KR20200137935A - Composition for preventing heart damage in case of heart arrest containing 8-Oxo-2'-deoxyguanosine as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing cardiac injury in case of cardiac arrest containing 8-oxo-2′-deoxyguanosine as an active component. The 8-oxo-2′-deoxyguanosine inhibits an increase of free radicals in cardiomyocytes, inhibits ion transporter activity, inhibits the expression of poly-ADP-ribose (PAR), HSP60, Bcl-2, P-53, and BAX, which are apoptosis factors, and reduces COX-2 which is an inflammatory factor and CTGF which is a fibrosis factor, thereby being able to be usefully used as the composition for preventing cardiac injury in case of cardiac arrest.

Description

Composition for preventing heart damage in case of heart arrest containing 8-Oxo-2'-deoxyguanosine as an active ingredient, containing 8-oxo-2'-deoxyguanosine as an active ingredient}

The present invention relates to a composition for preventing heart damage during cardiac arrest, and specifically relates to a composition for preventing heart damage during cardiac arrest, comprising 8-oxo-2'-dioxyguanosine as an active ingredient.

The heart in the human body is the central organ of the circulatory system that is the motive force for circulating blood, and acts as a pump that supplies blood to the whole body by repetitive contraction and relaxation. Heart diseases include congenital heart disease and acquired heart disease.

Heart surgery refers to an operation in which the heart is emptied by blocking blood flowing into the heart, and the heart is incised to see the inside directly with the naked eye. In such heart surgery, heart stop is essential because it is necessary to stop the beating of the heart, drain the blood from the heart, and secure the field of view.

When the heartbeat is completely stopped, the heart muscle is damaged because there is no coronary artery blood flow.In order to minimize the damage to the heart muscle during the cardiac arrest period, cardioplegic solution is given and surgery is performed under hypothermia.

Cardiac arrest fluid used for this purpose is injected after cardiopulmonary bypass, when the aortic blood vessel is blocked and blood flow to the heart is blocked. Cardiac arrest fluid usually contains a high concentration of potassium (mainly 20 to 25 mEq/L), and when cardiac arrest fluid is injected, immediate and continuous diastolic arrest is induced, thereby minimizing energy consumption of the myocardium.

However, during cardiac arrest, the myocardial muscle becomes ischemic and lacks oxygen, so cardiac function is liable to decrease. Therefore, in addition to cardiac arrest, the cardiac arrest fluid must have an action to protect the myocardium. St. Thomas' fluid is known as a conventional cardiac arrest fluid. St Thomas' fluid contains several ions such as Na ⁺ , K ⁺ , Mg ² and Ca ^{2 +,} and the concentration of each ion is close to that of the extracellular fluid.

For cardiac arrest, Thomas hospital solution based on high-concentration potassium was developed in the 1970s, and since then, continuous improvement research has been conducted. Research results have been published that high concentration of potassium cardiac arrest fluid can cause problems such as myocardial necrosis, and studies to overcome this have been continued as follows, but no significant effect has been obtained.

The high-concentration potassium (K ⁺ ) cardiac arrest fluid, which is used to stop the heart during cardiac surgery and protect the myocardium during cardiac arrest, causes a change in the ionic concentration of the myocardial cells, causing a change in the intracellular ionic concentration such as Ca ^{2 +} . Therefore, paradoxically, cardiomyocyte damage may occur after the use of cardiac arrest fluid (Front Physiol. 2013 Aug 28;4:228).

That is, cardiomyocytes exposed to cardiac arrest fluid break homeostasis, which is important for cell survival and death, and activate various enzymes in the cytoplasm to cause apoptosis and cell necrosis, resulting in damage to cardiomyocytes. Specifically, the activity of NBC (Sodium/Bicarbonate Cotransporter), which is an ion transporter of cardiomyocytes, increases, resulting in excessive ion imbalance, impairing various functions involved in cell homeostasis, and acidification of cardiomyocytes due to the increase in the activity of the NBC. Happens. Therefore, it is necessary to control the activity of NBC, an ion transporter, that occurs during administration of cardiac arrest fluid.

In Ca ^{2 +} channel antagonist for inhibiting an increase in calcium that occur during cardiac arrest verapamil, nifedifine or, Na ⁺ by using the Na ⁺ channel blocker for the ion adjustment was conducted also studies to create one cardioplegia (Ann Thorac Surg. 1983 Dec;36(6):654-63; Thorac Cardiovasc Surg.1985 Dec;33(6):354-9; Ann Thorac Surg.1985 Apr;39(4):324-8; Eur Heart J. 1983 May ;4 Suppl C:115-21.).

Meanwhile, when cardiomyocytes are exposed to cardiac arrest fluid, the expression of apoptosis factors increases, which can be a measure of apoptosis assay. According to a recent study, the activity of PARP (Poly-ADP-ribose polymerase) is an important process for cell survival when DNA damage is restored, but since it is a process that consumes a lot of ATP, it further reduces high-energy phosphate, resulting in apoptosis or necrosis. It is known to be followed (Mol. Med. 2014, 20, 313-328.). In addition, it has been reported that the reduction of mitochondrial heat shock protein 60 (HSP60) after cardiac arrest is helpful in preserving mitochondria (Front Physiol. 2017; 8: 324). Therefore, it can be seen that reducing the expression of the apoptosis factors PAR (Poly-ADP-ribose) and HSP60 helps to protect the myocardium by reducing the death of myocardial cells.

Since the myocardial protective action is not sufficient in the conventional cardiac arrest solution, the present inventors studied to provide a composition for preventing heart damage during cardiac arrest, which is useful for cardiac arrest. As a result, when 8-oxo-2'-dioxyguanosine is used together with cardiac arrest solution Inhibits the increase of free radicals in cardiomyocytes, inhibits ion transporter activity, inhibits the expression of apoptosis factors PAR (Poly-ADP-ribose), HSP60, Bcl-2, P-53 and BAX, and inflammation It was found to have an effect of reducing the factor COX-2 and the fibrosis factor CTGF.

