阅读说明：本技术 Chmp2b蛋白靶向抑制剂及在缺血性心脏病中的应用 (CHMP2B protein targeted inhibitor and application thereof in ischemic heart disease ) 是由 马恒 李天� 穆楠 余璐 于 2021-01-25 设计创作，主要内容包括：本发明涉及药物技术领域,具体涉及CHMP2B蛋白靶向抑制剂及其在制备预防或治疗缺血性心脏病药物中的应用。本发明为CHMP2B蛋白靶向抑制剂用于治疗缺血性心脏病提供了重要理论依据,为现有缺血性心脏病的预防和治疗提供了新的药物解决方案。同时为已知药物二甲双胍或其可药用盐发掘了新的制药用途,扩宽了该药物的治疗范围。(The invention relates to the technical field of medicines, in particular to a CHMP2B protein targeted inhibitor and application thereof in preparing medicines for preventing or treating ischemic heart diseases. The invention provides an important theoretical basis for the application of the CHMP2B protein targeted inhibitor in treating the ischemic heart disease, and provides a new drug solution for the prevention and treatment of the existing ischemic heart disease. Meanwhile, the new pharmaceutical application of the known medicament metformin or the pharmaceutically acceptable salt thereof is developed, and the treatment range of the medicament is widened.)

Application of CHMP2B protein targeted inhibitor in preparation of drugs for preventing or treating ischemic heart diseases.

2. The use as claimed in claim 1, wherein the CHMP2B protein targeted inhibitor is metformin or a pharmaceutically acceptable salt thereof.

3. The use according to claim 2, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

4. The use according to claim 3, wherein the metformin hydrochloride is administered in a daily dose of 100mg/kg body weight of mice.

5. The use according to claim 3, wherein the metformin hydrochloride is administered in a daily dose of not more than 2550mg/kg body weight of a human.

6. The use according to claim 1, wherein the ischemic heart disease is any one of acute myocardial infarction and chronic ischemic heart disease.

7. A pharmaceutical formulation for inhibiting CHMP2B protein expression, comprising metformin or a pharmaceutically acceptable salt thereof as an active ingredient, together with one or more pharmaceutically acceptable carriers, diluents or excipients as auxiliary ingredients.

8. The pharmaceutical formulation for inhibiting the expression of CHMP2B protein according to claim 7, wherein the pharmaceutical formulation is in an oral dosage form.

9. The pharmaceutical preparation for inhibiting CHMP2B protein expression according to claim 8, wherein the dosage form of the pharmaceutical preparation is oral tablet or capsule.

10. The pharmaceutical formulation for inhibiting CHMP2B protein expression according to claim 9, wherein the pharmaceutical formulation is a sustained release, delayed release, sustained release or controlled release pharmaceutical formulation.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a CHMP2B protein targeted inhibitor and application thereof in ischemic heart disease.

Background

Data of a Chinese cardiovascular disease report 2019 show that cardiovascular diseases are the leading causes of death of Chinese people, and are higher than tumors and other diseases. Among them, myocardial infarction caused by coronary heart disease is an important cause of death rate and disability rate of Chinese people. At present, the incidence rate of coronary heart disease is still rising and high, the quality of life of most patients after percutaneous coronary artery intervention is poor, and no cure method exists at present.

The myocardial cells are highly differentiated terminal cells, abnormal events or protein destabilization in the cell survival process are mainly completed by the cells, and protein degradation and reutilization are efficiently, reliably and accurately carried out. Abnormal accumulation of biological macromolecules in cardiomyocytes during the course of protection against injury can cause an imbalance in cellular homeostasis, ultimately leading to cell death. Autophagy, a physiological process effective in preventing the accumulation of abnormal proteins and organelles, is essential for maintaining the homeostasis of cell self-renewal and energy. Myocardial autophagy dysfunction can lead to significant exacerbation of myocardial ischemia reperfusion injury. Similarly, myocardial ischemia reperfusion injury can also disrupt myocardial autophagy function. Therefore, actively increasing the autophagy function of the myocardium during ischemia reperfusion is an effective cardioprotective strategy.

