阅读说明：本技术 一种大气污染物导致的心脏毒性评价方法 (Method for evaluating cardiotoxicity caused by atmospheric pollutants ) 是由 洪敏� 魏丽萍 吴伟娜 段怀龙 刘兵 李青梅 戴敏 于 2020-05-12 设计创作，主要内容包括：本发明公开了一种非治疗目的的大气污染物导致的心脏毒性评价方法,其包括检测离子通道的功能以评价心脏毒性的步骤。本发明所述的心脏毒性评价方法操作简单、快速、效率高、通量高、可重复性好、样品用量小,能够获知对于心脏电生理的影响,特别适用于心肌细胞功能的评价。(The invention discloses a method for evaluating cardiotoxicity caused by atmospheric pollutants for non-therapeutic purposes, which comprises the step of detecting the function of an ion channel to evaluate the cardiotoxicity. The cardiotoxicity evaluation method provided by the invention is simple and rapid to operate, high in efficiency, high in flux, good in repeatability and small in sample dosage, can know the influence on the heart electrophysiology, and is particularly suitable for evaluating the function of the myocardial cells.)

1. A method for assessing cardiotoxicity caused by atmospheric contaminants for non-therapeutic purposes, comprising the step of detecting the function of an ion channel to assess cardiotoxicity.

2. The cardiotoxicity evaluation method of claim 1, wherein the atmospheric contaminant is a PM2.5 contaminant.

3. The cardiotoxicity evaluation method of claim 1 or 2, wherein the ion channel is a cardiac-related ion channel.

4. The cardiotoxicity evaluation method according to any one of claims 1 to 3, wherein the ion channel is a sodium ion channel, a potassium ion channel, and/or a calcium ion channel.

5. The cardiotoxicity assessment method of any one of claims 1 to 4, wherein said ion channel is a sodium ion channel Nav1.5 and/or a hERG potassium ion channel.

6. The method for evaluating cardiotoxicity according to any of claims 1 to 5, wherein the function of the ion channel is detected by an electrophysiological technique;

preferably, the function of detecting the ion channel is performed by a patch clamp technique;

more preferably, said performing by patch clamp technique is performed by detecting a cell line expressing said ion channel by patch clamp technique, said expression preferably being exogenous expression or endogenous expression;

more preferably still, the cell line expressing the ion channel is a HEK293cell line expressing a sodium ion channel nav1.5 and/or a hERG potassium ion channel.

7. The cardiotoxicity assessment method according to any of claims 1 to 6, further comprising the steps of sample collection of said atmospheric contaminants, sample preparation of said atmospheric contaminants and/or component identification of said atmospheric contaminants sample;

and/or, the detection also comprises a step of dissolving the atmospheric pollutants, wherein the dissolving is ultrasonic dissolving, and the final concentration of the atmospheric pollutants in the sample is 10-300 μ g/mL, such as 30 μ g/mL or 100 μ g/mL;

and/or, detecting the function of said ion channel to obtain the effect of said atmospheric contaminant on the action potential of the electrophysiological effects of the heart, preferably cardiomyocytes.

8. A method for screening a drug for preventing and/or treating cardiotoxicity caused by atmospheric pollutants, comprising the step of screening a drug using the cardiotoxicity evaluation method according to any one of claims 1 to 7; the atmospheric pollutants are preferably PM2.5 pollutants.

9. The application of the ion channel in screening drugs for preventing and/or treating cardiotoxicity caused by atmospheric pollutants;

preferably:

the atmospheric pollutants are preferably PM2.5 pollutants; and/or, the ion channel is a cardiac-related ion channel; and/or the ion channel is a sodium ion channel, a potassium ion channel and/or a calcium ion channel; the sodium channel is preferably Nav1.5, and the potassium channel is preferably the hERG potassium channel.

10. Use of a cell line expressing an ion channel for screening a medicament for preventing and/or treating cardiotoxicity caused by atmospheric pollutants;

preferably:

the atmospheric pollutants are preferably PM2.5 pollutants; and/or, the ion channel is a cardiac-related ion channel; and/or the ion channel is a sodium ion channel, a potassium ion channel and/or a calcium ion channel; the sodium ion channel is preferably Nav1.5, and the potassium ion channel is preferably an hERG potassium ion channel;

more preferably, the ion channel expressing cell line is a HEK293cell line expressing Nav1.5 and/or hERG potassium channels.

