Application method based on Mongolian medicine compound preparation in isolated cold preservation of heart

Abstract

The invention discloses an application method of a Mongolian medicine compound preparation in isolated cold preservation of heart, and relates to the technical field of heart preservation, comprising a dose-effect experiment process and an animal experiment process; the dose-effect experiment process comprises the following steps: testing the influence of choerospondium-7 pills with different concentrations on the isolated heart, carrying out multi-channel electric marking on the heart, recording field potential and electric signals under stimulation, and determining the optimal onset concentration; the animal experiment process comprises the following steps: step 1, animal isolated heart modeling and grouping (the isolated heart is stored in UW liquid for 8 hours and stored in UW liquid and Mongolian medicine for 8 hours), step 2, animal isolated heart stimulation and position mapping, and step 3, index recording and data acquisition. The cold preservation liquid can be optimized, damage caused by cold preservation is reduced, the preservation effect of the donor heart is improved, the treatment effect of heart transplantation is improved to the maximum extent, ischemic myocardial damage is reduced to the maximum extent, and the isolated heart function is maintained.

Description

Technical Field

[0001] The invention relates to the technical field of heart preservation, and in particular to an application method of a Mongolian medicine compound preparation in in vitro cold preservation of a heart. Background Art

[0002] Heart transplantation is the only effective treatment to save the lives of patients with end-stage heart disease. However, due to the short cold preservation period of donor hearts, many donor hearts are forced to be abandoned due to geographical or traffic reasons within the limited donor range. In addition, during the process of heart transplantation, the myocardium of the isolated heart is in a resting state during cold preservation (i.e., conventional 4°C low temperature, static preservation in UW), although some basic metabolic activities are maintained. However, under low temperature conditions, the donor heart must undergo the process of ischemia, hypoxia, reperfusion and re-oxygenation, resulting in myocardial tissue damage, affecting cardiac electrical conduction and even malignant arrhythmias. The occurrence of malignant arrhythmias is the main cause of death after heart transplantation. Therefore, the limitations of cold preservation technology have always been the main bottleneck that plagues the promotion and application of heart transplantation. Therefore, how to minimize ischemic myocardial damage and maintain the function of the isolated heart is a key factor in determining the success or failure of heart transplantation. Researchers have been trying to add some compounds to the cold preservation UW solution to extend the storage period of the donor heart and reduce the damage caused by cold ischemia. Thereby improving the quality of life and life expectancy of patients.

[0003] During cold storage, myocardial cells are exposed to cold ischemia, hypoxia and other factors, resulting in cell edema, tissue glycolysis and increased lactic acid. The metabolites produced cannot be discharged and accumulate in the microenvironment, causing ATP depletion, Ca2+ homeostasis disorder, mitochondrial disorder and increased reactive oxygen species (ROS) levels, resulting in myocardial infarction, ventricular tachycardia and ventricular fibrillation and other malignant arrhythmias. This is the main cause of isolated heart function damage. More than 70% of potential donor hearts are abandoned worldwide, and a large part of the reason is the limitations of preservation technology. Therefore, how to minimize ischemic myocardial damage and maintain isolated heart function is a key factor in determining the success or failure of heart transplantation.

[0004] Mongolian medicine Guangzao-7-flavor pills are a clinical prescription commonly used for cardiovascular diseases. It is a traditional Mongolian medicine compound preparation with warming properties, and has significant effects on the treatment of cardiovascular diseases, with few side effects. Therefore, scientific methods are used to study its efficacy and mechanism of action, provide safe and effective medicines for clinical use, and provide a scientific basis for the next step of developing new optimized cold-preserved heart drugs. Therefore, a method for applying Mongolian medicine compound preparations in isolated cold-preserved hearts is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for applying a Mongolian medicine compound preparation in the isolated cold storage of a heart, so as to solve the problems raised in the above-mentioned background technology.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for applying a Mongolian medicine compound preparation to an isolated cold-preserved heart, the method comprising a dose-effect experiment process and an animal experiment process;

[0008] The dose-effect experiment process is to test the effects of different concentrations of Guangzao-7 pills on isolated hearts, and perform multi-channel electrogramming on the hearts, record the field potential and electrical signals under stimulation, and determine the optimal effective concentration;

[0009] The animal experiment process includes the following steps:

[0010] Step 1: Animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group);

[0011] Step 2: Stimulate the isolated heart of the animal and mark its position;

[0012] Step 3: Record indicators and collect data;

[0013] Step 4: Myocardial tissue detection;

[0014] Step 5: Data statistics.