It is an object of the present invention to provide a composition capable of preventing cardiac damage caused by the use of a cardiac arrest solution containing a high concentration of K ⁺ while at the same time providing a cardiac arrest solution containing the composition.

Another object of the present invention is to provide a composition capable of preventing heart damage caused by the use of a cardiac arrest solution containing a high concentration of K ⁺ and a kit including the cardiac arrest solution.

An aspect of the present invention is to provide a composition for preventing heart damage during cardiac arrest, comprising 8-oxo-2'-dioxyguanosine as an active ingredient.

Another aspect of the present invention is to provide the composition in the form of a cardiac arrest solution in which 8-oxo-2'-dioxyguanosine is added to cardiac arrest solution containing K ⁺ .

Another aspect of the present invention is to provide a cardiac arrest kit for cardiac surgery comprising the cardiac arrest fluid and the composition.

When 8-oxo-2'-deoxyguanosine according to the present invention is used with cardiac arrest fluid, it inhibits the increase of free radicals in cardiomyocytes, inhibits ion transporter activity, and is an apoptosis factor PAR (Poly-ADP- ribose), HSP60, Bcl-2, P-53, and BAX, and reduce COX-2, an inflammatory factor, and CTGF, a fibrosis factor, so that it can be usefully used as a composition for preventing heart damage during cardiac arrest.

FIG. 1 shows HEK 293T cells (human embryonic kidney cells) overexpressing Sodium/Bicarbonate Cotransporter (NBC), NBCe1, in HCO ₃ ^- solution and H ₂ O ₂ without Na ⁺ It is a graph showing the intracellular ion transporter (NBC) activity before and after simultaneously injecting.
Figure 2 shows HEK 293T cells (human embryonic kidney cells) overexpressing Sodium/Bicarbonate Cotransporter (NBC) NBCe1, H ₂ O ₂ was first injected and 1 hour later, Na ⁺ free HCO ₃ ^- solution was injected, and then cells It is a graph showing the change in the activity of the inner ion transporter.
Figure 3 is a control group, cardiac arrest fluid (HTK), 100 μg/mL oh8dG 10 min pretreatment followed by cardiac arrest fluid treatment and 100 μg/mL oh8dG 30 min pretreatment followed by cardiac arrest fluid treatment, measuring the changes in active oxygen in single myocardial cells in mice This is a confocal micrograph.
Figure 4 is a control group, cardiac arrest fluid, 100 μg/mL oh8dG 10 minutes pretreatment followed by cardiac arrest fluid treatment and 100 μg/mL oh8dG 30 minutes pretreatment followed by cardiac arrest fluid treatment. This is the graph shown.
5 is a control group, cardiac arrest fluid treatment, 100 μg/mL oh8dG treatment and 100 μg/mL oh8dG 1 hour pretreatment followed by cardiac arrest fluid treatment using BCECF-AM to measure NBC activity in single cardiomyocytes of mice, It is a graph showing the result of measuring the resting pH level.
6 is a graph showing the NBC activity in single cardiomyocytes of mice according to the control, cardiac arrest fluid treatment, 100 μg/mL oh8dG treatment, and 100 μg/mL oh8dG 1 hour pretreatment and then cardiac arrest fluid treatment.
7 is a graph showing a summary of Resting pH levels in single cardiomyocytes of mice according to the control, cardiac arrest fluid treatment, 100 μg/mL oh8dG treatment, and 100 μg/mL oh8dG 1 hour pretreatment and then cardiac arrest fluid treatment.
FIG. 8 shows the results of measuring NBC activity by treating HEK293T cells overexpressing NBCe1-B as a control and oh8dG at a concentration of 10 μg/mL or 100 μg/mL for 1 hour using BCECF-AM. It is a graph.
9 is a graph showing a summary of NBC activity according to treatment of HEK293T cells overexpressing NBCe1-B as a control and oh8dG at a concentration of 10 μg/mL or 100 μg/mL for 1 hour.
FIG. 10 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours, 100 μg/mL oh8dG for 1 hour, cardiac arrest for 2 hours, 100 μg/mL oh8dG for 2 hours, This is a picture of the degree of cell death measured by Tunel assay.
11 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours treatment with 100 μg/mL oh8dG for 1 hour and cardiac arrest for 2 hours with 100 μg/mL oh8dG for 2 hours, This is a graph that quantifies the result of measuring the degree of apoptosis by Tunel assay.
12 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours treatment with 100 μg/mL oh8dG for 1 hour and cardiac arrest for 2 hours with 100 μg/mL oh8dG for 2 hours, This is a picture of the level of PAR expression.
13 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours and 100 μg/mL oh8dG for 1 hour and cardiac arrest for 2 hours and 100 μg/mL oh8dG for 2 hours, This is a graph showing the quantification of the result of measuring the level of PAR expression.
14 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours treatment with 100 μg/mL oh8dG for 1 hour and cardiac arrest for 2 hours with 100 μg/mL oh8dG for 2 hours, This is a picture of HSP60 expression level.
15 is a control group immediately after cardiac arrest, cardiac arrest for 2 hours, cardiac arrest for 2 hours treatment with 100 μg/mL oh8dG for 1 hour and cardiac arrest for 2 hours with 100 μg/mL oh8dG for 2 hours, It is a graph showing the quantification of the result of measuring the HSP60 expression level.
Figure 16 is a cardiac arrest fluid treatment for 2 hours, 100 μg/mL oh8dG with a cardiac arrest solution for 2 hours treatment and 100 μg/mL oh8dG with a cardiac arrest solution for 2 hours treatment for 2 hours, Bcl-2, P This is a picture of the RNA expression level of -53 and BAX measured by gel electrophoresis.
FIG. 17 shows Bcl-2, P according to treatment with cardiac arrest for 2 hours, treatment with 100 μg/mL oh8dG for 1 hour and treatment with cardiac arrest for 2 hours and treatment with 100 μg/mL oh8dG for 2 hours It is a graph showing the quantification of the results of measuring the RNA expression levels of -53 and BAX by gel electrophoresis.
FIG. 18 shows the level of RNA expression of CTGF according to treatment with cardiac arrest for 2 hours, treatment with 100 μg/mL oh8dG for 1 hour and treatment with cardiac arrest for 2 hours and treatment with 100 μg/mL oh8dG for 2 hours. Is a photograph measured by gel electrophoresis.
FIG. 19 shows the level of RNA expression of CTGF as a result of 2 hours of cardiac arrest, 2 hours of cardiac arrest and 100 μg/mL oh8dG for 1 hour, and 100 μg/mL oh8dG with 2 hours of cardiac arrest and 2 hours of treatment. Is a graph showing the quantification of the results measured by gel electrophoresis.
Figure 20 is a cardiac arrest fluid treatment for 2 hours, 100 μg/mL oh8dG with a cardiac arrest solution for 2 hours treatment and 100 μg/mL oh8dG with a cardiac arrest fluid treatment for 1 hour, COX-2 and CTGF This is a photograph of the protein expression level measured by gel electrophoresis.
FIG. 21 is a protein of COX-2 according to treatment with cardiac arrest for 2 hours, treatment with 100 μg/mL oh8dG for 1 hour and treatment with cardiac arrest for 2 hours and treatment with 100 μg/mL oh8dG for 2 hours It is a graph showing the quantification of the result of measuring the expression level by gel electrophoresis.
FIG. 22 shows the protein expression level of CTGF according to treatment with cardiac arrest for 2 hours, treatment with 100 μg/mL oh8dG for 1 hour and treatment with cardiac arrest for 2 hours and treatment with 100 μg/mL oh8dG for 2 hours. Is a graph showing the quantification of the results measured by gel electrophoresis.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention,

