Use of bufalin in preparation of cold storage solution for isolated heart, cold storage solution for isolated heart and resuscitation method

Abstract

The application discloses application of saranid in preparation of an isolated heart cold preservation solution, the isolated heart cold preservation solution and a resuscitation method, relates to the technical field of heart preservation, and adopts multi-channel electrophysiological mapping technology and pressure sensing technology by taking rats as experimental animal models, sets up a control group (cold crystal cardioplegic solution+UW solution) and an experimental group (cold crystal cardioplegic solution+UW solution+saranid), and systematically evaluates the myocardial protection effect of the cold preservation isolated heart. The experimental results show that after the saranid is matched with the UW solution and the cold crystal cardioplegic solution, the mechanical function of the isolated heart can be improved, the systolic function and the blood pumping efficiency of the isolated heart are improved, the left ventricular pressure difference and the left ventricular work index are statistically improved compared with the control group, the preservation time limit of the isolated heart is also prolonged to a certain extent, under the standard cold preservation condition of 4 DEG C, the safe preservation time limit of the isolated heart in the experimental group can be prolonged to at least 8 hours, and the transplantation quality of the donor heart is significantly improved.

Description

Technical Field

[0001] This invention relates to the field of heart preservation technology, and in particular to the application of sand bufotoxin in the preparation of cryopreservation solution for isolated hearts, the cryopreservation solution for isolated hearts and the resuscitation method. Background Technology

[0002] Heart failure (HF) is a major cardiovascular disease in my country with high incidence, high hospitalization rate, and high disease burden. Despite continuous advancements in clinical diagnosis and treatment, the rate of negative events (such as in-hospital mortality and 30-day readmission rate) after HF treatment remains high, placing a heavy burden on patients' families and society. Heart transplantation is the only treatment for end-stage heart disease patients that can fundamentally improve cardiac function, prolong long-term survival, and enhance quality of life. The preservation quality of the ex vivo heart is a core factor determining the success or failure of heart transplantation surgery. Currently, the mainstream clinical method of "simple cryopreservation" uses UW solution (University of Wisconsin solution) and cold crystalloid cardioplegic solution as the core cryopreservation system. By lowering the heart temperature, it slows down myocardial cell metabolism, achieving a safe preservation period of 4-6 hours.

[0003] However, during ex vivo preservation of the heart, especially when using simple cryopreservation, cardiomyocytes face numerous adverse factors, inevitably leading to damage to cardiac function. On one hand, while simple cryopreservation can reduce metabolism, it cannot fully meet the physiological needs of cardiomyocytes in an ex vivo state. Under low temperatures, the stability of the cell membrane and the function of organelles are affected to varying degrees, leading to intracellular environmental disturbances and consequently affecting the heart's normal contractile and diastolic functions. On the other hand, prolonged ischemia during transport further exacerbates cardiomyocyte damage, triggering a series of complex pathophysiological changes. During heart transplantation, if the function of the ex vivo heart is severely impaired due to improper preservation, even if successfully transplanted into a recipient, it is difficult to quickly restore normal pumping function, leading to postoperative complications such as low cardiac output syndrome and arrhythmias, increasing surgical mortality and the incidence of postoperative complications. Therefore, developing a low-toxicity, highly stable ex vivo heart cryopreservation system that can significantly prolong preservation time and reduce myocardial damage has become a key technological bottleneck that urgently needs to be overcome in the field of heart transplantation.

[0004] Bufotalin (SBG) is a bufotalide compound extracted from the dried secretions of toads (such as the Chinese giant toad or the black-rimmed toad). Its CAS number is 464-74-4, and its molecular formula is C. 24 H 32O6, with a molecular weight of 416.50 g / mol, typically has a purity of ≥98% by HPLC. It is a white crystalline powder and requires refrigerated storage protected from light. Currently, it is commonly used in anti-tumor applications, inhibiting cancer cell progression through multiple pathways. No specific cases have been found regarding its cardioprotective effects. No significant cardiotoxic or hepatotoxic side effects have been observed at therapeutic doses for anti-tumor treatment, and the resource is abundant, with a content exceeding 2% in toad venom, indicating promising potential for drug development. Summary of the Invention

[0005] The purpose of this invention is to provide the application of sand bull venom in the preparation of cryopreservation solution for isolated hearts, the cryopreservation solution for isolated hearts and the resuscitation method, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the first aspect of the present invention proposes the application of sand bufotoxin in the preparation of cryopreservation solution for isolated hearts.

[0007] Preferably, the cryopreservation solution can improve the function of the isolated heart after cryopreservation and / or prolong the cryopreservation period of the isolated heart.

