CN111249309B - ALDH2 activated mitochondria preparation for treating myocardial ischemia reperfusion injury and preparation method and application thereof - Google Patents

Abstract

The invention relates to an acetaldehyde dehydrogenase 2 (ALDH 2) activated mitochondrial preparation for treating myocardial ischemia reperfusion injury and a preparation method and application thereof. According to the invention, the ALDH2 in the myocardial cell mitochondria is activated by the micromolecular activator Alda-1 for the first time, and the myocardial cell mitochondria is separated to carry out myocardial ischemia reperfusion injury transplantation, so that the ALDH2 activated mitochondria can obviously increase the productivity of the myocardial cells, reduce the apoptosis of the myocardial cells after myocardial ischemia reperfusion injury, and improve the myocardial ischemia reperfusion injury, and the mitochondrial preparation obtained by treating the separated myocardial cells with the Alda-1 can enhance the treatment effect, and the mitochondrial transplantation for clinically carrying out the ALHD2 activation in the process of recanalization of blood vessels of acute myocardial infarction patients can ensure myocardial energy supply, improve the myocardial contraction and relaxation function, reduce the reperfusion injury and inhibit the occurrence of heart failure.

Description

ALDH2 activated mitochondria preparation for treating myocardial ischemia reperfusion injury and preparation method and application thereof

Technical Field

The invention relates to the field of biological medicines, and in particular relates to an ALDH2 activated mitochondria preparation for treating myocardial ischemia-reperfusion injury, and a preparation method and application thereof.

Background

Ischemic cardiovascular diseases such as coronary heart disease and acute myocardial infarction are the most important killers threatening human health in the world at present, and the realization of timely blood vessel recanalization by clinical percutaneous coronary intervention can effectively improve the myocardial function of patients. However, myocardial ischemia reperfusion Injury (IR) occurs in patients receiving revascularization therapy, which is the major cause of death in nearly 10% of patients with acute myocardial infarction within 1 year after discharge and chronic heart failure in about 20% of surviving patients. The myocardial cell has a large amount of mitochondria to maintain the normal function of the heart, the insufficient oxygen supply of the myocardial cell caused by myocardial infarction seriously damages the structure and the function of the mitochondria of the myocardial cell by causing oxidative stress and accumulation of mitochondria intermediate metabolites, and the implementation of the revascularization on patients can effectively increase the oxygen supply of the myocardial cell to relieve the myocardial cell damage caused by blood deficiency, but the damaged mitochondria can cause abnormal recovery of the vitality and the contractile function of the myocardial cell, and the process can cause the re-damage of the mitochondria. Therefore, the process of carrying out mitochondrial transplantation on the patients with revascularization is expected to improve the energy metabolism of the myocardial cells of the patients, and further increase the regeneration and contraction capacity of the myocardial cells.

Journal literature (Populus Seifeng, Sunpong, Kingyupei, et al. development of mitochondrial transplantation in treating mitochondrial defect diseases [ J]The biochemical and biophysical progress, 2018,45(3):297-304.) discloses that the James topic group utilizes the isolated rabbit heart left anterior descending artery ligation mode to make a myocardial ischemia reperfusion injury model and evaluates the mitochondrial transplantation protection function. After 29min of myocardial ischemia, the injection concentration in the ischemic region was (7.7. + -. 1.5). times.10 ⁶ Intervention was performed in/mL of normal mitochondria. The results show that after 120min of re-perfusion, the left ventricular diastolic pressure and post-contraction of the mitochondrion transplantation treatment group are respectively and remarkably increased to 75% and 83% of the normal state, and the levels are close to the normal level. TTC staining shows that the myocardial infarction area at the mitochondrial transplantation position is obviously reduced compared with that of a model group, and the protection effect is obvious. Subsequently, it was also verified by some in vivo heart models in rabbits and pigs that mitochondrial transplantation had significant effects in the protection of cardiac ischemia/reperfusion injury. ".

Although the mitochondria transplantation has an extremely wide application prospect, how to improve the repair capability of the injured myocardial cells after the exogenous mitochondria transplantation is a key limiting factor of the mitochondria transplantation strategy.

