Experimental method for evaluating electrophysiological activity of traditional Chinese medicine monomeric compound acting on myocardial cells

Abstract

The invention discloses an experimental method for evaluating the electrophysiological activity of a traditional Chinese medicine monomeric compound acting on myocardial cells. The method comprises the following steps: constructing a cell-based drug evaluation platform, constructing a cardiac muscle cell electrophysiological model for drug evaluation, treating cardiac muscle cells with a traditional Chinese medicine monomer compound, and evaluating the electrophysiological activity of the cardiac muscle cells. A drug evaluation platform is established by combining a microelectrode array device and an electrophysiological recording instrument, electrophysiological signals of a traditional Chinese medicine monomer compound tanshinone IIA after myocardial cells are treated for a short time and a long time are accurately monitored in real time, and the influence of the tanshinone IIA on the myocardial cells is evaluated. The concentration of tanshinone IIA and the acting time of tanshinone IIA on myocardial cells are regulated and controlled, and the characteristic parameters of the electrophysiological signals in different states are comprehensively analyzed, so that the treatment effect is effectively evaluated, and a brand new research thought is provided for popularization of clinical application of traditional Chinese medicine monomeric compounds.

Description

Technical Field

[0001] The present invention relates to the field of drug monitoring and evaluation, and particularly to an experimental method for evaluating the electrophysiological activity of traditional Chinese medicine monomer compounds on cardiomyocytes. Background Art

[0002] Cardiovascular diseases remain the leading cause of high morbidity and mortality globally. With the gradual in - depth study of the pathogenesis of cardiovascular diseases, it is particularly important to develop and screen effective drugs to provide personalized and precise intervention and treatment for patients with cardiovascular diseases. In recent years, many natural compounds, including traditional Chinese medicine monomer compounds derived from Chinese herbal medicines, have attracted attention due to their potential in treating heart diseases. Tanshinone IIA is a lipophilic compound extracted from Salvia miltiorrhiza, with the molecular formula C 19 H 18 O 3 . In recent years, it has attracted much attention due to its significant cardiovascular protective effects shown in the research of ischemic heart disease, atherosclerosis, heart failure and other fields. However, despite the great therapeutic potential of tanshinone IIA, its specific effects on the heart are still unclear, which poses challenges to its clinical application.

[0003] Currently, a variety of methods have been developed to evaluate the effects of drugs on the heart. Although clinical diagnostic methods can achieve non - invasive detection of the heart and reflect heart function, their spatial resolution limits the exploration of the mechanisms of cardiovascular diseases. Although animal models can be used to accurately explore the early effects of drugs on the heart, they have problems such as high cost and low throughput. Cell - based electrophysiological models can non - invasively and quantitatively detect signal propagation between cells, and can achieve electrophysiological signal detection of large - scale cell networks, breaking through the spatio - temporal resolution of detection. The patch - clamp technique is the gold standard for detecting electrophysiological signals of cardiomyocytes, but this technique has limitations such as complex operation, cell damage, time - consuming, and low throughput. Optical recording methods allow simultaneous recording of electrophysiological signals of multiple cells, however, they have problems such as phototoxicity and the need for chemical labeling or gene modification of cells. Microelectrode arrays are a safer technology that can non - invasively and parallelly record electrophysiological signals of cell networks, and they provide a promising solution for evaluating the effects of drugs on the heart. Therefore, it is clearly in line with the actual needs to develop an experimental method based on a cell - based microelectrode array sensing system for real - time and accurate evaluation of the electrophysiological effects of tanshinone IIA on the heart. Summary of the Invention

[0004] The object of the present invention is to provide an experimental method for evaluating the electrophysiological activity of myocardial cells by a traditional Chinese medicine monomer compound, aiming at the deficiencies of low efficiency and poor effect in the existing methods for evaluating the effects of traditional Chinese medicine monomer compounds on the heart. This method can not only accurately and effectively monitor the electrophysiological signals of myocardial cells, but also preliminarily explore the mechanism of drug action on the heart.