[0008] Preferably, the improvement of isolated heart function after cold preservation includes improving myocardial contractile function.

[0009] Preferably, the concentration of bufotoxin in the cold preservation solution is 75-7500 ng / ml.

[0010] Preferably, the concentration of bufotoxin in the cold preservation solution is 750 ng / ml.

[0011] Preferably, the cryopreservation solution further includes one or more of UW solution and cryopreservation crystalloid solution.

[0012] To achieve the above objectives, a second aspect of the present invention provides an in vitro heart cryopreservation solution comprising bufotoxin.

[0013] Preferably, the concentration of bufotoxin in the cryopreservation solution for the ex vivo heart is 75-7500 ng / ml.

[0014] A third aspect of the present invention provides a method for resuscitating an isolated heart, comprising the following stages: First stage: Irrigate with room temperature oxygen-enriched KH solution for 10-20 minutes under conditions of 25±5℃ and 40±10 cmH2O. Second stage: Under the conditions of 30±5℃ and 60±10 cmH2O, use room temperature oxygen-enriched KH solution for irrigation for 15-25 minutes. The third stage: Under the conditions of 37±2℃ and 80±10 cmH2O, use room temperature oxygen-enriched KH solution for irrigation for 25-35 minutes.

[0015] Preferably, the isolated heart is preserved in the above-mentioned isolated heart cryopreservation solution.

[0016] Compared to existing technologies, the beneficial effects of this invention are as follows: Using rats as an experimental animal model, this invention employs multi-channel electrophysiological mapping technology and pressure sensing technology, establishing a control group (cold crystalloid cardioplegic solution + UW solution) and an experimental group (cold crystalloid cardioplegic solution + UW solution + bufotoxin) to systematically evaluate the cardioprotective effect of cryopreserved isolated hearts. Experimental results show that the combination of bufotoxin with UW solution and cold crystalloid cardioplegic solution improves the mechanical function of isolated hearts: enhancing the contractile function and pumping efficiency of isolated hearts, with key indicators including left ventricular pressure gradient and left ventricular work index showing statistically significant increases compared to the control group; furthermore, it extends the preservation time of isolated hearts: under standard 4°C cryopreservation conditions, the safe preservation time of isolated hearts in the experimental group can be extended to at least 8 hours, significantly improving the transplant adaptability quality of donor hearts.

[0017] In summary, the application of the sand bufotoxin described in this invention in the preparation of cryopreservation solution for isolated hearts has the technical advantages of controllable preparation cost and definite myocardial protection efficacy, providing an efficient and practical new solution for clinical cold preservation of donor hearts. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Figure 1 shows a comparison of left and right ventricular conduction between the UW and SBG+UW groups; Figure A shows the right ventricular activation time, Figure B shows the right ventricular conduction velocity, Figure C shows the right ventricular conduction dispersion, Figure D shows the left ventricular activation time, Figure E shows the left ventricular conduction velocity, and Figure F shows the left ventricular conduction dispersion. Figure 2 Figure 1 shows a comparison of cardiac pressure and functional parameters between the UW group and the SBG+UW group; Figure A is a representative graph of left ventricular systolic pressure; Figure B is a statistical graph of left ventricular systolic pressure; Figure C is a statistical graph of left ventricular diastolic pressure; Figure D is a statistical graph of left ventricular pressure gradient; Figure E is a statistical graph of left ventricular work.

[0020] Figure 3Figure 1 shows a comparison of ECG parameters between the UW group and the SBG+UW group; Figure A is a statistical graph of sinus recovery time; Figure B is a statistical graph of heart rate; Figure C is a statistical graph of PR interval; Figure D is a statistical graph of QRS complex duration; Figure E is a statistical graph of QT interval; Figure F is a statistical graph of intraventricular conduction time. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention. Example

[0023] This embodiment provides the application of bufotalin in the preparation of cryopreservation solution for isolated hearts. Simultaneously, using multi-channel electrophysiological mapping and pressure sensing technology, the cardioprotective effect of cryopreserved hearts was detected in the "cold crystalloid cardioplegic solution + UW solution" group (hereinafter referred to as the UW group) as a control, and the "cold crystalloid cardioplegic solution + UW solution + 750 ng / ml bufotalin" group (hereinafter referred to as the SBG+UW group). The aim is to explore the application potential of bufotalin in extending the safe cryopreservation period of isolated hearts and improving the quality of functional recovery after donor heart resuscitation.

