration (n=5).

[0042] Figure 5 Lyz2-AMPKγ2-CKO mice with macrophage-specific deletion of AMPKγ2 (Lyz2-AMPKγ2-CKO) exhibit exacerbated cardiac dysfunction associated with MI and macrophage infiltration. (A) Survival analysis of Lyz2-AMPKγ2-CKO and flox / flox MI mice; (B) Left ventricular systolic function (EF%) assessed by small animal ultrasound (n=3). #p<0.05, compared with flox / flox); (C) FS% of left ventricular systolic function evaluated by small animal ultrasound (n=3, # p<0.05, compared with flox / flox); (DE) F4 / 80 and CD11b co-staining, flow cytometry evaluation of macrophage infiltration in Lyz2-AMPKγ2-CKO and flox / flox MI mice ( # p<0.05, compared with flox / flox); (F) CD68 staining to evaluate macrophage infiltration in Lyz2-AMPKγ2-CKO and flox / flox MI mice; (G) HE staining to detect cardiomyocyte morphology, Masson staining to detect myocardial fibrosis, and WGA staining to detect cardiomyocyte hypertrophy in Lyz2-AMPKγ2-CKO and flox / flox MI mice (n=3).

[0043] Figure 6 Macrophage-specific overexpression of AMPKγ2 in MI mice (AAV-F4 / 80-GFP-AMPKγ2) alleviates cardiac dysfunction associated with MI and macrophage infiltration. (A) Left ventricular systolic function (EF%) assessed by small animal ultrasound (n=5, * p<0.05, compared with AAV-F4 / 80-GFP); (B) FS% of left ventricular systolic function evaluated by small animal ultrasound (n=5, * p<0.05, compared with AAV-F4 / 80-GFP); (CD) F4 / 80 and CD11b co-staining, flow cytometry evaluation of macrophage infiltration in AAV-F4 / 80-GFP-AMPKγ2 and AAV-F4 / 80-GFP MI mice ( # p<0.05, compared with AAV-F4 / 80-GFP); (E) CD68 staining to evaluate macrophage infiltration in AAV-F4 / 80-GFP-AMPKγ2 and AAV-F4 / 80-GFP MI mice (n=5); (F) CD68 staining to evaluate macrophage infiltration in AAV-F4 / 80-GFP-AMPKγ2 and AAV-F4 / 80

[0044] -GFP MI mice, myocardial fibrosis was detected by Masson staining, and cardiomyocyte hypertrophy was detected by WGA staining (n=5).

[0045] Figure 7 AMPKγ2 knockout can aggravate IFN-γ-induced macrophage migration and inflammation. (AB) are transwell experiments to study si-AMPKγ2-regulated macrophage migration (n=4, ** P < 0.01, compared with si-NC; #P<0.05, compared with IFN-γ+si-NC); (CD) Scratch assay to study the macrophage migration regulated by si-AMPKγ2 (n=3, ** P < 0.01, compared with si-NC; # P<0.05, compared with IFN-γ+si-NC); (EF) Western Blot analysis of the expression of inflammatory factors in RAW264.7si-AMPKγ2 (n=3, * P<0.05, ** p < 0.01, compared with si-NC; # P<0.05, ## P<0.01 compared with IFN-γ+si-NC); (G) Fluorescence quantitative PCR detection of the mRNA expression of IL-6 and MCP1, representative proteins of inflammatory factors in RAW264.7si-AMPKγ2 (n=3, * P < 0.05, compared with si-NC; # P < 0.05, compared with IFN-γ + si-NC).

