Application of MG53 in preparation of medicine for diagnosing / treating alcoholic cardiomyopathy

Abstract

The invention discloses an application of MG53 in preparation of a medicine for treating alcoholic cardiomyopathy. By constructing an alcoholic cardiomyopathy model mouse, it is detected that the concentration of cardiac muscle tissue of the mouse is reduced, and it is proved that MG53 can be used as a potential marker for diagnosing alcoholic cardiomyopathy; the survival rate of mice and cardiac function indexes of cardiac ultrasonic detection can be effectively improved through intervention treatment of the MG53 recombinant protein, and it is proved that MG53 can be used as a potential medicine for treating the alcoholic cardiomyopathy and has important significance in prevention and treatment of the alcoholic cardiomyopathy. Meanwhile, it is verified that the MG53 can be applied to potential drugs for treating alcohol-related lipid metabolism disorders including the alcoholic cardiomyopathy and myocardial remodeling related diseases, and a new drug, health care product or food additive preparation and application scheme is provided for treatment of alcoholic cardiomyopathy and other alcohol-related damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an application of MG53 in preparing a drug for diagnosing / treating alcoholic cardiomyopathy. Background Art

[0002] Alcoholic cardiomyopathy is an acquired dilated cardiomyopathy caused by long-term heavy drinking, characterized by damage to myocardial structure and function caused solely by excessive drinking. It is generally believed that daily intake of more than 80g of alcohol for 5 years will lead to the development of alcoholic cardiomyopathy. The pathogenesis of alcoholic cardiomyopathy involves a variety of different biological processes. First, the direct toxic effect of ethanol is a key factor. Ethanol can induce oxidative stress in the myocardium and activate the renin-angiotensin system, further aggravating myocardial damage. In addition, acetaldehyde, the main metabolite of ethanol, has been widely studied and found to directly cause cardiomyocyte damage by disrupting actin-myosin interactions and causing mitochondrial dysfunction. However, there is currently a lack of diagnostic indicators for alcoholic cardiomyopathy, and the treatment of alcoholic cardiomyopathy lacks specificity. In view of this, it is crucial to explore new diagnostic and therapeutic methods.

[0003] MG53 (Mitsugumin-53 or TRIM72), as a key myogenic factor in cell membrane repair, plays an important role in the protection of various myocardial damages such as ischemic cardiomyopathy and doxorubicin-related cardiomyopathy. Moreover, MG53, as an E3 ubiquitin ligase, participates in the regulation of various biological processes and signaling pathways. However, the role of MG53 in alcoholic cardiomyopathy and its mechanism remain to be elucidated. Exploring the role of MG53 in alcoholic cardiomyopathy will not only help us understand its biological role in alcohol metabolism, but also provide a theoretical basis for the development of new drugs and therapeutic drugs for alcoholic cardiomyopathy.

[0004] Whether MG53 has a role in the treatment of alcoholic cardiomyopathy and the specific targets involved in its repair have not been reported. Therefore, the development of new cardioprotective drugs targeting MG53 for the treatment of alcoholic cardiomyopathy is expected to provide a new strategy for the treatment of alcoholic cardiomyopathy. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to:

[0006] The first object of the present invention is to provide a method for detecting MG53 expression as a diagnostic marker for alcoholic cardiomyopathy;

[0007] The second object of the present invention is to provide a use of MG53 in preparing a drug for treating alcoholic cardiomyopathy.

[0008] The objective of the present invention is achieved through the following technical solutions:

[0009] First, MG53 expression was detected as a diagnostic marker for alcoholic cardiomyopathy.

[0010] In certain specific embodiments, the alcoholic cardiomyopathy includes disease states such as heart damage, heart function damage, arrhythmia, dilated cardiomyopathy and further development of heart failure caused by alcohol and aldehydes.

[0011] Secondly, the application of MG53 in the preparation of drugs for treating alcoholic cardiomyopathy.

[0012] A related commercial product prepared by MG53 recombinant protein, the commercial product includes a pharmaceutical composition, food, health care product or food additive.

[0013] Statements about drugs or pharmaceutical compositions:

[0014] Preferably, the medicine or pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers include (but are not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents or adsorption carriers.