At present, medicines for treating ischemic heart disease mainly depend on statins, nitrates, beta receptor antagonists and other medicines, but the medicines can not radically cure the ischemic heart disease. Therefore, the method has important clinical significance for exploring a new treatment strategy to improve the curative effect and improve the prognosis of patients with ischemic heart disease. The key factor for targeted regulation and control of ischemia reperfusion myocardial autophagy has a promising clinical application, and the unique treatment scheme conforms to the medical concept of precise target intervention.

Disclosure of Invention

The first purpose of the invention is to provide an application of a CHMP2B protein targeted inhibitor in preparation of a medicine for preventing or treating ischemic heart disease.

Charged multivesicular protein 2B (CHMP2B, Charged multivesicular body protein 2B) is an essential molecule for autophagy homeostasis as a component of the intracellular classifier complex (ESCT-III). Firstly, the invention discovers the relationship between CHMP2B overexpression and myocardial ischemia vulnerability in ischemia-reperfusion mice through in vivo and in vitro experimental researches.

In vivo experiments, Wild Type (WT) C57BL/6J mice are used as study objects, the CHMP2B protein is successfully over-expressed in the myocardium of the mice in a mode of thoracotomy, connection with a respirator and myocardial adenovirus point injection, pentobarbital sodium is injected into the abdominal cavity 3 days after injection, the respirator is connected, and the anterior descending branch of the coronary artery is ligated and ligated to remove the ligation, so that an in vivo myocardial ischemia reperfusion (MI/R) injury model is constructed. Wherein ischemia is 30min, reperfusion is 2h (signal pathway assay) or 4h (histology/morphology assay). Immunohistochemical result detection shows that compared with a blank control group, the myocardial CHMP2B protein is expressed in a large amount, LC3 II/I and p62 levels are obviously increased, the myocardial infarction area of (MI/R) mice is increased, and peripheral blood LDH and CK-MB levels are increased. Echocardiography results show significant reductions in left ventricular ejection fraction (EF%) and left ventricular minor axis shortening (FS%), with significant increases in MI/R mortality. Compared with the CHMP2B protein overexpression group, the MI/R + CHMP2B overexpression group has higher myocardial damage and mortality, and the results prove that: over-expression/accumulation of CHMP2B can lead to increased MI/R injury, with the development of myocardial ischemic vulnerability.

In vitro experiments rat H9c2 cardiomyocyte line was used as the subject with 95% N₂And 5% CO₂The oxygen deficiency and reoxygenation time is 15H and 1H to construct a myocardial cell oxygen deficiency and reoxygenation model (H/R). H/R leads to a decrease in cardiomyocyte viability and an increase in CK-MB activity and LDH levels in the cellular secretions. After the CHMP2B is transduced to over-express adenovirus for 48H, the CHMP2B is found to remarkably improve the protein levels of LC3 II/I, LAMP1 and p62 in cells, increase the apoptosis rate of myocardial cells under H/R and aggravate H/R injury.

According to the invention, through research, the accumulation of CHMP2B and Charged multicular body protein 2B in MI/R treated mice and H/R treated myocardial cells is increased, and the accumulated CHMP2B can induce the damage of autophagy flux, thereby enhancing the vulnerability to myocardial ischemia. Both in vivo and in vitro experiments indicate that up-regulation of CHMP2B protein levels inhibits cardiomyocyte autophagy flux, resulting in H/R cardiomyocyte degeneration and MI/R injury. Namely, the invention discovers and verifies for the first time that accumulation of CHMP2B damages the autophagy function of the heart and enhances the vulnerability of the heart system to ischemia-reperfusion injury. This finding suggests that accumulation of CHMP2B increases the risk of myocardial ischemia and is a potential target for the treatment of ischemic heart disease.