[0046] Figure 8 AMPKγ2 overexpression can reduce IFN-γ-induced macrophage migration and inflammation. (AB) are transwell experiments to study pc3.1-AMPKγ2-regulated macrophage migration (n=4, ** P < 0.01, compared with pc3.1-con; ## P<0.01, compared with IFN-γ+pc3.1-con); (CD) Scratch assay to study the macrophage migration regulated by pc3.1-AMPKγ2 (n=3, ** P < 0.01, compared with pc3.1-con; ## P<0.01, compared with IFN-γ+pc3.1-con); (EF) Western Blot analysis of the expression of inflammatory factors in RAW264.7 pc3.1-AMPKγ2 (n=3, * P<0.05, ** p < 0.01, compared with pc3.1-con; # P<0.05, ## P<0.01 compared with IFN-γ+pc3.1-con); (G) Fluorescence quantitative PCR detection of the mRNA expression of IL-6 and MCP1, representative proteins of inflammatory factors in RAW264.7 pc3.1-AMPKγ2 (n=3, ** p < 0.01, compared with pc3.1-con; ##P < 0.01, compared with IFN-γ + pc3.1-con). DETAILED DESCRIPTION

[0047] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0048] The experimental data of the present invention are all percentages. The chi-square test was used to compare the two sample rates, and the statistical analysis was performed using the SPSS 22.0 software package. P < 0.05 was considered statistically significant.

[0049] Example 1: After the C57BL / 6 mouse MI model was established, in the early inflammatory activation stage, IFN-γ expression was upregulated and AMPKγ2 expression was downregulated.

[0050] ①Establishment of MI model in C57BL / 6J mice.

[0051] Turn on the anesthesia machine and place the mouse under gas anesthesia. After the mouse loses consciousness, place it on the operating table, secure its limbs with tape, and connect its nose to the anesthesia mask (isoflurane to oxygen ratio of 1:5) to maintain continuous anesthesia. After routine alcohol disinfection, cut the skin at the 3rd and 4th ribs, separate the pectoralis major muscles, and squeeze the ribs on both sides to expose the heart. The coronary artery is visible 1-2 mm below the inferior edge of the left atrial appendage. After puncture and ligation, the anterior wall of the left ventricle quickly turns white. The chest cavity is quickly closed, a drainage tube is placed to expel air, and the muscles are sutured. An ultrasound examination is performed again 3 days after surgery to confirm the success of the MI myocardial infarction model.

[0052] ② Serum IFN-γ expression.

[0053] The ELISA method was used to detect the expression of IFN-γ in the serum of control mice and myocardial infarction mice to determine the degree of inflammation.

[0054] The results showed that compared with the sham group, the serum IFN-γ level began to increase 3 days after ischemia and reached a peak 7 days after ischemia, indicating the occurrence of post-MI inflammation ( Figure 1 A).

[0055] ③ IFN-γ expression in spleen.

[0056] The ELISA method was used to detect the expression of IFN-γ in the spleen of the control group mice and myocardial infarction mice to determine the degree of inflammation.

[0057] The results showed that compared with the sham group, the level of IFN-γ in the spleen began to increase 3 days after ischemia and reached a peak 21 days after ischemia, indicating the occurrence of post-MI inflammation ( Figure 1 B).

[0058] ④Western blot was used to detect the expression of AMPKγ2 protein in bone marrow-derived macrophages (BMDM) of MI mice at different time points.

[0059] To examine the expression of AMPKγ2 in bone marrow-derived macrophages (BMDM) from MI mice at different time points, protein was extracted from sham and MI-treated BMDM using RIPA buffer, and protein concentration was determined using a BCA colorimetric assay. AMPKγ2 protein expression was detected by Western blotting. Specifically, 40 μg of protein was boiled at 95°C for 5 minutes, then subjected to SDS-PAGE electrophoresis on a 10% separating gel, with the electrophoresis end time determined. The sample was transferred to a cellulose membrane at 90 V for 2 hours. Blocking was performed in 5% skim milk diluted in TBS-T at room temperature for 2 hours, followed by incubation with the primary antibody overnight at 4°C. Western blot detection was performed using anti-AMPKγ2 antibody (1:1000, Abcam, USA) and anti-GAPDH antibody (1:1000, Abcam, USA) as primary antibodies, and horseradish peroxidase-conjugated goat anti-rabbit antibody (Cell Signaling, USA) as secondary antibody. The cells were visualized using an ECL kit (GE, USA).