[0005] The object of the present invention is achieved by the following technical solutions:

[0006] An experimental method for evaluating the electrophysiological activity of myocardial cells by a traditional Chinese medicine monomer compound, comprising:

[0007] (1) Preparing a microelectrode array by microfabrication technology, fixing the microelectrode array on a printed circuit board, and bonding a glass culture chamber for cell culture above it to complete the assembly of the microelectrode array device; connecting the microelectrode array device to an electrophysiological signal recording system;

[0008] (2) Isolating and extracting primary myocardial cells from neonatal rats and culturing them on the microelectrode array device. On the 3rd day of culture, an electrophysiological model of myocardial cells for drug evaluation is established;

[0009] (3) Adding the traditional Chinese medicine monomer compound tanshinone IIA to the cell culture medium to treat the myocardial cells;

[0010] (4) Connecting the microelectrode array device cultured with myocardial cells treated with tanshinone IIA to an electrophysiological recording instrument, continuously recording the electrophysiological signals of the myocardial cells, extracting and statistically analyzing the characteristic parameters in the signals at a set time interval, and calculating the amplitude and firing frequency of the signals to evaluate the effect of the traditional Chinese medicine monomer compound tanshinone IIA on the electrophysiological signals of myocardial cells.

[0011] Furthermore, the substrate of the microelectrode array is glass, there are 32 electrodes, the diameter of the electrode sites is 30 μm, which is prepared by microfabrication technology and assembled with a customized printed circuit board and a glass culture chamber into a microelectrode array device; the electrophysiological signal recording system includes an electrical signal conditioning module, a data acquisition module and a host computer; the weak electrophysiological signals are amplified, noise is filtered, sampled through the electrical signal conditioning module, and the electrophysiological signals are transmitted to the host computer for acquisition, display and storage.

[0012] Furthermore, the seeding density of the primary myocardial cells of neonatal rats on the microelectrode array device is 3.0×10 5 ~5.0×10 5 cells / cm 2 ; the culture conditions of the cells in the incubator are: temperature 37 °C, culture atmosphere 5.0% (v / v) CO 2 , and the culture medium is changed every 24 h.

[0013] Further, tanshinone IIA was added to the cell culture medium to treat cardiomyocytes at a concentration of 10 - 200 μM for 0.5 h, 12 h, and 24 h.

[0014] Further, the electrophysiological signals of cardiomyocytes were continuously recorded for 0 - 180 min.

[0015] The beneficial effects of the present invention are that the experimental method of the present invention is simple to operate, can non-invasively and highly parallelly detect cell network electrophysiological signals, and can quickly and efficiently evaluate the early effects of drugs on the heart in the form of a cell model simulation. An experimental method for evaluating the electrophysiological activity of a traditional Chinese medicine monomer compound on cardiomyocytes of the present invention breaks through the spatio-temporal resolution of detection. Description of the Drawings

[0016] The present invention will be further described below in conjunction with the drawings and embodiments;

[0017] Figure 1 is a microscope image of the microelectrode array in the present invention;

[0018] Figure 2 is a schematic structural diagram of the cell-based drug evaluation platform in the present invention;

[0019] Figure 3 is the signal diagram corresponding to the construction of the cardiomyocyte electrophysiological model for drug evaluation;

[0020] Figure 4 is a statistical chart of the characteristic parameters of the electrophysiological signals of cardiomyocytes treated with different concentrations of tanshinone IIA for a short time;

[0021] Figure 5 is the electrophysiological signal recording diagram of cardiomyocytes treated with different concentrations of tanshinone IIA for a long time;

[0022] In the figure, the microelectrode array device 1, the electrical signal conditioning and data acquisition module 2, and the upper computer 3. Detailed Embodiments

[0023] The present invention provides an experimental method for evaluating the electrophysiological activity of a traditional Chinese medicine monomer compound on cardiomyocytes, including:

[0024] Building a cell-based drug evaluation platform, constructing a cardiomyocyte electrophysiological model for drug evaluation, treating cardiomyocytes with a traditional Chinese medicine monomer compound, and evaluating the electrophysiological activity of cardiomyocytes. The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0025] I. The method for building a cell-based drug evaluation platform includes the following steps:

[0026] The cell-based drug evaluation platform includes a microelectrode array device and an electrophysiological signal recording system. The microelectrode array is fabricated on a glass substrate by standard microfabrication techniques. Electrodes, leads, and pads are fabricated using photolithography, and then a 10-nm Ti layer and a 100-nm Au layer are deposited by magnetron sputtering. After removing the excess photoresist, a 5-μm-thick SU-8 layer is drawn to define the electrode positions and insulate the leads. As Figure 1 shown, the size of the microelectrode array is 2×2 cm 2 , and each consists of 32 electrodes with an electrode diameter of 30 μm and a center-to-center spacing of 300 μm. As Figure 2 shown, the microelectrode array is fixed on a printed circuit board, and a glass culture chamber is bonded on top to complete the assembly of the microelectrode array device 1. The microelectrode array device is connected to the electrophysiological signal recording system to build the drug evaluation platform. The electrophysiological signal recording system includes an electrical signal conditioning and data acquisition module 2 and a host computer 3; the electrical signal conditioning and data acquisition module is connected to the host computer through a signal line. The electrical signal conditioning and data acquisition module can amplify weak electrophysiological signals, filter out noise, sample, and finally transmit the electrophysiological signals to the host computer for acquisition, display, and storage.

[0027] II. Construction of an electrophysiological model of cardiomyocytes for drug evaluation, including the following steps:

[0028] Primary cardiomyocytes are isolated from the hearts of 1-day-old Sprague-Dawley rats. The ventricular tissue is excised and minced into approximately 1-mm 3 fragments in Hanks' balanced salt solution (HBSS). The tissue fragments are digested with 0.08% trypsin and 0.1% type II collagenase for 10 - 12 cycles (8 min per cycle). Subsequently, the cell suspension is collected and centrifuged at 800 rpm for 5 minutes. The cardiomyocytes are resuspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and filtered through a 70-μm cell strainer. After two differential adhesion steps (45 minutes each), cardiomyocytes are seeded onto fibronectin-coated electrodes at a density of approximately 3×10 5 cells per microelectrode array device. The device is maintained in a cell culture incubator at 37°C and 5% CO 2 , and the culture medium is renewed every 24 h. As Figure 3 shown, on the 3rd day of culture, the isolated cardiomyocytes develop towards the mature stage, and the recorded electrophysiological signals show consistency and rhythmicity, with the amplitude and discharge rate showing a relatively stable state, thus establishing an electrophysiological model of cardiomyocytes for drug evaluation.

[0029] III. Treatment of cardiomyocytes with traditional Chinese medicine monomer compounds, including the following steps:

[0030] When the cardiomyocytes cultured on the microelectrode array device exhibit consistent and stable rhythmic beating, drug assays are performed. Tanshinone IIA is dissolved in dimethyl sulfoxide (DMSO) as the stock solution, and then diluted to a concentration of 1 mM with DMEM. After heating in a 37 °C water bath, it is added to the cells at different doses (10 μM, 30 μM, 50 μM, 100 μM, 200 μM) for incubation, and the electrophysiological signals are recorded at 0.5 h, 12 h, and 24 h after treatment respectively.