It provides a composition for preventing heart damage during cardiac arrest containing 8-oxo-2'-deoxyguanosine as an active ingredient.

During heart surgery, cardiac arrest fluid containing high concentration of potassium (K ⁺ ) is used to stop the heart and reduce damage to the myocardium during cardiac arrest. However, when blood is reperfused to the heart after surgery, the cardiac arrest fluid causes damage to the heart. I can. This is because the activity of NBC (Sodium/Bicarbonate Cotransporter), which is an ion transporter of cardiomyocytes, increases due to the high concentration of potassium (K ⁺ ) contained in cardiac arrest fluid, and acidification of cardiomyocytes occurs.

Accordingly, for myocardial protection, a composition for preventing heart damage during cardiac arrest may be used together with cardiac arrest fluid. Specifically, the composition for preventing heart damage during cardiac arrest containing 8-oxo-2'-deoxyguanosine as an active ingredient, when used with cardiac arrest fluid, inhibits the increase of free radicals in cardiomyocytes and inhibits ion transporter activity, Heart damage can be prevented by inhibiting the expression of the apoptosis factors PAR (Poly-ADP-ribose), HSP60, Bcl-2, P-53 and BAX, and reducing the inflammatory factor COX-2 and the fibrosis factor CTGF. have.

On the other hand, when used with cardiac arrest, the concentration of the 8-oxo-2'-dioxyguanosine (oh8dG) is 1 to 1000 μg/mL, 3 to 700 μg/mL, 6 to 400 μg/mL, 10 In the case of to 100 μg/mL, 50 to 150 μg/mL, or 90 to 110 μg/mL, heart damage can be prevented, but this is only an example and is not limited thereto.

In addition, the type of cardiac arrest fluid may be HTK (Histidine-Tryptophan-Ketoglutarate), Hyperkalemic cardioplegia, Cold antegrade crystalloid cardioplegia, crystalloid cardioplegia, warm blood cardioplegia, high-KCl cardioplegic solution or del Nido (DN) solution, but is not limited thereto. Does not.

The amount of cardiac arrest fluid used may vary depending on factors such as the age, weight, and severity of the disease of the person undergoing heart surgery, but 0.1 to 10 mL/minute/heart weight (g), 0.3 to 7 mL/minute/heart weight (g), 0.5 to 4 mL/minute/heart weight (g), 0.8 to 2 mL/minute/heart weight (g), 0.2 to 3 mL/minute/heart weight (g) or 0.7 to 8 mL/minute/ It may be the heart weight (g), but this is only an example and is not limited thereto.

In addition, the heart damage during cardiac arrest may be a heart damage due to reperfusion after cardiac arrest for cardiac surgery, but is not limited thereto.

Furthermore, the composition may be administered simultaneously or sequentially with the cardiac arrest solution.In one embodiment of the present invention, the cardiac arrest solution HTK (Histidine-Tryptophan-Ketoglutarate) and 8-oxo-2'-dioxyguanosine (oh8dG) ) Were used together, and in other examples, HTK was used after 8-oxo-2'-dioxyguanosine (oh8dG) was pretreated (Experimental Examples 2 to 10).

And the composition may be a cardiac arrest solution in which 8-oxo-2'-dioxyguanosine is added to a cardiac arrest solution containing K ⁺ , wherein the concentration of K ⁺ is 1 to 100 mM, 3 to 70 mM, 5 to 40 mM , 7 to 20 mM, 4 to 50 mM, or 6 to 80 mM, but this is only an example and is not limited thereto. In addition, the cardiac arrest fluid containing K ⁺ may further include Na ⁺ , Mg ^{2 +} , Ca ²⁺ , histidine, tryptophan, ketoglutarate, and mannitol.

The composition for preventing heart damage during cardiac arrest according to the present invention has an excellent effect of inhibiting an increase in free radicals during cardiac arrest, and thus can prevent damage to heart tissues caused by free radicals.

In Experimental Example 1 of the present invention, it was confirmed that the activity of the ion transporter NBC (Sodium/Bicarbonate Cotransporter) was increased by H ₂ O ₂ as a kind of active oxygen, through which the activity generated during cardiac arrest by the use of cardiac arrest fluid It can be seen that oxygen can damage heart tissue (see FIGS. 1 and 2).

