CN116327939A

CN116327939A - Medical application of AMPK gamma 2 protein in preventing or treating heart failure after myocardial infarction

Info

Publication number: CN116327939A
Application number: CN202211332254.4A
Authority: CN
Inventors: 韩雅玲; 闫承慧; 侯晶津; 宋紫萍; 宋海旭; 刘丹
Original assignee: General Hospital of Shenyang Military Region
Current assignee: General Hospital of Shenyang Military Region
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-06-27

Abstract

The invention discloses medical application of AMPK gamma 2 protein in preventing or treating Heart Failure (HF) after Myocardial Infarction (MI), and belongs to the technical field of medical biology. The expression level of the AMPK gamma 2 protein or the active fragment thereof is related to HF caused by abnormal heart reconstruction due to excessive inflammatory reaction after MI, the left ventricular contractile function of MI mice with the AMPK gamma 2 knocked out by the whole body is obviously reduced, and the macrophage infiltration is obviously increased; whereas MI mice that overexpress AMPK gamma 2 systemically can alleviate MI and macrophage infiltrated cardiac insufficiency. Meanwhile, macrophage specific knockout of AMPK gamma 2 can exacerbate MI and macrophage infiltrated cardiac dysfunction; in contrast, macrophage-specific overexpression of AMPK gamma 2 reduces MI and macrophage-infiltrated cardiac dysfunction. The invention provides a new target for diagnosis and treatment of HF after MI, and has great clinical significance.

Description

Medical application of AMPK gamma 2 protein in preventing or treating heart failure after myocardial infarction

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to medical application of a regulatory subunit AMPK gamma 2 in AMP-activated kinase (AMPK), in particular to medical application of an AMPK gamma 2 protein in preventing or treating heart failure after myocardial infarction.

Background

Cardiovascular disease is the major health burden, which is still the most common cause of death worldwide, and global disease burden (GBD) studies estimated that the total number of deaths from cardiovascular disease increases by 21.1% during 2007 to 2017 worldwide, and cardiovascular disease can lead to 380 more than ten thousand deaths per year in europe. The Chinese cardiovascular health and disease research report 2021 also indicates that the incidence and prevalence of cardiovascular diseases in China are continuously increasing, and the cardiovascular diseases become the first cause of death of residents, wherein the incidence of coronary heart diseases is relatively large. Acute Myocardial Infarction (AMI) is the most serious disease type in coronary heart disease, and AMI mortality has generally increased since 2002-2018. Although percutaneous coronary intervention and coronary bypass can benefit patients with myocardial infarction, several clinical trials have shown that Heart Failure (HF) after Myocardial Infarction (MI) remains an unavoidable and intractable problem for several years, with death occurring within about 6 years after MI mostly associated with severe HF. HF is a common condition after MI, and the rate of HF incidence after MI increases dramatically with age. Effective development of protection of post-MI myocardium is therefore a necessary condition for preventing post-MI HF occurrence and improving 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 expands left ventricular remodeling after MI. "poor" or "pathological" reconstitution after MI would increase the risk of HF and significantly reduce patient survival, but its key regulatory mechanisms have not yet been elucidated and effective targeted therapeutic drugs have not been discovered so far. Therefore, the key mechanism of heart failure after MI is defined, and the search of new preventive and therapeutic targets has important clinical significance and social significance.

MI triggers a strong inflammatory response, critical to cardiac repair, and the extent of post-infarction remodeling depends on infarct size and quality of cardiac repair. Inflammatory pathways are involved in the expansion and fibrotic remodeling of the infarcted heart, driving key events in the postinfarct HF pathogenesis. Following Acute Myocardial Infarction (AMI), monocytes produced in the bone marrow and spleen enter the blood and recruit to the damaged heart tissue. Thereafter, the infiltrated monocytes differentiate into M1 macrophages, responsible for the removal of cellular debris from the damaged tissue. And then the chemotactic factors and the growth factors regulate and control M2 macrophages to carry out tissue repair through the secreted cytokines. Mononuclear macrophages are pleiotropic cells of the innate immune system, which are indispensable in both the initial inflammatory response to injury and in subsequent wound healing in the heart, but excessive inflammatory responses may lead to myocardial necrosis, and how to regulate and target macrophage migration to inhibit myocardial inflammation remains to be studied. Mononuclear macrophages in the heart have strong heterogeneity and plasticity after AMI. Thus, targeted therapeutic intervention by elucidating the molecular mechanisms of mononuclear macrophage phenotype transformation in ventricular remodeling may be an important research direction to improve post-MI outcome.

AMP-activated protein kinase (AMPK), a conserved serine/threonine kinase, is a key cellular energy sensor regulating bioenergy metabolism, and AMPK exists in almost all eukaryotes, exerting anti-inflammatory and antioxidant activities. AMPK can appear as a heterotrimeric complex, containing one catalytic alpha subunit and regulatory beta and gamma subunits. Each subtype has multiple isoforms (two α, two β and three γ), with the heart expressing predominantly α2, β2, γ1 and γ2 subtypes. AMPK has been widely reported to have cardioprotective effects in various cardiovascular diseases, such as phosphorylation of AMPK in ACE2 Ser680 in pulmonary endothelial cells, increasing ACE2 expression in the endothelium, protecting pulmonary arterial hypertension; AMPK activation improves insulin sensitivity by inhibiting adipogenesis (ACC 1, SREBP1 c), protein synthesis (mTORC 1) and lipolysis (HSL), and activating FAO (ACC 2), and is useful for preventing and treating type 2 diabetics and the like. In addition, AMPK can protect myocardial infarction and heart aging through metabolic and non-metabolic pathways. AMPK gamma subunits play a considerable role in AMPK activation. When AMP binds to the gamma subunit, the allosteric activating complex makes it a substrate for more phosphorylation of threonine 172 site, more phosphorylation by major upstream AMPK kinase in the activation loop of the alpha subunit, and gamma subunit favors the allosteric and downstream protective gene expression of AMPK. Furthermore, studies have shown that AMPK gamma 2 inhibits ribosome biogenesis through nuclear translocation, protecting against myocardial ischemia/reperfusion (I/R) injury. Mutations in AMPK gamma 2 are associated with several disease states including cardiomyopathy, walf-parkinson-white syndrome, and the like. However, it is not clear whether ampkγ2 is involved in the occurrence and progress of MI.