[0031] IV. Evaluating the electrophysiological activity of cardiomyocytes, including the following steps:

[0032] The microelectrode array device cultured with cardiomyocytes treated with tanshinone IIA is connected to an electrophysiological recording instrument, and the electrophysiological signals of cardiomyocytes are continuously recorded for 0 - 180 min; the Labview software is used to extract and statistically analyze the characteristic parameters in the signals at set time intervals, and calculate the amplitude and firing frequency of the signals. In the absence of drug treatment, the electrophysiological signals of cardiomyocytes are first recorded. Then, cardiomyocytes are treated with tanshinone IIA at concentrations of 10, 30, 50, 100, and 200 μM respectively, and the electrophysiological signals are monitored 0.5 h after treatment. Figure 4 It is a statistical chart of the electrophysiological signals of cardiomyocytes treated with tanshinone IIA at different concentrations (10, 30, 50, 100, and 200 μM) for a short time. The electrophysiological signals of cardiomyocytes treated with tanshinone IIA at different concentrations remain basically stable within 0.5 h, indicating that short-term treatment with tanshinone IIA has basically no effect on the electrophysiological signals of cardiomyocytes. Figure 5 It is the electrophysiological signal diagram of cardiomyocytes treated with tanshinone IIA at different concentrations for a long time. After 12 - 24 h of treatment, the amplitude of the recorded electrophysiological signals is significantly enhanced, and the signal amplitude is enhanced more significantly when the concentration is lower than 50 μM, indicating that long-term treatment of cardiomyocytes with tanshinone IIA has the potential to enhance cardiac contractility.

[0033] Obviously, the above embodiments are only examples given for clear illustration, and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An experimental method for evaluating the electrophysiological activity of myocardial cells by a monomeric compound of a traditional Chinese medicine, characterized in that: include: (1) preparing a microelectrode array by micromachining technology, fixing the microelectrode array on a printed circuit board, and bonding a glass culture chamber for cell culture on top to complete the assembly of the microelectrode array device; connecting the microelectrode array device to an electrophysiological signal recording system; (2) Primary cardiomyocytes were isolated and extracted from neonatal rats and cultured on a microelectrode array device. On the third day of culture, a cardiomyocyte electrophysiological model for drug evaluation was established; (3) adding tanshinone IIA monomer compound to the cell culture medium to treat cardiomyocytes; (4) The microelectrode array device in which cardiomyocytes were cultured and treated with Tanshinone IIA was connected to an electrophysiological recording instrument to continuously record the electrophysiological signals of the cardiomyocytes. The characteristic parameters in the signals were extracted and counted at set time intervals, and the amplitude and emission frequency of the signals were calculated to evaluate the effect of the Tanshinone IIA Chinese medicine monomer compound on the electrophysiological signals of the cardiomyocytes.

2. The experimental method for evaluating the electrophysiological activity of myocardial cells by a monomeric compound of traditional Chinese medicine according to claim 1, characterized in that: The microelectrode array substrate is glass, with 32 electrodes and an electrode site diameter of 30 μm. It is prepared by micromachining technology and assembled with a customized printed circuit board and a glass culture chamber into a microelectrode array device; the electrophysiological signal recording system includes an electrical signal conditioning module, a data acquisition module and a host computer; the electrical signal conditioning module amplifies weak electrophysiological signals, filters noise, samples, and transmits the electrophysiological signals to the host computer for collection, display and storage.

3. The experimental method for evaluating the electrophysiological activity of myocardial cells by a monomeric compound of a traditional Chinese medicine according to claim 1, characterized in that: The planting density of the neonatal rat primary cardiomyocytes on the microelectrode array device is 3.0×10 5 ~5.0×10 5 cells / cm 2 The culture conditions of the cells in the incubator were: temperature 37°C, culture atmosphere 5.0% (v / v) CO2, and the culture medium was replaced every 24 hours.

4. The experimental method for evaluating the electrophysiological activity of myocardial cells by a monomeric compound of traditional Chinese medicine according to claim 1, characterized in that: Tanshinone IIA was added to the cell culture medium to treat the cardiomyocytes at a concentration of 10-200 μM. The treatment time was 0.5 h, 12 h, and 24 h.

5. The experimental method for evaluating the electrophysiological activity of myocardial cells by a monomeric compound of traditional Chinese medicine according to claim 1, characterized in that: The time for continuously recording electrophysiological signals of cardiomyocytes was 0 to 180 minutes.

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