However, in an embodiment of the present invention, after the cardiomyocytes of mice were isolated, 8-oxo-2'-deoxyguanosine (oh8dG) was pretreated at a concentration of 100 μg/mL, and cardiac arrest fluid (HTK, Histidine-Tryptophan-Ketoglutarate ), it was confirmed that the increase in free radicals of the cardiomyocytes was suppressed compared to the case of treatment with only the control or cardiac arrest fluid, so it can be seen that the composition can prevent damage to the heart tissue during cardiac arrest (Experimental Example 2, see FIGS. 3 and 4).

In addition, the composition may exhibit an effect of reducing the ion transporter activity of cardiomyocytes.

In one embodiment of the present invention, cardiac arrest fluid was treated by isolating rat cardiomyocytes and pretreated with 8-oxo-2'-deoxyguanosine (oh8dG) at a concentration of 100 μg/mL for 1 hour, and then BCECF-AM was The activity of the ion transporter, NBC (Sodium/Bicarbonate Cotransporter), was measured. Compared with the control, cardiac arrest fluid, and 8-oxo-2'-deoxyguanosine (oh8dG) at a concentration of 100 μg/mL, respectively. It can be seen that the activity of NBC decreases (see Experimental Example 3, Figs. 5, 6 and 7).

In addition, when 8-oxo-2'-deoxyguanosine (oh8dG) was treated on tissue cells in which reactive oxygen was not generated, it was confirmed that NBC activity was decreased compared to the control (Experimental Example 4, FIGS. 8 and 9). Reference).

The composition may reduce apoptosis factors PAR, HSP60, Bcl-2, P-53, and BAX.

When cardiomyocytes are exposed to cardiac arrest fluid, the expression of apoptosis factors increases. It is known that inhibition of this can reduce the death of cardiomyocytes to protect the myocardium. Accordingly, in an embodiment of the present invention, the TUNEL assay, which is a method for confirming apoptosis, was performed, and 8-oxo-2'-dioxyguanosine at a concentration of 100 μg/mL of cardiac arrest fluid and 100 μg/mL than when only the control and cardiac arrest fluid were treated ( oh8dG), it can be seen that the degree of apoptosis of cardiac arrested cardiomyocytes decreases (see Experimental Example 5, FIGS. 10 and 11).

In addition, when treated with cardiac arrest fluid and 8-oxo-2'-deoxyguanosine (oh8dG) at a concentration of 100 μg/mL, PAR (Poly -ADP-ribose), HSP60, Bcl-2, P-53 and BAX can be confirmed to decrease (see Experimental Examples 6 to 8 and Figs. 12 to 17).

In addition, it can be seen that the expression of the inflammatory factor COX-2 and the factor involved in fibrosis, CTGF, are decreased (see Experimental Example 9, Experimental Example 10 and FIGS. 18 to 22).

Through the above results, it can be seen that the composition for preventing heart damage during cardiac arrest according to the present invention can prevent damage to the heart tissue during cardiac arrest.

Another aspect of the invention,

It provides a cardiac arrest kit for cardiac surgery comprising a cardiac arrest fluid and a composition for preventing heart damage during cardiac arrest.

The kit is a kit that allows the cardiac arrest solution and the composition to be used together to prevent cardiac damage caused by the use of cardiac arrest solution during heart surgery.

Here, the description of the cardiac arrest fluid, the composition, and heart damage are as described above.

In addition, the cardiac arrest solution and the composition may be administered simultaneously or sequentially. In one embodiment of the present invention, the cardiac arrest solution HTK (Histidine-Tryptophan-Ketoglutarate) and 8-oxo-2'-dioxyguanosine (oh8dG) These have been used together, and in other examples, HTK has been used after pretreatment with 8-oxo-2'-dioxyguanosine (oh8dG) (Experimental Examples 2 to 9).

Furthermore, cardiac arrest fluid and the composition may be mixed and administered.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

< Example 1> Preparation of the composition for preventing heart damage during cardiac arrest according to the present invention

The 8-oxo-2'-deoxyguanosine (oh8dG) contained in the composition for preventing heart damage during cardiac arrest according to the present invention may be commercially available, and is obtained by a conventionally prepared method. You can also use it.

In addition to 8-oxo-2'-deoxyguanosine, the composition includes K ⁺ , Na ⁺ , Mg ^{2 +} , Ca ^{2 +} , histidine, tryptophan, ketoglutarate, mannitol ( mannitol) may be further included, which is for illustrative purposes only, and is not limited thereto, and may further include a component widely known to those skilled in the art and commonly used.

Meanwhile, in the following experimental examples, the composition was used alone or concurrently or sequentially with cardiac arrest fluid containing K ⁺ , but when used alone, it was administered with a physiological salt solution (PSS) buffer.

< Experimental example 1> Measurement of cell ion transporter function change by free radicals

Changes in the function of the ion transporter of the cells due to free radicals were measured through the following experiments, and the results are shown in FIGS. 1 and 2.

When HCO ₃ ^- solution without Na ⁺ is injected, the pH of the cell decreases, and when HCO ₃ ^- solution containing Na ⁺ is injected, Na ⁺ and HCO ₃ ^- enter the cell together. HCO ₃ that has entered the cell ^- (Basic component) increases the pH in the cell. Such changes in intracellular pH can be measured using BCECF-AM (2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester), a fluorescent substance for pH measurement. . In addition, when EIPA (5-(N-Ethyl-N-isopropyl) Amiloride), which inhibits Sodium/Hydrogen Exchanger (NHE), is mixed with the HCO ₃ ^- solution, only the activity of NBC (Sodium/Bicarbonate Cotransporter) can be measured. .

Solution (HCO ₃ ^{- -)} HCO ₃ with no Na ⁺ mixture of EIPA in HEK293T cells in which the NBC a type of NBCe1 of over-expression for 24 h (human embryonic kidney cells) were injected into the injecting H ₂ O ₂ to the HEK293T cells In the case of not taking as a control, HCO ₃ ^- solution without Na ⁺ and H ₂ O ₂ were simultaneously injected into the HEK293T cells, and H ₂ O ₂ was first injected into the HEK293T cells and HCO without Na ⁺ 1 hour later ₃ ^-The case where the solution was injected was set as the experimental group.