[0024] This study explores the application of sand bull venom in prolonging the safe storage period of isolated hearts and improving the quality of functional recovery after donor heart resuscitation, specifically including: (1) The rats to be tested were anesthetized with isoflurane gas and an in vivo electrocardiogram (ECG) was performed to determine that the rats' cardiac status was normal; (2) Inject heparin sodium (3125U / kg), wait 15 minutes, quickly remove the heart, place it in a dish containing warm (37℃) oxygen-enriched (95% oxygen + 5% carbon dioxide) KH solution, and trim excess tissue. (3) With the assistance of forceps, quickly connect the aorta to the perfusion needle, fix the aorta with a clamp, fix it with surgical sutures, and re-perfuse the heart with warm (37°C) oxygenated (95% O2, 5% CO2) KH solution to restart the heart; (4) Transfer the heart to a constant flow system and rinse off the residual blood with warm (37°C) oxygen-enriched (95% oxygen + 5% carbon dioxide) KH solution within 3 minutes. (5) The control group first injected 10 mL of cold (4℃) cold crystal cardioplegic solution, and then injected 10 mL of cold UW solution; the experimental group first injected 10 mL of cold (4℃) cold crystal cardioplegic solution, and then injected 10 mL of cold (4℃) UW+SBG mixture. (6) The isolated hearts of the control group and the experimental group after step (5) and their perfusion needles were placed into centrifuge tubes containing cold preservation solution (UW solution) and (UW+SBG mixture) respectively; (7) The isolated rat hearts of both the control group and the experimental group were kept at 4℃ for 8 h. (8) After 8 hours of storage, remove the isolated heart and slowly inject 15 mL of room temperature (20℃) oxygen-enriched (95% oxygen + 5% carbon dioxide) KH solution into the syringe to rinse until the isolated heart shows peristalsis. (9) The isolated heart was resuscitated using a gradient constant pressure reperfusion method. The specific steps are as follows: First stage: The isolated heart was moved to a low constant pressure perfusion system (under the conditions of 25±5℃ and 40±10cm H2O) and perfused with room temperature oxygen-enriched KH buffer for 10-20 minutes. Second stage: The heart was then gradiented to a medium constant pressure perfusion system (under the conditions of 30±5℃ and 60±10cm H2O) and perfused with room temperature oxygen-enriched KH buffer for 15-25 minutes. Third stage: The heart was then gradiented to a high constant pressure perfusion system (under the conditions of 37±2℃ and 80±10cm H2O) and perfused with room temperature oxygen-enriched KH buffer for 25-35 minutes. (10) Record indicators and collect data.

[0025] In steps (5) and (6), the concentration of the sand bull venom used is 750 ng / mL.

[0026] In step (8), the rinsing time is generally 2 minutes and 30 seconds to 3 minutes and 30 seconds.

[0027] In step (10), after different groups of isolated heart samples have completed the specified treatment, their stable physiological state is maintained by the Langendorff isolated heart perfusion system. The electrophysiological signals and hemodynamic parameters of each group of samples are recorded synchronously by combining pressure sensing technology and multi-channel electrophysiological mapping technology. The electrophysiological signals include: sinus rhythm recovery time, heart rate, PR interval, QRS width, QT interval, intraventricular conduction time, etc.; the hemodynamic parameters include: left ventricular systolic pressure (LV-Sbp), left ventricular diastolic pressure (LV-Dbp), left ventricular pressure gradient (Pulse Pressure), left ventricular work (P*HR), etc.

[0028] In step (10), the measurement data are expressed as mean ± standard deviation, and the independent samples t-test is used to compare the two groups.

[0029] Experimental results are as follows Figure 1 As shown in the figure, Figure A is the right ventricular activation time, Figure B is the right ventricular conduction velocity, Figure C is the right ventricular conduction dispersion, Figure D is the left ventricular activation time, Figure E is the left ventricular conduction velocity, and Figure F is the left ventricular conduction dispersion. The experimental results showed that: the RV-AT (right ventricular activation time) values ​​in the UW group were more dispersed, with a relatively high average value, while the RV-AT values ​​in the SBG+UW group were generally lower, and the data points were relatively concentrated; the average RV-CV (right ventricular conduction velocity) values ​​in the UW group were relatively lower, while the RV-CV values ​​in the SBG+UW group were higher; the average RV-Dis (right ventricular conduction dispersion) values ​​in the UW group were higher, with dispersed data points, while the average RV-Dis values ​​in the SBG+UW group were significantly lower, and the data points were concentrated; the LV-AT (left ventricular activation time) values ​​in the UW group had a certain distribution range, with a relatively higher average value compared to the SBG+UW group; the average LV-CV (left ventricular conduction velocity) values ​​in the UW group were relatively lower, while the average LV-CV values ​​in the SBG+UW group were higher; the average LV-Dis (left ventricular conduction dispersion) values ​​in the UW group were higher, while the average LV-Dis values ​​in the SBG+UW group were relatively lower; from Figure 1 The results showed that, although the differences were not statistically significant, the SBG+UW groups all exhibited a trend of relatively shorter activation time, relatively faster conduction velocity, and relatively lower dispersion. This may suggest that treatment with the SBG+UW group helps to improve the electrical conduction velocity and electrical activation synchronicity of the left and right ventricles of the heart, thus having a positive impact on cardiac function.