Literature (zuirui, schlemongyang, royal, et al. ALDH2 alleviates myocardial ischemia-reperfusion injury by modulating the mitochondrial network-chinese scientific papers on-line) discloses: ALDH2 is an enzyme located within the mitochondrial matrix and has important cardioprotective effects. Increased mitochondrial division during myocardial ischemia-reperfusion results in the formation of large numbers of fragmented mitochondria, while inhibition of mitochondrial division reduces cell death. In order to study whether ALDH2 exerts a myocardial protection effect by regulating and controlling mitochondrial fusion and division, rat myocardial cells H9C2 are selected as a cell model, and ALDH2 activator Alda-1 is administered for treatment under the condition of anoxic-aerobic culture; observing the morphological change of mitochondria under a fluorescence microscope; and observing the apoptosis condition. The experiment shows that after the activity of ALDH2 is activated by using Alda-1, mitochondrion division under the anoxic-reoxygenation condition is obviously reduced, and apoptosis is reduced. It was shown that ALDH2 may exert a regulatory effect on apoptosis by modulating the mitochondrial network. However, the document proves that the activity of ALDH2 of H9C2 cardiac muscle cells is activated by Alda-1 in advance, the mitochondrion division of the H9C2 cardiac muscle cells under the condition of hypoxia-reoxygenation can be inhibited, and the apoptosis of the cardiac muscle cells can be reduced, namely, the mitochondrion with higher activity of ALDH2 can better cope with the hypoxia-reoxygenation, the mitochondrion damage is not easy to occur, and the method is based on in vitro cell experiment verification. Furthermore, there is literature (McCully J D, Cowan D B, Pacak C A, et al. injection of isolated mitochondria along early recovery for cardiac protection. am J Physiol Heart physical biol 2009,296(1): H94-H105) that the use of or addition of ROS scavenger MPG to mitochondria injected into ischemic areas during reperfusion of whole myocardial ischemia failed to exert cardioprotective effects.

Therefore, it is unknown whether or not the improvement of the function of the exogenous mitochondria can enhance the therapeutic effect of the ischemia reperfusion injury in mitochondrial transplantation.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a preparation capable of enhancing the treatment effect of ischemia-reperfusion injury of mitochondrial transplantation and a preparation method and application thereof.

In a first aspect, the invention provides an application of a mitochondrial preparation in preparing a medicament for preventing and treating myocardial ischemia-reperfusion injury, wherein the mitochondrial preparation is obtained by separating myocardial cells after ALDH2 is activated by Alda-1.

As a preferred example of the present invention, the preparation method of the mitochondrial preparation comprises:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-21 mu M, and drying for 11-13 hours;

c) and (5) after the intervention is finished, separating mitochondria, and dissolving the mitochondria in PBS to obtain the mitochondrial preparation.

More preferably, the final concentration of Alda-1 in step b) is 20. mu.M, and the intervention time is 12 hours.

In a second aspect, the present invention provides a mitochondrial preparation activated by ALDH2 for treating myocardial ischemia-reperfusion injury, and a preparation method of the mitochondrial preparation comprises:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-21 mu M, and drying for 11-13 hours;

c) and (5) after the intervention is finished, separating mitochondria, and dissolving the mitochondria in PBS to obtain the mitochondrial preparation.

As a preferred embodiment of the present invention, the final concentration of Alda-1 in step b) is 20. mu.M, and the intervention time is 12 hours.

In a third aspect, the present invention provides a method for preparing an ALDH2 activated mitochondrial preparation for treating myocardial ischemia reperfusion injury, comprising the steps of:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-21 mu M, and drying for 11-13 hours;

c) after the intervention is finished, mitochondria are separated and dissolved in PBS, and the mitochondrial preparation is obtained.

More preferably, the final concentration of Alda-1 in step b) is 20. mu.M, and the intervention time is 12 hours.

In a second aspect, the present invention provides a mitochondrial preparation activated by ALDH2 for treating myocardial ischemia-reperfusion injury, the preparation method of the mitochondrial preparation comprises:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-21 mu M, and drying for 11-13 hours;

c) and (5) after the intervention is finished, separating mitochondria, and dissolving the mitochondria in PBS to obtain the mitochondrial preparation.

As a preferred embodiment of the present invention, the final concentration of Alda-1 in step b) is 20. mu.M, and the intervention time is 12 hours.