[0015] A further improvement of the technical solution of the present invention is that the dose-effect experiment process specifically includes the following steps:

[0016] Set up different concentrations of Guangzao-7 pill solutions;

[0017] The isolated hearts of normal rats were placed in different concentrations of Guangzao-7 pill solutions;

[0018] The isolated rat hearts were subjected to Langendorff perfusion multi-channel electrolabeling in different concentrations.

[0019] Record the field potential and electrical signals of the left and right ventricles under sinus conditions and 6HZ stimulation conditions, and analyze indicators such as heart rate, ECG, and ventricular conduction time;

[0020] Determine the optimal effective concentration in isolated rat hearts.

[0021] The further improvement of the technical solution of the present invention is that: the step 1, animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group) specifically includes:

[0022] Take a number of animal hearts and divide them into two groups according to the number;

[0023] One group of hearts was stored at 4°C for 8 hours, and KH solution, UW solution and Mongolian medicine were added. The concentration of Mongolian medicine was the best effective concentration determined by the dose-effect time to prepare the control group.

[0024] Another group of hearts were kept cold at 4°C for 8 hours, and KH solution and UW solution were added to prepare the injury group.

[0025] A further improvement of the technical solution of the present invention is that: in the step 2, the isolated heart of the animal is stimulated, and the mapping position specifically includes: two 64-channel pen-type mapping electrodes respectively record LV and RV signals, and synchronously record the simulated electrocardiogram II lead signal (pseudo-ECG II) with the negative pole in the right atrium and the positive pole at the apex slightly to the left ventricle.

[0026] A further improvement of the technical solution of the present invention is that: the step three, recording indicators and data collection are specifically as follows: after completing the treatment, different groups respectively record the electrophysiological signals during Sinus, 6Hz, and S1S2 stimulation, and under cold storage conditions, the heart is fixed with 10% methanol tissue fixative to obtain the animal heart myocardial tissue.

[0027] The further improvement of the technical solution of the present invention is that: the step 4, myocardial tissue detection specifically includes: immunohistochemistry detection, fluorescent probe detection, ELISA detection;

[0028] The immunohistochemical assay is used to detect the expression of apoptosis-related genes Bax and Bcl-2;

[0029] The fluorescent probe method is used to detect ROS and ATP content, and a fully automated analyzer is used to detect CPK and LDH;

[0030] The ELISA test is used to detect myocardial cell IL-6, IL-1β, HIF-1α, and TNF-α.

[0031] A further improvement of the technical solution of the present invention is that: the step five, data statistics specifically includes: respectively counting the detection indicators and detection values ​​of the animal hearts in the control group and the injury group, analyzing and comparing the numerical difference between the control group and the injury group, and explaining the protective effect of Guangzao-7-flavor pills on cold-preserved hearts.

[0032] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0033] 1. The present invention provides an application method of a Mongolian medicine compound preparation in an isolated cold-preserved heart, which can optimize the cold preservation solution, reduce the damage caused by cold preservation, improve the preservation effect of the donor heart, maximize the therapeutic effect of heart transplantation, minimize ischemic myocardial damage, and maintain the function of the isolated heart.

[0034] 2. The present invention provides an application method of a Mongolian medicine compound preparation in an isolated cold-preserved heart, and uses scientific means to study the efficacy and mechanism of action of the Mongolian medicine compound preparation, thereby providing safe and effective medicines for clinical use and providing a scientific basis for the next step of developing new optimized cold-preserved heart drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of the process of using a Mongolian medicine compound preparation in isolated cold-preserved hearts;

[0036] Figure 2 This is a schematic diagram of the electrocardiogram results under sinus rhythm recorded by pseudo-ECG II;

[0037] Figure 3 This is a schematic diagram of the results of cardiac conduction recorded by electrical mapping electrodes under 6Hz apical pacing rhythm;

[0038] Figure 4 It is a schematic diagram of the field potential results and the right ventricular conduction results;

[0039] Figure 5 Schematic diagram of ventricular pressure detection in sinus rhythm recorded by electrical mapping electrodes. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with embodiments:

[0041] Example 1

[0042] like Figure 1 As shown, the present invention provides a method for applying a Mongolian medicine compound preparation in an isolated cold-preserved heart, and the method for applying a Mongolian medicine compound preparation in an isolated cold-preserved heart includes a dose-effect experiment process and an animal experiment process;

[0043] The dose-effect experiment process is to test the effects of different concentrations of Guangzao-7 pills on isolated hearts, and conduct multi-channel electrograms on the hearts to record the field potential and electrical signals under stimulation to determine the best effective concentration;

[0044] The dose-effect experiment process specifically includes the following steps:

[0045] Set up different concentrations of Guangzao-7 pill solutions;

[0046] The isolated hearts of normal rats were placed in different concentrations of Guangzao-7 pill solutions;

[0047] The isolated rat hearts were subjected to Langendorff perfusion multi-channel electrolabeling in different concentrations.