[0015] The medicine or pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

[0016] The administration route of the drug or pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or preventive effect, including but not limited to oral administration, intravenous injection, intramuscular injection, subcutaneous injection, sublingual dissolution, rectal irrigation, nasal spray, oral spray, topical skin administration or systemic transdermal administration.

[0017] The medicine or pharmaceutical composition of the present invention can also be used in combination with other medicines for treating alcoholic diseases including alcoholic cardiomyopathy and heart diseases. The combined use of multiple medicines can greatly increase the success rate of treatment.

[0018] Compared with the prior art, the present invention has at least the following advantages:

[0019] 1) The present invention constructs an alcoholic cardiomyopathy model mouse and detects a decrease in the expression of MG53 in the mouse myocardial tissue, proving that MG53 can be used as a potential marker for diagnosing alcoholic cardiomyopathy.

[0020] 2) The present invention can effectively improve the survival rate of mice induced by alcohol and cardiac function indexes detected by cardiac ultrasound by administering MG53 recombinant protein intervention treatment, which proves that MG53 can be used as a potential drug for the treatment of alcoholic cardiomyopathy and has important significance in the prevention and treatment of alcoholic cardiomyopathy;

[0021] 3) The present invention verifies that intervention with MG53 recombinant protein can effectively inhibit alcohol-induced myocardial tissue disorder and lipid droplet formation, and improve cardiomyocyte hypertrophy, that is, it verifies the potential drug application of MG53 for the treatment of alcohol-related lipid metabolism disorders and myocardial remodeling-related diseases including alcoholic cardiomyopathy, and provides new drug, health product or food additive preparation and application scheme for the treatment of alcoholic cardiomyopathy and other alcohol-related damages. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0023] Figure 1 The immunofluorescence detection and expression analysis of MG53 in the myocardial tissue of the alcoholic cardiomyopathy mouse model in Example 1 of the present invention; wherein (A) is an immunofluorescence detection diagram of MG53 in the myocardial tissue of the alcoholic cardiomyopathy mouse model, n=8; (B) is an expression analysis of MG53 by immunofluorescence content, n=8 (* represents p<0.05; Pair fed: control feed feeding; EtOH: 2.8% alcohol feed feeding);

[0024] Figure 2 Exogenous administration of MG53 recombinant protein to each group of mice improved the survival rate of mice induced by long-term alcohol, n=8 (* represents p<0.05, EtOH fed group compared with Pair fed group; # represents p<0.05, rhMG53 group compared with EtOH fed group; Pair fed: fed with control diet; EtOH fed: fed with 2.8% alcohol diet);

[0025] Figure 3 Exogenous administration of MG53 recombinant protein to each group of mice improved the cardiac function indicators detected by cardiac ultrasound under long-term alcohol induction; (A) is the image shown by cardiac ultrasound, n=8; (B) is the left ventricular ejection fraction LVEF statistically analyzed based on cardiac ultrasound, n=8; (C) is the left ventricular short axis shortening FS statistically analyzed based on cardiac ultrasound, n=8 (* represents p<0.05; Pair fed: fed with control diet; EtOH fed: fed with 2.8% alcohol diet);

[0026] Figure 4Exogenous administration of MG53 recombinant protein to each group of mice improved the myocardial pathological changes induced by long-term alcohol in mice; (A) is H&E staining and WGA staining of myocardial tissue, n=8;

[0027] (B) The area occupied by lipid droplets was statistically analyzed based on H&E staining of myocardial tissue, n=8; (C) The area of ​​single cardiomyocytes was statistically analyzed based on WGA staining of myocardial tissue, n=8 (* represents p<0.05; Pair fed: fed with control diet; EtOH fed: fed with 2.8% alcohol diet). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but it should not be understood as limiting the scope of protection of the present invention. Any equivalent changes in form made based on the concept of the present invention should be regarded as the scope of the present invention.

[0029] The present invention provides a general and / or specific description of the materials and test methods used in the experiment. The test methods or test methods involved are all conventional methods unless otherwise specified; the reagents or instruments used are all conventional products available on the market, prepared or used in a conventional manner, unless the manufacturer is specified.