Solution _{^{(Na + + HCO 3 - -}} ) HCO 3 containing Na ⁺ after water when the pH of each of HEK293T cells increased by implanting, was measured for these changes using BCECF-AM, pH changes over time (slope ), it was confirmed how much the activity of NBCe1 changes.

Without injection of H ₂ O ₂ in HEK293T cells Na ⁺ is HCO ₃ ^no-solution (HCO ₃ ^-) when the injection a more injection control, the H ₂ O ₂ at the same time or 1 hour before the NBCe1 activity was increased ( 1 and 2).

This indicates that the activity of NBC, which is an ion transporter, is increased by H ₂ O ₂ , a kind of active oxygen, and through the above results, the active oxygen increases the ion transport capacity of the chlorine/bicarbonate exchanger, causing excessive ion imbalance, and cardiomyocytes It can be seen that it can cause acidification of the heart and damage the heart tissue.

< Experimental example 2> single Cardiomyocyte Measurement of changes in active oxygen after separation

When the composition for preventing heart damage during cardiac arrest according to the present invention was treated to cardiomyocytes, the following experiment was performed to evaluate the change in free radicals, and the results are shown in FIGS. 3 and 4.

Depending on the use of cardiac arrest fluid, if free radicals increase during cardiac arrest, excessive ion imbalance may be caused and cardiac tissue may be damaged by acidification of cardiomyocytes. Therefore, suppressing the increase in free radicals can prevent damage to heart tissues.

According to the single cardiomyocyte isolation method, 7 mL of EDTA solution was injected into the right ventricle to 8-week-old male mice, and then only the heart was separated and transferred to a plate. After injecting 10 mL of EDTA solution into the left ventricle of the separated heart, 3 mL of Perfusion solution was injected into the left ventricle, and 30 to 40 mL of Collagenase solution was injected into the left ventricle, thereby confirming whether the myocardium became pale and soft. Thereafter, only the ventricle was cleanly cut and separated, and after tearing it into several pieces using forceps, it was immersed in a Stop solution, filtered through a 100 μm filter, and centrifuged at 40 g for 15 minutes to separate only cardiomyocytes.

The isolated cardiomyocytes were treated with untreated control, cardiac arrest solution HTK (Histidine-Tryptophan-Ketoglutarate) for 1 hour and HTK for 1 hour, but before that, oh8dG at a concentration of 100 μg/mL for 10 minutes or 30 minutes It was divided into four pretreated experimental groups. Treat the drug according to each experimental group at room temperature, and perform DCFDA staining to measure the amount of active oxygen in the cells, and use a confocal microscope to irradiate light with a wavelength of 495 nm and then use a confocal microscope. And measured. When the active oxygen increases, the intensity of the green light at the wavelength of 530 nm increases, so by measuring the amount of active oxygen in the cardiomyocytes through this, the increase or decrease of the active oxygen was confirmed.

When treated with cardiac arrest solution (HTK) than the control group, free radicals increased. And, compared to the case where only HTK was treated, when the cardiac arrest solution was treated after pretreatment with 100 μg/mL oh8dG, the generation of active oxygen decreased, but the increase of the active oxygen was suppressed as the oh8dG treatment time increased (see FIGS. 3 and 4 ).

This indicates that if only cardiac arrest fluid is treated without oh8dG, active oxygen is increased, but if cardiac arrest fluid is treated after pretreatment with 100 μg/mL oh8dG, the increase of free radical is suppressed. Through the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention suppresses an increase in free radicals, so that damage to the heart tissue during cardiac arrest may be prevented.

< Experimental example 3> Cardiomyocyte Cardiac arrest ( HTK) Confirmation of reduction in ion transporter activation by treatment of composition for preventing heart damage during cardiac arrest and change of ion transporter activity

In order to evaluate the effect of reducing the activation of the ion transporter during cardiac arrest of the composition for preventing cardiac damage during cardiac arrest of the present invention, the following experiment was performed, and the results are shown in FIGS. 5 to 7 below.

In the same manner as in Experimental Example 2, cardiomyocytes were isolated using 8-week-old male mice, respectively, as a control without cardiac arrest (HTK) treatment, and when treated with cardiac arrest solution for 5 minutes, oh8dG at a concentration of 100 μg/mL was used. The case of treatment for 1 hour and the case of pretreatment with oh8dG at a concentration of 100 μg/mL for 1 hour before the cardiac arrest solution was treated for 5 minutes were divided into experimental groups.

Thereafter, for the cardiomyocytes of the control group and the experimental group treated with the drug as described above, the activity of NBC was measured using BCECF-AM as in Experimental Example 1, and the pH value for the first minute of the measurement was averaged to the resting pH level in the cells. Was confirmed.

When treated with cardiac arrest fluid (HTK) than the control group, NBC activity was increased. In addition, NBC activity decreased when treated with 100 μg/mL oh8dG than in the control group or treated with only cardiac arrest fluid, and NBC activity decreased even more when cardiac arrest fluid was treated after pretreatment with 100 μg/mL oh8dG. (See Figs. 5 to 7).

This is the effect of decreasing the activity of NBC, an ion transporter of cardiomyocytes, compared to the case where only cardiac arrest fluid without oh8dG is treated, when 100 μg/mL oh8dG is treated or when cardiac arrest fluid is treated after pretreatment with 100 μg/mL oh8dG. Indicates that it is excellent. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention prevents excessive ion imbalance and acidification of cardiomyocytes by reducing ion transporter activity, so that damage to the heart tissue during cardiac arrest can be prevented.

< Experimental example 4> Evaluation of NBC activity control when treating a composition for preventing heart damage during cardiac arrest

In order to evaluate the control of the NBC activity of the composition for preventing heart damage during cardiac arrest of the present invention, the following experiment was performed, and the results are shown in FIGS. 8 and 9 below.

The case where oh8dG was not treated in HEK293T cells (human embryonic kidney cells) overexpressing NBCe1-B, a sodium/bicarbonate cotransporter (NBC) for 24 hours, was set as a control, and oh8dG or 100 at a concentration of 10 μg/mL in the HEK293T cells. A case where oh8dG at a concentration of μg/mL was treated for 1 hour each was set as an experimental group.