Disclosure of Invention

In view of the above problems, the present invention provides a medical use of AMPK γ2 protein for preventing or treating HF after MI. The inventor finds through a large number of experiments that after the MI of the C57BL/6 mouse, the expression of inflammatory factor INF-gamma is increased, and the expression of PRKAG2 gene mRNA and protein is obviously reduced; in vitro cell experiments simulating inflammatory stimuli also found that PRKAG2 gene mRNA and protein expression were significantly reduced. The contraction function of the left ventricle of MI mice with the AMPK gamma 2 knocked out in the whole body is obviously reduced, and the infiltration of macrophages is obviously increased; whereas MI mice that overexpress AMPK gamma 2 systemically can alleviate MI and macrophage infiltrated cardiac insufficiency. Meanwhile, macrophage specific knockout of AMPK gamma 2 can exacerbate MI and macrophage infiltrated cardiac dysfunction; in contrast, macrophage-specific overexpression of AMPK gamma 2 reduces MI and macrophage-infiltrated cardiac dysfunction. Cytology studies have found that si-AMPK gamma 2 results in increased macrophage migration and inflammation-associated protein expression upon INF-gamma stimulation, while overexpression of AMPK gamma 2 reduces macrophage migration and inflammation-associated protein expression. The above results indicate that AMPK gamma 2 protein can be used for preventing or treating HF caused by abnormal heart remodeling due to excessive inflammatory reaction after MI.

In order to achieve the above purpose, the present invention provides the following technical solutions.

The invention provides application of a preparation for specifically over-expressing AMPK gamma 2 protein or active fragment thereof in preparing a medicine for preventing and/or treating heart failure caused by abnormal heart reconstruction caused by excessive inflammatory reaction after myocardial infarction.

Further, the preparation for specifically overexpressing AMPK gamma 2 protein or an active fragment thereof comprises:

(1) AMPK gamma 2 protein, or modified AMPK gamma 2 protein derivatives, or AMPK gamma 2 protein analogues;

(2) A polynucleotide encoding AMPK gamma 2 protein;

(3) An expression construct comprising the AMPK gamma 2 protein of (1) or the polynucleotide of (2);

(4) An agonist of the AMPK gamma 2 protein described in (1);

(5) A nucleic acid molecule of AMPK gamma 2 protein or a recombinant vector or recombinant cell of the nucleic acid molecule;

(6) An agent capable of up-regulating the expression of AMPK gamma 2 protein or its activity.

The invention also provides the use of an agent for detecting the expression level of AMPK gamma 2 protein or an active fragment thereof in the manufacture of a product, characterized in that said product is used for the prediction of excessive inflammatory response after MI and/or the evaluation of the therapeutic effect, prognosis.

Further, AMPK gamma 2 protein or an active fragment thereof is used as a marker for predicting and/or treating effect and prognosis evaluation of excessive inflammatory response after MI.

Furthermore, the product can predict myocardial injury and inflammatory response after MI or evaluate the therapeutic effect or prognosis by detecting the expression level of AMPK gamma 2 protein or active fragment thereof in blood, tissue or cells below a reference value. Further, the product is a chip, a preparation or a kit.

The invention also provides a pharmaceutical composition comprising a formulation that specifically overexpresses AMPK gamma 2 protein or an active fragment thereof, and optionally a pharmaceutically acceptable carrier or excipient.

(2) A polynucleotide encoding AMPK gamma 2 protein;

(4) An agonist of the AMPK gamma 2 protein described in (1);

Further, the use of a pharmaceutical composition according to any of the preceding claims for the prevention and/or treatment of HF caused by abnormal cardiac remodeling due to excessive inflammatory response after MI.

The invention also provides application of the preparation for specifically over-expressing the AMPK gamma 2 protein or the active fragment thereof in preparing anti-inflammatory drugs.

In the invention, the active fragment of the AMPKγ2 protein refers to a fragment with the function of the AMPKγ2 protein, which can be a part of the AMPKγ2 protein or a fragment obtained by deleting, adding or replacing the amino acid sequence of the AMPKγ2 protein; the site which possibly influences the activity is avoided as required by a person skilled in the art, and other sites are modified by deletion, addition or replacement, so that the modified AMPK gamma 2 protein still has the activity or the function of the AMPK gamma 2 protein.

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.

In the present invention, the prevention and/or treatment of excessive inflammatory response after MI refers to inhibition or slowing of macrophage infiltration and increase in inflammatory factors.

In the present invention, the use of the expression level of AMPK γ2 protein or an active fragment thereof for prediction and/or evaluation means that when the expression level of AMPK γ2 protein or an active fragment thereof in blood, tissue or cells is lower than a reference value, myocardial damage and inflammatory response after MI can be predicted, or the therapeutic effect or prognosis thereof can be evaluated.

In the present invention, the expression level of AMPK γ2 protein or an active fragment thereof can be detected by a method well known in the art, for example, by amplifying mRNA of PRKAG2 by polymerase chain reaction and performing a quantitative reaction, or by detecting AMPK γ2 protein expression level by Western Blot.

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

In the present invention, the up/down regulation of protein expression in a tissue/cell means increasing or decreasing the protein level or mRNA level in a tissue/cell by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or by more than 100%. Wherein said up-or down-regulation is compared to non-intervening tissues or cells.

In the present invention, the agent capable of inhibiting down-regulation of expression of AMPK γ2 protein or an active fragment thereof or promoting up-regulation of expression of AMPK γ2 protein or an active fragment thereof is known in the art.