Secondly, the invention discovers that the interaction relationship between the protein Atrogin-1 and CHMP2B regulated by the metformin through research. Immunoprecipitation (IP) results confirmed that Atrogin-1 and CHMP2B have a clear binding effect with each other in H9c2 cardiomyocytes. Atrogin-1 expression can promote CHMP2B protein degradation. Atrogin-1, also known as MAFbx or Fbx32, is an E3 ubiquitin ligase that is specific in skeletal and cardiac muscle. So far, no relevant report on the internal relationship between Atrogin-1 and CHMP2B is found. The research proves that the CHMP2B is a substrate for Atrogin-1 degradation, and Atrogin-1 degrades CHMP2B through a ubiquitin-dependent pathway, thereby playing a key role in myocardial ischemia-reperfusion injury.

Finally, the research of the invention discovers a novel action mechanism that the metformin exerts myocardial protection effect by specifically inhibiting the expression of CHMP2B protein. The test result shows that: 1. the immunoblotting result shows that metformin can activate pAMPK level by exogenous metformin treatment in myocardial cells. Metformin further upregulated pAMPK levels compared to the H/R group. 2. The myocardial cells are treated by exogenous metformin, and immunoblotting results show that metformin can activate Atrogin-1 pathway and down-regulate CHMP2B pathway. 3. Under H/R and MI/R conditions, CHMP2B signal expression was up-regulated, and administration of metformin reduced CHMP2B accumulation. 4. Under H/R and MI/R conditions, autophagic flow was impeded, as evidenced by an upregulation of LC3 levels, an excessive accumulation of the substrate marker p62, and maintenance of autophagic flow functional homeostasis given metformin treatment. Increased myocardial infarct size in mice overexpressing myocardial CHMP2B and elevated levels of peripheral blood LDH and CK-MB, as compared to MI/R conditions. The echocardiogram result shows that the left ventricular ejection fraction (EF%) and the left ventricular short axis shortening rate (FS%) are obviously reduced, the MI/R death rate is obviously increased, and the exogenous metformin can be given to reduce the myocardial injury index and improve the cardiac function and the survival rate after ischemia.

The test results prove that the metformin can promote the degradation of accumulated CHMP2B by activating an AMPK/Atrogin-1 pathway, thereby playing a role in protecting ischemic myocardium. Specifically, metformin up-regulates Atrogin-1 by activating adenosine monophosphate activated protein kinase (AMPK), so that the interaction of Atrogin-1 and CHMP2B is enhanced, and CHMP2B protein is promoted in H/R and MI/R myocardial cellsThereby inhibiting accumulation-induced autophagy injury and ischemic vulnerability of CHMP 2B. That is, metformin therapy promotes Atrogin-1 degradation of CHMP2B via the AMPK-dependent pathway, maintaining steady state of myocardial autophagy flux at MI/R. On one hand, the result reveals a brand-new action mechanism of the metformin in myocardial ischemia protection, enriches the cognitive range of myocardial ischemia protection, and provides a new medicament for preventing and treating ischemic heart diseases; on the other hand, the application of the metformin in developing a CHMP2B protein targeted inhibitor is suggested. GermanyScholars report that: the number of neurons in hippocampal cytoplasmic CHMP2B positive granule vacuolated inclusion bodies was greater in patients with Alzheimer's disease than in normal controls (Brain Res.2019.PMID: 30414727 DOI:10.1016/j. brainnres.2018.11.008). This suggests that targeted inhibitors of CHMP2B protein may have some effect on the treatment of alzheimer's disease. Thus, as a variant of the invention, the invention also relates to the use of metformin for the preparation of a medicament for the treatment or prevention of diseases associated with the overexpression of CHMP2B protein.

According to the application of the invention, the metformin is metformin or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable salt of the metformin is preferably metformin hydrochloride, has better water solubility compared with metformin, and is beneficial to patients to take. The daily dose is 100mg/kg mouse body weight, or not more than 2550mg/kg human body weight.

According to the application of the invention, the ischemic heart disease is any one of acute myocardial infarction and chronic ischemic heart disease.