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for evaluating cardiotoxicity caused by atmospheric pollutants, in particular PM2.5 pollutants.

Background

Atmospheric pollutants include those substances that are emitted into the atmosphere by human activities or natural processes and have a deleterious effect on humans and the environment. PM2.5(Particulate Matter 2.5) is a Particulate contaminant having a diameter of 2.5 μm or less, which is produced in industrial production and daily production. International Standards such as National Air Quality Standards (NAAQS) use PM2.5 as a marker for routine atmospheric pollution monitoring. Due to its small volume, PM2.5 can be absorbed into the blood through the alveolar region, resulting in systemic toxicity. A number of epidemiological and animal studies have shown that PM2.5 has a number of toxic effects. Both domestic and foreign reports show that PM2.5 can cause cardiotoxicity in human bodies. In recent years, there have been some studies aimed at exploring the Mechanism of Cardiotoxicity, and the results showed that PM2.5 can cause oxidative stress (ROS), functional changes, and other cytological damage (ref: Wang, H., et al, cardio oxidation and Mechanism of cellular Matter 2.5(PM2.5) Exposure in offset rails Dual scientific. Med Sci Unit, 2017.23: p.3890-3896). However, some of the technical means adopt living animal experiments, and the method has high cost, low efficiency and large sample consumption; some methods for evaluating the influence of the activity of the cells by in vitro cell culture have the defects of long culture time, relatively simple evaluation index, limitation to myocardial cell damage, insufficient study on myocardial function and incapability of knowing the influence on the heart electrophysiology particularly. Therefore, a method for evaluating the cardiotoxicity caused by the atmospheric pollutants, particularly the PM2.5 pollutants, is urgently required to be found.

Disclosure of Invention

The invention aims to overcome the defects that the conventional method for evaluating cardiotoxicity caused by atmospheric pollutants (such as PM2.5 pollutants) is low in efficiency, large in sample dosage, long in culture time, relatively simple in evaluation index, limited in myocardial cell damage, insufficient in study on myocardial function, and particularly incapable of knowing the influence on cardiac electrophysiology, and the like, and provides a method for evaluating cardiotoxicity caused by atmospheric pollutants, particularly PM2.5 pollutants, and application thereof. The cardiotoxicity evaluation method disclosed by the invention evaluates cardiotoxicity by directly detecting the function of the ion channel, is simple and rapid to operate, has high efficiency, high flux, good repeatability and small sample dosage, can know the influence on the heart electrophysiology, and is particularly suitable for evaluating the function of the myocardial cells.

The cardiotoxicity caused by the atmospheric pollutants (such as PM2.5 pollutants) is different from the cardiotoxicity caused by other medicines and the like, the mechanism is complex, in the field, generally, in-vitro myocardial cell culture and other modes are adopted, the cardiotoxicity caused by the atmospheric pollutants (such as PM2.5 pollutants) is evaluated through activity change, and the modes have the defects of long operation time, relatively simple evaluation index, only limited myocardial cell damage, insufficient study on myocardial function, incapability of knowing the electrophysiology of the heart and the like. However, through a large number of experiments, the inventor firstly discovers a molecular mechanism of cardiotoxicity caused by the atmospheric pollutants, particularly the PM2.5 pollutants, that is, the inventor firstly discovers that the atmospheric pollutants, particularly the PM2.5 pollutants, generate cardiotoxicity by influencing the action of ion channels, and further causing the change of cardiac muscle function, so that the electrophysiological technology (such as patch clamp technology) is used for evaluating the possible influence of the atmospheric pollutants, particularly the PM2.5 pollutants, on the action potential of the heart to evaluate the cardiotoxicity caused by the atmospheric pollutants, particularly the PM2.5 pollutants, and overcomes the shortcomings of the cardiotoxicity evaluation method caused by the atmospheric pollutants, particularly the PM2.5 pollutants in the prior art.

In order to solve the above technical problems, a first aspect of the present invention provides a method for evaluating cardiotoxicity caused by atmospheric pollutants for non-therapeutic purposes, which includes the step of detecting the function of an ion channel to evaluate cardiotoxicity.

Preferably, the atmospheric pollutants are PM2.5 pollutants.