Compared with the prior art, the invention has the beneficial effects.

The expression level of the AMPK gamma 2 protein or the active fragment thereof is related to HF caused by abnormal heart reconstruction due to excessive inflammatory reaction after MI, the left ventricular contractile function of MI mice with the AMPK gamma 2 knocked out by the whole body is obviously reduced, and the macrophage infiltration is obviously increased; whereas MI mice that overexpress AMPK gamma 2 systemically can alleviate MI and macrophage infiltrated cardiac insufficiency. Meanwhile, macrophage specific knockout of AMPK gamma 2 can exacerbate MI and macrophage infiltrated cardiac dysfunction; in contrast, macrophage-specific overexpression of AMPK gamma 2 reduces MI and macrophage-infiltrated cardiac dysfunction. The invention provides a new target for diagnosis and treatment of HF after MI, and has great clinical significance.

Drawings

FIG. 1. After the establishment of the MI model in C57BL/6 mice, IFN-. Gamma.expression was up-regulated and AMPK-. Gamma.2 expression was decreased in the early inflammatory activation phase. Wherein (a) is an ELISA method for detecting serum IFN- γ levels in MI mice (n=5, ^** p<0.01, compared to sham group); (B) Spleen IFN- γ levels were measured for MI mice by ELISA (n=5, ^* p<0.05， ^** p<0.01, compared to sham group); (C-D) Western blot was used to detect AMPK gamma 2 protein expression in bone marrow-derived macrophages (BMDM) of MI mice at different time points (n=3, ^** p<0.01, compared to sham group); (E) PRKAG2 gene mRNA expression in MI mice BMDM was detected for fluorescent quantitative PCR (n=3, ^* p<0.05, compared to the control group).

FIG. 2. In vitro mimicking inflammatory stimuli, AMPKγ2 expression was decreased. Wherein (A-B) is representative protein IL-6, MCP1 and AMPK gamma 2 expression of inflammatory factors in macrophages (RAW 264.7) under the stimulation of IFN-gamma by Western blot detection (n=3, ^** p<0.01, compared to control group); (C) For fluorescent quantitative PCR detection of representative proteins of inflammatory factors IL-6, MCP1 and PRKAG2 gene mRNA expression in MI mice BMDM (n=3, ^** p<0.01， ^*** P<0.001, compared to control group); (D-E) detection of AMPK gamma 2 protein expression in different concentrations of IFN-gamma stimulated C57BL/6 mouse BMDM for Western blot (n=3, ^* p<0.05, compared to control group); (F-I) is Western blot to detect TNF-alpha and LPS with different concentrations, coCl ₂ Stimulation of ampkγ2 protein expression in C57BL/6 mouse BMDM (n=3, ^* p<0.05， ^** p<0.01, compared to the control group).

FIG. 3A systemic knockout of AMPKγ2 mice (AMPKγ2-KO) exacerbates MI and macrophage infiltrated cardiac insufficiency. Wherein (a) is a small animal ultrasound evaluation of left ventricular contractile function EF% (n=11, 10,9,9, ^* p<0.05， ^** p<0.01, compared to the control group; ^# p<0.05, and MI groupRatio of; (B) The left ventricular contractile function FS% was evaluated by ultrasound in small animals (n=11, 10,8,8, ^* p<0.05， ^** p<0.01, compared to sham; ^# p<0.05, to MI group); (C) Masson staining detects myocardial fibrosis, WGA staining detects cardiomyocyte hypertrophy (n=11, 10,9,9); (D) Inflammatory cell infiltration was detected for immunohistochemical staining of CD68 (n=11, 10,8,8).

Fig. 4. Cardiac insufficiency with Mi and macrophage infiltration was alleviated in mice with systemic overexpression of AMPKγ2MI (AAV-AMPKγ2 MI). Wherein (a) the left ventricular contractile function EF was evaluated for small animals by ultrasound (n=5, ^# p<0.05, compared to MI control group); (B) The left ventricular contractile function FS was evaluated ultrasonically for small animals (n=5, ^# p<0.05, compared to MI control group); (C) Myocardial fibrosis was detected for Masson staining, and cardiomyocyte hypertrophy was detected for WGA staining (n=5); (D) Inflammatory cell infiltration was detected for immunohistochemical staining of CD68 (n=5).

FIG. 5 Lyz A mice with specific loss of AMPKγ2 (Lyz-AMPKγ2-CKO) on macrophages exacerbate MI and macrophage infiltrated cardiac dysfunction. Wherein (A) is Lyz2-AMPKγ2-CKO and flox/flox MI mice survival analysis; (B) The left ventricular contractile function EF was evaluated ultrasonically for small animals (n=3, ^# p<0.05, compared to flox/flox); (C) The left ventricular contractile function FS was evaluated ultrasonically for small animals (n=3, ^# p<0.05, compared to flox/flox); (D-E) Co-staining with F4/80 and CD11b, flow cytometry evaluation of macrophage infiltration in Lyz2-AMPKγ2-CKO and flox/flox MI mice ^# p<0.05, compared to flox/flox); (F) Macrophage infiltration in Lyz2-AMPKγ2-CKO and flox/flox MI mice was assessed for CD68 staining; (G) For both Lyz2-ampkγ2-CKO and flox/flox MI mice, HE staining detected cardiomyocyte morphology, masson staining detected myocardial fibrosis, WGA staining detected cardiomyocyte hypertrophy (n=3).