The invention has the advantages that:

1. at present, the research on transplantation of mitochondria in China is relatively rare, and how to treat transplanted mitochondria to enhance the treatment effect of ischemia-reperfusion injury is much less involved. Based on abundant research experiments, the inventor of the application firstly activates ALDH2 in myocardial cell mitochondria through a small molecular activator Alda-1 of ALDH2, and separates the myocardial cell mitochondria to carry out ischemia reperfusion injury transplantation of the mitochondria after ALDH2 activation, finds that the mitochondria activated by ALDH2 can obviously increase the productivity of the myocardial cells, reduce the apoptosis of the myocardial cells after the myocardial ischemia reperfusion injury and improve the myocardial ischemia reperfusion injury, and shows that the preparation stored in PBS can enhance the treatment effect by firstly applying Alda-1 treatment to the separated myocardial cells and then extracting the mitochondria. The application of the ALHD2 activated mitochondrial transplantation in the process of recanalization of blood vessels of patients with acute myocardial infarction clinically can ensure myocardial energy supply, improve the myocardial contraction and relaxation function, reduce reperfusion injury and inhibit the occurrence of heart failure.

2. In the preparation process of the mitochondria preparation, appropriate cells and culture conditions are selected, the intervention time, concentration and time of Alda-1 are determined, the separated mitochondria are stored in PBS, the activation effect on the mitochondria is obvious, the activity of the mitochondria is well maintained, and excellent treatment effect is achieved.

Drawings

Figure 1 is a technical scheme of the study. Separating adult mouse myocardial cells by an enzyme digestion method, intervening with ALDH2 micromolecule activator Alda-1, respectively separating normal and ALDH2 activated myocardial cell mitochondria to construct a mouse ischemia reperfusion injury model, carrying out multi-point transplantation on the differently treated mitochondria during myocardial reperfusion, and detecting the cardiac function of the mouse and the functional change of a single myocardial cell after 24 hours of transplantation.

FIGS. 2 to 5: evaluation of the efficacy of ALDH2 activity-dependent mitochondrial transplantation in the treatment of myocardial ischemia reperfusion injury in mice. In each figure, C: control, I: IR group, M: IR + MITO (IR + mitochondrial transplantation), a: IR + a (mitochondrial transplantation after IR + ALDH2 activation). P <0.05, p <0.01, p <0.001, p < 0.0001.

FIG. 2: the transplantation of ALDH2 activated mitochondria can obviously increase the left ventricular ejection fraction of the heart of mice with ischemia-reperfusion injury and improve the cardiac function.

FIG. 3: transplantation of ALDH 2-activated mitochondria decreased cardiomyocyte apoptosis levels (green fluorescence) by significantly increasing the retention in cardiomyocytes.

FIG. 4: after the mitochondria of ischemic mice of different treatment groups are transplanted and then are perfused for 24 hours, exogenous fluorescence labeled mitochondria (red) and fluorescence intensity thereof are analyzed.

FIG. 5: analyzing the expression condition of myocardial apoptosis index clear-caspase 3 after different groups of mitochondria are transplanted.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings.

Example 1

The technical route is as follows:

1) adult mouse cardiomyocytes were isolated and 20 μ M Alda-1 was subjected to intervention to activate the mitochondrial enzyme ALDH2 for 12 hours.

2) After Mitotracker red fluorescence labeling of primary cardiomyocyte mitochondria, normal and ALDH2 activated cardiomyocyte mitochondria were isolated for use.

3) Gas anesthetized C57BL/6 mice, left anterior descending coronary ligation performed myocardial ischemia for 45 minutes, followed by release of the ligature to the left ventricular myocardium by multipoint transplantation of normal and ALDH2 activated mitochondria with reperfusion for 24 hours.

4) Detecting the cardiac function of the mice with ischemia-reperfusion injury after 24 hours, analyzing the positioning of mitochondria in myocardial cells, and analyzing the apoptosis level and myocardial infarction area of ischemia-reperfusion myocardium before and after different mitochondria transplantation.

The specific contents are as follows:

1 Material

The mice referred to in the following examples were purchased from the animal center for Calvin experiments in Changzhou, the mitochondrial isolation kit and the TUNNEL assay kit were purchased from Byunnan Biotech, Inc., Alda-1, Evans blue and TTC were provided by Sigma, USA, and the animal experiments were approved by the ethical Committee of subsidiary Zhongshan Hospital, university of Compound denier.