[0030] Example 1: Preparation of Alcoholic Cardiomyopathy Mouse Model and Detection of MG53 Expression in Myocardial Tissue

[0031] 1) Preparation of Alcoholic Cardiomyopathy Mouse Model

[0032] The animals used in the experiment were provided by the Experimental Animal Center of Daping Hospital of Army Medical University. Male C57BL / 6 mice weighing 22-25 g were kept in the SPF Animal Experimental Center of Army Specialty Medical Center, with an indoor temperature of 23-25 ​​°C and a relative humidity of 40%-60%. They were fed with a standardized experimental diet in a constant temperature environment for at least 1 week before the experiment, with unlimited drinking water. The surgical operations during the experiment were in accordance with the regulations of experimental animals of Army Medical University. The mice were randomly divided into a control group (normal) and a model group (alcoholic cardiomyopathy), with 8 mice in each group and 4 mice in each cage, and were fed with liquid feed bottles.

[0033] Control group (Pair fed): Lieber-DeCarli control liquid feed (0% alcohol) was used for adaptive feeding in the first week, and then only liquid feed was given every day. The mice were free to eat. The food intake was counted between 15:00 and 17:00 every afternoon, the feeding bottles were cleaned, the feed was changed, and the mice were observed;

[0034] Model group (alcoholic cardiomyopathy (EtOH fed)): Lieber-DeCarli alcohol liquid feed was used, and gradient alcohol content adaptive feeding was performed in the first week. The mice were fed with Lieber-DeCarli alcohol liquid feed containing 0.75% alcohol for the first 3 days, and with Lieber-DeCarli alcohol liquid feed containing 1.5% alcohol from the 4th to the 7th day. Thereafter, they were only fed with Lieber-DeCarli alcohol liquid feed containing 2.8% alcohol every day. The mice were allowed to eat freely, and the food intake was counted between 15:00 and 17:00 every day. The feeding bottles were cleaned, the feed was changed, and the mice were observed.

[0035] Lieber-DeCarli control liquid feed and Lieber-DeCarli alcohol liquid feed were purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0036] 2) Detection of MG53 expression in myocardial tissue

[0037] At the 8th week of modeling, mice in each group were anesthetized and killed; heart tissues were fixed with 4% paraformaldehyde, routinely paraffin-embedded, and sliced ​​for the detection of MG53 content and localization in tissue samples; the results are shown in Figure 1 As shown, the MG53 content in the heart tissue of mice in the alcoholic cardiomyopathy model group was lower than that in normal mice.

[0038] Example 2: Improvement of alcoholic cardiomyopathy by MG53

[0039] 1) Experimental Grouping

[0040] The animals used in the experiment were provided by the Experimental Animal Center of Daping Hospital of Army Medical University. Male C57BL / 6 mice weighing 22-25 g were kept in the SPF Animal Experimental Center of Army Specialty Medical Center, with an indoor temperature of 23-25 ​​°C and a relative humidity of 40%-60%. They were fed with a standardized experimental diet in a constant temperature environment for at least 1 week before the experiment, with unlimited drinking water. The surgical operations during the experiment were in accordance with the regulations of experimental animals of Army Medical University. The mice were randomly divided into blank control group, treatment control group, model group and drug treatment group, with 8 mice in each group and 4 mice in each cage, and fed with liquid feed feeding bottles.

[0041] Blank control group (Pair fed-Vehicle): Use Lieber-DeCarli control liquid feed (0% alcohol) and perform adaptive feeding in the first week. Thereafter, only liquid feed is given every day. Mice can eat freely. The food intake is counted between 15:00 and 17:00 every afternoon, the feeding bottles are cleaned, the feed is changed, and the condition of the mice is observed.

[0042] Treatment control group (pair fed-rhMG53): feeding and observation methods were the same as those of the blank control group. After the adaptive feeding, MG53 recombinant protein was administered at 1 mg / kg body weight by subcutaneous pump and observed every day.