FIG. 6 macrophage specific overexpression of AMPKγ2MI mice (AAV-F4/80-GFP-AMPKγ2) reduced MI and macrophage infiltrated cardiac dysfunction. Wherein (a) the left ventricular contractile function EF was evaluated for small animals by ultrasound (n=5, ^* p<0.05, compared to AAV-F4/80-GFP); (B) The left ventricular contractile function FS was evaluated ultrasonically for small animals (n=5, ^* p<0.05, compared to AAV-F4/80-GFP); (C-D) Co-staining with F4/80 and CD11b, flow cytometry evaluation of macrophage infiltration in AAV-F4/80-GFP-AMPK gamma 2 and AAV-F4/80-GFP MI mice ^# p<0.05, compared to AAV-F4/80-GFP); (E) Macrophage infiltration (n=5) in AAV-F4/80-GFP-ampkγ2 and AAV-F4/80-GFPMI mice was assessed for CD68 staining; (F) For AAV-F4/80-GFP-AMPKγ2 and AAV-F4/80

GFP MI two mice, masson staining to detect myocardial fibrosis, WGA staining to detect cardiomyocyte hypertrophy (n=5).

FIG. 7. IFN-gamma induced macrophage migration and inflammation may be exacerbated after AMPK gamma 2 knockout. Wherein (a-B) is a transwell experimental study of si-AMPK gamma 2-mediated macrophage migration (n=4, ^** P<0.01, compared to si-NC; ^# P<0.05, compared to IFN-. Gamma. + si-NC); (C-D) study si-ampkγ2 mediated macrophage migration for scratch test (n=3, ^** P<0.01, compared to si-NC; ^# P<0.05, compared to IFN-. Gamma. + si-NC); (E-F) detection of expression of raw264.7si-ampkγ2 inflammatory factor by Western Blot method (n=3, ^* P<0.05， ^** p<0.01, compared to si-NC; ^# P<0.05， ^## P<0.01 compared to IFN-. Gamma. + si-NC); (G) Representative proteins of the inflammatory factor IL-6, MCP1 gene mRNA expression of raw264.7si-AMPK gamma 2 were detected for fluorescent quantitative PCR (n=3, ^* P<0.05, compared to si-NC; ^# P<0.05, compared to IFN-. Gamma. + si-NC).

FIG. 8. AMPKγ2 overexpression can reduce IFN- γ induced macrophage migration and inflammation. Wherein (a-B) is a transwell experimental study pc3.1-ampkγ2 mediated macrophage migration (n=4, ^** P<0.01, compared to pc 3.1-con; ^## P<0.01, compared to IFN-. Gamma. + pc 3.1-con); (C-D) study pc3.1-ampkγ2 mediated macrophage migration for scratch test (n=3, ^** P<0.01, compared to pc 3.1-con; ^## P<0.01, compared to IFN-. Gamma. + pc 3.1-con); (E-F) detection of RAW 264.7pc 3.1-AMP for Western Blot methodKγ2 inflammatory factor expression (n=3, ^* P<0.05， ^** p<0.01, compared to pc 3.1-con; ^# P<0.05， ^## P<0.01 compared to IFN-. Gamma. + pc 3.1-con); (G) Representative proteins of RAW264.7 pc3.1-AMPK gamma 2 inflammatory factor IL-6, MCP1 gene mRNA expression were detected for fluorescent quantitative PCR (n=3, ^** p<0.01, compared to pc 3.1-con; ^## P<0.01, compared to IFN-. Gamma. + pc 3.1-con).

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The experimental data of the present invention are all percentages. The comparison of the two sample rates uses chi-square test and the statistical processing uses SPSS 22.0 package processing. P <0.05 is statistically different.

Example 1: after the establishment of the MI model of the C57BL/6 mice, the IFN-gamma expression is up-regulated in the early inflammatory activation stage, and the AMPK gamma 2 expression is reduced.

(1) Establishment of MI model of C57BL/6J mice.

Starting an anesthesia machine, putting the mice into the anesthesia machine for gas anesthesia, putting the mice on an operation table after the mice lose consciousness, fixing limbs by using rubberized fabric, connecting the nose with a mask of the anesthesia machine (the ratio of isoflurane to oxygen is 1:5), and continuously anesthetizing the mice. Conventional alcohol sterilization treatment, cutting the skin at 3, 4 ribs, separating pectoral large muscle, and squeezing the two ribs to expose the heart. The coronary artery is visible at the lower edge of the left auricle for 1-2 mm, the front wall of the left ventricle is quickly whitened after puncture ligation, the thoracic cavity is quickly closed, a drainage tube is placed for discharging air, and muscles are sutured. An ultrasound examination was again performed 3 days after the operation to determine whether the MI myocardial infarction model was successful.

(2) Serum IFN-gamma expression.

IFN-gamma expression in serum of mice in control group and myocardial infarction mice was detected by ELISA method to determine the degree of inflammation.

The results show that: IFN-gamma levels in serum began to rise after 3d ischemia compared to sham group, peaking after 7d ischemia, suggesting the onset of inflammation after MI (FIG. 1A).

(3) Spleen IFN-gamma expression.

IFN-gamma expression in the spleens of control mice and myocardial infarction mice was examined by ELISA to determine the degree of inflammation.

The results show that: IFN-gamma levels began to rise in the spleen after 3d ischemia compared to sham group, peaking after 21d ischemia, suggesting the onset of inflammation after MI (FIG. 1B).

(4) Western blot detects AMPK gamma 2 protein expression in Bone Marrow Derived Macrophages (BMDM) of MI mice at different time points.

To detect the expression of AMPK gamma 2 in Bone Marrow Derived Macrophages (BMDM) of MI mice at different time points, the protein concentration was determined using a BCA colorimetric kit using RIPA lysate to extract sham group and different time snack stem mouse BMDM proteins. The protein expression of AMPK gamma 2 is detected by a Western Blot method. The method comprises the following steps: 40 μg of protein was boiled at 95℃for 5min, and subjected to SDS-PAGE electrophoresis with 10% separation gel, and the electrophoresis termination time was determined. Transferring the sample onto a cellulose membrane at a voltage of 90V for 2 hours; primary antibody was added to 5% nonfat dry milk diluted with TBS-T after 2h of blocking at ambient temperature and incubated overnight at 4 ℃. anti-AMPKγ2 antibody (1:1000, abcam Co., U.S.A.) and anti-GAPDH antibody (1:1000, abcam Co., U.S.A.) were used as primary antibodies, horseradish peroxidase-labeled goat anti-rabbit antibody (Cell signaling Co., U.S.A.) was used as secondary antibodies, and Western Blot detection was performed and developed by using ECL kit (GE Co., U.S.A.).