According to the application of the invention, when the CHMP2B protein targeted inhibitor drug is used for preventing or treating ischemic heart disease, the CHMP2B protein targeted inhibitor drug can be used alone or combined with one or more drugs including statins, nitrate ester preparations, beta receptor antagonists and salicylates. The invention therefore also relates to the use of targeted inhibitors of the CHMP2B protein in combination with other existing drugs for the treatment of ischemic heart disease.

Still another object of the present invention is to provide a pharmaceutical preparation for inhibiting the expression of CHMP2B protein.

The pharmaceutical preparation according to the present invention comprises metformin or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers, diluents or excipients as auxiliary ingredients. The dosage form of the pharmaceutical preparation is preferably an oral dosage form, including but not limited to tablets, capsules, preferably tablets. The medicinal preparation is a normal-release, delayed-release, sustained-release or controlled-release medicinal preparation. The pharmaceutical preparation can be prepared according to the conventional pharmaceutical preparation technology.

The invention has the beneficial effects that:

1. according to the invention, the in vivo MI/R and in vitro H/R experiments prove that the inhibition of the accumulation of CHMP2B is beneficial to the steady state of myocardial autophagy flux for the first time, and further is beneficial to the reduction of MI/R or H/R induced myocardial cell death and myocardial dysfunction, so that the CHMP2B protein is a potential target for treating ischemic heart disease. The application of the CHMP2B protein targeted inhibitor in preparing the medicine for preventing or treating the ischemic heart disease provides a new medicine solution for treating the existing ischemic heart disease, and has important clinical significance for improving the prognosis of patients with the ischemic heart disease.

2. The invention discovers for the first time that metformin can degrade CHMP2B in time through an AMPK/Atrogin-1 dependent way, and effectively improves autophagy flow. The metformin is a new mechanism of myocardial protection effect of metformin, enriches the understanding of the myocardial protection mechanism, and suggests that metformin can be developed into CHMP2B protein targeted inhibitor for treating or preventing diseases related to CHMP2B protein overexpression. Therefore, the invention develops a new pharmaceutical application for the known medicament metformin or the pharmaceutically acceptable salt thereof and develops a new application field.

Drawings

FIG. 1A: representative immunoblots of Atg5, LC3 II/I, LAMP1 and p62 proteins from H9c2 cells;

FIG. 1B: cell viability, CK-MB activity and LDH levels;

FIG. 2A: western blot maps of CHMP2B for the blank control group, model group, and treatment group;

FIGS. 2B-2C: analyzing the expression conditions of CHMP2B mRNA in peripheral blood of three Han coronary heart disease patients and three Han healthy people by a microarray method;

FIG. 3A: representative immunoblots of CHMP2B, LC3 II/I, LAMP1, and p62 proteins;

FIG. 3B: immunoblot graphs representative of CHMP2B, LC3 II/I, and p62 proteins following bafilomycin A1(10nM) or DMSO treatment of cardiomyocytes in the group of blank adenovirus and CHMP 2B-overexpressing adenovirus;

FIG. 3C: the apoptosis rate is measured by using a flow cytometry V-FITC/PI method, and the B2+ B4 quadrant represents the apoptosis rate;

FIG. 4A: representative immunoblots for each set of p-AMPK α, Atrogin-1, and CHMP 2B;

FIG. 4B: immunoblotches representative of p-AMPK α, Atrogin-1 and CHMP2B for each group 12H after metformin (2.5mmol/L) or Compound C (20 μmol/L) treatment with H9C2 cells;

FIG. 4C: measuring the relative expression quantity of mRNA of Atrogin-1 by qRT-PCR;

FIG. 5A: h9c2 cells are given plasmid tags of Myc-Atrogin-1 and Flag-CHMP2B, and the binding condition of Atrogin-1 and CHMP2B proteins is detected by using an Immunoprecipitation (IP) method;

FIGS. 5B-5C: h9c2 cells were treated with MG-132 (10. mu.M) for 12 and 24H;

FIG. 5D: 5C quantitative gray value result analysis;

FIG. 5E: IP results show that: atrogin-1 and CHMP2B protein are combined in the myocardium of C57 mouse;