Preferably, the ion channel is a cardiac-related ion channel.

Preferably, the ion channel is a sodium ion channel, a potassium ion channel, and/or a calcium ion channel.

Preferably, the ion channel is sodium ion channel Nav1.5 and/or hERG potassium channel.

Preferably, the function of detecting the ion channel is performed by an electrophysiological technique.

Preferably, the function of detecting the ion channel is performed by patch clamp techniques.

More preferably, said performing by patch clamp technique is performed by detecting by patch clamp technique a cell line expressing said ion channel, said expression preferably being exogenous expression or endogenous expression.

More preferably still, the cell line expressing the ion channel is a HEK293cell line expressing a sodium ion channel nav1.5 and/or a hERG potassium ion channel.

Preferably, the method for assessing cardiotoxicity further comprises the steps of collecting a sample of the atmospheric pollutant, preparing a sample of the atmospheric pollutant, and/or identifying a component of the sample of the atmospheric pollutant.

Preferably, the detection method further comprises a step of dissolving the atmospheric pollutants, wherein the dissolving is ultrasonic dissolving, and the final concentration of the atmospheric pollutants in the sample is 10-300 μ g/mL, such as 30 μ g/mL or 100 μ g/mL. The inventor finds in the experimental process that when the final concentration of the sample is below 10 mug/mL, the IC50 value cannot be obtained, and when the final concentration is above 300 mug/mL, the test can fail because of cell rupture.

Preferably, the function of the ion channel is examined to obtain the effect of the atmospheric contaminants on the action potential of the electrophysiological effects of the heart, preferably cardiomyocytes.

In a preferred embodiment of the present invention, the method for evaluating cardiotoxicity comprises the following steps: (1) collecting a PM2.5 sample and preparing the sample; (2) identifying components of the PM2.5 sample; (3) evaluating the functions of ion channels participating in action potentials in an hERG-HEK293cell system and a Nav1.5-HEK293 cell system respectively by adopting a patch clamp technology, and calculating IC₅₀Value, thereby evaluating cardiotoxicity.

In a preferred embodiment of the present invention, the method for evaluating cardiotoxicity comprises the following steps: (1) collecting PM2.5 samples in the atmosphere by adopting a glass fiber filter membrane, a KB-1000 microcomputer particulate matter large-flow sampler and a QG-1000 large-flow PM2.5 particulate matter cutter;

(2) cutting the sampled glass filter membrane into small pieces, performing ultrasonic treatment in ultrapure water for 6 times, performing ultrasonic treatment for 30 minutes each time, then filtering by adopting 12 layers of sterile gauze, and preparing the final suspension into powder by adopting vacuum drying;

(3) extracting the prepared PM2.5 sample by adopting n-hexane/acetone extracting solution, detecting by adopting gas chromatography-mass spectrometry (DSQII-MS), digesting the residual extracted sample by adopting nitric acid/hydrochloric acid solution at 100 ℃ for about 2 hours, and detecting metal components in the sample by adopting ICP-MS7700 inductively coupled plasma mass spectrometry;

(4) the ion channels of hERG-HEK293cells were analyzed using the Patchliner NPC1 and Patch clamp Amplifier EPC10(HEKA, Germany) analysis systems;

(5) the ion channels of Na v1.5-HEK293 cells were analyzed using the Patchliner NPC16 and Patch clamp Amplifier EPC10(HEKA, Germany) analysis systems;

(6) and measuring the peak tail current after the analysis is finished. Each concentration sample under each cell was replicated three times. The percent peak tail current was calculated by dividing the peak cell tail current measurement for PM2.5 treated cells by the peak cell tail current measurement for cells that did not contain PM 2.5.

In order to solve the above technical problems, a second aspect of the present invention provides a method for screening a drug for preventing and/or treating cardiotoxicity caused by atmospheric pollutants, comprising the step of screening the drug using the cardiotoxicity evaluation method according to the first aspect of the present invention.

Preferably, the atmospheric pollutants are preferably PM2.5 pollutants.

In order to solve the technical problems, the third aspect of the invention provides the application of the ion channel in screening drugs for preventing and/or treating cardiotoxicity caused by atmospheric pollutants.

Preferably, the atmospheric pollutants are preferably PM2.5 pollutants.

Preferably, the ion channel is a cardiac-related ion channel.

