CN111249309A - ALDH2 activated mitochondria preparation for treating myocardial ischemia reperfusion injury and preparation method and application thereof - Google Patents
ALDH2 activated mitochondria preparation for treating myocardial ischemia reperfusion injury and preparation method and application thereof Download PDFInfo
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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
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 hypoxia caused by myocardial infarction can effectively increase oxygen supply of the myocardial cell to relieve myocardial cell injury caused by ischemia by causing oxidative stress and accumulation of mitochondrial intermediate metabolites so as to lead the myocardial cell hypoxia structure and function to be seriously injured, but the injured mitochondria can cause abnormal recovery of myocardial cell vitality and contraction function, and the process can cause the injured mitochondria to be injured again. Therefore, the process of carrying out mitochondrial transplantation on the patient with revascularization is expected to improve the energy metabolism of the myocardial cells of the patient, 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 manufacture 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.106Intervention was performed by normal mitochondria at/mL. The results show that after reperfusion for 120min, the left ventricular diastolic pressure and post-contraction of the mitochondria transplantation treatment group are obviously increased to 75% and 83% of the normal state respectively, and the level is 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, the process of the present invention,in addition, as proved by people in heart models of rabbits and pigs, the mitochondria transplantation has obvious effect on the protection of cardiac ischemia/reperfusion injury. ".
Although the mitochondrial transplantation has an extremely wide application prospect, how to improve the repair capability of the damaged myocardial cells after the exogenous mitochondrial transplantation is a key limiting factor of the mitochondrial 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 fission during myocardial ischemia-reperfusion leads to the formation of large numbers of fragmented mitochondria, while inhibition of mitochondrial fission 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 cell models, and ALDH2 activator Alda-1 is given 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 activation of ALDH2 activity of H9C2 cardiomyocytes by Alda-1 in advance can inhibit mitochondrion division of H9C2 cardiomyocytes under the condition of hypoxia-reoxygenation and reduce apoptosis of the cardiomyocytes, namely mitochondria with higher ALDH2 activity can better cope with hypoxia-reoxygenation, mitochondrial 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 fails to exert cardioprotective effects during reperfusion of global myocardial ischemia by using or adding ROS scavenger MPG to mitochondria for injection into the ischemic area.
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 separated from 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 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.
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 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.
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 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.
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 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.
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 experiences, the inventor firstly activates ALDH2 in myocardial cell mitochondria through a small molecular activator Alda-1 of ALDH2, separates the myocardial cell mitochondria to carry out ischemia reperfusion injury transplantation of the mitochondria after ALDH2 activation, and 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 ischemia reperfusion injury and improve the myocardial ischemia reperfusion injury, and shows that the preparation obtained by treating with Alda-1 and then extracting the mitochondria after the separation of the myocardial cells can enhance the treatment effect. The mitochondrial transplantation for realizing ALHD2 activation during the blood vessel recanalization of a patient 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 an 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 by ALDH2 micromolecule activator Alda-1, respectively separating normal and ALDH2 activated myocardial cell mitochondria, constructing a mouse ischemia reperfusion injury model, carrying out multi-point transplantation on the differently treated mitochondria during myocardial reperfusion, and detecting the heart function of the mouse and the functional change of single myocardial cells 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 survival in cardiomyocytes.
FIG. 4: after 24 hours of reperfusion after the mitochondria transplantation of ischemic mice of different treatment groups, 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 ALDH 2-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 culture of animal Heart disease research Res.2016; 119: 909. sup.920) were isolated by enzymatic digestion, cultured in 5. mu.g/mL laminin-coated plates, and incubated in CM primary cardiomyocyte medium (see above) at 37 ℃ for 12 hours with a final concentration of 20. mu.M of Alda-1 added to the culture. 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 the normal and ALDH2 activated myocardial cell mitochondria 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, pectoralis major and pectoralis minor muscles were separated, pleura and pericardium were opened with hemostats, the anterior left descending branch of the heart was subjected to slipknot ligation ischemia for 45min with 6-0 silk thread by extrusion, and then the heart was extruded 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 points6Mitochondria, 100 microliters of normal or ALDH2 activated mitochondria were co-transplanted per mouse. 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 of the heart without ligation. 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 positioning 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, anesthetized and fixed on a small animal operating table, the heart was exposed quickly by opening the chest, and the anterior descending branch of the mice was re-ligated with 6-0 silk thread while the left atrial appendage was exposed. The insulin syringe absorbs 1 percent Evans blue dye solution and injects the Evans blue dye solution into the left auricle of the heart until the non-ligation 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 is removed and left and right ventricles are left intact. Transferring the heart to a refrigerator at-80 deg.C, freezing for 30min, taking out the frozen hardened heart, and rapidly cutting the hardened heart transversely into 4-5 pieces 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 areas of myocardial infarction (not stained by Evans blue and TTC stains), red areas of myocardial ischemia (not stained by Evans blue but by TTC stains), and blue areas of non-ischemia (stained by Evans blue and TTC stains). Infarct size is calculated as a percentage of the area at risk.
3 results
Assessment of cardiac function in mice of different intervention groups was normalized to the left ventricular myocardial ejection fraction (EF%) and the short axis shortening rate of left asphyxia (FS%) in the 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 the IR mice, Evans blue/TTC staining is used in the research to evaluate the myocardial infarction area (% risk zone) of different intervention groups, myocardial tissue frozen section staining is carried out by using Mitotracker red fluorescence labeled mitochondria, and TUNNEL apoptosis is used for detecting the apoptosis level of myocardial cells, 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 the mitochondria in the myocardium and reduce the apoptosis level of the myocardial cells (figure 3).
To further confirm the presence of mitochondria after transplantation in mouse myocardium, fluorescence intensity of mouse myocardium was examined by immunofluorescence labeling mitochondria in studies after 24 hours of reperfusion transplantation, and fig. 4 results show that mitochondrial transplantation after ALDH2 activation significantly increased mitochondrial fluorescence intensity (i.e., mitochondrial retention) of 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, and compared with the simple mitochondrial transplantation, the method can obviously improve the cardiac function and reduce the ischemia reperfusion injury of mice.
Example 2
The following processing is performed in the meantime with the above research, and 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: mitochondrial transplantation after IR + ALDH2 activation was performed in the same manner as in example 1 except that the mitochondria were extracted directly after isolation of cardiomyocytes, 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 table 1, and compared with the control group C, the values of EF and FS in the mice in the intervention group 2 are significantly increased, the difference is statistically significant (P <0.05), the values of EF and FS in the mice in the intervention group 1 and the intervention group 2 are not significantly 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 (7)
1. An application of a mitochondrial preparation in preparing a medicament for preventing and treating myocardial ischemia-reperfusion injury, wherein the mitochondrial preparation is separated from myocardial cells after ALDH2 is activated by Alda-1.
2. The use according to claim 1, wherein the preparation of the mitochondrial preparation is carried out by:
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.
3. Use according to claim 2, wherein the final concentration of Alda-1 in step b) is 20 μ Μ, the intervention time is 12 hours.
4. An ALDH2 activated mitochondrial preparation for treating myocardial ischemia reperfusion injury, which is prepared by 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 18-22 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.
5. The mitochondrial preparation of claim 4 wherein the Alda-1 final concentration in step b) is 20 μ Μ with an intervention time of 12 hours.
6. 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.
7. The method according to claim 6, wherein the final concentration of Alda-1 in step b) is 20 μ Μ and the intervention time is 12 hours.
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