FIG. 6A: immunohistochemistry results showed that CHMP2B protein was overexpressed (scale: 100. mu.M);

FIG. 6B: c57 mice were injected with representative immunoblots of Ad-CHMP2B, p-AMPK α, Atrogin-1, CHMP2B, p62, and LC3 II/I72 h prior to MI/R;

FIG. 6C: left Ventricular Ejection Fraction (LVEF) and left ventricular minor axis contraction rate (LVFS);

FIG. 6D: LDH level;

FIG. 6E: CK-MB activity;

FIG. 6F: an arrhythmia index;

FIG. 7A: representative immunoblot pictures of Ad-CHMP2B, p-AMPK α, Atrogin-1, CHMP2B, p62, and LC3 II/I;

fig. 7B-7C: left Ventricular Ejection Fraction (LVEF) and left ventricular minor axis contraction rate (LVFS);

FIG. 7D: LDH level;

FIG. 7E: CK-MB activity;

FIG. 7F: an arrhythmia index;

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

The first embodiment is as follows: experimental study on relationship among myocardial CHMP2B protein level, myocardial autophagy function and myocardial ischemia injury

1. Materials and Experimental design

1.1 in vivo experiments

1.1.1 Experimental materials

Male C57BL/6J mice (3-4 months of age) were purchased from the fourth university of military medical laboratory animal center. The study was approved by the animal ethics laboratory Committee of the fourth university of military medicine (IACUC-20200602). Animals were fed on a 12:12 hour day/night schedule with free diet.

1.1.2 Experimental groups and dosing

Mice were randomly divided into treatment and control groups. Metformin hydrochloride (100mg/kg/d) of Solarbiol company in Beijing was administered to the treatment groups by intraperitoneal injection, and the administration was continued for 7 days with physiological saline as a control. Control mice were given an equal amount of saline.

1.1.3 in vivo ischemia reperfusion surgery (MI/R)

Mice were anesthetized with 2% isoflurane, intubated with trachea, and mechanically ventilated with a small animal ventilator (chengtai alliance ltd, china). The left lateral aspect was opened and Left Anterior Descending (LAD) was ligated with 7-0 nylon suture, ischemic for 30 minutes, 2 hours (signal molecule assay) or 4 hours (morphological assay) after reperfusion.

1.2 in vitro experiments

1.2.1 Experimental materials

H9c2 cells were a subcloned cell line derived from embryonic myocardial tissue from BD1X rats purchased from Shanghai cell Bank, Chinese academy of sciences. Modified Du's medium (DMEM, Gibco, USA), 10% fetal bovine serum (Gibco, USA), penicillin streptomycin mixture (Gibco, USA) and 1% (Solarbiol, Beijing) were used. Cell culture at 37 ℃ with 5% CO₂The culture under the conditions of (1). The fluid was changed every two days and experiments were performed when the cells reached 70-80% confluence.

1.2.2 Experimental groups and dosing

H9c2 cells were randomly divided into treatment, model and blank controls. The treatment group is treated with metformin hydrochloride (hereinafter, each metformin is used as a substitute, 2.5mmol/L) for 12h, and the model group and the blank control group are treated with DMEM medium for 12h, wherein the gas environment of the model group is an anoxic reoxygenation environment.

1.2.3 hypoxic/reoxygenation injury (H/R)

In addition to the blank control group, H9c2 cells in each of the other groups first contained 95% N₂And 5% CO₂Is treated in an anoxic incubation tray (Billups-Rothenberg, USA) for 15 hours and then reoxidized (95% air and 5% CO)₂) For 1 hour.