[0030] Experimental results are as follows Figure 2 As shown, Figure A is a representative graph of systolic blood pressure; Figure B is a statistical graph of left ventricular systolic blood pressure; Figure C is a statistical graph of left ventricular diastolic blood pressure; Figure D is a statistical graph of left ventricular pressure gradient; and Figure E is a statistical graph of left ventricular work. The experimental results show that: compared to the UW group, the SBG+UW group exhibited larger waveform amplitude and more regular rhythm, reflecting superior electrical activity intensity and rhythm stability of the heart. Compared to the UW group, the SBG+UW group had higher left ventricular systolic pressure, potentially indicating that the pressure changes in the isolated heart during a single cardiac cycle were more significant after SBG+UW treatment, with higher systolic pressure peaks and more complete diastolic pressure decline. There was no significant difference between the average LV-Sbp (left ventricular systolic pressure) of the UW group and the average LV-Dbp (left ventricular diastolic pressure) of the SBG+UW group. The difference between left ventricular systolic and diastolic pressure reflects the fluctuation amplitude of ventricular pressure during a single cardiac cycle, providing a direct indication of cardiac pumping function. The SBG+UW group's Pulse... The average pressure (left ventricular pressure gradient) in the SBG+UW group was higher than that in the UW group, indicating that the SBG+UW treatment can significantly enhance the effective pressure gradient of isolated heart contraction, and has a better protective and recovery effect on the contractile function of isolated heart. The average P*HR (left ventricular work) of the heart in the SBG+UW group was significantly higher than that in the UW group, reflecting that the SBG+UW treatment can significantly improve the work capacity of the heart with each beat, and shows a better cardiac function state compared with the UW group.

[0031] Experimental results are as follows Figure 3 As shown, Figure A is a statistical graph of sinus rhythm recovery time in the UW group and the SBG+UW group; Figure B is a statistical graph of heart rate; Figure C is a statistical graph of PR interval; Figure D is a statistical graph of QRS width; Figure E is a statistical graph of QT interval; and Figure F is a statistical graph of intraventricular conduction time. Experimental results: There were no significant differences in sinus rhythm recovery time, heart rate, PR interval, QRS width, QT interval, and intraventricular conduction time.

[0032] The above experimental results demonstrate that the present invention can improve cardiac contraction and pumping function by adding sand bufotoxin to the cryopreservation solution of isolated hearts, thereby achieving targeted protection of isolated heart function, ultimately improving the preservation quality of isolated hearts, and effectively extending the cryopreservation time of isolated hearts in the transplantation process.

[0033] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of sand bufotoxin in the preparation of cryopreservation solution for isolated hearts.

2. The application according to claim 1, characterized in that, The cryopreservation solution can improve the function of isolated hearts after cryopreservation and / or prolong the cryopreservation period of isolated hearts.

3. The application according to claim 2, characterized in that, The improvement of isolated heart function after cryopreservation includes improving myocardial contractile function.

4. The application according to any one of claims 1-3, characterized in that, The concentration of bufotoxin used in the cold preservation solution is 75-7500 ng / mL.

5. The application according to claim 4, characterized in that, The concentration of bufotoxin used in the cold preservation solution was 750 ng / mL.

6. A cryopreservation solution for isolated hearts, characterized in that, The cryopreservation solution for the ex vivo heart includes sand bufotoxin.

7. The cryopreservation solution for isolated hearts according to claim 6, wherein the concentration of bufotoxin in the cryopreservation solution for isolated hearts is 75-7500 ng / mL.

8. A method for resuscitating an isolated heart, characterized in that, Includes the following stages: First stage: Irrigate with room temperature oxygen-enriched KH solution for 10-20 minutes under conditions of 25±5℃ and 40±10 cmH2O. Second stage: Under the conditions of 30±5℃ and 60±10 cmH2O, use room temperature oxygen-enriched KH solution for irrigation for 15-25 minutes. The third stage: Under the conditions of 37±2℃ and 80±10 cmH2O, use room temperature oxygen-enriched KH solution for irrigation for 25-35 minutes.

9. The resuscitation method according to claim 8, characterized in that, The ex vivo heart is preserved using the ex vivo heart cryopreservation solution as described in claim 6 or 7.

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