[0060] The results showed that compared with the sham group, the protein level of AMPKγ2 decreased in a time-dependent manner within 28 days after MI ( Figure 1 CD).

[0061] ⑤ Fluorescence quantitative PCR was used to detect the expression of PRKAG2 gene mRNA in BMDM of MI mice.

[0062] Tissue RNA was extracted using a Promega kit, and reverse transcription was performed using a TakaRa reverse transcription kit to obtain cDNA, followed by quantitative PCR using the SYBGreen method.

[0063] The quantitative PCR primer sequences are as follows:

[0064] .

[0065] The results showed that compared with the sham group, the expression level of AMPKγ2 gene mRNA was significantly decreased after MI ( Figure 1 E).

[0066] Example 2: In vitro simulated inflammatory stimulation reduced the expression of AMPKγ2.

[0067] ①Western blot was used to detect the expression of IL-6, MCP1 and AMPKγ2, representative proteins of inflammatory factors, in macrophages (RAW264.7) stimulated by IFN-γ.

[0068] The specific method is shown in Example 1. IL-6 antibody (1:1000, Abcam, USA), MCP1 antibody (1:1000, Abcam, USA), AMPKγ2 antibody (1:1000, CST, USA), and GAPDH antibody (1:1000, Abcam, USA) were used as primary antibodies.

[0069] The results showed that compared with the control group, IFN-γ (20 ng / mL) stimulated the expression of L-6 and MCP1 in RAW264.7 cells, while the expression of AMPKγ2 protein decreased significantly, suggesting that inflammatory stimulation can reduce the expression of AMPKγ2 ( Figure 2 AB).

[0070] ② Fluorescence quantitative PCR was used to detect the mRNA expression of IL-6, MCP1 and PRKAG2, representative proteins of inflammatory factors, in BMDM (RAW264.7) of MI mice under IFN-γ stimulation.

[0071] Tissue RNA was extracted using a Promega kit, and reverse transcription was performed using a TakaRa reverse transcription kit to obtain cDNA, followed by quantitative PCR using the SYBGreen method.

[0072] The quantitative PCR primer sequences are as follows:

[0073] .

[0074] The results showed that compared with the control group, IFN-γ (20 ng / mL) stimulated the expression of L-6 and MCP1 gene mRNA in RAW264.7 cells, while the expression of PRKAG2 gene mRNA was significantly decreased ( Figure 2 C), suggesting that inflammatory stimulation can reduce the expression of AMPKγ2.

[0075] ③Western blot was used to detect the expression of AMPKγ2 protein in BMDM of C57BL / 6 mice stimulated with different concentrations of IFN-γ.

[0076] The specific method is shown in Example 1.

[0077] The results showed that compared with the control group, the expression of AMPKγ2 protein decreased in an IFN-γ dose-dependent manner and was statistically significant ( Figure 2 DE).

[0078] ④Western blot was used to detect the expression of AMPKγ2 protein in BMDM of C57BL / 6 mice stimulated by different concentrations of TNF-α and LPS, and CoCl2.

[0079] The specific method is shown in Example 1.

[0080] The results showed that compared with the control group, AMPKγ2 protein expression was decreased under other inflammatory stimuli ( Figure 2 FI). These results suggest that AMPKγ2 is associated with macrophage inflammation and may be a regulatory factor for MI.

[0081] Example 3: Systemic AMPKγ2 knockout mice (AMPKγ2-KO) exacerbate cardiac dysfunction with MI and macrophage infiltration.

[0082] ① Small animal ultrasound was used to evaluate the cardiac contractile function of mice.

[0083] Mice were anesthetized with isoflurane, and cardiac diastolic and systolic function were assessed using a Vevo 2100 small animal cardiac ultrasound system. Physiological parameters, including electrocardiogram (ECG) and respiration, were recorded simultaneously. The heart rate was maintained at approximately 450 beats / min. After the heart rate stabilized for 1 minute, a coupling agent was applied to the chest for ultrasound examination and image acquisition. The systolic function indicators, EF% and FS%, were measured and analyzed using the small animal ultrasound system's built-in cardiac function analysis software.