The results show that: the protein levels of ampkγ2 decreased in 28D after MI in a time-dependent manner compared to sham group (fig. 1C-D).

(5) Fluorescent quantitative PCR detects PRKAG2 gene mRNA expression in MI mouse BMDM.

Tissue RNA was extracted using Promega kit, and cDNA was obtained by reverse transcription using TakaRa reverse transcription kit, followed by quantitative PCR using SYBGreen method.

The quantitative PCR primer sequences were as follows:

。

the results show that: the mRNA expression level of AMPK gamma 2 gene was significantly reduced after MI compared to sham group (fig. 1E).

Example 2: in vitro simulated inflammatory stimuli, AMPK gamma 2 expression was decreased.

(1) Western blot detects the expression of IL-6, MCP1 and AMPK gamma 2 by macrophages (RAW 264.7) stimulated by IFN-gamma.

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), GAPDH antibody (1:1000, abcam, USA) were used as primary antibodies.

The results show that: compared to the control group, IFN-. Gamma.stimulation (20 ng/mL) and L-6 and MCP1 were up-regulated in RAW264.7, whereas AMPKγ2 protein expression was significantly decreased, suggesting that inflammatory stimulation could decrease AMPKγ2 expression (FIGS. 2A-B).

(2) Fluorescent quantitative PCR (Fluorescence) detection of representative proteins IL-6, MCP1 and PRKAG2 gene mRNA expression of inflammatory factors under IFN-gamma stimulation in BMDM (RAW 264.7) of MI mice.

The quantitative PCR primer sequences were as follows:

。

the results show that: compared to the control group, IFN-. Gamma.stimulation (20 ng/mL) and L-6 and MCP1 gene mRNA expression were up-regulated in RAW264.7, while PRKAG2 gene mRNA expression was significantly decreased (FIG. 2C), suggesting that inflammatory stimulation may decrease AMPK. Gamma.2 expression.

(3) Western blot detects that IFN-gamma at different concentrations stimulates the expression of AMPK gamma 2 protein in BMDM of C57BL/6 mice.

The specific method is shown in example 1.

The results show that: AMPK gamma 2 protein expression was decreased in a dose-dependent manner with IFN-gamma compared to the control group, and had statistical significance (fig. 2D-E).

(4) Western blot detection of different concentrations of TNF-alpha and LPS, coCl ₂ Stimulation of AMPK gamma 2 protein expression in C57BL/6 mice BMDM.

The specific method is shown in example 1.

The results show that: AMPK gamma 2 protein expression was reduced under other inflammatory stimuli compared to the control group (fig. 2F-I). Together, the above results suggest that ampkγ2 is associated with macrophage inflammation and may be a regulator of MI.

Example 3: systemic knockout of ampkγ2 mice (ampkγ2-KO) exacerbates MI and macrophage infiltrated cardiac insufficiency.

(1) Mice were evaluated for cardiac contractile function by ultrasound.

Mice were anesthetized with isoflurane and tested for diastolic and systolic function using a Vevo2100 small animal heart sonicator. Synchronously recording physiological parameters such as electrocardio and respiration of the mice, maintaining the heart rate at about 450 times/min, coating a couplant on the front chest after the heart rate is stable for 1min, performing ultrasonic examination, collecting images, and measuring and analyzing by using a small animal ultrasonic instrument self-provided heart function analysis software to obtain shrinkage function indexes EF% and FS%.

The results show that: the shrinkage function index EF% and FS% were significantly decreased after 28d in the AMPKγ2-KO group and the AMPKγ2-KO MI group compared to the respective control groups (FIG. 3A-B), suggesting that the loss of AMPKγ2 may cause cardiac dysfunction in mice.

(2) Masson staining detects myocardial fibrosis and WGA staining detects cardiomyocyte hypertrophy.

The Masson staining kit was used, and the specific steps were as follows:

1) Paraffin sections were placed in xylene I for 10min, xylene II for 10min, absolute ethanol I for 10min, absolute ethanol II for 5min,95% ethanol for 3min,90% ethanol for 3min,85% ethanol for 3min, respectively, and sections were placed in distilled water;

2) Placing the dewaxed slice in ponceau for 10min;

3) Flushing with 0.2% glacial acetic acid;

4) Slicing and covering molybdic acid for 2min;

5) Flushing with 0.2% glacial acetic acid;

6) The sections were washed with 0.2% glacial acetic acid for 80s by staining with cymbidium;

7) Respectively placing the slices in absolute ethyl alcohol I for 5min and absolute ethyl alcohol II for 5min for dehydration;

8) Placing the slice in xylene I and xylene II for 10min for transparency;

9) Taking out the slice from the dimethylbenzene, slightly airing, and sealing the neutral resin;

10 Microscopic observation.

The WGA staining kit was used, the specific steps were as follows:

1) Dewaxing and rehydrating paraffin sections;

2) Repairing the antigen retrieval liquid, and boiling for 40min;

3) WGA staining solution is used for shading and staining for 30min, and washing the slice with running water;

4) DAPI staining, washing sections with running water;

5) And (5) airing, and sealing the fluorescent sealing piece with liquid.

The results show that: the ampkγ2-KO group had an increased degree of myocardial fibrosis and cardiomyocyte hypertrophy compared to WT; the ampkγ2-KO MI group also increased the degree of myocardial fibrosis and cardiomyocyte hypertrophy compared to the WT MI group (fig. 3C).

(3) Immunohistochemical staining of CD68 detected inflammatory cell infiltration.