2. Molecular, morphological and functional experiments

2.1 immunoblotting and immunoprecipitation

Rabbit anti-p-AMPK α (#2535), AMPK α (#5832), CHMP2B (#76173), α -tubulin (#9099), p62(#16177), LC3B (#3868), and atg5(#12994) antibodies were purchased from Cell Signaling Technology, inc (Danvers, USA); rabbit anti-Atrogin-1 (Muscle thiophy box F gene 1, ab168372) and LAMP1(ab24170) antibodies were purchased from Abcam, USA; sheep anti-CHMP 2B (AF7509) was purchased from R & D System, Inc., USA, MG-132(10 μ M) and Bafilomycin-A1 (Baf-A1,10nM) were purchased from MCE, USA. Immunoprecipitation an immunoprecipitation kit from Invitrogen, usa was used. The enhanced chemiluminescence assay kit was purchased from Millipore, germany.

2.2 immunohistochemistry and immunofluorescence

Immunofluorescence and immunohistochemical staining were performed on cardiac sections, and immunofluorescence experiments were performed after H9c2 cells were fixed, according to the methods reported in the prior art (Yan et al circulation 136(22), 2162-2177; Zhao et al Ann Transl Med 8(10), 647).

2.3 cell viability

Cell viability was determined using the Solarbiol CCK-8 kit. mu.L of H9c2 cell culture medium was added to 10. mu.L of CCK-8 solution. After incubation at 37 ℃ for 1 hour, absorbance at 450nm was measured using a microplate reader (Molecular Devices, CA, USA).

2.4 myocardial injury detection

Blood samples were taken 24 hours after MI/R and assayed spectrophotometrically for CK-MB activity and LDH concentration.

2.5 adenovirus transfection

H9c2 cells were transfected with Ad-CHMP2B (GemmaPharma, Shanghai). Adenovirus titers of 1.0X 10 were used in this study¹⁰pfu/mL, infection ratio (MOI) of 100: 1. 48h after transfection, metformin treatment was given.

The Rattus Flag-CHMP2B plasmid and the Myc-Atrogin-1 plasmid (RR212771, OriGene, Rockville, Md., USA) were used for IP experiments. Complete Open Reading Frames (ORFs) of Rattus CHMP2B containing Not I and Kpn I cleavage sites at the 5 'and 3' ends were constructed by DNA synthesis (Shanghai Biotech Co., Ltd.). The synthetic cDNA was inserted into the pFLAg-CMV-2 vector. GFP-mRFP-LC3 virus is an adenovirus tool for studying autophagy flux (20812860). Cells were adenovirus (1.0X 10) with GFP-mRFP-LC3¹⁰pfu/mL, Henken, Shanghai, China) at an MOI of 100: 1. Cells were injected with metformin 48h after transfection.

2.6 flow cytometry analysis

Cells were harvested with Tyrisin (Solarbiol, China, Beijing) and washed 3 times with PBS. Staining was performed with annexin V-FITC/PI apoptosis detection kit (BD Biosciences, San Jose, Calif., USA) and further analyzed by FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif., USA).

2.7 qRT-PCR

RNA was extracted and PCR was quantified in real time. Primer sequences used in this study were as follows (5 '-3')/Atrogin-1 forward GGGAGTACTAAGGAGCGCCA, reverse TCTGGACCAGCGTGCATAAG; actin goes forward GTCCCTCACCCTCCCAAAAG and backward GCTGCCTCAACCTCAACCC.

2.8 echocardiography and electrocardiogram

Echocardiography was performed using Vevo 2100(visualsonics, Canada). Mice were anesthetized with 2% isoflurane. Electrodes are connected to the four limbs of the mouse to obtain a high-resolution two-dimensional model image of the Left Ventricle (LV), and then the left ventricular shortening fraction (LVFS) and the ejection fraction (LVEF) are analyzed.

3. Data processing

Data are presented as mean ± mean standard deviation (SEM). Statistical differences between groups were analyzed using one-way analysis of variance and Dunnett's test. All statistical analyses were performed using GraphPad Prism 8.0(San Diego, CA, USA). p.ltoreq.0.05 is considered statistically different.