[0084] The results showed that compared with the respective control groups, the contractile function indexes EF% and FS% in the AMPKγ2-KO group and the AMPKγ2-KO MI group were significantly decreased 28 days later ( Figure 3 AB), suggesting that AMPKγ2 deficiency can lead to abnormal cardiac function in mice.

[0085] ②Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect myocardial hypertrophy.

[0086] Use the Masson staining kit, the specific steps are as follows:

[0087] 1) Place paraffin sections in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 10 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 3 minutes, 90% ethanol for 3 minutes, and 85% ethanol for 3 minutes, and then place the sections in distilled water.

[0088] 2) Place the dewaxed sections in Ponceau for 10 minutes;

[0089] 3) Rinse with 0.2% glacial acetic acid;

[0090] 4) Cover the sections with molybdic acid for 2 minutes;

[0091] 5) Rinse with 0.2% glacial acetic acid;

[0092] 6) Stain with phenylamino blue for 80 seconds and rinse with 0.2% glacial acetic acid;

[0093] 7) Dehydrate the sections in ethanol I for 5 minutes and ethanol II for 5 minutes respectively.

[0094] 8) Place the sections in xylene I and xylene II for 10 minutes to clear them.

[0095] 9) Remove the slices from the xylene and allow them to dry slightly, then mount them with neutral gum.

[0096] 10) Microscope observation.

[0097] Use the WGA staining kit, the specific steps are as follows:

[0098] 1) Dewaxing and rehydration of paraffin sections;

[0099] 2) Repair with antigen retrieval solution and boil in water for 40 minutes;

[0100] 3) Stain with WGA staining solution in the dark for 30 minutes, then rinse the sections with running water;

[0101] 4) DAPI staining and washing the sections with running water;

[0102] 5) Allow to dry and seal the slides with fluorescent mounting solution.

[0103] The results showed that compared with WT, myocardial fibrosis and cardiomyocyte hypertrophy were increased in the AMPKγ2-KO group; compared with WT MI group, myocardial fibrosis and cardiomyocyte hypertrophy were also increased in the AMPKγ2-KO MI group ( Figure 3 C).

[0104] ③ Immunohistochemical staining of CD68 was used to detect inflammatory cell infiltration.

[0105] The specific steps are as follows:

[0106] 1) Place paraffin sections in xylene I for 20 minutes, xylene II for 20 minutes, ethanol I for 10 minutes, ethanol II for 10 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, and 85% ethanol for 5 minutes. Place the sections in distilled water for 5-10 minutes.

[0107] 2) Place the slices in the prepared antigen retrieval solution (antigen retrieval solution: distilled water = 1:49) and cook at 100°C for 40 minutes. Seal the container and allow to cool naturally.

[0108] 3) Immunohistochemistry was performed according to the immunohistochemistry kit: 1 drop or 50 μL of peroxidase blocking solution (reagent A) was added at room temperature for 10 minutes, and the sections were washed three times with PBS. 1 drop or 50 μL of normal non-immune animal serum (reagent B) was added at room temperature for 10-30 minutes. The serum was removed without washing the sections. 1 drop or 50 μL of LCD68 primary antibody diluent (prepared fresh, 100-fold dilution) was added overnight, and the sections were rewarmed for 30 minutes. The sections were washed three times with PBS for 3 minutes each. 50 μL of secondary antibody (reagent C) was added at room temperature for 30-60 minutes, and the sections were washed three times with PBS for 3 minutes each. 50 μL of streptavidin-peroxidase solution (reagent D) was added at room temperature for 10 minutes, and the sections were washed three times with PBS for 3 minutes each. DAB color development solution (prepared fresh, used within 30 minutes) was added for 3-10 minutes. The color change was observed under a microscope and the reaction was terminated by rinsing with running water.

[0109] 4) Place the dewaxed sections in hematoxylin for 10 minutes and then wash the sections with water;

[0110] 5) Differentiate the slices in 75% ethanol and 10% hydrochloric acid for 30 seconds, then wash the slices with water.