The method comprises the following specific steps:

1) Paraffin sections are respectively placed in xylene I for 20min, xylene II for 20min and absolute ethyl alcohol I for 10min, absolute ethyl alcohol II for 10min,95% ethyl alcohol for 5min,90% ethyl alcohol for 5min and 85% ethyl alcohol for 5min, and the sections are placed in distilled water for 5-10min;

2) The sections were placed in the prepared antigen retrieval solution (antigen retrieval solution: distilled water=1:49) boiling the slices at 100 ℃ for 40min, sealing and naturally cooling;

3) Immunohistochemistry was performed according to the immunohistochemical kit: dripping 1 drop or 50 mu L of peroxidase blocking solution (reagent A) at room temperature for 10min, washing with PBS for 3 times, dripping 1 drop or 50 mu L of normal non-immune animal serum (reagent B) at room temperature for 10-30min, removing serum, cleaning slices, adding 1 drop or 50 mu L of primary antibody diluent (prepared in use, 100-time diluted) for overnight, rewarming for 30min, washing with PBS for 3 times, adding 50 mu L of secondary antibody (reagent C) each time for 3min, washing with PBS for 30-60min at room temperature for 3 times, dripping 50 mu L of streptomycin-biotin-peroxidase solution (reagent D) each time for 3min, dripping DAB color development solution (prepared in use, used up in 30 min) each time for 3-10min, observing color change under a lens, and stopping reaction after running water washing for 3min;

4) Placing the dewaxed slice in hematoxylin for 10min, and washing the slice with water;

5) Placing the slices in 75% ethanol and 10% hydrochloric acid for differentiation for 30s, washing the slices with water;

6) Placing the slices in ammonia water for 1min to turn blue, washing the slices with water;

7) Placing the slices in low concentration alcohol for 5-10s, respectively, and dehydrating in absolute ethanol I and absolute ethanol II for 5min;

8) Placing the slice in xylene I and xylene II for 10min for transparency;

10 Microscopic observation.

The results show that: compared with WT, the infiltration of inflammatory cells in the AMPKgamma 2-KO group is enhanced; compared to WT MI group, ampkγ2-KO MI group inflammatory cell infiltration was more pronounced (fig. 3D), suggesting that ampkγ2 deficiency may increase inflammation.

In conclusion, whole body knockout of ampkγ2 mice (ampkγ2-KO) exacerbates MI and macrophage infiltrated cardiac insufficiency.

Example 4: mice that overexpress ampkγ2MI systemically (AAV-ampkγ2 MI) alleviate MI and macrophage infiltrated cardiac insufficiency.

(1) Mice overexpressing ampkγ2MI (AAV-ampkγ2 MI) were established.

Male 8-week-old C57BL/6 mice were modeled as described in example 1, and after MI modeling was successful, 40 male 8-week-old C57BL/6JMI mice were divided into the following 2 groups by the random table method: AAV-CMV-GFP and AAV-CMV-AMPK gamma 2 experimental groups, 20 in each group, were injected with AAV-CMV-GFP and AAV-CMV-AMPK gamma 2, respectively, from the tail vein.

(2) Mice were evaluated for cardiac contractile function by ultrasound.

See in particular example 3.

The results show that: compared with MI+AAV-CMV-GFP mice, the contraction function of the MI+AAV-CMV-AMPK gamma 2 mice is improved, and the mice have remarkable statistical significance (figures 4A-B), which suggests that the overexpression of AMPK gamma 2 can alleviate heart dysfunction caused by MI.

(3) Masson staining detects myocardial fibrosis and WGA staining detects cardiomyocyte hypertrophy.

See in particular example 3.

The results show that: compared to mi+ AAV-CMV-GFP mice, mi+ AAV-CMV-AMPK gamma 2 mice reduced myocardial fibrosis and cardiomyocyte hypertrophy (fig. 4C), suggesting that overexpression of AMPK gamma 2 may result in improved cardiac tissue inflammation.

(4) Immunohistochemical staining of CD68 detected inflammatory cell infiltration.

See in particular example 3.

The results show that: AAV-CMV-ampkγ2 improved infiltration of CD68 in MI-3D, suggesting that ampkγ2 may protect cardiac remodeling after MI by inhibiting macrophage recruitment (fig. 4D).

Taken together, mice that overexpress ampkγ2MI systemically (AAV-ampkγ2 MI) alleviate MI and macrophage infiltrated cardiac insufficiency.

Example 5: lyz2 mice with specific loss of AMPKγ2 (LyZ 2-AMPKγ2-CKO) on macrophages exacerbate MI and macrophage infiltrated cardiac dysfunction.

(1) Mice were evaluated for cardiac contractile function by ultrasound.

See in particular example 3.

The results show that: compared with flox/flox mice, lyZ-ampkγ2-CKO mice were significantly reduced in contractile function (fig. 5B-C), with significant statistical significance, suggesting that macrophage-specific loss of ampkγ2 can exacerbate cardiac dysfunction.

(2) F4/80 and CD11b co-staining, flow cytometry assessed macrophage infiltration in LyZ2-AMPKγ2-CKO and flox/flox MI mice.

The method comprises the following specific steps:

1) Taking 100 μl of cell suspension;

2) Adding a proper amount of surface antibody, and incubating for 15min at room temperature in a dark place;

3) Adding 1-2mL of flow type dyeing buffer solution, centrifuging for 5min at 300g, and discarding the supernatant;

4) Cells were resuspended with 500 μl flow staining buffer, detected and analyzed on the machine.

The results show that: the macrophage infiltration enhancement was further exacerbated in LyZ-AMPKγ2-CKO mice compared to flox/flox mice (FIGS. 5D-E).

(3) CD68 staining evaluation of macrophage infiltration in LyZ2-AMPKγ2-CKO and flox/flox MI mice.

See in particular example 3.

The results show that: the LyZ2-AMPKγ2-CKO group showed enhanced inflammatory cell infiltration compared to flox/flox MI (FIG. 5F).