Since, Sodium / Hydrogen Exchanger (NHE) 5- (N-Ethyl-N-isopropyl) HCO 3 for Amiloride (EIPA) in the Na ⁺ that ^{inhibit-solution} (Na ⁺ + HCO ₃ ^-) and Na ⁺ is not HCO ₃ ^- solution (HCO ₃ ^-) was mixed with a flow over the course of the experiment. BCECF-AM was used to measure the intracellular pH change, and it was confirmed how much the activity of NBCe-1B changes through the intracellular pH change (slope) over time.

When treated with 10 μg/mL oh8dG than the control, the activity of NBCe-1B decreased, and more than that, when treated with 100 μg/mL oh8dG, the activity of NBCe-1B decreased (see FIGS. 8 and 9).

This indicates that treatment with oh8dG has an effect of reducing NBC activity, and in particular, it is confirmed that NBC activity can be reduced regardless of active oxygen. In other words, even in tissue cells in which the active oxygen was not increased by the cardiac arrest fluid treatment, the effect of reducing NBC activity was exhibited by the oh8dG treatment. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention can prevent damage to tissues due to excessive ionic imbalance and acidification of cells even in tissues in which reactive oxygen is not generated.

< Experimental example 5> Cardiac arrest Cardiomyocyte Assessment of the degree of apoptosis

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed as follows to evaluate the effect of the composition for preventing heart damage during cardiac arrest of the present invention on reducing the degree of apoptosis during cardiac arrest, and the results are shown in FIGS. 10 and 11 .

The TUNEL assay is a well-known test method for confirming apoptosis, and the degree of apoptosis can be confirmed by measuring DNA fragmentation resulting from apoptosis.

The heart was isolated from 8-week-old male rats, and only the left ventricle was divided into 4 cases and placed in an ep-tube (eppendorf tube). 1) Immediately after cardiac arrest, 2) When cardiac arrest fluid was treated for 2 hours, 3) When oh8dG at a concentration of 100 μg/mL was treated for 1 hour with the cardiac arrest fluid treatment, and 4) 100 μg/mL with the cardiac arrest fluid treatment In the case of treatment with the concentration of oh8dG for 2 hours, in the case of treatment with oh8dG, 3) and 4), pieces were placed in a mold, filled with OCT compound, and frozen in liquid nitrogen. In all four cases, a tissue cut into slices was attached to a slide glass using a cryo secion machine. TransDetect ^® Fluorescein staining for TUNEL assay The experiment was conducted using the product of TUNEL cell apoptosis detection kit (FA201-01).

A guideline was drawn on the outside of the tissue with a hydrophobic pen, washed three times with PBS for 10 minutes each, and the tissue was fixed for 30 minutes at room temperature using 4% paraformaldehyde. Then, after washing again 3 times with PBS for 10 minutes, treated with Permeabilization solution for 5 minutes at room temperature, and washed again with PBS 3 times. Labeling solution and TdT (Terminal deoxynucleotidyl transferase) were mixed at 1:25 and treated on the tissue under light blocking for 1 hour at 37°C. After washing this again with PBS, DAPI solution containing the mounting solution was added at room temperature. Attached for 5 minutes. The stained slides were confirmed at a wavelength of 488 nm using a fluorescence microscope.

TUNEL positive cells (tissue damage scale) increased 2 hours after cardiac arrest than immediately after cardiac arrest without cardiac arrest (HTK) treatment, the control group, but TUNEL positive cells were treated with 100 μg/mL oh8dG with cardiac arrest fluid. The cells effectively decreased, but the longer the oh8dG treatment time, the more decreased (see FIGS. 10 and 11). This indicates that treatment with only cardiac arrest fluid without 100 μg/mL oh8dG increases the degree of apoptosis, but treatment with 100 μg/mL oh8dG decreases the degree of apoptosis.

From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention reduces apoptosis when used together with cardiac arrest fluid, and thus it can be seen that damage to the heart tissue during cardiac arrest can be suppressed. In the following experimental examples, assays for apoptosis factors involved in apoptosis were performed.

< Experimental example 6> Cardiac arrest Cardiomyocyte Apoptosis factor PAR Whether to decrease Measure

The expression of apoptosis factors is increased by exposure to cardiac arrest fluid.In order to evaluate the effect of reducing poly-ADP-ribose, apoptosis factor PAR (Poly-ADP-ribose) through treatment of the composition for preventing heart damage during cardiac arrest of the present invention, the following experiment Was performed, and the results are shown in FIGS. 12 and 13.

When PARP (Poly-ADP-ribose polymerase) is activated in DNA-damaged cells, ATP of cells can be depleted and this can lead to apoptosis, so the level of expression of PAR can be a measure of apoptosis.

Heart tissue was removed from 8-week-old male rats, 1) immediately after cardiac arrest, 2) treated with cardiac arrest fluid (HTK) for 2 hours, 3) oh8dG at a concentration of 100 μg/mL for 1 hour with the cardiac arrest solution treatment The experiment was conducted by dividing into the case of treatment and 4) the case where oh8dG at a concentration of 100 μg/mL was treated for 2 hours together with the cardiac arrest solution treatment. The heart tissue in each case was frozen in liquid nitrogen using an OCT compound (Optimal Cutting Temperature compound), and the frozen tissue was micro-sectioned (10 μm thick).

A tissue section was attached to the slide, and a guideline was drawn around the tissue using a hydrophobic pen, and washed three times with PBS for 10 minutes. The tissue was fixed at -20°C for 10 minutes using methanol, glycine was treated at 4°C, and the blocking solution was treated for 1 hour. Thereafter, the PAR antibody was mixed with the blocking solution at 1:100, and then treated on the tissue and placed at 4°C for 24 hours. After washing 3 times with an incu-buffer for 10 minutes, FITC, a secondary antibody, was attached for 1 hour at room temperature, and after washing 3 times with PBS for 10 minutes, a mounting solution with DAPI for staining the nuclei was applied. Treated and covered with a cover-slip Using a confocal microscope, the stained slide was measured for expression of PAR at wavelengths of DAPI (450 nm) and FITC (488 nm).