[0111] 6) Place the sections in ammonia water for 1 minute to debluing, then wash the sections with water;

[0112] 7) Dehydrate the sections in low-concentration alcohol for 5-10 seconds, anhydrous ethanol I, and anhydrous ethanol II for 5 minutes.

[0113] 8) Place the sections in xylene I and xylene II for 10 minutes to clear them.

[0114] 9) Remove the slices from the xylene and allow them to dry slightly, then mount them with neutral gum.

[0115] 10) Microscope observation.

[0116] The results showed that compared with WT, the inflammatory cell infiltration in the AMPKγ2-KO group was enhanced; compared with the WT MI group, the inflammatory cell infiltration in the AMPKγ2-KO MI group was more obvious ( Figure 3 D), suggesting that AMPKγ2 deficiency can increase inflammation.

[0117] In conclusion, systemic AMPKγ2 knockout mice (AMPKγ2-KO) exacerbate cardiac dysfunction with MI and macrophage infiltration.

[0118] Example 4: Systemic overexpression of AMPKγ2 in MI mice (AAV-AMPKγ2 MI) alleviates cardiac dysfunction caused by MI and macrophage infiltration.

[0119] ① Establishment of innately overexpressed AMPKγ2 MI mice (AAV-AMPKγ2 MI mice).

[0120] An MI injury model was established in male 8-week-old C57BL / 6 mice according to the method in Example 1. After the MI model was established, 40 male 8-week-old C57BL / 6JMI mice were divided into the following two groups using a random table method: AAV-CMV-GFP and AAV-CMV-AMPKγ2 experimental groups, with 20 mice in each group, and AAV-CMV-GFP and AAV-CMV-AMPKγ2 were injected into the tail vein, respectively.

[0121] ② Small animal ultrasound was used to evaluate the cardiac contractile function of mice.

[0122] See Example 3 for details.

[0123] The results showed that compared with MI+AAV-CMV-GFP mice, the contractile function of MI+AAV-CMV-AMPKγ2 mice was improved, with statistically significant ( Figure 4 AB), suggesting that overexpression of AMPKγ2 can alleviate cardiac dysfunction caused by MI.

[0124] ③Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect myocardial hypertrophy.

[0125] See Example 3 for details.

[0126] The results showed that compared with MI+AAV-CMV-GFP mice, MI+AAV-CMV-AMPKγ2 mice had reduced myocardial fibrosis and cardiomyocyte hypertrophy ( Figure 4 C), suggesting that overexpression of AMPKγ2 can improve cardiac tissue inflammation.

[0127] ④ Immunohistochemical staining of CD68 was used to detect inflammatory cell infiltration.

[0128] See Example 3 for details.

[0129] The results showed that AAV-CMV-AMPKγ2 improved the infiltration of CD68 in MI-3d, suggesting that AMPKγ2 may protect cardiac remodeling after MI by inhibiting the recruitment of macrophages. Figure 4 D).

[0130] In conclusion, systemic overexpression of AMPKγ2 in MI mice (AAV-AMPKγ2 MI) alleviates cardiac dysfunction in MI and macrophage infiltration.

[0131] Example 5: Lyz2-macrophage-specific AMPKγ2-deficient mice (LyZ2-AMPKγ2-CKO) exacerbate cardiac dysfunction caused by MI and macrophage infiltration.

[0132] ① Small animal ultrasound was used to evaluate the cardiac contractile function of mice.

[0133] See Example 3 for details.

[0134] The results showed that compared with flox / flox mice, the contractile function of LyZ2-AMPKγ2-CKO mice was significantly improved ( Figure 5 BC) were significantly decreased, with significant statistical significance, suggesting that macrophage-specific deletion of AMPKγ2 can aggravate cardiac dysfunction.

[0135] ②F4 / 80 and CD11b were co-stained, and macrophage infiltration in LyZ2-AMPKγ2-CKO and flox / flox MI mice was evaluated by flow cytometry.