(4) LyZ2-AMPKγ2-CKO and flox/flox MI mice, HE staining to detect cardiomyocyte morphology, masson staining to detect myocardial fibrosis, WGA staining to detect cardiomyocyte hypertrophy.

Masson, WGA staining is described in example 3.

HE staining evaluates cardiomyocyte morphology:

1) Taking myocardial tissue, fixing with 4% formaldehyde, embedding with conventional paraffin, and slicing with 5 μm;

2) Slices were conventionally dewaxed with xylene, washed with ethanol to each stage: xylene (I) 5 min- & gt xylene (II) 5 min- & gt 100% ethanol 2 min- & gt 95% ethanol 1 min- & gt 80% ethanol 1 min- & gt 75% ethanol 1 min- & gt distilled water washing 2min;

3) Hematoxylin staining for 5min, washing with tap water;

4) Ethanol hydrochloride differentiation is carried out for 30s;

5) Soaking in tap water for 15min;

6) Placing eosin solution for 2min;

7) Conventional dehydration, transparency and sealing sheet: 95% ethanol for 1min, 100% ethanol (I) for 1min, 100% ethanol (II) for 1min, xylene (I) for 1min, xylene (II) for 1min, and neutral resin sealing;

8) The morphology was observed under a microscope and stored photographic for statistical analysis.

The results show that: lyZ2-AMPKγ2-CKO mice had further exacerbations of myocardial hypertrophy and myocardial fibrosis compared to flox/flox mice (FIG. 5G).

Taken together, macrophage-specific depletion of AMPK gamma 2 exacerbates MI and macrophage-infiltrated cardiac dysfunction.

Example 6: macrophage specific over-expression AMPKγ2MI mice (AAV-F4/80-GFP-

Ampkγ2) reduces MI and macrophage infiltrated cardiac dysfunction.

(1) Mice were evaluated for cardiac contractile function by ultrasound.

See in particular example 3.

The results show that: compared with MI+AAV-F4/80-GFP mice, the contraction functions of the MI+AAV-F4/80-GFP-AMPKγ2 mice are improved, and the mice have remarkable statistical significance (figures 6A-B), so that macrophage-specific overexpression of AMPKγ2 can alleviate cardiac dysfunction caused by MI.

(2) F4/80 and CD11b co-staining, flow cytometry evaluated macrophage infiltration in MI+ AAV-F4/80-GFP and MI+ AAV-F4/80-GFP-AMPK gamma 2 mice.

See in particular example 5.

The results show that: the MI+ AAV-F4/80-GFP-AMPKγ2 group improved inflammatory cell infiltration compared to the MI+ AAV-F4/80-GFP group (FIGS. 6C-D).

(3) CD68 staining evaluates macrophage infiltration in MI+ AAV-F4/80-GFP and MI+ AAV-F4/80-GFP-AMPKγ2 mice.

See in particular example 3.

The results show that: the MI+ AAV-F4/80-GFP-AMPKγ2 group improved inflammatory cell infiltration compared to MI+ AAV-F4/80-GFP (FIG. 6E).

(4) MI+AAV-F4/80-GFP and MI+AAV-F4/80-GFP-AMPKγ2 mice, myocardial fibrosis was detected by Masson staining, and cardiomyocyte hypertrophy was detected by WGA staining.

See in particular example 3.

The results show that: MI+ AAV-F4/80-GFAMPKγ2 mice improved cardiac hypertrophy and cardiac fibrosis compared to MI+ AAV-F4/80-GFP mice (FIG. 6F).

In conclusion, macrophage-specific overexpression of AMPK γ2 rescues cardiac remodeling following myocardial infarction.

Example 7: the AMPK gamma 2 knockout aggravates IFN-gamma induced macrophage migration and inflammation.

(1) transwell experiments studied si-AMPK gamma 2 mediated macrophage migration.

The method comprises the following specific steps:

1) RAW264.7 is planted in a six-hole plate;

2) After 24 hours of adherence, gently blowing and digesting the cells by using prepared pancreatin, and placing the cells into a 15mL centrifuge tube;

3) Centrifuging at 5min and 3000bpm in a normal temperature centrifuge;

4) Discarding the supernatant, adding 2mL of serum-free DMEM into each sample centrifuge tube, and blowing and mixing uniformly;

5) Taking a 12-well plate, adding 500 mu L of 10% serum DMEM into each well, taking out a small chamber, adding 200 mu L of serum-free cell suspension into the upper chamber of each well, and incubating the lower chamber with 500 mu L of 10% serum DMEM;

6) After 24h, observing the cell state and the number of the migrated cells, taking down the cell from the 12-well plate, flushing the cell with PBS for 2 times and 5min each time, then adding 1mL of paraformaldehyde solution into each well, and putting the cell into the paraformaldehyde solution for fixing for 30min;

7) Taking a giemsa staining kit, adding the giemsa staining reagent into a 24-well plate according to the solution A: liquid B (1:1) ratio, staining for 30min, adding giemsa staining liquid into a 24-well plate according to liquid A: dyeing the solution B (1:3) for 10min;

8) Then PBS washes for 2 times, each for 3min;

9) Taking down the round patch of the small chamber by using a 1mL syringe needle, placing the lower surface of the small chamber on a glass slide upwards, sealing the patch by neutral resin, and smearing nail polish along four edges after air drying to ensure that a specimen cannot fall off;

10 And recorded in optical photomicrographs.

The results show that: si-ampkγ2 resulted in more cell migration under IFN- γ stimulation (fig. 7A-B), indicating that the absence of ampkγ2 aggravates IFN- γ -induced macrophage infiltration.

(2) Scratch test study si-AMPK gamma 2 mediated macrophage migration.