Preferably, the ion channel is a sodium ion channel, a potassium ion channel and/or a calcium ion channel; the sodium channel is preferably Nav1.5, and the potassium channel is preferably the hERG potassium channel.

Preferably, the screening is generally performed by detecting the function of the ion channel, which is generally to obtain the effect of the atmospheric contaminants on the electrophysiological effects of the heart, preferably the action potential of the cardiomyocytes, can be performed mainly by using electrophysiological techniques, e.g. by patch clamp techniques. The detection of the cell line expressing the ion channel by the patch clamp technique, preferably by the patch clamp technique, is preferably performed by exogenous expression or endogenous expression. The cell line expressing the ion channel is preferably a HEK293cell line expressing a sodium ion channel Nav1.5 and/or a hERG potassium ion channel. Wherein the detection further comprises a step of dissolving the atmospheric pollutants, the dissolving is ultrasonic dissolving, and the final concentration of the atmospheric pollutants in the sample is 10-300 μ g/mL, such as 30 μ g/mL or 100 μ g/mL.

In order to solve the above technical problems, the fourth aspect of the present invention provides the use of a cell line expressing an ion channel for screening a drug for preventing and/or treating cardiotoxicity caused by atmospheric pollutants.

Preferably, the atmospheric pollutants are preferably PM2.5 pollutants.

Preferably, the ion channel is a cardiac-related ion channel.

Preferably, the ion channel is a sodium ion channel, a potassium ion channel and/or a calcium ion channel; the sodium channel is preferably Nav1.5, and the potassium channel is preferably the hERG potassium channel.

More preferably, the ion channel expressing cell line is a HEK293cell line expressing Nav1.5 and/or hERG potassium channels.

Preferably, the screening is generally performed by detecting the function of the ion channel, which is generally to obtain the effect of the atmospheric contaminants on the electrophysiological effects of the heart, preferably the action potential of the cardiomyocytes, can be performed mainly by using electrophysiological techniques, e.g. by patch clamp techniques. The detection of the cell line expressing the ion channel by the patch clamp technique, preferably by the patch clamp technique, is preferably performed by exogenous expression or endogenous expression. The cell line expressing the ion channel is preferably a HEK293cell line expressing a sodium ion channel Nav1.5 and/or a hERG potassium ion channel. Wherein the detection further comprises a step of dissolving the atmospheric pollutants, the dissolving is ultrasonic dissolving, and the final concentration of the atmospheric pollutants in the sample is 10-300 μ g/mL, such as 30 μ g/mL or 100 μ g/mL.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the cardiotoxicity evaluation method disclosed by the invention evaluates cardiotoxicity by directly detecting the function of the ion channel, is simple and rapid to operate, has high efficiency, high flux, good repeatability and small sample dosage, can know the influence on the heart electrophysiology, and is particularly suitable for evaluating the function of the myocardial cells.

Drawings

FIG. 1 shows a graph of the results of PM2.5 samples on inhibition of Nav1.5 potassium sodium channels in HEK293 cells. Percent peak tail current of cells-peak tail current of PM2.5 added sample ÷ peak tail current of no PM2.5 seen. Half maximal inhibitory concentration IC was calculated using GraphPad Prism 5.0₅₀。

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

EXAMPLES assessment of cardiotoxicity of PM2.5 samples in contaminated and non-contaminated areas

1. Experimental sample

PM2.5 samples were collected using Whatman glass fiber filters (Whatman, UK), KB-1000 microcomputer particulate mass flow samplers (Qingdao Jinshida electronics technologies Co., Ltd.), and QG-1000 mass flow PM2.5 particulate cutters (Qingdao Jinshida electronics technologies Co., Ltd.). And taking the Jiading area and the Yanpu area as a pollution area and a non-pollution area respectively for sampling.

2. Sample preservation and preparation

The samples were stored at-20 ℃ protected from light prior to analysis. Cutting the sampled glass filter membrane into small pieces, performing ultrasonic treatment in ultrapure water for 6 times, performing ultrasonic treatment for 30 minutes each time, filtering with 12 layers of sterile gauze, and vacuum drying the final suspension to obtain powder.