2 method

2.1 mitochondrial preparation

Adult Mouse (C57BL/6) cardiomyocytes (see A Simplicied, Langendorff-Free Method for Concomitant Isolation of Viable Cardiac Myocytes and Nonmyocytes From the Adult Mouse heart Res.2016; 119: 909. sup.920) were isolated by enzymatic digestion, cultured in 5. mu.g/mL laminin-coated plates, and cultured in CM primary cardiomyocyte medium (see above) for 12 hours at 37 ℃ by addition of a final concentration of 20. mu.M of Alda-1 to the culture system. And then incubating normal and Alda-1 treated myocardial cells by using a Mitotracker red fluorescent probe working solution, incubating for 30 minutes in an incubator at 37 ℃, separating by using a Biyun antenna mitochondrial extraction kit to obtain normal and ALDH2 activated myocardial cell mitochondria, dissolving in PBS, storing at 0-4 ℃, and counting for later use.

2.2 ischemia-reperfusion injury model construction

Adult C57BL/6 mice were previously subjected to a chest depilation treatment, 2% isoflurane gas was used to inhale anesthetized mice, left chest open surgery was performed to separate the pectoralis major from the pectoralis minor, the pleura and pericardium were opened with hemostats, the heart was squeezed out to ligate the anterior left descending branch with 6-0 silk thread for ischemia for 45min, and then the heart was squeezed out twice to release the ligature to perform myocardial reperfusion. At the moment, according to the scale of a 1ml insulin syringe, 25 microlitres are pushed into each small grid at four points on the surface of the left ventricle of the cardiac muscle, each point is pushed into each small grid, and the injection concentration is 10 at four points ⁶ Mitochondria, per cellMice were co-transplanted with 100 microliters of normal or ALDH2 to activate mitochondria. The IR group was injected with the same volume of PBS only, and the control group was sham operated, with open chest extrusion of the heart and threading with no ligature. And 3, performing ultrasonic cardiogram after 24 hours of reperfusion to detect the heart function of the mouse, performing histopathology to detect the myocardial structural change of the mouse, performing immunofluorescence to detect the location of mitochondria after transplantation, and performing Evans blue/TTC staining to detect the infarct size of the myocardium of the mouse.

2.3 Evans blue/TTC staining

After 24 hours of reperfusion, mice of different treatment groups were subjected to intraperitoneal injection of 0.2ml of 1% pentobarbital sodium, the mice were anesthetized and fixed on a small animal operating table, the heart was exposed quickly by opening the chest, the anterior descending branch of the mice was rebunched using 6-0 silk thread, and the left atrial appendage was exposed at the same time. The insulin syringe absorbs 1 percent Evans blue dye solution and injects the mixture into the left auricle until the non-ligature area of the heart is filled with the dye solution. The heart was removed and washed three times in sterile saline to remove excess stain. Non-myocardial tissue was removed and left and right ventricles were left intact. The heart was transferred to a-80 ℃ freezer and frozen for 30min, and then the frozen hardened heart was removed and rapidly cut evenly into 4-5 transverse slices on ice. The heart slices were placed in 1% TTC staining solution, protected from light and water bath at 37 ℃ for 30min, and then the hearts were flattened and fixed in 4% paraformaldehyde. And taking pictures under a stereoscopic microscope for observation. White for myocardial infarction areas (not stained by Evans blue and TTC stain), red for ischemic areas (not stained by Evans blue but TTC stain), and blue for non-ischemic areas (stained by Evans blue and TTC stain). Infarct size is calculated as a percentage of the area at risk.

3 results

Cardiac function assessment of mice in different intervention groups was normalized to the left ventricular myocardial ejection fraction (EF%) and the short axis shortening rate (FS%) of left asphyxia in mice. As shown in fig. 2, the results showed that the myocardial EF and FS values were significantly decreased in the IR group mice compared to the control group C, and that mitochondrial transplantation after ALDH2 activation could significantly increase the EF and FS values after IR.