The expression of PAR increased after 2 hours after treatment with cardiac arrest than immediately after cardiac arrest without cardiac arrest (HTK) treatment as a control group. And, compared to the case where only cardiac arrest fluid was treated, when oh8dG at a concentration of 100 μg/mL was treated with cardiac arrest fluid, the expression of PAR decreased, but as the oh8dG treatment time increased, the expression of PAR decreased (see FIGS. 12 and 13). .

This indicates that apoptosis increases when only cardiac arrest fluid is treated without oh8dG, but apoptosis decreases when treated with 100 μg/mL oh8dG. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention is used together with cardiac arrest fluid to reduce the expression of apoptosis factors, so that damage to the heart tissue during cardiac arrest can be prevented.

< Experimental example 7> Cardiac arrest Cardiomyocyte Apoptosis factor HSP60 Whether to decrease Measure

The expression of apoptosis factors is increased by exposure to cardiac arrest fluid.In order to evaluate the effect of reducing the apoptosis factor HSP60 (heat shock protein 60) through the treatment of the composition for preventing heart damage during cardiac arrest of the present invention, the following experiment was performed. Was performed, and the results are shown in FIGS. 14 and 15.

HSP60 is a protein that indicates intracellular stress, and it is well known that the reduction of HSP60 in mitochondria after cardiac arrest helps to preserve mitochondria, so the level of expression of HSP60 can be a measure of apoptosis.

As in Experimental Example 6, 1) immediately after cardiac arrest, 2) when the cardiac arrest solution was treated for 2 hours, 3) when oh8dG at a concentration of 100 μg/mL was treated for 1 hour together with the cardiac arrest solution treatment, and 4) the cardiac arrest solution treatment and Together, a heart tissue was prepared when oh8dG at a concentration of 100 μg/mL was treated for 2 hours. Heart tissue in each case was frozen in liquid nitrogen using an OCT compound (Optimal Cutting Temperature compound), and the frozen tissue was cryo-sectioned.

Tissue sections were attached to the slide, fixed at room temperature with 4% PFA, washed twice with PBS for 10 minutes, treated with 0.5% triton-X for 5 minutes at room temperature, and treated with glycine at 4°C. After that, the blocking solution was treated for 1 hour. After mixing the HSP60 antibody 1:100 with the blocking solution, the tissue was treated and placed at 4°C for 24 hours. After washing with an incu-buffer, Rhodamine, a secondary antibody, was applied at room temperature for 1 hour, a mounting solution with DAPI was treated, and the tissue was covered with a cover-slip. Using a confocal microscope, the stained slide was measured for expression of HSP60 at wavelengths of DAPI (450 nm) and Rhodamine (595 nm).

The expression of HSP60 increased after 2 hours after treatment with cardiac arrest than immediately after cardiac arrest without cardiac arrest (HTK) treatment as a control group. And, compared to the case where only the control and cardiac arrest fluid were treated, the expression of HSP60 decreased when 100 μg/mL oh8dG was treated with cardiac arrest fluid, but the expression of HSP60 decreased as the oh8dG treatment time increased (see FIGS. 14 and 15). .

This indicates that apoptosis increases when only cardiac arrest fluid is treated without oh8dG, but apoptosis decreases when treated with 100 μg/mL oh8dG. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention is used together with cardiac arrest fluid to reduce the expression of apoptosis factors, so that damage to the heart tissue during cardiac arrest can be prevented.

< Experimental example 8> Cardiac arrest Cardiomyocyte Apoptosis factor Bcl -2, P-53 or BAX Whether to decrease Measure

Expression of apoptosis factors is increased by exposure to cardiac arrest fluid, and the effect of reducing Bcl-2 (B-cell lymphoma 2), P-53 or BAX, which is apoptosis factor, through treatment of the composition for preventing heart damage during cardiac arrest of the present invention In order to evaluate, the following experiment was performed, and the results are shown in FIGS. 16 and 17.

Bcl-2, p53, and BAX are well known as proteins that control apoptosis, and their expression level can be a measure of apoptosis, so the level of RNA expression was measured by gel electrophoresis.

In 8-week-old male rats, myocardial tissue was divided into 3 cases and placed in ep-tubes. 1) When treated with cardiac arrest solution (HTK) for 2 hours, 2) When treated with oh8dG at a concentration of 100 μg/mL for 1 hour with the cardiac arrest solution treatment, and 3) when treated with the cardiac arrest solution at a concentration of 100 μg/mL In all cases where oh8dG was treated for 2 hours, RNA was isolated from the myocardial tissue using a Hybrid-Ribo ^EX extraction system.

The isolated RNA was measured for its concentration with ND-1000, a spectrophotometer. And cDNA was synthesized under conditions of 1 hour at 42°C and 5 minutes at 94°C using an RT-PCR kit, and primers of GAPDH, Bcl-2, P-53 and BAX were mixed with the cDNA for 5 minutes at 95°C, It was synthesized under the conditions of 45 cycles for 30 seconds at 95°C, 1 minute at 58°C and 1 minute at 72°C. The resulting PCR product was loaded on a 1% agarose gel, electrophoresed at 100 mV for 24 minutes, and then the electrophoretic band was confirmed using GelDoc ^XR .

Compared to 2 hours after treatment with cardiac arrest, when 100 μg/mL oh8dG was treated with cardiac arrest, the level of RNA expression of Bcl-2, P-53 or BAX decreased, but the longer oh8dG treatment time The level of RNA expression of Bcl-2 or P-53 was further reduced (see FIGS. 16 and 17).

This indicates that apoptosis is reduced when treated with 100 μg/mL oh8dG than when only cardiac arrest fluid is treated without oh8dG. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention is used together with cardiac arrest fluid to reduce the expression of apoptosis factors, so that damage to the heart tissue during cardiac arrest can be prevented.