The method comprises the following specific steps:

1) RAW264.7 species into 6 well plates;

2) After 24h of cell adhesion, the cell density was observed, and a cross was drawn in the longitudinal and transverse directions with a small gun head of 1. Mu.L when the cell density was 80%. Then washing the cells 3 times with PBS for 2min each to wash out dead cells;

3) At this time, photographing and recording the initial condition of 0h in an inverted microscope;

4) After 24h, the cells were rinsed 3 times with PBS for 2min each to wash away dead cells, at which time the migration was recorded for 24h by taking a photograph with an inverted microscope.

The results show that: si-AMPK γ2 resulted in more cell migration and accelerated migration areas under IFN- γ stimulation (fig. 7C-D).

(3) The Western Blot method detects the expression of representative proteins IL-6, MCP1 and AMPK gamma 2 of RAW264.7si-AMPK gamma 2 inflammatory factors.

See in particular example 2.

The results show that: in RAW264.7, the expression of IL-6 and MCP1 was increased in the si-AMPKγ2 group compared to the si-NC, and at the same time, the expression of IL-6 and MCP1 was increased in the si-AMPKγ2+IFN- γ group compared to the si-NC+IFN- γ group, which was statistically significant (FIG. 7E-F). The results suggest that ampkγ2 knockout may exacerbate inflammation of RAW.

(4) Fluorescent quantitative PCR detects the expression of representative protein IL-6 and MCP1 gene mRNA of RAW264.7si-AMPK gamma 2 inflammatory factor.

See in particular example 2.

The results show that: the expression of the IL-6 and MCP1 genes in the si-AMPKγ2 group is up-regulated in RAW264.7 compared with the si-NC, and the expression of the IL-6 and MCP1 genes in the si-AMPKγ2+IFN- γ group is up-regulated in RAW264.7 compared with the si-NC+IFN- γ, which is of significant statistical significance. The results suggest that ampkγ2 knockout may exacerbate INF- γ -induced inflammation of RAW from RNA levels (fig. 7G).

Example 8: AMPK gamma 2 overexpression can reduce IFN-gamma induced macrophage migration and inflammation.

(1) transwell experiments studied pc3.1-ampkγ2 mediated macrophage migration.

See in particular example 7.

The results show that: pc3.1-ampkγ2 significantly reduced more cell migration under IFN- γ stimulation (fig. 8A-B), suggesting that overexpression of ampkγ2 reduces IFN- γ induced macrophage infiltration.

(2) Scratch test study pc3.1-ampkγ2 mediated macrophage migration.

See in particular example 7.

The results show that: pc3.1-ampkγ2 decreased more cell migration and decreased migration area under IFN- γ stimulation (fig. 8C-D).

(3) The Western Blot method detects expression of representative proteins IL-6, MCP1 and AMPK gamma 2 of RAW 264.7pc3.1-AMPK gamma 2 inflammatory factors.

See in particular example 2.

The results show that: there was no significant change in pc3.1-AMPKγ2 group IL-6 and MCP1 expression compared to pc3.1-con, but there was a significant statistical significance in increasing pc3.1-AMPKγ2+ IFN- γIL-6 and MCP1 expression compared to pc3.1-con+ IFN- γ (FIG. 7E-F). The results suggest that the inflammation of RAW caused by INF- γ can be reduced after ampkγ2 is overexpressed.

(4) Fluorescent quantitative PCR detects the expression of representative protein IL-6 and MCP1 gene mRNA of RAW264.7 pc3.1-AMPK gamma 2 inflammatory factor.

See in particular example 2.

The results show that: the IL-6 and MCP1 gene mRNA expression in the pc3.1-AMPKγ2 group did not significantly change compared to pc3.1-con, but the IL-6 and MCP1 gene mRNA expression in the pc3.1-AMPKγ2+IFN- γ group was significantly statistically reduced compared to pc3.1-con+IFN- γ. The results suggest that overexpression of ampkγ2 can reduce the inflammation of RAW caused by INF- γ from RNA level (fig. 8G).

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a formulation that specifically overexpresses AMPK gamma 2 protein or an active fragment thereof for the preparation of a medicament for the prevention and/or treatment of heart failure caused by abnormal heart remodeling due to excessive inflammatory reaction after myocardial infarction.

2. The use according to claim 1, wherein the preparation of specific overexpression of AMPK γ2 protein or an active fragment thereof comprises:

(2) A polynucleotide encoding AMPK gamma 2 protein;

(4) An agonist of the AMPK gamma 2 protein described in (1);

3. Use of an agent for detecting the expression level of AMPK gamma 2 protein or an active fragment thereof in the preparation of a product, characterized in that said product is used for the prediction of excessive inflammatory response after myocardial infarction and/or for the evaluation of the therapeutic effect, prognosis.

4. The use according to claim 3, wherein AMPK gamma 2 protein or an active fragment thereof is used as a marker for predicting and/or treating effect and prognosis evaluation of excessive inflammatory reaction after myocardial infarction.

5. The use according to claim 4, wherein the product predicts myocardial damage and inflammatory response after MI or evaluates its therapeutic effect or prognosis by detecting that the expression level of AMPK gamma 2 protein or an active fragment thereof in blood, tissue or cells is below a reference value.

6. The use according to claim 3, wherein the product is a chip, a formulation or a kit.

7. A pharmaceutical composition comprising a formulation that specifically overexpresses AMPK gamma 2 protein or an active fragment thereof, and optionally a pharmaceutically acceptable carrier or excipient.

8. The use according to claim 7, wherein the preparation of the specific overexpression of AMPK γ2 protein or an active fragment thereof comprises:

(2) A polynucleotide encoding AMPK gamma 2 protein;

(4) An agonist of the AMPK gamma 2 protein described in (1);

9. Use of a pharmaceutical composition according to any one of claims 7-8 for the prevention and/or treatment of heart failure due to abnormal heart remodeling caused by excessive inflammatory reactions after myocardial infarction.

10. The application of a preparation for specifically over-expressing AMPK gamma 2 protein or active fragment thereof in preparing anti-inflammatory drugs.