[0048] Record the field potential and electrical signals of the left and right ventricles under sinus conditions and 6HZ stimulation conditions, and analyze indicators such as heart rate, ECG, and ventricular conduction time;

[0049] Determine the optimal effective concentration in isolated rat hearts.

[0050] The animal experiment process includes the following steps:

[0051] Step 1: Animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group);

[0052] Step 2: Stimulate the isolated heart of the animal and mark its position;

[0053] Step 3: Record indicators and collect data;

[0054] Step 4: Myocardial tissue detection;

[0055] Step 5: Data statistics.

[0056] The first step, animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group) specifically includes:

[0057] Take a number of animal hearts and divide them into two groups according to the number;

[0058] One group of hearts was stored at 4°C for 8 hours, and KH solution, UW solution and Mongolian medicine were added. The concentration of Mongolian medicine was the best effective concentration determined by the dose-effect time to prepare the control group.

[0059] Another group of hearts were kept at 4°C for 8 hours and added with KH solution and UW solution to make the injury group;

[0060] In step 2, the animal's isolated heart is stimulated, and the mapping positions specifically include: two 64-channel pen-type mapping electrodes record LV and RV signals respectively, and simultaneously record the pseudo-ECG II lead signal, with the negative electrode in the right atrium and the positive electrode in the apex slightly to the left ventricle;

[0061] The experimental results are as follows Figure 2As shown, A is a representative ECG graph; B is a heart rate graph; C is a PR interval graph; D is a QRS width graph; E is a QT interval graph; F is a ventricular effective refractory period graph; G is a vERP / QT graph; H is a ventricular conduction time graph; I is a CV*vERP graph;

[0062] Experimental results: From the perspective of ECG morphology, the ECG morphology of the UW+GZ7 group was more normal, and the ST segment elevation phenomenon was obvious in the UW group; from the perspective of various indicators, the UW+GZ7 group was more normal. Compared with the UW+GZ7 group, the PR interval was prolonged, the QRS wave was wider, the QT was longer, and the intraventricular conduction time was longer; compared with the UW group, the vERP / QT of the UW+GZ7 group was larger, and the susceptibility to arrhythmias was lower.

[0063] Step 3, recording indicators and data collection, specifically includes: after completing the treatment, different groups respectively record the electrophysiological signals during Sinus, 6Hz, S1S2 stimulation, and under cold storage conditions, fix the heart with 10% methanol tissue fixative to obtain the animal heart myocardial tissue;

[0064] The experimental results are as follows Figure 3 As shown, Figure A: left ventricular conduction, conduction dispersion isochronous diagram under 6Hz stimulation; Figure B: left ventricular conduction velocity statistics under 6Hz stimulation; Figure C: left ventricular conduction dispersion statistics under 6Hz stimulation;

[0065] The experimental results showed that the UW+GZ7 group significantly accelerated ventricular conduction velocity and reduced left ventricular conduction dispersion.

[0066] The experimental results are as follows Figure 4 As shown, Figure A: Representative diagram of right ventricular field potential signal under spontaneous rhythm; Figure B: Statistical diagram of right ventricular conduction under 6Hz stimulation;

[0067] The experimental results: Compared with the UW+GZ7 group, the right ventricular field potential signal of the UW group was more discrete and the signal amplitude was smaller; B) The above statistics are data of 6 animals in each group. When the right ventricle of the UW+GZ7 group stimulated the apex, 5 animals could be analyzed (considered to be conductive), while only 3 animals in the UW group;

[0068] The experimental results are as follows Figure 5 As shown, Figure A: Representative graph of systolic pressure under different storage conditions; Figure B: Statistical graph of left ventricular systolic pressure under different storage conditions; Figure C: Statistical graph of left ventricular diastolic pressure under different storage conditions; Figure D: Statistical graph of left ventricular pressure difference under different storage conditions;

[0069] The experimental results showed that there was no significant difference in systolic blood pressure, diastolic blood pressure and pressure difference between the two groups.