The myocardial infarction area and the apoptosis level are also reference indexes for evaluating the influence of mitochondrial transplantation on the heart function of IR mice, Evans blue/TTC staining is used in the research to evaluate the myocardial infarction area (% risk zone) of different intervention groups, the Mitotracker red fluorescence labeled mitochondria are used for staining myocardial tissue frozen sections, TUNNEL apoptosis is used for detecting the myocardial apoptosis level, and the research result shows that compared with the IR group, the ALDH2 activated mitochondrial transplantation can obviously reduce the myocardial infarction area of the IR mice, increase the survival amount of mitochondria in the myocardium and reduce the apoptosis level of the myocardial cells (figure 3).

To further confirm the persistence of mitochondria after transplantation in mouse myocardium, fluorescence intensity of mouse myocardium was examined by immunofluorescence labeling mitochondria and reperfusion transplantation 24 hours after transplantation in the study, the results in fig. 4 show that mitochondria transplantation after ALDH2 activation significantly increases mitochondrial fluorescence intensity (i.e., mitochondrial retention) in mouse myocardium.

While the expression level of cleared-caspase 3 represents the myocardial apoptosis, this example detects the expression of cleared-caspase 3 in the myocardium of different intervention groups (fig. 5), and the results show that the mitochondrial transplantation after activation of ALDH2 can reverse the apoptosis level of IR myocardium of mice to be significantly increased.

The research results show that the mitochondrial transplantation after the activation of ALDH2 can obviously inhibit apoptosis, improve the myocardial EF value and the FS value, obviously improve the cardiac function and reduce the ischemia-reperfusion injury of mice compared with the simple mitochondrial transplantation.

Example 2

The following processing is performed in the same time as the above research, and the supplementary contents are as follows:

intervention group 1: mitochondrial transplantation after IR + ALDH2 activation, which is different from example 1 in that the treatment time of Alda-1 is 8 h;

intervention group 2: mitochondrial transplantation after IR + ALDH2 activation, which is different from example 1 in that the treatment time of Alda-1 is 10 h;

intervention group 3: mitochondrial transplantation after IR + ALDH2 activation, which is different from example 1 in that the treatment time of Alda-1 is 14 h;

intervention group 4: mitochondrion transplantation after IR + ALDH2 activation was performed in the same manner as in example 1 except that myocytes were centrifuged and mitochondrion was extracted directly, dissolved in PBS containing 20. mu.M Alda-1, and injected.

The statistical results of EF% and FS% of mice in each intervention group are shown in the following table, compared with the control group C, the EF and FS values of the mice in the intervention group 2 are obviously increased, the difference is statistically significant (P <0.05), the EF and FS values of the mice in the intervention group 1 and the intervention group 3 are not obviously increased, and the difference is not statistically significant (P > 0.05). The results show that the method disclosed by the invention is appropriate in treatment, the activity of ALDH2 in mitochondria is improved to the greatest extent, and the activity of the mitochondria is kept.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims (4)

1. An application of a mitochondrial preparation in preparing a medicament for preventing and treating myocardial ischemia-reperfusion injury, the mitochondrial preparation is separated from myocardial cells after ALDH2 is activated by Alda-1, and the preparation method of the mitochondrial preparation comprises the following steps:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-21 mu M, and intervening for 11-13 hours;

c) and (5) after the intervention is finished, separating mitochondria, and dissolving the mitochondria in PBS to obtain the mitochondrial preparation.

2. Use according to claim 1, wherein the final concentration of Alda-1 in step b) is 20 μ Μ, the intervention time is 12 hours.

3. A method for preparing an ALDH2 activated mitochondrial preparation for treating myocardial ischemia reperfusion injury, comprising the steps of:

a) culturing isolated cardiomyocytes with a cell survival rate of more than 80% in a culture dish coated with laminin in advance by using a primary cardiomyocyte culture medium under a culture condition of 37 ℃;

b) adding Alda-1 into the culture system after the cells adhere to the wall normally, wherein the final concentration is 18-22 mu M, and intervening for 11-13 hours;

c) and (5) after the intervention is finished, separating mitochondria, and dissolving the mitochondria in PBS to obtain the mitochondrial preparation.

4. The preparation method according to claim 3, wherein the final concentration of Alda-1 in step b) is 20 μ M and the intervention time is 12 hours.

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