[0136] The specific steps are as follows:

[0137] 1) Take 100 μL of cell suspension;

[0138] 2) Add an appropriate amount of surface antibody and incubate at room temperature in the dark for 15 minutes;

[0139] 3) Add 1-2 mL of flow cytometry staining buffer, centrifuge at 300 g for 5 minutes, and discard the supernatant;

[0140] 4) Resuspend the cells in 500 μL of flow cytometry staining buffer and analyze them.

[0141] The results showed that compared with flox / flox mice, the enhanced macrophage infiltration in LyZ2-AMPKγ2-CKO mice was further aggravated ( Figure 5 DE).

[0142] ③CD68 staining was used to evaluate macrophage infiltration in LyZ2-AMPKγ2-CKO and flox / flox MI mice.

[0143] See Example 3 for details.

[0144] The results showed that compared with flox / flox MI, the infiltration of inflammatory cells in the LyZ2-AMPKγ2-CKO group was enhanced ( Figure 5 F).

[0145] ④ In LyZ2-AMPKγ2-CKO and flox / flox MI mice, HE staining was used to detect cardiomyocyte morphology, Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect cardiomyocyte hypertrophy.

[0146] For details of Masson and WGA staining, see Example 3.

[0147] HE staining to evaluate cardiomyocyte morphology:

[0148] 1) Myocardial tissue was obtained, fixed with 4% formaldehyde, embedded in conventional paraffin, and sliced into 5 μm sections;

[0149] 2) Sections were routinely dewaxed with xylene and washed with various levels of ethanol and then water: xylene (I) for 5 min → xylene (II) for 5 min → 100% ethanol for 2 min → 95% ethanol for 1 min → 80% ethanol for 1 min → 75% ethanol for 1 min → distilled water for 2 min;

[0150] 3) Hematoxylin staining for 5 minutes, then rinsing with tap water;

[0151] 4) Hydrochloric acid-ethanol differentiation for 30 seconds;

[0152] 5) Soak in tap water for 15 minutes;

[0153] 6) Place in eosin solution for 2 minutes;

[0154] 7) Conventional dehydration, transparency, and mounting: 95% ethanol for 1 minute → 95% ethanol for 1 minute → 100% ethanol (I) for 1 minute → 100% ethanol (II) for 1 minute → xylene (I) for 1 minute → xylene (II) for 1 minute → mounting with neutral resin;

[0155] 8) Observe the morphology under a microscope and take photos for statistical analysis.

[0156] The results showed that compared with flox / flox mice, myocardial hypertrophy and myocardial fibrosis were further aggravated in LyZ2-AMPKγ2-CKO mice ( Figure 5 G).

[0157] In conclusion, macrophage-specific deletion of AMPKγ2 exacerbates cardiac dysfunction caused by MI and macrophage infiltration.

[0158] Example 6: Macrophage-specific overexpression of AMPKγ2 in MI mice (AAV-F4 / 80-GFP-

[0159] AMPKγ2) attenuates cardiac dysfunction in MI and macrophage infiltration.

[0160] ① Small animal ultrasound was used to evaluate the cardiac contractile function of mice.

[0161] See Example 3 for details.

[0162] The results showed that compared with MI+AAV-F4 / 80-GFP mice, the contractile function of MI+AAV-F4 / 80-GFP-AMPKγ2 mice was improved, with statistically significant ( Figure 6 AB), suggesting that macrophage-specific overexpression of AMPKγ2 can alleviate cardiac dysfunction caused by MI.

[0163] ②F4 / 80 and CD11b were co-stained, and macrophage infiltration in MI+AAV-F4 / 80-GFP and MI+AAV-F4 / 80-GFP-AMPKγ2 mice was evaluated by flow cytometry.

[0164] See Example 5 for details.

[0165] The results showed that compared with MI+AAV-F4 / 80-GFP, the MI+AAV-F4 / 80-GFP-AMPKγ2 group improved the infiltration of inflammatory cells ( Figure 6 CD).

[0166] ③CD68 staining was used to evaluate macrophage infiltration in MI+AAV-F4 / 80-GFP and MI+AAV-F4 / 80-GFP-AMPKγ2 mice.