4. Conclusion

(1) Metformin can prevent H/R-induced myocardial autophagy disorder and cell injury

In H/R treated H9c2 cells, the expression levels of model groups Atg5, LC3 II/I, LAMP1 and p62 were significantly increased (FIG. 1A), indicating that autophagic flow was impeded. In vitro fluorescence results confirmed the accumulation of autophagosomes. Compared with the model group, the metformin-treated group significantly reduced the level of H/R cardiomyocyte p62, which is shown by the levels of Atg5, LC3 II/I, LAMP2 (fig. 1A) and a decrease in yellow fluorescence. In addition, the metformin-treated group protected H/R-induced myocardial cell injury, increased cell viability, and inhibited CK-MB activity and LDH concentration levels (fig. 1B). In fig. 1, M: metformin; v: administering DMEM; H/R: oxygen deficiency and reoxygenation; RLU: a relative luminous value; compared with blank control group^*p is less than or equal to 0.05, and the ratio of p to model group^#p ≤0.05。

(2) CHMP2B was over-accumulated in cardiomyocytes at H/R. CHMP2B mRNA is up-regulated in peripheral blood of patients with coronary heart disease compared to healthy humans

CHMP2B levels were significantly elevated (approximately 3.8-fold) in H/R-induced autophagy-impaired cardiomyocytes compared to the blank control group (fig. 2A). The metformin treated group inhibited CHMP2B levels and restored cardiomyocyte autophagy flux levels (fig. 2A).

(3) Over-expression of CHMP2B decreased the level of cardiomyocyte autophagy, exacerbating the vulnerability of myocardial MI/R.

We evaluated the relationship between autophagy inhibition and CHMP2B accumulation. H9c2 cells were infected with blank adenovirus (Ad-Con) and CHMP2B overexpression adenovirus (Ad-CHMP2B), respectively, for 48H. Adenovirus-mediated overexpression of CHMP2B significantly increased protein levels of LC3 II/I, LAMP1 and p62 in cells (fig. 3A). Autophagy markers were determined after treatment with bafilomycin A1 (Baf-A1). Ad-Con group LC3 II/I and p62 protein levels increased 48h after Baf-A1 treatment. However, no significant changes were shown on cardiomyocytes overexpressing CHMP2B (fig. 3B), suggesting that excessive CHMP2B accumulation has contributed to autophagy disorders. Inhibition of CHMP 2B-related autophagy flux enhanced H/R-induced cardiomyocyte apoptosis (fig. 3C). These results indicate that while CHMP2B mediates autophagy under basal conditions, excessive CHMP2B accumulation inhibits autophagy and enhances H/R-induced cardiomyocyte death.

Furthermore, we analyzed the clinical data of patients with coronary heart disease by the GEO database (serial number: GSE71226) of the national center for Biotechnology information. We analyzed these data using volcano plots from total RNA samples extracted from peripheral blood of chinese han-nationality coronary disease patients and healthy controls. The chip results showed that the CHMP2B RNA levels were up-regulated in patients with coronary heart disease compared to healthy people. These results indicate that accumulation of CHMP2B is associated with autophagy dysfunction and ischemic injury.

(4) Metformin inhibits CHMP2B accumulation in H/R-treated cardiomyocytes via AMPK

The metformin treated group significantly enhanced phosphorylation of AMPK in H/R-treated cardiomyocytes, enhanced expression of Atrogin-1, a muscle-specific ubiquitin ligase, and inhibited accumulation of CHMP2B in H/R cardiomyocytes (fig. 4A). In addition, metformin up-regulates the expression levels of Atrogin-1 protein and mRNA via the AMPK pathway (FIGS. 4B and 4C), while inhibiting the level of CHMP 2B. However, these effects were blocked by AMPK activity Compound C (CC, 20. mu.M) (FIGS. 4B-4C). AMPK inhibitors result in decreased cardiomyocyte autophagy function, which results in the blockade of autophagy flow. We also found that Atrogin-1 and CHMP2B proteins co-localize in cardiomyocytes. Metformin treatment significantly enhanced the fluorescent co-localization of Atrogin-1 and CHMP2B during H/R injury. These results indicate that Atrogin-1 interacts directly with CHMP2B and that metformin regulates this process by activating AMPK under H/R conditions.