[0043] Model group (EtOH fed-Vehicle): Lieber-DeCarli alcohol liquid feed was used, and gradient alcohol content adaptive feeding was performed in the first week. The mice were fed with Lieber-DeCarli alcohol liquid feed containing 0.75% alcohol for the first 3 days, and with Lieber-DeCarli alcohol liquid feed containing 1.5% alcohol from the 4th to the 7th day. Thereafter, they were only fed with Lieber-DeCarli alcohol liquid feed containing 2.8% alcohol every day. The mice were allowed to eat freely, and the food intake was counted between 15:00 and 17:00 every day. The feeding bottles were cleaned, the feed was changed, and the mice were observed.

[0044] Treatment group (EtOH fed-rhMG53): feeding and observation methods were the same as the model group. After the adaptive feeding, human MG53 recombinant protein (purchased from Nearshore Biotechnology, LOT 20210826) was administered at 1 mg / kg body weight by subcutaneous pump and observed every day.

[0045] Lieber-DeCarli control liquid feed and Lieber-DeCarli alcohol liquid feed were purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0046] The results are as follows Figure 2 As shown, MG53 recombinant protein intervention can effectively improve the survival rate of alcohol-induced mice.

[0047] 2) Cardiac function test by cardiac ultrasound

[0048] At the end of the 8th week of modeling (modeling method as described above), the chest area of ​​mice in each group was depilated. After anesthesia, the mice were fixed on the operation board, and the long axis section of the left ventricle was measured using a small animal ultrasound probe, and the image was intercepted. Then, the left ventricular ejection fraction (LVEF) and the left ventricular short axis shortening rate (FS) were calculated based on the ultrasound test results. Each mouse was measured 3 times, and the average value was taken and the standard deviation was calculated. The results are shown in Figure 3 The results showed that long-term alcohol feeding induced a significant decline in cardiac function in mice, but the mice fed with MG53 recombinant protein had a significant improvement in cardiac function in time with alcohol feeding. This shows that supplementing with an appropriate concentration of MG53 recombinant protein can effectively improve cardiac dysfunction caused by long-term alcohol intake.

[0049] 3) Preparation of myocardial tissue pathological sections and H&E and WGA staining

[0050] At the end of the 8th week of modeling (modeling method as described above), the myocardial tissues of mice in each group were rinsed with ice-cold phosphate-buffered saline (PBS) to remove blood, fixed with 4% phosphate-buffered paraformaldehyde at 4°C for 24 hours, and then embedded in paraffin. Myocardial tissue paraffin sections (4 mm) were dried in a 70°C oven for 24 hours, dewaxed and rehydrated in xylene, 100% ethanol, 95% ethanol, 75% ethanol and PBS, and then stained with H&E staining kit and WGA probe, respectively. Subsequently, decolorization was performed, the plates were sealed with neutral resin, and the stored images were observed and scanned under an optical microscope; Image J software was used to quantify the area of ​​myocardial area occupied by lipid droplets in H&E images and the area of ​​single myocardial cells in WGA images, respectively.

[0051] The experimental results are as follows Figure 4 As shown, MG53 can effectively improve myocardial tissue pathological damage induced by long-term alcohol intake, reduce the area occupied by lipid droplets in myocardial tissue, and inhibit cardiomyocyte hypertrophy, which proves that MG53 recombinant protein plays a key role in the treatment of alcoholic cardiomyopathy and can be used as a potential drug for the treatment of alcohol-related myocardial remodeling and lipid metabolism disorders including alcoholic cardiomyopathy.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Application of detecting MG53 expression in the preparation of products for diagnosing alcoholic cardiomyopathy.

2. The use according to claim 1, characterized in that: The MG53 is downregulated in alcoholic cardiomyopathy.

3. The use according to claim 1, characterized in that: The product includes a reagent or a drug for detecting the expression level of MG53 in alcoholic cardiomyopathy.

4. The use according to claim 1, characterized in that: The alcoholic cardiomyopathy includes disease states such as heart damage, heart function damage, arrhythmia, dilated cardiomyopathy and further development into heart failure caused by alcohol and aldehydes.

5. Application of MG53 inhibitors in the preparation of drugs for treating alcoholic cardiomyopathy.

6. The use according to claim 5, characterized in that: The drug can be administered orally, sublingually, transdermally, by injection, instillation, mucosa, spray, infusion, rectum or parenterally.

7. The use according to claim 5, characterized in that: The dosage of the drug is 1 mg / kg.

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