[0167] See Example 3 for details.

[0168] The results showed that compared with MI+AAV-F4 / 80-GFP, MI+AAV-F4 / 80-GFP-AMPKγ2 group improved inflammatory cell infiltration ( Figure 6 E).

[0169] ④ In MI+AAV-F4 / 80-GFP and MI+AAV-F4 / 80-GFP-AMPKγ2 mice, Masson staining was used to detect myocardial fibrosis, and WGA staining was used to detect cardiomyocyte hypertrophy.

[0170] See Example 3 for details.

[0171] The results showed that compared with MI+AAV-F4 / 80-GFP mice, MI+AAV-F4 / 80-GFAMPKγ2 mice had improved myocardial hypertrophy and myocardial fibrosis ( Figure 6 F).

[0172] In conclusion, macrophage-specific overexpression of AMPKγ2 rescues cardiac remodeling after myocardial infarction.

[0173] Example 7: AMPKγ2 knockout can aggravate IFN-γ-induced macrophage migration and inflammation.

[0174] ①Transwell experiment to study macrophage migration regulated by si-AMPKγ2.

[0175] The specific steps are as follows:

[0176] 1) Seed RAW264.7 cells in a six-well plate;

[0177] 2) After 24 hours of adherence, gently dissipate the cells with prepared trypsin and place them in a 15 mL centrifuge tube.

[0178] 3) Centrifuge in a normal temperature centrifuge at 3000 bpm for 5 minutes;

[0179] 4) Discard the supernatant and add 2 mL of serum-free DMEM to each sample centrifuge tube and mix thoroughly by pipetting.

[0180] 5) Take a 12-well plate and add 500 μL of 10% serum DMEM to each well. Remove the chambers and add 200 μL of serum-free cell suspension to the upper chamber of each well. Incubate the lower chamber with 500 μL of 10% serum DMEM.

[0181] 6) After 24 hours, observe the cell status and number of migrated cells, remove the chamber from the 12-well plate, rinse the chamber twice with PBS for 5 minutes each time, then add 1 mL of paraformaldehyde solution to each well and place the chamber in the paraformaldehyde solution for 30 minutes.

[0182] 7) Take the Giemsa staining kit and add Giemsa staining reagent to a 24-well plate at a ratio of solution A:solution B (1:1) for 30 minutes. Then add Giemsa staining solution to the 24-well plate at a ratio of solution A:solution B (1:3) for 10 minutes.

[0183] 8) Rinse twice with PBS, 3 minutes each time;

[0184] 9) Remove the circular chamber sticker using a 1 mL syringe needle and place the chamber bottom up on a glass slide. Seal the slide with neutral resin. Once air-dried, apply nail polish along the four edges to prevent the specimen from falling off.

[0185] 10) Take photos under an optical microscope.

[0186] The results showed that si-AMPKγ2 led to more cell migration under IFN-γ stimulation ( Figure 7 AB), indicating that loss of AMPKγ2 aggravated IFN-γ-induced macrophage infiltration.

[0187] ②Scratch test was used to study the macrophage migration regulated by si-AMPKγ2.

[0188] The specific steps are as follows:

[0189] 1) Seed RAW264.7 cells into 6-well plates;

[0190] 2) After 24 hours of cell attachment, observe the cell density. When the cell density reaches 80%, use a 1 μL pipette tip to draw a cross along the longitudinal and transverse directions. Then, rinse the cells three times with PBS for 2 minutes each to remove dead cells.

[0191] 3) At this time, take a photo under an inverted microscope to record the initial conditions at 0h;

[0192] 4) After 24 hours, rinse the cells three times with PBS for 2 minutes each time to remove dead cells. At this time, take photos under an inverted microscope to record the migration status of the cells over the 24 hours.

[0193] The results showed that si-AMPKγ2 led to more cell migration and accelerated migration area under IFN-γ stimulation ( Figure 7 CD).

[0194] ③Western Blot method was used to detect the expression of representative proteins of inflammatory factors such as IL-6, MCP1 and AMPKγ2 in RAW264.7 si-AMPKγ2.