(5) Degradation of CHMP2B by Atrogin-1

We confirmed the interaction of Atrogin-1 with CHMP2B by in vitro experiments. In H9c2 cells, flag-tagged CHMP2B was associated with myc-tagged Atrogin-1 protein (FIG. 5A). Proteasome inhibitor mg-132(10 μ M) treatment of cardiomyocytes resulted in ubiquitination of CHMP2B (FIG. 5B) and a time-dependent effect (FIG. 5D). Our in vivo experiments demonstrated the interaction between Atrogin-1 and CHMP2B (FIGS. 5C-5E). These results show that: CHMP2B is a ubiquitination degradation substrate for Atrogin-1 in cardiomyocytes.

(6) Accumulation of CHMP2B in vivo can reduce autophagy levels and exacerbate MI/R injury

3-4 month old C57 wild-type mice were injected ventricles with CHMP2B over-expressed adenovirus (Ad-CHMP2B) or adenovirus blanks (Ad-Con) for 48 hours and the infected myocardial tissue was labeled with GFP. Immunofluorescence and immunohistochemistry results show that an in vivo model of Ad-CHMP2B overexpression was successfully established (fig. 6A). We found that over-expressed CHMP2B had no effect on AMPK phosphorylation and Atrogin-1 expression. However, LC3 II/I and p62 levels were significantly elevated in myocardial tissue and maintained at higher levels during MI/R (FIG. 6B). In vivo experiments, overexpression of CHMP2B significantly aggravated myocardial MI/R injury, as evidenced by a decrease in ejection fraction and LVFS (FIG. 6C), upregulation of LDH and CK-MB (FIGS. 6D-F), and an elevated arrhythmia index (FIG. 6F). Over-expressed CHMP2B significantly increased mortality in MI/R injured mice. We also found that CHMP2B overexpression decreased autophagy levels and that metformin cardioprotection up-regulated autophagy levels under CHMP2B treatment.

(7) Metformin enhances the myocardial AMPK/Atrogin-1 pathway and prevents accumulation of CHMP2B in MI/R injury

In vivo MI/R injury inhibited AMPK phosphorylation and reduced Atrogin-1 levels, but absent myocardial CHMP2B levels, compared to control. In addition, myocardium LC3 II/I and p62 accumulated in MI/R hearts, indicating impaired autophagic flow (fig. 7A). Metformin therapy can significantly improve MI/R heart AMPK phosphorylation level and Atrogin-1 level, and effectively inhibit CHMP2B accumulation. These effects were associated with a decrease in LC3 II/I and p62 levels, indicating that levels of autophagy were activated (FIG. 7A). In vivo, metformin therapy enhances the co-localization of Atrogin-1 and CHMP2B to the myocardium at MI/R. Metformin therapy inhibited myocardial contractile dysfunction (fig. 7B-D), protected the heart from MI/R injury (fig. 7D, F), reduced the severity index of arrhythmia (fig. 7F), and significantly reduced mortality in MI/R injured mice.

EXAMPLE 2 preparation of metformin tablet

The metformin hydrochloride is mixed with conventional auxiliary materials, and the metformin hydrochloride tablet is prepared according to a conventional method. It is administered orally at a dose of 0.25g for adult, 3 times daily.

EXAMPLE 3 preparation of metformin sustained-release tablet

The metformin hydrochloride is mixed with conventional auxiliary materials, and the metformin hydrochloride sustained-release tablet is prepared according to a conventional method. It is administered orally at a dose of 0.5g for adult once and 1 time daily.

EXAMPLE 4 preparation of metformin enteric-coated tablet

The metformin hydrochloride is mixed with conventional auxiliary materials, and the metformin hydrochloride enteric-coated tablet is prepared according to a conventional method. It is administered orally at a dose of 0.25g for adult, 3 times daily.

Example 5 preparation of metformin capsules

The metformin hydrochloride is mixed with conventional auxiliary materials, and the metformin hydrochloride capsule is prepared according to a conventional method. It is administered orally at a dose of 0.25g for adult, 3 times daily.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.