[0070] Step 4: myocardial tissue testing specifically includes: immunohistochemistry testing, fluorescent probe testing, and ELISA testing;

[0071] Immunohistochemistry was used to detect the expression of apoptosis-related genes Bax and Bcl-2;

[0072] Fluorescence probe detection was used to detect ROS and ATP content, and a fully automated analyzer was used to detect CPK and LDH;

[0073] ELISA assays were used to detect cardiomyocyte IL-6, IL-1β, HIF-1α, and TNF-α;

[0074] Step five, data statistics specifically include: separately counting the test indicators and test values ​​of the animal hearts in the control group and the injury group, analyzing and comparing the numerical differences between the control group and the injury group, and explaining the protective effect of Guangzao-7-flavor pills on cold-preserved hearts.

[0075] In summary, the effects of the present invention are: being able to optimize the cold preservation solution, reduce the damage caused by cold preservation, improve the preservation effect of the donor heart, maximize the therapeutic effect of heart transplantation, minimize ischemic myocardial damage, and maintain the function of the isolated heart. Scientific means are used to study the efficacy and mechanism of action of the Mongolian medicine compound preparation, provide safe and effective medicines for clinical use, and provide a scientific basis for the next step of developing new optimized cold preservation heart drugs.

[0076] The above generally describes the present invention in detail, but it is obvious to a person skilled in the art that some modifications or improvements can be made to the present invention. Therefore, modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.

Claims

1. A method for using a Mongolian medicine compound preparation in isolated cold-preserved heart, characterized in that: The method for applying the Mongolian medicine compound preparation in the isolated cold-preserved heart includes a dose-effect experiment process and an animal experiment process; The dose-effect experiment process is to test the effects of different concentrations of Guangzao-7 pills on isolated hearts, and perform multi-channel electrogramming on the hearts, record the field potential and electrical signals under stimulation, and determine the optimal effective concentration; The animal experiment process includes the following steps: Step 1: Animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group); Step 2: Stimulate the isolated heart of the animal and mark its position; Step 3: Record indicators and collect data; Step 4: Myocardial tissue detection; Step 5: Data statistics.

2. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 1, characterized in that: The dose-effect experiment process specifically includes the following steps: Set up different concentrations of Guangzao-7 pill solutions; The isolated hearts of normal rats were placed in different concentrations of Guangzao-7 pill solutions; The isolated rat hearts were subjected to Langendorff perfusion multi-channel electrolabeling in different concentrations. Record the field potential and electrical signals of the left and right ventricles under sinus conditions and 6HZ stimulation conditions, and analyze indicators such as heart rate, ECG, and ventricular conduction time; Determine the optimal effective concentration in isolated rat hearts.

3. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 2, characterized in that: The step 1, animal isolated heart modeling and grouping (isolated heart UW solution preservation for 8 hours, UW solution + Mongolian medicine preservation for 8 hours group) specifically includes: Take a number of animal hearts and divide them into two groups according to the number; One group of hearts was stored at 4°C for 8 hours, and KH solution, UW solution and Mongolian medicine were added. The concentration of Mongolian medicine was the best effective concentration determined by the dose-effect time to prepare the control group. Another group of hearts were kept cold at 4°C for 8 hours, and KH solution and UW solution were added to prepare the injury group.

4. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 3, characterized in that: The second step is to stimulate the isolated heart of the animal, and the mapping positions specifically include: two 64-channel pen-type mapping electrodes respectively record LV and RV signals, and simultaneously record the simulated electrocardiogram II lead signal (pseudo-ECG II) with the negative pole in the right atrium and the positive pole at the apex slightly to the left ventricle.

5. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 4, characterized in that: The step three, recording indicators and data collection, is specifically as follows: after completing the treatment, the electrophysiological signals of different groups during Sinus, 6Hz, and S1S2 stimulation are recorded respectively, and under cold storage conditions, the heart is fixed with 10% methanol tissue fixative to obtain the animal heart myocardial tissue.

6. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 5, characterized in that: The step 4, myocardial tissue detection specifically includes: immunohistochemistry detection, fluorescent probe detection, ELISA detection; The immunohistochemical assay is used to detect the expression of apoptosis-related genes Bax and Bcl-2; The fluorescent probe method is used to detect the content of ROS and ATP, and a fully automated analyzer is used to detect CPK and LDH; The ELISA test is used to detect myocardial cell IL-6, IL-1β, HIF-1α, and TNF-α.

7. The method for applying a Mongolian medicine compound preparation in isolated cold storage of a heart according to claim 6, characterized in that: The step 5, data statistics specifically includes: respectively counting the detection indicators and detection values ​​of the animal hearts in the control group and the injury group, analyzing and comparing the numerical difference between the control group and the injury group, and explaining the protective effect of Guangzao-7-flavor pills on cold-preserved hearts.

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