[0195] See Example 2 for details.

[0196] The results showed that compared with si-NC, the expression of IL-6 and MCP1 in the si-AMPKγ2 group increased in RAW264.7 cells. At the same time, compared with si-NC+IFN-γ, the expression of IL-6 and MCP1 in the si-AMPKγ2+IFN-γ group increased, which was statistically significant ( Figure 7 EF). The results suggest that AMPKγ2 knockout can aggravate inflammation in RAW cells.

[0197] ④ Fluorescence quantitative PCR was used to detect the mRNA expression of IL-6 and MCP1 genes, representative proteins of inflammatory factors of RAW264.7 si-AMPKγ2.

[0198] See Example 2 for details.

[0199] The results showed that compared with si-NC, the mRNA expression of IL-6 and MCP1 genes in RAW264.7 cells was upregulated in the si-AMPKγ2 group. At the same time, compared with si-NC+IFN-γ, the mRNA expression of IL-6 and MCP1 genes in RAW264.7 cells in the si-AMPKγ2+IFN-γ group was upregulated, with statistical significance. The results suggest that AMPKγ2 knockout can aggravate the inflammation of RAW cells caused by INF-γ at the RNA level ( Figure 7 G).

[0200] Example 8: Overexpression of AMPKγ2 can reduce IFN-γ-induced macrophage migration and inflammation.

[0201] ①Transwell experiment to study macrophage migration regulated by pc3.1-AMPKγ2.

[0202] See Example 7 for details.

[0203] The results showed that pc3.1-AMPKγ2 significantly reduced the migration of more cells under IFN-γ stimulation ( Figure 8 AB), indicating that overexpression of AMPKγ2 alleviates IFN-γ-induced macrophage infiltration.

[0204] ②The scratch test was used to study the macrophage migration regulated by pc3.1-AMPKγ2.

[0205] See Example 7 for details.

[0206] The results showed that pc3.1-AMPKγ2 reduced the migration of more cells and the migration area under IFN-γ stimulation ( Figure 8 CD).

[0207] ③Western Blot method was used to detect the expression of representative proteins of inflammatory factors such as IL-6, MCP1 and AMPKγ2 in RAW264.7 pc3.1-AMPKγ2.

[0208] See Example 2 for details.

[0209] The results showed that compared with pc3.1-con, the expression of IL-6 and MCP1 in the pc3.1-AMPKγ2 group did not change significantly. However, compared with pc3.1-con+IFN-γ, the expression of IL-6 and MCP1 in the pc3.1-AMPKγ2+IFN-γ group increased with statistical significance ( Figure 7 EF). The results suggest that overexpression of AMPKγ2 can alleviate INF-γ-induced inflammation in RAW cells.

[0210] ④ Fluorescence quantitative PCR was used to detect the mRNA expression of IL-6 and MCP1 genes, representative proteins of inflammatory factors in RAW264.7 pc3.1-AMPKγ2.

[0211] See Example 2 for details.

[0212] The results showed that compared with pc3.1-con, the mRNA expression of IL-6 and MCP1 genes in the pc3.1-AMPKγ2 group did not change significantly. However, compared with pc3.1-con+IFN-γ, the mRNA expression of IL-6 and MCP1 genes in RAW264.7 cells in the pc3.1-AMPKγ2+IFN-γ group decreased, which was statistically significant. The results suggest that overexpression of AMPKγ2 can reduce INF-γ-induced RAW inflammation at the RNA level ( Figure 8 G).

[0213] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details based on all the teachings disclosed, and these changes are all within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a preparation that specifically overexpresses AMPKγ2 protein in the preparation of a medicament for preventing and / or treating heart failure caused by abnormal cardiac remodeling due to excessive inflammatory response after myocardial infarction; the preparation that specifically overexpresses AMPKγ2 protein comprises: (1) AMPKγ2 protein; (2) a polynucleotide encoding an AMPKγ2 protein; (3) Recombinant vector or recombinant cell of nucleic acid molecule of AMPKγ2 protein.

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