3. PM2.5 component identification

Extracting the prepared PM2.5 sample by using n-hexane/acetone extracting solution, detecting by using gas chromatography-mass spectrometry (DSQII-MS, USA), digesting the residual sample by using nitric acid/hydrochloric acid solution at 100 ℃ for about 2 hours, and detecting the metal components by using ICP-MS7700 inductively coupled plasma mass spectrometry (Agilent, USA).

4. Detection of hERG (human Ether-a-go-go Related Gene) ion channel, an ion channel associated with cardiac potassium efflux, and Nav1.5 ion channel, an ion channel associated with cardiac sodium influx. hERG-HEK293CELLs (purchased from (S.A. R.L CREA-CELL) and Nav1.5-HEK293 CELLs (prepared as described in Cummins TR; Aglioc F; Rengathan M; Herzog; Dib-HajSD; Waxman SG. "Nav1.3 channels: rapid reprinting and slow activated displacement qualitative sensitivity expression after expression a macromolecular CELL line and in physiological sensors) were analyzed using a Patchliner NPC16(Nanion, Germany) and a Patch clamp Amplifier EPC10(HEKA, Germany) analysis system and were first subjected to lysis in a pH adjustment buffer of the sample of the colloidal ion of the glucose sample of pH adjustment of the colloidal ion of the glucose of the colloidal ion of the pH of the colloidal ion of the glucose in a 1.3. Soft2. A. B. A. B. of the analysis system of the same kind of glucose of the same kind of the same, and a similar to the same kind of the same, and a similar to the same, and a, and similar to the same, respectively, and similar to the same, respectively, and similar, respectively, are analyzed using the same, and similar to the same, respectively, and similar, respectively, and similar, and their respective, were subjected to the same, respectively, and their respective, were subjected to the same, and their respective, respectively, and their respective, were subjected to the same, respectively, and their respective, respectively, and their respective, were subjected to the same, and their respective, were subjected to the same, and their respective, respectively, and their respective, respectively, to the same, respectively, to the same, and their respective, respectively, and their respective, were subjected to the same, respectively, and their respective, respectively, and their respective, were subjected to a, and their respective, so that the final concentration was 10, 30, 100, 300. mu.g/mL. For each analysis, 2mL of test sample was added to Nav1.5-HEK293 cells and hERG-HEK293cells for peak tail current determination. Each concentration sample under each cell was replicated three times. The percent peak tail current was calculated by dividing the peak cell tail current measurement for PM2.5 treated cells by the peak cell tail current measurement for cells that did not contain PM 2.5.

5. Results obtained

(1) The main chemical components of the PM2.5 samples in the polluted area and the non-polluted area are different from each other, and the concentrations of PAH (Polycyclic Aromatic Hydrocarbons), metal components, particularly inorganic salt ions in the air PM2.5 in the polluted area are higher than those of the samples in the non-polluted area. The ratio of each component in the two regions is basically consistent, and the PAH, the metal component and the inorganic salt ion with the highest abundance are fluoranthene, aluminum/manganese and nitrate ions respectively.

(2) The results of the hERG-HEK293cell line assay are shown in Table 1, which shows that in the PM2.5 concentration range of 10 μ g/mL to 300 μ g/mL, samples from the contaminated area can cause concentration-dependent inhibition of the hERG potassium channel on the hERG-HEK293cells, about 50% inhibition can be achieved at 300 μ g/mL, and samples from the non-contaminated area have no inhibition on the hERG potassium channel.

TABLE 1 inhibition of hERG potassium channel in HEK293cells by PM2.5 samples

Data are presented as mean ± standard deviation of triplicate samples.

(3) The results of the assay of the Nav1.5-HEK293 cell line are shown in FIG. 1. It is shown that both contaminated and uncontaminated zone source PM2.5 samples can inhibit the nav1.5 sodium channel. Contaminated and uncontaminated zone source samples at concentrations of 10. mu.g/mL to 1000. mu.g/mL for inhibition of Nav1.5 sodium channel IC₅₀Respectively 90. mu.g/mL and 200. mu.g/mL. The inhibition of the sample in the contaminated area is more than twice that in the non-contaminated area.

In general analysis, the different effects of the PM2.5 samples in the contaminated and uncontaminated regions on the ion channel may be due to differences in PAH, metal content, and inorganic salt ion content, especially the concentration of metal content in the contaminated region samples is significantly higher than that in the uncontaminated region by a factor of two.