< Experimental example 9> Cardiac arrest Cardiomyocyte CTGF Whether to decrease Measure

In order to evaluate the effect of reducing CTGF (connective tissue growth factor) through the treatment of the composition for preventing heart damage during cardiac arrest of the present invention, the same experiment as in Experimental Example 8 was performed, except that the primer of CTGF was used. It is shown in Figures 18 and 19.

Since CTGF is involved in fibrosis and growth, differentiation and migration of fibroblasts, the level of RNA expression was measured by gel electrophoresis.

Compared to 2 hours after treatment with cardiac arrest fluid, when 100 μg/mL oh8dG is treated with cardiac arrest fluid, the level of RNA expression of CTGF decreases, but the longer oh8dG treatment time, the more the level of RNA expression of CTGF decreases. (See Figs. 18 and 19).

This indicates that the incidence of fibrosis is reduced when treated with 100 μg/mL oh8dG than when only cardiac arrest fluid is treated without oh8dG. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention is used together with the cardiac arrest fluid to reduce the occurrence of fibrosis of the heart, so that damage to the heart tissue during cardiac arrest can be prevented.

< Experimental example 10> Cardiac arrest Cardiomyocyte COX-2 or CTGF Whether to decrease Measure

In order to evaluate the effect of reducing COX-2 (cyclooxygenase-2) or CTGF (connective tissue growth factor) through treatment of the composition for preventing heart damage during cardiac arrest of the present invention, the following experiment was performed, and the results are shown in FIGS. It is shown in 22.

COX-2 is an inflammatory factor that increases the level of expression when there is inflammation, and CTGF is involved in fibrosis and growth, differentiation and migration of fibroblasts. Their protein expression levels were measured by gel electrophoresis.

Myocardial tissues isolated from 8-week-old male rats were divided into 3 cases and placed in ep-tubes. 1) When treated with cardiac arrest solution (HTK) for 2 hours, 2) When treated with oh8dG at a concentration of 100 μg/mL for 1 hour with the cardiac arrest solution treatment, and 3) when treated with the cardiac arrest solution at a concentration of 100 μg/mL In all cases where oh8dG was treated for 2 hours, 200 uL of lysis buffer was added after drug treatment, and the cells were broken by sonication.

This was put into a centrifuge again and centrifuged at 4° C. and 12000 g for 15 minutes, and then only the supernatant was obtained and quantified according to the standard through Bradford assay, and the absorbance was measured at 595 nm. Samples were quantified through the measured absorbance, mixed with a sample buffer (2x laemmli sample buffer + 2-Mercaptoethanol), and heated at 37° C. for 15 minutes.

After 10 μL of each sample was loaded on 8% SDS-gel, electrophoresis was performed at 120 V for 15 minutes, and electrophoresis was performed again at 150 V for 30 minutes. The electrophoresed gel was placed on 3M paper and PVDF membrane, inserted into a plate, and transferred to a transfer buffer at 300 mA for 90 minutes. The transferred membrane was cut to fit the size, immersed in non-fat dry milk for 1 hour, and the primary antibodies COX-2, CTGF, and β-actin were attached, and stored at 4°C for 24 hours. After 24 hours, after washing three times with TBST for 10 minutes, HRP secondary antibody was added for 1 hour according to each antibody (COX-2: Rabbit, CTGF: Mouse). After that, after washing 4 times with TBST again for 10 minutes, ECL was attached and expression was confirmed using a developer and fixer in a dark room.

Compared to 2 hours after the cardiac arrest fluid was treated, when 100 μg/mL oh8dG was treated with cardiac arrest fluid, the protein expression levels of COX-2 and CTGF were decreased (see FIGS. 20 to 22).

This indicates that the incidence of fibrosis and inflammation was reduced when treated with 100 μg/mL oh8dG than when only cardiac arrest fluid was treated without oh8dG. From the above results, it can be seen that the composition for preventing heart damage during cardiac arrest of the present invention is used together with cardiac arrest fluid to reduce the occurrence of fibrosis and inflammation of the heart, so that damage to the heart tissue during cardiac arrest can be prevented.

Claims (11)

A composition for preventing heart damage during cardiac arrest, comprising 8-oxo-2'-deoxyguanosine as an active ingredient.
The method of claim 1,
The composition for preventing heart damage during cardiac arrest, characterized in that the heart damage during cardiac arrest is a heart damage due to reperfusion after cardiac arrest for cardiac surgery.
The method of claim 1,
The composition for preventing heart damage during cardiac arrest is a composition for preventing heart damage during cardiac arrest, characterized in that it can be administered simultaneously or sequentially with the cardiac arrest solution.
The method of claim 1,
The composition for preventing heart damage during cardiac arrest is a cardiac arrest solution in which 8-oxo-2'-dioxyguanosine is added to the cardiac arrest solution containing K ⁺ . The composition for preventing heart damage during cardiac arrest.
The method of claim 4,
A composition for preventing heart damage during cardiac arrest, characterized in that the concentration of K ⁺ is 1 to 100 mM.
The method of claim 1,
The composition for preventing heart damage during cardiac arrest, characterized in that it suppresses the increase of free radicals, the composition for preventing heart damage during cardiac arrest.
The method of claim 1,
The composition for preventing heart damage during cardiac arrest is characterized in that it reduces the activity of the ion transporter of cardiomyocytes, and the composition for preventing heart damage during cardiac arrest
The method of claim 1,
The composition for preventing heart damage during cardiac arrest is a composition for preventing heart damage during cardiac arrest, which reduces at least one selected from the group consisting of PAR, HSP60, Bcl-2, P-53, BAX, COX-2 and CTGF.
Cardiac arrest kit for cardiac surgery comprising a cardiac arrest fluid and a composition for preventing heart damage during cardiac arrest according to claim 1.
The method of claim 9,
Cardiac arrest kit for cardiac surgery, characterized in that the cardiac arrest fluid and the composition can be administered simultaneously or sequentially.
The method of claim 9,
Cardiac arrest kit for cardiac surgery, characterized in that the cardiac arrest solution and the composition are mixed and administered.

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